CD4(+) T cell anergy prevents autoimmunity and generates regulatory T cell precursors

Abstract

The role of anergy, an acquired state of T cell functional unresponsiveness, in natural peripheral tolerance remains unclear. In this study, we found that anergy was selectively induced in fetal antigen-specific maternal CD4(+) T cells during pregnancy. A naturally occurring subpopulation of anergic polyclonal CD4(+) T cells, enriched for self antigen-specific T cell antigen receptors, was also present in healthy hosts. Neuropilin-1 expression in anergic conventional CD4(+) T cells was associated with hypomethylation of genes related to thymic regulatory T cells (Treg cells), and this correlated with their ability to differentiate into Foxp3(+) Treg cells that suppressed immunopathology. Thus, our data suggest that not only is anergy induction important in preventing autoimmunity but also it generates the precursors for peripheral Treg cell differentiation.

Figure 1

Maternal polyclonal CD4⁺ T cells specific for fetal Ag accumulate during gestation with an anergic phenotype. Wild-type B6 females were bred to Act2W-transgenic syngeneic B6 male mice, and then 2W1S:I-A^b tetramer-binding T cells were tracked at various times during and after gestation. (a) Representative flow cytometry plots showing CD4⁺ T cells from virgin or d18 pregnant females (WT × WT and WT × Act2W) that were immobilized and stained using a combination of PE and APC 2W1S:I-A^b tetramers for cells pooled from spleen and lymph nodes (inguinal, axillary, brachial, cervical, mesenteric and pancreatic). (b) Number of 2W1S:I-A^b tetramer-binding T cells at various time-points during pregnancy. Filled circles indicate virgin mice (c) Representative 2W1S:I-A^b tetramer-binding T cells stained for CD44 and Foxp3. (d) Identification of anergic (CD73^hiFR4^hi) and Teff/mem (CD73^loFR4^lo) cells within the Foxp3⁻CD44^hi fraction of 2W1S:I-A^b tetramer-binding CD4⁺ polyclonal T cells. (e, f) Number and percentage of 2W1S:I-A^b tetramer-binding anergic and Teff/mem cells during pregnancy. (g) Representative Ki67 and Nrp1 staining for 2W1S:I-A^b tetramer-binding Teff/mem and anergic cells on day 18 of pregnancy (h) Percentage of 2W1S:I-A^b tetramer-binding anergic and Teff/mem cells that express Ki67 during pregnancy. (i) Pregnant females on day 18 of gestation were injected with 100 µg of 2W1S peptide for two hours. Tetramer enriched cells were stained intracellularly for IL-2. Mean data are representative of 3 to 4 independent experiments, n = 3 to 6 mice each experiment. Error bars represent the SEM. One-way ANOVA; * p < 0.05 **p < 0.01, *** p < 0.001, **** p < 0.0001 in (c, f, g, i). Unpaired one-tailed Student t-Test (j). Circles indicate individual mice.

Figure 2

Foxp3⁻CD44^hiCD73^hiFR4^hi anergic phenotype CD4⁺ polyclonal T cells accumulate in the secondary lymphoid organs. Cells pooled from spleen and lymph nodes (inguinal, axillary, brachial, cervical, mesenteric and pancreatic) (a) Representative analysis of Foxp3⁻CD44^hi CD4⁺ polyclonal T cells for the expression of CD73 and FR4. (b) Percent Foxp3⁻CD44^lo (naive), Foxp3⁺ (T_reg cell), Foxp3⁻CD44^hiCD73^loFR4^lo (Teff/mem) and Foxp3⁻CD44^hiCD73^hiFR4^hi (anergic) of total CD4⁺ T cells in various mouse strains as indicated. Chi-Square test (c) Change in the mean total number and percentage of anergic CD4⁺ cells from secondary lymphoid organs per mouse over time, with Pearson correlation coefficient (r²) as indicated. (d) Number and percentage of anergic-phenotype CD4⁺ polyclonal T cells from Aire^−/− and age-matched Aire^+/− littermate controls. (e) Sorted polyclonal naive, Teff/mem, anergic, and T_reg cell CD4⁺ T cell subsets from Foxp3^DTR donors stimulated with a combination of anti-CD3 and CD28 for 8 or 96 h (as indicated) and then culture supernatants examined for secretion of IL-2 by ELISA. (f, g) Pooled spleen and lymph node CD4⁺ polyclonal T cell subsets stimulated with 1 µg/ml of PMA and 1µM ionomycin (PMA+Iono.) for 4 h or left unstimulated and then stained for intracellular IL-2, IFN-γ, TNF, IL-17a, IL-10, and IL-21. Mean data are representative of 2 independent experiments, n = 4 to 5 mice each experiment. Error bars represent the SEM. One-way ANOVA (e) and Unpaired one-tailed Student’s t-Test (d, g). * p < 0.05, **p < 0.01, *** p < 0.0001. Points denote individual mice.

Figure 3

Anergic polyclonal CD4⁺ T cells are quiescent at steady state, yet show signs of continuous Ag encounter. Cells pooled from spleen and lymph nodes (inguinal, axillary, brachial, cervical, mesenteric and pancreatic) (a) Ki67 expression on Foxp3⁻CD44^lo naive (shaded histograms), Foxp3⁻CD44^hiCD73^loFR4^lo Teff/mem, Foxp3⁻CD44^hiCD73^hiFR4^hi anergic, and Foxp3⁺ T_reg cell (all three open tracings, as indicated) polyclonal CD4⁺ T cells from 8 to 11 week old B6 mice. The bar graphs below indicate the mean percentage of Ki67 expressing cells in each group. (b) Representative CD69, PD1, and CTLA4 expression, with bar graphs representing the mean percent CD69⁺ and MFI for PD1 and CTLA4. (c) CD4⁺ T cell subsets analyzed for Nr4a1 gene activation, CD5, and Neuropilin-1 in transgenic Nur77^GFP reporter gene mice, with bar graphs below indicating the MFI. Mean data are representative of 2 independent experiments, n = 2 to 3 mice each experiment. Error bars represent the SEM. One-way ANOVA; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, non-significant (ns) (d) Gene expression data for individual sample groups are shown as the fold change (log base 2) relative to the mean of all samples, where red indicates up-regulation and blue indicates down-regulation of transcripts.

Figure 4

Reversal of anergy in polyclonal CD4⁺ T cells gives rise to Foxp3⁺ T_reg cells. Cells pooled from spleen and lymph nodes (inguinal, axillary, brachial, cervical, mesenteric and pancreatic) or as specified. Sorted naive, Teff/mem, anergic, and T_reg cell Foxp3^DTR polyclonal CD4⁺ T cells were transferred (10⁵) into syngeneic lymphopenic Tcra^−/− B6 mice and analyzed 21 d later. (a) Weights of mice over time, relative to start. (b) Number of donor-derived spleen and lymph node CD4⁺ T cells recovered on day 21. Donor-derived CD4⁺ T cell expression of (c) CD73 and FR4, or (d) CD44 and Foxp3 on day 21. (e) Percent and number of Foxp3⁺ T_reg cells among total donor CD4⁺ T cells on day 21. (f, g) Sorted naive and anergic Foxp3^DTR polyclonal CD4⁺ T cells were transferred (5 × 10⁵) into syngeneic lymphoreplete B6 mice and analyzed 30 d later. (f) Representative flow cytometry staining and (g) Percentage and number of Foxp3⁺ T_reg cella among donor derived CD4⁺ T cells on day 30. Mean data shown are representative of 3 (a–e) and 2 independent experiments (f–g), with n = 2 to 3 mice per group. Error bars represent the SEM. One-way ANOVA; * p < 0.05 ** p < 0.01, *** p < 0.001, **** p < 0.0001, non-significant (ns). Points denote individual mice.

Figure 5

Depletion of T_reg cells derived from anergic cells leads to autoimmunity in lymphopenic mice. Sorted naive or anergic syngeneic Foxp3^DTR polyclonal CD4⁺ T cells were transferred (10⁵) into syngeneic lymphopenic Tcra^−/− hosts and treated with either PBS or diphtheria toxin (DT), as indicated. Mice were monitored for weight loss and the experiment stopped if ~20% weight loss was observed. (a) Change in body weight of Tcra^−/− mice receiving anergic T cells from Foxp3^GFP mice and then treated with DT as a toxicity control (‘DT Control’), or else anergic or naive T cells from Foxp3^DTR mice adoptively transferred in the presence or absence of DT. (b) Total donor-derived CD4⁺ T cells recovered on day 21. (c) Day 21 spleen and LN cells from adoptive transfer mice treated with DT (or PBS control) were stimulated with 1 µg/ml of PMA and 1µM ionomycin for 4 h and then examined for intracellular T-bet and IFN-γ levels in donor-derived naive and anergic CD4⁺ T cells. (d) Number and percentage of T-bet⁺ cells among total donor CD4⁺ T cells on day 21. Mean data shown are representative of 3 independent experiments, with n = 4 to 7 mice per group in total. Error bars represent the SEM. One-way ANOVA; * p < 0.05, ** p < 0.01, *** p < 0.0001. Points denote individual mice. (e) Sera obtained from recipient mice on day 21 were used to probe various Rag^−/− tissue extracts (He = heart, Ki = kidney, Pa = pancreas, Li = liver, Lu = lung, Gu = gut, Sa = salivary gland) in an immunoblot. Detectable serum antibody reactivity to tissue antigen(s) is indicated in red.

Figure 6

T_reg cells generated from anergic CD4⁺ polyclonal T cells prevent arthritis and colitis. (a–c) Lymphopenic Tcra^−/− B6G7F1 mice were reconstituted with 1.5 × 10⁶ sorted syngeneic Teff/mem, anergic, or T_reg cell CD4⁺ polyclonal T cells from a Foxp3^DTR mouse for a period of 21 d. On day 21, 10⁴ naive KRN transgenic CD4⁺ T cells were transferred into these mice (as well as into control WT and Tcra^−/− B6G7F1 hosts) and then recipients were monitored over 12 d for arthritis development as described, with the (a) number of KRN cells recovered, and (b) clinical arthritis index score on day 12 after KRN cell transfer as indicated. (c) CD73 and FR4 expression on KRN T cells on day 33. (d–f) A total of 2 × 10⁵ congenic–marked naive polyclonal CD4⁺ T cells were adoptively transferred into Tcra^−/− B6 mice either alone or in combination with 4 × 10⁵ Teff/mem, anergic, or T_reg cell CD4⁺ polyclonal cells sorted from Foxp3^GFP mice, and then recipients were monitored over 8 weeks for weight loss. (d) Number of donor naive cells recovered at the end of week 8. (e) Change in body weight of Tcra^−/− B6 mice receiving naive cells alone, or naive plus Teff/mem, anergic or T_reg cells. (f) Representative H&E staining of the colon (all panels same magnification, 200µm). Mean data shown are representative of 3 (a–c) or 2 (d–f) independent experiments, with n = 2 to 3 mice per group. Error bars represent the SEM. (a–c, d). Cells pooled from spleen and lymph nodes (inguinal, axillary, brachial, cervical, mesenteric and pancreatic). One-way ANOVA; * p < 0.05, ** p < 0.01, *** p < 0.001 **** p < 0.0001, non-significant (ns). Points denote individual mice.

Figure 7

Polyclonal anergic CD4⁺ T cells demonstrate unique gene methylations. Sorted naive, Teff/mem, anergic, and T_reg cell populations were examined for CpG methylation status using bisulfite sequencing. Average methylation for each CpG probed is shown in the top row, followed by rows representing sequencing reactions of individual amplicons. Methylation patterns shown are for (a) Tnfrsf18 (GITR) exon 5, (b) Ctla4 exon 2, (c) Ikzf4 (Eos) intron 1b, and (d) Foxp3 intron 1. Data are pooled from 3 independent sequencing experiments, utilizing T cell subsets sorted from a total of 6 males and 6 females. (e–h) Foxp3⁺ Treg cells sorted on day 21 after transfer of polyclonal anergic cells (10⁵) into syngeneic lymphopenic Tcra^−/− B6 mice were examined for CpG methylation status using bisulfite sequencing. (e) Tnfrsf18 exon 5, (f) Ctla4 exon 2, (g) Ikzf4 intron 1b, and (h) Foxp3 intron 1.

Figure 8

Anergic cells expressing neuropilin-1 are enriched in T_reg cell precursors. Sorted Neuropilin-1 positive (Nrp1⁺) and negative (Nrp1⁻) anergic populations were examined for CpG methylation status using bisulfite sequencing. Average methylation for each CpG probed is shown in the top row followed by rows representing successful sequencing reactions of individual amplicons (pooled from n = 3 individual sequencing experiments, using sorted T cell subsets from 6 mice). Methylation patterns shown are for (a) Tnfrsf18 exon 5, (b) Ctla4 exon 2, (c) Ikzf4 intron 1b, and (d) Foxp3 intron 1. (e) Sorted Nrp1⁺ and Nrp1⁻ anergic cells from Foxp3^GFP polyclonal CD4⁺ T cells were transferred (10⁵) into syngeneic lymphopenic Tcra^−/− B6 mice and analyzed 21 d later. (f) Total CD4⁺ T cells recovered and (g) Representative flow cytometry plots for Foxp3⁺ T_reg cell proliferation and Neuropilin-1 expression in cells recovered on day 21 is shown from pooled spleen and lymph nodes cells (inguinal, axillary, brachial, cervical, mesenteric and pancreatic). (h) The percentage and number of Foxp3⁺ T_reg cells on day 21 after Nrp1⁺ and Nrp1⁻ polyclonal anergic cell transfer. Mean data shown are representative of 3 independent experiments, with n = 2 to 3 mice per group. Error bars represent the SEM. Student’s t-test (f, h). * p < 0.01, ** p < 0.001. Points denote individual mice.