Conventional CD4+ T cells regulate IL-22-producing intestinal innate lymphoid cells - PubMed (original) (raw)

Conventional CD4+ T cells regulate IL-22-producing intestinal innate lymphoid cells

L L Korn et al. Mucosal Immunol. 2014 Sep.

Abstract

The innate and adaptive immune systems in the intestine cooperate to maintain the integrity of the intestinal barrier and to regulate the composition of the resident microbiota. However, little is known about the crosstalk between the innate and adaptive immune systems that contribute to this homeostasis. We find that CD4+ T cells regulate the number and function of barrier-protective innate lymphoid cells (ILCs), as well as production of antimicrobial peptides (AMPs), Reg3γ and Reg3β. RAG1-/- mice lacking T and B cells had elevated ILC numbers, interleukin-22 (IL-22) production, and AMP expression, which were corrected by replacement of CD4+ T cells. Major histocompatibility class II-/- (MHCII-/-) mice lacking CD4+ T cells also had increased ILCs, IL-22, and AMPs, suggesting that negative regulation by CD4+ T cells occurs at steady state. We utilized transfers and genetically modified mice to show that reduction of IL-22 is mediated by conventional CD4+ T cells and is T-cell receptor dependent. The IL-22-AMP axis responds to commensal bacteria; however, neither the bacterial repertoire nor the gross localization of commensal bacteria differed between MHCII+/- and MHCII-/- littermates. These data define a novel ability of CD4+ T cells to regulate intestinal IL-22-producing ILCs and AMPs.

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Conflict of interest statement

Disclosure

The authors have no conflicts of interest to declare.

Figures

Figure 1. IL-22-dependent innate responses are enhanced in RAG1−/− mice and reduced after restoration of adaptive immunity

RAG1−/− mice were reconstituted with 50 x106 cells from WT spleen and MLNs; recipients were analyzed eight weeks later (RAG−/− +Total). Un-manipulated RAG1−/− and RAG1+/− mice were controls. A) Total mRNA from tissue sections from terminal ileum, cecum, or proximal colon was reverse transcribed and analyzed by real-time PCR. Data were analyzed using the CT method normalized to a RAG1+/− for each experiment and GAPDH. Error bars show SEM, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Data pooled from three experiments. B) Representative plots showing T cells (TCRβ+) and B cells (CD19+) in Si-LP and spleen. C) Bar graph of representative IgA ELISA small intestine luminal contents. Data in B and C are representative of the three experiments.

Figure 2. CD4+ T cells are sufficient to reduce IL-22-dependent innate responses

RAG1−/− mice received 10 x106 sorted CD4+ T cells; recipients were analyzed six to eight weeks later (RAG−/− + CD4). A) Representative flow cytometric analysis of transferred cells, ILCs, and Ki-67 staining on ILC3 cells in the Si-LP of RAG1+/−, RAG1−/− , and RAG1−/− +CD4 mice. B) Representative IgA in the small intestinal lumen eight weeks after transfer of CD4+ T cells. C) Si-LP and Si-IEC ILC numbers. D) The percentage of RORγt+, ILC3 cells among total ILCs. E) Ki-67+ percentage among ILC3 cells. F) Representative flow cytometric analysis of cytokine production by Si-LP ILCs; plots are gated on lineage-cd90.2+ cells. G) Cytokine production by Si-LP ILCs. H) Reg3g, Reg3b, and IL-22 mRNA expression in total terminal ileum (small intestine) tissue, analyzed by quantitative real-time PCR as in figure 1. I) Representative staining of MHCII on Si-LP ILC3 cells in RAG1−/− mice (solid light gray line), RAG1+/− mice (dotted dark gray line), and RAG1−/− +CD4 mice (solid black line). MHCII-negative T cell (light gray filled) and MHCII-positive dendritic cell (thin gray line) controls from RAG1+/− mice are also depicted. J) Percentage of ILC3 cells that are MHCII+. Data in C, D, E, G, and H (IL-22 and Reg3g) were pooled from four experiments. Data in H (Reg3b) and J were each pooled from two of the four experiments. Error bars show SEM, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Figure 3. Transferred CD4+ T cells preferentially produce IFNγ

Cytokine production by endogenous or transferred sorted CD4+ T cells (RAG+/- or RAG−/− +CD4, respectively) from mice analyzed six to eight weeks after cell transfer. A) Representative flow cytometic analysis from Si-LP; plots are gated on CD4+TCRβ+ cells. B) Cytokine production by Si-LP CD4+ T cells. Data are pooled from either two (IL-2, IL-10) or all four (IFNγ, IL-17, IL-22) of the experiments shown in Figure 2. Error bars show SEM, *P<0.05, **P<0.01, ****P<0.0001

Figure 4. CD4+ T cell regulation of ILCs is independent of IFNγ

RAG1−/− mice received 10 x106 sorted IFNγ −/− CD4+ T cells; recipients were analyzed five or six weeks later (RAG−/− +IFNγ −/− CD4). (A) Percent of transferred or endogenous CD4+ T cells in the Si-LP producing IFNγ. C) Numbers of Si-LP and ILCs in Si-LP of indicated mice. D) Percentage of ILC3 cells that are Ki-67+. E) Cytokine production by Si-LP ILCs in the indicated mice. Data in A-E are pooled from two experiments.

Figure 5. IL-22-dependent innate responses are enhanced in the absence of TCR-stimulated CD4+ T cells

A) Representative plots of CD4+ and CD8+ T cells in the Si-LP of MHCII+/-, MHCII−/− , K14 (β2m WT or +/−), and β2m−/− (all MHCII+/−) mice. B) ILC numbers and C) ILC-derived IL-22 after PMA/ionomycin stimulation in the indicated mice. D) Reg3g and IL-22 mRNA expression in total intestinal tissue, analyzed by quantitative real-time PCR as in figure 1. Error bars show SEM, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Data are pooled from six or seven experiments.

Figure 6. Depletion of CD4+ T cells in WT mice

Mice were treated with CD4-depleting antibody (GK1.5) or an isotype control for six weeks prior to Si-LP analysis. A) Flow cytometry of CD4+ T cells in the Si-LP of treated mice. (B-F) Graphs show B) ILC numbers, C) the percentage of ILC3 cells that are Ki-67+, D) the percentage of ILC3 cells that are MHCII+, E) cytokine production by ILCs after stimulation with PMA and ionomycin. F) Reg3g, Reg3b, and IL-22 mRNA expression in total terminal ileum (small intestine) tissue, analyzed by quantitative real-time PCR. Data are pooled from two independent experiments, *P<0.05.

Figure 7. Impact of CD4+ T cells and adaptive immunity on the large intestine microbiota

A) Reg3g, Reg3b, and IL-22 mRNA expression in total terminal ileum (small intestine) tissue of antibiotic-treated or control mice, analyzed by quantitative real-time PCR as in figure 1, pooled from two experiments. Error bars show SEM, **P<0.01, ***P<0.001. B) ILC numbers and IL-22 production in antibiotic-treated or control RAG1−/− mice, pooled from two experiments. C) Quantitative PCR of major bacterial groups in MHCII+/− and MHCII−/− littermates (MHCII+/− n=9–12, MHCII−/− n=13–14), analyzed using genomic reference DNA standards. D) Principle coordinate plot of a representative MHCII litter, with +/− genotype shown in red and −/− in blue. E) Principle coordinate analyses of all MHCII+/− or MHC−/− mice sequenced, colored by genotype (+/− in red and −/− in blue), mother (purple and green), and litter (blue and brown tones). F-H) The same analyses performed for MHCII mice, applied to RAG1+/− or RAG1−/− littermates (for E, RAG1+/− n=47, RAG1−/− n=35).

Figure 8. Large intestine bacterial localization is not altered by the presence of CD4+ T cells

A) Representative images of FISH staining of large intestine demonstrating localization scores: 1-no bacterial contact with epithelium and preserved mucus layer; 2-bacterial penetration into mucus layer without epithelial contact; 3-epithelia contact; 4-extensive epithelial contact. Images were acquired and analyzed in a blinded manner, with three to six images acquired per large intestine. Image scores were averaged to acquire a single score per mouse. B) Localization scores from MHCII+/− and MHCII−/− mice. C) Localization scores from RAG1+/− mice, RAG1−/− mice, and six-week CD4+ T cell-reconstituted RAG1−/− mice. Error bars show SEM.

Figure 9. Tregs are not sufficient to reduce IL-22 dependent innate responses

RAG1−/− mice that received 0.5–1×106 Tregs were analyzed six weeks after transfer. A) Representative plots from Si-LP showing Treg purity. B) Si-LP ILC numbers and C) percentage of ILCs that are RORγt+ pooled from two experiments. D) ILC-derived cytokines after stimulation with PMA/ionomycin from the same experiments shown in B-C. E) Reg3g, Reg3b, and IL-22 mRNA expression in total terminal ileum (small intestine) tissue, analyzed by quantitative real-time PCR pooled from two experiments, analyzed as in figure 1. Error bars show SEM, **P<0.01, ***P<0.001, ****P<0.0001

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Conventional CD4+ T cells regulate IL-22-producing intestinal innate lymphoid cells - PubMed (original) (raw)