Thymic stromal lymphopoietin (TSLP) is a cytokine that acts directly on CD4+ T cells and dendritic cells to promote progression of asthma, atopic dermatitis, and allergic inflammation. However, a direct role for TSLP in CD8+ T-cell primary responses remains controversial and its role in memory CD8+ T cell responses to secondary viral infection is unknown. Here, we investigate the role of TSLP in both primary and recall responses in mice using two different viral systems. Interestingly, TSLP limited the primary CD8+ T-cell response to influenza but did not affect T cell function nor significantly alter the number of memory CD8+ T cells generated after influenza infection. However, TSLP inhibited memory CD8+ T-cell responses to secondary viral infection with influenza or acute systemic LCMV infection. These data reveal a previously unappreciated role for TSLP on recall CD8+ T-cell responses in response to viral infection, findings with potential translational implications.
Influenza virus infection accounts for significant morbidity and mortality (Davlin, 2016; Budd, 2016), and understanding factors controlling the immune response to influenza is important for developing strategies for enhancing immunity and designing new therapies and vaccines. The absence of CD8+ T cells delays influenza clearance (Moskophidis and Kioussis, 1998), demonstrating the importance of these cells in the control of infection by this virus. The main cellular targets of influenza are pulmonary epithelial cells, which once infected produce multiple inflammatory mediators that can alter the immune response to influenza infection. One of these mediators is TSLP, a pleiotropic cytokine with a range of actions, affecting cellular maturation, survival, and recruitment of cells. Although TSLP was initially reported to act on T cells indirectly through dendritic cells (Soumelis et al., 2002; Ito et al., 2005), it was later shown to also act directly on both mouse and human CD4+ and CD8+ T cells (Rochman et al., 2007; Al-Shami et al., 2005; Shane and Klonowski, 2014; Rochman and Leonard, 2008). TSLP has additional effects on B cells, neutrophils, mast cells, and eosinophils (West et al., 2016; Rochman et al., 2009; Corren and Ziegler, 2019). TSLP signals via a receptor comprising a TSLP-specific binding protein, TSLPR, and the IL-7 receptor α chain, IL-7Rα (CD127), thereby activating JAK1, JAK2, and STAT5 (Rochman et al., 2010). TSLP is expressed at barrier surfaces, and has been extensively studied in the context of T helper 2 (TH2) type responses and shown to promote the progression of TH2-mediated diseases, including asthma, atopic dermatitis, and allergic inflammation (Al-Shami et al., 2005; Divekar and Kita, 2015; Yoo et al., 2005; Zhou et al., 2005), as well as immune responses to the intestinal pathogen, Trichuris muris (Taylor et al., 2009), but the role of TSLP on CD8+ T-cell responses is less-well characterized. There are conflicting reports of the role of TSLP on CD8+ T cells during primary influenza infection (Shane and Klonowski, 2014; Plumb et al., 2012; Yadava et al., 2013), and the effects of TSLP on memory CD8+ T cells and secondary responses to acute viral infections have not been characterized. Here, we used an adoptive co-transfer model of WT and TSLPR-deficient mice (the gene encoding TSLPR is the Crlf2 gene, so these mice are designated as Crlf2-/-) virus-specific CD8+ T cells to analyze the direct actions of TSLP on CD8+ T cells during both primary and secondary responses to influenza virus infection, as well as the role of this cytokine in naïve and memory CD8+ T-cell homeostasis. We also assessed the role of TSLP in the context of an acute systemic infection caused by LCMV.
To assess the role of TSLP on CD8+ T-cell responses during influenza infection, we adoptively transferred P14 T cells (TCR transgenic CD8+ T cells specific for LCMV glycoprotein 33, gp33) into WT mice. We then infected these mice intranasally one day later with influenza strain PR8-33, which represents the PR8 strain genetically modified to express gp33 (Mueller et al., 2010), and then examined TSLPR expression over time in lungs and spleen (see schematic, upper part of Figure 1A). TSLPR was expressed on naïve (CD44low) CD8+ T cells, with high expression on virus-specific CD8+ T cells in both the lungs and spleen by day six post-infection (Figure 1A), with a subsequent decrease evident at days 14 and 33 (Figure 1A), suggesting that TSLP might directly act on virus-specific CD8+ T cells, and indeed increased Tslp mRNA expression has been observed during influenza infection (Shane and Klonowski, 2014; Yadava et al., 2013).
To determine whether there was a direct effect of TSLP on virus-specific CD8+ T cells during influenza infection, we co-transferred equal numbers of congenically-labeled naïve WT and Crlf2-/- P14 T cells into WT recipient mice, infected them intranasally with PR8-33, and assessed TSLPR expression as well as WT and Crlf2-/- T-cell numbers and function, both at the peak of the response (day 8) and after the formation of memory cells (>day 30 p.i.) (see schematic in Figure 1B, upper panel and transferred cells lower panel). TSLPR was highly expressed on the virus-specific WT CD8+ T cells but not Crlf2-/- cells in all tissues assessed at day 8 p.i.: lungs, mediastinal (draining) lymph node, spleen, and bronchoalveolar lavage (BAL) fluid, with highest expression seen in the BAL fluid (Figure 1C). At day 8 p.i., there was a modest increase in Crlf2-/- T cells compared to WT cells in the lungs, mediastinal lymph node, and spleen, but not in BAL fluid (Figure 1D and Figure 1—figure supplement 1); the results were qualitatively similar when the experiment was performed using Th1.1+/Thy1.1+ WT P14T cells and Thy1.1+/Thy1.2+ Crlf2-/-cells (Figure 1—figure supplement 1A) or with Th1.1+/Thy1.2+ WT P14T cells and Thy1.1+/Thy1.1+ Crlf2-/- cells (Figure 1—figure supplement 1B), with composite results shown in (Figure 1D). Thus, the difference was due to WT versus Crlf2-/- differences rather than differences that might exist between Thy1.1+/Thy1.1+ and Thy1.1+/Thy1.2+ genetic backgrounds. Some variation in the expression of CD44 (Figure 1—figure supplement 2A) and KLRG1 (Figure 1—figure supplement 2B) was observed, but the differences were modest, and there were similar levels of granzyme B (Figure 1—figure supplement 2C) and percentages of WT and Crlf2-/- cells expressing IFN-γ, TNF-α, or both cytokines after ex-vivo stimulation with cognate peptide (GP33) (Figure 1—figure supplement 2D). Because TSLP signals via a dimer of the TSLP-specific receptor chain (TSLPR) and IL-7Rα, we considered the possibility that the absence of TSLPR leads to the compensatory induction of IL-7Rα (CD127), which might result in an IL-7-dependent increase in the number of Crlf2-/- cells, but expression of IL-7Rα was similar in WT and Crlf2-/- cells (Figure 1E and F). The increased cellularity in lungs, lymph node, and spleen (Figure 1D) indicated that TSLP negatively regulates CD8+ T cell effector responses during acute pulmonary influenza infection; however, the proportions of WT and Crlf2-/- memory CD8+ T cells were not significantly different during the memory phase in lungs, lymph nodes, spleen, and BAL fluid (Figure 1G and H).
TSLP has been shown to play a direct role in the survival and homeostasis of naïve CD8+ T cells both in vivo and in vitro, inducing enhanced BCL-2 expression and higher proliferation, with decreased apoptosis in naïve CD8+ T cells in vitro and higher survival/homeostasis of these cells after cell transfer into naïve hosts (Rochman and Leonard, 2008). We therefore investigated whether TSLP could enhance memory CD8+ T-cell survival/homeostasis. We isolated P14 CD8+ T cells from the spleens of naïve P14 mice or from the spleens of mice that were seeded with naïve P14 T cells and then infected with PR8-33 virus for more than 30 days to create memory P14 T cells. When naïve P14 T cells were cultured without anti-CD3 + anti-CD28, cell survival was >90% at 4 hr in the absence or presence of TSLP (Figure 2A, 1st and 2nd lanes). Survival was lower at 24 hr in medium, but TSLP significantly enhanced the survival (Figure 2A, 3rd and 4th lanes). However, when analogous cells were stimulated with anti-CD3 + anti-CD28, basal survival was higher and TSLP no longer further increased survival (Figure 2A, last two lanes). When we performed the same analysis on memory CD8+ T cells, cell survival was also lower at 24 than at 4 hr, without a statistically significant effect of TSLP (Figure 2B). We next examined the role of TSLP in naïve and memory CD8+ T-cell homeostasis in vivo. We co-transferred equal numbers of congenically marked WT and Crlf2-/- naïve P14 cells into WT mice (Figure 2C) and found fewer Crlf2-/- than WT cells at days 9–11 post-transfer (Figure 2D, left panels), consistent with a previous study that suggested that TSLP is important for naïve CD8+ T-cell homeostasis (Rochman and Leonard, 2008). In contrast, both WT and Crlf2-/- P14 memory CD8+ T cells persisted similarly following transfer into naïve hosts (Figure 2D, right panels).
Given the differential impact of TSLP on naïve versus memory CD8+ T cells, we sought to elucidate the mechanism and performed RNA-sequencing analysis on naïve and memory CD8+ T cells stimulated in vitro with or without TSLP and in the absence (Figure 3A and Supplementary file 1) or presence (Figure 3B and Supplementary file 2) of TCR stimulation. The gating strategy for the memory CD8+ T cells is shown in Figure 3—figure supplement 1. As expected, TSLP treatment of naïve cells and memory cells resulted in a number of genes up- or down-regulated in each treatment group. There were more down-regulated than up-regulated genes with TSLP except at the 4 hr time point without TCR activation, suggesting that TSLP was a mediator of gene repression. In naïve cells, the number of upregulated genes was lower at 24 than at 4 hr, independent of TCR stimulation, but in memory cells, the number of upregulated genes was higher at 24 than at 4 hr (Figure 3A and B), again indicating differences in the effect of this cytokine on naïve and memory T cells. We analyzed the genes in naïve versus memory CD8+ T cells without TCR stimulation at 24 hr (overlap is shown in the Venn diagram in Figure 3C; list of genes in Supplementary file 3), and found that only a few genes were shared between naïve cells and memory cells, indicating distinctive effects of TSLP on these different cell types. Ten genes including Alcam, Nfil3, Bcat1, Olfr613, Nr4a2, Bloc1s3, Zfp488, Cpox, Zfp457, and Ap1s3 were down-regulated in both naïve and memory CD8+ T cells (Figure 3C, genes in blue in Figure 3D), whereas five genes including Bcl2, Pole2, Socs3, Nek6, and Tfrc were up-regulated in both cell types (Figure 3C; genes in red in Figure 3D). Up-regulation of Bcl2 is consistent with higher cell viability after TSLP stimulation in vitro (Figure 2). Interestingly, seven genes were down-regulated in naïve CD8+ T cells but up-regulated in memory CD8+ T cells (Hspa1b, Gadd45g, H2-ab1, Isg15, Socs2, Fcer1g, and Sfxn2) (genes in black in Figure 3D), consistent with some potentially opposing/distinctive actions for TSLP in naïve versus memory CD8+ T cells. Moreover, there were a number of genes significantly up- or down- regulated by TSLP in memory cells relative to naïve cells (Figure 3E and Supplementary file 3). One of the induced genes, Hk2, encodes a key rate-limiting enzyme in glycolysis, a process that is active in effector T cells and promotes T cell proliferation (Robey and Hay, 2006; O'Neill et al., 2016). Three of the genes whose expression was diminished by TSLP were Runx2, Egr2, and Panx1. RUNX2 is a transcription factor that promotes long-term persistence of antiviral CD8+ memory T cells (Olesin et al., 2018), EGR2 is critical for normal differentiation of naïve T cells and for regulating antigen-specific immune responses to influenza viral infection (Du et al., 2014), and PANX1 was reported to influence memory T cell maintenance (Wanhainen et al., 2019), collectively indicating that there are distinctive effects of TSLP on naïve and memory T cells. The potential role(s) of these genes during influenza infection remains to be further elucidated.
To further understand the biological consequences of TSLP on memory cells, we next co-transferred equal numbers of congenically marked WT and Crlf2-/- memory P14 cells (>30 days after influenza infection) into naïve WT mice and infected these mice with PR8-33 intranasally the following day (schematic in Figure 4A, left; CD44 expression on WT and Crlf2-/- memory P14 cells and ratio of transferred cells Figure 4A, right panels). On day 8 p.i., virus-specific secondary effector CD8+ T cells in BAL fluid, lungs, lymph nodes, and spleen expressed high levels of TSLPR (Figure 4B). There tended to be higher expression than observed on primary effector CD8+ T cells at day 8 p.i. (Figure 1C), but the highest TSLPR expression was still observed on BAL fluid cells (Figure 4B). When we assessed the secondary effector P14 responses on day 8 p.i., effector P14 T cells were present in all tissues, with a markedly increased proportion of Crlf2-/- P14 T cells in lungs, lymph nodes, spleen, and BAL fluid (Figure 4C and D). These data demonstrate that TSLP constrains secondary CD8+ T-cell responses during influenza infection. However, the percentage of cells expressing both IFNγ and TNFα was similar in WT and Crlf2-/- mice after ex-vivo stimulation with cognate peptide (GP33), with only modest differences in the percentage of cells producing only IFNγ or TNFα alone (Figure 4—figure supplement 1A–C).
We next assessed the effects of TSLP signaling on CD8+ T-cell recall responses at day 8 after secondary infection by RNA-Seq. The gating strategy for WT and Crlf2-/- P14 cells is shown in Figure 4—figure supplement 2. Compared with WT cells, 20 genes were induced and nine genes were repressed in Crlf2-/- cells (Figure 4E and Supplementary file 4); we confirmed that Crlf2 gene expression was absent in Crlf2-/- cells (Figure 4F). Two genes whose expression was markedly increased were Eps8l1 (epidermal growth factor receptor pathway substrate 8-like 1) and Eaf2. Eps8l1 has been reported to upregulate cell cycle genes, induce chemokines and enhance migration of some cancer cells (Yang et al., 2019; Huang et al., 2018; Zeng et al., 2018), whereas Eaf2 acts as an upstream modulator of non-canonical Wnt signaling, and has been suggested to suppress oxidative stress–induced apoptosis through inhibition of caspase 3 production and activation of Wnt3a signaling (Feng and Guo, 2018). By enhancing migration and decreasing apoptosis the upregulation of these genes may help to explain the greater numbers of Crlf2-/- cells we observed in the recall CD8+ T-cell responses (Figure 4C and D). Several Ifitm (interferon induced transmembrane) family members were also more highly expressed in Crlf2-/- cells (Figure 4G and Supplementary file 4). These proteins confer cellular resistance to many viruses in both mice and humans (Zhao et al., 2018; Liao et al., 2019; Bailey et al., 2014), and IFITM3 is known to contribute to the control of influenza A virus (Brass et al., 2009). Thus, the induction of Ifitm family genes might protect cells, limiting cell death after viral infection and leading to greater numbers of Crlf2-/- cells after secondary infection, helping to explain the negative effect of TSLP on the expansion of CD8+ T cells in response to influenza virus infection.
We next investigated whether the effects of TSLP on primary CD8+ T-cell responses that we observed with influenza (where infection is at a barrier surface where TSLP is expressed) might extend to a systemic infection such as acute LCMV infection. Using the same adoptive co-transfer method of WT and Crlf2-/- P14 cells (Figure 5A) that we used for influenza infection (Figure 4A), we found that virus-specific P14 CD8+ T cells expressed more TSLPR at day 8 after LCMV infection in all tissues analyzed (blood, lungs, lymph node, and spleen) (Figure 5B), analogous to what we observed after influenza infection in lungs, lymph node, spleen, and BAL fluid (Figure 1C). Moreover, TSLP protein was also increased in the lungs at day 8 after LCMV infection (Figure 5—figure supplement 1). At day 8 p.i. with LCMV, WT, and Crlf2-/- P14 T cell numbers were similar in most tissues examined, but Crlf2-/- cells were modestly more abundant in the spleen (Figure 5C). At a memory time point, CD8+ T cells also expressed TSLPR (Figure 5D), with more Crlf2-/- than WT P14 cells in the blood and lymph nodes of mice (Figure 5E), indicating that TSLP influences CD8+ memory T cell numbers in at least some tissues after acute LCMV infection.
To determine if TSLP also limited the secondary response to an acute systemic infection, analogous to what we observed with influenza infection, we next adoptively transferred equal numbers of LCMV memory WT and Crlf2-/- P14 cells into naïve mice and infected these mice with LCMV Armstrong intraperitoneally (Figure 5F). At day 8 p.i., there were more Crlf2-/- secondary effector P14 T cells than corresponding WT effector P14 T cells in blood, lungs, inguinal lymph nodes, and spleen, with Crlf2-/- P14 T cells making up greater than 60% of the P14 population (Figure 5G). Thus, TSLP negatively regulated the secondary CD8+ T cell response to either an acute local lung infection (influenza) or an acute systemic infection (LCMV), revealing a previously unappreciated role for TSLP in directly modulating memory CD8+ T cell recall responses.
TSLP has been studied extensively in the context of TH2-type immunity, and we previously demonstrated that TSLP can act directly on CD4+ and CD8+ T cells, but its roles in CD8+ T cell responses during viral infection remain poorly understood. Although TSLP is induced by viral infection of CD8+ T cells, there have been conflicting reports regarding its actions on CD8+ T cells during the primary response to influenza infection. One study using Crlf2-/- mice indicated that TSLP does not affect the control of influenza infection nor affect virus-specific CD8+ T cell responses during primary infection (Plumb et al., 2012). Another report concluded that TSLP enhances CD8+ T-cell responses during primary influenza infection, but that this was not due to a direct action on CD8+ T cells and instead was an indirect effect on CD8+ T-cell responses resulting from TSLP-induced IL-15 production by dendritic cells (Yadava et al., 2013). Finally, a third study used an adoptive co-transfer model of WT and Crlf2-/- TCR transgenic ovalbumin-specific CD8+ T cells (OT-I cells) and found that after infection with an OVA-expressing influenza virus, there were fewer Crlf2-/- OT I cells during the primary infection, indicating that TSLP enhances primary CD8+ T-cell responses (Shane and Klonowski, 2014). Thus, the reported roles of TSLP in CD8+ T-cell responses during primary influenza infection have been somewhat variable, possibly at least in part due to variability in the experimental models/design/animal facilities. Here, we found that TSLP acts directly on CD8+ T cells to limit their responses in most tissues during primary influenza infection, with more virus-specific Crlf2-/- cells than virus-specific WT cells. We additionally show that after acute systemic infection with LCMV Armstrong virus, TSLP had a modest effect on primary CD8+ T-cell responses, with only a slight increase in Crlf2-/- cells in the spleen at day 8 p.i. Thus, TSLP can limit CD8+ T cell responses during primary viral infections, but the effect varied based on the tissue assessed and the type of viral infection. The apparent differences in the various studies of TSLP’s impact on CD8+ T cells during primary influenza infection may be due at least in part to the tissues sampled (e.g., Yadava et al., 2013 only assessed responses in BAL fluid). Additionally, the use of influenza viruses with differing pathogenicity in mice might affect the results, as Shane and Klonowski, 2014 used a strain of influenza, x31, which is less pathogenic than the PR8 strain that we used. Overall, the effect of TSLP on CD8+ T cells during the effector phase of a primary acute infection may vary according to the viral infection and tissues analyzed, but our data support the potential for an inhibitory effect by TSLP.
Knowledge of the action of TSLP on memory CD8+ T-cell responses has been limited. We found that TSLP does not affect the development/maintenance of memory CD8+ T cells after primary influenza infection. In contrast, however, we observed differences after systemic acute LCMV infection, with increased Crlf2-/- virus-specific cells in the blood and lymph nodes at a memory time point post-infection. Importantly, TSLP limited memory CD8+ T cell recall responses, with enhanced cellular responses in multiple tissues of Crlf2-/- T cells following either secondary influenza infection or LCMV systemic infection. Interestingly, RNA-Seq data indicated that TSLP suppresses several genes that are related to cell cycle, apoptosis, or protection from virus in influenza infection, with an increased number of virus-specific Crlf2-/- CD8+ T cells.
Thus, while TSLP appears to have variable direct effects on primary CD8+ T cell responses, possibly depending on the context of infection, here we reveal that TSLP differentially affects naïve and memory cell homeostasis. TSLP enhances naïve CD8+ T cell survival in vitro and homeostasis in vivo, but memory CD8+ T cell responses are negatively controlled by TSLP, highlighting a key difference between the two cell types. We also found that TSLP uniformly diminished the CD8+ T cell responses to secondary acute viral infection in all tissues examined in both pulmonary influenza infection and acute LCMV systemic infection, underscoring a greater effect for TSLP on secondary CD8+ T cell responses than primary responses. These findings have potential implications for better controlling secondary responses to viral infection.
Six to ten week old female C57BL/6 mice were obtained from The Jackson Laboratory. P14 TCR transgenic mice were provided by Dr. Dorian McGavern (NINDS/NIH) and were bred to C57BL/6 Thy1.1+ congenic mice from Jackson Laboratories (B6.PL-Thy1a/CyJ) and with Crlf2-/- mice (Al-Shami et al., 2004) in our facility to create congenically-labeled WT and Crlf2-/- P14 mice. All experiments were performed under protocols approved by the National Heart, Lung, and Blood or the National Institute of Neurological Disorders and Stroke Animal Care and Use Committee and followed National Institutes of Health guidelines for the use of animals in intramural research.
Recombinant influenza virus expressing the LCMV gp33-41 epitope (KAVYNFATM) inserted into the NA of A/PR/8/34 (H1N1) (PR8-33) was kindly provided by Dr. Rafi Ahmed (Emory University) (Mueller et al., 2010).
Naïve WT and Crlf2-/- P14 T cells were isolated for use in vitro and in vivo from the spleens of naïve WT and Crlf2-/- P14 mice and purified using a negative selection CD8+ T- cell kit (Stem Cell Technologies). To generate memory CD8+ T cells for use in vivo or in vitro, 5 × 104 (influenza infections) or 105 (LCMV infections) WT or Crlf2-/- naïve P14 cells were injected i.v. into naïve C57BL/6 mice, and the following day the mice were infected with either 103 EID50 of PR8-33 i.n. or 2 × 106 pfu LCMV Armstrong i.p. At >30 days p.i. the spleens were harvested and memory WT and Crlf2-/- P14 cells purified using a negative selection CD8+ T cell kit (Stem Cell Technologies). A sample of the cells were stained with Thy1.1, Thy1.2, CD8, CD44, Vα2, and Live/Dead stain to determine the number of P14 T cells. Equal numbers of naïve or memory WT and Crlf2-/- P14 were combined; verification that equal numbers of WT and Crlf2-/- were added was determined by flow cytometry using the same antibodies listed above, and 2.5 × 104 (influenza) or 103 (LCMV) naive P14 T cells or 104 (influenza) or 103 (LMCV) memory P14 T cells of each population (WT and Crlf2-/-) were co-transferred i.v. into naive C57BL/6 mice. On the day following cell transfer, the mice were infected with either 103 EID50 of PR8-33 i.n. or 2 × 106 pfu LCMV Armstrong i.p. For the assessment of the homeostasis of naïve and memory P14 cells in vivo, equal numbers of naïve or memory WT and Crlf2-/- P14 T cells (~1–2×106 total cells) were co-transferred into naïve C57BL/6 mice in Figure 2D.
Lymphocytes were isolated from tissues as previously described (Masopust et al., 2001), with some modifications. Briefly, bronchoalveolar lavage (BAL) of the airways was performed with 1 ml of PBS containing 1% BSA prior to perfusion of the lungs with PBS. Lungs were treated with 1 mg/ml Collagenase plus 1 mg/ml DNase (Sigma-Aldrich) in 3 ml of RPMI for 45 min at 37° C. Single cell suspensions were obtained by pushing digested lungs, spleens, and lymph nodes through 40 μM mesh screens (BD Biosciences). Lung lymphocytes were purified by centrifuging (2000 rpm, 4°, for 20 min) on a 44/67% Percoll gradient (Sigma-Aldrich).
Naïve and memory WT and Crlf2-/- P14 T cells were obtained and purified as described above in ‘Cell transfers and infections’. 1 × 106 CD8+ T cells were plated in 0.75 ml/well in 48 well plates or 2 × 106 CD8+ T cells were plated in 1.5 ml/well total volume in 24 well plates with either medium or 100 ng/ml TSLP (R&D Systems) with or without stimulation. For in vitro stimulation of naïve cells, the plates were pre-coated with 2 μg/ml anti-CD3ε (BioXcell) and 1 μg/ml of soluble anti-CD28 was added. For in vitro stimulation of memory cells, 1 μg/ml of soluble anti-CD3 was added.
Single-cell suspensions were stained with anti-mouse Thy1.1-APC, BV421, BV605, FITC, or PE (OX-7), Thy1.2-PerCP, PerCp-Cy5.5 or BV510 (53–2.1), CD8-PE, APC or BV421 (53–6.7), CD44-FITC, PerCP, or BV421 (IM7), KLRG1-BV421 (2F1/KLRG1), Vα2-PE or APC (B20.1), CD127-APC (A7R34), TNF-α- PE-cy7 (MP6-XT22), IFN-γ- PE-cy7, BV421 or Alexa Fluor 647 (XMG1.2), Annexin V-FITC, and TruStain FcX all were purchased from Biolegend. 7-AAD was purchased from BD Pharmingen. Polyclonal anti-mouse TSLPR-FITC was from R&D Systems and anti-Granzyme B-V450 from BD Horizon (clone GB11). Intracellular staining for granzyme B was performed directly ex vivo or IFN-γ, TNF-α after a 5 hr in vitro stimulation at 37° with 0.1 μg/ml of gp33 peptide for P14 cells or 0.2 μg/ml of PR8 NP peptide for non-P14 CD8+ T cells in the presence of GolgiStop and GolgiPlug (BD Biosciences) and cells were fixed and permeablized with Cytofix Cytoperm and Perm wash (BD Biosciences). Cells were analyzed on a LSR II, BD Fortessa or Canto II (BD Immunocytometry Systems), and sorting was done on a BD FACS Aria (BD Immunocytometry Systems). Dead cells were excluded by gated on Live/Dead NEAR IR (Invitrogen).
Mouse lungs were excised and homogenized using a Minibead beater (Biospec), cleared by centrifugation, and samples were immediately frozen. IFNγ and TNFα protein was determined using the Mouse Inflammation Panel (13-plex) (BioLegend), according to the manufacturer’s protocol.
RNA was isolated from sorted P14 T cells at the indicated time points using the Zymo RNA miniprep kit (Zymo Research), and 500 ng RNA was used for RNA-Seq library preparation with the Kapa mRNA HyperPrep Kit (KK8580, Kapa Biosystems) and indexed with NEXTflex DNA Barcodes-24. After the final amplification, samples were loaded onto 2% E-Gel pre-cast gels (ThermoFisher), and 250 to 400 bp DNA fragments were excised and purified with Zymoclean Gel DNA Recovery Kit (Zymo Research). After quantification by Qubit (Invitrogen), barcoded samples were mixed and sequenced on an Illumina HiSeq 3000 system. Sequenced reads (50 bp, single-end or paired-end) were obtained with the Illumina CASAVA pipeline and mapped to the mouse genome mm10 (GRCm38, Dec. 2011) using Bowtie 2.2.6 and Tophat 2.2.1. Raw counts that fell on exons of each gene were calculated and normalized by RPKM (Reads Per Kilobase per Million mapped reads). Differentially expressed genes were identified with the R Bioconductor package ‘edgeR’, and expression heat maps were generated with the R package ‘pheatmap’.
Data are presented as mean ± SEM. Two-tailed paired students t-test was used for statistical analysis. All statistical analyses were performed using Prism v7 (GraphPad Software, La Jolla, CA). Differences were considered significant when p<0.05.
A role for thymic stromal lymphopoietin in CD4(+) T cell developmentJournal of Experimental Medicine 200:159–168.https://doi.org/10.1084/jem.20031975
A role for TSLP in the development of inflammation in an asthma modelJournal of Experimental Medicine 202:829–839.https://doi.org/10.1084/jem.20050199
IFITM-Family proteins: the cell's First Line of Antiviral DefenseAnnual Review of Virology 1:261–283.https://doi.org/10.1146/annurev-virology-031413-085537
Update: Influenza Activity - United States and WorldwideMMWR Morb Mortal Wkly Rep.
Influenza Activity - United States, 2015-16 Season and Composition of the 2016-17 Influenza VaccineMMWR Morb Mortal Wkly Rep.
Recent advances in epithelium-derived cytokines (IL-33, IL-25, and thymic stromal lymphopoietin) and allergic inflammationCurrent Opinion in Allergy and Clinical Immunology 15:98–103.https://doi.org/10.1097/ACI.0000000000000133
Eaf2 protects human Lens epithelial cells against oxidative stressinduced apoptosis by wnt signalingMolecular Medicine Reports 17:2795–2802.https://doi.org/10.3892/mmr.2017.8246
TSLP-activated dendritic cells induce an inflammatory T helper type 2 cell response through OX40 ligandJournal of Experimental Medicine 202:1213–1223.https://doi.org/10.1084/jem.20051135
Functional involvement of Interferon-Inducible transmembrane proteins in antiviral immunityFrontiers in Microbiology 10:1097.https://doi.org/10.3389/fmicb.2019.01097
Cutting edge: direct action of thymic stromal lymphopoietin on activated human CD4+ T cellsJournal of Immunology 178:6720–6724.https://doi.org/10.4049/jimmunol.178.11.6720
New insights into the regulation of T cells by γc family cytokinesNature Reviews Immunology 9:480–490.https://doi.org/10.1038/nri2580
The role of thymic stromal lymphopoietin in CD8+ T cell homeostasisJournal of Immunology 181:7699–7705.https://doi.org/10.4049/jimmunol.181.11.7699
TSLP regulates intestinal immunity and inflammation in mouse models of helminth infection and colitisJournal of Experimental Medicine 206:655–667.https://doi.org/10.1084/jem.20081499
Spontaneous atopic dermatitis in mice expressing an inducible thymic stromal lymphopoietin transgene specifically in the skinJournal of Experimental Medicine 202:541–549.https://doi.org/10.1084/jem.20041503
Thymic stromal lymphopoietin as a key initiator of allergic airway inflammation in miceNature Immunology 6:1047–1053.https://doi.org/10.1038/ni1247
Ellen A RobeyReviewing Editor; University of California, Berkeley, United States
Tadatsugu TaniguchiSenior Editor; Institute of Industrial Science, The University of Tokyo, Japan
In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.
The role of the cytokine TSLP on CD4+ T cells responses has been extensively studied, but less is known about its' role in CD8+ responses. This paper shows that CD8+ T cells lacking the TSLP receptor are impaired in their response to viral infection, particularly during secondary challenge responses. They also show that naïve and memory T cells show distinct transcriptional responses to TSLP, providing insight into the function of this important cytokine in CD8+ T-cell response.
Decision letter after peer review:
Thank you for sending your article entitled "Thymic stromal lymphopoietin limits primary and recall CD8+ T cell anti-viral responses" for peer review at eLife. Your article is being evaluated by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation is being overseen by Tadatsugu Taniguchi as the Senior Editor.
As you and your coauthors noted, there is a notable discrepancy between your study and the work of Shane et al., which demonstrated that TslprKO CD8 T cells were reduced at memory timepoints in the broncheoalveolar lavage and lungs using the OT-I TCR transgenic and OVA-expressing influenza virus. While the authors suggest that differences in models could explain the discrepancy between the two studies, it is surprising that these groups arrived at diametrically opposed results using the influenza model (albeit different strains). While the current results with TslprKO mice are strengthened by using the LCMV model, there is concern that there may be unexpected differences between TslprKO mouse lines used by the two groups (Ziegler lab and Leonard lab lines). Of particular concern is the possibility that passenger mutations in one of these lines could have created a confounding variable. For example, see PMCIDs PMC4364188 [Dock8 mutation in Nlrp10KO mice, Eisenbarth group], PMC3326475 [Dock2 mutation in Irf5KO mice, Bhattacharya group], and PMC3660499 [Sox13 mutation in B6.SJL congenic mice, Cyster group]. Both Shane et al. and Ebina-Shibuya, West, et al. appear to have maintained independent crosses of WT and TslprKO mice to P14, which could in theory allow a confounding mutation to be maintained in the TslprKO P14 line. Because of the relatively subtle nature of the observed numerical changes, it is also possible that the observed differences between congenically distinct donor cells arise not from TSLPR, but from background genetic differences between Thy1.1/Thy1.1 and Thy1.1/Thy1.2, which could have been addressed by using co-transfer of congenically distinct WT P14 T cells to a separate group of mice as a control. The best way to address all of these concerns would be to block TSLP signaling using the M702 antibody (see Kuan and Ziegler, Nat Immunol, 2018). This would provide independent validation of the effects of TSLP signaling loss, with the additional benefit of avoiding changes in CD8+ T cell development. Could the authors either singly transfer WT P14 or cotransfer WT and TslprKO P14 to recipients and treat with control Ig or anti-TSLP antibodies as an independent means to validate their findings? In the case of single transfer, donor WT P14s would be expected to increase in mice treated with anti-TSLP abs. In the case of cotransfer, donor WT P14s would be expected to expand equally to TslprKO P14s in mice treated with anti-TSLP abs. If the authors had Thy1.1+ Thy1.2- TslprKO P14 mice available as a separate line, this would be another means to satisfy these points (study currently uses Thy1.1+ Thy1.2+ TslprKO P14 mice). I realize that such experiments are challenging in our current environment, but the reviewers consider addressing these issues critical for publication.https://doi.org/10.7554/eLife.61912.sa1
As you and your coauthors noted, there is a notable discrepancy between your study and the work of Shane et al., which demonstrated that TslprKO CD8 T cells were reduced at memory timepoints in the broncheoalveolar lavage and lungs using the OT-I TCR transgenic and OVA-expressing influenza virus. While the authors suggest that differences in models could explain the discrepancy between the two studies, it is surprising that these groups arrived at diametrically opposed results using the influenza model (albeit different strains). While the current results with TslprKO mice are strengthened by using the LCMV model, there is concern that there may be unexpected differences between TslprKO mouse lines used by the two groups (Ziegler lab and Leonard lab lines). Of particular concern is the possibility that passenger mutations in one of these lines could have created a confounding variable. For example, see PMCIDs PMC4364188 [Dock8 mutation in Nlrp10KO mice, Eisenbarth group], PMC3326475 [Dock2 mutation in Irf5KO mice, Bhattacharya group], and PMC3660499 [Sox13 mutation in B6.SJL congenic mice, Cyster group]. Both Shane et al. and Ebina-Shibuya, West, et al. appear to have maintained independent crosses of WT and TslprKO mice to P14, which could in theory allow a confounding mutation to be maintained in the TslprKO P14 line.
We agree that it is possible that a passenger or other mutation could be influencing the results in one or both of the studies, although both of the Tslpr KO mouse strains have been shown to behave similarly, for example related to Ova-induced allergic inflammation (PMID 16172260 and 16142237). Although the basis for the differences remains unclear, it is possible, as we suggested, that differences in the model and the strains of influenza could affect the outcome, and moreover the animals were in animal facilities at different institutions, so that differences in the microbiome might be another contributing factor as well.
Because of the relatively subtle nature of the observed numerical changes, it is also possible that the observed differences between congenically distinct donor cells arise not from TSLPR, but from background genetic differences between Thy1.1/Thy1.1 and Thy1.1/Thy1.2, which could have been addressed by using cotransfer of congenically distinct WT P14 T cells to a separate group of mice as a control. The best way to address all of these concerns would be to block TSLP signaling using the M702 antibody (see Kuan and Ziegler, Nat Immunol, 2018). This would provide independent validation of the effects of TSLP signaling loss, with the additional benefit of avoiding changes in CD8+ T cell development. Could the authors either singly transfer WT P14 or cotransfer WT and TslprKO P14 to recipients and treat with control Ig or anti-TSLP antibodies as an independent means to validate their findings? In the case of single transfer, donor WT P14s would be expected to increase in mice treated with anti-TSLP abs. In the case of cotransfer, donor WT P14s would be expected to expand equally to TslprKO P14s in mice treated with anti-TSLP abs. If the authors had Thy1.1+ Thy1.2- TslprKO P14 mice available as a separate line, this would be another means to satisfy these points (study currently uses Thy1.1+ Thy1.2+ TslprKO P14 mice). I realize that such experiments are challenging in our current environment, but the reviewers consider addressing these issues critical for publication.
Two different approaches were suggested: using a blocking antibody (M702) to TSLP or using Thy1.1+ Thy1.2-Tslpr KO P14 mice as a second line. Regarding the first approach, we have concerns that the experiment could be technically challenging, as it would require multiple injections of an antibody, where one would also have to assure that a sufficient level of blocking activity was achieved. Moreover, an experiment using a TSLP blocking antibody would block the actions of TSLP on all cell types and thus would not allow one to infer the direct actions of TSLP on CD8+ T cells. Accordingly, we prefer the second approach, and in fact, we already had performed the requested experiment. We apologize for having erred in our labeling of Figure 1D and the cartoon schematic in Figure 1B; although all but one of the included experiments were as indicated, one experiment was in fact included that used Thy1.1+ Thy1.2-Tslpr KO P14 mice. In the appended figure, the data in Figure 1D are now divided to show the data where recipients were Thy1.1+ Thy1.2+Tslpr KO P14 mice in panel A and the experiment in which recipients were Thy1.1+ Thy1.2-Tslpr KO P14 mice in panel B. As you can see, the trend is the same, providing confirmation of the validity of our findings as requested. Figure 1D represents the composite of these data.https://doi.org/10.7554/eLife.61912.sa2
- Warren J Leonard
- Dorian B McGavern
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
This work was supported by the Division of Intramural Research, National Heart, Lung, and Blood Institute and the Division of Intramural Research, National Institute of Neurological Disorders and Stroke. Next generation sequencing was performed in the NHLBI Sequencing core. RE-S. was supported by a Public Interest Incorporated Foundation Scholarship from the MSD Life Science Foundation. We thank Dr. Rosa Nguyen for assisting with harvesting mouse tissues. We thank Dr. Rafi Ahmed, Emory University for valuable suggestions/critical comments.
Animal experimentation: All experiments were performed under protocols approved by the National Heart, Lung, and Blood Institute Animal Care and Use Committee (Protocol H-0087R4) or the National Institute of Neurological Disorders and Stroke Animal Care and Use Committee (Protocol 1295-17) and followed National Institutes of Health guidelines for the use of animals in intramural research.
- Tadatsugu Taniguchi, Institute of Industrial Science, The University of Tokyo, Japan
- Ellen A Robey, University of California, Berkeley, United States
- Received: August 8, 2020
- Accepted: December 6, 2020
- Version of Record published: January 13, 2021 (version 1)
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.