Immune tolerance. Group 3 innate lymphoid cells mediate intestinal selection of commensal bacteria-specific CD4⁺ T cells

Abstract

Inflammatory CD4(+) T cell responses to self or commensal bacteria underlie the pathogenesis of autoimmunity and inflammatory bowel disease (IBD), respectively. Although selection of self-specific T cells in the thymus limits responses to mammalian tissue antigens, the mechanisms that control selection of commensal bacteria-specific T cells remain poorly understood. Here, we demonstrate that group 3 innate lymphoid cell (ILC3)-intrinsic expression of major histocompatibility complex class II (MHCII) is regulated similarly to thymic epithelial cells and that MHCII(+) ILC3s directly induce cell death of activated commensal bacteria-specific T cells. Further, MHCII on colonic ILC3s was reduced in pediatric IBD patients. Collectively, these results define a selection pathway for commensal bacteria-specific CD4(+) T cells in the intestine and suggest that this process is dysregulated in human IBD.

Fig. 1. ILC3 expression of MHCII is controlled by a transcriptional pathway previously associated with thymic epithelial cells

A) Mesenteric lymph node (mLN) cells from naïve mice were gated as CD45⁺ lineage (x-axis; CD3, CD5, CD8, NK1.1, y-axis; B220, CD11c, CD11b) negative, CD25⁺ and CD127⁺ and further divided by expression of ST2 (ILC2; red) or CCR6 (ILC3; blue). MHCII expression was determined on ILC3s in mice deficient in B) CIITA, C) CIITA promoter regions (pIII/pIV, pIV) or D) IFN-γ and IFN-γR1. All data representative of at least 3 independent experiments with n=2-3 mice per group. SPF, specific pathogen free.

Fig. 2. MHCII⁺ ILC3s induce deletion of commensal bacteria-specific CD4⁺ T effector cells in the intestine and associated lymph nodes

Total Vβ5⁺ (OT-II) or Vβ8.3⁺ (CBir1) CD4⁺ T cell numbers determined in A) the thymus and B) cLPL of control (H₂0) or antibiotic (ABX)-treated OT-II and CBir1 transgenic mice crossed with MHCII^ΔILC3 or H2-Ab1^fl/fl littermate controls. C) Colon histology (scale bar 200μm) and D) frequencies of (CD45⁺ B220^- CD3^-) Ly6C⁺ Ly6G⁺ neutrophils in the cLPL of CBir1^MHCIIΔILC3 and CBir1^H2-Ab1fl/fl mice. E) Immunofluorescence imaging of mLN sections stained for CD3 (blue), RORγt (green), MHCII (red) or DAPI (grey). White arrows indicate RORγt⁺ cell clusters. Scale bar = 200μm. Insert demonstrates colocalization of RORγt⁺ MHCII⁺ ILCs and CD3⁺ T cells. Insert scale bar = 10μm. F) MHCII expression was restricted to RORγt⁺ ILC3s (MHCII^ILC3+ mice) by crossing Rorc^Cre mice with IAβ^bSTOP^fl/fl (MHCII^neg) mice and G) MHCII levels were determined on B220⁺ B cells, CD11b⁺ CD11c^hi dendritic cells (DCs), CD11b⁺ F4/80⁺ macrophages (Macs) or Lin^neg CD25⁺ CD127⁺ CCR6⁺ ILC3s in the mLN of heterozygote littermates (IAβ^bSTOP^+/fl, MHCII^pos), MHCII^neg or MHCII^ILC3+ mice. (H-K) MHCII^neg and MHCII^ILC3+ received pre-activated CD45.1⁺ CBir1 transgenic CD4⁺ T cells and were injected with CBir1 peptide i.p. every 2 days. Frequencies and numbers of transferred T cells were analyzed in the mLN (H,I), siLPL and cLPL 9 days post transfer (J,K). All data representative of at least 4 independent experiments with n=2-3 mice per group. Results are shown as the mean +/- s.e.m. * p < 0.05, ** p < 0.01, *** p < 0.001 (two-tailed students t-test).

Fig. 3. MHCII⁺ ILC3s directly induce cell death of commensal bacteria-specific CD4⁺ T cells

(A-D) Activated CBir1 CD4⁺ T cells were co-cultured with sort-purified CCR6⁺ ILC3s in the presence or absence of CBir1 antigen or an anti-MHCII neutralizing antibody and A) relative cell recovery (%) was quantified relative to T cells cultured alone, B) frequency of active Caspase-3 expressing cells were assessed and C) frequencies of Annexin V⁺ Dead cell exclusion dye-negative (pre-apoptotic) cells were quantified. D) Expression of Nur77 by CBir1 CD4⁺ T cells and E) expression of Bim by pre-activated CBir1 CD4⁺ T cells following 24h (D) or 48h (E) co-culture with antigen-pulsed MHCII⁺ ILC3s in the presence or absence of an anti-MHCII neutralizing antibody. Mean fluorescent intensity (MFI) values are shown in italics. (F-G) Bim^+/+ or Bim^-/- CBir1 T cells were co-cultured with ILC3s in the presence or absence of CBir1 antigen for 48h and F) relative cell recovery (%) and G) frequencies of Annexin V⁺ pre-apoptotic cells were quantified. H) Heat map of selected candidate genes from mLN-derived CCR6⁺ ILC3s. I) Relative cell recovery (%) of wildtype CBir1 CD4⁺ T cells co-cultured with antigen-pulsed ILC3s in the presence or absence of exogenous rIL-2 or rIL-7. (J-K) Activated CBir1 CD4⁺ T cells with wildtype or (WT) or constitutively active (CA) STAT-5 signaling were co-cultured with ILC3s in the presence or absence of CBir1 antigen for 48h and J) relative cell recovery (%) and K) frequencies of Annexin V⁺ pre-apoptotic cells were quantified. L) WT CBir1 CD4⁺ T cells or CBir1 STAT5-CA CD4⁺ T cells were adoptively transferred into MHCII^neg or MHCII^ILC3+ mice. Mice were administered CBir1 antigen and numbers of FoxP3^- CD45.1⁺ CBir1 T cells in the cLPL were quantified 9 days post-transfer. In vitro assay data are representative of at least 2-3 independent experiments with 2-3 biological replicates per experiment. Array data are representative of a single experiment with 4 biological replicates. All in vivo data are representative of at least 2 independent experiments with at least n=3 mice per group. Results are shown as the mean +/- s.e.m. * p < 0.05, ** p < 0.01, *** p < 0.001 (two-tailed students t-test).

Fig. 4. ILC3-intrinsic MHCII is dysregulated in pediatric Crohn's disease patients and is associated with increased intestinal Th17 cells

A) Lamina propria cells were isolated from colon biopsies from non-IBD control patients and ILCs were identified as CD45⁺ and lineage (x-axis; CD3, CD5, CD14, FcεRI, y-axis; CD11b CD11c, and CD19) negative, CD127⁺ and further divided by expression of ST2L (ILC2s; red) and c-kit (ILC3s; blue) or as lacking expression of both markers (ILC1s; black). Expression of MHC class II (HLA-DR) was then determined on ILC subsets in representative biopsies from B) non-IBD patients or C) pediatric Crohn's disease (CD) patients, and the D) frequencies and E) mean fluorescent intensity of HLA-DR expression on ILC3s was quantified. ILC3 HLA-DR MFI was correlated with F) frequencies of IL-17A⁺ CD4⁺ Th17 cells in colon biopsies and G) commensal bacterial-specific IgG was quantified in the sera of pediatric Crohn's disease patients. (B-E) Representative of n=31 non-IBD and n=31 CD patients or F) n=27 and G) n=21 pediatric Crohn's disease patients. Results are shown as the mean +/- s.e.m. Statistical analyses between patient groups are performed using a Mann-Whitney test * p < 0.05, ** p < 0.01, *** p < 0.001. Correlative analyses were compared by parametric Pearson's rank correlation coefficient (r).