Small intestine CD11c+ CD8+ T cells suppress CD4+ T cell-induced immune colitis

Abstract

The large (LI) and small intestine (SI) differ in patterns of susceptibility to chronic mucosal inflammation. In this study, we evaluated whether this might, in part, reflect differences in resident mucosal CD11c(+) T cells. These cells comprised 39-48% (SI) and 12-17% (LI) of the intraepithelial compartment, most of which were T-cell receptor-αβ(+). In the SI, the majority of these cells were CD103(+) CD8(+) NK1.1(-), whereas the opposite phenotype prevailed in the LI. In transfer models of CD4(+) T cell-induced colitis, small numbers (2.5 × 10(5)) of SI CD11c(+) CD8(+) T cells suppressed proinflammatory cytokine-producing CD4(+) T cells in mesenteric lymph nodes and mucosa-associated lymphoid compartments (SI and LI) and protected mice from chronic inflammation. On a per-cell basis, the regulatory function of SI CD11c(+) T cells in CD4(+) T cell colitis was potent compared with other reported regulatory CD4(+) or CD8(+) T cells. In contrast, neither LI CD11c(+) T cells nor SI CD11c(-) T cells were effective in such immunoregulation. SI CD11c(+) CD8(+) T cells were similarly effective in suppressing CD4(+)CD45RB(hi) T cell colitis, as evidenced by inhibition of intracellular proinflammatory cytokine expression and histological inflammation. These findings indicate that SI CD11c(+) CD8(+) T cells are a distinct intestinal T cell population that plays an immunoregulatory role in control of proinflammatory CD4(+) T cells and maintenance of intestinal mucosal homeostasis.

Fig. 1.

CD11c⁺ T cells mainly present in the intestine. A: fluorescent-activated cell sorting (FACS) analysis of CD11c vs. CD3 staining of the cells from spleen (SPN), thymus, and intraepithelial lymphocytes (IELs) isolated from small (SI) and large intestine (LI). Nos. in quadrants are percentages of each cell populations among total lymphocytes. B: comparison of percentages of CD11c⁺ CD3⁺ T cells in the SI and LI from a representative experiment. P values were determined by Student's t-test. C: the expression of CD103, NK1.1, and CD8α on the CD11c⁺ T cells in SI and LI. D: T-cell receptor (TCR)-γδ vs. TCR-β expression on CD11c⁻ T cells and CD11c⁺ T cells in SI and LI. FACS plots show data from one experiment, representative of at least three individual experiments with more than 15 mice in total.

Fig. 2.

CD11c⁺ T cells are derivative of T cells and do not possess antigen presenting cell (APC) function. A: transfer of SPN CD45.1⁺ T cell into RAG2^−/− mice resulted in significant accumulation of CD11c⁺ T cells in SI IEL, while the same population was absent from SI of RAG2^−/− mice. B: 96% of CD11c⁺ T cells in T-cell-transferred RAG2^−/− recipients are CD45.1 positive. C: proliferation of carboxyfluorescein diacetate succinamidyl ester (CFSE)-labeled responder OT-II CD4⁺ T cells to stimulation by peptide pulsed CD11c⁺ or CD11c⁻ cells from SI and LI. SPN dendritic cells (DC) were used as positive control. DC, SI CD11c⁺ or CD11c⁻ T cells, and LI CD11c⁺ or CD11c⁻ T cells were sorted from C57BL/6 and pulsed with OVA_323–339 peptide added to the coculture with OT-II CD4⁺ T cells. Ratios of divided CD4⁺ T cells were calculated to determine response based on reduction of CFSE fluorescence intensity. The data are representative of three individual experiments. WT, wild type.

Fig. 3.

Effects of SI and LI CD11c⁺CD3⁺ T cells on Gαi2^−/− T-cell-induced mucosal inflammation. FACS-sorted SI and LI CD11c⁺ T cells were cotransferred with colitogenic Gαi2^−/− CD4⁺ T cells into immunodeficient RAG2^−/− mice to observe their effects on T-cell-induced mucosal inflammation. A: the percentages of body weight change of RAG^−/− mice that received Gαi2^−/− T cells or were cotransferred with SI and LI CD11c⁺ T cells. The weight of each individual mouse at different time points was compared with its original body weight before receiving Gαi2^−/− T cells or CD11c⁺ T cells. The percentages of weight change of mice in each group were analyzed by using one-way ANOVA. Values are means ± SE. *P values showing the significant difference among three groups of mice. B: histological scoring results for mucosal inflammation in different segments of intestine of Gαi2^−/− T-cell recipients from three groups, including Gαi2^−/− T cells (n = 5), Gαi2^−/− T cells cotransferred with SI CD11c⁺ T cells (n = 7), or LI CD11c⁺ T cells (n = 3). C: percentages and absolute nos. of CD8α⁺ T cells in the intestine of Gαi2^−/− T-cell recipients. The tabulated data were derived from two individual experiments.

Fig. 4.

Intracellular cytokines produced by lymphocytes from SPN, mesenteric lymph node (MLN), and IELs of Gαi2^−/− T-cell recipients. A: staining of intracellular cytokines (IL-17, TNF-α, IFN-γ, and IL-2) produced by SPN CD4⁺ T lymphocytes. Analysis was performed in CD3⁺ T-cell gate. B: TNF-α and IL-17 production by CD4⁺ T cells from MLNs of Gαi2^−/− T-cell recipients. Nos. indicate the percentages of cytokine producing CD4⁺ T cells in total T-cell gate. C: IL-17 production by SI and LI IELs from RAG2^−/− T-cell recipients. IL-17-producing T cells in IELs of the recipients from each group were analyzed. The percentages of IL-17-producing CD3⁺ T cells were labeled. The data are representative of five Gαi2^−/− T-cell-only recipients, seven SI CD11c⁺ T cells, and three LI CD11c⁺ T-cell cotransferred Gαi2^−/− T-cell recipients from different set of experiments. One-way ANOVA test was used to analyze the statistic significance among three groups of mice. No significant difference was found with all cytokines producing CD4⁺ T cells in SPN (P > 0.05). CD4⁺ T cells from MLNs of three groups of mice showed significant difference in IL-17 (P = 0.001) and TNF-α expression (P < 0.05).

Fig. 5.

SI CD11c⁺ CD8⁺ T cells are protective in CD4⁺CD45RB^hi T-cell colitis. CD11c⁺ and CD11c⁻ CD8⁺ T cells were isolated from SI IELs by sorting and cotransferred, respectively, with isolated CD4⁺CD45RB^hi T cells into C.B-17/scid mice to evaluate their protective functions. A: the percentages of body weight loss of T-cell recipients. The percentages of body weight loss of each individual mouse were compared with its original body weight before T-cell transfer. B: representative histological examination results showed the lymphocyte infiltration in the LI of unprotected and T-cell-only recipients. C: histological scoring of inflammation in proximal and distal colon. Student's t-test was used to compare the two groups. D: representative results of serum cytokine detection from one out of two sets of individual experiments with six mice in each group. One-way ANOVA test was used to analyze the statistic significance among three groups of mice. MCP-1, monocyte chemoattractant protein-1.

Fig. 6.

Increased CD11c⁺ CD8⁺ T cells in the intestine were correlated with the colitis protection in CD4⁺CD45RB^hi T-cell recipients. The lymphocytes were isolated from spleen (SP), SI, and LI of T-cell recipients and analyzed in lymphocytes gated by flow cytometry for CD11c vs. CD8 expression. Top: SP lymphocytes. Middle: IEL and lamina propria (LPL) cells from SI. Bottom: IEL and LPL from LI. Nos. are the percentages of total gated lymphocytes. These data show an increased accumulation of CD11c⁺CD8⁺ T cells in the protected T-cell recipients. The data are representative of six mice in each group of two individual experiments.