Local Inflammatory Cues Regulate Differentiation and Persistence of CD8+ Tissue-Resident Memory T Cells

Abstract

Many pathogens initiate infection at mucosal surfaces, and tissue-resident memory T (Trm) cells play an important role in protective immunity, yet the tissue-specific signals that regulate Trm differentiation are poorly defined. During Yersinia infection, CD8⁺ T cell recruitment to areas of inflammation within the intestine is required for differentiation of the CD103^-CD69⁺ Trm subset. Intestinal proinflammatory microenvironments have elevated interferon (IFN)-β and interleukin-12 (IL-12), which regulated Trm markers, including CD103. Type I interferon-receptor- or IL-12-receptor-deficient T cells functioned similarly to wild-type (WT) cells during infection; however, the inability of T cells to respond to inflammation resulted in defective differentiation of CD103^-CD69⁺ Trm cells and reduced Trm persistence. Intestinal macrophages were the main producers of IFN-β and IL-12 during infection, and deletion of CCR2⁺ IL-12-producing cells reduced the size of the CD103^- Trm population. These data indicate that intestinal inflammation drives phenotypic diversity and abundance of Trm cells for optimal tissue-specific immunity.

Keywords: CD103; IL-12; bacterial infection; tissue-resident memory T cells.

Figure 1. Ex vivo stimulation with cytokines negatively regulates TGF-β-mediated upregulation of CD103

Mice received WT OT-I T cells (A–C) or an equal number of WT and Ifnar1^−/− OT-I T cells (D–E) and were infected with Yptb-OVA. Seven days post infection, cells from the MLNs and spleen were cultured with the indicated cytokines for 20 hours and CD103 and CD69 expression were analyzed. (A) Representative histograms of CD103 expression on OT-I T cells stimulated with the indicated cytokines in the absence (shaded histograms) or presence of TGF-β (open histograms). Numbers represent the percent of cells within the CD103⁺ gate. Mean percent CD103⁺ cells (B) and CD69 MFI (C) from technical replicates and representative of 3 experiments. Error bars represent SD. (D) Representative histograms of CD103 expression on in vivo primed WT (shaded histogram) and Ifnar1^−/− (open histogram) OT-I T cells cultured with TGF-β and IFN-β. Numbers represent the percent of cells within the CD103⁺ gate. (E) Mean percent CD103⁺ cells from triplicate samples from one representative experiment of 2. Error bars represent SD. *p≤0.05, **p≤0.005, ***p≤0.0005 by unpaired t-test. See also Figure S1.

Figure 2. Yersinia infection induces IFN-β and IL-12 upregulation in proinflammatory microenvironments in vivo

IFNβ-YFP and IL12p40-YFP mice were infected with Yptb-OVA and tissues were isolated 7 days post infection. (A) Expression of YFP (green), Epcam (red), and DNA (blue) in control uninfected intestine (left panels) and Yptb-OVA infected intestine (right panels). Scale bars, 250μm. (B) The number of YFP⁺ cells/villus in uninfected control and Yptb-infected IFN-β-YFP and IL-12p40-YFP mice. *p<0.0001 by the Mann-Whitney test. (C) Infiltrating CD8β⁺ T cells (red) are found in areas of cytokine expression (green). Scale bars, 40μm. All images are representative of 2–4 mice for each condition. See also Figure S2.

Figure 3. LP Trm populations differentially express STAT4-regulated genes

Mice received OT-I T cells and were infected with Yptb-OVA and T cells were analyzed at 9 and >30 days post infection. (A) CD103⁺ and CD103⁻ OT-I T cells were sorted from the LP at the indicated timepoints and expression of Tbet was determined by qRT-PCR. Values are expressed relative to actb expression. Mean and SD represent technical replicates, representative of 2 experiments. (B) T-bet expression in CD103⁺ and CD103⁻ LP and splenic (SP) OT-I T cells at 30 days post infection. Numbers are the percent of cells in each quadrant. (C) Percent T-bet^hi of CD103⁺ and CD103⁻ LP Trm populations. Symbols represent individual mice from one experiment and are representative of 2 experiments. (D) CD103⁺ and CD103⁻ T cells were sorted from the LP at the indicated timepoints and gene expression was determined by qRT-PCR. Values are expressed relative to actb expression and the fold change in gene expression in CD103⁻/CD103⁺ populations is shown. The dashed line indicates equivalent expression by both populations. (E) OT-I T cells from the LP and spleen were analyzed for expression of CD103 and NKG2D (top panel) or CD244 (bottom panel) at >30 days post infection. Numbers represent percent of cells in each quadrant. Representative plots of at least 6 mice from 2 or more experiments. (F) MFI of NKG2D (top panel) and CD244 (bottom panel) on CD103⁺ and CD103⁻ LP OT-I T cells at >30 days post infection. Lines connect populations from individual mice. *p<0.05 by paired t-test. See also Figure S3.

Figure 4. Cytokine receptor-deficient T cells expand, enter the intestine, and show enhanced early expression of Trm markers

Mice received 5×10³ WT and 5×10³ Il18r1^−/−, Ifnar1^−/−, or Il12rb2^−/− OT-I T cells and were infected with Yptb-OVA by oral gavage. T cells were analyzed at 9 days post infection. (A) Ratio of WT to Il18r1^−/−, Ifnar1^−/−, or Il12rb2^−/− OT-I T cells in the spleen (left panel) and LP (right panel). Ratios do not significantly differ from 1.0 using a one sample t-test. (B) Representative histograms of CD103 (top panels) and CD69 (bottom panels) expression on LP T cells. Numbers (top panels) are the percent in the CD103⁺ gate. (C) Percent CD103⁺ of WT and cytokine receptor-deficient OT-I T cells isolated from the LP. Lines connect data points from individual mice. (D) CD69 expression on LP cells expressed as the ratio of CD69 MFI on WT/cytokine receptor-deficient T cells. (E) Representative plot of CD103 and CD244 expression on WT and Il12rb2^−/− LP OT-I T cells. Data are pooled from 2 or more experiments (A,C,D) or from one experiment and representative of at least two experiments (B,E). *p<0.005 by paired t-test.

Figure 5. Cytokine receptor-deficient T cells localize to proinflammatory microenvironments and display normal effector function

Mice received 5×10³ WT and 5×10³ Il12rb2^−/− OT-I T cells and were infected with Yptb-OVA by oral gavage. T cells were analyzed at 9 days post infection (A–E). (A) Representative histogram of CXCR3 expression on splenic WT and Il12rb2^−/− OT-I T cells. (B) Localization of GFP⁺ CD45.2⁺ WT (green and red) and CD45.2⁺ Il12rb2^−/− (red) OT-I T cells in the ileum. Representative image from one of 4 mice. (C) Ratio of WT to Il12rb2^−/− LP OT-I T cells within individual T cell clusters containing at least 10 cells and non-clustered T cells from individual fields. Data are pooled from 4 mice. p=0.14 by Mann-Whitney. (D) LP cells were restimulated with SIINFEKL peptide in the presence of Brefeldin A for 4 hours and IFNγ production was analyzed. IFNγ⁺ gates were drawn using unstimulated controls. Representative plots of 5 mice from 3 experiments. (E) Granzyme B expression in LP T cells, representative of 5 mice from 3 experiments. (F) Day 7 effector CD8⁺ T cells from WT and Il12rb2^−/− mice were transferred into WT mice that were infected with Yptb 3 days earlier. Two days after T cell transfer, CFU were enumerated in the ileum (left panel) and spleen (right panel) and compared to infected mice receiving no T cells. Data are pooled from 4 experiments and geometric mean is shown. *p<0.05, **p<0.005 by the Mann-Whitney test. See also Figure S4.

Figure 6. Cytokine receptor-deficient T cells show impaired differentiation into CD103⁻CD69⁺ LP Trm cells

Mice received 5×10³ WT and 5×10³ Il18r1^−/−, Ifnar^−/−, or Il12rb2^−/− OT-I T cells and were infected with Yptb-OVA by oral gavage. (A) Ratio of WT to Il18r1^−/−, Ifnar^−/−, or Il12rb2^−/− OT-I T cells in the LP on 9 and >30 days post infection. (B) Representative CD103 expression on LP T cells. (C) Percent CD103⁺ of LP OT-I T cells at >30 days post infection. Lines connect data points from individual mice. (D) CD69 expression on LP cells expressed as the ratio of CD69 MFI on WT/cytokine receptor-deficient T cells. (E) WT and Il12rb2^−/− OT-I T cells were analyzed for expression of NKG2D (top panels) and CD244 (bottom panels) on CD103⁺ and CD103⁻subsets. Numbers are percent of cells in each quadrant. Plots are representative of at least 5 mice from 2 or more experiments. (F) Absolute number of OT-I T cells in the CD103⁻CD69⁺ (left) and CD103⁺CD69⁺ (right) subsets at 9 and >30 days post infection. Numbers represent the fold difference in the number of WT and Il12rb2^−/− OT-I T cells at each timepoint. (G) MFI of Bcl-2 in the CD103⁻CD69⁺ and CD103⁺CD69⁺ subsets at 9 days post infection. *p<0.05, **p<0.005 by the Mann-Whitney (A), paired t-test (C,E,G), or ratio paired t-test (F). Data are pooled from 2 or more experiments (A,D,F) or from one experiment and representative of at least two experiments (B,C,E,G). See also Figure S5.

Figure 7. IL-12 production by CCR2⁺ populations drives differentiation of CD103⁻ CD69⁺ LP Trm cells

(A) IFNβ-YFP and IL12p40-YFP mice were infected with Yptb-OVA and tissues were isolated 7 days post infection. Expression of YFP (green) in CD11b⁺ (blue) and CD11c⁺ (red) cells in the intestinal LP. Images are representative of 3 mice. (B–D) WT:CCR2-DTR or Il12p35^−/−:CCR2-DTR mixed bone marrow chimeric mice received 1×10⁴ WT OT-I T cells and were infected with Yptb-OVA. Mice were given daily injections of DT beginning at 5 days post infection. Twelve days after infection, OT-I T cells in the LP and spleen were analyzed. (B) Representative histograms of CD103 (left) and CD69 (right) expression on OT-I T cells from mice containing WT (filled histogram) or Il12p35^−/− (open histogram) monocytes/monocyte-derived cells. Numbers are the percent of the population in the CD103⁺ gate or the CD69 MFI. (C) Percent of LP OT-I T cells expressing CD103. Data are pooled from 2 experiments. (D) CD103 and CD244 expression on OT-I T cells from the LP (top panels) and spleen (bottom panels). Numbers are percent of cells in each quadrant. Representative of 3 or more mice per group pooled from 2 experiments. *p<0.05 by unpaired t-test. See also Figure S6.