Opsonization-Enhanced Antigen Presentation by MR1 Activates Rapid Polyfunctional MAIT Cell Responses Acting as an Effector Arm of Humoral Antibacterial Immunity

Abstract

Mucosa-associated invariant T (MAIT) cells are innate-like antimicrobial T cells recognizing a breadth of important pathogens via presentation of microbial riboflavin metabolite Ags by MHC class Ib-related (MR1) molecules. However, the interaction of human MAIT cells with adaptive immune responses and the role they may play in settings of vaccinology remain relatively little explored. In this study we investigated the interplay between MAIT cell-mediated antibacterial effector functions and the humoral immune response. IgG opsonization of the model microbe Escherichia coli with pooled human sera markedly enhanced the capacity of monocytic APC to stimulate MAIT cells. This effect included greater sensitivity of recognition and faster response kinetics, as well as a markedly higher polyfunctionality and magnitude of MAIT cell responses involving a range of effector functions. The boost of MAIT cell responses was dependent on strongly enhanced MR1-mediated Ag presentation via increased FcγR-mediated uptake and signaling primarily mediated by FcγRI. To investigate possible translation of this effect to a vaccine setting, sera from human subjects before and after vaccination with the 13-valent-conjugated Streptococcus pneumoniae vaccine were assessed in a MAIT cell activation assay. Interestingly, vaccine-induced Abs enhanced Ag presentation to MAIT cells, resulting in more potent effector responses. These findings indicate that enhancement of Ag presentation by IgG opsonization allows innate-like MAIT cells to mount a faster, stronger, and qualitatively more complex response and to function as an effector arm of vaccine-induced humoral adaptive antibacterial immunity.

FIGURE 1.

IgG-opsonization increases the magnitude of the MR1-restricted MAIT cell response to E. coli. Representative flow cytometry data (A), and mean percentage (B) of expression of IFN-γ, TNF, IL-17A, CD69, CD107a, and GzB in Vα7.2⁺CD161⁺ MAIT cells stimulated for 24 h with THP-1 cells fed nonopsonized or IVIg-opsonized E. coli at the microbial dose of 1 (n = 30–38). (C) Concentration of IFN-γ, TNF, and IL-17A in the supernatant of the coculture after stimulation with nonopsonized and IVIg-opsonized E. coli for 24 h (n = 4–7). (D) MR1 and IL-12/IL-18 dependency of IFN-γ, TNF, IL-17A, CD107a, and GzB production. (E) MR1/IL-12/IL-18 dependency of IFN-γ, TNF, CD107a, and GzB production (n = 30–38). (F) Concentration of IL-12p70 and IL-18 in the supernatant of the 24-h coculture (n = 4–7). n = individual human donor cells tested in independent experiments. The lines and error bars represent mean and SE. In (D), response dependency (percentage) = ([cytokine⁺ in presence of isotype control or without blocking − cytokine⁺ in presence of blocking)/(cytokine⁺ in presence of isotype control or without blocking)] × 100. *p < 0.05, **p < 0.005, ****p < 0.0001 by the paired t test to determine significant differences between paired samples in (B) and (D) (left graph). *p < 0.05, **p < 0.01. and the Wilcoxon signed-rank test to detect significance in (C), [(D), center and right graph], (E), and (F).

FIGURE 2.

MAIT cell polyfunctionality and T-bet expression are increased by IVIg-opsonized E. coli stimulation. Polyfunctional profile of Vα7.2⁺CD161⁺ MAIT cells responding to nonopsonized or IgG-opsonized E. coli in terms of the number of functions displayed (A), and combinations of IFN-γ, TNF, IL-17A, and GzB production (B). (A)–(C) show the frequency of MAIT cells expressing all combinations of functions or cytokines (n = 12). Representative flow cytometry histograms (D), and combined data and ratio (E) of RORγt, T-bet, and PLZF expression in MAIT cells unstimulated or in the presence of nonopsonized or IVIg-opsonized E. coli for 24 h (n = 3–6). The fluorescence minus one (FMO) control is showed as gray dashed line in (D). The lines and error bars represent mean and SE. *p < 0.05, **p < 0.01, ***p < 0.001 by the Wilcoxon signed-rank test to detect significance in (C). *p < 0.05, **p < 0.005 by the Friedman test followed by Dunn post hoc test to determine significant differences between multiple, paired samples in (E).

FIGURE 3.

MAIT cell responses to IgG-opsonized E. coli display enhanced dose sensitivity and kinetics. (A) Percentage of IFN-γ⁺CD69⁺ and relative normalized Vα7.2 expression in Vα7.2⁺CD161⁺ MAIT cells stimulated by THP-1 cells fed nonopsonized or IVIg-opsonized E. coli over a range of microbial doses and at different concentrations of IVIg. Data shown representative of five independent experiments. (B) Percentage of IFN-γ⁺CD69⁺ and TNF⁺ MAIT cells over 24 h of incubation, with MAIT cell harvest every 2 h (n = 3). The lines and error bars represent mean and SE.

FIGURE 4.

Enhanced MAIT cell responses to IgG-opsonized bacteria are dependent on FcγR binding. Representative flow cytometry data of the expression of IFN-γ, TNF, IL-17A, GzB, and CD107a (A), and percentage of IFN-γ⁺CD69⁺ and TNF⁺CD69⁺ MAIT cells (B) in the presence of THP-1 cells fed nonopsonized or IVIg-opsonized E. coli and with or without purified Fc fragment for 24 h (n = 6). (C) Percentage of the boost inhibition because of the Fc fragment for the indicated cytokines. Representative example of TNF and CD69 expression (D) and percentage of IFN-γ⁺CD69⁺ and TNF⁺CD69⁺ MAIT cells (E) in the presence of THP-1 cells fed nonopsonized, IVIg-opsonized, or deglycosylated IVIg-opsonized E. coli (n = 6). The lines and error bars represent mean and SE. *p < 0.05 by the Wilcoxon signed-rank test performed to detect significance in (B). *p < 0.05, **p < 0.005 by the Friedman test followed by Dunn post hoc test to determine significant differences between multiple, paired samples in (C) and (E).

FIGURE 5.

Increase in MR1-mediated Ag presentation by IVIg-opsonized bacteria is dependent on bacterial uptake, MR1 ER egress, and lysosomal acidification. Representative flow cytometry histogram (A), and combined donor data (B) on MR1, HLA-DR, and HLA-A2 expression in THP-1 cells fed with nonopsonized or IVIg-opsonized E. coli for 3 h (n = 6–12). The MR1 isotype control staining is shown as gray dashed line in (A). (C) Kinetics of MR1 expression on THP-1 cells in presence of nonopsonized or IVIg-opsonized E. coli. The MR1 isotype control staining is shown for both stimulations (n = 2–4). (D) MR1 expression in THP-1 cells unstimulated or in the presence of nonopsonized or IVIg-opsonized E. coli and with indicated compound for 3 h (n = 6–12). (E) The degree of inhibition of the MAIT cell response stimulated by IVIg-opsonized E. coli for 24 h, in presence of the indicated compound. For the THP-1 wash conditions, THP-1 cells were incubated for 30 min in presence of the compound, then washed, resuspended in fresh medium, and cocultured with MAIT cells for 24 h. The lines and error bars represent mean and SE. *p < 0.05, **p < 0.005, ****p < 0.0001 by the Friedman test followed by Dunn post hoc test to detect significant differences between multiple, paired samples in (B). *p < 0.05, ***p < 0.001 by the Wilcoxon signed-rank test to determine significance in (D).

FIGURE 6.

Sera from human subjects after vaccination with the 13-valent S. pneumoniae vaccine support enhanced MAIT cell activation. Representative flow cytometry plots (A) of IFN-γ, TNF, and CD69 expression and combined data (B) of MAIT cell responses determined as percentage IFN-γ⁺CD69⁺, TNF⁺CD69⁺, CD107a⁺, GzB⁺, and mean fluorescence intensity (MFI) of the Vα7.2 TCR in Vα7.2⁺CD161⁺ MAIT cells in the presence of THP-1 cells incubated with sera-opsonized S. pneumoniae for 24 h (n = 12). *p < 0.05, **p < 0.01 by the Wilcoxon signed-rank test to determine significance in (B).