Defective differentiation of regulatory FoxP3+ T cells by small-intestinal dendritic cells in patients with type 1 diabetes

Abstract

Objective: The gut environment modulates the pathogenesis of type 1 diabetes (T1D), but how it affects autoimmunity toward pancreatic β-cells, a self-tissue located outside the intestine, is still unclear. In the small intestine, lamina propria dendritic cells (LPDCs) induce peripheral differentiation of FoxP3(+) regulatory T (Treg) cells. We tested the hypothesis that the intestinal milieu impinges on human T1D by affecting differentiation of FoxP3(+) Treg cells.

Research design and methods: We collected duodenal biopsies of 10 T1D patients, 16 healthy subjects, and 20 celiac individuals and performed a fluorescent-activated cell sorter analysis to measure percentages of various immune cell subsets, including CD4(+) and CD8(+) T cells, NK cells, γδ T cells, CD103(+)CD11c(+) LPDCs, and CD4(+)CD25(+)FoxP3(+)CD127(-) Treg cells. In parallel, we assessed the tolerogenic function (i.e., capacity to induce differentiation of FoxP3(+) Treg cells) by LPDCs of T1D patients and control subjects.

Results: Our analysis revealed a significant reduction in the percentage of intestinal CD4(+)CD25(+)FoxP3(+)CD127(-) Treg cells in T1D patients compared with healthy subjects (P = 0.03) and celiac individuals (P = 0.003). In addition, we found that LPDCs from T1D patients completely lacked their tolerogenic function; they were unable to convert CD4(+)CD25(-) T cells into CD4(+)CD25(+)FoxP3(+)CD127(-) Treg cells.

Conclusions: Our data indicate that T1D patients have a reduced number of intestinal FoxP3(+) Treg cells as a result of their defective differentiation in the gut. These findings suggest that intestinal immune regulation is not only calibrated to tolerate commensal bacteria and food components but also is instrumental in maintaining immune tolerance toward pancreatic β-cells and preventing T1D.

Figures

FIG. 1.

The relative percentages of total CD4+ T cells, CD8+ T cells, NK cells, and γδ T cells in the lamina propria of the small-intestinal mucosa were comparable in patients with T1D, HC subjects, and patients with CD. Lamina propria cells were isolated from the small-intestinal mucosa; simultaneously stained with Pacific Blue–conjugated anti-human CD4, APC-conjugated anti-human NKG2D, FITC-conjugated anti-human TCRδ1, and AmCyan-conjugated anti-human CD3 monoclonal antibodies; and FACS analyzed. Means of percentages ± SEM of CD4+ T cells (CD3+CD4+ cells), CD8+ T cells (CD3+CD8+ cells), γδ T cells (CD3+ TCRδ1+ cells), and NK cells (CD3− NKG2D+ cells) out of total lamina propria lymphocytes (left) and the means of percentages ± SEM of CD4+ T cells, CD8+ T cells, and γδ T cells out of CD3+ T cells (right) are indicated.

FIG. 2.

Selective reduction of FoxP3+ Treg cells in the gut of T1D patients. A: The percentages of CD4+CD25+FoxP3+CD127− Treg cells out of total lymphocytes were measured in single cell suspensions isolated from the small-intestinal mucosa (duodenal biopsies) of patients with T1D (n = 10), HC subjects (n = 16), and individuals with CD (n = 20). The CD4+CD25+FoxP3+CD127− Treg cells in gut mucosa were identified by multiparametric FACS analysis. Data are presented as means ± SEM of all experiments. *P = 0.03; **P = 0.003. B: Blood samples were collected from the same individuals simultaneously with the duodenal biopsy. PBMCs were isolated and analyzed as in A. Data are presented as means ± SEM of all experiments. NS, not significant; P > 0.05. C: The percentages of CD4+CD25+FoxP3+CD127− Treg cells were compared among patients with T1D (n = 5), patients with T1D and CD (T1D+CD, n = 5), HC subjects (n = 16), and patients with CD only (n = 20). Groups were compared using two-way ANOVA. ***P = 0.0001 (T1D vs. nondiabetic). D: Gut CD4+CD25+FoxP3+CD127− T cells were bona fide Treg cells and showed a suppressive capacity similar to that of their blood counterparts. A suppression assay was used to test 105 CD4+CD25+ Treg cells or CD4+CD25− Teff cells isolated from the mucosa of normal surgical samples of small intestine or PBMCs of HC subjects. Briefly, Treg or Teff cells were added at 1:1 ratio to responder CFSE-labeled CD4+CD25− T cells stimulated with plate-bound anti-CD3 monoclonal antibody, soluble anti-CD28 monoclonal antibody, and rhIL-2. The percentage of suppression was measured by the percent reduction of proliferation of responder Teff cells (measured by mean of CFSE dilution). Data are expressed as means ± SEM of four independent experiments.

FIG. 3.

CD103+CD11c+ LPDCs are present in the small intestine of T1D patients but fail to induce FoxP3+ Treg cell differentiation. A: Percentages of CD103+CD11c+ LPDCs in the gut mucosa of patients with T1D, HC subjects, and patients with CD. Cells were gated on the DC region (see Supplementary Fig. 4). B: Percentages of cells that express the gut receptor CD103 among various gut lymphocyte subsets (CD4+ T cells, CD8+ T cells, and CD11c+ DCs) in the three groups. C: The capacity of small-intestinal CD11c+CD103+ LPDCs of T1D patients and control subjects (healthy or celiac individuals [CTRs]) to induce CD4+CD25+FoxP3+CD127− Treg cell differentiation was evaluated in conversion assays. BMDCs or small-intestinal CD11c+CD103+ LPDCs were obtained from T1D patients and CTRs and added at 1:10 ratio (DC to T cells) to autologous naive CD4+CD25− T cells isolated from PBMC and stimulated with soluble anti-human CD3 monoclonal antibody in the presence of rhIL-2 and rhTGF-β. Cells were collected after 4 days and FACS analyzed to measure percentages of CD4+CD25+FoxP3+CD127− Treg cells. One representative experiment out of three (T1D) or four (CTR) is shown (cells gated on CD4+CD25+CD127− cells). D: Means ± SEM of CD4+CD25+FoxP3+CD127− Treg cell percentages obtained in conversion assays with BMDCs or LPDCs in three (T1D) or four (CTR) independent experiments are shown. *P = 0.028. (A high-quality color representation of this figure is available in the online issue.)