Dissecting the role of retinoic acid receptor isoforms in the CD8 response to infection

Abstract

Vitamin A deficiency leads to increased susceptibility to a spectrum of infectious diseases. The studies presented dissect the intrinsic role of each of the retinoic acid receptor (RAR) isoforms in the clonal expansion, differentiation, and survival of pathogen-specific CD8 T cells in vivo. The data show that RARα is required for the expression of gut-homing receptors on CD8(+) T cells and survival of CD8(+) T cells in vitro. Furthermore, RARα is essential for survival of CD8(+) T cells in vivo following Listeria monocytogenes infection. In contrast, RARβ deletion leads to modest deficiency in Ag-specific CD8(+) T cell expansion during infection. The defective survival of RARα-deficient CD8(+) T cells leads to a deficiency in control of L. monocytogenes expansion in the spleen. To our knowledge, these are the first comparative studies of the role of RAR isoforms in CD8(+) T cell immunity.

FIGURE 1

Analysis of activation and proliferation of RARα^fl/fl, RARβ^fl/fl, RARγ^fl/fl, and control CD8⁺ T cells in vitro. (A) Representative graph of CD25 (upper panels) and CD69 (lower panels) in activated CD8⁺ T cells at 18 h postactivation. Filled: naive control. Blue line: activated CD8⁺ T cell. (B) Mean fluorescence intensity of CD25 (left panel) and CD69 (right panel) of CD8⁺ T cells at 18 h postactivation. Horizontal lines stand for the mean value of mean fluorescence intensity in indicated strain. (C and D) CD8⁺ T cells were purified from spleen and peripheral lymph node of RARα^fl/fl, RARβ^fl/fl, RARγ^fl/fl, and control mice and activated with plate-bound anti-CD3, with or without anti-CD28, at the indicated concentrations for 72 h before analysis. (C) Representative CFSE profile of CD8⁺ T cells 72 h postactivation. Data are gated on live CD8⁺ T cells. (D) Quantification of the proportion of CD8⁺ T cells undergoing proliferation in various concentrations of anti-CD3, with or without anti-CD28. Shown is the percentage of CFSE^low cells of live CD8⁺ T cells. Data shown are representative of at least four experiments (one mouse/group in triplicate wells in each experiment) with similar results. Data shown are mean ± SD.

FIGURE 2

Impaired survival of RARα^fl/fl, but not RARβ^fl/fl, RARγ^fl/fl, or control, CD8⁺ T cells when activated in vitro. CD8⁺ T cells were purified from spleen and peripheral lymph node of RARα^fl/fl, RARβ^fl/fl, RARγ^fl/fl, and control mice, activated with plate-bound anti-CD3 (10 μg/ml) + anti-CD28 (1 μg/ ml) for 48 h, washed, resuspended in fresh media with 100 U/ml IL-2 for an additional 48 h, and analyzed at the indicated time points. (A) Representative near-infrared staining of CD8⁺ T cells on day 1 (upper panels) and day 4 (lower panels) postactivation. (B) Proportion of live CD8⁺ T cells measured by Live/Dead Near-IR staining. (C) Total live CD8⁺ T cell number at indicated time points. Data shown are representative of at least five experiments (one mouse/group in triplicate wells in each experiment) with similar results. Data shown are mean ± SD.

FIGURE 3

Impaired IL-2 and IFN-γ production by RARα^fl/fl CD8⁺ T cells upon activation. Representative FACS plots of IL-2 (A) and IFN-γ (B) in CD8⁺ T cells by intracellular staining at 24 h postactivation with no stimulation (left panels), anti-CD3 stimulation (middle panels), or anti-CD3 + anti-CD28 stimulation (right panels). Data shown are gated on live CD8⁺ T cells. Numbers in boxes indicate percentage of IL2⁺ (A) or IFNγ⁺ (B) CD8⁺ T cells. (C) Quantification of percentage of IL2⁺ (left panel) or IFNγ⁺ (right panel) among CD8⁺ T cells. Data shown are mean ± SEM pooled from two experiments with n = 5–6 mice/group.

FIGURE 4

Analysis of RA-induced α4β7 and CCR9 expression in activated CD8⁺ T cells from RARα^fl/fl, RARβ^fl/fl, RARγ^fl/fl, and control mice. CD8⁺ T cells from different strains were activated as in Fig. 2, except that RA was in culture at various concentrations until analysis at 96 h. Representative α4β7 (A) and CCR9 (B) expression on activated CD8⁺ T cells in the presence of 0 nM (top panels) or 100 nM (bottom panels) RA. Data shown are gated on live CD8⁺ T cells. Numbers indicate percentage of α4β7⁺ (A) and CCR9⁺ (B) CD8⁺ T cells. (C) Quantification of α4β7 (left panel) and CCR9 (right panel) on CD8⁺ T cells from different strains at various RA concentrations. Data shown are representative of four experiments (one mouse/group in triplicate wells in each experiment) with similar results. Data shown are mean ± SD.

FIGURE 5

Defective CD8⁺ T cell response against L. monocytogenes–OVA infection and bacterial burden control in RARα^fl/fl mice. (A) Representative FACS plots of MHC-I OVA-tetramer staining in naive and infected control, RARα^fl/fl, RARβ^fl/fl, and RARγ^fl/fl mice splenocytes. Representative profile of CD8⁺MHCII⁻ T cells gated on live cells (upper panels). Representative profile of CD44 and OVA-tet on gated CD8⁺MHCII⁻ T cells (lower panels). Gating strategy was determined with naive mice as negative control. (B) Quantification of proportion (left panel) and total number (right panel) of CD8⁺ T cells in spleens of infected mice on day 5 postinfection. (C) Proportion of CD44^highOVA-tet⁺ cells among CD8⁺ T cells (left panel) and total number of CD44^highOVA-tet⁺ CD8⁺ T cells (right panel) in the spleen of infected mice on day 5. (D) Total number of CD8⁺granzyme B⁺ (upper left panel), CD8⁺IFNγ⁺ (upper right panel), and CD8⁺TNFα⁺ (lower left panel) T cells in the spleen of infected mice on day 5. (E) Bacterial load in spleen of day-5 infected mice. Data shown in (B), (C), (D), and (E) are pooled from four independent experiments (n ≥ 11 mice/group). Horizontal lines in (B)–(D) stand for mean value in corresponding strain. Dashed lines in (E) indicate lowest number of bacteria that can be detected in our system.