Cbf-β is required for the development, differentiation, and function of murine mucosal-associated invariant T cells

Mucosal-associated invariant T (MAIT) cells are an evolutionarily conserved subset of T cells that respond to microbial threats via semi-invariant T cell receptor (TCR)-mediated recognition of microbial riboflavin-derivative antigens presented by MHC class I-related (MR1) molecules [1]. MAIT cells mediate functions that link innate and acquired immunity in a broad spectrum of diseases, including infection, cancer, allergy, and autoimmunity [2]. MAIT cells are abundant in human nonmucosal tissues, where they account for up to 10% of blood T cells and 45% of liver T cells, but are much less abundant in mice [3].

Recently, the availability of MR1-loaded tetramers has allowed the study of MAIT cells in mice [3, 4] and facilitated the mapping of murine thymic MAIT cell development, revealing that MAIT cells originate from CD4⁺CD8⁺ precursors and undergo maturation through the following three stages: stage 1, CD24⁺CD44^– cells; stage 2, CD24^–CD44^– cells; and stage 3, CD24^–CD44⁺ cells [4]. Stage 3 MAIT cells further differentiate into the following effector lineages in the thymus: IFN-γ–secreting T-bet⁺ MAIT1 cells and IL-17–secreting RORγt⁺ MAIT17 cells [5]. The specific cytokines produced by MAIT1 and MAIT17 cells and the relative frequencies of these cell types have a major impact on the pathogenesis of cancer and autoimmune diseases. Therefore, to develop MAIT cell-based therapies, it is crucial to identify novel targets for regulating MAIT cell development and differentiation. Although transcription factors such as PLZF, SATB1, miR-181a/b-1, miR-155, Drosha, and the adapter molecule SAP have been shown to regulate MAIT cell development and differentiation [6, 7], a significant gap in our knowledge of MAIT cell regulation remains.

Core binding factor subunit beta (Cbf-β) is the non-DNA binding subunit of the heterodimeric transcription factors referred to as core binding factors (CBFs), and RUNX1 is one of three DNA-binding subunits that can form a heterodimer with Cbf-β. RUNX1 and Cbf-β form heterodimers in the nucleus, where Cbf-β plays a crucial role in enhancing RUNX1 DNA binding, contributing to transcriptional regulation. Studies report that the Cbf-β/RUNX1 complex regulates diverse signaling pathways to maintain homeostasis in a wide range of immune cells. For example, RUNX1 has been shown to regulate the transition of immune cells from the CD4^–CD8^– stage to the CD4⁺CD8⁺ stage and from the CD4⁺CD8⁺ stage to the mature single-positive stage [8]. Additionally, Cbf-β controls the homing and differentiation of prethymic progenitor cells [9], and mice with reduced Cbf-β levels have defects in natural killer (NK) cell development and in T-cell specification [10]. Given the role of Cbf-β in the development, differentiation, and function of innate and adaptive immune cells, we hypothesized that MAIT cells are also regulated by Cbf-β.

To test this hypothesis, we generated CD4cre Cbf-β^fl/fl mice, in which the entire Cbf-β gene was floxed, resulting in a T-cell-specific knockout of Cbf-β (Cbf-β KO) beginning at late stages of thymic T-cell development. We validated the deletion of Cbf-β in thymic CD4⁺ T cells by RT‒qPCR (Fig. S1a). By flow cytometry, we found that the frequencies and absolute cell numbers of single-positive populations (CD4⁺ or CD8⁺) were significantly decreased and that the double-positive population (CD4⁺CD8⁺) was significantly increased in most peripheral organs of Cbf-β KO mice compared to their control littermates, while we did not detect an increase in double-positive cells in the thymus in Cbf-β KO mice (gating strategy in Figs. S1b, S2a, b). Our findings corroborate previous studies showing that RUNX1 depletion has a similar effect on the frequencies of conventional T-cells [8].

Next, we sought to understand how Cbf-β regulates MAIT cell development. We analyzed Cbf-β expression during the three stages of MAIT cell development using a previously published scRNA-seq dataset [7]. We observed a significant increase in Cbf-β expression in stage 3 MAIT cells (Fig. 1a), which led us to investigate the MAIT cell frequency in various organs of Cbf-β KO mice. Using flow cytometry, we found a significant lack of MAIT cells in all organs of Cbf-β KO mice by staining MAIT cells with MR1 5-OP-RU tetramers, with MR1 tetramers loaded with 6-FP as negative control tetramers (Fig. 1b, c). This finding suggests a possible role for Cbf-β in MAIT cell development.

Fig. 1.

Cbf-β is required for murine MAIT cell development, differentiation, and function. a Analysis of Cbf-β expression in scRNA-seq data from Koay et al. [7]. b The MAIT cell frequencies in different organs of CD4cre Cbf-β^fl/fl (Cbf-β KO) mice were compared to those in littermate control mice through analysis of the indicated tetramers. Cells were gated on live single cells. c The percentage (top) and absolute number (bottom) of MAIT cells from b. d Enriched thymic MAIT cells were gated and stained with the indicated antibodies to determine developmental stages in Cbf-β KO mice and littermate control mice. e MAIT cells from different organs of Cbf-β KO mice and littermate control mice were stained with T-bet and RORγt to identify and quantify MAIT1 and MAIT17 cells, respectively. Stimulated hepatic MAIT cells isolated from UBC Cre-ERT2 Cbf-β^fl/fl mice after tamoxifen induction were stained with f an anti-CD69 antibody to measure activation and g an anti-GZMB antibody to measure cytotoxicity. UBC + Cbf-β indicates mice with inducible Cbf-β knockdown, and UBC-Cbf-β indicates mice without inducible knockdown. The data are representative of at least 2 independent experiments. Each dot represents one mouse. At least three mice were used for each group. The data were analyzed by an unpaired t test. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.00001 compared to WT

Next, we analyzed the developmental stages of enriched thymic MAIT cells from Cbf-β KO mice using anti-CD24 and anti-CD44 antibodies. We found a defect in MAIT cell progression from stage 1 to stage 2 and a significant lack of stage 3 MAIT cells in Cbf-β KO mice (Fig. 1d). This finding corroborates the enhanced Cbf-β expression in stage 3 MAIT cells that we observed in previously published scRNA-seq datasets (Fig. 1a) [7] and led us to hypothesize that the lack of thymic stage 3 MAIT cells may inhibit the differentiation of MAIT cells into MAIT1 (T-bet⁺) and MAIT17 (RORγt⁺) cells. To test this hypothesis, we analyzed the frequencies of MAIT1 and MAIT17 cells in the thymus and peripheral organs of control and Cbf-β KO mice based on T-bet and RORγt expression. We found significantly lower frequencies and absolute numbers of MAIT17 and MAIT1 cells in the thymus and a significant reduction in frequencies and absolute numbers of MAIT17 cells in the spleen, lymph nodes, liver, and lungs; but a significant increase in frequency of double-negative (T-bet^-RORγt^-) MAIT cells in all tested organs except for the lymph nodes in Cbf-β KO mice relative to control mice (Fig. 1e).

To bypass the early MAIT cell developmental defect in KO mice and delineate the impact of Cbf-β on MAIT cell function, we employed a tamoxifen-inducible UBC Cre-ERT2 Cbf-β^fl/fl mouse model. Cbf-β knockdown was validated in thymic CD4⁺ T cells by RT‒qPCR 9 days after tamoxifen treatment (Fig. S3). We observed that PMA/ionomycin-activated hepatic MAIT cells from mice with inducible Cbf-β knockdown had reduced CD69 and granzyme B (GZMB) expression, indicating reduced MAIT cell activation and cytotoxicity, respectively (Fig. 1f, g). Furthermore, we analyzed IFN-γ, IL-17, and TNF-α expression in stimulated hepatic MAIT cells by flow cytometry and observed no significant differences in cytokine expression between Cbf-β KO mice and control mice (Fig. S4). We also observed an increasing trend in MAIT cell exhaustion (as determined by increased PD-1 expression) and a decreasing trend in MAIT cell proliferation (as determined by decreased Ki67 expression) in knockdown mice, but these changes were not significant (Fig. S5).

Overall, we found that Cbf-β deficiency in murine MAIT cells impaired MAIT cell development and interrupted MAIT1 and MAIT17 cell differentiation. Additionally, inducible deletion of Cbf-β inhibited the activation and cytotoxicity of peripheral MAIT cells. We realize that there are some limitations to our current study. We were unable to elucidate the Cbf-β signaling pathways involved in MAIT cell differentiation and function, given the technically difficultity of the dramatic reduction in MAIT cells in Cbf-β KO mice. However, our work elucidates the mechanism of MAIT cell development and identifies a novel target, Cbf-β, for modulation of MAIT cells for applications in MAIT cell-based immunotherapies.

Author contributions

QSM and LZ conceived and designed the project. TL, JST, KS, QY, and JW harvested and processed mouse tissues. TL and JST designed and performed the flow cytometry experiments. JST, TL, and IL analyzed the data. JST, TL, and QSM wrote the manuscript with feedback from all authors.

Funding

Henry Ford Immunology Research Program (T71016 to QSM, T71017 to LZ).

Competing interests

The authors declare no competing interests.

Supplementary information

The online version contains supplementary material available at 10.1038/s41423-022-00920-y.