Human mucosal-associated invariant T (MAIT) cells possess capacity for B cell help

Abstract

Mucosal-associated invariant T (MAIT) cells are an innate-like T cell subset, restricted by the nonclassic MHC class I-related protein MR1 and enriched at mucosal sites. Human studies have shown an association between MAIT cells and pathogen-specific antibody responses. In this study, we investigate the effect of human MAIT cells on B cells ex vivo. We found that supernatants from microbe- or cytokine-stimulated MAIT cells, when added to purified autologous B cells, increase frequencies of plasmablasts and promote IgA, IgG, and IgM production. We found effects to be mostly MR1-dependent and that the increases in plasmablasts are likely a result of increased differentiation from memory B cells. Furthermore, microbe-activated MAIT cell supernatant contains multiple cytokines known to stimulate B cells, including IL-6, -10, and -21. This study thus provides the first direct evidence of a newly identified role of MAIT cells in providing help to B cells.

Keywords: B cell help; MR-1; antibody; plasmablast.

Figure 1.. MR1-dependent activation of MAIT cells by E. coli.

(A) Gating strategy of PBMCs stimulated overnight with formaldehyde-fixed BL21, 1100-2, or BSV18 E. coli, with or without anti-MR1 antibody (10 µg/ml) at 0.1, 1, 10, and 100 MOI, with 1.25 µg/ml anti-CD28. Levels of TNF-α and IFN-γ in (B and C) MAIT (Vα7.2⁺CD161⁺⁺) and (D and E) non-MAIT (Vα7.2⁺CD161⁻) T cell populations were measured by flow cytometry. Data are from a single donor and are representative of 2 independent experiments.

Figure 2.. B cell phenotypic changes after addition of E. coli-stimulated MAIT cell supernatant.

We cultured B cells from different subjects in the presence of supernatant from subject-matched MAIT cells and THP-1 cells (A) or MAIT/THP-1/BL21 (B). Of CD38⁺⁺CD24⁻ cells, we measured HLA-DR (C) in B cells stimulated with MAIT/THP-1 (blue) or MAIT/THP-1/BL21 supernatant. Frequency of CD38⁺⁺CD24⁻ cells after addition of MAIT supernatant with THP-1 (D) and primary monocytes (E) as APCs, frequency of CD138⁻HLA-DR⁺ of CD38⁺⁺CD24⁻ B cells (F), the effect of addition of anti-MR1 (10 µg/ml) and/or anti-IL-12 (5 µg/ml) and anti-IL-18 (5 µg/ml) during MAIT/THP-1/BL21 culture on the ability of resulting supernatant to differentiate B cells (G), the effect of different E. coli strains, with or without added anti-MR1 in MAIT/THP-1/E. coli supernatant (H). Data are means ± sem, and significance was determined by Mann-Whitney U tests. Data are representative of 3 independent experiments. *P ≤ 0.05; **P ≤ 0.01; ****P ≤ 0.00001.

Figure 3.. MR1 is essential for induction of B cell antibody production by MAIT/THP-1/BL21 supernatant.

We cultured B cells from different subjects for 7 d in supernatant from subject-matched MAIT cells and THP-1 cells incubated overnight with BL21. (A–C) IgA, IgG, and IgM levels for the different conditions, with anti-MR1 (10 µg/ml), anti-IL-12/18, or both at 5 µg/ml each during initial MAIT/THP-1/BL21 stimulation or anti-CD40L (20 µg/ml) added to supernatant after MAIT culture and before addition to B cells. Data are means ± sem, and significance was determined by 1-way ANOVA using Tukey’s multiple-comparisons test. Data are representative of 2 independent experiments. *P ≤ 0.05; **P ≤ 0.01; ****P ≤ 0.00001.

Figure 4.. Induction of antibody production and plasmablast differentiation by TCR- and cytokine-stimulated MAIT cells.

(A–D) We cocultured Vα7.2⁺CD161⁺⁺ MAIT cells and B cells from different subjects in the presence of several combinations of IL-2, -12, and -15 for 7 d and measured antibody levels and B cell phenotype. Data are means ± sem, and were analyzed by 2-way ANOVA, with Fisher’s LSD test, comparing MAIT+B cells with B cells only. We also cultured Vα7.2⁺CD161⁺⁺ T cells for 24 h with plate-bound anti-CD3/CD28 stimulation and/or recombinant IL-12/18, and supernatant with IL-12/18 blocking antibodies was applied to B cells for 7 d, followed by ELISA (E–G) and flow cytometry (H) for B cell phenotype. Data shown are mean ± sem, and significance was determined by 1-way ANOVA using Tukey’s multiple comparisons test. Data are representative of 2 independent experiments. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.00001.

Figure 5.. Activated MAIT supernatant induces plasmablast differentiation from memory B cells.

(A) B cells from multiple subjects were sorted and then pooled into CD3⁻CD19⁺ B cell populations based on IgD, CD27, CD38, and CD24 expression. (B–E) Supernatant from cocultures of different combinations of MAIT cells from multiple subjects, THP-1 cells, and BL21 (100 MOI per THP-1 cell) were added to different populations of sort-purified B cells, each population containing 150,000 IgD⁺CD27⁻ cells per well plus either no extra cells (B), 150,000 added IgD⁺CD27⁺CD38⁻ cells (C), 150,000 added IgD-CD27⁺CD38⁻ cells (D), or 150,000 IgD⁻CD27⁺CD38⁺⁺ cells (E), and cultured for 7 d. After 7 d, B cells were assayed for the frequency of CD38⁺⁺CD24⁻ plasmablasts. Data are s ± sem, and representative of 2 independent experiments. Significance was determined by 1-way ANOVA using Tukey’s multiple comparisons test. ****P ≤ 0.00001.

Figure 6.. Cytokine profile of ex vivo-stimulated MAIT cells.

Vα7.2⁺ primary T cells from multiple subjects were cultured overnight with THP-1 cells in the presence or absence of fixed E. coli (strain BL21) at an MOI of 100 per THP-1 cell and with or without anti-MR1 (10 µg/ml) and cytokine levels were measured with a T-helper-specific multiplex cytokine array. Data shown are means ± sem, significance was determined by Mann-Whitney U tests and are representative of 2 independent experiments. **P ≤ 0.01; ***P ≤ 0.001.