Oral infection drives a distinct population of intestinal resident memory CD8(+) T cells with enhanced protective function

Abstract

The intestinal mucosa promotes T cell responses that might be beneficial for effective mucosal vaccines. However, intestinal resident memory T (Trm) cell formation and function are poorly understood. We found that oral infection with Listeria monocytogenes induced a robust intestinal CD8 T cell response and blocking effector T cell migration showed that intestinal Trm cells were critical for secondary protection. Intestinal effector CD8 T cells were predominately composed of memory precursor effector cells (MPECs) that rapidly upregulated CD103, which was needed for T cell accumulation in the intestinal epithelium. CD103 expression, rapid MPEC formation, and maintenance in intestinal tissues were dependent on T cell intrinsic transforming growth factor β signals. Moreover, intestinal Trm cells generated after intranasal or intravenous infection were less robust and phenotypically distinct from Trm cells generated after oral infection, demonstrating the critical contribution of infection route for directing the generation of protective intestinal Trm cells.

Figure 1. Oral L. monocytogenes infection generates a protective mucosal T cell response

(A) The LLO₉₁-specific CD8 T cell response was quantified in the blood after oral L. monocytogenes infection. Data are representative of at least two independent experiments with at least four mice per group (mean and s.e.m.) (B) The LLO₉₁-specific CD8 T cell response in tissues at 9 dpi mice. Representative contour plots are gated on CD8αβ⁺ T cells. The numbers within plots correspond to the percentage of cells within gates. (C) Spleens were surgically removed (splenectomized) or mice underwent a sham surgery (control) and recovered for 2 weeks prior to oral infection. LLO₉₁-specific CD8 T cells were enumerated at 12 dpi. Data are representative of at least two independent experiments with at least three mice per group (mean and s.e.m), (D) Integrin α₄β₇ expression was determined on LLO₉₁-specific CD8 T cells at 7 dpi. Data are representative of at least two independent experiments. (E) Mice were orally immunized with 2×10⁹ CFU L. monocytogenes and received 100μg DATK-32 (anti-α₄β₇) or Rat IgG_2A injections daily for the first 14 days and every 5 days thereafter. Thirty days after primary infection, mice were rechallenged with 2×10¹⁰ CFU L. monocytogenes and the bacterial burden was determined 3 days following challenge infection. Data are pooled from two experiments with at least 12 mice per group (mean and s.e.m.). See also Figure S1.

Figure 2. Memory T cells are generated early in intestinal tissues after oral L. monocytogenes infection

(A and B) MPEC (A) and SLEC (B) phenotype was determined for LLO₉₁-specific CD8 T cells following oral L. monocytogenes infection. * P < 0.05, ** P < 0.01, *** P < 0.001 (unpaired two-tailed t-test). P value indicators (*) are color matched to the tissue compared to the spleen. Data are representative of at least two independent experiments with at least three mice per group (mean and s.e.m.). See also Figure S2. (C) Representative contour plots gated on LLO₉₁-specific CD8 T cells depicting identification of MPEC and SLEC 12 dpi. The numbers within plots correspond to the percentage of cells within gates. (D) Quantification of SLEC and MPEC LLO₉₁-specific CD8 T cells at 12 dpi following oral infection. ** P < 0.01 (unpaired two-tailed t-test). Data are representative of at least two independent experiments with at least three mice per group (mean and s.e.m.).

Figure 3. In situ events regulate MPEC accumulation within intestinal tissues

(A – D) Balb/c mice were treated with 100μg DATK-32 or Rat IgG_2a daily from 7 to 11 dpi. The LP and IEL were harvested at 12 dpi and LLO₉₁-specific CD8 T cells were identified (A and B) and analyzed for CD127 and KLRG1 expression (C and D). Representative contour plots gated on CD8β⁺ T cells (A) or LLO₉₁-specific CD8 T cells (C) are shown. The numbers within plots correspond to the percentage of cells within gates. NS, not significant; * P ≤ 0.05 (unpaired two-tailed t-test). Data represents five mice per group (mean and s.e.m.). See also Figure S3. (E) Naïve Balb/c mice were challenged with 2×10¹⁰ CFU Lm and indicated tissues were harvested 4 days following infection. Alternatively, mice orally immunized with 2×10⁹ CFU L. monocytogenes were challenged with 2×10¹⁰ CFU L. monocytogenes 15 days following primary infection and indicated tissues were harvested 4 days following secondary infection. Both groups were treated with 100μg DATK-32 daily following the challenge infection. ** P < 0.01 (Mann-Whitney two-tailed test). Data represents at least six mice per group (mean and s.e.m.) and is representative of two independent experiments.

Figure 4. CD103 expression identifies mucosal memory precursor CD8 T cells

(A) Balb/c mice were orally infected with L. monocytogenes and LLO₉₁-specific CD8 T cells from the indicated tissues were analyzed at 12 dpi. Representative contour plots gated on either LLO₉₁-specific CD8 T cells with an MPEC (top row) or SLEC (bottom row) phenotypes demonstrate CD103 and CD69 staining. Numbers in plots correspond to the percentage of cells within gated quadrants. (B) Graphical quantification of the data presented in (A). * P < 0.05, *** P < 0.001 (unpaired two-tailed t-test). Data are representative of at least two independent experiments with five mice per group (mean and s.e.m.). See also Figure S4.

Figure 5. Intranasal infection leads to distinct Trm populations in the intestine

(A) C57Bl/6 mice were infected with i.n. influenza virus expressing ovalbumin and the LP and IEL of the small intestine were harvested 10 and 25 days later to identify ova-specific CD8α⁺ TCRβ⁺ cells. NS, not significant; ** P < 0.01 (unpaired two-tailed t-test). See also Figure S5. (B) Effector subset phenotype was determined for ova-specific CD8 T cells in the LP and IEL at 10 dpi (EEC - CD127^neg KLRG1^neg). CD103 expression was determined on ova-specific effector subsets in the LP and IEL at 10 dpi. NS, not significant; * P < 0.05, ** P < 0.01, *** P < 0.001 (unpaired two-tailed t-test). (C) Balb/c mice were infected with i.n. or oral L. monocytogenes and the LP and IEL of the small intestine were harvested 30 – 32 days later. Cells are gated on CD8α⁺ TCRβ⁺ cells. (D) LLO₉₁-specific CD8 T cells from i.n. infection were examined for MPEC phenotype and CD103 and CD69 co-expression. All data are representative of at least two independent experiments with three to four mice per group (mean and s.e.m.).

Figure 6. CD103 expression provides a selective advantage for epithelial accumulation but is not required for retention

Equal numbers of B6 (CD45.1/.2) and Itgae^−/− (CD45.1) OT-I T cells were mixed and transferred into naïve B6 mice (CD45.2) prior to infection. (A) OT-I T cells from B6 and Itgae^−/− donors were quantified at 9 and 29 dpi using congenic markers in the spleen, MLN, LP, IEL, and lumen. OT-I IELs are presented as a ratio of Itgae^−/− to B6 OT-I T cells and normalized to the LP ratio. NS, not significant; * P < 0.05, *** P < 0.001 (left panels, paired two-tailed t-test; right panel, unpaired two-tailed t-test). See also Figure S6. (B) Mice orally infected with L. monocytogenes were examined at 7, 9, and 113 dpi and presented as in (A). NS, not significant; ** P < 0.005 (unpaired two-tailed t-test). Data from (A) and (B) are representative of three similar experiments with at least four mice per group (mean and s.e.m.). (C and D) B6 or Itgae^−/− OT-I cells from the intestinal epithelium (C) and lumen (D) at 9 dpi were examined for CD127 and KLRG1 expression. Representative contour plots are shown. (C) NS, not significant; ** P < 0.005, *** P < 0.001 (unpaired two-tailed t-test). Data are representative of three similar experiments with at least four mice per group (mean and s.e.m.). (D) Data represents the pooled luminal contents of at least four mice per group.

Figure 7. TGFβ regulates rapid MPEC and Trm generation and is required for intestinal Trm maintenance

Equal numbers of CD44^lo CD8 T cells from Tgfbr2^fl/fl distal promoter (d) Lck-cre (KO) CD45.1 OT-I and Tgfbr2^fl/fl (WT) CD45.1/.2 OT-I were co-transferred into CD45.2 B6 mice and infected one day later. (A) Representative dot plots are gated on donor cells after oral L. monocytogenes infection. The numbers within plots correspond to the percentage of cells within gates. (B) Donor derived cells were quantified in the indicated tissues after oral L. monocytogenes infection. Absolute number and ratios of donor cells are depicted graphically with mean and s.e.m of 4 mice per group. See also Figure S7. (C and D) Donor derived OT-I T cells were analyzed for CD69 and CD103 (C) and CD127 and KLRG1 (D) expression in the indicated tissues. Representative contour plots are gated on donor OT-I T cells as indicated at 9 dpi. The numbers within plots correspond to the percentage of cells within gates. Graphs depict the mean and s.e.m of 4 mice per group. See also Figure S7.