Mucosal-associated invariant T (MAIT) cells are promising innate-like lymphocytes with potential for use in anti-tumor immunotherapy. Existing MAIT cell expansion protocols are associated with potentially decremental phenotypic changes, including increased frequency of CD4+ MAIT cells and higher inhibitory receptor expression. In this study, we compared the effect on expansion of human MAIT cells of a serum replacement, Physiologix XF SR (Phx), with traditional serum FBS for supplementing RPMI 1640 media. Using flow cytometry, we found that Phx supported a significantly higher proliferative capacity for MAIT cells and resulted in a lower frequency of CD4+ MAIT cells, which have been associated with reduced Th1 effector and cytolytic functions. We saw that culturing MAIT cells in Phx led to better survival of MAIT cells and lower frequency of PD-1+ MAIT cells than FBS-supplemented media. Functionally, we saw that Phx supplementation was associated with a higher frequency of IFN-γ+ MAIT cells after stimulation with Escherichia coli than FBS-supplemented RPMI. In conclusion, we show that MAIT cells cultured in Phx have higher proliferative capacity, lower expression of inhibitory receptors, and higher capacity to produce IFN-γ after E. coli stimulation than FBS-supplemented RPMI. This work shows that expanding MAIT cells with Phx compared with FBS-supplemented RPMI results in a more functionally desirable MAIT cell for future anti-tumor immunotherapy.
T cells require specific nutrients and environment for optimal function and proliferation (1, 2), and recent advances in our understanding of immunometabolism have uncovered cellular mechanisms that enable T cells for rapid cytokine production (3). These mechanisms include the switch from glycolytic and oxidative phosphorylation following T cell activation (1, 2, 4). In particular, glucose is a crucial nutrient for the efficient production of cytokines, and uptake of glucose is rapidly induced after T cell activation (5, 6). For swift production of energy, the glucose transporter GLUT1 increases in expression in murine conventional CD4+ T cells following TCR stimulation (7). In addition, unconventional innate-like T cells, such as murine NK T cells, also require glucose for optimal cytokine production (8).
Mucosal-associated invariant T (MAIT) cells are an innate-like T cell that rapidly produces effector molecules such as IFN-γ and granzyme B following activation (9, 10). MAIT cells can be activated in a TCR-dependent manner through interaction with microbially derived ligand 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil (5-OP-RU) loaded onto MHC class I–related gene protein (MR1) or in a TCR-independent manner with IL-12 and IL-18 (11). Previously, it was shown that MAIT cells have a lower metabolic rate than conventional T cells and show significantly higher glycolytic activity following TCR and cytokine stimulation than non-MAIT CD8+ effector memory cells (12). GLUT1 expression in MAIT cells increases following TCR and cytokine (IL-12 and IL-18) stimulation, and 2-NBDG expression increases following TCR stimulation but not cytokine stimulation (13). When glycolysis is disrupted with glucose analog 2-deoxy-d-glucose, stimulated MAIT cells produced significantly lower IFN-γ but not TNF-α (13). These studies suggest that cytokine production in MAIT cells may vary, depending on the nutrient composition of their environment.
MAIT cells have the potential to be an HLA-independent, pan-cancer immunotherapy (14, 15), and recent work has examined methods for expanding MAIT cells while preserving cytolytic function (16). The majority of studies on expanded MAIT cells have used FBS as the major component of media supplementation, which is relatively inexpensive and effective for functional and proliferative experiments. Although others have shown effective proliferation of MAIT cells using 5-amino-6-(d-ribitylamino)uracil with methylglyoxal, IL-2, IL-7, and the serum replacement CTS (16, 17). These methods prove useful but have been shown to increase the frequency of CD4+ MAIT cells, which have been shown to produce fewer effector molecules than CD8+ MAIT cells following Escherichia coli stimulation (18).
The goal of this study was to examine the expansion ability of MAIT cells in culture supplemented with a novel serum replacement reagent, Physiologix XF SR (Phx; detailed below), previously shown to enhance chimeric Ag receptor T cell survival and function in vivo while being able to expand and maintain a metabolically unstressed conventional T cell in vitro (19). We compared the proliferation, function, and exhaustion markers of MAIT cells cultured using Phx in comparison with FBS as a supplement to conventional RPMI media. We found that use of Phx was associated with greater proliferative capacity and increased IFN-γ production in MAIT cells following stimulation.
Materials and Methods
Human subjects and processing of PBMCs for assays
We obtained blood from discarded leukocyte filters and apheresis cones from healthy anonymous blood donors, and we isolated PBMCs by density gradient centrifugation using Ficoll-Paque PLUS (Cytiva). We froze isolated PBMCs <10 × 106 cell/ml in a medium containing 30% FBS, 60% RPMI 1640 (Life Technologies), and 10% DMSO. We thawed 1-ml aliquots of frozen PBMCs rapidly in a 37°C water bath and centrifuged them in 10 ml warm RPMI 1640 media. We washed PBMCs twice in warm RPMI 1640 before proceeding to assays comparing RPMI 1640 supplemented with either FBS (Life Technologies) or Phx (Nucleus Biologics).
Media culturing test
We thawed PBMCs from cryovials and washed them as described above and seeded 2.5 × 106 cells/ml in RPMI 1640 with 10% FBS or 2% Phx in five separate wells corresponding to time of incubation. We performed extracellular staining with fixable viability dye eFluor 780 (eBioscience), anti-CD3-PerCP-cyanine 5.5 (Cy5.5) (BioLegend), anti-CD8-BV605 (BioLegend), anti-CD4-BV510 (BioLegend), anti-Vα7.2-PE-Cy7 (BioLegend), anti-LAG-3-BV786 (BioLegend), anti-CD69-allophycocyanin (BioLegend), anti-CD25-PE-Cy5 (BD Biosciences), anti-PD-1 Alexa Fluor 700 (BioLegend), anti-CD161-BV605 (BioLegend), anti-CD69-PE-Cy5 (BioLegend), and anti-human PE-MR1-5-OP-RU tetramer (National Institutes of Health [NIH] Tetramer Core Facility). We collected data using a five-laser LSRFortessa flow cytometer (BD Biosciences), and the flow data were analyzed using FlowJo software version 10 (BD Biosciences, Ashland, OR).
E. coli stimulation
We thawed PBMCs from cryovials and washed them as described above, and we seeded 2.5 × 106 cells/ml in RPMI 1640 with 10% FBS or 2% Phx. We stimulated PBMCs with 10 multiplicities of infection (MOIs) of strain 1100-2 fixed E. coli for 20 h and blocked extracellular transport with brefeldin A for a total of 4 h. We performed extracellular staining with fixable viability dye eFluor 780 (eBioscience), anti-CD3-BUV395 (BD Biosciences), anti-CD8-BV605 (BioLegend), anti-CD4-BV510 (BioLegend), anti-Vα7.2-PE-Cy7 (BioLegend), anti-LAG-3-BV786 (BioLegend), anti-CD25-BV650 (BioLegend), anti-PD-1-PerCP-Cy-5.5 (BioLegend), anti-CD161-PE/Dazzle-594 (BioLegend), anti-CD69-PE-Cy5 (Invitrogen), anti-CD161-allophycocyanin (BioLegend), and anti-human PE-MR1-5-OP-RU tetramer (NIH Tetramer Core Facility). The PBMCs were fixed and permeabilized using an Foxp3/transcription factor kit (eBioscience) and stained intracellularly with anti–granzyme B Alexa Fluor 700 (BioLegend), anti-TNF-α-eFluor-450 (Invitrogen), anti-IFN-γ-FITC (BioLegend), and anti-T-bet-BV711 (BioLegend). We collected data using a five-laser LSRFortessa flow cytometer (BD Biosciences), and the flow data were analyzed using FlowJo software version 10 (BD Biosciences).
Expansion of MAIT cells
We thawed PBMCs from cryovials and washed as described above and seeded 1 × 106 cells/ml in a volume of 1 ml in a 24-well plate. The media we compared were RPMI 1640 with 5%, 10%, or 20% FBS, 2% Phx, and Immunocult-XF T cell expansion medium (STEMCELL Technologies) with 8% CTS serum (Invitrogen) (hereinafter referred to as CTS), with all media receiving 1% penicillin-streptomycin. We used a method of MAIT cell expansion as previously described that used the Immunocult-XF T cell expansion base medium with 8% CTS (17). We further supplemented the media with 5 ng/ml rhIL-2 (PeproTech) and 10 ng/ml rhIL-7 (PeproTech). We stimulated proliferation of MAIT cells with 10 nM 5-OP-RU on days 0, 5, and 10, and 50% of the media was exchanged every 2–3 d with fresh cytokines. On days 0, 7, and 14, 100 µl was analyzed for MAIT cell count, frequency, activation, and inhibitory receptor markers. For extracellular staining, we used Zombie ultraviolet fixable viability dye (BioLegend), anti-CD3-BUV395 (BD Biosciences), anti-CD8-BV605 (BioLegend), anti-CD4-BUV496 (BD Biosciences), anti-Vα7.2-BV711 (BioLegend), anti-LAG-3-BV786 (BioLegend), anti-CD69-BUV563 (BD Biosciences), anti-CD161-PE-Dazzle-594 (BioLegend), anti-TIM-3-BV421 (BioLegend), and anti-human MR1-5-OP-RU tetramer (NIH Tetramer Core Facility). Sample data were acquired with a five-laser Cytek Aurora flow cytometer (Cytek) and analyzed using FlowJo software version 10 (BD Biosciences).
E. coli stimulation of expanded MAIT cells
We cultured 2,000–5,000 MAIT cells from MAIT cells expanded in RPMI 1640 with 10% FBS or 2% Phx with 10,000–25,000 THP-1 cells to serve as APCs in RPMI with 10% FBS without cytokines overnight. We then stimulated the coculture of MAIT cells and THP-1 with 10 MOIs of strain 1100-2 fixed E. coli for 20 h and blocked extracellular transport with brefeldin A for a total of 4 h. For extracellular staining, we used Zombie ultraviolet fixable viability dye (BioLegend), anti-CD3-BUV395 (BD Biosciences), anti-CD8-BV605 (BioLegend), anti-CD4-BUV496 (BD Biosciences), anti-Vα7.2-PE-Cy7 (BioLegend), anti-LAG-3-BV786 (BioLegend), anti-CD69-BUV563 (BD Biosciences), anti-CD161-PE-Dazzle-594 (BioLegend), anti-TIM-3-BV421 (BioLegend), anti-CD25-BV650 (BD Biosciences), and anti-human PE-MR1-5-OP-RU tetramer (NIH Tetramer Core Facility). The cells were fixed and permeabilized using Foxp3/transcription factor kit (eBioscience) and stained intracellularly with anti-granzyme-B Alexa Fluor 700 (BioLegend), anti-TNF-α eFluor 450 (Invitrogen), and anti-IFN-γ-FITC (BioLegend). Sample data were acquired with a five-laser Cytek Aurora flow cytometer (Cytek) and analyzed using FlowJo software version 10 (BD Biosciences).
For comparisons of FBS and Phx, a paired Wilcoxon rank-sum test was used. GraphPad Prism 8.3.0 software was used for statistical analysis of the flow cytometry data, and p < 0.05 was considered statistically significant.
Ligand-activated MAIT cells in Phx-supplemented RPMI had greater proliferative capacity and lower expression of inhibitory receptors TIM-3 and PD-1
We first examined the ability of Phx to proliferate MAIT cells in vitro using a published method (17) of activating MAIT cells with the ligand 5-OP-RU three times over the course of 10 d (Supplemental Fig. 1A). We defined MAIT cells as live CD3+ Vα7.2+ MR1-tetramer+ (Fig. 1A). We supplemented RPMI 1640 with 2% Phx or 10% FBS. We saw a significant increase in MAIT frequency and MAIT cell count in Phx compared with FBS-supplemented RPMI on day 7 (p < 0.0001) and day 14 (p < 0.0001) (Fig. 1B–1D). Next, we examined the proportion of CD4+ and CD4−CD8− (double-negative [DN]) MAIT cells, given that CD4+ and DN MAIT cells are known to have reduced Th1 effector and cytolytic functions compared with CD8+ MAIT cells (18, 20). We saw significantly fewer CD4+ MAIT cells in Phx than in FBS-supplemented RPMI at day 7 (p = 0.0008) and day 14 (p < 0.0001) and significantly more DN MAIT cells in Phx than in FBS-supplemented RPMI (Fig. 1E, 1F). We also examined the expression of inhibitory receptors, which have been associated with reduced functionality among MAIT cells in colon tumors (21). We found that MAIT cells cultured in Phx have significantly fewer TIM-3+ MAIT cells after day 7 and significantly fewer TIM-3+ and PD-1+ MAIT cells at day 14 than in FBS-supplemented RPMI, though we saw significantly more LAG-3+ MAIT cells in Phx than in FBS-supplemented RPMI at day 7 (Fig. 1G–1K). Inhibitory receptor expression often coincides with activation in MAIT cells (10), and we see significantly lower CD69 expression in MAIT cells at day 14 in Phx than in FBS-supplemented RPMI (Supplemental Fig. 1B).
We saw that expansion with Phx resulted in significantly higher MAIT cell frequency and count than that with 5% FBS, 10% FBS, 20% FBS, and Immunocult-XF with 8% CTS, a medium used in prior publications of MAIT cell expansion (16, 22) (Supplemental Fig. 1C, 1D). Although CTS resulted in a higher number of CD3+ T cells (Supplemental Fig. 1E), the use of Phx resulted in a significantly higher proportion of MAIT cells at day 14 (Supplemental Fig. 1C). We further characterized CD25 (IL-2 receptor α) expression as a phenotypic marker associated with CD4+ MAIT cells (18), and we saw in these ligand-stimulated MAIT cells significantly higher CD25 expression in Phx than in 10% FBS at day 7 but no significant differences at day 14 (Supplemental Fig. 1F). Last, when we stimulated Phx-expanded MAIT cells with E. coli, we saw no differences in MAIT cell subset frequencies, but observed lower expression of inhibitory receptors TIM-3, LAG-3, and PD-1, compared with MAIT cells expanded in FBS-supplemented RPMI (Supplemental Fig. 2A–2F). We also noted that in response to E. coli stimulation, Phx-expanded MAIT cells have non–statistically significant higher IFN-γ and TNF-α production (p = 0.06) than FBS-expanded MAIT cells (Supplemental Fig. 2G, 2H), though no differences in granzyme B were seen. Overall, Phx supplementation of MAIT cell expansion resulted in greater proliferative capacity, a lower proportion of CD4+ MAIT cells, and lower frequency of TIM-3+ and PD-1+ MAIT cells than FBS-supplemented RPMI.
Nonactivated MAIT cells cultured in Phx-supplemented RPMI had better persistence and lower inhibitory receptor expression
To determine the impact of Phx on conventional and unconventional T cell survival, we cultured primary T cells from 10 blood donors for 8 d. We did not see any significant differences in CD3+ frequency or total CD3+ cells between Phx- and FBS-supplemented RPMI at any time point measured (Fig. 2A–2C). However, when we examined the impact on MAIT cells, we saw a significantly higher frequency and count of MAIT cells at all time points in Phx- compared with FBS-supplemented RPMI (Fig. 2D, 2E). Among subsets of MAIT cells, there were no significant changes in CD4+ MAIT cell frequency; in these nonactivated MAIT cells, those that were Phx supplemented had lower DN frequency, higher CD8+ MAIT cell frequency, and lower CD25 expression than FBS-supplemented RPMI (Supplemental Fig. 3A–3D).
We then looked at expression of inhibitory receptor PD-1 because it has been implicated in exhausted MAIT cells in tumors (23). We saw that there was a significantly lower frequency of PD-1+ MAIT cells in the Phx at all time points (Fig. 2F, 2G). Last, we saw significantly lower LAG-3+ and TIM-3+ expression on MAIT cells in Phx- than in FBS-supplemented RPMI (Fig. 2H, 2I). Overall, we saw no differences in frequency or counts of conventional T cells between Phx- or FBS-supplemented RPMI, but we noted significantly higher frequency and count of MAIT cells with lower expression of inhibitory receptors in Phx- than in FBS-supplemented RPMI.
E.coli–stimulated MAIT cells in Phx-supplemented RPMI had greater IFN-γ expression than in FBS-supplemented RPMI
We next looked at the effect of Phx on the effector molecule production of MAIT cells following stimulation with E. coli, a relevant microbe with the capacity to produce the MAIT-stimulating ligand. We saw no changes in total MAIT cell frequency after stimulation with E. coli between Phx- and FBS-supplemented RPMI (Fig. 3A). We saw a significantly lower amount of CD8+ MAIT cells following E. coli stimulation in Phx than in FBS-supplemented RPMI, whereas there were no differences in CD4+ or DN MAIT cell frequencies (Supplemental Fig. 4A–4C). We looked at activation markers and saw no differences in frequency of CD69+ and CD25+ MAIT cells between Phx- and FBS-supplemented RPMI following E. coli stimulation (Fig. 3B, 3C). We also did not see any significant differences in the frequency of LAG-3+ or PD-1+ MAIT cells, but we observed a lower frequency of TIM-3+ MAIT cells (Fig. 3D–3F). Notably, we saw significantly higher IFN-γ expression following E. coli stimulation in Phx- than in FBS-supplemented RPMI, driven mainly by CD4+ and CD8+ MAIT cells, because DN MAIT cells showed no differences (Fig. 3G, 3H, and Supplemental Fig. 4D–4F) but no significant differences in TNF-α or granzyme B expression (Fig. 3I, 3J, and Supplemental Fig. 4G–4I) or in the expression of T-bet, which is implicated in the regulation of IFN-γ expression (Fig. 3K). Taken together, Phx supplementation of E. coli–stimulated MAIT cells resulted in lower frequency of TIM-3+ MAIT cells and higher IFN-γ expression than FBS supplementation, but no differences were seen in activation markers, LAG-3 and PD-1, or T-bet expression.
In this study, we present a method to expand MAIT cells isolated from peripheral blood using a novel serum substitute. We show that, compared with FBS, Phx supplementation results in greater expansion and a higher cytokine potential following E. coli stimulation while maintaining a more biologically similar phenotype with lower expression of inhibitory receptor expression in expanded MAIT cells.
The potential use of expanded MAIT cells for anti-tumor immunotherapy has been suggested after recent studies demonstrating MR1 to be an important candidate target for human cancers (14, 24) and demonstration of anti-tumor effects in various mouse models of MAIT cell immunotherapy (25, 26). CD4+ MAIT cells are associated with decreased effector function, mainly IFN-γ and granzyme B, compared with CD8+ MAIT cells ex vivo (18), and studies of MAIT cells from patients with colorectal cancer have suggested that CD8+ MAIT cells are depleted in the circulation and enriched in tumors (27, 28). Our MAIT cell expansion protocol was able to mitigate the increase in frequency of CD4+ MAIT cells, thus possibly offering improved anti-tumor activity over previously published protocols for MAIT cell expansion, which resulted in an increased frequency of CD4+ MAIT cells (16). Although we do not know the mechanism by which Phx may preferentially expand CD8+ MAIT cells, it is possible that Phx may lack the appropriate metabolic signals for proliferation of CD4+ MAIT cells, which are found in higher frequency in lymphoid tissues such as tonsils and thymus than in blood (29, 30), and may require specific TCR interactions or certain lymphoid-like conditions. The ability to efficiently expand MAIT cells ex vivo while maintaining a functional phenotype may support the development of new MAIT cell–based tumor immunotherapies.
The addition of a serum substitute such as Phx provides differential nutrients for MAIT cells in culture compared with FBS and was associated with decreased expression of inhibitory receptors and an increased capacity for IFN-γ production after E. coli stimulation but not cytokine stimulation. Previous studies have shown that MAIT cell production of granzyme B and IFN-γ is affected by glucose availability, most strikingly following a combination of TCR and cytokine stimulation, suggesting that the combination of both pathways is required (12, 13). Others have demonstrated that Phx has 166% higher carnosine than human serum and could switch T cells from a glycolytic to an oxidative metabolic state (19), which could preferentially expand CD8+ MAIT cells and increase functionality by reducing stress on the cells during the ex vivo expansion. Although the exact composition of Phx is unknown, studies using mass spectrometry demonstrated that Phx showed higher levels of metabolites such as glucose-1-phosphate and pyruvate than human serum, both of which can be used to generate ATP through the TCA cycle for oxidative phosphorylation purposes (2, 19).
Inhibitory receptors such as PD-1 and TIM-3 are part of the subset of receptors that are associated with T cell exhaustion and decreased effector function (31), as seen with MAIT cells in hepatic carcinoma (23) and colorectal cancer (21). Our data show that Phx is associated with lower TIM-3 and PD-1 expression during ligand-activated MAIT cell proliferation and lower PD-1, TIM-3, and LAG-3 in unstimulated MAIT cell culture. These data suggest that expanding and culturing MAIT cells with Phx-supplemented RPMI may result in lower levels of MAIT cell exhaustion, which is desirable for anti-tumor immunotherapy.
In conclusion, we show that use of Phx-supplemented RPMI results in a higher proliferation and IFN-γ production from MAIT cells than FBS-supplemented RPMI. In addition, we saw that Phx was associated with decreased PD-1, TIM-3, and LAG-3 in MAIT cell culture, and lower TIM-3 and PD-1 expression in MAIT cell proliferation, compared with FBS-supplemented RPMI. Our findings have the potential to inform development strategies for ex vivo proliferation and maintenance of a functional phenotype of MAIT cells that could be used in future immunotherapies against cancers.
The authors have no financial conflicts of interest.
We thank Associated Regional and University Pathologists for the blood samples. We also thank the staff of the University of Utah Flow Cytometry Core. We thank Nucleus Biologics for providing some of the Physiologix XF SR used in this study.
This work was supported in part by the National Institutes of Health (AI130378 to D.T.L. and TL1TR002540 to D.L.).
The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
The online version of this article contains supplemental material.
Abbreviations used in this article:
mucosal-associated invariant T
multiplicity of infection
MHC class I–related gene protein
National Institutes of Health
Physiologix XF SR