Differentiation of distinct long-lived memory CD4 T cells in intestinal tissues after oral Listeria monocytogenes infection

Abstract

Mucosal antigen-specific CD4 T-cell responses to intestinal pathogens remain incompletely understood. Here we examined the CD4 T-cell response after oral infection with an internalin A 'murinized' Listeria monocytogenes (Lm). Oral Lm infection induced a robust endogenous listeriolysin O (LLO)-specific CD4 T-cell response with distinct phenotypic and functional characteristics in the intestine. Circulating LLO-specific CD4 T cells transiently expressed the 'gut-homing' integrin α₄β₇ and accumulated in the intestinal lamina propria and epithelium where they were maintained independent of interleukin (IL)-15. The majority of intestinal LLO-specific CD4 T cells were CD27^- Ly6C^- and CD69⁺ CD103^- while the lymphoid LLO-specific CD4 T cells were heterogeneous based on CD27 and Ly6C expression and predominately CD69^-. LLO-specific effector CD4 T cells transitioned into a long-lived memory population that phenotypically resembled their parent effectors and displayed hallmarks of residency. In addition, intestinal effector and memory CD4 T cells showed a predominant polyfunctional Th1 profile producing IFNγ, TNFα, and IL-2 at high levels with minimal but detectable levels of IL-17A. Depletion of CD4 T cells in immunized mice led to elevated bacterial burden after challenge infection highlighting a critical role for memory CD4 T cells in controlling intestinal intracellular pathogens.

Figure 1

Induction of robust LLO-specific mucosal CD4 T cells. (a–c) C57Bl/6 (B6) mice were orally infected with 2×10⁹ cfu InlA^M rLm and LLO-specific CD4 T cells were quantified and examined for α₄β₇ expression from the blood at the indicated times. Representative zebra plots and histograms are gated on live CD4 T cells (a) or live LLO-I-A^b+ CD4 T cells (c). Absolute numbers of circulating LLO-specific CD4 T cells were calculated per 1 ml of blood (b). Data are representative of 3 experiments with at least 3 mice per group. (d–h) B6 mice were orally infected with InlA^M rLm and LLO-specific CD4 T cells were quantified from the spleen, mesenteric lymph nodes (MLN), lamina propria (LP), and intraepithelial lymphocyte (IEL) compartment after infection. (d) Representative zebra plots are gated on live CD4 T cells at 9 (primary) and 60 (memory) days post infection (dpi), or 7 days after a secondary challenge of mice that were immunized 60 days previously (secondary). Numbers within quadrants correspond to the percentage of cells within gates. (e) The graph shows pooled data from 5 independent experiments with 15 – 17 mice total at 9 dpi (primary). (f) The graph shows pooled data from 3 independent experiments with 12 mice total between 50–90 dpi (memory). (g) The graphs showed pooled data from 3 independent experiments with 8 – 9 mice total at 7 days after secondary infection of mice immunized 60 days previously (secondary). (h) Absolute numbers of LLO-specific CD4 T cells were quantified in the spleen, MLN, LP and IEL with tetramer enrichment and counting beads at the indicated time post infection. Mice were given a secondary challenge infection with oral InlA^M rLm at 33 dpi. Data are representative of 3 independent experiments. All graphs depict the mean ± SEM.

Figure 2

Intestinal CD4 T cells display distinct phenotypic characteristics. B6 mice were orally infected with InlA^M rLm and LLO-specific CD4 T cells were analyzed for the indicated markers from the spleen, MLN, LP, and IEL compartment after infection. (a) Representative dot plots with adjunct histograms are gated on LLO-I-A^b+ CD4 T cells at 9 (primary) and 60 (memory) dpi, or 7 days after a secondary challenge of mice that were immunized 60 days previously (secondary). For each dot plot, LLO-I-A^b+ CD4 T cells from the LP (dark blue) are overlaid onto LLO-I-A^b+ CD4 T cells from the spleen (light orange). Numbers within plots are the percentage of cells within gated quadrants and are color coded to the tissue they derive from. (b, e) Representative histograms are gated on live LLO-I-A^b+ CD4 T cells at 9 (primary) and 60 (memory) dpi, or 7 days after a secondary challenge of mice that were immunized 60 days previously (secondary). The graphs display pooled data from 9 dpi (primary), between 50–90 dpi (memory), and at 7 days after secondary infection of mice immunized 60 days previously (secondary). The graphs show the mean ± SEM of pooled data from 2 – 7 independent experiments with 6 – 27 mice total. (c, d) LLO-specific CD4 T cells were enumerated from the blood at the indicated days after infection and examined for α₄β₇, Ly6C, and CD27 expression. (c) Line graph depicts the mean ± SEM of 3 mice per group and representative of 2 independent experiments. (d) Representative zebra plots from 7 dpi mice are gated on LLO-I-A^b+ CD4 T cells that do or do not express α₄β₇.

Figure 3

Polyfunctional LLO-specific CD4 Th1 cells in intestinal tissues. Single cell suspensions isolated from the spleen, MLN, LP or IEL during the primary (9 dpi), memory (60 dpi), or secondary (60 + 7 dpi) responses were stimulated with LLO_190-201 peptide in the presence of brefeldin A, with BD leukocyte activation cocktail (PMA/Iono), or with brefeldin A alone (unstim). (a) IFNγ and IL-17A expression was determined by intracellular cytokine staining after 5 h at 37°C and 5% CO₂. Representative zebra plots are gated on live CD45⁺ CD4⁺ T cells and numbers within quadrants correspond to the percentage of cells within gates. Data are representative of 3 experiments with at least 3 mice per group. (b) Pie charts display the multifunctional nature of CD4 T cells. Multifunctionality was determined amongst an IFNγ⁺ CD4⁺ parent population. Numbers in pie charts represent the mean percentage of IFNγ⁺ cells that produced the indicated cytokines. Data depict the mean of 3 mice per group and are representative of 2 experiments. (c) Representative zebra plots are presented to demonstrate the multifunctional CD4 T cell response. Numbers within gates represent the mean percentage of concatenated cells that produced the indicated cytokines and are representative of 2 experiments.

Figure 4

Maintenance of CD4 memory is IL-15 independent. B6 wild-type (WT) and IL-15 deficient (IL-15 KO) mice were orally infected with InlA^M rLm and LLO-specific CD4 T cell populations were examined in the spleen, MLN, LP, and IEL for frequency (a) and number (b) at the indicated times after infection. Data are representative of 2–4 individual time-course experiments with 3–8 mice per time point and depict the mean ± SEM. * p < 0.05, ** p < 0.01 (unpaired Student’s t-test).

Figure 5

Memory CD4 T cells provide protection from secondary oral InlA^M rLm infection. Naive or immunized mice were challenged with oral InlA^M rLm infection. Some immunized mice were also treated with anti-CD4 (clone GK1.5) to deplete CD4 T cells. The small intestines and MLN were harvested 4 days after challenge infection and Lm burden was quantified on agar plates supplemented with streptomycin. Pooled data from two individual experiments with 5 mice per experiment are shown. The bar graph depicts the mean and individual mice are shown for each group. * p < 0.05, ** p < 0.01, *** p < 0.001 (Mann-Whitney test).