Inflammatory cytokines induce sustained CTLA-4 cell surface expression on human MAIT cells

Abstract

Mucosal-associated invariant T (MAIT) cells acquire effector function in response to proinflammatory signals, which synergize with T cell receptor (TCR)-mediated signals. We asked if cell intrinsic regulatory mechanisms exist to curtail MAIT cell effector function akin to the activation-induced expression of inhibitory receptors by conventional T cells. We examined human MAIT cells from blood and oral mucosal tissues by RNA-seq and found differential expression of immunoregulatory genes, including CTLA-4, by MAIT cells isolated from tissue. Using an ex vivo experimental set-up we demonstrate that inflammatory cytokines were sufficient to induce CTLA-4 expression on the MAIT cell surface in the absence of TCR signals. Even brief exposure to the cytokines IL-12, IL-15 and IL-18 was sufficient for sustained CTLA-4 expression by MAIT cells. These data suggest that control of CTLA-4 expression is fundamentally different between MAIT cells and conventional T cells. We propose that this mechanism serves to limit MAIT cell-mediated tissue-damage.

Introduction

Mucosal-associated invariant T (MAIT) cells acquire effector function when exposed to bacterial metabolites (presented by MR1) in the presence of inflammatory cues, both of which are present during bacterial infections (1, 2). Importantly, inflammatory cues are sufficient to activate MAIT cells and synergize with TCR signals to induce expression of effector molecules including granzyme B and interferon-γ (IFN-γ) (3–5). Inflammatory cytokine-driven activation of MAIT cells has been demonstrated using primary human MAIT cells in short term ex vivo experiments with IL-12, IL-15, and IL-18 (3, 5, 6), but also directly ex vivo in the context of viral infections including dengue, influenza and hepatitis C (7, 8). There is also evidence that infection with the parasite Plasmodium falciparum can induce some MAIT cell activation in humans, presumably in the absence of T cell receptor (TCR) agonist signals (9). This inflammation-driven, TCR-independent mechanism of activation is akin to the phenomenon of bystander-activation in conventional memory T cells (10, 11). The physiological significance and the consequences of inflammation-driven activation of MAIT cells are still largely unclear. However, the distinction between TCR-mediated versus inflammation-driven T cell activation is important for conventional T cells, since TCR signals control the expression of co-stimulatory and co-inhibitory receptors.

The co-inhibitory molecule CTLA-4 is induced on conventional T cells following TCR stimulation and constitutively expressed on regulatory T cells (12, 13). CTLA-4 is noteworthy among co-inhibitory molecules in that it shares its ligands B7–1 (CD80) and B7–2 (CD86) with the co-stimulatory molecule CD28. CTLA-4 binds these ligands with greater avidity and affinity than CD28, which allows the inhibitory signal to outcompete the stimulatory signal and turn off the T cell response (13). It has been proposed that CTLA-4-mediated inhibition can act cell-intrinsically via interaction of phosphatases with its cytoplasmic domain as well as cell-extrinsically by reducing B7–1 and B7–2 availability and thus interfering with the co-stimulatory function of CD28 (13, 14).

We asked if CTLA-4 or other immunoregulatory mechanisms are in place to curtail MAIT cell effector function following activation of MAIT cells by either TCR-mediated signals or inflammatory cytokines. We report here that surface expression of CTLA-4 on MAIT cells is induced by inflammatory cytokines independently of the TCR. This indicates that the signals controlling surface CTLA-4 expression on MAIT cells occur independently of the TCR – calcineurin – NFAT signaling axis (15) required for CTLA-4 expression by conventional human T cells (16) and thus, control of CTLA-4 expression is fundamentally different between MAIT cells and conventional T cells.

Material and Methods

Study approval and patient cohort:

All participants provided signed informed consent and protocols were approved by the Fred Hutchinson Cancer Research Center IRB. Surgical procedures included gingivectomy/gingivoplasty, osseous surgery, implant uncovering and tooth extractions. Participants (n=39) were between 14 and 83 years old (mean = 52).

Cell isolation from mucosal tissues:

Cells were isolated from tissues and blood as previously described (3).

Pathology assessment and scoring:

A small portion of each OM sample was embedded in Tissue-Tek O.C.T. (Sakura Finetek, Thermo Fisher) and stored at −80°C. Frozen tissue blocks were cut into 8μm sections and slides were stained with hematoxylin and eosin (H&E). Histologic sections were then evaluated blinded and scored according to the following criteria: severity of inflammation (1–5), location of inflammation, type of inflammatory infiltrate, presence of epithelial lesions. It is noteworthy that even gingival tissues that could be considered “healthy” have a minimal/basal level of inflammation that is referred to as homeostatic inflammation (17). Thus, tissues that received a score of 1 or 2 were defined as “minimally inflamed” and tissues that received a score of 3 to 5 were defined as “inflamed”.

Flow cytometry:

All flow cytometric stains were conducted at room temperature (RT). For phenotypic identification, bulk PBMCs or mononuclear cells isolated from tissues were initially stained with Live/Dead Cell Stain (Invitrogen) for 15 minutes in PBS. MR1 tetramer (BV421, NIH tetramer core) staining was performed as previously described (18). Afterwards we conducted surface staining with optimized antibody cocktails for 20 minutes in FACS Buffer (PBS containing 2% FBS). Cells were then washed with FACS buffer and fixed in PBS containing 1% paraformaldehyde (Sigma-Aldrich). For samples requiring intracellular staining, cells were fixed and permeabilized using the Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher) according to the manufacturer’s instructions.

Antibodies used for Cell Sorting:

LIVE/DEAD Fixable Aqua Viability Dye (Invitrogen), CD3 Pacific Blue (clone OKT3, BioLegend), CD8 PerCPCy5.5 (clone SK1, BD Biosciences), CD4 ECD (clone SFCI12T4D11, Beckman Coulter), CD161 PECy5 (clone DX12, BD Biosciences), Vα7.2 PE (clone 3C10, BioLegend).

Antibodies used for analysis following PBMC stimulation:

LIVE/DEAD Fixable Blue Viability Dye (Invitrogen), CD25 BV421 (clone M-A251, BD), CD3 BUV661 (clone UCHT1, BD Biosciences), CD8 BUV805 (clone SK1, BD Biosciences), CD4 APCR700 (clone RPA-T4, BD Biosciences), CD161 BV605 (clone DX12, BD), Vα7.2 PE (clone 3C10, BioLegend), IL-7Rα BV786 (clone HIL-7R-M21, BD Biosciences), Foxp3 Alexa Fluor 488 (clone 259D/C7, BD Biosciences), Helios PerCP-eFluor 710 (clone 22F6, Invitrogen), CTLA-4 PECF594 (BNI3, BD Biosciences).

Instruments:

Sorting experiments were performed on a FACS Aria II (BD Biosciences) and phenotyping for CTLA-4 induction assays were performed on FACSSymphony (BD Biosciences). Data was analyzed using Flowjo software v10.4.1 (BD Biosciences).

RNA-seq:

We performed RNA-seq on 100 – 1000 sorted CD8⁺ MAIT cells from blood or tissue samples as previously described (18). In total, 22 samples were sequenced, 10 from blood and 11 from tissue. Single-read sequencing of the libraries was carried out on a HiSeq2500 sequencer (Illumina) with 58-base pair reads, using TruSeq v4 Cluster and SBS kits (Illumina) with a target depth of 5 million reads. Reads were aligned to the University of California Santa Cruz (UCSC) Human genome assembly version 19 in Galaxy using Bowtie and TopHat (Tophat for Illumina tool, v.1.5.0). A quality filter was applied to retain libraries prepared with at least 225 cells, in which the fraction of unpaired reads examined compared to total FASTQ reads was >75%, the median coefficient of variation of coverage was less than 0.8, and the library had at least 500,000 reads. 16 of the 22 sequenced samples passed these quality filters. Genes with a false discovery rate of less than 0.05 and expression fold-change of greater than 2 between two blood and tissue samples were considered differentially expressed using the linear models for microarray data (Limma) R package (19, 20).

Ex vivo stimulation of PBMCs:

Cryopreserved PBMCs were thawed and left untreated in RP10 or stimulated with 100ng/mL cytokines (IL-12 (Thermo Fisher), IL-15 (Thermo Fisher), and IL-18 (MBL International), anti-CD3/CD28 beads (Thermo Fisher), or a combination of both treatments as previously described (3). 1×10⁶ PBMCs were resuspended per well in 96-well plates for 6 – 24 hours at 37°C. Indicated samples were treated with anti-MR1 antibody (Low Endotoxin, Azide-Free clone 26.5, Biolegend) at a concentration of 50μg/mL. To assess CTLA-4 expression after brief stimulation, PBMCs were stimulated for 6 hours and then anti-CD3/CD28 beads were removed using magnetic extraction following manufacturer’s protocol (Thermo Fisher). Cytokines were removed by washing cell twice before resuspending in RP10.

Ex vivo stimulation of OM:

Cells from OM were resuspended at a concentration of 8×10³−1×10⁵ T cells per condition (CD3-expressing cells in the single cell suspension were quantified using Guava easyCyte (Millipore)) and left untreated or stimulated with IL-12/15/18 and anti-CD3/CD28 beads for 7 hours in the presence of Golgi Plug (BD Biosciences). Intracellular straining of IFNγ and CTLA-4 was done using Foxp3/Transcription Factor buffer set (eBioscience).

Statistical analysis

Statistical analysis was performed with Prism 8 (GraphPad) as specified in figure legends.

Results

MAIT cells are present in oral mucosal tissue

To identify cell intrinsic regulatory mechanisms of MAIT cells that are physiologically relevant, we examined MAIT cells isolated from human oral mucosal tissues (OM). We specifically used gingival tissues obtained from routine periodontic surgeries yielding tissues that range from healthy/minimally inflamed to severely inflamed. To assess the extent of inflammation, a piece of each analyzed tissue was saved for a blinded evaluation by a pathologist (see Methods). We assigned tissues with a score of 1 and 2 to a “minimally inflamed” group and tissues with a score of 3 – 5 to an “inflamed” group. We use the term “minimally inflamed” for tissues with a score of 1 or 2, because even healthy gingival tissues have a minimal/basal level of inflammation that is referred to as homeostatic inflammation (17). The remaining tissue was processed to acquire a single-cell suspension for subsequent analysis by flow cytometry. MAIT cells were identified as live, CD3⁺, Vα7.2⁺ (the invariant TCRα chain that allowed initial identification of MAIT cells (21, 22)) and CD161^hi cells in the OM and matched blood (Fig 1A).

Figure 1: CD8⁺ MAIT cells are present in oral mucosal tissues.

(A) Representative FACS plots of Vα7.2⁺CD161^hi MAIT cells of the total CD3⁺ T cell compartment from donor-matched blood and oral mucosa (OM). (B) CD4 and CD8 expression within blood (black) and OM (grey) MAIT cells. (C) MAIT cell frequencies in matched blood (black circles) and minimally inflamed or inflamed OM (white squares) (minimally inflamed OM n=9, inflamed OM n=18) (D) CD69 and CD103 expression on CD8⁺ MAIT cells from inflamed OM (n= 11). Wilcoxon matched-paired signed rank tests were performed. *p ≤ 0.05 **p ≤0.01 ***p ≤ 0.001.

In some samples MAIT cells were also identified using the MR1 tetramer (23) in addition to using the Vαα7.2⁺CD161^hi definition, which yielded a largely but not fully congruent population of MAIT cells (Supplement 1). To provide consistency with previous studies of MAIT cells from human tissues including our own (18), we used the Vα7.2⁺CD161^hi definition of MAIT cells in this study and acknowledge that this population does not include all MR1-restricted cells. We next assessed the distribution of CD4 and CD8 co-receptor expression within the MAIT cell population in the OM. We found that a majority expressed CD8 (Fig 1B) similar to MAIT cells from blood (24), lymph (18) and other mucosal barrier tissues (25) including buccal tissue (26), indicating that the general distribution of MAIT cell subsets in the OM is comparable to other tissues. MAIT cell frequencies (as a % of CD3⁺ T cells) in both minimally inflamed (pathology score 1 and 2) and inflamed OM tissues (scores of 3 – 5) were overall similar to donor-matched blood (lines between data points indicate donor-matched blood and tissue) (Fig 1C), although we observed a slight, but statistically significant decrease of MAIT cell frequencies in inflamed tissues. We next examined if MAIT cells in inflamed OM tissues (scores of 3 – 5) expressed CD69 and CD103, which are biomarkers that can indicate recent activation (CD69) and/or tissue-residence (CD69, CD103) (27). We found that CD8⁺ MAIT cells in inflamed OM expressed one or both of these proteins indicating a possible tissue-resident phenotype similar to conventional CD8⁺ T cells (Fig 1D, Supplement 2A). The presence of CD8⁺ MAIT cells with a tissue-resident phenotype also suggests that MAIT cells remain in tissues for long periods of time and may require cell intrinsic immunoregulatory mechanisms.

Distinct transcriptional profiles of CD8⁺ MAIT cells isolated from oral mucosa and donor-matched blood

To identify immunomodulatory genes expressed by MAIT cells in the OM, we examined the CD8⁺ MAIT cell population in the blood vs. minimally inflamed and inflamed OM inflamed OM by RNA-seq. PCA analysis did not reveal any striking differences between CD8⁺ MAIT cells isolated from minimally inflamed versus inflamed OM tissues (Fig 2A). In contrast, the differences between donor-matched blood and tissue MAIT cells yielded 56 differentially expressed genes between CD8⁺ MAIT cells from OM and blood (an arbitrary patient ID is listed at the bottom of the heatmap to allow pairing of donor-matched blood and tissue) (Fig 2B). Finally, we found that CD8⁺ MAIT cell isolated from the OM had a gene expression signature (compared to blood) indicative of tissue-residence (28) (Supplement 2B), which aligned with our flow cytometry data (Fig 1D).

Figure 2: CD8⁺ MAIT cells in oral mucosa express transcripts associated with immunomodulatory function including CTLA-4.

CD8⁺ MAIT cells were sorted from minimally inflamed or inflamed oral mucosa (OM) and donor-matched blood for bulk RNA-sequencing. (A) Principal component (PC) analysis of global gene expression of OM samples (blue circles = minimally inflamed OM, purple circles = inflamed OM; n=4 donors, each). (B) Heatmap showcasing top 56 differentially expressed genes using fold-change cutoff of at least 2 (FDR ≤ 0.05). (C) Volcano plot showing FDR on Y axis and log2 fold change on X axis with genes associated with immunoregulatory function highlighted in orange. (D) Unscaled Log2 expression counts of transcripts are shown to visualize expression levels. K-means clustering was applied (k=2) to the samples to assess sample separation. In (B) and (D) samples are identified by OM inflammation score (Group: blue, minimally inflamed; purple, inflamed) and tissue (Source: red, blood; OM, teal).

We next screened the differentially expressed genes between CD8⁺ MAIT cells from OM (minimally inflamed and inflamed tissues combined) and blood for genes associated with regulatory or inhibitory functional properties (Fig 2C). We found genes that negatively regulate TCR co-stimulation or activation including CTLA4 and TIGIT (29) as well as genes (IRF4 and BATF) that mediate expression of inhibitory receptors in conventional CD8⁺ T cells (30). CTLA4 was one of the most highly differentially expressed genes suggesting it may play a key role in regulating MAIT cell function similar to its role in conventional T cells. Finally, we compared the expression levels of transcripts encoding for proteins with well-defined costimulatory or coinhibitory function as well as the gene expression levels of the signaling components for IL-12, IL-15 and IL-18 in blood vs. OM CD8⁺ MAIT cells. Applying k-means clustering to our samples we found near uniform separation between blood and OM CD8⁺ MAIT cells (Fig. 2D). We also observed that OM CD8⁺ MAIT cells had higher expression of genes with inhibitory as well as costimulatory function (Fig. 2D). Further, components of the IL-12, IL-15 and IL-18 signaling pathways appeared to be expressed at comparable levels suggesting that MAIT cells in the tissues maintain responsiveness to these cytokines (Fig. 2D). These data indicate that there is heterogeneity in the expression of costimulatory and coinhibitory molecules in the OM MAIT cell population and that OM MAIT cells retain their ability to respond to activating cytokines (3).

Inflammatory cytokines are sufficient to trigger CTLA-4 expression on the MAIT cell surface

CTLA-4 protein is expressed by conventional T cells following TCR stimulation and constitutively expressed by regulatory T cells (31, 32). Importantly, expression of CTLA-4 protein on the cell surface is regulated by the phosphorylation status of its intracellular domain with most of the protein being located intracellularly in resting T cells (33). Since expression of CTLA-4 on the cell surface is critical for exerting its regulatory function, we assessed the cell surface expression of CTLA-4 on CD8⁺ MAIT cells in blood and OM directly ex vivo. Analysis by flow cytometry revealed significantly increased expression of CTLA-4 on the cell surface of CD8⁺ MAIT cells isolated from OM compared to blood (lines between data points indicate donor-matched blood and tissue), which was observed in minimally inflamed and inflamed tissues (Fig 3A, B). Regulatory T cells (Treg) were included as a positive control and conventional memory CD8⁺ T cells (defined as non-MAIT CD45RA⁻) were included as a reference population (Fig 3A–C). These data demonstrate ex vivo CTLA-4 protein expression on the cell surface of a MAIT cell population in OM tissues and highlight a potential regulatory mechanism for MAIT cell activation.

Figure 3: Inflammatory cytokines induce cell surface expression of CTLA-4 on CD8⁺ MAIT cells (A–C).

Ex vivo surface CTLA-4 expression in CD8⁺ MAIT cells, conventional memory CD8⁺ T cells (non-MAIT CD45RA⁻), and regulatory T cells (Treg; CD3⁺CD4⁺IL-7RαloCD25^hiFoxP3⁺) in donor-matched blood and OM. (A) Representative gating for surface CTLA-4 expression. (B-C) CTLA-4 expression in CD8⁺ MAIT and conventional memory CD8⁺ T cells (B) and Treg (C) sourced from blood (circles) and OM (squares). Light blue symbol fill indicates donors from which OM was minimally inflamed. (D) Peripheral blood mononuclear cells (PBMCs) were stimulated with media alone (“no stim,” black symbols), recombinant cytokines IL-12, −15, and −18 (100ng/mL, each; red symbols), anti-CD3/CD28 beads (“TCR,” blue symbols), or anti-CD3/CD28 beads in combination with recombinant cytokines IL-12, −15, and −18 (orange symbols). Surface expression of CTLA-4 was measured in CD8⁺ MAIT cells (left), conventional memory CD8⁺ T cells (center), and Treg (right). (E) PBMCs were left untreated (black symbols) or treated with IL-12, −15, and −18 (green symbols) in the absence (circles) or presence (squares) of MR1-blocking antibody (50µg/mL). Two-way ANOVA with Dunnett’s multiple comparisons test were performed in (D) while Wilcoxon matched-paired signed rank tests were used in B and C. *p ≤ 0.05 **p ≤0.01 ****p ≤ 0.001.

We next wanted to define which signals are necessary to induce CTLA-4 expression on the MAIT cell surface. To address this, peripheral blood mononuclear cells (PBMCs) from healthy donors were stimulated for 24 hours ex vivo with either anti-CD3/CD28 beads, a combination of the cytokines IL-12/15/18 or both signals. IL-18 is a potent inducer of IFNγ together with IL-12 or IL-15 in conventional memory T cells and natural killer (NK) cells (34, 35). Similarly, we previously reported that IL-18 elicited IFNγ production by MAIT cells in the presence of IL-12 or IL-15, but not by itself (3). Since we furthermore observed that a combination of all 3 cytokines elicited the strongest IFNγ and granzyme B response (3) and our RNAseq data indicated that OM CD8⁺ MAIT cells express the necessary receptors (Fig. 2D), we chose to stimulate MAIT cells with a combination of these 3 cytokines to assess CTLA-4 expression. CTLA-4 expression on CD8⁺ MAIT cells was compared to conventional memory CD8⁺ T cells (CD45RO⁺) and Tregs. All three T cell populations increased expression of CTLA-4 after incubation with anti-CD3/CD28 beads (Fig 3D), but only CD8⁺ MAIT cells also induced surface expression of CTLA-4 following incubation with IL-12/15/18 for 24 hours (Fig 3D). Stimulation of T cells with anti-CD3/CD28 beads resulted in TCR downregulation, which affected subsequent detection by flow cytometry (data not shown). Thus, the anti-CD3/CD28 experimental condition serves primarily as a positive control for conventional T cells but cannot be directly compared to the IL-12/15/18 stimulation condition in regards to its ability to induce CTLA-4 surface expression. Importantly, addition of an anti-MR-1 blocking antibody did not affect CTLA-4 expression in IL-12/15/18 stimulated MAIT cells formally excluding the possibility that MAIT cells received a TCR signal during the culture (Fig 3E). Together, these data provide strong evidence that inflammatory signals are sufficient to induce expression of CTLA-4 on the cell surface of MAIT cells.

Brief exposure to inflammatory cytokines is sufficient to induce sustained CTLA-4 expression by MAIT cells

To determine whether cytokine-induced CTLA-4 surface expression was maintained following cytokine withdrawal, we incubated PBMCs for 6 hours with either IL-12/15/18, anti-CD3/CD28 beads or both stimuli (Fig 4A). After 6 hours the stimuli were removed by washing and cells were either analyzed (Fig 4B) or cultured for another 12hrs or 18hrs without further stimulation before examining CTLA-4 surface expression (Fig 4C, D). We found that CTLA-4 was not expressed after 6hrs of stimulation (Fig. 4B), but this brief (6hrs) exposure to cytokines in the absence of a TCR signal was sufficient to induce surface expression of CTLA-4 on CD8⁺ MAIT cells 12hrs (Fig 4C) and 18hrs later (Fig 4D). These data suggest that a program for surface expression of CTLA-4 is quickly exerted and at least temporarily sustained after exposure to inflammatory stimuli.

Figure 4: Brief exposure to inflammatory cytokines is sufficient to induce CTLA-4 expression by MAIT cells.

(A) Outline of the experimental workflow. (B) CTLA-4 expression on cell surface of CD8⁺ MAIT cells after 6 hour stimulation of PBMCs either (B) immediately, or (C) 12 hours and (D) 18 hours post removal of beads and cytokines. n = 4 (E) Single-cell suspensions isolated from the oral mucosa were stimulated with recombinant cytokines IL-12, −15, and −18 (100ng/mL, each) and anti-CD3/CD28 “TCR” beads for 7 hours and assayed for CTLA-4 and IFNγ with intracellular cytokine staining. (Left) representative gating for IFNγ and total CTLA-4 expression in CD8⁺ MAIT cells. (Center CTLA-4 expression in OM CD8⁺ MAIT cells after stimulation. (Right) IFNγ expression in CTLA-4⁻ and CTLA-4⁺ OM CD8⁺ MAIT cells after TCR and IL-12, −15, and −18 stimulation. (n=6 donors). Two-way ANOVA with Dunnett’s multiple comparisons test were performed in C while Wilcoxon matched-paired signed rank tests were used in E. *p ≤ 0.05 **p ≤0.01 ****p ≤ 0.001.

Finally, we sought to determine if CTLA-4⁺ MAIT cells isolated from OM tissue could respond to activating stimuli when CTLA-4 is bypassed by direct engagement of CD28. To test this, we stimulated cells isolated from the OM with anti-CD3/CD28 beads and cytokines (IL-12/15/18) for 7 hours. We chose 7 hours to minimize de novo expression of CTLA-4 in these experiments and used both stimuli to induce maximum MAIT effector function (Fig 4B). For this ex vivo stimulation experiment, we assessed overall CTLA-4 expression (including intracellular CTLA-4) to facilitate acquisition of a sufficient number of CTLA-4-expressing MAIT cells. The detection of MAIT cells was again affected by the anti-CD3/CD28 bead stimulation (data not shown). We found that MAIT cells that expressed CTLA-4 had reduced production of IFNγ after stimulation (Fig 4E) even though CTLA-4 itself was not engaged by anti-CD3/CD28 beads.

Discussion

Immunoregulatory mechanisms that serve to limit the effector response of conventional T cells are well documented (13, 36). We asked if similar immunoregulatory mechanisms are in place to curtail or prevent MAIT cell effector function following activation of MAIT cells by either TCR-mediated signals or inflammatory cytokines. We considered that the signals eliciting expression of immunoregulatory proteins such as CTLA-4 could differ between MAIT cells and conventional T cells. CTLA-4 is a protein with critical immunoregulatory properties and expressed on regulatory T cells as well as conventional CD4⁺ and CD8⁺ T cells following activation via their TCR (13). In addition to cell intrinsic inhibition that is mediated by CTLA-4 via interaction of phosphatases with its cytoplasmic domain, it has been suggested that CTLA-4 could also act cell-extrinsically. In this scenario, CTLA-4 inhibits priming of other T cells in trans by competing for availability of B7–1 and B7–2 on APCs (14). Whether the role of CTLA-4 on MAIT cells is to restrain their own proinflammatory responses or to preclude neighboring T cells from receiving co-stimulation similar to what has been proposed for Tregs will require further studies. CTLA-4 must be expressed on the cell surface for these cell-intrinsic and -extrinsic immunoregulatory mechanisms, but most of the protein is located intracellularly in resting T cells (33). Importantly, we report here that cell surface expression of CTLA-4 on MAIT cells is induced by inflammatory cytokines independently of a TCR signal (Fig 3). A TCR-independent induction of CTLA-4 cell surface expression on MAIT cells is noteworthy as it indicates that CTLA-4 expression is regulated by distinct signaling pathways in MAIT and conventional T cells. Expression of CTLA-4 is strictly dependent on the transcription factor family “nuclear factor of activated T cells” (NFAT) in conventional T cells as shown by the loss of CTLA-4 transcription when inhibitors of NFAT were present or point mutations were introduced to the NFAT consensus sequence of the promoter region (16). NFAT itself is activated by calcineurin following a TCR-triggered Ca²⁺ influx (15). Interestingly, an alternative, calcineurin-independent NFAT-activation pathway exists in thymocytes (37) and further studies are required to pinpoint whether CTLA-4 expression in MAIT cells is calcineurin-dependent or not. Studies that will compare the chromatin accessibility of the CTLA-4 promoter and nearby regions in MAIT cells to conventional T cells (16) will be of particular interest. Finally, it is important to note that other cells are activated by IL-12, IL-15 and IL-18 (including NK cells and memory CD8⁺ T cells(34)) and cytokines secreted by these cells may also act on MAIT cells and contribute to the induction of CTLA-4 expression on the MAIT cell surface. We limited our stimulation experiments to 24 hours to minimize such indirect effects. Importantly, our main conclusion that this expression occurs in the absence of a TCR signal is not affected by possible indirect effects since TCR signals were prevented by the addition of anti-MR1 antibody. We have also not observed MAIT cell proliferation in this experimental set-up indicating that CTLA-4 expression is not dependent on MAIT cells entering cell cycle.

Our data also raise the question of why CTLA-4 expression is regulated by inflammatory signals in MAIT cells. Importantly, our data demonstrate that even a brief exposure to inflammatory stimuli is sufficient to induce CTLA-4 expression (Fig 4). This suggests that MAIT cells may have a temporally restricted effector phase that is followed by a phase that is refractory to stimulation, which could be essential to ensure that inflammatory responses eventually subside in tissues. It is also important to consider that MAIT cells are located in mucosal tissues with a microbiome and thus are presumably exposed to their antigen. Since a TCR plus costimulatory signal alone is not sufficient to activate MAIT cells, but synergizes with inflammatory cues to elicit a potent effector response (3), inflammation-driven induction of CTLA-4 expression could be in place to minimize responses against commensal-derived antigen by preventing co-stimulation. For example, viral infections or physical tissue damage elicit inflammatory responses, but in these cases a misdirected immune-response against commensal-derived antigen would impair resolution of inflammation and prevent return to tissue homeostasis. Thus, we propose that this cytokine-driven, TCR-independent regulation of CTLA-4 expression ensures that MAIT cells in inflamed tissues restrain deleterious inflammatory responses against commensal organisms.

Finally, our ex vivo MAIT cell stimulation data (Fig 4) also suggest that CTLA-4 expression by MAIT cells in tissues may be a biomarker for overall hyporesponsiveness given that CTLA-4⁺ MAIT cells failed to produce cytokines even when CTLA-4 itself was not engaged by stimulating MAIT cells with anti-CD3/CD28 antibody coated beads in the presence of IL-12/15/18. Continual TCR signals as encountered in the context of chronic infections or cancer eventually lead to T cell dysfunction including the loss of effector function (38). Whether CTLA-4 expressing MAIT cells are just temporarily hyporesponsive or in a dysfunctional state similar to conventional T cells remains to be determined. Similarly, if functionally hyporesponsive MAIT cells have lost their ability to interact with other immune subsets such as B cells (39) or can still provide some help will need to be addressed in future studies.

In summary, our data provide strong evidence that inflammatory cytokines induce sustained CTLA-4 cell surface expression on MAIT cells in a TCR-independent manner. We propose that this TCR-independent induction of CTLA-4 expression in MAIT cells serves to limit MAIT cell-mediated tissue-damage, which may be of interest for therapeutic intervention for a wide range of inflammation driven tissue pathologies including autoimmune lesions and tumors.