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


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.

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Figure 1: Maternal polyclonal CD4+ T cells specific for fetal antigen accumulate during gestation and display an anergic phenotype.
Figure 2: Foxp3CD44hiCD73hiFR4hi anergic-phenotype CD4+ polyclonal T cells accumulate in the secondary lymphoid organs.
Figure 3: Anergic polyclonal CD4+ T cells are quiescent at steady state yet show signs of continuous antigen encounter.
Figure 4: Reversal of anergy in polyclonal CD4+ T cells gives rise to Foxp3+ Treg cells.
Figure 5: Depletion of Treg cells derived from anergic cells leads to autoimmunity in lymphopenic mice.
Figure 6: Treg cells generated from anergic CD4+ polyclonal T cells prevent arthritis and colitis.
Figure 7: Polyclonal anergic CD4+ T cells demonstrate unique gene methylations.
Figure 8: Treg cell precursor populations show enrichment for Nrp1-expressing anergic cells.


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We thank J.A. Bluestone (University of California, San Francisco) for spleen and lymph node cells from Foxp3-Cre-GFP × R26-YFP mice and discussions; D. Mathis and C. Benoist (Harvard Medical School) and the Institut de Genetique et de Biologie Moleculaire et Cellulaire (Strasbourg, France) for B6.g7 mice and KRN B6 mice; A. Rudensky (Memorial Sloan-Kettering Cancer Center) for B6 Foxp3DTR knock-in mice; S.S. Way (University of Cincinnati) for B6 Foxp3GFP and Foxp3DTR CD45.1 mice; J.A. Bluestone (University of San Francisco) for cells from the spleen and all lymph nodes of Foxp3-Cre-GFP × R26-YFP mice; S.C. Jameson, M. Mescher and M.A. Farrar for discussions; P.J. Titcombe for technical support; and N. Shah, T. Martin and J. Motl for assistance in cell sorting. Supported by the Rheumatology Research Foundation (Within Our Reach: Finding a Cure for Rheumatoid Arthritis campaign grant to D.L.M.) and the US National Institutes of Health (01 AI35296 to D.L.M., B.T.F., K.A.H. and M.K.J.).

Author information

L.A.K. and D.L.M. designed the experiments and analyzed the data; L.A.K. performed most of the experiments; S.E.S., S.L.N., W.Y.L., L.O.B., N.Z., G.L.S., D.M., K.E.P. and J.L.L. performed experiments or provided technical help; M.G.O.S. scored histology slides; B.T.F., K.A.H. and M.K.J. provided scientific input; D.L.M. conceived of the study and directed the research; and L.A.K. and D.L.M. wrote the manuscript.

Correspondence to Daniel L Mueller.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 A Foxp3CD44hiCD73hiFR4hi anergic compartment shows enrichment for autoreactive insulin-specific CD4+ polyclonal T cells.

(a) InsB10-23:I-Ag7 and HEL:I-Ag7 tetramer-binding CD4 polyclonal T cells (pulled-down and detected as previously reported) in NOD and B6.g7 mice were stained for Foxp3 and CD44. (b) CD73 and FR4 expression on the Foxp3CD44hi tetramer-binding CD4+ polyclonal T cells. (c) Percent and number of Foxp3CD44hiCD73hiFR4hi anergic CD4 polyclonal T cells for each tetramer-binding specificity. Mean data shown are representative of 2 independent experiment, n = 5 to 7 animals per experiment. Error bars represent the SEM. Unpaired student’s t-test (c); * p < 0.05, ** p < 0.01. Points denote individual mice.

Supplementary Figure 2 KRN CD4+ T cells cause arthritis in lymphopenic Tcra−/− mice following reversal of anergy.

CD73hiFR4hi KRN transgenic CD4+ T cells made anergic by adoptive transfer into WT B6G7F1 hosts for 6 days were subsequently recovered by flow cytometric cell sorting and transferred (104) into either WT or Tcra−/− B6G7F1 hosts. (a) CD73 and FR4 expression on donor KRN cells recovered from the WT and Tcra−/− hosts 15 days later. (b) Percent change in the body weight of WT and Tcra−/− mice receiving anergic KRN T cells. (c) Arthritis Clinical Index score for WT and Tcra−/− mice receiving anergic KRN T cells. Data shown are representative of 2 independent experiments, n = 2 to 3 animals per experiment. Error bars represent the SEM. Unpaired student’s t-test (b) or Mann-Whitney U-test (c) at day 15. * p < 0.0001

Supplementary Figure 3 Gating strategy and purity after sorting.

(a) Polyclonal CD4+ T cells from Foxp3DTR mice were first isolated by MACS CD4 negative selection, and then the naive, Teff/mem, anergic and Treg cell subsets were physically sorted by flow cytometry using the gating strategy shown. Arrowheads indicate the subpopulations collected. (b) Post-sort purity for naive, Teff/mem, anergic and Treg cells.

Supplementary Figure 4 Experimental design for Figure 5.

Sorted naive or anergic syngeneic Foxp3DTR 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.

Supplementary Figure 5 Reversal of anergy in polyclonal CD4+ T cells results in autoantibody production after ablation of newly generated Treg cells.

Lymphopenic Tcra−/− mice were given an adoptive transfer (105) of either naive or anergic syngeneic Foxp3DTR polyclonal CD4+ T cells, followed by every other day treatment with diphtheria toxin (DT). Sera were recovered from mice 21 days later and used to probe various tissue extracts (as indicted). (a) Sera taken from Rag−/− (top), Tcra−/− (middle), and WT B6 (bottom) mice were assayed as a negative control for autoantibody generation. (b) Sera obtained from three separate adoptive transfer recipients of naive T cells or (c) anergic T cells. Arrowheads indicate self antigen-binding by serum antibodies that are uniquely present with recipients of anergic CD4+ T cells. m = marker, He = heart, Ki = kidney, Pa = pancreas, Li = liver, Lu = lung, Gu = gut, Sa = salivary gland. Summary data are shown in figure 5e. Note that an irrelevant 60 Kd background band (most prominent in lung extracts) was demonstrated in all blots even in the absence of serum antibody (not shown), and this band is disregarded.

Supplementary Figure 6 Quantification and frequency of Treg cells recovered in models of arthritis and colitis from polyclonal anergic cells.

(a-c) Experimental design for the KRN model of arthritis (a). The number and percentage of Treg cells recovered on day 33 of reconstitution (b-c). 3 independent experiments. 1-3 mice per group. (d-e) Experimental design for colitis experiment (d). Percentage and number of Treg cells recovered on week 8 (e). 2 independent experiments. 2 mice per group. Mean data shown. Error bars represent the SEM. One-Way ANOVA (c); * p < 0.05, ** p < 0.001, *** p < 0.0001, ns (non-significant). Points denote individual mice.

Supplementary Figure 7 Formerly Foxp3-expressing cells make up a very small fraction of anergic cells.

(a) CD4+ T cells from the spleen and all lymph nodes of Foxp3-Cre-GFP x R26-YFP were gated on CD44 and Foxp3GFP, then the naive and Treg cells are gated on YFP. (b) Foxp3CD44hi anergic and Teff/mem cells were analyzed for exFoxp3 cells by YFP expression. (c) Percent and number of exFoxp3 cells in naive, Teff/mem and anergic cells is shown. Mean data shown. Error bars represent the SEM. One-Way ANOVA (c); * p < 0.05, ** p < 0.01, *** p < 0.0001, ns (non-significant). Points denote individual mice.

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Kalekar, L., Schmiel, S., Nandiwada, S. et al. CD4+ T cell anergy prevents autoimmunity and generates regulatory T cell precursors. Nat Immunol 17, 304–314 (2016).

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