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Id2 reinforces TH1 differentiation and inhibits E2A to repress TFH differentiation

Nature Immunology volume 17, pages 834–843 (2016)Cite this article

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Abstract

The differentiation of helper T cells into effector subsets is critical to host protection. Transcription factors of the E-protein and Id families are important arbiters of T cell development, but their role in the differentiation of the TH1 and TFH subsets of helper T cells is not well understood. Here, TH1 cells showed more robust Id2 expression than that of TFH cells, and depletion of Id2 via RNA-mediated interference increased the frequency of TFH cells. Furthermore, TH1 differentiation was blocked by Id2 deficiency, which led to E-protein-dependent accumulation of effector cells with mixed characteristics during viral infection and severely impaired the generation of TH1 cells following infection with Toxoplasma gondii. The TFH cell–defining transcriptional repressor Bcl6 bound the Id2 locus, which provides a mechanism for the bimodal Id2 expression and reciprocal development of TH1 cells and TFH cells.

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Figure 1: Differential expression of Id2 and Id3 defines the TH1 and TFH cell subsets.
Figure 2: Knockdown of Id2 results in enhanced TFH differentiation.
Figure 3: Id2 is necessary for the generation of CD4+ TH1 cells during infection.
Figure 4: Increased E2A binding in the absence of Id2 regulates expression of key helper T cell genes.
Figure 5: E proteins drive CXCR5 expression and inhibit the formation of TH1 cells.
Figure 6: Bcl6 inhibits Id2 expression.

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Change history

  • 02 June 2016

    In the version of this article initially published online, the label above the far right plot in Figure 3g ('Blc6') was incorrect. The correct label is 'Bcl6'. The error has been corrected for the print, PDF and HTML versions of this article.

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Acknowledgements

We thank the members of the Goldrath and Crotty laboratories for discussions; B. Yu for assistance with bioinformatics analysis; and I. Bilic and M. Busslinger for E2A Bio-ChiP-seq data. Supported by the US National Institutes of Health (1F31AG043222-01A1 to L.A.S., AI108651 to L.-F.L., AI067545 to A.W.G., AI109976 to A.W.G. and S.C.), the Fonds de la recherche Québec–Santé (Postdoctoral Training Award to S.B.), The Damon Runyon Cancer Research Foundation (Fraternal Order of Eagles Fellowship DRG-2069-11 to J.P.S.-B.) and the Leukemia and Lymphoma Society (K.D.O. and A.W.G.).

Author information

Author notes

  1. Laura A Shaw and Simon Bélanger: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biological Sciences, University of California San Diego, La Jolla, California, USA

    Laura A Shaw, Kyla D Omilusik, Sunglim Cho, John Goulding, Li-Fan Lu & Ananda W Goldrath

  2. Division of Vaccine Discovery, La Jolla Institute for Allergy & Immunology, La Jolla, California, USA

    Simon Bélanger, J Philip Nance & Shane Crotty

  3. Division of Signaling and Gene Expression, La Jolla Institute for Allergy & Immunology, La Jolla, California, USA

    James P Scott-Browne

  4. Institute for Cancer Genetics, Columbia University Medical Center, New York, New York, USA

    Anna Lasorella

  5. Department of Medicine, University of California San Diego, La Jolla, California, USA

    Shane Crotty

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Contributions

L.A.S. and S.B. performed experiments; K.D.O., Su.C., J.P.S.-B., J.P.N., J.G., A.L. and L.-F.L. provided intellectual input and generated new reagents or performed experiments; and L.A.S., S.B., Sh.C. and A.W.G. conceived of the study, analyzed and interpreted data, and wrote the manuscript.

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Integrated supplementary information

Supplementary Figure 1 Id2 and Id3 define polyclonal TH1 and TFH cell subsets.

Id2YFP/+ (a) or Id3GFP/+ (b) mice were analyzed 7 days after LCMV infection. TH1 (SLAM+CXCR5− or CXCR5−PD-1−), TFH (SLAMloCXCR5+ or CXCR5+PD-1−) or GC TFH (CXCR5+PD-1+) differentiation for the indicated antigen-experienced (CD49d+CD11a+) CD4+ T cell populations was analyzed by flow cytometry and quantified. *p<0.01, **p<0.001 and ***p<0.0001 (two-tailed unpaired Student's t test). Data are representative of three experiments (a,b), each with n = 3 mice per group (mean ± s.e.m.).

Supplementary Figure 2 Knockdown of Id2 results in increased TFH differentiation.

SMARTA CD4+ T cells transduced with the indicated shRNAmir-RV were transferred into B6 SMARTA CD4+ T cells were transduced with the indicated shRNAmir-RV. (a) RNA was isolated from shRNAmir-RV+ CD4+ T cells and Id2 expression was determined by qRT-PCR. shRNAmir-RV+ CD4+T cells were transferred into B6 mice and analyzed 6 (b-e) or 3 (f-i) days after LCMV infection. (b,f) Quantitation of shRNA+ SMARTA CD4+ T cells. (c-e) GC TFH (CXCR5+PSGL-1−) cell development was analyzed by flow cytometry (c) and quantified as a fraction of SMARTA CD4+ T cells (d) or total splenocytes (e). (g-i) TFH (CXCR5+CD25−) and TH1 (CD25+CXCR5−) differentiation was analyzed by flow cytometry (g) and quantified as a fraction of SMARTA CD4+ T cells (h) or total splenocytes (i). (j,k) OT-II CD4+ T cells transduced with the indicated shRNAmir-RV were transferred into B6 mice and analyzed 11 days after footpad immunization with NP-OVA in alum. (j) TFH (CXCR5+PD-1+) and (k) GC TFH (CXCR5+Bcl6+) differentiation was analyzed by flow cytometry and quantified as a fraction of OT-II CD4+ T cells or total lymph node cells. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (two-tailed unpaired Student's t test). Data are pooled from two (a) experiments or representative of two (j,k), four (b-e) or five (f-i) independent experiments with n=6-14 mice per group (mean ± s.e.m.).

Supplementary Figure 3 Expression of Id proteins orchestrates CD4+ T cell differentiation.

(a-c) Id2+/+ CD4-Cre+ (Id2+/+) or Id2fl/fl CD4-Cre+ (Id2−/−) SMARTA CD4+ T cells were transferred into B6 mice and analyzed 4 (a) and 7 (a-c) days after LCMV infection. (a) Flow cytometric analysis of CXCR5 and SLAM expression quantified as gMFI on days 4 and 7. (b) Flow cytometric analysis of granzyme B and TCF1 expression (left panels), quantification as a frequency of SMARTA CD4+ T cells (right panels). (c) Flow cytometric analysis of IFN-γ and T-bet expression in total SMARTA CD4+ T cells. gMFI of T-bet expression and total number of IFN-y+ SMARTA CD4+ T cells is shown. (d) Id2+/+ CD4-Cre+ and Id2fl/fl CD4-Cre+ mice were analyzed 7 days after LCMV infection, SLAMhiCXCR5−, SLAMmidCXCR5mid and SLAMloCXCR5+ cells were analyzed by flow cytometry (left panels) and quantified as a frequency of antigen-experienced (CD49d+CD11a+) CD4+ T cells (middle panel) or as total splenic numbers (right panel). (e) SMARTA CD4+ T cells transduced with the indicated shRNAmir-RV were transferred into B6 mice and analyzed 3 days after LCMV infection. Quantitation of Bcl6 expression in SMARTA TH1 (CXCR5−PD-1−) and TFH (CXCR5+PD-1+) cells is shown. (f) Id2+/+ CD4-Cre+ or Id2fl/fl CD4-Cre+ SMARTA CD4+ T cells were transferred into Bcl6fl/fl CD4-Cre+ mice and analyzed 8 days after LCMV infection. SLAMhiCXCR5−, SLAMmidCXCR5mid and SLAMloCXCR5+ cells were analyzed by flow cytometry (left panels) and quantified as a frequency of SMARTA CD4+ T cells (middle panel) or as total numbers (right panel) *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (two-tailed unpaired Student's t test). Data are representative of one (e) or three (a-d,f) independent experiments with n=3-10 mice per group (mean ± s.e.m.).

Supplementary Figure 4 Expression of Id3 limits unregulated differentiation of TFH cells and GC TFH cells.

(a) Id3+/+ CD4-Cre+ (Id3+/+) or Id3fl/fl CD4-Cre+ (Id3−/−) SMARTA CD4+ T cells were transferred into B6 mice and analyzed 7 days after LCMV infection. (a) Flow cytometric analysis of CXCR5+Bcl6hi (top), SLAMhiCXCR5− (TH1) and SLAMloCXCR5+ (TFH) (middle); or PD-1−CXCR5− (TH1), PD-1−CXCR5+ (TFH) and PD-1+CXCR5+ (GC TFH) (bottom) populations and quantification as a frequency of SMARTA CD4+ T cells (right panels). (b) Id3+/+ CD4-Cre+ and Id3fl/fl CD4-Cre+ mice were analyzed 7 days after LCMV and PD-1−CXCR5− (TH1), PD-1−CXCR5+ (TFH) and PD-1+CXCR5+ (GC TFH) expression was analyzed by flow cytometry (left) and quantified as a frequency of antigen-specific (gp66-77) CD4+ T cells (right). (c-d) NIP CD4+ T cells transduced with the indicated RV were transferred into B6 mice and analyzed 6 days (c) or 3 days (d) after LCMV infection. (c) TFH (CXCR5+SLAMlo) differentiation was analyzed by flow cytometry and quantified as a frequency of NIP CD4+ T cells. (d) Early TFH (CXCR5+Bcl6+) differentiation was analyzed by flow cytometry and quantified as a frequency of NIP CD4+ T cells. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (two-tailed unpaired Student's t test). Data are pooled from two (c,d) experiments or representative of two (a,b) independent experiments with n=8-10 mice per group (mean ± s.e.m.).

Supplementary Figure 5 E proteins drive CXCR5 expression and inhibit the formation of TH1 cells.

(a) Id2+/+ CD4-Cre+ or Id2fl/fl CD4-Cre+ SMARTA CD4+ T cells were transduced with the indicated shRNAmir-RV and expanded in vitro for 4 days. Graph indicates relative mRNA expression of Tcf3 by dsRED+ SMARTA CD4+ T cells. (b) Gene expression of E proteins and related genes of interest in TH1 and TFH SMARTA at day 3 after acute LCMV infection measured by RNA-Seq. Cd8a and Cd19 are included as negative controls. Data are from ref. 32 (GSE67336). SMARTA (c) or NIP (d-k) CD4+ T cells transduced with the indicated RV were transferred into B6 mice and analyzed 3 days (c-f) or 6 days (g-k) after LCMV infection. (c) CXCR5 expression by SMARTA TH1 (CXCR5−Bcl6−) and TFH (CXCR5+Bcl6+) cells. (d,g) Quantitation of RV+ NIP CD4+ T cells. (e) CXCR5 expression by NIP TH1 (CXCR5−Bcl6−) and TFH (CXCR5+Bcl6+) cells. (f) Quantitation of Tbet expression by NIP TH1 (CD25+CXCR5−) and TFH (CXCR5+CD25−) cells. (h-j) TFH (CXCR5+SLAMlo) and TH1 (SLAM+CXCR5−) differentiation was analyzed by flow cytometry (h) and quantified as a fraction of NIP CD4+ T cells (i) or total splenocytes (j). (k) CXCR5 expression by NIP TH1 (SLAM+CXCR5 −) and TFH (CXCR5+SLAMlo) cells.. **p<0.01, ***p<0.0001 (two-tailed unpaired Student's t test). Data are pooled from two (i,j) experiments or are representative of two (a,c) or three (d-h,k) independent experiments with n=8-10 mice per group (mean ± s.e.m.).

Supplementary Figure 6 Bcl6 inhibits Id2 expression.

(a) Bcl6fl/fl CD4-Cre+ SMARTA CD4+ T cells transduced with the indicated vectors were transferred into B6 mice. (b) WT, Bcl6fl/WT CD4-Cre+ (Bcl6+/–), Bcl6fl/fl CD4-Cre+ (Bcl6−/−) SMARTA CD4+ T cells were transferred into B6 mice. Gates used to sort IL-2Rα+ and IL-2Rα− SMARTA CD4+ T cells 3 days after LCMV infection are indicated. (c) Sequences of the primers used in the study. (d) Model for the role of Id and E proteins in orchestrating CD4+ T cell differentiation. Data are representative of 2 independent experiments.

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Shaw, L., Bélanger, S., Omilusik, K. et al. Id2 reinforces TH1 differentiation and inhibits E2A to repress TFH differentiation. Nat Immunol 17, 834–843 (2016). https://doi.org/10.1038/ni.3461

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