Immune tolerance of food is mediated by layers of CD4+ T cell dysfunction

Abstract

Gastrointestinal health depends on the adaptive immune system tolerating the foreign proteins in food^1,2. This tolerance is paradoxical because the immune system normally attacks foreign substances by generating inflammation. Here we addressed this conundrum by using a sensitive cell enrichment method to show that polyclonal CD4⁺ T cells responded to food peptides, including a natural one from gliadin, by proliferating weakly in secondary lymphoid organs of the gut-liver axis owing to the action of regulatory T cells. A few food-specific T cells then differentiated into T follicular helper cells that promoted a weak antibody response. Most cells in the expanded population, however, lacked canonical T helper lineage markers and fell into five subsets dominated by naive-like or T follicular helper-like anergic cells with limited capacity to form inflammatory T helper 1 cells. Eventually, many of the T helper lineage-negative cells became regulatory T cells themselves through an interleukin-2-dependent mechanism. Our results indicate that exposure to food antigens causes cognate CD4⁺ naive T cells to form a complex set of noncanonical hyporesponsive T helper cell subsets that lack the inflammatory functions needed to cause gut pathology and yet have the potential to produce regulatory T cells that may suppress it.

Extended Data Figure 1.. Gliadin antibody production after feeding depends on Tfh cells.

Gliadin antibody amounts determined by Enzyme-Linked Immunosorbent Assay in serial dilutions of serum from Bcl6f^l/fl (n=6 mice) or Lck^Cre Bcl6^fl/fl mice (n=6 mice) on a gliadin-containing diet, wild-type C57BL/6 mice on a gliadin-free diet (n=2 mice), or C57BL/6 mice on a gliadin-free diet mice 2 weeks after subcutaneous injection of gliadin in complete Freund’s adjuvant (n=2 mice). Wild-type and Bcl6f^l/fl mice have the capacity to produce Bcl-6 and Tfh cells. Lck^Cre Bcl6^fl/fl mice have a T cell-specific defect in Bcl-6 and cannot produce Tfh cells. Values for each dilution from Bcl6f^l/fl or Lck^Cre Bcl6^fl/fl mice were compared by Student’s t-test. The data show that Tfh-sufficient mice on a gliadin-containing diet make small amounts of gliadin antibody, while Tfh-deficient mice do not.

Extended Data Figure 2.. Th^lin- cells induced by peptide feeding are stable after cessation of peptide feeding and lack markers of Th2, Tr1, and Th3 cells.

a, Flow cytometry plots of 2W:I-A^b tetramer-binding CD4⁺ T cells from mice fed with 2W peptide 3 times and analyzed 2 days or 1 week after cessation of feeding, with a gate on Treg cells. b, Percentages of Treg (left) or Th^lin- (right) cells in 2W:I-A^b tetramer-binding populations from mice fed with 2W peptide 3 times and analyzed at the indicated times after cessation of feeding (n=6 mice for 2 days, n=5 mice for 1 wk, n= 4 mice for 2 wks, n=3 mice for 3 wks). c, Flow cytometry plots of 2W:I-A^b tetramer-binding Th^lin- cells from Il4^CD2 (left plot) or wild-type (middle and right plots) mice fed with 2W peptide 3 times and analyzed 2 days after cessation of feeding for expression of human CD2 (a marker of IL-4 and Th2 cells in Il4^CD2 mice), LAG-3 (a marker of Tr1 cells), or LAP (a marker of Th3 cells) (n=4 or 5 mice). The scatterplot shows the percentages of cells with these markers in the 2W:I-A^b tetramer-binding Th^lin- populations from individual mice.

Extended Data Figure 3.. Experimental scheme used to identify CD4⁺ T cell subsets and a heatmap of protein expression assessed by flow cytometry.

a, Experimental scheme used to identify 2W:I-A^b, TOXO:I-A^b, or LLO:I-A^b tetramer-binding Th^lin- cells from CD45.1 mice fed the peptides on days 0, 2, and 4 and analyzed 2 days after the last feeding, 2W:I-A^b, TOXO:I-A^b, or LLO:I-A^b tetramer-binding Th1 and Th17 cells from mice immunized 7 days earlier with the peptides in complete Freund’s adjuvant (CFA), or tetramer-negative Treg, naïve, Tfh, and anergic T cells. b, Heatmap of the log2 values of mean fluorescence intensity (MFI) for each PE-labeled antibody for a given T cell subset divided by the MFI for that antibody for naïve T cells. These normalized values were used for the PCA shown in Fig. 2a.

Extended Data Figure 4.. Flow cytometry histograms of protein expression by CD4⁺ T cell subsets.

a, Histograms of antibody staining of the indicated proteins on or in the indicated subsets identified in Extended Data Figure 3a. b, Histograms of antibody staining of the indicated proteins on or in Glp:I-A^b tetramer-binding Th^lin- cells identified as in Extended Data Figure 3a from mice on a gliadin-containing diet for 1 week. The other subsets were identified as in Extended Data Figure 3a except that the Th1/Th17 cells were isolated from the non-tetramer-binding CD4⁺ T cell population.

Extended Data Figure 5.. RNA sequencing data used for definition of clusters of CD4⁺ T cells from mice fed with peptides.

a, Heat map of signature genes that defined the 11 clusters shown in Fig. 2b with yellow bars indicating larger amounts of mRNA. b, UMAPs shown in Fig. 2b with a border around cluster 5 and cells with larger amounts of Tbx21 (top) or Foxp3 (bottom) mRNA shown in purple.

Extended Data Figure 6.. RNA sequencing data used for definition of clusters of CD4⁺ T cells from mice on a gliadin-containing diet.

a, Single-cell RNA sequencing analysis of Glp:I-A^b tetramer-binding T cells from mice exposed to a gliadin-containing diet for 1 week (Gliadin), 2W:I-A^b tetramer-binding T cells from mice infected with 2W-expressing Listeria monocytogenes (Lm), or naïve CD4⁺ T cells (Naïve). UMAPs identifying 9 T cell clusters in merged populations (left panel) and in each population (right panel) are shown. b, c, Expression levels of signature genes from each cluster are shown as violin plots (b) or a heat map (c). Note that the total Glp:I-A^b tetramer-binding T cell population was included in the analysis not just the CD44^hi subset. In this analysis, clusters 1 and 2 contained naïve T cells defined by expression of Sell, Ccr7, and Ly6C1. The Lm-induced 2W:I-A^b-specific effector cell population, known to contain Th1 and Tfh cells, consisted of cells in clusters 0, 3, 5, and 6. Clusters 0 and 3 contained Th1 cells as evidenced by expression of the Th1 markers Cxcr3 and Itga4, with cells in cluster 3 expressing markers of more advanced Th1 differentiation (Ccr2, Nkg7, and Ly6C2)^–. Cluster 5 contained Tfh cells as evidenced by expression of the Tfh markers Bcl6 and Cxcr5. Cluster 6 was defined by cells expressing interferon-induced genes (Gbp2, Stat1, Ifi47, Ly6A, and Igtp) and may contain Tfh-related cells based on proximity in UMAP space to cluster 5. The Glp:I-A^b tetramer-binding population from mice on a gliadin-containing diet contained some naïve cells in clusters 1 and 2 and cells in clusters 4, 7, and 8 that were not found in the Lm-induced effector population. Cluster 7 contained Treg cells based on expression of Foxp3, while the cells in cluster 8 were Tfh-like cells based on expression of Bcl6 and Cxcr5 but were different from the genuine cluster 5 Tfh cells induced by Listeria infection. The cells in cluster 4 were defined by expression of naïve cell markers Sell and Ccr7 and markers of TCR signaling such as Nr4a1, which were also expressed by the Treg cells in cluster 7 and the Tfh-like cells in cluster 8. Glp:I-A^b tetramer-binding cells in clusters 4 and 8 also expressed Izumor1. Cluster 4 in the Glp:I-A^b tetramer-binding population shown in Extended Data Figure 6a likely corresponds to Th^lin- naïve-like cluster 0 or 1 in the peptide-feeding experiment shown in Fig. 2b, while cluster 8 in the Glp:I-A^b tetramer-binding population likely corresponds to Tfh-like cluster 3 in the peptide feeding experiment. The minor cluster 8 (Th^lin- Treg-like type 1 interferon-signaled) and cluster 9 (Th^lin- Tfh-like ongoing TCR signaling) subsets that were detected in the peptide feeding experiment (Fig. 2b) were not detected in the Glp:I-A^b tetramer-binding population shown in Extended Data Figure 6a, perhaps because fewer cells were analyzed in this case. Thus, the cognate T cell population induced by an antigen in a natural diet and that induced by peptide feeding by gavage contained Treg cells and Th^lin- cells with naïve-like and Tfh-like subsets.

Figure 1.. Food antigen-specific T cells mainly become Treg or Th^lin- cells in the GLA SLO.

a, b, CD4⁺ T cells from SLO of mice on gliadin-free (GFD) (a) or gliadin-containing diets (GCD) for 1 week (b). c, d, Glp:I-A^b tetramer⁺ cell numbers (n=6 mice) (c) and frequencies of subsets in the GCD group (d) (n=5 mice, 3 independent experiments). e-h, (e) CD4⁺ T cells from SLO of mice fed with the peptides alone or with cholera toxin on days 0, 2, and 4 and analyzed 2 days after the last feeding (3X protocol) with tetramer⁺ cell numbers (f, n=6 mice for 2W+CT, 2W, and TOXO, and n=5 for LLO, 3 independent experiments) and subset frequencies (g, n=4 mice for 2W+CT, n=6 for 2W and TOXO, and n=5 for LLO, 3 independent experiments). h, 2W:I-A^b tetramer⁺ cell numbers in the 2W and 2W+CT groups shown in g (n=5 mice for 2W+CT, n=6 in 2W, 3 independent experiments). i, 2W:I-A^b tetramer⁺ cell numbers in the SLO of mice fed on the 3X protocol or on days 0, 2, 4, 6, 8, and 10 (6X) with 2W peptide alone and analyzed 2 days after the last feeding (n=5 mice, 2 independent experiments). j, 2W:I-A^b tetramer⁺ cell numbers in mice fed on the 3X protocol with 2W peptide alone (closed square, n=6 mice, 3 independent experiments) or with FTY720 treatment (open square, n=7 mice, 3 independent experiments). k, CD4⁺ T cells in the indicated tissues from mice fed with 2W, TOXO, and LLO peptides on the 3X protocol. l, Frequencies of subsets among the tetramer⁺ populations shown in k (n=9 mice for Non-GLA and GLA, n=5 for SI and Colon, 3 independent experiments). Error bars indicate mean ± SEM. Data in f and l were compared by one-way ANOVA and post hoc Tukey’s test, and in c, h, i, and j by two-tailed Student’s t test.

Fig. 2.. The Th^lin- population is mixture of naïve-like and Tfh-like cells that are related to anergic T cells.

a, Principal Components Analysis plot based on flow cytometry measurements of proteins expressed by naïve, Treg, Anergic, Tfh, Th1 and Th17, and Th^lin- cells identified as shown in Extended Data Fig. 3a. b, c, Single-cell RNA sequencing analysis of CD44^hi 2W:I-A^b, TOXO:I-A^b, or LLO:I-A^b tetramer-binding T cells from the SLO of mice fed on days 0, 2, and 4 (3X) or 0, 2, 4, 6, 8, and 10 (6X) with the 3 peptides alone or days 0, 2, and 4 with the 3 peptides plus cholera toxin (3X+CT) or CD44^lo tetramer-negative CD4⁺ T cells (naïve). Tetramer-binding T cells from the fed groups were sorted 2 days after the last feeding. b, UMAPs identifying T cell clusters. Ovals delineate individual clusters 2, 4, 5, and 6, or clusters 7 and 10 containing proliferating cells, or the set of Th^lin- clusters 1, 2, 3, 8, and 9. c, Violin plots showing expression levels of signature genes.

Fig. 3.. The Th^lin- population is hyporesponsive and generates peripheral T reg cells.

a, IL-2 production by 2W:I-A^b tetramer-binding Th1 and Th17 cells from mice immunized with 2W peptide in complete Freund’s adjuvant or Th^lin- cells from mice fed 3 times with 2W peptide, 2 hours after intravenous injection of 2W peptide. Mean fluorescent intensity values ± SEM of IL-2 antibody staining are shown. Gray histograms are from CD44^lo CD4⁺ T cells. b, CD44^lo CD4⁺ T cells or Th^lin- cells sorted from 2W:I-A^b, TOXO:I-A^b, and LLO:I-A^b tetramer-enriched samples from Foxp3^RFP Rorc^eGFP Tbx21^Zsgreen reporter mice fed 3 times with 2W, TOXO, and LLO were transferred into CD90.1 SM1 TCR transgenic Rag^−/− mice, which were immunized with polyI:C and peptides and analyzed after 7 days. Frequencies of T-bet⁺ Th1 cells in recipient mice among tetramer-binding cells from the indicated donors are shown (n=5 mice, 2 independent experiments). c, CD44^lo CD4⁺ T cells and Th^lin- cells sorted as in b were transferred into CD45.1 mice, which were fed 3 times with peptides. Frequencies of subsets among tetramer-binding cells from the indicated donors 2 days after the last feeding are shown (n=5 mice, 3 independent experiments). d, Th^lin- cells sorted as in b were transferred into CD45.1 mice, which were fed 3 times with peptides without (None) or with injection of IL-2 antibody (IL-2 ab) during feeding. Frequencies of Treg cells among donor-derived tetramer-binding cells in the 2 groups are shown (n=6 mice, 2 independent experiments). e, 2W peptide was fed and IL-2 antibody was injected as indicated. Treg cell frequencies among CD44^hi 2W:I-A^b+ T cell populations analyzed 2 days after the last feeding (n=6 mice, 2 or 3 independent experiments) are shown. Error bars indicate mean ± SEM. Data in e were compared by one-way ANOVA and post hoc Tukey’s test, and in c and d by two-tailed Student’s t test.

Figure 4.. Treg cells inhibit Th1 cell formation during feeding by sequestering IL-2 and after feeding and priming.

a-c, 2W:I-A^b tetramer⁺ cell numbers in Foxp3^DTR mice that were not (gray bars) or were (blue bars) treated with diphtheria toxin (DT) and fed with 2W peptide 3 times or were treated with DT, fed 2W peptide 3 times, and treated with IL-2 antibody during peptide feeding (red bars) and analyzed 2 days after the last feeding (a) with the frequencies (b) and numbers (c) of cells in subsets in each group (n=4 mice, 3 independent experiments). d, Footpad thickness of mice fed with nothing or with 2W peptide on days 0, 2, and 4, primed with 2W peptide in complete Freund’s adjuvant, and injected with 2W peptide in the footpad 1 day before measurement (n=4 mice, 1 experiment). e, 2W:I-A^b tetramer⁺ cell numbers in SLO of mice fed no 2W peptide, or on days 0, 2, and 4 with 2W peptide alone or with cholera toxin (CT) and enriched with 2W:I-A^b tetramer on day 6 (open bars, n=4 mice, 2 independent experiments), or from mice from those groups primed systemically with 2W peptide plus polyI:C 2 days after the last feeding and enriched with 2W:I-A^b tetramer 7 days later (gray bars, n=5 mice, 2 independent experiments). Fold increases in post-peptide/polyI:C priming numbers compared to the pre-priming numbers are shown. f, Frequencies of indicated subsets among 2W:I-A^b tetramer⁺ cells in the post-peptide/polyI:C primed groups shown in e (n=5 mice, 2 independent experiments). g, T-bet⁺ Th1 cell frequencies among 2W:I-A^b tetramer⁺ cells from naïve or 2W peptide-fed Foxp3^DTR mice that were (red bars, n=4 mice, 2 independent experiments) or were not (blue bars, n=5 mice, 2 independent experiments) treated with DT before immunization with 2W peptide plus poly I:C and analyzed 7 days later. Error bars indicate mean ± SEM. Data were compared by one-way ANOVA and post hoc Tukey’s test.