1. Immunology and Inflammation
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The kinase PDK1 is critical for promoting T follicular helper cell differentiation

  1. Zhen Sun
  2. Yingpeng Yao
  3. Menghao You
  4. Jingjing Liu
  5. Wenhui Guo
  6. Zhihong Qi
  7. Zhao Wang
  8. Fang Wang
  9. Weiping Yuan
  10. Shuyang Yu  Is a corresponding author
  1. State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, China
  2. State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, and Center for Stem Cell Medicine, Chinese Academy of Medical Sciences and Peking Union Medical College, China
Research Article
Cite this article as: eLife 2021;10:e61406 doi: 10.7554/eLife.61406
9 figures, 1 table and 1 additional file

Figures

PDK1 supports Tfh cell differentiation and effector functions.

(A) Flow cytometry analysis of PDK1 expression in naïve CD4+ T (CD62LhiCD44lo), Th1, and Tfh cells from C57BL/6J mice on 8 dpi. Quantification of PDK1 MFI is shown on the right (n = 8). (B) Quantitative RT-PCR analysis of Pdk1 abundance in naïve CD4+ T cells, from WT and Pdk1fl/fl::Cd4-Cre mice (n = 3). (C, D) Flow cytometry analysis of CD44+CXCR5+ Tfh cells and CD44+CXCR5- Th1 cells gated on total CD4+ T cells (top panel), or PD-1hiCXCR5+ GC Tfh cells (middle panel) and Bcl-6hiCXCR5+ GC Tfh cells (bottom panel) gated on CD44+CD62L-CD4+ T cells from spleens of WT and Pdk1fl/fl::Cd4-Cre mice on 8 dpi with representative contour plots and cumulative data in (C) and (D), respectively (n ≥ 6). (E, F) Expression of PD-1, ICOS, and Bcl-6 on Tfh cells (CD4+CD44+CXCR5+) was analyzed by flow cytometry with representative histograms and quantification data in (E) and (F), respectively (n = 6). (G) Chemotaxis transwell assay for Tfh cells. Splenocytes from WT and Pdk1fl/fl::Cd4-Cre mice on 8 dpi were added to a transwell plate and migration in the presence of CXCL13 was assessed (n ≥ 5). (H, I) Flow cytometry analysis of splenic PNA+Fas+ GC B cells (top panel) and B220CD138+ plasma cells (bottom panel) from WT and Pdk1fl/fl::Cd4-Cre mice on 8 dpi with representative contour plots and cumulative data in (H) and (I), respectively (n = 4). (J) Confocal microscopy analysis of GC histology in spleen sections from WT and Pdk1fl/fl::Cd4-Cre mice on 8 dpi. Green: PNA, red: IgD, blue: CD4; scale bar: 10 μm. (K) LCMV-specific IgG concentration of sera from WT and Pdk1fl/fl::Cd4-Cre mice on day 8 (top panel) and 56 (bottom panel) post-infection was measured by ELISA (n ≥ 7). Data are representative of at least two independent experiments (A, B, E–G, H–I, K) or pooled from three independent experiments (C, D). Error bars represent SD. *p<0.05, **p<0.01, and ***p<0.001 (Student’s t-test).

Figure 1—source data 1

PDK1 supports Tfh cell differentiation and effector functions.

https://cdn.elifesciences.org/articles/61406/elife-61406-fig1-data1-v1.xlsx
Figure 2 with 1 supplement
PDK1 is essential for Tfh cell differentiation upon protein immunization.

(A, B) Flow cytometry analysis of CD44+CXCR5+ Tfh cells and CD44+CXCR5 Th1 cells gated on splenic CD4+ T cells (top panel) or PD-1hiCXCR5+ GC Tfh cells (middle panel) and Bcl-6hiCXCR5+ GC Tfh cells (bottom panel) gated on splenic CD44+CD62L-CD4+ T cells from WT and Pdk1fl/fl::Cd4-Cre mice on day 8 post-KLH immunization with representative contour plots and cumulative data in (A) and (B), respectively (n = 3). (C, D) Expression of PD-1, ICOS, and Bcl-6 on Tfh cells (CD4+CD44+CXCR5+) was analyzed by flow cytometry with representative histograms and quantification data in (C) and (D), respectively (n = 3). (E, F) Flow cytometry analysis of splenic PNA+Fas+ GC B cells (top panel) and B220-CD138+ plasma cells (bottom panel) from WT and Pdk1fl/fl::Cd4-Cre mice on day 8 post-KLH immunization with representative contour plots and cumulative data in (E) and (F), respectively (n = 3). Data are representative of two independent experiments. Error bars represent SD. *p<0.05, **p<0.01, and ***p<0.001 (Student’s t-test).

Figure 2—source data 1

PDK1 is essential for Tfh cell differentiation and GC responses upon KLH immunization.

https://cdn.elifesciences.org/articles/61406/elife-61406-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
Analysis of T helper cells upon KLH immunization.

(A) Splenocytes from WT and Pdk1fl/fl::Cd4-Cre mice on day 8 post-KLH immunization were restimulated with PMA and Ionomycin at 37℃ for 5 hr. Then the cells were surface stained, followed by intracellular staining of IFNγ, IL-4, and IL-17a (n = 6). (B) Cumulative data on frequency of IFNγ+, IL-4+, and IL-17a+ cells gated on splenic CD4+CD44+CD62L T cells from WT and Pdk1fl/fl::Cd4-Cre mice (n = 6). Data are pooled from two independent experiments. Error bars represent SD. *p<0.05, **p<0.01, and ***p<0.001 (Student’s t-test).

Figure 2—figure supplement 1—source data 1

Analysis of distinct T helper subsets upon KLH immunization.

https://cdn.elifesciences.org/articles/61406/elife-61406-fig2-figsupp1-data1-v1.xlsx
PDK1 intrinsically regulates Tfh cell differentiation.

(A) Generation of bone marrow (BM) chimeric mice. BM cells from WT or Pdk1fl/fl::Cd4-Cre mice (CD45.2+) were mixed with WT (CD45.1+CD45.2+) competitor cells at a 1:1 ratio, and transferred to lethally irradiated WT recipients (CD45.1+CD45.2+). After 9 weeks reconstitution, the recipients were infected with LCMV and analyzed 8 days later. (B) Analysis of chimerism by flow cytometry. WT and Pdk1fl/fl::Cd4-Cre cells (CD45.2+) in CD4+ T cells of chimera mice were determined. (C–E) Flow cytometry analysis of competitive contributions by CD45.2+ cells to the total CD44+CXCR5+ Tfh (C), PD-1hiCXCR5+ GC Tfh (D), and Bcl-6hiCXCR5+ Tfh (E) cell population from recipients with representative contour plots and cumulative data (n = 4). (F, G) Detection of PD-1, ICOS, and Bcl-6 expression on CD44+CXCR5+ Tfh cells from recipients by flow cytometry with representative histograms and quantification data in (F) and (G), respectively (n = 4). Data are representative of two independent experiments. Error bars represent SD. *p<0.05, **p<0.01, and ***p<0.001 (Student’s t-test).

Figure 3—source data 1

PDK1 intrinsically programs Tfh cell differentiation.

https://cdn.elifesciences.org/articles/61406/elife-61406-fig3-data1-v1.xlsx
Figure 4 with 1 supplement
PDK1 is required for both early differentiation and late maintenance of Tfh cells.

(A) Schematic of the SMARTA cell transfer system used for characterization of early Tfh cell commitment. SMARTA CD4+ T cells from Pdk1fl/fl::Rosa26CreER::SMARTA mice were transferred into C57BL/6J (CD45.2+) host mice, followed by Tamoxifen treatment for four consecutive days, LCMV infection, and analyzed on 3 dpi. (B) Quantitative RT-PCR analysis of Pdk1 abundance in donor-derived CXCR5+ Tfh cells from recipients on 3 dpi as in (A) (n = 6). (C) Flow cytometry analysis of Bcl-6+CXCR5+ Tfh cells gated on SMARTA CD4+ T cells from recipients on 3 dpi with representative contour plots and cumulative data (n = 3). (D) Contour plots represents BrdU+ cells gated on donor-derived activated CD4+ T cells (top panel) and CXCR5+ Tfh cells (bottom panel) from WT and Pdk1fl/fl::Rosa26CreER::SMARTA mice on 3 dpi. Cumulative data on frequency of BrdU+ cells are shown on the right (n = 6). (E) Quantitative RT-PCR analysis of selected genes in donor-derived CXCR5+ Tfh cells from recipients as in (A) (n = 3). (F) Schematic of the Tamoxifen-induced deletion system used for characterization of late Tfh cell differentiation. WT and Pdk1fl/fl::Rosa26CreER mice were treated with Tamoxifen from day 4 to day 7 post-LCMV infection and analyzed on 8 dpi. (G) Quantitative RT-PCR analysis of Pdk1 abundance in Tfh cells from WT and Pdk1fl/fl::Rosa26CreER mice on 8 dpi as in (F) (n ≥ 5). (H) Flow cytometry analysis of CD44+CXCR5+ Tfh cells (top panel) gated on CD4+ T cells and Bcl-6+CXCR5+ GC Tfh cells (bottom panel) gated on CD44+CD62L-CD4+ T cells on 8 dpi with representative contour plots and cumulative data (n ≥ 5). (I) Contour plots represents BrdU+ cells gated on activated CD4+CD44+ T cells (top panel) and CD44+CXCR5+ Tfh cells (bottom panel) from WT and Pdk1fl/fl::Rosa26CreER mice on 8 dpi. Cumulative data on frequency of BrdU+ cells are shown on the right (n ≥ 5). Data are representative of at least three independent experiments (B–C, E, G–H) or pooled from two independent experiments (D). Error bars represent SD. *p<0.05, **p<0.01, and ***p<0.001 (Student’s t-test).

Figure 4—source data 1

PDK1 is essential for Tfh cell differentiation at both early and late stages.

https://cdn.elifesciences.org/articles/61406/elife-61406-fig4-data1-v1.xlsx
Figure 4—figure supplement 1
Detection of apoptosis of activated CD4+ T cells and Tfh cells.

(A) Contour plots represents Caspase-3+ cells gated on donor-derived activated CD4+ T cells (top panel) and CXCR5+ Tfh cells (bottom panel) from WT and Pdk1fl/fl::Rosa26CreER::SMARTA mice on 3 dpi. Cumulative data on frequency of Caspase-3+ cells are shown on the right (n = 6). (B) Contour plots represents Caspase-3+ cells gated on activated CD4+CD44+ T cells (top panel) and CD44+CXCR5+ Tfh cell (bottom panel) from WT and Pdk1fl/fl::Rosa26CreER mice on 8 dpi. Cumulative data on frequency of Caspase-3+ cells are shown on the right (n ≥ 5). Data are pooled from two independent experiments (A) or representative of at least three independent experiments. Error bars represent SD. *p<0.05, **p<0.01, and ***p<0.001 (Student’s t-test).

Figure 4—figure supplement 1—source data 1

Analysis of apoptosis of CD4 T and Tfh cells.

https://cdn.elifesciences.org/articles/61406/elife-61406-fig4-figsupp1-data1-v1.xlsx
ICOS-dependent PDK1 promotes transcriptional program for Tfh cells.

(A) RNA-seq analysis of Pdk1fl/fl::Cd4-Cre or WT Tfh cells sort-purified on 8 dpi. Volcano plot shows genes upregulated (red) or downregulated (blue) in Pdk1fl/fl::Cd4-Cre Tfh cells compared with WT cells. (B) KEGG pathway analysis of differentially expressed genes in Pdk1fl/fl::Cd4-Cre Tfh cells relative to their expression in WT Tfh cells. (C) GSEA of the Tfh cell gene signature in Pdk1fl/fl::Cd4-Cre Tfh cells relative to their expression in WT Tfh cells. (D) Quantitative RT-PCR analysis of selected genes in Pdk1fl/fl::Cd4-Cre and WT Tfh cells. Relative expression was normalized to WT cells (n ≥ 4). (E) GSEA of ‘Up-regulated under anti-CD28’, ‘Down-regulated under anti-CD28’, ‘Up-regulated under anti-ICOS-L’, and ‘Down-regulated under anti-ICOS-L’ gene sets in WT and Pdk1fl/fl::Cd4-Cre Tfh cells. (F, G) Flow cytometry analysis of p-AKTT308 level on CD4+ T cells from WT SMARTA cells, cultured in medium without any stimulus (blank), or stimulated with anti-CD3e + anti-CD28, anti-CD3e + anti-ICOS, anti-CD3e + anti-CD28 + anti-ICOS, anti-ICOS, and anti-CD25. Representative histogram plot and cumulative data are shown in (F) and (G), respectively (n = 3). Data are representative of at least two independent experiments (D, F, G). Error bars represent SD. *p<0.05, and ***p<0.001 (Student’s t-test).

Figure 5—source data 1

ICOS-dependent PDK1 activity regulates Tfh cell transcriptional files.

https://cdn.elifesciences.org/articles/61406/elife-61406-fig5-data1-v1.xlsx
Figure 6 with 1 supplement
PDK1 deficiency impaired Tfh cell differentiation via mTORC1 and mTORC2 signal-dependent TCF1 expression.

(A) GSEA of ‘Raptor-activated genes’, ‘Raptor-suppressed genes’, ‘Rictor-activated genes’, and ‘Rictor-suppressed genes’ gene sets in WT and Pdk1fl/fl::Cd4-Cre Tfh cells. (B) Flow cytometry analysis of AKT, p-AKTT308, and p-AKTS473 levels on Pdk1fl/fl::Cd4-Cre and WT Tfh cells on 8 dpi by flow cytometry with representative histograms and quantification data (n = 4). (C) Flow cytometry analysis of FoxO1, p-FoxO1/3a, STAT3, p-STAT3S727, Hif1α, p-S6, GSK3β, p-GSK3βS9, and TCF1 levels on Pdk1fl/fl::Cd4-Cre and WT Tfh cells on 8 dpi by flow cytometry with representative histograms and quantification data (n ≥ 4). (D, E) Flow cytometry analysis of Tfh populations from recipients adoptively transferred with STAT3-CA, TCF1, Bcl-6, or CXCR5 retrovirus-infected SMARTA cells on 8 dpi by flow cytometry with representative contour plots and cumulative data in (D) and (E), respectively (n ≥ 3). Data are representative of at least two independent experiments. Error bars represent SD. *p<0.05, **p<0.01, and ***p<0.001 (Student’s t-test).

Figure 6—source data 1

PDK1 regulates Tfh cell differentiation via mTORC1 and mTORC2 signal-dependent TCF1 expression.

https://cdn.elifesciences.org/articles/61406/elife-61406-fig6-data1-v1.xlsx
Figure 6—figure supplement 1
Analysis of p-PKCζ/λ level in Tfh cells and detection of overexpression level of relative genes in primed CD4+ cells via retrovirus transduction.

(A) Flow cytometry analysis of p-PKCζ/λ level on Pdk1fl/fl::Cd4-Cre and WT Tfh cells on 8 dpi by flow cytometry with representative histograms and quantification data (n = 3). (B) Quantitative RT-PCR analysis of Tcf7, Stat3, Bcl6, or Cxcr5 in GFP+ cells transduced with indicated retrovirus (n = 3). Data are representative of at least two independent experiments. Error bars represent SD. *p<0.05, **p<0.01, and ***p<0.001 (Student’s t-test).

Figure 6—figure supplement 1—source data 1

Analysis of p-PKCζ/λ level and validation of overexpression efficiency.

https://cdn.elifesciences.org/articles/61406/elife-61406-fig6-figsupp1-data1-v1.xlsx
Working model of PDK1 in regulating Tfh cell differentiation.

Left panel: In PDK1-sufficient cells, AKT gets activated by phosphorylation at Thr308 and Ser473. p-AKT activates mTORC1, and mTORC1 further phosphorylates S6 and supports Hif1α expression, promoting protein synthesis, proliferation, and metabolization. mTORC1 also phosphorates STAT3 to induce TCF1 expression. In addition, p-AKT also guards TCF1 activity through the inactivation of GSK3β, an inhibitor of TCF1 and β-catenin. Enhanced TCF1 contributes to Tfh cell differentiation, GC responses, and humoral immunity. Right panel: In PDK1-deficient cells, AKT remains inactivated by loss of phosphorylation at Thr308 and Ser473, which contributes to impaired mTORC1 activity and activities of downstream molecules, inducing p-STAT3-dependent TCF1 expression. In addition, inactivation of p-AKT leads to compromised p-GSK3β, resulting in increased GSK3β activity and subsequently inhibition on TCF1 level. Decreased TCF1 leads to impaired Tfh cell differentiation, GC responses, and humoral immunity.

Author response image 1
Analysis of PD-1, ICOS, and Bcl-6 expression in naive (A), and Th1 (B) cells.
Author response image 2
Analysis of transduction efficiency.

Tables

Appendix 1—key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Genetic reagent (M. musculus)Mouse: C57BL/6J (CD45.2 and CD45.1)Jackson LaboratoryRRID: IMSR_JAX:000664
Genetic reagent (M. musculus)Mouse: B6. Cg-Tg(Cd4-cre)1Cwi/BfluJ (Cd4-Cre)Jackson LaboratoryRRID: IMSR_JAX:022071
Genetic reagent (M. musculus)Mouse: B6. Cg-Ndor1Tg(UBC-cre/ERT2)1Ejb/2J (Rosa26CreER)Jackson LaboratoryRRID: IMSR_JAX:008085
Genetic reagent (M. musculus)Mouse: B6. SMARTAR. AhmedEmory University
Genetic reagent (M. musculus)Mouse: B6. Pdk1fl/flW. YuanChinese Academy of Medical Sciences and Peking Union Medical College
Cell line (H. sapiens)HEK293T
(human embryonic kidney cells)
ATCCCat # CRL-3216, RRID: CVCL_0063
Biological sample (M. musculus)Primary mouse splenocytesChina Agricultural UniversityFreshly isolated from mice
Biological sample (M. musculus)Primary mouse bone marrow cellsChina Agricultural UniversityFreshly isolated from mice
Biological sample (M. musculus)Primary mouse serumChina Agricultural UniversityFreshly isolated from mice
AntibodyRat monoclonal anti-mouse CD19-PE/Cy7Thermo Fisher ScientificCat # 25-0193-82, RRID: AB_657663FACS (1:100)
AntibodyRat monoclonal anti-mouse CD25-PEThermo Fisher ScientificCat # 12-0251-83; RRID: AB_465608FACS (1:100)
AntibodyRat monoclonal anti-mouse CD4-PE/Cy7Thermo Fisher ScientificCat # 25-0041-82; RRID: AB_469576FACS (1:100)
AntibodyRat monoclonal anti-mouse CD4-APC/eFluor 780Thermo Fisher ScientificCat # 47-0041-82; RRID: AB_11218896FACS (1:100)
AntibodyRat monoclonal anti-mouse CD4-BV510BD BiosciencesCat # 563106; RRID: AB_2687550IF
(1:100)
AntibodyRat monoclonal anti-mouse/human CD44-FITCThermo Fisher ScientificCat # 11-0441-82; RRID: AB_465045FACS (1:100)
AntibodyRat monoclonal anti-mouse/human CD44-APCThermo Fisher ScientificCat # 17-0441-83; RRID: AB_469391FACS (1:100)
AntibodyRat monoclonal anti-mouse CD45.1-APCThermo Fisher ScientificCat # 17-0453-82; RRID: AB_469398FACS (1:100)
AntibodyMouse monoclonal anti-mouse CD45.1- Percp/Cy5.5Thermo Fisher ScientificCat # 45-0453-82; RRID: AB_1107003FACS (1:100)
AntibodyMouse monoclonal anti-mouse CD45.2- APCThermo Fisher ScientificCat # 17-0454-82; RRID: AB_469400FACS (1:100)
AntibodyMouse monoclonal anti-mouse CD45.2- eFluor 506Thermo Fisher ScientificCat # 69-0454-82 RRID: AB_2637105FACS (1:100)
AntibodyRat monoclonal anti-mouse CD62L- BV510BioLegendCat # 104441; RRID: AB_2561537FACS (1:100)
AntibodyRat monoclonal anti-mouse CD62L- APCThermo Fisher ScientificCat # 17-0621-83; RRID: AB_469411FACS (1:100)
AntibodyRat monoclonal anti-mouse/human CD45R-FITCThermo Fisher ScientificCat # 11-0452-86; RRID: AB_465056FACS (1:100)
AntibodyRat monoclonal anti-mouse CD45R- PerCP/Cy5.5Thermo Fisher ScientificCat # 45-0451-82; RRID: AB_1107002FACS (1:100)
AntibodyRat monoclonal anti-mouse/human CD45R- BiotinThermo Fisher ScientificCat # 13-0452-86; RRID: AB_466451FACS (1:100)
AntibodyRat monoclonal anti-mouse IgD- APCThermo Fisher ScientificCat # 17-5993-82; RRID: AB_10598660IF
(1:100)
AntibodyRat monoclonal anti-mouse/human GL7- eFluor 450Thermo Fisher ScientificCat # 48-5902-82; RRID: AB_10870775FACS (1:100)
AntibodyArmenian hamster monoclonal anti-mouse PD-1- PEThermo Fisher ScientificCat # 12-9985-82; RRID: AB_466295FACS (1:100)
AntibodyRat monoclonal anti-mouse TCR Vα2-PEThermo Fisher ScientificCat # 12-5812-82; RRID: AB_465949FACS (1:100)
AntibodyRat monoclonal anti-mouse/rat Foxp3-PerCP/Cy5.5Thermo Fisher ScientificCat # 45-5773-82
; RRID: AB_914351
FACS (1:100)
AntibodyRat monoclonal anti-mouse/rat Foxp3-APCThermo Fisher ScientificCat # 17-5773-82
; RRID: AB_469457
FACS (1:100)
AntibodyRat monoclonal anti-mouse CD138-PEBD BiosciencesCat # 553714; RRID: AB_395000FACS (1:100)
AntibodyRat monoclonal anti-mouse CD138-BV421BD BiosciencesCat # 562610; RRID: AB_11153126FACS (1:100)
AntibodyArmenian hamster monoclonal anti-mouse Fas-PEBD BiosciencesCat # 561985;
RRID: AB_10895586
FACS (1:100)
AntibodyMouse monoclonal anti-mouse/human Bcl6-PEBD BiosciencesCat # 561522; RRID: AB_10717126FACS
(1:40)
AntibodyRat monoclonal anti-mouse SLAM-PEBioLegendCat # 115904; RRID: AB_10895586FACS (1:100)
AntibodyRat monoclonal anti-mouse SLAM-APCBioLegendCat # 115910; RRID: AB_493460FACS (1:100)
AntibodyRat monoclonal anti-mouse/human ICOS-PE/Cy7BioLegendCat # 313520; RRID: AB_10643411FACS (1:100)
AntibodyRat monoclonal anti-mouse IFN-γ-FITCThermo Fisher ScientificCat # 11-7311-82;
RRID: AB_465412
FACS (1:100)
AntibodyRat monoclonal anti-mouse IL-17a-PEBD BiosciencesCat # 559502;
RRID: AB_397256
FACS (1:100)
AntibodyRat monoclonal anti-mouse IL-4-PE/Cy7Thermo Fisher ScientificCat # 25-7041-80;
RRID: AB_2573519
FACS (1:100)
AntibodyRabbit monoclonal anti-mouse/human TCF1Cell Signaling TechnologyCat # 2203;
RRID: AB_2199302
FACS (1:100)
AntibodyRabbit monoclonal anti-mouse/rat/human PDK1Cell Signaling TechnologyCat # 3062;
RRID: AB_2236832
FACS (1:100)
AntibodyRabbit monoclonal anti-mouse/human Hif1aCell Signaling TechnologyCat # 36169;
RRID: AB_2799095
FACS (1:100)
AntibodyRabbit monoclonal anti-mouse/rat/human FoxO1Cell Signaling TechnologyCat # 2880;
RRID: AB_2106495
FACS (1:100)
AntibodyRabbit monoclonal anti-mouse/rat/human p-AKTT308Cell Signaling TechnologyCat # 13038;
RRID: AB_2629447
FACS (1:100)
AntibodyRabbit monoclonal anti-mouse/rat/human p-AKTS473Cell Signaling TechnologyCat # 4060;
RRID: AB_2315049
FACS (1:100)
AntibodyRabbit monoclonal anti-mouse/rat/human p-S6S235/236Cell Signaling TechnologyCat # 4858;
RRID: AB_916156
FACS (1:100)
AntibodyRabbit polyclone anti-mouse/rat/human p-FoxO1/3aCell Signaling TechnologyCat # 9464;
RRID: AB_329842
FACS (1:100)
AntibodyRabbit polyclone anti-mouse/rat/human p-PKCζ/λCell Signaling TechnologyCat # 9378;
RRID: AB_2168217
FACS (1:100)
AntibodyRabbit polyclone anti-mouse/rat/human AKTBeyotime BiotechnologyCat # AA326FACS (1:100)
AntibodyRabbit monoclonal anti-mouse/human GSK3βBeyotime BiotechnologyCat # AF1543FACS (1:100)
AntibodyRabbit monoclonal anti-mouse/rat/human p-GSK3βS9Cell Signaling TechnologyCat # 5558
RRID: AB_10013750
FACS (1:100)
AntibodyRabbit polyclone anti-mouse/rat/human p-STAT3S727Cell Signaling TechnologyCat # 9134
RRID: AB_331589
FACS (1:100)
AntibodyRabbit monoclonal anti-mouse/rat/human STAT3Cell Signaling TechnologyCat # 4904
RRID: AB_331269
FACS (1:100)
AntibodyDonkey polyclonal anti-rabbit IgG (minimal x-reactivity)-FITCBioLegendCat # 406403;
RRID: AB_893531
FACS (1:1000)
AntibodyDonkey polyclonal anti-rabbit IgG (minimal x-reactivity)-AF647BioLegendCat # 406414;
RRID: AB_2563202
FACS (1:1000)
Transfected construct (M. musculus)MIGR1 (MSCV-IRES-GFP) (plasmid)This paperN/ARetrovirus construct to transfect
Transfected construct (M. musculus)MIGR1-Tcf7 overexpressing (plasmid)This paperN/ARetrovirus construct to transfect
Transfected construct (M. musculus)MIGR1-Bcl6 overexpressing (plasmid)This paperN/ARetrovirus construct to transfect
Transfected construct (M. musculus)MIGR1-STAT3-CA (constitutive-active) (plasmid)This paperN/ARetrovirus construct to transfect
Transfected construct (M. musculus)MIGR1-Cxcr5 overexpressing (plasmid)This paperN/ARetrovirus construct to transfect
Sequence-based reagentTcf7_FThis paperPCR primersCCCTTCCTGCGGATATAGAC
Sequence-based reagentTcf7_RThis paperPCR primersGGTACACCAGATCCCAGCAT
Sequence-based reagentCxcr5_FThis paperPCR primersCATGGGCTCCATCACATACA
Sequence-based reagentCxcr5_RThis paperPCR primersGGCATGAATACCGCCTTAAA
Sequence-based reagentBcl6_FThis paperPCR primersAGACGCACAGTGACAAACCA
Sequence-based reagentBcl6_RThis paperPCR primersAGTGTGGGTCTTCAGGTTGG
Sequence-based reagentIcos_FThis paperPCR primersTGCCGTGTCTTTGTCTTCTG
Sequence-based reagentIcos_RThis paperPCR primersCTTCCCTTGGTCTTGGTGAG
Sequence-based reagentPdcd1_FThis paperPCR primersCTGGTCATTCACTTGGGCTG
Sequence-based reagentPdcd1_RThis paperPCR primersAAACCATTACAGAAGGCGGC
Sequence-based reagentMaf_FThis paperPCR primersAGCAGTTGGTGACCATGTCG
Sequence-based reagentMaf_RThis paperPCR primersTGGAGATCTCCTGCTTGAGG
Sequence-based reagentHif1a_FThis paperPCR primersCCTTAACCTGTCTGCCACTTTG
Sequence-based reagentHif1a_RThis paperPCR primersTCAGCTGTGGTAATCCACTCTC
Sequence-based reagentGzmb_FThis paperPCR primersCAAAGACCAAACGTGCTTCC
Sequence-based reagentGzmb_RThis paperPCR primersCTCAGCTCTAGGGACGATGG
Sequence-based reagentId2_FThis paperPCR primersGTCCTTGCAGGCATCTGAAT
Sequence-based reagentId2_RThis paperPCR primersTTCAACGTGTTCTCCTGGTG
Sequence-based reagentPrdm1_FThis paperPCR primersACAGAGGCCGAGTTTGAAGAGA
Sequence-based reagentPrdm1_RThis paperPCR primersAAGGATGCCTCGGCTTGAA
Sequence-based reagentGata3_FThis paperPCR primersCTTATCAAGCCCAAGCGAAG
Sequence-based reagentGata3_RThis paperPCR primersCATTAGCGTTCCTCCTCCAG
Sequence-based reagentStat3_FThis paperPCR primersCAATACCATTGACCTGCCGAT
Sequence-based reagentStat3_RThis paperPCR primersGAGCGACTCAAACTGCCCT
Peptide, recombinant proteinKLHSigma–AldrichCat# H7017
Peptide, recombinant proteinGP61-80 (GLNGPDIYKGVYQFKSVEFD)Synthesized by ChinaPeptidesN/A
Peptide, recombinant proteinrecombinant murine IL-2R and DCat # 212–12
Peptide, recombinant proteinrecombinant murine IL-7R and DCat # 217–17
Commercial assay or kitPhosflow Lyse/Fix buffer, 5XBD BiosciencesCat # 558049;
RRID: AB_2869117
Commercial assay or kitPhosflow Perm buffer IBD BiosciencesCat # 557885
RRID: AB_2869104
Commercial assay or kitCaspase-3 Staining KitThermo Fisher ScientificCat # 88-7004-42; RRID: AB_2574939
Commercial assay or kitFixation/Permeabilization Solution KitBD BiosciencesCat # 554714
RRID: AB_2869008
Commercial assay or kitDynabeads M-280 StreptavidinThermo Fisher ScientificCat # 60210
Commercial assay or kitLipofectamine 2000 ReagentThermo Fisher ScientificCat # 11668019
Commercial assay or kitRNeasy Mini KitQiagenCat # 74106
Commercial assay or kitFastQuant RT KitTiangenCat # KR106-02
Commercial assay or kitSuperReal PreMix Plus SYBR GreenTiangenCat # FP205-02
Chemical compound, drugFreund’s Adjuvant, CompleteSigma–AldrichCat # F5881
Chemical compound, drugTamoxifenSigma–AldrichCat # T5648
Chemical compound, drugCorn OilSigma–AldrichCat # C8267
Chemical compound, drugPMASigma–AldrichCat # P8139
Chemical compound, drugIonomycinSigma–AldrichCat # I0634
Chemical compound, drugPolybreneSigma–AldrichCat # H9268
Software, algorithmFlowjo v10.5TreestarRRID: SCR_008520
Software, algorithmGraphpad Prism 8GraphpadRRID: SCR_002798
Software, algorithmAdobe IllustratorAdobeRRID: SCR_010279
Software, algorithmGSEAhttp://www.broadinstitute.org/gsea/RRID: SCR_003199
Other7-AADBD BiosciencesCat # 559925
RRID: AB_2869266
OtherPNA-FITCVector LaboratoriesCat # FL-1071; RRID: AB_2315097FACS (1:500)
OtherPNA-BiotinVector LaboratoriesCat# BA-0074; RRID: AB_2336190IF (1:50)
OtherStreptavidin-APC/eFluor 780Thermo Fisher ScientificCat # 47-4317-82; RRID: AB_10366688FACS (1:500)
OtherStreptavidin-eFluor 450Thermo Fisher ScientificCat # 48-4317-82; RRID: AB_10359737FACS (1:500)

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