Autoimmune CD4+ T cells fine-tune TCF1 expression to maintain function and survive persistent antigen exposure during diabetes

Abstract

Self-reactive T cells experience chronic antigen exposure but do not exhibit signs of exhaustion. Here, we investigated the mechanisms for sustained, functioning autoimmune CD4⁺ T cells despite chronic stimulation. Examination of T cell priming showed that CD4⁺ T cells activated in the absence of infectious signals retained TCF1 expression. At later time points and during blockade of new T cell recruitment, most islet-infiltrating autoimmune CD4⁺ T cells were TCF1⁺, although expression was reduced on a per T cell basis. The Tcf7 locus was epigenetically modified in circulating autoimmune CD4⁺ T cells, suggesting a pre-programmed de novo methylation of the locus in early stages of autoimmune CD4⁺ T cell differentiation. This mirrored the epigenetic profile of recently recruited CD4⁺CD62L⁺ T cells in the pancreas. Collectively, these data reveal a unique environment during autoimmune CD4⁺ T cell priming that allows T cells to fine-tune TCF1 expression and maintain long-term survival and function.

Keywords: CD4(+) T cell; TCF1; autoimmunity; exhaustion; type 1 diabetes.

Figure 1.. Islet infiltrating T cells include populations with features of exhaustion.

(A and B) Uniform Manifold Approximation and Projection (UMAP) representation, feature plots, and CITE-seq oligo-tagged antibodies based on scRNA-seq of islet T cells. Darker purple denotes higher expression. (C) Frequency of clusters. (D) TCF1 and Tox populations among CD11a^hi islet T cells based on flow cytometric analysis. Numbers represent the average and standard deviation of the population frequency, n=4. (E) Frequency of TCF1 negative cells. (F, G) PD-1, Lag3, Tim3, and BrdU staining on islet T cells, n=5–7. (H) IL-21 (CD4⁺), IFNγ, and IL-2 staining on CD4⁺Foxp3⁻CD3⁺ or CD8⁺CD3⁺ islet T cells, n=7–8. Mean±SD, Mann-Whitney or One-way ANOVA with Tukey’s multiple comparison test, * p<0.05, ** p<0.01, *** p<0.001. See also Figure S1,S2.

Figure 2.. Lower frequency of T cells with a signature of exhaustion in autoimmune T cells compared to T cells in chronic infection.

scRNAseq data from Xia et al. were analyzed in parallel with data from Figure 1. (A) UMAP projection of merged samples (left) and feature plots showing expression of key genes (right). (B) UMAP projections and pie charts of cluster frequency of CD4⁺ T cells. (C) Expression of Pdcd1 (PD-1), Tigit, and Lag3 in clusters 4 (Itgal⁺Tcf7⁺Tox⁻) and 7 (Itgal⁺Tcf7⁺Tox⁺).

Figure 3.. TCF1 is retained in autoimmune CD4⁺ T cells during priming.

(A) Frequency and MFI of TCF1 in CD4⁺Foxp3⁻ 2.5HIP tetramer⁺ T cells in the islets and pancreatic lymph nodes (pLN) of female NOD mice (n=7–13). (B,C) Naive BDC2.5 TCR transgenic T cells were transferred into congenic 8-week-old NOD recipients (n=12). Frequency of TCF1 (B) and Ki67 (C) were analyzed at 7 days, 2- and 6-weeks post transfer. (D) Naïve BDC2.5 T cells were transferred into the immunized congenic recipients 24 hours after immunization and analyzed at day 7. Mean±SD, Mann-Whitney or One-way ANOVA with Tukey’s multiple comparison test, * p<0.05, ** p<0.01, *** p<0.001. See also Figure S3.

Figure 4.. FTY720 blockade reveals functional stability of islet infiltrating CD4⁺ T cells.

(A) Frequency and number of CD4⁺CD3⁺ T cells in islets of FTY720 treated mice. (B,C) Representative flow plots and frequencies of CD62L and CD11a population in CD4⁺Foxp3⁻CD3⁺ islet cells. (D,E) Frequencies of PD-1⁺, TCF1⁺ and TCF1 RFI (E), and percent Ki67⁺ cells (F) in CD4⁺CD3⁺CD11a^hi cells (n=6–8). (G) Experimental design and diabetes incidence of FTY720 and αPD-1 treated mice, n=8–15. (H,I) CD4⁺CD5⁺CD25⁻GITR⁻ (H) or CD4⁺CD5⁺ T cells (I) were sorted from the islets and transferred into NOD.scid recipients which were then monitored for diabetes. (K,J) Representative flow plots (K) and cumulative analysis of IFNγ, Tbet, Ki67, and BrdU gated on islet CD4⁺CD11a^hiPD-1⁺TCF1⁺ effector T cells, (n=5–6). Mean ±SD, Mann-Whitney, * p<0.05, Log-rank or One-way ANOVA with Tukey’s multiple comparison, * p<0.05, ** p<0.01, **** p<0.0001. See also Figure S4.

Figure 5.. Distal lymph nodes serve as a reservoir for non-differentiated autoimmune CD4⁺ T cells.

(A-C) Inguinal lymph nodes were exposed to violet laser and RFP-converted T cells were traced to axillary LN, pancreatic LN, and islets, n=6. (A) Representative flow plots. (B) Percent converted RFP CD4⁺CD3⁺Foxp3⁻ T cells recovered 24 hours post conversion. (C) CD11a and PD-1 expression on GFP and RFP (converted) T cells. (D-E) A full compartment (pooled LNs and Spleen) of CD44^hi or CD62L⁺CD44^lo CD4⁺ and CD8⁺ T cells were sorted from Thy1.1 donor mice and transferred to Thy1.2 congenic recipients. Two weeks post transfer the frequency of donor cells were analyzed in spleens (E) and islets (F) of recipient mice. (G) Frequency of donor cells within CD11a^hi islet effector T cells. Mean ±SD, Mann-Whitney or One-way ANOVA with Bonferroni correction, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. See also Figure S5.

Figure 6.. Islet infiltrating CD62L⁺ CD4⁺ T cells are epigenetically distinct from lymph node naïve cells.

(A-F) The CD4⁺Foxp3⁻ T cells in the pancreatic islets were sorted into 3 populations (CD62L⁺CD11a^lo, CD62L⁻CD11a^lo, and CD11a^hi) while naïve CD62L⁺CD44^lo CD4⁺ T cells were sorted from the lymph nodes and subjected to WGEM-seq, n=4. (A) Top 100 differentially hypermethylated or hypomethylated loci between islet CD11a^hi and ndLN CD62L⁺CD44^lo naïve T cells. The heatmap is scaled by percent methylation and represent the number of standard deviations from the mean rlog value. (B) Total numbers of DMRs and shared DMRs between islet-infiltrating CD11a^hi and CD62L⁺ hyper- and hypomethylated DMRs are shown. University of California Santa Cruz (UCSC) Genome browser plots of the (C) Tcf7, (D) Lef1, (E) Ccl5, and (F) IL21 loci with encode candidate cis-regulatory elements (cCREs) and mammalian conservation tracks. Differentially methylated regions (DMRs) are marked with gray shading and each column within the accompanying heatmap depicts the methylation status of individual CpG sites within the selected DMR. (G,H) CD62L⁺ and CD11a^hi CD4⁺Foxp3⁻ T cells were sorted from islets and lymph nodes of prediabetic NOD.FoxP3-GFP female mice. (G) Ccl5 mRNA expression was measured by qPCR. (H) T cells were stimulated in vitro and Il21 mRNA expression was measured by qPCR. n=3–5, mean ±SD, Mann-Whitney, * p<0.05, ** p<0.01. See also Figure S6.

Figure 7.. Epigenetic pre-programing of autoimmune CD4⁺ T cells.

CD62L⁺CD44-CD4⁺CD25⁻ T cells were sorted from the spleens of BDC2.5 TCR transgenic mice and transferred to congenially labeled 8-week-old NOD mice (n=4) and analyzed 5 weeks post transfer. (A,B) scRNA/ATACseq combined UMAP clustering of CD4⁺Foxp3⁻ populations obtained from the NOD mice and BDC2.5 transfer model. (C) Select gene expression analysis overlaid onto the scRNA/ATACseq UMAP projection. Select clusters were analyzed for expression of Ccl5 (D), Tcf7 (E), and Lef1 (F) RNA transcripts. Peak analysis showing Tcf7 locus for BDC2.5 T cells (G). Transcription factor footprint analysis for TCF1 (H), BATF (I). (J) KEGG pathway analysis of genes associated with significant peak changes in NODis-1 and NODis-3 populations. Transcription factor footprint analysis for JUN::FOS (K), BATF (L) in NOD clusters. See also Figure S7.