Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Jul;17(7):834-43.
doi: 10.1038/ni.3461. Epub 2016 May 23.

Id2 Reinforces TH1 Differentiation and Inhibits E2A to Repress TFH Differentiation

Affiliations
Free PMC article

Id2 Reinforces TH1 Differentiation and Inhibits E2A to Repress TFH Differentiation

Laura A Shaw et al. Nat Immunol. .
Free PMC article

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.

Conflict of interest statement

COMPETING FINANCIAL INTERESTS

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Differential Id2 and Id3 expression defines TH1 and TFH cell subsets
Id2YFP/+ (a) or Id3GFP/+ (b) SMARTA CD4+ T cells were transferred into B6 mice and analyzed 7 days after LCMV infection. TH1 (SLAM+CXCR5 or CXCR5PD-1), TFH (SLAMloCXCR5+ or CXCR5+PD-1) or GC TFH (CXCR5+PD-1+) cell development in the indicated SMARTA CD4+ T cell populations was analyzed by flow cytometry and quantified. (c) Id2YFP/+Id3GFP/+ SMARTA CD4+ T cells were transferred into B6 mice and analyzed 0, 4 or 7 days after LCMV infection. Flow cytometric analysis of Id2-YFP and Id3-GFP expression (bottom plots) in SLAM+CXCR5 (black lines) or CXCR5+SLAMlo (dashed lines) populations (top plots). (d) Id2YFP/+ reporter mice were immunized subcutaneously with phycoerythrin (PE) emulsified in TiterMax Gold Adjuvant and analyzed by histology 13 days later. Draining lymph node sections were stained with IgD (blue) and CD4 (red). Id2-YFP reporter expression is shown in green. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (two-tailed unpaired Student’s t test). Data are representative of three experiments (a–c), each with n = 3 mice per group, or are representative of two experiments (d), each with n = 2 mice per group (mean ± s.e.m.).
Figure 2
Figure 2. Id2 knockdown results in increased TFH differentiation
SMARTA CD4+ T cells transduced with the indicated shRNAmir-RV were transferred into B6 mice and analyzed 6 (a–f) or 3 days (g–i) after LCMV infection. (a–c) TFH (CXCR5+SLAMlo) and TH1 (SLAM+CXCR5) differentiation was analyzed by flow cytometry (a) and quantified as a fraction of SMARTA CD4+ T cells (b) or total splenocytes (c). (d–f) GC TFH (CXCR5+Bcl6+) differentiation was analyzed by flow cytometry (d) and quantified as a fraction of SMARTA CD4+ T cells (e) or total splenocytes (f). (g–i) TFH (CXCR5+Bcl6+) and TH1 (CXCR5Bcl6) cell development was analyzed by flow cytometry (g) and quantified as a fraction of SMARTA CD4+ T cells (h) or total splenocytes (i). (j–m) OT-II CD4+ T cells transduced with the indicated shRNAmir-RV were transferred into Bcl6fl/fl CD4-Cre+ mice and analyzed 11 days after footpad immunization with NP-OVA in alum. (j) Quantitation of OT-II CD4+ T cells. GC B cells (Fas+PNA+) (k) and plasma cells (CD138+IgD) (l) were analyzed by flow cytometry and quantified as a fraction of B cells. (m) NP-specific IgG titers. The dotted line indicates NP-specific titers in naive mice. *p<0.05, **p<0.001, ***p<0.0001 (two-tailed unpaired Student’s t test). Data are pooled from four (a–f) five (g–i) or two (j–m) independent experiments with n=6-14 mice per group (mean ± s.e.m.).
Figure 3
Figure 3. Id2 is necessary for the generation of TH1 CD4+ helper cells during infection
(a–d,g) Id2+/+ CD4-Cre+ (Id2+/+) or Id2fl/fl CD4-Cre+ (Id2−/−) SMARTA CD4+ T cells were analyzed 4 (a,c,d,g) or 7 (b) days after LCMV infection. (a,b) SLAMhiCXCR5 (green), SLAMmidCXCR5mid (purple) and SLAMloCXCR5+ (blue) cells were analyzed (left panels) and quantified as a frequency of SMARTA CD4+ T cells (middle panels) and total cell numbers (right panels). PSGL-1 and Ly6C (c) and IL-2Rα or Bcl6 (d) expression on the indicated subsets, histogram color (d) corresponds to the gates drawn in (a). (e,f) Id2+/+ or Id2−/− cells were transferred into Bcl6fl/fl CD4-Cre+ mice and analyzed 8 (e) days after LCMV infection. (e) Analysis and quantification of GC B cells (Fas+PNA+) or plasma cells (CD138+IgD) as a normalized frequency of B cells. (f) LCMV-specific IgG titers at the indicated time points. (g) viSNE analysis of total splenic CD4+ T cells (left panel) or SMARTA CD4+ T cells (right panels), with visualization of SLAM and Bcl6 expression by color gradient. (h) Id2+/+ CD4-Cre+ or Id2fl/fl CD4-Cre+ mice were infected with T. gondii and analyzed 7 days after infection. Analysis and quantification of IFNγ, T-bet and Foxp3 expression by lamina propria CD4+ T cells. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (two-tailed unpaired Student’s t test). Data are representative of two (f,h) or three (a–d,g) experiments, each with n= 5–10 mice per group, or are pooled from two (e) independent experiments each with n= 10 mice per group (mean ± s.e.m.)
Figure 4
Figure 4. Increased E2A binding in the absence of Id2 regulates expression of key T helper genes
Id2+/+ CD4-Cre+ (Id2+/+) or Id2fl/fl CD4-Cre+ (Id2−/−) SMARTA CD4+ T cells were transferred into B6 hosts and infected with LCMV. (a) Microarray analysis was performed on the indicated TH1 and TFH populations sorted 7 days after LCMV infection. (b and c) Comparisons between the indicated populations are shown as expression-by-expression plots with FC ≥1.4, CV ≤ 0.10 and expression ≥40. (d) Expression of putative E2A-target genes identified by ChIP-seq between Id2+/+ TH1 (purple) and Id2−/− TH1 (grey). White bars indicate genes not significantly differentially regulated. (e-h) Volcano plots for the indicated data sets. (e) Differentially regulated Id2+/+ TH1 genes from Id2+/+ TH1 vs Id2+/+ TFH mean-class expression (MCE) plot (b). (f) Differentially regulated Id2+/+ TH1 genes from Id2+/+ TH1 vs Id2−/− TH1 MCE plot (c). (g) Differentially regulated Id2+/+ TFH genes from Id2+/+ TFH vs Id2+/+ TH1 MCE plot (b). (h) Differentially regulated Id2−/− TH1 genes from Id2−/− TH1 vs Id2+/+ TH1 MCE plot (c). (i) Comparisons between Id2+/+ and Id2−/− TFH are shown as expression-by-expression plots with FC ≥1.4, CV ≤0.10 and expression ≥40. (j) Frequency of differentially regulated genes (FC ≥1.4) between Id2+/+ TH1 and Id2−/− TH1 populations, which are also E2A targets as indicated by E2A ChIP-seq. Black bar indicates the background frequency of E2A targets. Data are representative of 2 independent experiments each with n= 5 mice per group.
Figure 5
Figure 5. E proteins drive CXCR5 expression and inhibit TH1 formation
(a) Id2+/+ CD4-Cre+ (Id2+/+) or Id2fl/fl CD4-Cre+ (Id2−/−) SMARTA CD4+ T cells were transduced with the indicated shRNAmir-RV, transferred into B6 mice and analyzed 7 days after LCMV infection. TH1 cells were analyzed by flow cytometry and quantified as a fraction of transduced SMARTA CD4+ T cells. (b) CXCR5 expression in GFP+ CD4+ T cells and pooled quantitation. (c) Quantitation of CXCR5 expression by RV+ SMARTA TFH (CXCR5+Bcl6+) cells 3 days after LCMV. (d–k) RV transduced NIP CD4+ T cells were transferred into B6 mice and analyzed 3 (d–g) or 6 days (h–k) after LCMV infection. (d–g) TFH (CXCR5+Bcl6+) and TH1 (CXCR5Bcl6) differentiation was analyzed by flow cytometry (d) and quantified as a fraction of NIP CD4+ T cells (f) or total splenocytes (g). (e) Quantitation of CXCR5 expression in NIP TFH (CXCR5+Bcl6+) cells. (h–k) TH1 (PSGL-1+CXCR5), TFH (CXCR5+PSGL-1+) and GC TFH (CXCR5+PSGL-1) cell development was analyzed by flow cytometry (h) and quantified as a fraction of NIP CD4+ T cells (j) or total splenocytes (k). (I) Quantitation of CXCR5 expression in NIP TFH (CXCR5+SLAMlo) cells. (l) CXCR5 expression by GFP+Ame+ CD4+ T cells and quantitation. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001(two-tailed unpaired (a–k) or paired (l) Student’s t test). Data are representative of two (a,c), three (d,e) or four (h,i) independent experiments or pooled from two (f,g,j,k), four (l) or nine (b) individual experiments each with n= 3-8 mice per group (mean ± s.e.m.).
Figure 6
Figure 6. Bcl6 inhibits Id2 expression
(a) BCL6, H3K27Ac, H3K4me1 and H3K4me3 density tracks of ID2 in human GC TFH cells. Arrows above the BCL6 tracks indicate primers used in ChIP-qPCR analysis. Sequence conservation with mouse is shown at the bottom. (b) PD-1hi GC TFH cells were isolated from human tonsil cells, chromatin was prepared and ChIP was performed for BCL6 at the indicated loci. (c) Bcl6fl/fl CD4-Cre+ SMARTA CD4+ T cells transduced with the indicated vectors were transferred into B6 mice. IL-2Rα+ SMARTA CD4+ T cells were sorted 3 days after LCMV infection and Id2 expression was tested by qRT-PCR. (d) WT (Bcl6+/+), Bcl6fl/WT CD4-Cre+ (Bcl6+/−), Bcl6fl/fl CD4-Cre+ (Bcl6−/−) SMARTA CD4+ T cells were transferred into B6 mice. IL-2Rα+ or IL-2Rα SMARTA cells were sorted and Id2 expression was tested by qRT-PCR. *p<0.05, **p<0.01, ***p<0.0001 (two-tailed unpaired Student’s t test). Data are pooled from two (c,d) or four (b) independent experiments, with n= 5 mice per group (b) (mean ± s.e.m.). For c and d, 3 mice were pooled to create a data point.

Similar articles

See all similar articles

Cited by 27 articles

See all "Cited by" articles

References

    1. Zhu J, Yamane H, Paul WE. Differentiation of effector CD4 T cell populations (*) Annual review of immunology. 2010;28:445–489. - PMC - PubMed
    1. Crotty S. T follicular helper cell differentiation, function, and roles in disease. Immunity. 2014;41:529–542. - PMC - PubMed
    1. Vahedi G, et al. Helper T-cell identity and evolution of differential transcriptomes and epigenomes. Immunological reviews. 2013;252:24–40. - PMC - PubMed
    1. Nurieva RI, et al. Bcl6 mediates the development of T follicular helper cells. Science. 2009;325:1001–1005. - PMC - PubMed
    1. Yu D, et al. The transcriptional repressor Bcl-6 directs T follicular helper cell lineage commitment. Immunity. 2009;31:457–468. - PubMed

Publication types

MeSH terms

Substances

Feedback