1. Immunology and Inflammation

Regulation of positive and negative selection and TCR signaling during thymic T cell development by capicua

  1. Soeun Kim
  2. Guk-Yeol Park
  3. Jong Seok Park
  4. Jiho Park
  5. Hyebeen Hong
  6. Yoontae Lee  Is a corresponding author
  1. Department of Life Sciences, Pohang University of Science and Technology (POSTECH), Republic of Korea
  2. Institute of Convergence Science, Yonsei University, Republic of Korea
https://doi.org/10.7554/eLife.71769
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Cite this article
  1. Soeun Kim
  2. Guk-Yeol Park
  3. Jong Seok Park
  4. Jiho Park
  5. Hyebeen Hong
  6. Yoontae Lee
(2021)
Regulation of positive and negative selection and TCR signaling during thymic T cell development by capicua
eLife 10:e71769.
https://doi.org/10.7554/eLife.71769
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Abstract

Central tolerance is achieved through positive and negative selection of thymocytes mediated by T cell receptor (TCR) signaling strength. Thus, dysregulation of the thymic selection process often leads to autoimmunity. Here, we show that Capicua (CIC), a transcriptional repressor that suppresses autoimmunity, controls the thymic selection process. Loss of CIC prior to T-cell lineage commitment impairs both positive and negative selection of thymocytes. CIC deficiency attenuated TCR signaling in CD4+CD8+ double-positive (DP) cells, as evidenced by a decrease in CD5 and phospho-ERK levels and calcium flux. We identified Spry4, Dusp4, Dusp6, and Spred1 as CIC target genes that could inhibit TCR signaling in DP cells. Furthermore, impaired positive selection and TCR signaling were partially rescued in Cic and Spry4 double mutant mice. Our findings indicate that CIC is a transcription factor required for thymic T cell development and suggests that CIC acts at multiple stages of T cell development and differentiation to prevent autoimmunity.

Editor's evaluation

This paper focuses on the transcriptional regulation of the T cell receptor (TCR) signaling cascade and would be of interest to those studying T cell development and differentiation. The authors employ a conditional deletion of the Capicua (Cic) gene, a transcriptional repressor previously shown to be involved in regulating autoimmunity and follicular helper T (Tfh) cell differentiation, and now show that loss of CIC in hematopoietic cells leads to defects in TCR-β selection as well as in positive and negative selection of developing thymocytes. The overall conclusions are well supported by the findings.

https://doi.org/10.7554/eLife.71769.sa0

Introduction

T cells play a crucial role in the adaptive immune system’s defense against external invasion. To distinguish between self and non-self, T cells use their T cell receptors (TCRs) to recognize peptide-loaded major histocompatibility complex (MHC) molecules and respond to many types of antigens with an enormous TCR repertoire. T cells with a specific TCR are generated in the thymus through a series of processes, ranging from random rearrangement of TCR gene segments to selection processes that entail apoptosis of inappropriate cells (Klein et al., 2014).

T cell development in the thymus is tightly regulated to prevent the generation of nonfunctional or self-reactive T cells. Progenitor cells from bone marrow (BM) become CD4-CD8- double-negative (DN) cells and undergo a process called β-selection, which selects only T cells with a functional TCRβ chain. DN cells that have passed β-selection then become CD4+CD8+ double-positive (DP) cells and undergo positive selection, which selects T cells that bind to self-peptide ligands loaded onto MHC (self-pMHC) molecules. These CD4+CD8+ DP cells then develop into CD4+CD8- single-positive (CD4+ SP) or CD4-CD8+ single-positive (CD8+ SP) T cells. Negative selection, also known as clonal deletion, selectively removes T cells that bind with high affinity to self-pMHC during the DP and SP stages. Because the intensity and duration of TCR signaling based on TCR affinity to self-pMHC are the major determinants of selection (Gascoigne et al., 2016), defects in the TCR signaling component lead to abnormal T cell development and alteration of the TCR repertoire (Fu et al., 2010; Sakaguchi et al., 2003). Impairment of thymic selection caused by decreased TCR signaling destroys central tolerance, and consequently induces autoimmunity accompanied by the expansion of the CD44hiCD62Llo activated effector/memory T cell population (Fu et al., 2010; Sakaguchi et al., 2003; Sommers et al., 2002).

Capicua (CIC) is an evolutionarily conserved transcriptional repressor that regulates the receptor tyrosine kinase (RTK) signaling pathway in Drosophila and mammals (Jiménez et al., 2000; Jiménez et al., 2012). CIC is expressed in two different isoforms: long (CIC-L) and short (CIC-S), which differ in their amino termini (Lee, 2020). CIC recognizes specific octameric DNA sequences (5ʹ-T(G/C)AATG(A/G)(A/G)–3ʹ) within its target gene promoter region and represses its expression (Kawamura-Saito et al., 2006; Shin and Hong, 2014; Weissmann et al., 2018). Several genomic and transcriptomic analyses have identified CIC target genes, including ETV1, ETV4, ETV5, SPRY4, SPRED1, DUSP4, and DUSP6, in various cell types of cells (Fryer et al., 2011; Weissmann et al., 2018; Yang et al., 2017). Activation of the RTK/RAS/MAPK signaling pathway phosphorylates and inactivates CIC via dissociation from target gene promoters, cytoplasmic translocation, and/or proteasomal degradation (Jiménez et al., 2012; Keenan et al., 2020; Lee, 2020). CIC also mediates the ERK-DUSP6-negative feedback loop to maintain ERK activity within the physiological range in mammals (Ren et al., 2020).

We previously reported that murine CIC deficiency spontaneously induced lymphoproliferative autoimmune-like phenotypes (Park et al., 2017). Hematopoietic lineage cell-specific (Vav1-Cre-mediated knockout) and T-cell-specific (Cd4-Cre-mediated knockout) Cic-null mice commonly exhibit autoimmune-like symptoms including hyperglobulinemia, increased serum anti-dsDNA antibody levels, and tissue infiltration of immune cells, accompanied by increased frequency of CD44hiCD62Llo T and follicular helper T (Tfh) cells in the spleen (Park et al., 2017). However, these phenotypes were more severe in Cicf/f;Vav1-Cre mice than in Cicf/f;Cd4-Cre mice. Moreover, enlargement of secondary lymphoid organs was observed in Cicf/f;Vav1-Cre mice but not in Cicf/f;Cd4-Cre mice (Park et al., 2020; Park et al., 2017). These results indicate that deletion of Cic alleles in hematopoietic stem and progenitor cells leads to more severe peripheral T-cell hyperactivation and autoimmunity than the Cd4-Cre-mediated Cic deletion in CD4+CD8+ DP thymocytes. We also reported enhanced peripheral T cell hyperactivation in Cicf/f;Vav1-Cre mice relative to Cicf/f;Cd4-Cre mice were caused by CIC deficiency in T cells rather than in other types of immune cells (Park et al., 2020), suggesting that this abnormality could be caused by impaired control of early thymic T cell development in Cicf/f;Vav1-Cre mice. It was reported that the frequency of CD4-CD8-CD44+CD25- DN1 cells was increased in adult stage-specific Cic-null mice (Tan et al., 2018). Taken together, these studies suggest that CIC plays a crucial role in thymic T cell development.

In this study, we found that CIC regulates thymic T cell development from the CD4-CD8- DN stage and positive and negative selection of thymocytes, primarily during the CD4+CD8+ DP stage. TCR signaling was significantly attenuated in DP cells of Cicf/f;Vav1-Cre mice, thereby impairing both positive and negative selections in Cicf/f;Vav1-Cre mice. We also identified Spry4, Dusp4, Dusp6, and Spred1 as CIC target genes that could potentially contribute to the reduced TCR signaling strength and impaired thymic selection processes in Cicf/f;Vav1-Cre mice. Our findings demonstrate that CIC is a critical regulator of TCR signaling in DP cells and thymic T cell development.

Results

Changes in the frequencies of thymic T cell subsets over development in Cicf/f;Vav1-Cre mice

To examine the role of CIC in thymic T cell development, we first evaluated the levels of CIC in multiple subsets of developing thymocytes using homozygous FLAG-tagged Cic knock-in (CicFLAG/FLAG) mice (Park et al., 2019). Flow cytometry for FLAG-CIC revealed that CIC levels were relatively high in CD4-CD8- DN and immature CD8+ single positive (ISP) cells than in cells at later developmental stages, such as CD4+CD8+ DP and CD4+ or CD8+ SP subsets (Figure 1A). We then determined the frequency and number of thymic T cell subsets in Cicf/f (WT), Cicf/f;Cd4-Cre, and Cicf/f;Vav1-Cre mice at 7 weeks of age. The total number of thymocytes was comparable among WT, Cicf/f;Cd4-Cre, and Cicf/f;Vav1-Cre mice (Figure 1B). However, T cell development during the DN stage was abnormal in Cicf/f;Vav1-Cre mice, as evidenced by an increased frequency of CD44hiCD25hi DN2 and CD44loCD25hi DN3 subsets at the expense of the CD44loCD25lo DN4 subset (Figure 1C). As expected, these changes were not detected in Cicf/f;Cd4-Cre mice (Figure 1C), because Cd4-Cre was not expressed in DN cells (Lee et al., 2001). We also found a slight increase in the frequency of total thymic γδT cells, which are derived from DN3 thymocytes (Ciofani and Zúñiga-Pflücker, 2010), in Cicf/f;Vav1-Cre mice (Figure 1—figure supplement 1A). However, the frequencies of mature and type 1 and 17γδT cells were comparable among WT, Cicf/f;Cd4-Cre, and Cicf/f;Vav1-Cre mice (Figure 1—figure supplement 1B and C).

Figure 1 with 3 supplements see all
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Altered T cell development in Cicf/f;Vav1-Cre mice.

(A) Capicua (CIC) protein levels in thymic T cell subsets. Thymocytes of CicFLAG/FLAG mice (N = 7) were subjected to flow cytometry using anti-FLAG antibody. Representative histograms of CIC-FLAG expression are shown in the left panel for each cell population. The difference in mean fluorescence intensity (ΔMFI) of the CIC-FLAG signal was calculated by subtraction of the MFI value of the isotype control from that obtained by anti-FLAG antibody staining. DN1: CD4-CD8-CD44hiCD25lo, DN2: CD4-CD8-CD44hiCD25hi, DN3: CD4-CD8-CD44loCD25hi, DN4: CD4-CD8-CD44loCD25lo, ISP: immature CD8+ single positive cells (CD4-CD8+TCRβloCD24hi), DP: CD4+CD8+, SM: semi-mature (CD69+TCRβhi), and M: mature (CD69-TCRβhi). Lineage (CD11b, CD11c, CD19, NK1.1, Gr-1, γδTCR, and TER119)-negative (Lin-)-gated cells were analyzed for DN cell populations. (B–D) Flow cytometric analysis of thymocytes from 7-week-old Cicf/f, Cicf/f;Cd4-Cre, and Cicf/f;Vav1-Cre mice. (B) Total numbers of thymocytes for each genotype. N = 11, 7, and 12 for Cicf/f, Cicf/f;Cd4-Cre, and Cicf/f;Vav1-Cre mice, respectively. (C) Proportions of DN1-4 subsets for each genotype. N = 8, 7, and seven for Cicf/f, Cicf/f;Cd4-Cre, and Cicf/f;Vav1-Cre mice, respectively. Lin--gated cells were used for analysis of DN cell populations. (D) Frequencies and numbers of DN, DP, CD4+ SP, and CD8+ SP cells for each genotype. N = 11, 7, and 12 for Cicf/f, Cicf/f;Cd4-Cre, and Cicf/f;Vav1-Cre mice, respectively. (E) Flow cytometric analysis of thymocytes isolated from 1-week-old Cicf/f and Cicf/f;Vav1-Cre mice using CD4 and CD8 markers. Total thymocyte numbers, and the frequencies and numbers of DN, DP, CD4+ SP, and CD8+ SP subsets in mice of each genotype, as well as representative plots are presented. N = 5 and 4 for Cicf/f and Cicf/f;Vav1-Cre mice, respectively. Data are representative of two independent experiments. Bar graphs represent the mean and SEM. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. One-way ANOVA with Tukey’s multiple comparison test (B–D) and unpaired two-tailed Student’s t-test (E) were used to calculate the corresponding p values. See also Figure 1—source data 1.

Figure 1—source data 1

Raw data for Figure 1.

https://cdn.elifesciences.org/articles/71769/elife-71769-fig1-data1-v2.xlsx

We previously reported that the frequency of DN, DP, and SP cells was comparable between WT and Cicf/f;Vav1-Cre mice at 9 weeks of age (Park et al., 2017). However, at 7 weeks of age, Cicf/f;Vav1-Cre mice exhibited a mild block in the formation of SP cells in the thymus, and the frequency of DP cells was significantly increased in Cicf/f;Vav1-Cre mice compared to WT and Cicf/f;Cd4-Cre mice, whereas that of SP cells decreased (Figure 1D). This difference was not statistically significant when calculated using the cell numbers (Figure 1D). To clarify whether CIC deficiency affected thymic SP cell formation, we performed the same analysis using WT and Cicf/f;Vav1-Cre mice at 1 week of age. The frequency and number of CD4+ and CD8+ SP cells were significantly decreased in 1-week-old Cicf/f;Vav1-Cre mice (Figure 1E), suggesting that CIC regulates SP cell development in the thymus. Consistent with this result, both CD4+ and CD8+ T cell populations were substantially reduced in the spleens of 1-week-old Cicf/f;Vav1-Cre mice (Figure 1—figure supplement 2A). In accordance with the observations made at 7 weeks of age (Figure 1C), a partial block in the DN3-to-DN4 transition was also found in Cicf/f;Vav1-Cre mice at 1 week of age (Figure 1—figure supplement 2B). Total SP thymocytes include peripheral T cells that are recirculated into the thymus as well as SP cells matured from DP thymocytes. The proportion of recirculating CD24loCD73+ CD4+ SP cells (Owen et al., 2019) in the thymus slightly increased in Cicf/f;Vav1-Cre mice that were 1 week old (Figure 1—figure supplement 3A), and was similar between WT and Cicf/f;Vav1-Cre mice at 9 weeks old (Figure 1—figure supplement 3B). These results suggest that the decreased frequency of CD4+ SP thymocytes in 1-week-old Cicf/f;Vav1-Cre mice was not due to reduced accumulation of recirculating CD4+ SP cells in the thymus, and that the disappearance of the decrease in the frequency of SP thymocytes in Cicf/f;Vav1-Cre mice at 9 weeks old (Park et al., 2017) might not have resulted from the differential accumulation of recirculating peripheral T cells in the thymus between WT and Cicf/f;Vav1-Cre mice. Together, these data demonstrate that CIC is involved in the regulation of thymic T cell development.

Stable CIC expression in DP cells of Cicf/f;Cd4-Cre mice

After obtaining the results shown in Figure 1D, we wondered why the frequency of DP cells was comparable between WT and Cicf/f;Cd4-Cre mice because Cic alleles were supposed to be deleted in DP cells by Cd4-Cre (Lee et al., 2001). Western blotting for CIC in multiple developing thymic T cell subsets from WT, Cicf/f;Cd4-Cre, and Cicf/f;Vav1-Cre mice provided the answer. Although CIC expression disappeared in all tested thymic T cell subsets of Cicf/f;Vav1-Cre mice, CIC was still substantially expressed in the DP cells of Cicf/f;Cd4-Cre mice (Figure 2A). This appeared to be caused by CIC protein stability, because the loxP site-flanked genomic regions containing exons 9–11 of Cic were completely removed in DP cells by Cd4-Cre and Vav1-Cre (Figure 2B). These data suggest that the impaired SP cell formation in the thymus of Cicf/f;Vav1-Cre mice is attributed to the loss of CIC in DN and/or DP cells rather than that in SP cells, because CIC expression was not detected in SP thymocytes from Cicf/f;Cd4-Cre and Cicf/f;Vav1-Cre mice (Figure 2A).

Figure 2
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Stable capicua (CIC) expression in double-positive (DP) cells of Cicf/f;Cd4-Cre mice.

(A) Western blotting for detection of CIC levels in double-negative 1–2 (DN1-2), DN3, DN4, immature CD8+ single-positive (ISP), double-positive (DP), CD4+ SP, and CD8+ SP cells from Cicf/f, Cicf/f;Cd4-Cre, and Cicf/f;Vav1-Cre mice. Lin--gated DN1-2, DN3, DN4, ISP, DP, CD4+ SP, and CD8+ SP (TCRβhi) cells were sorted from mice of each genotype. (B) PCR analysis of Cic knock-out efficiency in Lin--gated DN, DP, CD4+ SP, and CD8+ SP (TCRβhi) cells from Cicf/f, Cicf/f;Cd4-Cre, and Cicf/f;Vav1-Cre mice. Genomic DNA was extracted from sorted DN, DP, CD4+ SP, and CD8+ SP cells and subjected to PCR amplification of a part of the floxed Cic allele. Upper panel, schematic of the Cic floxed allele. Arrowheads indicate the primers used for PCR. Lower panel, representative agarose gel image of PCR products. The arrow indicates the PCR products corresponding to the amplified part of the floxed Cic allele. See also Figure 2—source data 1.

Figure 2—source data 1

Original and labelled files for western blot and PCR gel images.

https://cdn.elifesciences.org/articles/71769/elife-71769-fig2-data1-v2.zip

Impaired thymic positive selection in Cicf/f;Vav1-Cre mice

Defects in thymic positive selection that occurs during the CD4+CD8+ DP developmental stage often result in a decrease in the CD4+ and CD8+ SP cell populations (Fischer et al., 2005; Lesourne et al., 2009; Neilson et al., 2004; Wang et al., 2012). Therefore, we examined thymic positive selection in Cicf/f;Vav1-Cre mice. First, we analyzed thymocytes from 7-week-old WT, Cicf/f;Cd4-Cre, and Cicf/f;Vav1-Cre mice for the expression of CD69 and TCRβ using flow cytometry. The frequency of CD69+TCRβhi cells, which represent post-positive-selection thymocytes (Fu et al., 2009), were significantly decreased in Cicf/f;Vav1-Cre mice compared with that of WT or Cicf/f;Cd4-Cre mice (Figure 3A). This reduction was also observed in the thymus of Cicf/f;Vav1-Cre mice at 1 week of age (Figure 1—figure supplement 2C). Next, we determined the effect of CIC deficiency on thymic positive selection in mice expressing the MHC class I-restricted H-Y TCR transgene specific to the H-Y male antigen (Kisielow et al., 1988) or the MHC class II-restricted OT-II TCR transgene specific to ovalbumin (Barnden et al., 1998) at 7–9 weeks of age. Defects in SP thymocyte development were much more severe in TCR-transgenic hematopoietic lineage cell-specific Cic-null (H-Y;Cicf/f;Vav1-Cre or OT-II;Cicf/f;Vav1-Cre) mice than in non-transgenic polyclonal Cicf/f;Vav1-Cre mice: CIC deficiency dramatically reduced the populations of CD8+ and CD4+ SP cells in female H-Y TCR transgenic and OT-II TCR transgenic mice, respectively (Figure 3B and C, Figure 3—figure supplement 1A). The frequency of CD24loTCRβhi ISP cells among total CD8+ SP thymocytes was significantly increased in female H-Y;Cicf/f;Vav1-Cre mice (Figure 3—figure supplement 1B), excluding the possibility that the decreased frequency of CD8+ SP thymocytes could result from decreased ISP cell formation in female H-Y;Cicf/f;Vav1-Cre mice. Taken together, these results indicate that CIC controls the positive selection of thymocytes.

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Defective positive selection in the absence of capicua (CIC).
Figure 3—source data 1

Raw data for Figure 3.

https://cdn.elifesciences.org/articles/71769/elife-71769-fig3-data1-v2.xlsx

Impaired thymic negative selection in Cicf/f;Vav1-Cre mice

Most thymocytes expressing autoreactive TCRs are eliminated through negative selection during the DP and SP developmental stages (Klein et al., 2014). The strong TCR signal induced by the interaction of a self-reactive TCR with the self-pMHC molecule triggers apoptosis of autoreactive T cells (Hogquist and Jameson, 2014). Interestingly, we found that the expression of Bcl2, a representative anti-apoptotic gene (Vaux et al., 1988) and an inhibitor of negative selection (Williams et al., 1998), was markedly increased in DP cells but not in DN and SP cells from Cicf/f;Vav1-Cre mice (Figure 4A). Furthermore, the TCR stimulation-induced expression of Nur77, an orphan receptor that promotes apoptosis and negative selection of thymocytes (Calnan et al., 1995), was significantly reduced in DP cells from Cicf/f;Vav1-Cre mice compared to that in WT mice (Figure 4B). To determine whether CIC regulates thymic negative selection, we analyzed thymocytes from male H-Y;Cicf/f and H-Y;Cicf/f;Vav1-Cre mice by flow cytometry. As previously reported (Kisielow et al., 1988), massive negative selection of DP and CD8+ SP thymocytes was observed in male H-Y TCR transgenic mice (Figure 4C), in which the male-specific H-Y autoantigens are expressed in thymic antigen-presenting cells. The CD8+ SP population and total thymic cell number significantly increased in male H-Y;Cicf/f;Vav1-Cre mice compared to male H-Y;Cicf/f mice (Figure 4C and D, Figure 4—figure supplement 1A), whereas the frequency of CD24loTCRβhi ISP cells among all CD8+ SP thymocytes, which were less than 10%, was comparable between male H-Y;Cicf/f and H-Y;Cicf/f;Vav1-Cre mice (Figure 4—figure supplement 1B). Overall, our data demonstrate that CIC is required for negative selection mediated by TCR activation-induced apoptosis.

Figure 4 with 2 supplements see all
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Defective negative selection in the absence of capicua (CIC).

(A) qRT-PCR quantification of Bcl2 expression in double-negative (DN), double-positive (DP), CD4+ single-positive (4SP), and CD8+ SP (8SP, TCRβhi) cells of Cicf/f and Cicf/f;Vav1-Cre mice. N = 3 for each group. (B) Flow cytometric analysis of Nur77 expression in DP thymocytes from Cicf/f and Cicf/f;Vav1-Cre mice. Freshly isolated thymocytes were treated with plate-coated anti-CD3 (5 μg/ml) and anti-CD28 (10 μg/ml) antibodies for 2 hr and subsequently subjected to flow cytometry. Representative histograms for Nur77 expression in DP thymocytes of Cicf/f (black line) and Cicf/f;Vav1-Cre (red line) mice overlaid with isotype (gray shaded) and unstimulated (dotted line) control histograms (left), the frequency of Nur77+ DP cells (middle), and Nur77-derived mean fluorescence intensities (MFIs) of total and Nur77+ DP cells (right) are presented. N = 3 for each genotype. Data are representative of two independent experiments. (C and D) Thymocytes from male H-Y;Cicf/f (N = 3) and H-Y;Cicf/f;Vav1-Cre (N = 6) mice were analyzed for CD4 and CD8 expression. (C) Representative flow cytometry plots (left) and frequencies (top, right) and numbers (bottom, right) of DN, DP, CD4+ SP (4SP), and CD8+ SP (8SP) cells are shown. (D) Total thymocyte numbers. Data are representative of two independent experiments. Bar graphs represent the mean and SEM. *p < 0.05 and **p < 0.01. Unpaired two-tailed Student’s t-test was used to calculate the corresponding p values. See also Figure 4—source data 1.

Figure 4—source data 1

Raw data for Figure 4.

https://cdn.elifesciences.org/articles/71769/elife-71769-fig4-data1-v2.xlsx

Changes in the frequency of the variable (V) segments of the TCRβ chain in CIC-deficient CD4+ SP thymocytes

Developing T cells mature into SP cells through thymic selection processes. Consequently, numerous T cells with various TCRs constitute the TCR repertoire. As positive and negative selection determine the fate of T cells based on their TCR, defects in these processes can lead to changes in the TCR repertoire (Lu et al., 2019; Martínez-Riaño et al., 2019). To find out whether CIC deficiency alters the TCR repertoire of SP thymocytes, we analyzed the frequency of the variable (V) segments of the TCRβ (Vβ) chain on CD4+ non-Treg (CD4+CD25-Foxp3-) and Treg (CD4+CD25+Foxp3+) cells from WT and Cicf/f;Vav1-Cre mice by flow cytometry using antibodies against 10 different Vβ chains. Among the 10 Vβ chains tested, the frequency of CD4+ non-Treg cells expressing eight different Vβ chains was significantly changed: the frequency of Vβ5.1/5.2, 9, 11, 12, and 13 chain-expressing CD4+ non-Treg cells was decreased in Cicf/f;Vav1-Cre mice, whereas that of Vβ 6, 7, and 8.1/8.2 chain-expressing CD4+ non-Treg cells was increased (Figure 4—figure supplement 2A). In the Treg cell compartment, the frequencies of Vβ7 and Vβ9 were significantly changed in Cicf/f;Vav1-Cre mice compared to WT mice (Figure 4—figure supplement 2B). These data suggest that impaired thymic selection due to CIC deficiency might change the TCR repertoire of thymic SP cell populations.

Attenuated TCR signaling in CIC-deficient DP thymocytes

Because both positive and negative selection are mediated by TCR signaling strength (Klein et al., 2014), we investigated whether CIC regulates the TCR signaling pathway. First, we analyzed the surface expression of CD5, which is strongly correlated with TCR signaling intensity (Azzam et al., 1998), in DP and SP thymocytes from WT, Cicf/f;Cd4-Cre, and Cicf/f;Vav1-Cre mice. CD5 levels were substantially decreased in DP cells from Cicf/f;Vav1-Cre mice compared to those from WT or Cicf/f;Cd4-Cre mice, whereas this change was subtle in the CD4+ SP cell population and absent in the CD8+ SP cell population (Figure 5A). Consistent with previous results (Figure 3A, Figure 1—figure supplement 2C), the frequency of CD69+ post-selection DP thymocytes was significantly decreased in Cicf/f;Vav1-Cre mice (Figure 5B). Decreased CD5 expression was mostly observed in CD69- pre-selection DP thymocytes from Cicf/f;Vav1-Cre mice (Figure 5C). These results were recapitulated in TCR transgenic mice (Figure 5—figure supplement 1). Next, we examined the activation of the TCR signaling pathway in thymocytes from WT, Cicf/f;Cd4-Cre, and Cicf/f;Vav1-Cre mice upon TCR stimulation. As with CD5 levels, calcium influx sharply decreased in DP cells from Cicf/f;Vav1-Cre mice, but not in SP thymocytes (Figure 5D). We also observed a moderate decrease in calcium influx in DP cells from Cicf/f;Cd4-Cre mice (Figure 5D), suggesting that CIC sensitively regulates TCR activation-induced calcium influx in DP thymocytes. To further evaluate CIC regulation of TCR signaling in DP thymocytes, we assessed the activation of key TCR signaling cascade components in DP cells of WT, Cicf/f;Cd4-Cre, and Cicf/f;Vav1-Cre mice after TCR stimulation by treatment with anti-CD3 and anti-CD4. Among the components tested, including ZAP-70, PLCγ, ERK, JNK, and p38, phospho-ERK levels were significantly decreased in DP cells from Cicf/f;Vav1-Cre mice compared to those from WT and Cicf/f;Cd4-Cre mice (Figure 5E and F). Together, these results demonstrate that CIC deficiency attenuates TCR signaling by inhibiting calcium influx and ERK activation, especially in DP thymocytes.

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Attenuated TCR signaling in capicua (CIC)-deficient double-positive (DP) thymocytes.

(A) Thymocytes from 7-week-old Cicf/f, Cicf/f;Cd4-Cre, and Cicf/f;Vav1-Cre mice were FACS-gated into the DP, CD4+ single-positive (SP), and CD8+ SP (TCRβhi) cell population, and the CD5 mean fluorescence intensity (MFI) was measured. Representative histograms of CD5 expression in each cell population (left) and calculated CD5 MFI values (right) are shown (N = 4 per group). (B and C) DP cells from Cicf/f (N = 4) and Cicf/f;Vav1-Cre (N = 3) mice were analyzed for CD69 and CD5 expression. (B) Representative flow cytometry plots (left) and frequencies (right) of CD69+ DP cells are shown. (C) Representative histograms of CD5 expression in CD69+ or CD69- DP cells (left) and CD5 MFI values in each cell population (right) are shown. (D) TCR stimulation-induced Ca2+ influx in DP, CD4+ SP, and CD8+ SP (TCRβhi) thymocytes from 7-week-old Cicf/f, Cicf/f;Cd4-Cre, and Cicf/f;Vav1-Cre mice. Data are representative of at least three independent experiments. (E and F) Western blot analysis of TCR cascade component activation in DP thymocytes from 7-week-old Cicf/f, Cicf/f;Cd4-Cre, and Cicf/f;Vav1-Cre mice. Sorted DP cells were stimulated with soluble anti-CD3 and anti-CD4 antibodies for the times indicated. (E) Representative western blot images are shown. (F) Signal densities were measured using ImageJ software and are presented as ratios of phosphorylated to total forms for each protein (N = 3). Graphs represent the mean and SEM. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. #p < 0.05 and ##p < 0.01 (comparison between Cicf/f;Cd4-Cre and Cicf/f;Vav1-Cre mice). One-way (A) or two-way (F) ANOVA with Tukey’s multiple comparison test and unpaired two-tailed Student’s t-test (B and C) were used to calculate the corresponding p values. See also Figure 5—source data 1, Figure 5—source data 2.

Figure 5—source data 1

Raw data for Figure 5.

https://cdn.elifesciences.org/articles/71769/elife-71769-fig5-data1-v2.xlsx
Figure 5—source data 2

Original and labeled files for western blot images.

https://cdn.elifesciences.org/articles/71769/elife-71769-fig5-data2-v2.zip

Abnormal thymic T cell development and reduced TCR signaling intensity in Cicf/f;Vav1-Cre mice are T cell-intrinsic

Although most of the cells that make up thymocytes are developing T cells, other types of immune cells, including B cells and dendritic cells, also exist in small proportions and participate in the regulation of T cell development (Klein et al., 2014). Because CIC expression was abolished in whole immune cells of Cicf/f;Vav1-Cre mice, it is unclear whether abnormal thymic T cell development and decreased TCR signaling in Cicf/f;Vav1-Cre mice were T cell-intrinsic or -extrinsic. To clarify this issue, we generated mixed BM chimeric mice by transferring the same number of BM cells from Thy1.1/Thy1.2 heterozygous WT and Thy1.1/Thy1.1 homozygous Cicf/f;Vav1-Cre mice into irradiated Thy1.2/Thy1.2 homozygous WT recipient mice (mixed WT:Cicf/f;Vav1-Cre BM chimera), and analyzed thymic T cells in the chimeras after 8 weeks of reconstitution (Figure 6A). Similar to the observations in Cicf/f;Vav1-Cre mice (Figure 1C–E, Figure 1—figure supplement 2B), the frequency of DN4 and CD4+ SP cells was significantly lower in the CIC-deficient cell compartment than in the WT cell counterpart in the same mixed WT:Cicf/f;Vav1-Cre BM chimeric mice (Figure 6B and C). The frequency of CD69+TCRβhi cells was also significantly lower in the CIC-deficient cell compartment than in the WT cell compartment (Figure 6D), indicating that the impaired positive selection in Cicf/f;Vav1-Cre mice was T cell-intrinsic. Furthermore, similar to the results in Figure 5A, CD5 levels were most strongly reduced in DP thymocytes derived from Cicf/f;Vav1-Cre BM cells (Figure 6E).

Figure 6 with 1 supplement see all
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Altered T cell development and TCR intensity in Cicf/f;Vav1-Cre mice are caused by CIC loss in T cells.

(A) Schematic of the generation and analysis of mixed bone marrow (BM) chimeric mice. Equal numbers of BM cells from Cicf/f;Thy1.1/1.2 (WT) and Cicf/f;Vav1-Cre;Thy1.1/1.1 (KO) mice were mixed and transferred to irradiated B6 (Thy1.2/1.2) recipient mice. Representative FACS plot showing the thymocytes of different origin (left) and their frequencies (right) are presented (N = 10). (B–D) Flow cytometric analysis of thymocytes from mixed BM chimeras (N = 4) for the frequencies of (B) double-negative (DN) subsets based on CD44 and CD25 expression, (C) DN, double-positive (DP), CD4+ single-positive (SP), and CD8+ SP cells, and (D) post-positive selection subsets (CD69+TCRβhi). The CD69+TCRβhi cell population is highlighted by the red box in the flow cytometry plots in (D). (E) Flow cytometric analysis of surface expression levels of CD5 in DP, CD4+ SP, and CD8+ SP (TCRβhi) thymocytes derived from WT and KO BM cells in the same BM chimeric mice (N = 4). Data are representative of two independent experiments. Graphs represent the mean and SEM. *p < 0.05, **p < 0.01, and ****p < 0.0001. Unpaired two-tailed Student’s t-test was used to calculate the corresponding p values. See also Figure 6—source data 1.

Figure 6—source data 1

Raw data for Figure 6.

https://cdn.elifesciences.org/articles/71769/elife-71769-fig6-data1-v2.xlsx

As an alternative approach to examine T cell intrinsic function of CIC in the regulation of thymic T cell development and selection processes, we generated and analyzed the proximal Lck promoter (pLck)-driven Cre-mediated T cell-specific Cic null (Cicf/f;pLck-Cre) mice. Although pLck-Cre is expressed during the DN2 cell stage (Lee et al., 2001), CIC was substantially expressed in DN3 and DN4 cells from Cicf/f;pLck-Cre mice (Figure 6—figure supplement 1A). Moreover, a small amount of CIC still existed in DP thymocytes from Cicf/f;pLck-Cre mice (Figure 6—figure supplement 1A). The frequency of DN thymocytes was comparable between WT and Cicf/f;pLck-Cre mice at 7 weeks of age (Figure 6—figure supplement 1B), concurrent with the expression of a substantial amount of CIC in DN3 and DN4 cells from Cicf/f;pLck-Cre mice (Figure 6—figure supplement 1A). Although there was no significant difference observed in the frequencies of DP and SP subsets between WT and Cicf/f;pLck-Cre mice, there was a tendency for a slight increase in the frequency of DN and DP thymocytes and a decrease in the frequency of CD4+ SP cells (Figure 6—figure supplement 1C), similar to those in Cicf/f;Vav1-Cre mice at 7 weeks of age (Figure 1D). Furthermore, the frequency of CD69+TCRβhi cells and CD5 levels in DP thymocytes were significantly decreased in Cicf/f;pLck-Cre mice compared to WT mice. However, the fold decrease was subtle in Cicf/f;pLck-Cre mice than in Cicf/f;Vav1-Cre mice (Figure 6—figure supplement 1D and E). Overall, Cicf/f;pLck-Cre mice exhibited milder defects in positive selection and TCR signaling in DP thymocytes compared to Cicf/f;Vav1-Cre mice, which was probably due to the incomplete removal of CIC expression in DP thymocytes of Cicf/f;pLck-Cre mice (Figure 6—figure supplement 1A). Taken together, these data suggest that defects in thymic T cell development and TCR signaling in Cicf/f;Vav1-Cre mice are T cell-intrinsic.

Identification of CIC target genes responsible for attenuated TCR signaling in CIC-deficient DP thymocytes

To understand how CIC regulates the thymic selection process and TCR signaling in DP cells at the molecular level, we analyzed the gene expression profiles of DP thymocytes from WT and Cicf/f;Vav1-Cre mice by RNA sequencing. A total of 482 differentially expressed genes (DEGs; fold change >2 and p-value < 0.05), including 263 upregulated and 219 downregulated genes, were identified in CIC-deficient DP cells (Supplementary file 1). Gene Ontology (GO) analysis revealed that genes involved in the inactivation of MAPKs and anti-apoptotic processes were significantly enriched among the upregulated DEGs (Supplementary file 2), consistent with the characteristics found in CIC-deficient DP thymocytes. We also identified several known CIC target genes among the DEGs, including Etv1, Etv4, Etv5, Spry4, Dusp6, Dusp4, and Spred1 (Fryer et al., 2011; Weissmann et al., 2018; Yang et al., 2017; Figure 7A and Supplementary file 1). Of these, Spry4, Dusp4, Dusp6, and Spred1 are of particular interest because they are negative regulators of ERK activation (Kidger and Keyse, 2016; Sasaki et al., 2003; Wakioka et al., 2001). Murine SPRY4 suppresses Ras-independent ERK activation by binding to Raf1 (Sasaki et al., 2003), whereas it represses the insulin receptor and EGFR-induced ERK signaling upstream of Ras in humans (Leeksma et al., 2002). SPRED1 also inhibits the activation of ERK by suppressing Raf activation (Wakioka et al., 2001). DUSP6 specifically dephosphorylates activated ERK1/2, whereas DUSP4 acts on ERK and other MAPKs, such as JNK and p-38 (Caunt and Keyse, 2013). Additionally, SPRY4 suppresses calcium mobilization in HEK293T cells by inhibiting phosphatidylinositol 4,5-biphosphate (PIP2) hydrolysis induced by VEGF-A without affecting PLCγ phosphorylation (Ayada et al., 2009). Thus, we investigated the association between derepression of Spry4, Dusp4, Dusp6, and Spred1 and attenuated TCR signaling in CIC-deficient DP thymocytes. First, we measured the levels of Spry4, Dusp4, Dusp6, and Spred1 in DN, DP, CD4+ SP, and CD8+ SP thymocytes from WT and Cicf/f;Vav1-Cre mice by qRT-PCR. The expression of all four genes was significantly upregulated in DP cells from Cicf/f;Vav1-Cre mice relative to those from WT mice (Figure 7B), verifying the RNA sequencing data (Supplementary file 1 and Figure 7A). The expression of all four genes was also upregulated in CD4+ and CD8+ SP thymocytes from Cicf/f;Vav1-Cre mice; Spry4 and Dusp6 were more dramatically upregulated in DP cells than in SP cells (Figure 7B). Considering the different gene expression changes in each cell type, as well as the previously known functional significance in the regulation of ERK activation and calcium influx, two of the four CIC target genes, Spry4 and Dusp6, were selected for further investigation of their effect on TCR signaling in DP thymocytes.

Figure 7 with 1 supplement see all
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Identification of capicua (CIC) target genes regulating TCR signaling in double-positive (DP) thymocytes.

(A) Volcano plot showing differentially expressed genes (DEGs) in CIC-deficient DP thymocytes (fold change, > 2; adjusted P value, < 0.05). CIC target genes and Bcl2 are indicated at the corresponding dots. (B) qRT-PCR quantification of Spry4, Dusp4, Dusp6, and Spred1 expression in double-negative (DN), DP, CD4+ single-positive (4SP), and CD8+ SP (8SP) thymocytes from Cicf/f and Cicf/f;Vav1-Cre mice. N = 3 for each group. (C–E) Effects of SPRY4 and DUSP6 overexpression on TCR signaling in DP cells. Thymocytes were infected with retroviruses co-expressing GFP and either SPRY4 or DUSP6, and subjected to flow cytometry for (C) ERK activation, (D) Ca2+ influx, and (E) CD5 expression in GFP+ DP thymocytes. Three independent experiments were performed. (F) Thymocytes from 7-week-old Cicf/f, Cicf/f;Vav1-Cre, and Spry4-/-;Cicf/f;Vav1-Cre mice were analyzed for surface expression of CD69 and TCRβ. Representative FACS plots (left) and the frequency of CD69+TCRβhi cells (right) are shown. The CD69+TCRβhi cell population is highlighted by the red box in the FACS plots. N = 4, 6, and four for Cicf/f, Cicf/f;Vav1-Cre, and Spry4-/-;Cicf/f;Vav1-Cre mice, respectively. (G) CD5 levels in DP thymocytes from mice used in (F). Representative FACS plots (left) and CD5 mean fluorescence intensities (MFIs; right) are shown. N = 3, 5, and three for Cicf/f, Cicf/f;Vav1-Cre, and Spry4-/-;Cicf/f;Vav1-Cre mice, respectively. Bar graphs represent the mean and SEM. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Unpaired two-tailed Student’s t-test (B) and one-way ANOVA with Tukey’s multiple comparison test (C, E, F and G) were used to calculate the corresponding p values. See also Figure 7—source data 1.

Figure 7—source data 1

Raw data for Figure 7.

https://cdn.elifesciences.org/articles/71769/elife-71769-fig7-data1-v2.xlsx

We infected thymocytes with retroviruses expressing Spry4 or Dusp6 and analyzed phospho-ERK and CD5 levels and calcium influx by flow cytometry. The levels of phospho-ERK were greatly decreased in both SRPY4- and DUSP6-overexpressing DP thymocytes treated with anti-CD3 (Figure 7C), confirming their inhibitory effect on ERK activation (Groom et al., 1996; Muda et al., 1996; Sasaki et al., 2003). In contrast, calcium influx induced by TCR stimulation was almost completely suppressed in DP thymocytes by SPRY4 overexpression, but not by DUSP6 overexpression (Figure 7D). Moreover, CD5 expression was substantially reduced in SRPY4-overexpressing cells, but not in DUSP6-overexpressing cells (Figure 7E). These data imply that Spry4 derepression might critically contribute to attenuated TCR signaling in CIC-deficient DP thymocytes.

Finally, to determine whether impaired thymic selection and TCR signaling in Cicf/f;Vav1-Cre mice were caused by derepression of Spry4, we generated Cic and Spry4 double mutant (Spry4-/-;Cicf/f;Vav1-Cre) mice and analyzed thymic T cells in WT, Cicf/f;Vav1-Cre, and Spry4-/-;Cicf/f;Vav1-Cre mice at 7–8 weeks of age. The decreased frequency of CD69+TCRβhi cells and CD5 levels of DP thymocytes in Cicf/f;Vav1-Cre mice were significantly, but only partially, rescued in Spry4-/-;Cicf/f;Vav1-Cre mice (Figure 7F and G), suggesting a partial recovery of the CIC deficiency-mediated defective positive selection process by loss of SPRY4. However, the decreased DN4 cell frequency and Nur77 expression in DP thymocytes were not rescued in Spry4-/-;Cicf/f;Vav1-Cre mice (Figure 7—figure supplement 1A and B), indicating that Spry4 derepression alone is not sufficient to induce abnormal DN cell development in Cicf/f;Vav1-Cre mice and suppressed TCR activation-induced apoptosis in CIC-deficient DP thymocytes. Taken together, our findings demonstrate that CIC tightly controls thymic T cell development and TCR signaling by repressing multiple CIC target genes, including Dusp4, Dusp6, Spred1, and Spry4, in a cell type- and/or developmental stage-specific manner.

Discussion

Our study uncovered the role of CIC in thymic T cell development via in-depth analyses of thymocytes in various CIC-deficient mouse models. CIC deficiency partially suppressed the DN3-to-DN4 transition. However, the decreased DN4 cell population did not prevent the formation of thymic DP cells in Cicf/f;Vav1-Cre mice. Thus, this defect was not sufficient to significantly affect the transition from the DN to DP stage of thymic T cell development in Cicf/f;Vav1-Cre mice. Interestingly, similar to Cicf/f;Vav1-Cre mice, ERK-deficient mice also exhibit a partial block in DN3-to-DN4 maturation without defects in maturation to the DP stage of development (Fischer et al., 2005). Because TCR stimulation-induced ERK activation was markedly suppressed in CIC-deficient DP thymocytes (Figure 5E and F), we inferred that the formation of the abnormal DN subset in the thymus of Cicf/f;Vav1-Cre mice was, at least in part, caused by reduced ERK activity in DN cells deficient in CIC. Consistent with this inference, the Spry4, Dusp4, Dusp6, and Spred1 genes, involved in the inhibition of ERK activation (Kidger and Keyse, 2016; Sasaki et al., 2003; Wakioka et al., 2001), were significantly derepressed in CIC-deficient DN cells (Figure 7B). Further study on how CIC controls pre-TCR signaling is critical to better understand CIC regulation of thymic T cell development during the DN stage.

Our previous study showed that the frequency of thymic CD4+ and CD8+ SP cells was comparable between WT and Cicf/f;Vav1-Cre mice at 9 weeks of age (Park et al., 2017). However, thymic T cell subset analysis at a younger age revealed a significant decrease in the frequency of SP thymocytes in Cicf/f;Vav1-Cre mice (Figure 1D and E). This phenotypic change was not due to the enhanced accumulation of circulating peripheral T cells in the thymus of Cicf/f;Vav1-Cre mice at 9 weeks of age (Figure 1—figure supplement 3B). This difference could be explained by the differential effects of positive and negative selection on the formation of SP thymocytes during ontogeny. It is known that negative selection is inefficient early in ontogeny and becomes more efficient with age (He et al., 2013), implying that the process of positive selection might predominantly determine the size of the thymic SP cell population during neonatal life. Since Cicf/f;Vav1-Cre mice display defects in both positive and negative selection, it is conceivable that the decreased frequency of SP thymocytes in 1-week-old Cicf/f;Vav1-Cre mice were caused by a defect in positive selection, which was attenuated by ineffective negative selection at 7 weeks of age or older.

CIC deficiency, especially in DP thymocytes, disrupts the positive and negative selection of thymocytes, as evidenced by the impaired thymic selection process in Cicf/f;Vav1-Cre mice but not in Cicf/f;Cd4-Cre mice, which express significant amounts of CIC proteins in DP thymocytes. Moreover, CIC loss substantially suppressed TCR signaling in DP thymocytes, but not in SP cells. Although Spry4, Dusp4, Dusp6, and Spred1 were all significantly derepressed in SP and DP cells from Cicf/f;Vav1-Cre mice, among these four CIC target genes, Spry4 and Dusp6 were more dramatically derepressed in DP cells than in SP cells in the absence of CIC (Figure 7B). These results suggest that Spry4 and Dusp6 may be CIC target genes primarily responsible for the CIC deficiency-mediated dysregulation of TCR signaling in DP cells and thymic selection processes. However, removal of the Spry4 alleles only partially recovered the TCR signal intensity and positive selection, indicating that CIC regulates multiple target genes, including Spry4, to control TCR signaling and thymic T cell development. Notably, the hyperactivation of peripheral T cells and expansion of the Tfh cell population in Cicf/f;Vav1-Cre mice were also not rescued in Spry4-/-;Cicf/f;Vav1-Cre mice (Figure 7—figure supplement 1C and D). These findings suggest that the partial recovery of the TCR signal intensity and positive selection process by loss of SPRY4 was insufficient to ameliorate the abnormal peripheral T cell phenotypes in Cicf/f;Vav1-Cre mice or that derepression of CIC target genes other than Spry4 could lead to T cell hyperactivation and enhanced Tfh cell formation in Cicf/f;Vav1-Cre mice. Concordantly, we have shown that CIC deficiency promotes Tfh cell differentiation via Etv5 repression (Park et al., 2017). In contrast, CIC deficiency-induced thymic T cell phenotypes were not rescued in Cicf/f;Etv5f/f;Vav1-Cre mice (data not shown). Overall, these findings suggest that CIC regulates various target genes with differential effects to broadly control thymic T cell development and peripheral T cell activation and differentiation.

This study provides insight into how CIC controls autoimmunity. Because defects in thymic selection lead to the breakdown of central tolerance (Xing and Hogquist, 2012), it is conceivable that CIC deficiency during thymic T cell development generates autoreactive T cells, inducing autoimmunity. Therefore, CIC potentially suppresses autoimmunity by controlling the thymic selection process, as well as Tfh cell differentiation (Park et al., 2017). The more severe autoimmune-like phenotypes in Cicf/f;Vav1-Cre mice than in Cicf/f;Cd4-Cre mice (Park et al., 2017) highlight the significant contribution of the impaired thymic selection process to CIC deficiency-induced autoimmunity. To clarify the importance of each regulatory step in the suppression of autoimmunity, it will be necessary to better understand the molecular mechanisms underlying CIC regulation of thymic T cell development and Tfh cell differentiation. Additionally, studies on the role of CIC in T cell development and Tfh cell differentiation in humans should be conducted in the future to improve our understanding of the pathogenesis of autoimmune diseases such as systemic lupus erythematosus.

Materials and methods

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Strain, strain background (Mus musculus)C57BL/6 JThe Jackson LaboratoryRRID:IMSR_JAX:000664
Strain, strain background (M. musculus)B6.Cg-Commd10Tg(Vav1-icre)A2Kio/JThe Jackson LaboratoryRRID:IMSR_JAX:008610
Strain, strain background (M. musculus)B6.Cg-Tg(Cd4-cre)1Cwi/BfluJThe Jackson LaboratoryRRID:IMSR_JAX:022071
Strain, strain background (M. musculus)B6NTac.Cg-Tg(Lck-cre)1Cwi/MmncLee et al., 2001RRID:MMRRC_037396-UNC
Strain, strain background (M. musculus)CicfloxedLu et al., 2017; Park et al., 2017RRID:IMSR_JAX:030555
Strain, strain background (M. musculus)CicFLAG/FLAGPark et al., 2019PMID:30810242
Strain, strain background (M. musculus)B6.Cg-Tg(TcraH-Y,TcrbH-Y)71VboKisielow et al., 1988RRID:MGI:3588781
Strain, strain background (M. musculus)B6.Cg-Tg(TcraTcrb)425Cbn/JThe Jackson LaboratoryRRID:IMSR_JAX:004194
Strain, strain background (M. musculus)B6.Cg-Foxp3tm2Tch/JThe Jackson LaboratoryRRID:IMSR_JAX:006772
Strain, strain background (M. musculus)Spry4-/-This paperN/Agenerated using Spry4tm1a(KOMP)Mbp embryonic stem cells obtained from the UC Davis KOMP repository.
Cell line (Homo-sapiens)Platinum-E (Plat-E) Retroviral Packaging Cell LineCell BiolabsCat# RV-101, RRID:CVCL_B488
AntibodyAnti-mouse CD3ε (Armenian Hamster monoclonal)BioLegendTonbo BiosciencesCat# 100329, RRID:AB_1877171Cat# 50–0031, RRID:AB_2621730FC (1:300)
AntibodyAnti-mouse CD4 (Rat monoclonal)BiolegendBD BiosciencesCat# 100552, RRID:AB_2563053Cat# 562891, RRID:AB_2737870FC (1:300)
AntibodyAnti-mouse CD5 (Rat monoclonal)Biolegend eBioscienceCat# 100625, RRID:AB_2563928Cat# 45-0051-80, RRID:AB_914332FC (1:300)
AntibodyAnti-mouse CD8α (Rat monoclonal)BioLegendCat# 100723, RRID:AB_389304Cat# 100721, RRID:AB_312760FC (1:300)
AntibodyAnti-mouse CD11b (Rat monoclonal)BioLegendCat# 101211, RRID:AB_312794FC (1:300)
AntibodyAnti-mouse CD11c (Armenian Hamster monoclonal)BioLegendCat# 117309, RRID:AB_313778FC (1:300)
AntibodyAnti-mouse CD19 (Rat monoclonal)BD BiosciencesCat# 561738, RRID:AB_10893995FC (1:300)
AntibodyAnti-mouse CD24 (Rat monoclonal)BioLegendCat# 101819, RRID:AB_572010FC (1:300)
AntibodyAnti-mouse CD25 (Rat monoclonal)Tonbo BiosciencesCat# 75–0251, RRID:AB_2621943FC (1:300)
AntibodyAnti-mouse CD44 (Rat monoclonal)BD BiosciencesCat# 553133, RRID:AB_2076224Cat# 561860, RRID:AB_10895375FC (1:300)
AntibodyAnti-mouse CD62L (Rat monoclonal)BD BiosciencesCat# 560516, RRID:AB_1645257FC (1:300)
AntibodyAnti-mouse CD69 (Armenian Hamster monoclonal)BioLegendCat# 104513, RRID:AB_492844Cat# 104545, RRID:AB_2686969FC (1:300)
AntibodyAnti-mouse CD73 (Rat monoclonal)Biolegend eBioscienceCat# 127223, RRID:AB_2716102Cat# 12-0731-81, RRID:AB_763516FC (1:300)
AntibodyAnti-mouse CD90.1 (Mouse monoclonal)eBioscienceCat# 45-0900-82, RRID:AB_2573662FC (1:300)
AntibodyAnti-mouse CD90.2 (Rat monoclonal)BioLegendCat# 140303, RRID:AB_10642686FC (1:300)
AntibodyAnti-mouse CXCR5 (Rat monoclonal)BD BiosciencesCat# 551960, RRID:AB_394301FC (1:100)
AntibodyAnti-mouse TCRβ (Armenian Hamster monoclonal)Tonbo BiosciencesCat# 35–5961, RRID:AB_2621723FC (1:300)
AntibodyAnti-mouse TCR Vβ2 (Rat monoclonal)BioLegendCat# 127908, RRID:AB_1227784FC (1:300)
AntibodyAnti-mouse TCR Vβ5.1/5.2 (Mouse monoclonal)BD BiosciencesCat# 553189, RRID:AB_394697FC (1:300)
AntibodyAnti-mouse TCR Vβ6 (Rat monoclonal)BioLegendCat# 140004, RRID:AB_10643583FC (1:300)
AntibodyAnti-mouse TCR Vβ7 (Rat monoclonal)BioLegendCat# 118308, RRID:AB_893628FC (1:300)
AntibodyAnti-mouse TCR Vβ8.1/8.2 (Rat monoclonal)BioLegendCat# 118408, RRID:AB_1134109FC (1:300)
AntibodyAnti-mouse TCR Vβ8.3 (Armenian Hamster monoclonal)BD BiosciencesCat# 553664, RRID:AB_394980FC (1:300)
AntibodyAnti-mouse TCR Vβ9 (Mouse monoclonal)BioLegendCat# 139804, RRID:AB_10641563FC (1:300)
AntibodyAnti-mouse TCR Vβ11 (Rat monoclonal)BioLegendCat# 125907, RRID:AB_1227781FC (1:300)
AntibodyAnti-mouse TCR Vβ12 (Mouse monoclonal)BioLegendCat# 139704, RRID:AB_10639729FC (1:300)
AntibodyAnti-mouse TCR Vβ13 (Mouse monoclonal)BioLegendCat# 140704, RRID:AB_10639945FC (1:300)
AntibodyAnti-mouse TCRγ/δ (Armenian Hamster monoclonal)eBioscienceCat# 17-5711-81, RRID:AB_842757FC (1:300)
AntibodyAnti-mouse NK-1.1 (Mouse monoclonal)BioLegendCat# 108709, RRID:AB_313396FC (1:300)
AntibodyAnti-mouse TER-119 (Rat monoclonal)eBioscienceCat# 17-5921-81, RRID:AB_469472FC (1:300)
AntibodyAnti-mouse Gr-1 (Rat monoclonal)eBioscienceCat# 17-5931-81, RRID:AB_469475FC (1:300)
AntibodyAnti-mouse TCR H-Y (Mouse monoclonal)eBioscienceCat# 11-9930-81, RRID:AB_465452FC (1:300)
AntibodyAnti-mouse PD-1 (Rat monoclonal)eBioscienceCat# 11-9981-81, RRID:AB_465466FC (1:300)
AntibodyAnti-mouse Nur77 (Mouse monoclonal)eBioscienceCat# 12-5965-82, RRID:AB_1257209FC (1:100)
AntibodyAnti-T-bet (Mouse monoclonal)eBioscienceCat# 12-5825-80, RRID:AB_925762FC (1:100)
AntibodyAnti-RORγt (Rat monoclonal)eBioscienceCat# 12-5825-80, RRID:AB_925762FC (1:100)
AntibodyAnti-Foxp3 (Rat monoclonal)eBioscienceCat# 17-5773-80, RRID:AB_469456FC (1:100)
AntibodyAnti-DYKDDDDK(flag) Tag antibody (Rat monoclonal)BioLegendCat# 637309, RRID:AB_2563147FC (1:100)
AntibodyPE Donkey anti-rabbit IgG (min. x-reactivity) antibody (Donkey Polyclonal)BioLegendCat# 406421, RRID:AB_2563484FC (1:100)
AntibodyAnti-CIC (Rabbit polyclonal)Kim et al., 2015PMID:25653040WB (1:1000)
AntibodyPLCγ1 (D9H10) XP Rabbit mAb antibody (Rabbit monoclonal)Cell Signaling TechnologyCat# 5690, RRID:AB_10691383WB (1:1000)
AntibodyPhospho-PLC 1 (Tyr783) antibody (Rabbit polyclonal)Cell Signaling TechnologyCat# 2821, RRID:AB_330855WB (1:500)
AntibodyZap-70 (99F2) Rabbit mAb antibody (Rabbit monoclonal)Cell Signaling TechnologyCat# 2705, RRID:AB_2273231WB (1:1000)
AntibodyPhospho-Zap-70 (Tyr319)/Syk (Tyr352) antibody (Rabbit polyclonal)Cell Signaling TechnologyCat# 2701, RRID:AB_331600WB (1:500)
AntibodySAPK/JNK antibody (Rabbit polyclonal)Cell Signaling TechnologyCat# 9252, RRID:AB_2250373WB (1:2000)
AntibodyPhospho-SAPK/JNK (Thr183/Tyr185) antibody (Rabbit polyclonal)Cell Signaling TechnologyCat# 9251, RRID:AB_331659WB (1:1000)
Antibodyp44/42 MAPK (Erk1/2) antibody (Rabbit polyclonal)Cell Signaling TechnologyCat# 9102, RRID:AB_330744WB (1:2000)
AntibodyPhospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) antibody (Rabbit monoclonal)Cell Signaling TechnologyCat# 4370, RRID:AB_2315112WB (1:1000)FC (1:100)
AntibodyAnti-p38 MAPK antibody (Rabbit polyclonal)Cell Signaling TechnologyCat# 9212, RRID:AB_330713WB (1:2000)
AntibodyPhospho-p38 MAPK (Thr180/ Tyr182) antibody (Rabbit polyclonal)Cell Signaling TechnologyCat# 9211, RRID:AB_331641WB (1:1000)
Antibodyβ-Actin Antibody (Mouse monoclonal)Santa Cruz BiotechnologyCat# sc-47778, RRID:AB_626632WB (1:1000)
Antibodyα-Tubulin antibody (A-6) (Mouse monoclonal)Santa Cruz BiotechnologyCat# sc-398103, RRID:AB_2832217WB (1:1000)
AntibodyPurified NA/LE Hamster Anti-Mouse CD3e (Armenian Hamster monoclonal)BD BiosciencesCat# 553057, RRID:AB_394590(10 µg/mL)
AntibodyAffiniPure Goat Anti-Armenian Hamster IgG (H + L) (Goat polyclonal)Jackson ImmunoResearch LabsCat# 127-005-160, RRID:AB_2338972(25 µg/mL)
Recombinant DNA reagentpMIGR1-GFP (plasmid)Pear et al., 1998RRID:Addgene_27490
Recombinant DNA reagentpMIGR1-SPRY4-GFP (plasmid)This paperN/ACDS of mouse Spry4 was inserted into pMIGR1-GFP.
Recombinant DNA reagentpMIGR1-DUSP6-GFP (plasmid)This paperN/ACDS of mouse Dusp6 was inserted into pMIGR1-GFP.
Recombinant DNA reagentpCL-Eco (plasmid)Naviaux et al., 1996RRID:Addgene_12371Retrovirus packaging vector
Sequence-based reagentprimers used in qRT-PCR This paper See Supplementary file 3 for sequence information.
Peptide, recombinant proteinStreptavidinSouthern BiotechCat# 7100–01
Peptide, recombinant proteinStreptavidineBioscienceBD BiosciencesCat# 45-4317-80, RRID:AB_10260035Cat# 554067, RRID:AB_10050396FC (1:100)
Commercial assay or kitFixable Viability Dye eFluor 780eBioscienceCat# 65-0865-14
Commercial assay or kitGhost Dye Violet 510Tonbo BiosciencesCat# 13–0870
Commercial assay or kitFoxp3/ Transcription Factor Staining Buffer SeteBioscienceCat# 00-5523-00
Commercial assay or kitBD Cytofix Fixation BufferBD BiosciencesCat# 554655, RRID:AB_2869005
Commercial assay or kitFuGENE HD Transfection ReagentPromegaCat# E2311
Commercial assay or kitRiboExGeneAllCat# 301–002
Commercial assay or kitGoScript Reverse Transcriptase KitPromegaCat# A5001
Commercial assay or kitSYBR Green Realtime PCR Master MixTOYOBOCat# TOQPK-201
Commercial assay or kitEasySep Mouse Streptavidin RapidSpheres Isolation KitStem Cell TechnologiesCat# 19,860
Commercial assay or kitBCA Protein Assay KitPierceCat# 23,225
Commercial assay or kitClarity Western ECL SubstrateBio-RadCat# 1705061
Commercial assay or kitSuperSignal West Dura Extended Duration SubstrateThermo ScientificCat# 34,076
Chemical compound, drugIndo-1, AM, cell permeantInvitrogenCat# I1203
Chemical compound, drugIonomycin from Streptomyces conglobatusSigma-AldrichCat# I9657-1MG
Chemical compound, drugHexadimethrine bromideSigma-AldrichCat# H9268-10G
Software, algorithmFlowJoTree Star Inc.RRID:SCR_008520, https://www.flowjo.com/solutions/flowjo
Software, algorithmImageJNIHRRID:SCR_003070, https://imagej.nih.gov/ij/
Software, algorithmGraphPad Prism 7GraphPad SoftwareRRID:SCR_002798, https://www.graphpad.com/scientific- software/prism/

Mice

All mice were maintained on a C57BL/6 background. Cic-floxed (Lu et al., 2017; Park et al., 2017), Vav1-Cre (de Boer et al., 2003), Cd4-Cre (Lee et al., 2001), pLck-Cre (Lee et al., 2001), FLAG-tagged Cic knock-in (CicFLAG/FLAG) (Park et al., 2019), H-Y TCR transgenic (Kisielow et al., 1988), and OT-II TCR transgenic (Barnden et al., 1998) mice have been described previously. Spry4-/- mice were generated using Spry4tm1a(KOMP)Mbp embryonic stem cells obtained from the UC Davis KOMP repository. All experiments were conducted with age-matched mice, and 7–9 week-old mice were used unless otherwise indicated. Mice of both sexes were randomly allocated to the experimental groups. All mice were maintained in a specific pathogen-free animal facility under a standard 12 hr light/12 hr dark cycle. Mice were fed standard rodent chow and provided with water ad libitum. All experiments were approved by the Institutional Animal Care and Use Committee of Pohang University of Science and Technology.

Cell line

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The Platinum-E (Plat-E) retroviral packaging cell line (Cell Biolabs) was grown in Dulbecco’s modified Eagle’s medium (DMEM, Welgene) supplemented with 10% fetal bovine serum (FBS, Welgene) and penicillin/streptomycin (Gibco). The cells were cultured in a 37 °C incubator with 5% CO2. Mycoplasma contamination was routinely tested using the e-Myco plus Mycoplasma Detection Kit (INtRON Bio).

Flow cytometry

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Surface staining was performed using the following fluorescence-labeled antibodies against: CD3 (145–2 C11; Tonbo Biosciences), CD4 (RM4-5; Biolegend, BD Biosciences), CD5 (53–7.3; Biolegend, eBioscience), CD8α (53–6.7; Biolegend), CD11b (M1/70; Biolegend), CD11c (N418, Biolegend), CD19 (1D3; BD Biosciences), CD24 (M1/69; Biolegend), CD25 (PC61.5; Tonbo Biosciences), CD44 (IM7, BD Biosciences), CD62L (MEL-14; BD Biosciences), CD69 (H1.2F3; Biolegend), CD73 (TY/11.8; eBioscience), CD90.1 (HIS51; eBioscience), CD90.2 (53–2.1; Biolegend), CXCR5 (2G8; BD Biosciences), TCRβ (H57-597; Tonbo Biosciences), TCR Vβ2 (B20.6; Biolegend), TCR Vβ5.1/5.2 (MR9-4; BD Biosciences), TCR Vβ6 (RR4-7; Biolegend), TCR Vβ7 (TR310; Biolegend), TCR Vβ8.1/8.2 (KJ16-133.18; Biolegend), TCR Vβ8.3 (1B3.3; BD Biosciences), TCR Vβ9 (MR10-2; Biolegend), TCR Vβ11 (KT11; Biolegend), TCR Vβ12 (MR11-1; Biolegend), TCR Vβ13 (MR12-4; Biolegend), TCRγ/δ (GL-3; eBioscience), NK-1.1 (PK136; Biolegend), TER-119 (TER-119; eBioscience), Gr-1 (RB6-8C5; eBioscience), TCR H-Y (T3.70; eBioscience), and PD-1 (RMP1-30; eBioscience). For CXCR5 staining, cells were incubated with biotinylated anti-CXCR5 for 30 min and then sequentially incubated with APC- or PerCP- Cy5.5-labelled streptavidin (eBioscience) with other surface antibodies. Live/dead staining was performed using Fixable Viability Dye (FVD) eFluor 780 (eBioscience) or Ghost Dye Violet 510 (Tonbo Biosciences). Intracellular staining was performed using the Foxp3 staining buffer set (eBioscience). For intracellular staining, fluorochrome-labeled antibodies to FLAG (L5; Biolegend), T-bet (4B10; eBioscience), RORγt (B2D; eBioscience), Foxp3 (FJK-16s; eBioscience), and Nur77 (12.14; eBioscience) were used. For phospho-ERK staining, cells were stained with FVD for 30 min on ice, fixed with BD Cytofix for 30 min at room temperature, and permeabilized with cold methanol for at least 30 min at –20 °C. Permeabilized cells were stained with the p-ERK antibody (Cell Signaling Technology), and fluorochrome-labeled antibodies to surface markers and secondary antibody against rabbit IgG (Biolegend) were added. The stained cells were analyzed using an LSRII Fortessa flow cytometer (BD Biosciences) or CytoFLEX LX (Beckman Coulter). Data were analyzed using FlowJo software (TreeStar).

Cell sorting

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Single-cell suspensions of thymocytes were stained for surface markers including lineage (TCRγ/δ, NK-1.1, TER-119, Gr-1, CD11b, CD11c, CD19), lin/DN (CD4-CD8-), ISP (CD4-CD8+TCRβloCD24hi), DP (CD4+CD8+TCRβlo), CD4+ SP (CD4+CD8-TCRβhi), and CD8+ SP (CD4-CD8+TCRβhi) cells were sorted. To sort DN3 (lin-CD4-CD8-CD44loCD25hi) and DN4 (lin-CD4-CD8-CD44loCD25lo) cells, CD8- thymocytes were obtained by negative selection using the EasySep Mouse Streptavidin Rapid Spheres Isolation Kit (Stem Cell Technologies) and cell sorting was performed. A MoFlo-XDP (Beckman Coulter) was used for cell sorting.

Generation of BM chimeric mice

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To create a mixed BM chimera, 1 × 106 BM cells from each donor were mixed and injected intravenously into C57BL/6 recipient mice that had been irradiated (10 Gy). After 8 weeks of recovery, the mice were sacrificed, and thymi were harvested and homogenized to prepare single-cell suspensions. The cells were then stained using flow cytometry.

Analysis of Nur77 expression

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To measure Nur77 expression, freshly isolated thymocytes at 1 × 107 cells/ml were incubated with plate-coated anti-CD3 (5 μg/ml) and anti-CD28 (10 μg/ml) for 2 hr, followed by staining of surface markers (CD4, CD8, and TCRβ) and intracellular staining of Nur77. Samples were analyzed using an LSRII Fortessa flow cytometer or CytoFLEX LX. Data were analyzed using FlowJo software.

In vitro TCR stimulation

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Total thymocytes or sorted DP cells were rested for 30 min in Roswell Park Memorial Institute (RPMI) medium (Welgene) at 37 °C. Cells were then washed with T-cell medium (TCM, RPMI supplemented with 10% FBS [Welgene], 1% penicillin/streptomycin [Gibco], and 0.1% β-mercaptoethanol [Gibco]) and incubated with biotin-conjugated anti-CD3 (60 μg/ml, 145–2 C11) and anti-CD4 (60 μg/ml, GK1.5) for 20 min on ice. Cells were washed and incubated for 5 min on ice with streptavidin (60 μg/ml, SouthernBiotech) for cross-linking and incubated for the indicated time at 37 °C. To analyze p-ERK levels in virus-transduced thymocytes by flow cytometry, unconjugated anti-CD3 (10 μg/ml, 145–2 C11) and goat anti-hamster IgG (25 μg/ml, Jackson Immunoresearch) were used to stimulate TCR. The cells were incubated for 2 min at 37 °C for TCR stimulation. One milliliter of Cold PBS was added at the end of stimulation and cell pellets were lysed for western blotting or further stained with anti-p-ERK antibody for flow cytometry.

Calcium influx measurement

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Freshly isolated thymocytes or virus-transduced thymocytes were incubated with 4 μM Indo-1-AM (Invitrogen) in TCM at 37 °C for 40 min. Cells were washed twice with TCM and incubated with soluble anti-CD3 (10 μg/ml) and fluorochrome-conjugated antibodies for surface markers (CD4, CD8, and TCRβ) in TCM on ice for 20 min. The cells were then washed with TCM and warmed before cross-linking. Goat anti-hamster IgG (25 μg/ml) was added to cross-link anti-CD3, and the signals were measured by flow cytometry. Ionomycin was added to ensure that the T cells were effectively loaded with Indo-1. The emission wavelength ratios of Ca2+-bound to unbound Indo-1 were analyzed using an LSRII Fortessa flow cytometer.

Plasmids and retroviral transduction

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The coding sequences (CDSs) of mouse Spry4 or Dusp6 with enzyme sites XhoI/EcoRI were amplified by PCR and cloned into the MigR1 retroviral vector (Pear et al., 1998). Viruses were generated through transient cotransfection of Plat-E cells with cloned retroviral vectors and the pCL-Eco helper plasmid (Imgenex) (Naviaux et al., 1996). Briefly, 2.5 × 106 Plat-E cells were plated in 100 mm plates. The next day, cells were transfected with 3.6 μg of retroviral vector and 2.4 μg of pCL-Eco using FuGENE HD transfection reagent (Promega). Retrovirus-containing supernatants were harvested twice at 48 h and 72 h after transfection and filtered with a 0.22 μm syringe filter (Millipore). For retroviral transduction, 107 thymocytes from wild-type adult mice were mixed with 0.5 ml of the viral supernatant and 1.5 ml of fresh TCM in the presence of 4 μg/ml polybrene (Sigma) and seeded in a well of a six-well plate for spin-infection at 1000 g for 90 min at 25 °C. After spin infection, the cells were incubated for 48 hr before harvesting.

Western blotting

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Sorted cells or stimulated cells were lysed in RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM PMSF, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 x Roche Complete Protease Inhibitor Cocktail, and 1 x Roche Phosphatase Inhibitor Cocktail). Protein concentrations were measured using a BCA kit (Pierce). Equal amounts of protein samples were separated by 9% SDS-PAGE and transferred onto nitrocellulose membranes (Bio-Rad). The following primary antibodies were used: anti-CIC (homemade) (Kim et al., 2015), anti-PLCγ1 (#5690, Cell Signaling), anti-p-PLCγ1 (#2821, Cell Signaling), anti-ZAP-70 (#2705, Cell Signaling), anti-p-ZAP-70 (#2701, Cell Signaling Technology), anti-JNK (#9252, Cell Signaling), anti-p-JNK (#9251, Cell Signaling), anti-ERK (#9102, Cell Signaling), anti-p-ERK (#4370, Cell Signaling), anti-p38 (#9212, Cell Signaling), anti-p-p38 (#9211, Cell Signaling), anti-β-actin (#sc-47778, Santa Cruz), and anti-α-tubulin (#sc-398103, Santa Cruz). Membranes were incubated with secondary antibodies conjugated to horseradish peroxidase (HRP) and developed using Clarity Western ECL Substrate (Bio-Rad) or SuperSignal West Dura (Thermo Scientific). Images were acquired using an ImageQuant LAS 500 instrument (GE Healthcare).

RNA isolation, cDNA synthesis, and qRT-PCR

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Total RNA was extracted from sorted cells using RiboEx (GeneAll) and 0.2–1 μg was subjected to cDNA synthesized using the GoScript Reverse Transcription system (Promega) according to the manufacturer’s instructions. SYBR Green real-time PCR master mix (TOYOBO) was used for qRT-PCR analysis. The primers used for qRT-PCR are listed in Supplementary file 3.

RNA sequencing and data analysis

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Thymi from Cicf/f and Cicf/f;Vav1-Cre mice were dissected and homogenized into single-cell suspensions for cell sorting. DP thymocytes were sorted on the basis of surface markers of lin-CD4+CD8+TCRβlo, and total RNA was extracted with RiboEx. The library for mRNA sequencing was generated using the TruSeq Stranded Total RNA LT Sample Prep Kit (Illumina), and sequencing was performed using NovaSeq 6,000 (Illumina). Trimmed reads were mapped to the mouse reference genome (mm10 RefSeq) using HISAT2, and the transcripts were assembled using StringTie. DEGs were generated using edgeR and genes with fold changes > 2 and P-values < 0.05, were selected for Gene Ontology (GO) analyses on the basis of biological processes using the DAVID website (https://david.ncifcrf.gov/).

Statistical Analysis

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Statistical analyses were performed using GraphPad Prism 7 (https://www.Graphpad.com, La Jolla, CA, USA). All experiments were independently performed more than three times. Two-tailed Student’s t-tests were used to obtain P-values between the two groups. One-way or two-way ANOVA with Tukey’s multiple comparison test was used to calculate P-values among the three different groups. Statistical significance was set at P < 0.05. Error bars indicate standard error of the mean (SEM).

Data availability

The Gene Expression Omnibus (GEO) accession number for the RNA sequencing data of DP thymocytes reported in this paper is GSE173909. All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 1, 2, 3, 4, 5, 6, and 7.

The following data sets were generated
    1. Lee Y
    2. Kim S
    (2021) NCBI Gene Expression Omnibus
    ID GSE173909. RNA Sequencing Analysis of Gene Expression Profiles in WT and CIC-deficient DP Thymocytes.
    https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE173909

References

    1. Vaux DL
    2. Cory S
    3. Adams TM
    (1988)
    Bcl-2 promotes the survival of haemotopoeitic cells and cooperates with c-myc to immortalize pre-B cells
    Nature 258:1955–1957.

Decision letter

  1. Juan Carlos Zúñiga-Pflücker
    Reviewing Editor; University of Toronto, Sunnybrook Research Institute, Canada
  2. Tadatsugu Taniguchi
    Senior Editor; Institute of Industrial Science, The University of Tokyo, Japan
  3. Juan Carlos Zúñiga-Pflücker
    Reviewer; University of Toronto, Sunnybrook Research Institute, Canada

Our editorial process produces two outputs: (i) public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.

Decision letter after peer review:

Thank you for submitting your article "Regulation of positive and negative selection and TCR signaling during thymic T cell development by capicua" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, including JC Zúñiga-Pflücker as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Tadatsugu Taniguchi as the Senior Editor.

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

1) The main shared concerns were about how the data is presented and interpreted; additional information/discussion is required to fully appreciate the quality and appropriateness of the conclusions. The discussion disproportionately focuses on potential defects in beta-selection and thymic Treg differentiation that were only superficially studied in this manuscript. At this stage, perhaps the interpretation of these results should be tempered.

2) Please address the recommendations to the authors listed below by the reviewers, with an emphasis on Ref #3 concerns, as well as the corrected highlighted by Ref #2.

Reviewer #1 (Recommendations for the authors):

1. The main conclusion is that TCR signaling strength is affected by the increased expression of antagonists of the ERK/MAPK signaling pathway, as well as calcium, which are clearly supported by the observations shown. One would also predict that γ/δ TCR signaling to be affected during development, and the authors should show whether gd T cell development or differentiation is also affected, in particular as it pertains to the preTCR vs gd-TCR checkpoint.

2. One surprising finding is that in the bone marrow chimera experiments, when using equal numbers of WT and Cic-deficient cells, that the thymocytes seem to have equal contributions of WT and Cic-deficient cells, as shown in Figure 6A. However, the actual frequencies of Wt vs Cic-/- cells are not shown and should be included. If these are similar, it would be curious given the defect in preTCR signaling , which typically accounts for a large number of downstream DP thymocytes. This should be further discussed in the text.

Reviewer #2 (Recommendations for the authors):

– Figure 1C: FACS plots are not representative of the graphs (seems like CD4-cre mice display an intermediate phenotype).

– Figure 1D has no legend.

– Figure 1D : FACS plot doesn't reflect the graphs (increased DP cell population in CD4-cre mice).

– Figure 1E: These results suggest that a portion of the SP thymocytes in older mice may be recirculating SP cells. The authors should do an analysis of SP thymocyte maturation (show TCRβ vs CD24 for example) to investigate this. Also, they should determine if the DN3/4 block is seen in young mice.

– Figure 2: Is CIC protein absent in DN1/2 thymocytes of Vav-cre mice?

– Figure 5 : It seems odd that Calcium flux is affected by CIC deletion and not PLCγ phosphorylation. Do the authors have a theory why?

– Figure 6 : To avoid transferring other immune cells like B and DC and answer the question the author are asking with this experiment, bone marrow cells must be lineage depleted before injection. Was this the case? (method section mention transfer of whole BM).

– pLCK model is interesting to discriminate impact of CIC deletion on DN cells from DP cells. Authors should describe this model and show if they recapitulate the DP/SP phenotype without the DN stages being affected.

– Since the authors base their explanation of the auto-immune phenotype on impaired thymocyte selection in the Vav-cre model, why is there no impact on thymocyte development but still signs of autoimmunity in CD4-cre mice, as mentioned in the previous paper? Authors should discuss these discrepancies.

Reviewer #3 (Recommendations for the authors):

1) Spry4 (and DUSP6) as a target responsible for much of the phenotype observed in CIC-deficient thymocytes is intriguing. However, it is not clear to this reviewer how the over-expression studies were performed. What is the efficiency of retroviral transduction in, presumably adult, thymocytes? Given nearly full suppression of p-ERK and calcium induction after activation in cells reported to be over-expressing Spry4 (or DUSP6), presumably the transduced cells were tracked – what marker was used?

2) There is a rather striking decrease in CD5 on DP cells on TCR transgenic CIC-deficient DP cells. Is this due solely to differences in the proportion of DP cells undergoing thymic selection (e.g. CD69+) or because of differences in the strength of signal itself? Indeed, differences in CD5 on polyclonal DP cells from Cicf/f;Vav1-Cre versus control DP are also quite striking. While the (presumably) post-selection DP cells expressing higher levels of CD5 seem to express similar levels of this molecule, the bulk population (presumably pre-selection) is significantly lower. What do the authors make of this? Are the majority of DP cells sensing no TCR stimulation at all? It seems as if this might be the case even with ex vivo stimulation; at least based on the provided histogram, a larger percentage of DP thymocytes appear to fail to upregulate Nur77 after in vitro stimulation while those cells that do upregulate Nur77 seem to do so similar levels whether CIC-deficient or control. Further, why is TCR signaling more impacted at the DP stage than at the SP stage in the absence of CIC?

3) There appear to be differences in the extent to which CIC impacts CD4+ and CD8+ SP thymocyte numbers at different stages of ontogeny. The authors imply that mature T cells that have recirculated to the thymus in adult mice (the 7 week old mice presented in this manuscript or as previously reported in 9 week old mice) may mask any striking differences in the relative proportions and numbers of CD4+ and CD8+ SP thymocytes in CIC-deficient as compared to control mice. In younger mice, there are significantly fewer mature thymocytes in the absence of CIC. Whether this is due to differences in the recirculated mature T cell population is less clear than implied; this could be due to differences in the selection processes that accompany T cell development at different stages of ontogeny. One would need to use appropriate markers (e.g. CD73) or reporters (Rag-GFP) to make this distinction.

4) Careful explanation of the experimental set up and conclusions from the TCR sequencing studies would be appreciated. I do not understand the argument for the longer CDR3 sequences in the CIC KO conventional CD4 T cell populations as being 'pre-selection-like'; what does this imply? It appears as if the main conclusion of the TCR sequencing data is that the differences in the repertoire predominantly lie in the Treg population; outside of TCR sequence analysis this subset is not analyzed in the current manuscript. Are there overt differences in thymic Treg development in the absence of CIC?

5) Gating strategies and representative flow plots, as well as clear descriptions of the gates in the figure legends, for all analyses would be appreciated. It is not always clear, for example, if lineage+ cells have been removed from DN gates, whether mature T cells have been gated on TCRbhi cells, whether the conventional CD4+ SP population used for TCR sequencing includes CD25+ Treg progenitors, etc. In addition, representative histograms are not always provided for MFI analysis; this is important to understand, for example with the CIC-Flag tag, the extent to which expression is heterogenous in a population; clear statements about the population for which MFI is calculated (e.g. for Figure 4B, is the MFI calculated for the population in the positive gate or for the total population) should be added.

6) Consider splitting some of the data onto separate graphs (e.g. Figure 3C and D) as it is very difficult to appreciate the noted significant differences in terms of percentages and cell numbers when the symbols are against the x axis, for example.

7) Please revisit the appropriateness of the t test for assessing statistical significance across three mouse strains.

8) Please ensure that biological and experimental replicates are clearly noted for each experiment. For example, how many mice were used for the TCR sequencing experiments?

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "Regulation of positive and negative selection and TCR signaling during thymic T cell development by capicua" for further consideration by eLife. Your revised article has been reviewed by 3 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Tadatsugu Taniguchi as the Senior Editor.

The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:

The remaining required revisions are clearly outlined within the detailed reviewers comments below.

Reviewer #1 (Recommendations for the authors):

The authors have comprehensively addressed most of my initial concerns, however additional points need to be clarified. In particular, the differences in pLck-cre vs Vav-cre mice should be better addressed more clearly.

Reviewer #2 (Recommendations for the authors):

The authors have provided satisfactory responses to points #1, 2, 3, 4, 5 and 7 but this reviewer still has a problem with the answer to comment #6: The authors claim that , as they expected, no defect was observed in DP/SP frequencies of CIC f/f pLCK-cre mice. However, in contrast to the CD4-cre model, CIC depletion seems complete in DP thymocytes of CIC f/f pLCK-cre mice (Figure S6). Do the authors have an explanation for why they don't observe the same DP/SP phenotype (defects) in the Vav-cre and pLCK-cre models?

Reviewer #3 (Recommendations for the authors):

I appreciate the author responses to previous questions and critiques; the manuscript is improved though some outstanding issues remain.

1. The integration of some of the new data is unconventional. Additional analysis (including supplemental figures) of Treg development in Cic cKO mice as well as mature T cell recirculation to the thymus appears to be added to the discussion rather than the Results section. Following this, the explanation for differences in the phenotypes of 1, 7, and 9 week-old mice based on the absence of a recirculated T cell phenotype in WT vs Cic cKO mice at 9 weeks is not clear to me.

2. The authors now make it clear that the TCR sequencing datasets are n=1. While their data interpretation is consistent with their hypothesis, I am concerned about making conclusions on this sample set.

3. Additional information is provided for the thymocyte transduction protocol and subsequent analysis; yet, ambiguities remain. It appears as if the thymocytes (from adult mice) were transduced in the absence of incubation with cytokines, and the transduction rate seems rather high for this population as described. Perhaps more details are needed. In addition, though the authors show a representative example of the GFP in a Supplementary file, given a BD Cytofix followed by cold methanol protocol is reported prior to p-ERK staining, I wonder about the extent to which GFP is preserved for this staining condition (these reagents have been reported quench fluorescence under some conditions and for at least some GFP variants).

4. Some gating strategies were clarified while others are still ambiguous to this reviewer. For example, in some cases CD8 SP analyses include pre-gating on TCRb+ cells. This does not seem to be the case in all figures, however. For example, for the quantification of CD8 SP cells in 4C, I wonder if these are ISPs and the interpretation of the results is skewed.

https://doi.org/10.7554/eLife.71769.sa1

Author response

Essential revisions:

1) The main shared concerns were about how the data is presented and interpreted; additional information/discussion is required to fully appreciate the quality and appropriateness of the conclusions. The discussion disproportionately focuses on potential defects in beta-selection and thymic Treg differentiation that were only superficially studied in this manuscript. At this stage, perhaps the interpretation of these results should be tempered.

We appreciate this kind and helpful suggestion. We have shortened our discussion on the topic of beta-selection and thymic Treg cells, and tempered the interpretation of the results.

2) Please address the recommendations to the authors listed below by the reviewers, with an emphasis on Ref#3 concerns, as well as the corrected highlighted by Ref #2.

We have addressed every suggestion raised by the three reviewers by conducting additional experiments and data analyses. We hope that our point-by-point responses to the reviewers’ comments are clear and satisfactory.

Reviewer #1 (Recommendations for the authors):

1. The main conclusion is that TCR signaling strength is affected by the increased expression of antagonists of the ERK/MAPK signaling pathway, as well as calcium, which are clearly supported by the observations shown. One would also predict that γ/δ TCR signaling to be affected during development, and the authors should show whether gd T cell development or differentiation is also affected, in particular as it pertains to the preTCR vs gd-TCR checkpoint.

As suggested, we analyzed thymic γδT cells in 7-week-old Cicf/f;Vav1-Cre mice. We found a slight increase in the frequency of total γδT cells in the thymus of Cicf/f;Vav1-Cre mice. However, the frequencies of mature thymic CD24loCD44hi γδT cells and T-bet-expressing type 1 and RORγt-expressing type 17 γδT cells were comparable between WT and Cicf/f;Vav1-Cre mice. These results are presented in Figure 1—figure supplement 1 and are described in the Results section (page 6, lines 122-126).

2. One surprising finding is that in the bone marrow chimera experiments, when using equal numbers of WT and Cic-deficient cells, that the thymocytes seem to have equal contributions of WT and Cic-deficient cells, as shown in Figure 6A. However, the actual frequencies of Wt vs Cic-/- cells are not shown and should be included. If these are similar, it would be curious given the defect in preTCR signaling , which typically accounts for a large number of downstream DP thymocytes. This should be further discussed in the text.

We thank the reviewer for pointing out this important issue and apologize for the confusion caused. We analyzed the frequencies of Thy1.1/Thy1.2 WT and Thy1.1/Thy1.1 CIC-deficient thymocytes in mixed bone marrow chimeric mice. The results are presented next to the representative FACS plot in Figure 6A. We found that the frequency of CIC-deficient cells was significantly lower than that of WT cells, which may support the idea of defective pre-TCR signaling in CIC-deficient thymocytes.

Reviewer #2 (Recommendations for the authors):

– Figure 1C: FACS plots are not representative of the graphs (seems like CD4-cre mice display an intermediate phenotype).

– Figure 1D has no legend.

– Figure 1D : FACS plot doesn't reflect the graphs (increased DP cell population in CD4-cre mice).

We thank the reviewer for these thoughtful comments. We replaced the FACS plots in Figure 1C and D with more representative images. A legend for Figure 1D has been added at the correct position.

– Figure 1E: These results suggest that a portion of the SP thymocytes in older mice may be recirculating SP cells. The authors should do an analysis of SP thymocyte maturation (show TCRβ vs CD24 for example) to investigate this. Also, they should determine if the DN3/4 block is seen in young mice.

We appreciate this valuable suggestion. Reviewer #3 also asked a similar question about the issue of recirculating SP cells (Reviewer #3’s comment no. 3). We examined the frequency of CD24loCD73+ recirculating CD4+ SP cells in the thymus of WT and Cicf/f;Vav1-Cre mice at 9 weeks of age. The proportion of this cell population was comparable between WT and Cicf/f;Vav1-Cre mice, suggesting that the similar frequency of SP thymocytes in WT and Cicf/f;Vav1-Cre mice at 9 weeks of age did not result from an accumulation of higher numbers of recirculated peripheral SP cells in the thymus of Cicf/f;Vav1-Cre mice. The corresponding data are presented in Figure 1—figure supplement 3. We also included the interpretation of these data in the Discussion section (pages 17 and 18, lines 385-404). To address the second question, we examined the frequency of DN subsets in the thymus of 1-week-old mice. In agreement with results from 7-week-old mice, a partial block of the DN3-to-DN4 transition was observed in 1-week-old Cicf/f;Vav1-Cre mice. The results are presented in Figure 1—figure supplement 2B.

– Figure 2: Is CIC protein absent in DN1/2 thymocytes of Vav-cre mice?

Accordingly, we examined CIC protein levels in DN1/2 cells by western blotting and confirmed the absence of CIC in DN1/2 thymocytes of Cicf/f;Vav1-Cre mice. This result has been added to Figure 2A.

– Figure 5 : It seems odd that Calcium flux is affected by CIC deletion and not PLCγ phosphorylation. Do the authors have a theory why?

We thank the reviewer for this valuable question. It has been reported that SPRY4 suppresses VEGF-A-induced PIP2 breakdown and calcium flux without affecting PLCγ activation (Ayada et al., 2009). We have mentioned this in the corresponding Results section of the revised manuscript (page 14, lines 325-326).

– Figure 6 : To avoid transferring other immune cells like B and DC and answer the question the author are asking with this experiment, bone marrow cells must be lineage depleted before injection. Was this the case? (method section mention transfer of whole BM).

As mentioned in the Methods section, we used whole BM from WT and Cicf/f;Vav1-Cre mice (50:50) to generate mixed BM chimeras. We agree that transferring lineage-depleted BM cells is a standard way to generate BM chimeras. However, we believe that it is still valid to examine T cell-intrinsic functions of CIC in mice transferred with a 50:50 mixture of whole BM from WT and Cicf/f;Vav1-Cre mice, because WT and CIC-deficient hematopoietic stem and progenitor cells develop into T cell subsets within the same thymic environment. Numerous studies have successfully determined the cell-intrinsic function of a specific gene product by examining mixed BM chimeric mice transferred with whole BM cells (Huang et al., 2021; Redd et al., 2018; Ulges et al., 2015).

– pLCK model is interesting to discriminate impact of CIC deletion on DN cells from DP cells. Authors should describe this model and show if they recapitulate the DP/SP phenotype without the DN stages being affected.

Accordingly, we analyzed the frequency of DN, DP, and SP cells in the thymus of WT and Cicf/f;pLck-Cre mice at 7 weeks of age, and presented the results in Figure 6—figure supplement 1D and E. As expected, thymic T cell development was normal during the DN stage in Cicf/f;pLck-Cre mice. However, a decrease in the frequency of SP thymocytes was not unambiguously observed in 7-week-old Cicf/f;pLck-Cre mice. This result is consistent with our conclusion that Cicf/f;pLck-Cre mice have a milder defect in TCR signaling and positive selection than Cicf/f;Vav1-Cre mice (Figure 6—figure supplement 1A and B).

– Since the authors base their explanation of the auto-immune phenotype on impaired thymocyte selection in the Vav-cre model, why is there no impact on thymocyte development but still signs of autoimmunity in CD4-cre mice, as mentioned in the previous paper? Authors should discuss these discrepancies.

We appreciate this important question. In our previous study (Park et al., 2017), we showed that both Cicf/f;Vav1-Cre and Cicf/f;Cd4-Cre mice had a systemic autoimmune-like phenotype with expansion of the follicular helper T (Tfh) cell population and that derepression of Etv5 promoted Tfh cell differentiation in CIC-deficient mice. Since excessive formation and/or hyperactivation of Tfh cells is closely associated with the onset of autoimmunity (Crotty, 2019), systemic autoimmunity in Cicf/f;Cd4-Cre mice could be attributed to the Etv5 derepression-mediated promotion of Tfh cell differentiation. We found that CIC deficiency-induced autoimmune-like symptoms were significantly reduced in Cicf/f;Etv5f/f;Cd4-Cre mice (unpublished data), validating our previous conclusion. Our findings of defective thymic T cell development in Cicf/f;Vav1-Cre mice explain why Cicf/f;Vav1-Cre mice have a more severe autoimmune-like phenotype than Cicf/f;Cd4-Cre mice with normal thymic T cell development. We have mentioned this in the Discussion section (page 20, lines 444-458).

Reviewer #3 (Recommendations for the authors):

1) Spry4 (and DUSP6) as a target responsible for much of the phenotype observed in CIC-deficient thymocytes is intriguing. However, it is not clear to this reviewer how the over-expression studies were performed. What is the efficiency of retroviral transduction in, presumably adult, thymocytes? Given nearly full suppression of p-ERK and calcium induction after activation in cells reported to be over-expressing Spry4 (or DUSP6), presumably the transduced cells were tracked – what marker was used?

We apologize for the lack of clarity. We have thus eliminated any ambiguities regarding the experimental procedure performed for analyzing retrovirus-transduced thymocytes. We infected thymocytes from adult WT mice with retrovirus co-expressing GFP and either SPRY4 or DUSP6, and subsequently analyzed the levels of phospho-ERK and calcium flux in GFP-positive DP cells. The virus infection efficiency was approximately 20%. We clarified this point in the corresponding figure legend and Supplementary file 4I (gating strategy for GFP-positive DP thymocytes).

2) There is a rather striking decrease in CD5 on DP cells on TCR transgenic CIC-deficient DP cells. Is this due solely to differences in the proportion of DP cells undergoing thymic selection (e.g. CD69+) or because of differences in the strength of signal itself? Indeed, differences in CD5 on polyclonal DP cells from Cicf/f;Vav1-Cre versus control DP are also quite striking. While the (presumably) post-selection DP cells expressing higher levels of CD5 seem to express similar levels of this molecule, the bulk population (presumably pre-selection) is significantly lower. What do the authors make of this? Are the majority of DP cells sensing no TCR stimulation at all? It seems as if this might be the case even with ex vivo stimulation; at least based on the provided histogram, a larger percentage of DP thymocytes appear to fail to upregulate Nur77 after in vitro stimulation while those cells that do upregulate Nur77 seem to do so similar levels whether CIC-deficient or control. Further, why is TCR signaling more impacted at the DP stage than at the SP stage in the absence of CIC?

Questions about:

1) CD5 levels in WT and CIC-deficient CD69- and CD69+ DP cells:

To directly address the reviewer’s question, we examined the frequency of CD69- and CD69+ DP cells in WT and CIC-deficient mice. We also compared CD5 levels in CD69- and CD69+ DP cells between WT and CIC-deficient mice. As expected, the frequency of CD69+ DP cells was lower in CIC-deficient mice than in WT mice (Figure 5B, Figure 5—figure supplement 1B and E). Importantly, a substantial decrease in CD5 levels was detected in CIC-deficient CD69- DP cells, but not in CD69+ DP cells, compared to WT cells (Figure 5C, Figure 5—figure supplement 1C and F). As most DP cells were CD69- (more than 95% of total DP cells; Figure 5B, Figure 5—figure supplement 1B and E), decreased CD5 levels in CIC-deficient DP cells probably resulted from attenuated TCR signaling in DP cells rather than a decrease in the frequency of CD69+ DP cells in CIC-deficient mice.

2) Histograms of Nur77 expression in DP thymocytes:

As the reviewer pointed out, Nur77 expression analysis in DP cells resulted in a two-peaked histogram (Figure 4B, Figure 7—figure supplement 1B). However, there was a difference between WT and CIC-deficient DP cells at the second peak representing an upregulation of Nur77 upon TCR stimulation (Figure 4B, Figure 7—figure supplement 1B). For Nur77 expression analysis, we treated thymocytes at 1×107 cells/ml with plate-coated anti-CD3 (5 μg/ml) and anti-CD28 (10 μg/ml) antibodies for 2 h to stimulate the TCR, followed by intracellular staining of Nur77. By conducting additional experiments, we found that the appearance of the two peaks depended on the experimental conditions used. We performed the same experiments using various concentrations of thymocytes. The appearance of two peaks was reproduced when thymocytes were employed at 1×107 cells/ml (Author response image 1A). Interestingly, the first peak corresponding to the Nur77-negative DP cell population gradually disappeared as the concentration of thymocytes was reduced (Author response image 1A). Therefore, we concluded that the histogram of Nur77 expression could be modified by the experimental conditions employed, such as concentrations of thymocytes, anti-CD3, and anti-CD28, as well as the incubation time taken. We would also like to mention that another research group using a method similar to ours obtained a similar histogram pattern for Nur77-expressing cells (Thien et al., 2010) (Author response image 1B).

Author response image 1
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Histograms of Nur77 expression in double-positive (DP) thymocytes (A) Flow cytometric analysis of Nur77 expression in DP thymocytes.

Various concentrations of freshly isolated thymocytes were incubated with plate-coated anti-CD3 (5 μg/ml) and anti-CD28 (10 μg/ml) antibodies for 2 h, followed by staining of surface markers (CD4, CD8, and TCRβ) and intracellular staining of Nur77. Representative histograms are presented. (B) Independent experiment showing two-peaked histograms of Nur77-expressing cells. Adapted from Thien et al. (2010).

3) DP cell-specific regulation of TCR signaling by CIC:

We do agree that this is an important question. We believe that the qRT-PCR data presented in Figure 7B may partially explain this question. CIC deficiency more dramatically derepressed Spry4 and Dusp6, CIC target genes responsible for inhibition of TCR signaling, in DP cells than in SP cells (Figure 7B), potentially affecting the TCR signaling pathway in DP cells more drastically than in SP cells. Moreover, our data showed that TCR signaling was weakly activated in DP cells compared to SP cells, when treated with the same concentration of anti-CD3 antibody (Figure 5A and D). In this regard, it can be inferred that the CIC deficiency-mediated derepression of CIC target genes may have had a significant inhibitory effect on TCR signaling in DP cells with weak activation of TCR signaling, but a negligible inhibitory effect on TCR signaling in SP cells with strongly activated TCR signaling. We have included this in the Discussion section (pages 18 and 19, lines 405-431).

3) There appear to be differences in the extent to which CIC impacts CD4+ and CD8+ SP thymocyte numbers at different stages of ontogeny. The authors imply that mature T cells that have recirculated to the thymus in adult mice (the 7 week old mice presented in this manuscript or as previously reported in 9 week old mice) may mask any striking differences in the relative proportions and numbers of CD4+ and CD8+ SP thymocytes in CIC-deficient as compared to control mice. In younger mice, there are significantly fewer mature thymocytes in the absence of CIC. Whether this is due to differences in the recirculated mature T cell population is less clear than implied; this could be due to differences in the selection processes that accompany T cell development at different stages of ontogeny. One would need to use appropriate markers (e.g. CD73) or reporters (Rag-GFP) to make this distinction.

We thank the reviewer for this valuable suggestion. Accordingly, we analyzed the frequency of recirculated mature CD4+ T cells in the thymus of WT and Cicf/f;Vav1-Cre mice at 9 weeks of age using CD73 as a recirculating T cell marker. Unexpectedly, the frequency of thymic CD24loCD73+CD4+ SP cells was comparable between WT and Cicf/f;Vav1-Cre mice (Figure 1—figure supplement 3). Thereafter, we seriously considered the reviewer’s comment that the decreased thymic SP cell population in 1-week-old Cicf/f;Vav1-Cre mice could have resulted from differences in the selection processes that accompany T cell development at different stages of ontogeny. Based on a detailed research of the literature, we realized that negative selection is inefficient early in ontogeny and increases with age (He et al., 2013; Huseby et al., 2001). Since Cicf/f;Vav1-Cre mice have defects in both positive and negative selection, it is conceivable that the decreased frequency of SP thymocytes in 1-week-old Cicf/f;Vav1-Cre mice was caused by a defect in positive selection, and that this effect was attenuated by ineffective negative selection at 7 weeks of age or older. We discuss this interesting and reasonable possibility in the Discussion section (pages 17 and 18, lines 385-404).

4) Careful explanation of the experimental set up and conclusions from the TCR sequencing studies would be appreciated. I do not understand the argument for the longer CDR3 sequences in the CIC KO conventional CD4 T cell populations as being 'pre-selection-like'; what does this imply? It appears as if the main conclusion of the TCR sequencing data is that the differences in the repertoire predominantly lie in the Treg population; outside of TCR sequence analysis this subset is not analyzed in the current manuscript. Are there overt differences in thymic Treg development in the absence of CIC?

Previous studies have demonstrated that T cells with shorter CDR3 sequences are enriched in mature post-selection T cell populations (Hou et al., 2019; Lu et al., 2019). Based on this knowledge, we suggest that the increased frequency of longer CDR3 sequences in the TCR repertoires of CIC-deficient CD4+ SP thymocytes might have been the result of defective selection processes. As suggested, we analyzed thymic Treg cell populations in 7-week-old WT and Cicf/f;Vav1-Cre mice. Consistent with a defect in negative selection in Cicf/f;Vav1-Cre mice, the frequency of thymic CD25+Foxp3+ Treg and CD25-Foxp3lo progenitor cells was significantly increased in Cicf/f;Vav1-Cre mice. The corresponding data are presented in Figure 4—figure supplement 2 and are mentioned in the Discussion section (page 19, lines 440-441).

5) Gating strategies and representative flow plots, as well as clear descriptions of the gates in the figure legends, for all analyses would be appreciated. It is not always clear, for example, if lineage+ cells have been removed from DN gates, whether mature T cells have been gated on TCRbhi cells, whether the conventional CD4+ SP population used for TCR sequencing includes CD25+ Treg progenitors, etc. In addition, representative histograms are not always provided for MFI analysis; this is important to understand, for example with the CIC-Flag tag, the extent to which expression is heterogenous in a population; clear statements about the population for which MFI is calculated (e.g. for Figure 4B, is the MFI calculated for the population in the positive gate or for the total population) should be added.

We appreciate this thoughtful suggestion. Accordingly, we provided gating strategies for all flow cytometry experiments in Supplementary file 4 and described the gates in the figure legends. We also show representative flow plots and histograms for all flow cytometry and MFI analyses, including histograms for CIC-FLAG expression in various thymic T cell subsets (Figure 1A). To answer some of the reviewer’s specific questions, we removed lineage+ cells for all DN cell analyses, and excluded CD25+ Treg progenitors from the conventional CD4+ SP population for TCR repertoire analysis of non-Treg cells. We presented the graphs for Nur77-associated MFI for both total and Nur77+ DP cells in Figure 4B and Figure 7—figure supplement 1B.

6) Consider splitting some of the data onto separate graphs (e.g. Figure 3C and D) as it is very difficult to appreciate the noted significant differences in terms of percentages and cell numbers when the symbols are against the x axis, for example.

We thank the reviewer for this thoughtful suggestion. As suggested, the frequency and number of DN, DP, 4SP, and 8SP cells in Figure 3C, 3D, and 4C are displayed in separate graphs for reasons of clarity and comprehensibility.

7) Please revisit the appropriateness of the t test for assessing statistical significance across three mouse strains.

Accordingly, we used one-way or two-way ANOVA with Tukey’s multiple comparison test for reanalyzing the statistical significance across three groups of mice or samples.

8) Please ensure that biological and experimental replicates are clearly noted for each experiment. For example, how many mice were used for the TCR sequencing experiments?

As per the reviewer’s suggestion, we clearly indicated biological and experimental replicates for each experiment in the corresponding figure legends and/or method sections.

References:

Ayada, T., Taniguchi, K., Okamoto, F., Kato, R., Komune, S., Takaesu, G., and Yoshimura, A. (2009). Sprouty4 negatively regulates protein kinase C activation by inhibiting phosphatidylinositol 4,5-biphosphate hydrolysis. Oncogene 28, 1076–1088.

Crotty, S. (2019). T Follicular Helper Cell Biology: A Decade of Discovery and Diseases. Immunity 50, 1132–1148.

He, Q., Morillon, Y.M., Spidale, N.A., Kroger, C.J., Liu, B., Sartor, R.B., Wang, B., and Tisch, R. (2013). Thymic Development of Autoreactive T Cells in NOD Mice Is Regulated in an Age-Dependent Manner. J. Immunol. 191, 5858–5866.

Hou, X., Zeng, P., Zhang, X., Chen, J., Liang, Y., Yang, J., Yang, Y., Liu, X., and Diao, H. (2019). Shorter TCR β-chainsare highly enriched during thymic selection and antigen driven slection. Front. Immunol. 10, 299.

Huang, H., Zhou, P., Wei, J., Long, L., Shi, H., Dhungana, Y., Chapman, N.M., Fu, G., Saravia, J., Raynor, J.L., et al. (2021). in vivo CRISPR screening reveals nutrient signaling processes underpinning CD8+ T cell fate decisions. Cell 184, 1245-1261.e21.

Huseby, E.S., Sather, B., Huseby, P.G., and Goverman, J. (2001). Age-dependent T cell tolerance and autoimmunity to myelin basic protein. Immunity 14, 471–481.

Lu, J., Van Laethem, F., Bhattacharya, A., Craveiro, M., Saba, I., Chu, J., Love, N.C., Tikhonova, A., Radaev, S., Sun, X., et al. (2019). Molecular constraints on CDR3 for thymic selection of MHC-restricted TCRs from a random pre-selection repertoire. Nat. Commun. 10, 1–14.

Park, S., Lee, S., Lee, C.G., Park, G.Y., Hong, H., Lee, J.S., Kim, Y.M., Lee, S.B., Hwang, D., Choi, Y.S., et al. (2017). Capicua deficiency induces autoimmunity and promotes follicular helper T cell differentiation via derepression of ETV5. Nat. Commun. 8, 1–13.

Redd, P.S., Lu, C., Klement, J.D., Ibrahim, M.L., Zhou, G., Kumai, T., Celis, E., and Liu, K. (2018). H3K4me3 mediates the NF-κB p50 homodimer binding to the pdcd1 promoter to activate PD-1 transcription in T cells. Oncoimmunology 7, 1–11.

Thien, C.B.F., Dagger, S.A., Steer, J.H., Koentgen, F., Jansen, E.S., Scott, C.L., and Langdon, W.Y. (2010). c-Cbl promotes T cell receptor-induced thymocyte apoptosis by activating the phosphatidylinositol 3-kinase/Akt pathway. J. Biol. Chem. 285, 10969–10981.

Ulges, A., Klein, M., Reuter, S., Gerlitzki, B., Hoffmann, M., Grebe, N., Staudt, V., Stergiou, N., Bohn, T., Brühl, T.J., et al. (2015). Protein kinase CK2 enables regulatory T cells to suppress excessive TH2 responses in vivo. Nat. Immunol. 16, 267–275.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:

The remaining required revisions are clearly outlined within the detailed reviewers comments below.

Reviewer #1 (Recommendations for the authors):

The authors have comprehensively addressed most of my initial concerns, however additional points need to be clarified. In particular, the differences in pLck-cre vs Vav-cre mice should be better addressed more clearly.

Thank you for this thoughtful suggestion. As per your suggestion, we carefully examined the phenotypes of Cicf/f;pLck-Cre mice compared to those of WT, Cicf/f;Cd4-Cre, and Cicf/f;Vav1-Cre mice, and quantified CIC levels in developing thymic T cell subsets from WT, Cicf/f;Cd4-Cre, Cicf/f;Vav1-Cre, and Cicf/f;pLck-Cre mice using ImageJ software (Figure 6—figure supplement 1A). It was apparent that Cicf/f;pLck-Cre mice exhibited normal DN cell development (Figure 6—figure supplement 1B), but there were defects in positive selection and TCR signaling in DP cells (Figure 6—figure supplement 1C-E). However, these defects were milder in Cicf/f;pLck-Cre mice than in Cicf/f;Vav1-Cre mice (Figure 6—figure supplement 1C-E), which could be attributed to the incomplete removal of CIC expression in DP thymocytes of Cicf/f;pLck-Cre mice (Figure 6—figure supplement 1A). To more clearly explain the phenotypes of Cicf/f;pLck-Cre mice in comparison with those of Cicf/f;Vav1-Cre mice, data were reorganized and the text in the corresponding Results section was amended accordingly (pages 13 and 14, lines 298-320).

Reviewer #2 (Recommendations for the authors):

The authors have provided satisfactory responses to points #1, 2, 3, 4, 5 and 7 but this reviewer still has a problem with the answer to comment #6: The authors claim that , as they expected, no defect was observed in DP/SP frequencies of CIC f/f pLCK-cre mice. However, in contrast to the CD4-cre model, CIC depletion seems complete in DP thymocytes of CIC f/f pLCK-cre mice (Figure S6). Do the authors have an explanation for why they don't observe the same DP/SP phenotype (defects) in the Vav-cre and pLCK-cre models?

Thank you for raising this issue and apologies for how we carried out data analysis and interpretation. As described in the response to Reviewer #1’s comment, we quantified CIC levels in developing thymic T cell subsets from WT, Cicf/f;Cd4-Cre, Cicf/f;Vav1-Cre, and Cicf/f;pLck-Cre mice (Figure 6—figure supplement 1A). Approximately 24% of CIC proteins were still expressed in DP cells from Cicf/f;pLck-Cre mice compared to WT cells (Figure 6—figure supplement 1A), indicating incomplete depletion of CIC in DP cells of Cicf/f;pLck-Cre mice. Similar to the findings in 7-week-old Cicf/f;Vav1-Cre mice (Figure 1D), a slight increase in the frequency of DN and DP thymocytes, and a decrease in the frequency of CD4+ SP cells was observed in 7 week-old Cicf/f;pLck-Cre mice (Figure 6—figure supplement 1C). It is worth noting that the DP/SP phenotypes were also mild in Cicf/f;Vav1-Cre mice at 7 weeks old when compared to WT mice (a significant but slight decrease in the frequency of CD4+ SP thymocytes only) (Figure 1D). Therefore, it would be hard to expect that Cicf/f;pLck-Cre mice will show significant changes in the frequency of DP and SP cells at 7 weeks old compared to WT mice because CIC depletion is incomplete in DP thymocytes of Cicf/f;pLck-Cre mice. We rearranged the figure panels in Figure 6—figure supplement 1 and accordingly edited the text in the corresponding Results section (pages 13 and 14, lines 298-320) to make our conclusion clearer.

Reviewer #3 (Recommendations for the authors):

I appreciate the author responses to previous questions and critiques; the manuscript is improved though some outstanding issues remain.

1. The integration of some of the new data is unconventional. Additional analysis (including supplemental figures) of Treg development in Cic cKO mice as well as mature T cell recirculation to the thymus appears to be added to the discussion rather than the Results section. Following this, the explanation for differences in the phenotypes of 1, 7, and 9 week-old mice based on the absence of a recirculated T cell phenotype in WT vs Cic cKO mice at 9 weeks is not clear to me.

We appreciate this comment. Accordingly, we moved the data on the recirculating CD4+ SP cells in the thymus to the Results section (page 7, lines 142-153). We also analyzed the frequency of recirculating thymic CD4+ SP cells in 1-week-old WT and Cicf/f;Vav1-Cre mice and presented the data in Figure 1—figure supplement 3A. These modifications allowed us to more logically explain why we focused on the regulatory function of CIC in thymic T cell development.

The frequency and number of SP thymocytes were significantly decreased in Cicf/f;Vav1-Cre mice at 1 week old (Figure 1E), whereas these defects were attenuated and disappeared with age (Figure 1D) (Park et al., 2017). To determine whether the disappearance of the decrease in the frequency of SP thymocytes in Cicf/f;Vav1-Cre mice at 9 weeks old was due to increased accumulation of recirculating SP cells in the thymus of Cicf/f;Vav1-Cre mice compared to WT mice, we analyzed the frequency of recirculating CD24loCD73+CD4+ SP cells in the thymus of 9-week-old WT and Cicf/f;Vav1-Cre mice. The comparable frequency of thymic recirculating CD4+ SP cells between 9-week-old WT and Cicf/f;Vav1-Cre mice (Figure 1—figure supplement 3B) suggests that the SP cell population recirculated into the thymus from the periphery was not the cause of the phenotypic changes in Cicf/f;Vav1-Cre mice with age.

2. The authors now make it clear that the TCR sequencing datasets are n=1. While their data interpretation is consistent with their hypothesis, I am concerned about making conclusions on this sample set.

We appreciate your concern regarding the reliability of data obtained from only one sample set. To validate the TCR repertoire analysis results, we performed flow cytometric analysis of thymic CD4+ non-Treg and Treg cells from WT and Cicf/f;Vav1-Cre mice using antibodies specific to various TCRβ V segments. To our surprise, the results were markedly different from our previous conclusions based on TCR repertoire sequencing analysis. Among the 10 different TCRβ V segments tested, the usage frequencies of eight TCRβ V segments in non-Treg cells were significantly different between WT and Cicf/f;Vav1-Cre mice, whereas only two TCRβ V segments were differentially used in the thymic Treg cell compartments of WT and Cicf/f;Vav1-Cre mice. The data are presented in Figure 4—figure supplement 2. Considering your concerns and these findings, we decided to exclude the TCR repertoire sequencing analysis data from the text. Accordingly, we revised the corresponding sections of the manuscript (page 11, lines 228-246). We believe that this decision does not affect the main conclusion of this study, and that the new data (Figure 4—figure supplement 2) still support our findings that CIC regulates thymic selection processes.

3. Additional information is provided for the thymocyte transduction protocol and subsequent analysis; yet, ambiguities remain. It appears as if the thymocytes (from adult mice) were transduced in the absence of incubation with cytokines, and the transduction rate seems rather high for this population as described. Perhaps more details are needed. In addition, though the authors show a representative example of the GFP in a Supplementary file, given a BD Cytofix followed by cold methanol protocol is reported prior to p-ERK staining, I wonder about the extent to which GFP is preserved for this staining condition (these reagents have been reported quench fluorescence under some conditions and for at least some GFP variants).

As suggested, we have expounded on the procedures of retroviral transduction of thymocytes and subsequent analysis in the Methods section of the revised manuscript (pages 22-23, lines 511-513; page 24, lines 554-555; page 25, lines 579-583). As pointed out, thymocytes from adult C57/BL6 mice were transduced with retroviral supernatant and incubated for 48 h in the absence of additional cytokines. Based on the frequency of GFP+ thymocytes in three independent experiments, the transduction efficiency varied in each trial (Author response image 2).

Author response image 2
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Efficiency of retroviral transduction of thymocytes.

FACS plots showing the frequency of GFP+ thymocytes transduced with retrovirus co-expressing GFP and control, SPRY4, or DUSP6. Three independent experiments were performed.

We agree with the reviewer’s opinion that the GFP signal can be lost depending on the intracellular staining methods. We detected the loss of GFP signal when performing intracellular staining of transcription factors in thymocytes of Foxp3GFP mice using the Foxp3 staining buffer set from eBioscience (data not shown). However, we did not observe this phenomenon when performing intracellular staining of p-ERK in thymocytes transduced with GFP-expressing retrovirus using BD cytofix and cold methanol. To address the reviewer’s question, we investigated the extent of preserved GFP signal after permeabilization of thymocytes transduced with GFP-expressing retrovirus with the Foxp3 staining buffer set or cold methanol. Consistent with our previous observations, cell permeabilization with cold methanol did not significantly affect GFP signal in retrovirus-transduced thymocytes, whereas the Foxp3 staining buffer set (eBioscience) led to a dramatic decrease in the GFP signal (Author response image 3).

Author response image 3
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Comparison of the extent of preserved GFP signal after cell permeabilization by different methods.

(A) Flow cytometric analysis of GFP expression in live thymocytes infected with GFP-expressing retrovirus. Representative FACS plots are presented. No perm: without permeabilization, eBioscience: the Foxp3 staining buffer set, and methanol: cold methanol. (B) Relative expression levels of GFP in thymocytes before and after permeabilization with the Foxp3 staining buffer set (eBioscience) or cold methanol. N=2 for each group.

4. Some gating strategies were clarified while others are still ambiguous to this reviewer. For example, in some cases CD8 SP analyses include pre-gating on TCRb+ cells. This does not seem to be the case in all figures, however. For example, for the quantification of CD8 SP cells in 4C, I wonder if these are ISPs and the interpretation of the results is skewed.

We appreciate this thoughtful comment. To address your concern, we analyzed the proportion of CD24hiTCRβlo ISP cells among total CD8+ SP thymocytes from female and male H-Y;Cicf/f and H-Y;Cicf/f;Vav1-Cre mice; and presented the data in Figure 3—figure supplement 1B and Figure 4—figure supplement 1B, respectively. CD24hiTCRβlo ISP cells constituted less than 10% of total CD8+ SP thymocytes in male H-Y mice, and their frequency was comparable between male H-Y;Cicf/f and H-Y;Cicf/f;Vav1-Cre mice (Figure 4—figure supplement 1B). Therefore, we concluded that the expansion of the thymic CD8+ SP cell population in male H-Y;Cicf/f;Vav1-Cre mice was primarily attributed to a defect in negative selection. In female mice, the frequency of ISP cells was significantly increased in H-Y;Cicf/f;Vav1-Cre mice compared to H-Y;Cicf/f mice (Figure 3—figure supplement 1B), demonstrating that the decreased frequency of CD8+ SP thymocytes was due to a defect in positive selection rather than attenuated ISP cell formation.

Reference:

Park, S., Lee, S., Lee, C.G., Park, G.Y., Hong, H., Lee, J.S., Kim, Y.M., Lee, S.B., Hwang, D., Choi, Y.S., et al. (2017). Capicua deficiency induces autoimmunity and promotes follicular helper T cell differentiation via derepression of ETV5. Nat. Commun. 8, 1–13.

https://doi.org/10.7554/eLife.71769.sa2

Article and author information

Author details

  1. Soeun Kim

    Department of Life Sciences, Pohang University of Science and Technology (POSTECH), Pohang, Republic of Korea
    Contribution
    Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Validation, Visualization, Writing - original draft, Writing - review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6425-0899

  2. Guk-Yeol Park

    Department of Life Sciences, Pohang University of Science and Technology (POSTECH), Pohang, Republic of Korea
    Contribution
    Investigation
    Competing interests
    No competing interests declared

  3. Jong Seok Park

    Department of Life Sciences, Pohang University of Science and Technology (POSTECH), Pohang, Republic of Korea
    Contribution
    Investigation
    Competing interests
    No competing interests declared

  4. Jiho Park

    Department of Life Sciences, Pohang University of Science and Technology (POSTECH), Pohang, Republic of Korea
    Contribution
    Investigation
    Competing interests
    No competing interests declared

  5. Hyebeen Hong

    Department of Life Sciences, Pohang University of Science and Technology (POSTECH), Pohang, Republic of Korea
    Contribution
    Investigation
    Competing interests
    No competing interests declared

  6. Yoontae Lee

    1. Department of Life Sciences, Pohang University of Science and Technology (POSTECH), Pohang, Republic of Korea
    2. Institute of Convergence Science, Yonsei University, Seoul, Republic of Korea
    Contribution
    Conceptualization, Funding acquisition, Project administration, Supervision, Writing - original draft, Writing - review and editing
    For correspondence
    yoontael@postech.ac.kr
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6810-3087

Funding

Samsung Science and Technology Foundation (SSTF-BA1502-14)

  • Yoontae Lee

National Research Foundation of Korea (NRF-2021R1A2C3004006)

  • Yoontae Lee

National Research Foundation of Korea (NRF-2017R1A5A1015366)

  • Yoontae Lee

National Research Foundation of Korea (NRF-2017H1A2A1042705)

  • Hyebeen Hong

Brain Korea 21

  • Jong Seok Park
  • Jiho Park

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank Dr. Jaeho Cho and the Lee lab members for their helpful discussions and comments on this study. This work was supported by grants from the Samsung Science and Technology Foundation under project number SSTF-BA1502-14 and the National Research Foundation (NRF) of Korea (NRF-2021R1A2C3004006 and –2017 R1A5A1015366). JSP and JP were supported by BK21. HH was supported by a Global PhD Fellowship (NRF-2017H1A2A1042705).

Ethics

Animal experimentation: All experiments were approved by the Institutional Animal Care and Use Committee of Pohang University of Science and Technology (POSTECH-2019-0081). All experiments were carried out in accordance with the approved guidelines. Mouse sacrifice was performed under isoflurane anesthesia, and every effort was made to minimize suffering.

Senior Editor

  1. Tadatsugu Taniguchi, Institute of Industrial Science, The University of Tokyo, Japan

Reviewing Editor

  1. Juan Carlos Zúñiga-Pflücker, University of Toronto, Sunnybrook Research Institute, Canada

Reviewer

  1. Juan Carlos Zúñiga-Pflücker, University of Toronto, Sunnybrook Research Institute, Canada

Version history

  1. Received: June 29, 2021
  2. Preprint posted: July 12, 2021 (view preprint)
  3. Accepted: December 10, 2021
  4. Accepted Manuscript published: December 13, 2021 (version 1)
  5. Version of Record published: December 23, 2021 (version 2)

Copyright

© 2021, Kim et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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  1. Soeun Kim
  2. Guk-Yeol Park
  3. Jong Seok Park
  4. Jiho Park
  5. Hyebeen Hong
  6. Yoontae Lee
(2021)
Regulation of positive and negative selection and TCR signaling during thymic T cell development by capicua
eLife 10:e71769.
https://doi.org/10.7554/eLife.71769

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