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. 2021 Dec 24;10:e50324. doi: 10.7554/eLife.50324

Human MAIT cells respond to and suppress HIV-1

Chansavath Phetsouphanh 1,2,†,✉, Prabhjeet Phalora 1,†, Carl-Philipp Hackstein 1,†, John Thornhill 3, C Mee Ling Munier 2, Jodi Meyerowitz 1, Lyle Murray 1, Cloete VanVuuren 4, Dominique Goedhals 5, Linnea Drexhage 6, Rebecca A Russell 6, Quentin J Sattentau 6, Jeffrey YW Mak 7,8, David P Fairlie 7,8, Sarah Fidler 3, Anthony D Kelleher 2, John Frater 1, Paul Klenerman 1,✉
Editors: Stipan Jonjic9, Tadatsugu Taniguchi10
PMCID: PMC8752121  PMID: 34951583

Abstract

Human MAIT cells sit at the interface between innate and adaptive immunity, are polyfunctional and are capable of killing pathogen infected cells via recognition of the Class IB molecule MR1. MAIT cells have recently been shown to possess an antiviral protective role in vivo and we therefore sought to explore this in relation to HIV-1 infection. There was marked activation of MAIT cells in vivo in HIV-1-infected individuals, which decreased following ART. Stimulation of THP1 monocytes with R5 tropic HIVBAL potently activated MAIT cells in vitro. This activation was dependent on IL-12 and IL-18 but was independent of the TCR. Upon activation, MAIT cells were able to upregulate granzyme B, IFNγ and HIV-1 restriction factors CCL3, 4, and 5. Restriction factors produced by MAIT cells inhibited HIV-1 infection of primary PBMCs and immortalized target cells in vitro. These data reveal MAIT cells to be an additional T cell population responding to HIV-1, with a potentially important role in controlling viral replication at mucosal sites.

Research organism: Human

Introduction

Mucosal-associated invariant T-cells (MAIT cells) are innate-like T cells that rapidly produce cytokines upon activation and express a semi-invariant T-cell antigen receptor (TCR) (Birkinshaw et al., 2014; Zinser et al., 2018). Human MAIT cells are typically defined by expression of the Vα7.2 TCR (rearranged in-combination with Jα33) in combination with phenotypic markers, including high levels of the C-type lectin CD161 and IL18R (Kurioka et al., 2017). MAIT cells are restricted by the evolutionary conserved non-polymorphic MHC-related protein MR1, which presents microbially derived vitamin B metabolites (Birkinshaw et al., 2014; Eckle et al., 2015; Kjer-Nielsen et al., 2018). Recognition of MR1-bound ligands from riboflavin synthesizing bacteria by the MAIT TCR leads to release of cytokines such as IFNγ, TNFα, and IL-17, as well as triggering their cytolytic function (Kurioka et al., 2017; Kurioka et al., 2015; Leeansyah et al., 2015).

MAIT cells have been extensively studied in the context of HIV-1 infection. Early and non-reversible loss of CD161++/MAIT cell frequencies has been observed in HIV-1 infection (Salou et al., 2017; Cosgrove et al., 2013), and this loss has been confirmed in several other human studies (Juno et al., 2019a, Sortino, 2018; Spaan et al., 2016; Khaitan et al., 2016; Fernandez et al., 2015; Eberhard et al., 2014), as well as SIV infection of rhesus macaques (Vinton et al., 2016; Juno et al., 2019b). Depletion of MAIT cells in peripheral blood during HIV-1 infection may be caused by several factors. Firstly, down-regulation of CD161 expression may lead to an underestimation of CD161++ Vα7.2+ MAIT cells in blood. However, use of MR1/5-OP-RU tetramers and qPCR for the specific TCR has independently confirmed previous findings that MAIT cells are indeed depleted in the blood during HIV-1 infection (Fernandez et al., 2015; Ussher et al., 2018). Secondly, up-regulation of tissue homing markers α4β7 (Juno et al., 2019b) and chemokine receptors (CXCR6+, CCR2+, CCR5+, CCR6+, and CCR9+) and the identification of MAIT cells in affected tissues demonstrate that they can migrate into tissues during infection (D’Souza et al., 2018), although recovery of MAIT cell numbers in blood is not reproducibly seen upon viral suppression with Antiretroviral Therapy (ART). Increased bacterial translocation from the gut during HIV-1 infection and MAIT cell migration into these sites may lead to activation-induced cell death following activation via their TCR (Cosgrove et al., 2013).

It has been shown that MAIT cells have the ability to sense viral infections through specific cytokine-driven mechanisms, including IL-12, IL-15, IL-18, Type I interferons (van Wilgenburg et al., 2016; Ussher et al., 2014) and most recently TNF (Provine et al., 2021). These mechanisms have been defined in vitro and activation of MAIT cells in response to acute and persistent virus infections (Dengue, HCV, and Influenza) and vaccines has been clearly demonstrated in vivo in humans (van Wilgenburg et al., 2018; van Wilgenburg et al., 2016; Loh et al., 2016; Provine et al., 2021). Importantly, such activation is associated with protection against death in a lethal influenza challenge model in mice (van Wilgenburg et al., 2018), which provides proof-of-principle that such TCR-independent activity has an important biological role. While previous experiments showed that HIV-1 infection leads to a decrease of MAIT cells (Cosgrove et al., 2013), it remains to be determined if MAIT cells display any kind of antiviral activity in the context of HIV-1 infection.

Here, we investigated to what extent and by which mechanisms HIV-1 could activate MAIT cells and whether this resulted in measurable anti-HIV-1 activity. We assessed MAIT cells in the peripheral blood of HIV-1+ subjects during different stages of infection and, given the importance of the gastrointestinal (GI) tract in HIV-1 pathogenesis, in rectal and ileal tissue samples to define activation and redistribution. We established a model using HIVBAL to activate MAIT cells in vitro, showing that this process is TCR independent but dependent on IL-12 and IL-18 stimulation. Upon activation, MAIT cells were able to upregulate HIV-1 restriction factors and reduce levels of infection. These data, taken with emerging data from the field, suggest that MAIT cells should be included in the repertoire of antiviral populations activated during HIV-1 infection, with a potentially important role for control at mucosal sites.

Results

MAIT cells are activated by HIV-1 in vivo

To first address MAIT cell activation during HIV-1 infection, PD-1, granzyme B (GzmB), and TIM-3 protein levels were measured pre- and post-ART in donors with Primary and Chronic HIV-1 infection (PHI and CHI respectively) (Figure 1A and B). Expression of all three molecules was very low in healthy controls ex vivo (median: PD-1 = 0.85%, GzmB = 0%, and TIM-3 = 0.15%). Moderate levels of GzmB+ MAIT cells were detected during PHI at baseline (10.06%), which significantly decreased after ART (2.27%, p < 0.05). Granzyme B expression on MAIT cells was even higher in CHI donors, with a median of 55% (IQR: 41.2–93.9%) of cells being positive; however, these levels dramatically decreased following 1 year of ART (22.45%, p < 0.001). Activation-induced inhibitory receptors PD-1 and TIM-3 followed a similar pattern of expression to granzyme B during acute and chronic HIV-1 infection. There was a ~ 2.7-fold decrease in PD-1 and ~2.3-fold decrease of TIM-3 expressing MAIT cells after ART in PHI donors (p < 0.05 and p < 0.01, respectively). This was also observed in CHI, whereby lower percentages of PD-1 and TIM-3 in MAIT cells were detected post-ART (~6.4-fold [p < 0.01] and ~10.8 fold [p < 0.01], respectively). TIM-3 was also expressed at elevated levels in Elite and Viraemic controllers (EC and VC respectively), compared to Healthy controls (HC) (Figure 1—figure supplement 1), indicating some ongoing activation even with low levels of virus (Figure 1—source data 1).

Figure 1. Increased activation and inhibitory marker expression on MAIT cells during HIV-1 infection.

(A) Representative histograms showing upregulation of the activation/inhibitory markers PD-1, Granzyme B (GzmB) and TIM-3 in MAIT cells in chronic HIV-1 infection (CHI) compared to a healthy control (HC). (B) Increased expression of PD-1, GzmB, and TIM-3 on CD8+ CD161++ and Vα7.2+ MAIT cells during PHI and CHI chronic at baseline (BL) and 1 year post-ART (1 yr-Tx). Data points are biological replicates, shown as mean and standard deviation. *p < 0.05, **p < 0.01, *** p < 0.001, **** p < 0.0001; two-tailed t-tests.

Figure 1—source data 1. Marker expression on MAIT cells during HIV-1 infection.
(B) GzmB, PD-1, and TIM-3 expression levels on MAIT cells during PHI and CHI HIV-1 infection. Fig. Suppl.(1) TIM-3 expression levels on MAIT cells in EC and VC.

Figure 1.

Figure 1—figure supplement 1. TIM-3 expression on MAIT cells in LTNP.

Figure 1—figure supplement 1.

Elevated but not statistically significant expression of TIM-3 on MAIT cells from Elite vs.
Viraemic Controllers. Data points are biological replicates.
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To further assess the impact of HIV-1-induced activation on MAIT cells we tracked cell frequencies in blood in specific patient groups. While MR1-Tetramers loaded with the MAIT cell ligand 5-OP-RU represent the most reliable way to identify MAIT cells in the blood, they can also be identified as CD161++ Vα7.2+ cells, especially within the CD8+ population (Kurioka et al., 2017). In order to test whether this would also be the case in the gut, we co-stained CD161 and Vα7.2 with 5-OP-RU-loaded or control MR1-tetramers and assessed whether CD161++ Vα7.2+ cells stained positive for the tetramers (Figure 2—figure supplement 1). Within the CD8+ and double-negative (DN) populations, almost all CD161++ Vα7.2+ cells reacted with the 5-OP-RU-loaded tetramer but not the 6FP-loaded control tetramer, suggesting that the combination of high CD161 expression with Vα7.2 can be used to identify MAIT cells within the CD8 and DN T-cell populations in the gut. CD161++ Vα7.2+ expression on CD8+ T cells was comparable with the MR1-5-OP-RU+ tetramer and could identify the majority of MAIT cells within this compartment.

As previously noted, loss of CD8+ MAIT cells was observed in HIV-1+ cohorts regardless of disease stage when compared to uninfected controls (Figure 2A&B). Looking at specific clinically defined populations, MAIT cell percentages were also very low in Long-Term Non-Progressors (LNTP), with a ~ 9 fold decrease in Elite controllers (ECs) and a ~ 17-fold decrease in Viraemic controllers (VCs) when compared to healthy controls (Figure 2B). Interestingly, a higher percentage of MAITs was observed in EC compared to those with chronic HIV-1 infection (median of 0.76% and 0.5% respectively, p < 0.05) indicating a potential relationship between MAIT cell frequency and divergent clinical outcomes. No recovery of MAIT cells in blood was observed in either CHI or PHI groups 1 year post-ART (Figure 2C&D). This observation was also consistent during long-term ART (up to 5 years), where MAIT cell percentages did not increase from 1 year post-ART (Figure 2E). Analysis of rectal vs blood derived MAIT frequencies in ART-treated patients revealed a clear positive correlation between the two compartments (rho = 0.69, p < 0.05) (Figure 2F), with relative enrichment of the cells in rectal tissue (Figure 2G). This indicates that both compartments are impacted in parallel and the decline in frequency in blood is unlikely to be accounted for by redistribution to the gut. These data taken together confirm and extend existing studies, indicating that MAIT cells are strongly activated in vivo by HIV-1 infection, varying according to levels of viral replication (Figure 2—source data 1).

Figure 2. Frequency of MAITs cells in blood and intestine during HIV-1 infection.

(A) Representative dot-plot showing loss of CD8+ MAIT cells, gated on CD161++ and Vα7.2+, in CHI compared to HC. (B) Loss of MAIT cells in peripheral blood in HIV-1+ donors at different HIV-1 stages PHI, CHI, EC (Elite Controllers), and VC (Viraemic controllers). (C) No recovery of MAIT cells post-ART in CHI. (D) No recovery of MAIT cells post-ART in PHI. (E) No recovery of MAIT cells following long-term ART. (F) Higher percentage of MAIT cells in rectal and illeal tissue compared to blood in matched PHI-treated donors. (G) MAIT cell percentages in the rectum compared to terminal ileum of PHI-treated donors. Data points are biological replicates, shown as mean and standard deviation. Spearman’s correlation was used to calculate rho and p value. *p < 0.05, **p < 0.01, *** p < 0.001, **** p < 0.0001; two-tailed t-tests.

Figure 2—source data 1. Frequency of MAITs cells in blood and tissue during HIV-1 infection.
(B) MAIT cell frequencies in HC compared to PHI, CHI, EC, and VCs at baseline. (C) MAIT cell frequencies during CHI at baseline and 1 year post-ART. (D) MAIT cell frequencies during PHI at baseline and 1 year post-ART. (E) longitudinal MAIT cell frequencies during CHI at 1, 3, and 5 years post-ART. (F) Comparison of MAIT cell frequencies between GALT and PBMC. (G) Presence of MAIT cells in rectum and terminal ileum of PHI ART-treated subjects.

Figure 2.

Figure 2—figure supplement 1. CD161++ Vα7.2+ identifies MAIT cells in tissue- comparable to MR1-5OPRU tetramers.

Figure 2—figure supplement 1.

(A) Representative dot plot of CD161 and Vα7.2 versus MR1-5OPRU tetramer staining in T cell compartments. (B) Comparison of MAIT cell percentages identified using the two staining methods. (C) Proportion of MR1-5OPRU and MR1-Ac-6-FP tetramer-bound cells within CD8+, DN, and CD4+ populations. Data points are biological replicates.
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HIV-1 activates MAIT cells in vitro

We next addressed whether we could model HIV-1 activation of MAIT cells using an in vitro system to further define the mechanism of activation, which can vary between viral systems (Provine et al., 2021; van Wilgenburg et al., 2018), and which has not been previously demonstrated. THP-1 cells, a monocytic cell line, were infected with R5 tropic lab strain HIVBAL for 6 hr and then incubated overnight with enriched CD8+ T cells from peripheral blood from healthy donors. IFNγ production from the CD161++ Va7.2+ population was measured to determine MAIT cell activation and formaldehyde fixed E. coli was included as a positive control (Figure 3A).

Figure 3. MAIT cells are activated by HIV-1 in an IL-12 and IL-18-dependent manner and display anti- HIV-1 activity.

(A) Bar plots showing the percentage of MAIT cells expressing IFN-γ upon in vitro stimulation with fixed E. coli or HIVBAL in the presence or absence of blocking antibodies directed against IL-12 and IL-18. (B) Reduced frequency of GFP positive CEM-GXR cells following infection with HIVBAL (MOI = 0.2) and pre-treatment with stimulated supernatant from MAIT cells. Shown are representative dot plots (left) and cumulative column bars (right). (C) Inhibition of HIVJRFL-GFP infection in primary human PBMCs or CEM-CCR5 cells by addition of control or IL-12/18-treated supernatants obtained from MACS-enriched CD8s (left) or FACS-sorted MAIT cells (right). *p < 0.05, paired t-tests. Data were pooled from three independent experiments; error bars indicate the standard deviation.

Figure 3—source data 1. MAIT cells are activated by HIV-1 in an IL-12 and IL-18-dependent manner.
(A) percentage of MAIT cells expressing IFN-γ upon in vitro stimulation with fixed E. coli or HIVBAL in the presence or absence of blocking antibodies directed against IL-12 and IL-18. (B) frequency of GFP positive CEM-GXR cells following infection with HIVBAL (MOI = 0.2) and pre-treatment with stimulated supernatant from MAIT cells. (C) HIVJRFL-GFP infection in primary human PBMCs or CEM-CCR5 cells by addition of control or IL-12/18-treated supernatants obtained from MACS-enriched CD8s (top) or FACS-sorted MAIT cells (bottom).

Figure 3.

Figure 3—figure supplement 1. Inactivated HIV-1 does not stimulate MAIT cells.

Figure 3—figure supplement 1.

(A) Increased IFN-γ expression from MAIT cells after infection with HIVBAL but not with an inactivated HIVJRFL virus. Data points are biological replicates.
Figure 3—figure supplement 2. Inhibition of HIV-1 is increased with higher volumes of stimulated supernatant.

Figure 3—figure supplement 2.

(A) Titration of IL-12/18 stimulated MAIT cell supernatants on CEM-GXR cells infected with HIVBAL. Plotted are the means from two biological replicates.
Figure 3—figure supplement 3. Increased expression of restriction factor and pro-apoptosis markers in MAIT cells stimulated with IL-12 and IL-18.

Figure 3—figure supplement 3.

(A) Dot plots showing increased intracellular activation and apoptosis marker expression (caspase 3) in MAIT cells after IL-12/18 stimulation. Data points are biological replicates.
Figure 3—figure supplement 4. MAIT cell anti-viral activity is not dependent on IFNγ.

Figure 3—figure supplement 4.

(A) Reduced p24 expression in Jurkat-Tat-R5 cells following incubation with stimulated supernatants from MAIT cells compared to unstimulated control supernatants. Addition of an IFNγ blocking antibody did not fully rescue the block to infection. Data points are biological replicates. Bar plot shown as mean and standard deviation. *p < 0.05; two-tailed t-tests. ns = not significant.
Figure 3—figure supplement 5. Inhibition of HIV-1 is not dependent on cell contact.

Figure 3—figure supplement 5.

(A) MAIT cells and CEM-GXR cells in co-culture or physically separated in transwell plates were infected with HIVBAL and the number of GFP+ cells was measured. Data points are biological replicates. Bar plot shown as mean and standard deviation.
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In this system, HIVBAL was able to activate MAIT cells to similar levels as E. coli (15.98% and 19.65%; respectively) (Figure 3A), whereas an inactivated HIVJRFL primary viral isolate did not have this effect (Figure 3—figure supplement 1). Since IL-12 and IL-18 in combination have been shown to be important in TCR-independent triggering of MAIT cells by some viruses such as Dengue (van Wilgenburg et al., 2018 van Wilgenburg et al., 2016), we sought to analyze this in our system. Blocking antibodies to IL-12 and IL-18 inhibited MAIT cell activation with E. coli, as has been previously shown (Ussher et al., 2014; Kurioka et al., 2018), and importantly also blocked HIVBAL-mediated activation of MAIT cells (Figure 3A). This shows that HIV-1 can activate MAIT cells, similar to other viruses, in an IL12- and IL18-dependent manner.

MAIT cells possess antiviral activity against HIV-1

Since HIVBAL was able to activate MAIT cells in vitro, we sought to determine if viral stimulation of MAIT cells had an impact on HIV-1 infection. To that end, we set up an in vitro infection model, utilizing the CEM-GXR-GFP reporter cell line. Supernatants from unstimulated and IL-12/18 stimulated CD8+ enriched T cells were added to the reporter cells for 6 hr, followed by infection with HIVBaL and GFP expression was measured 4 days post infection (Figure 3B). GFP expression was highest in the positive control samples containing only HIVBAL (6.57%, p < 0.01) or HIVBAL plus the unstimulated supernatant (6.35%). However, this was significantly reduced when stimulated supernatant was added (2.75%, p < 0.01). This inhibition was also observed when a HIVJRFL-GFP virus was used instead (Figure 3C). The suppressive effect of MAIT cell-derived supernatants was evident irrespective of whether supernatants were derived from stimulated whole CD8s (Figure 3C, left) or from pure sorted MAIT cells (Figure 3C, right) or of the target cells used in the assay. The inhibition by the stimulated supernatants was also clearly titratable (Figure 3—figure supplement 2). Thus, IL12/18 stimulated MAIT cells are able to inhibit HIV-1 infection in vitro across a range of different reporter systems.

Activated MAIT cells secrete effector molecules

Having observed that activated MAIT cells can exert an antiviral effect on HIVBAL, we next sought to determine which effector molecules were important for mediating this effect. IL-12/18-stimulated PBMCs from healthy donors showed increased expression of granzyme B (198-fold), IFN-γ(57-fold)and Caspase 3 (9-fold) in the MAIT cell population (CD8+ CD161++ Vα7.2+) (Figure 3—figure supplement 3). To first assess whether IFN-γ contributes to the anti-HIV-1 phenotype, Jurkat-tat-R5 cells were infected with HIVBAL in the presence of IFNγ blocking antibodies and levels of p24 was measured. However, there was no impact of the blocking antibodies on infection levels indicating that IFN-γ is not required for the inhibitory phenotype in our model system (Figure 3—figure supplement 4). Next, as we observed high GzmB-expression in MAITs from HIV-1-infected individuals (Figure 1) and in vitro upon stimulation (Figure 3—figure supplement 3) we sought to determine whether viral inhibition was dependent upon cell contact. However, CEM-GXR cells either co-cultured with MAIT cells or separated in transwell plates showed no suppression of HIV-1 (Figure 3—figure supplement 5) suggesting that direct cell contact is not necessary for the MAIT-derived inhibition of HIV-1 infection (Figure 3—source data 1).

Antiviral chemokines are secreted by MAIT cells in response to IL12/18 stimulation

Interestingly, IL-12/18-stimulated MAIT cells also upregulated production of the antiviral chemokine MIP-1β (CCL4) (Figure 3—figure supplement 3). This chemokine is a ligand for the HIV-1 entry receptor CCR5 and hence can restrict HIV-1 infection by blocking viral entry. Expression of CCL4 indicated a potential novel antiviral function for MAIT cells. To examine this further, sorted MAIT cells were stimulated with IL-12/18 overnight and supernatants were analyzed using an ELISA to measure CCL4 (Figure 4A) or a cytometric bead array (CBA) to measure CCL3 (MIP1α) and CCL5 (RANTES) (Figure 4B), the other two known ligands for the CCR5 receptor. Higher levels of all three chemokines were expressed by stimulated MAIT cells compared to the unstimulated controls (CCL4 p = 0.125, CCL3 p = 0.0156, CCL5 p = 0.0078), although these differences were not statistically significant for CCL4. To further verify these findings, we measured intracellular expression levels of these chemokines by FACS. CD8+ enriched T cells were incubated overnight with IL-12 and IL-18 and chemokine expression from the CD161++ Va7.2+ population was assessed (Figure 4C). Although only CCL4 was detectable by this method, the results showed a significant increase in CCL4 expression in IL-12/18 stimulated MAIT cells compared to the unstimulated controls (p = 0.0169) confirming data from the stimulated MAIT supernatants (Figure 4A and B). Further, gating on all CCL4+ cells within the total CD8+ T cells, revealed that CD161++ Vα7.2+ cells are highly enriched within the CCL4-producing subset (Figure 4D), accounting on average for 60% of this population. Hence, MAIT cells likely represent the major source of CCL4 in our model system.

Figure 4. MAIT cell derived antiviral restriction factors are essential for suppressing HIV-1 in vitro.

(A) MAIT cells were FACS-sorted and the CCL4 (MIP-1β) concentration was measured in the supernatants by ELISA after 20 hr post stimulation with IL-12/18. (B) MAIT cells were FACS-sorted and the concentrations of CCL3 (MIP1α) and CCL5 (RANTES) were measured in the supernatants by cytometric bead array (CBA) after 20 hr post stimulation with IL-12/18. (C) Representative FACS-plots (left) and bar plots (right) depicting the expression of CCL4 by MAITs after incubation with IL-12/18 for 20 hr. MAIT cells were identified as CD161++ Vα7.2+ cells within MACS-enriched CD8s. (D) Representative FACS dot plots (left) and bar plots (right) showing the percentage of MAIT cells as identified by co-expression of Vα7.2 with high levels of CD161 within MACS-enriched CD8s and within all CCL4-expressing CD8 T cells from the same culture. CD8 T cells were stimulated with IL-12/18 for 20 hr. (E) Recovery of GFP-positive CEM-GXR cells following blocking of restriction factors (CCL3/4/5), after treatment with IL12/18 stimulated supernatant from CD8 cells and infection with HIVBAL. *p < 0.05, **p < 0.05, paired t-tests. Data were pooled from two independent experiments; error bars indicate the standard deviation.

Figure 4—source data 1. MAIT-cell-derived antiviral restriction factors are essential for suppressing HIV-1.
CCL4 (MIP-1β) concentration was measured in the supernatants by ELISA after 20 hr post stimulation with IL-12/18. (B) Concentrations of CCL3 (MIP1α) and CCL5 (RANTES) were measured in the supernatants by cytometric bead array (CBA) after 20 hr post stimulation with IL-12/18. (C) Expression of CCL4 by MAITs after incubation with IL-12/18 for 20 hr. MAIT cells were identified as CD161++ Vα7.2+ cells within MACS-enriched CD8s. (D) Percentage of MAIT cells as identified by co-expression of Vα7.2 with high levels of CD161 within MACS-enriched CD8s and within all CCL4-expressing CD8 T cells from the same culture. CD8 T cells were stimulated with IL-12/18 for 20 hr. (E) GFP-positive CEM-GXR cells following blocking of restriction factors (CCL3/4/5), after treatment with IL12/18 stimulated supernatant from CD8 cells and infection with HIVBAL.

Figure 4.

Figure 4—figure supplement 1. TCR-induced expression of CXCL12/SDF-1 by activated MAIT cells.

Figure 4—figure supplement 1.

(A) Intracellular expression (gMFI) of CXCL12 is increased with addition of 5-OPRU and decreased with a blocking antibody against MR1 (α MR1). Data points are biological replicates. Bar plot shown as mean and standard deviation. *p < 0.05, **p < 0.01; two-tailed t-tests.
Figure 4—figure supplement 2. MAIT cells do not inhibit infection by a CXCR4 tropic virus.

Figure 4—figure supplement 2.

(A) Representative FACS plots showing infection of CEM-GXR cells with the CXCR4 tropic virus HIVLAI following pre-treatment with unstimulated or IL-12/18-stimulated MAIT cell supernatants. B. Bar graph showing HIVLAI infection of CEM-GXR cells after incubation with unstimulated or IL-12/18-stimulated MAIT cell supernatants. Data points are biological replicates. ns = not significant, paired two-tailed t-test.
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Subsequent profiling of the effector molecules produced by MAITs showed that they are also capable of producing CXCL12/SDF-1, the ligand for the alternative HIV-1 entry co-receptor, CXCR4. In contrast to CCL3, 4 and 5, CXCL12 induction required TCR signaling, as it was only observed in co-culture of MAIT cells with 5-OP-RU-loaded THP-1 cells (Figure 4—figure supplement 1). In accordance with these results, no anti-HIV-1 effect of IL-12/18-stimulated supernatants was observed when the CXCR4 tropic HIV-1LAI virus was used instead of HIVBAL to infect the target cells (Figure 4—figure supplement 2). Therefore, upon IL12/18 stimulation MAIT cells significantly increase expression of known HIV-1 restriction factors.

Antiviral chemokines mediate the inhibition of HIV-1 infection by MAIT cells

As MAIT cells were able to produce three CCR5 binding factors, we aimed to determine their role in the anti-HIV-1 effect of MAIT cells. Using our CEM-GXR-GFP reporter cell - HIVBAL system, we added blocking antibodies to CCL3/4/5 and measured levels of viral infection. Strikingly, the addition of these blocking antibodies neutralized the inhibitory effect of the IL12/18-stimulated supernatant and restored GFP expression to levels equivalent to the control condition (Figure 4E). Taken together these data indicate that the anti-viral activity of MAIT cells in relation to HIV-1 is mediated at least in part by expression of the chemokines CCL3, CCL4, and CCL5 (Figure 4—source data 1).

Discussion

The impact of HIV-1 on MAIT cell populations was initially noted by two groups in 2012, and depletion of MAIT cells in blood of HIV-1-infected individuals has been subsequently broadly confirmed with effects seen in acute, chronic and long-term treated infection, and reproduced here (Eberhard et al., 2016; Cosgrove et al., 2013; Spaan et al., 2016; Paquin-Proulx et al., 2017; Leeansyah et al., 2013). However, while such clinical studies have focused on the impact of HIV-1 on MAIT cells, none have described how MAIT cells can be activated by HIV-1 and most importantly a potential impact of MAIT cells on HIV-1 (Ussher et al., 2018). Given recent data indicating that viruses can activate MAIT cells efficiently in vivo (van Wilgenburg et al., 2016) and provide protection against lethal challenge in a mouse model (van Wilgenburg et al., 2018), we aimed to define whether MAIT cells can respond to and suppress HIV-1.

To address this, we first defined activation of MAIT cells in vivo. We observed strongly increased inhibitory receptor and granzyme B expression. Granzyme B-positive MAIT cells were detected during acute HIV-1 infection (PHI), up to 10-fold higher than HIV-1- controls. During chronic infection the majority of MAIT cells were granzyme B+, more than 90% for some individuals. These data are highly congruent with those seen during acute dengue and influenza infections (Loh et al., 2016; van Wilgenburg et al., 2016). This trend was similar when inhibitory receptors PD-1 and TIM-3 were examined. Higher levels of both inhibitory receptors were observed in CHI and to a lesser degree in PHI, and reduction of both receptors occurred following ART. This response to treatment is similar, although not identical to that seen in other virus infections. Residual MAIT cells in chronic HCV-infection showed an activated phenotype with high levels of granzyme B, HLA-DR, PD-1 and CD69 expression (Hengst et al., 2016) and granzyme B levels remain high after successful cure (van Wilgenburg et al., 2016). MAIT cells were not reinvigorated following HCV clearance and remained dysfunctional (Hengst et al., 2016). Sustained expression of granzyme B and PD-1 was also observed during tuberculosis (TB) and HIV-1/TB co-infection (Jiang et al., 2014; Saeidi et al., 2015). Blockade of PD-1 on MAIT cells during TB infection resulted in significantly higher IFNγ expression when stimulated with BCG. This is yet to be proven in HIV-1 infection and it remains to be seen whether immune checkpoint blockade can rescue MAIT cell function following chronic activation.

MAIT cells are known to migrate toward mucosal sites, recognise bacterial metabolites and contribute to barrier immunity (Voillet et al., 2018). One possible hypothesis for low MAIT cell frequencies in peripheral blood is that they may migrate into mucosal tissues following infection and immune activation. MAIT cell frequencies in rectal tissue of treated PHI donors were not found to be substantially different from HIV-1- controls (Figure 2F). In this study, higher percentages were found within the rectum of HIV-1+ individuals, compared to terminal ileum. In pigtail macaques, MAIT cells exhibited lower expression of the gut homing marker α4β7 and were not enriched in the gut prior to SIV infection. Following infection, they upregulated α4β7 and their frequencies increased within the rectum (Juno et al., 2019b). The authors postulated that this may prevent depletion of MAIT cells during chronic infection. Here, a positive correlation was observed when blood and tissue MAIT cells were compared, although MAIT cell percentages were lower overall in blood. This suggests that there is a general reduction of MAIT cells in both compartments. If the impact of HIV-1 on MAIT cells was primarily redistribution to gut, then an inverse relationship between frequencies at the two sites would be expected.

To elucidate whether HIV-1 can activate MAIT cells, an R5 tropic lab strain virus (HIVBAL) was used to stimulate THP1 monocytes co-cultured with CD8+ T cells. IFN-γ expression in MAIT cells was observed when HIV-1BAL was used to stimulate THP-1 antigen presenting cells but had no anti-HIV-1 effect. The THP-1 monocytic cell line was used as it has been extensively trialled by our group and many others as an effective APC for MAIT cells in microbial and viral infections (Ussher et al., 2018; van Wilgenburg et al., 2018). MAIT cell activation was dependent on IL-12 and IL-18 produced by THP1 cells via innate sensing (van Wilgenburg et al., 2016; ). THP1 sensing of HIV-1BAL may be through TLR7/8 or cytosolic RIG-like receptors (Diget et al., 2013; Guo et al., 2014), which in turn activate THP-1 cells to secrete inflammatory cytokines such as IL-12 and IL-18 (Bandera et al., 2018). Inactivated virus was incapable of activating THP-1 cells. This could be due to the cross-linking of nucleocapsid P7 protein by aldrithiol-2, which may not allow single stranded HIV-1 RNA within virions to bind to TLR7/8 (Rossio et al., 1998). We note that inactivation of influenza and HCV also impacted on MAIT cell recognition in vitro (van Wilgenburg et al., 2018), even in a macrophage culture where true infection and replication does not occur.

IFN-γ production dependent on IL-12 and IL-18 has also been observed in other infection models (Okamura et al., 1998), as well as dengue virus infection (Fagundes et al., 2011). It must be noted that stimulation of MAIT cells by IL-12 and IL-18 not only activates the cells independent of TCR signalling to produce pro-inflammatory cytokines and cytotoxic granules, but it also increases caspase-3 expression, a pro-apoptotic marker. It has been shown that IL-12 can induce apoptosis of CD8+ T cells in the absence of antigenic stimulation (Fan et al., 2002) and it has also been observed that MAIT cells show PLZF-dependent enhanced capacity for apoptotic death following stimulation (Gérart et al., 2013). Overall HIV-1-induced depletion of MAITs could very likely be due to activation induced apoptosis, as observed in other disease settings (Wakao et al., 2017; Hinks, 2016).

HIV-1 restriction factors CCL3 (MIP-1α), CCL4 (MIP-1β), and CCL5 (RANTES) were detected in supernatants of purified MAIT cells stimulated in vitro. This fits with recent data exploring the broad activity of MAIT cells following diverse stimuli in mice and humans (Hinks et al., 2019; Leng et al., 2019; Leng et al., 2019). Such chemokines may play a role in cellular chemoattraction following infection consistent with the early recruitment role for MAIT cells observed in infectious models (Meierovics et al., 2013; van Wilgenburg et al., 2018). Stimulated supernatants containing these restriction factors were able to inhibit HIV-1 infection of target cells in a specific manner, and the inhibition observed was not dependent on cell contact. Loss of viral inhibition was observed when stimulated supernatants were treated with restriction factor-blocking antibodies before addition of target cells. Collectively, this data suggests that MAIT cells can be activated by HIV-1 via a TCR-independent pathway. IL-12 and IL-18 are necessary for MAIT cell activation and secretion of restriction factors in vitro, and activation induced cell death may explain declining numbers of MAIT cells during HIV-1 infection.

Both MAIT cells and THP-1 cells can produce a broad range of factors following stimulation as has been recently described (Hinks et al., 2019; Lamichhane et al., 2020; Leng et al., 2019). However, even though factors such as IFNγ could potentially possess anti-HIV-1 activity, as reported for iNKT cells (Paquin-Proulx et al., 2016), we were able to block the suppressive activity very effectively with anti-chemokine antibodies, suggesting that at least in our models, these are the most potent effectors. There has been no significant difference in HIV-1 specific IFNγ response reported in both progressors and long-term non-progressor patients with chronic HIV-1 infection (Roff et al., 2014; Zanussi et al., 1996). This is consistent with original in vitro studies which revealed no antiviral effect of IFNγ and even enhancement of infection in primary cells (Yamamoto et al., 1986; Mackewicz et al., 1994) and several clinical trials that revealed no impact of this cytokine in vivo (Roff et al., 2014).

These data provide novel evidence that MAIT cells can respond to HIV-1 and that part of this activation includes an antiviral function. It is not known whether activation of MAIT cells in a TCR-independent manner can lead to cell killing as this will likely depend on the nature of the interaction, including a potential role for cell surface interactions analogous to those involved in NK cell activation. In the GI tract it is also possible that high local levels of the MAIT cell ligand (5 OP-RU) could pre-sensitise MAIT cells via the TCR to enhance functionality and responsiveness to cytokines, increasing the levels of antiviral chemokines. Even in the absence of killing it is clearly evident that CCR5 levels play an important role in the long-term outcome of HIV-1 infection (McLaren et al., 2015) and thus this effect could certainly contribute to control of HIV-1 levels in vivo. More intriguingly, since MAIT cells are present in relevant tissues in persistent infection, including under therapy, it will be important to consider their role in strategies for cure.

Overall, these data, taken with other findings, suggest that the strong activation of MAIT cells by HIV-1 in natural infection can be accompanied by relevant antiviral functions. The recent data showing that MAIT cells can protect against lethal viral infection in mice (where they exist in much lower frequencies) provides some proof-of-principle that such activity can play an important role in vivo, in conjunction with other cell types. In the absence of a small animal model for HIV-1 where MAIT cells can be depleted, this sort of proof will be hard to reproduce for this infection, but further evidence should be sought to support this hypothesis.

Materials and methods

Participant samples

Participants with Primary HIV-1 (PHI) were recruited as either part of the HEATHER (HIV-1 Reservoir targeting with Early Antiretroviral Therapy) cohort or from the SPARTAC (Short Pulse Antiretroviral Therapy at HIV-1 Seroconversion) trial. For inclusion in the HEATHER cohort, participants with identified PHI commenced ART within 3 months of diagnosis, and did not have co-infection with Hepatitis B or C. For our study, cryopreserved PBMCs were used from the closest pre-therapy sample to seroconversion (baseline) and from a sample 9–15 months after commencement of ART (1 year). Only Baseline samples were used from the SPARTAC trial, which was a multi-centre, randomised controlled trial of short course ART during PHI, the full design of which is described elsewhere (Fidler et al., 2013).

Participants with Chronic HIV-1 (CHI) were recruited in Bloemfontein, located within the Mangaung Metropolitan Municipality in the Free State province of South Africa. Most participants had advanced HIV-1 disease progression (as reflected by a CD4 T cell count <350 cells/µL). All participants were tested for HIV-1 using a point-of- care ‘HIV-1 rapid test’ or laboratory-based HIV-1 ELISA. Follow-up samples were collected at 6- and 12 months post-ART initiation.

Long-term non-progressor (LTNP) samples were collected at various sites across New South Wales, Australia; samples were processed and stored at St. Vincent’s Centre of Applied Medical Research, Darlinghurst. Eligible subjects were HIV-1+, asymptomatic, diagnosed at least 8 years prior to enrolment, treatment naïve, and had an absolute CD4+ T cell count ≥500 cells/µL. Elite controllers (EC) had an undetectable viral load (median <1.7 Log) whilst viraemic controllers (VC) had a detectable viral load (median <5.8 Log) (Table 1).

Table 1. Participant cohort characteristics.

CD4 T cell count and HIV-1 (log) viral load.

Patient cohort Sample Number CD4+ T cells (Count/μL) median (IQR) Plasma viral load (log10 copies/mL) median (IQR)
Healthy Donors 12 - -
PHI (SPARTAC) 8 596 (437–755) 5.04 (4.51–5.45)
PHI (HEATHER) 12 524 (437–656) 4.34 (3.19–4.88)
HEATHER GUT 11 - < 1.3
CHI 12 360 (72–646) 4.90 (3.55–5.59)
EC LTNP 9 780 (615–1013) 1.70 (1.60–1.70)
VC LTNP 10 633 (442–800) 5.18 (5.03–5.37)
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Processing of tissue samples

Rectal and terminal ileum biopsies (up to 12 from each site) were collected at endoscopy and immediately placed in complete media [RPMI-1640 media with 5% heat-inactivated fetal bovine serum (FBS), 0.04 mg/mL gentamicin, 100 IU/mL penicillin, 0.1 mg/mL streptomycin and 2 mM L-glutamine] and processed within 3 hours of sampling. Briefly, samples were washed in 1 mM dithiothreitol (DTT) solution and then with PGA solution (Hanks’ Balanced Salt Solution with 0.04 mg/mL gentamicin, 100 IU/mL penicillin and 0.1 mg/mL streptomycin). Biopsy samples subsequently underwent collagenase and mechanical digestion using Collagenase D (1 mg/mL) for 30 min and a gentle MACS dissociator (Miltenyi Biotec), respectively. The resulting cell suspension was then strained using a 70 µM filter, washed with a penicillin/streptomycin, glutamine and amphotericin (PGA) solution containing 500 ml Hank’s Balanced Salt Solution (HBSS) without Ca2+ and Mg2+, 5 ml penicillin (100 IU/mL) /streptomycin (0.1 mg/mL), 2 ml Gentamicin, (10 mg/mL) Amphotericin B (50 uL). The washed cells were then used for staining.

Flow cytometry

Frozen PBMC were thawed using R10 medium (RPMI + L-glutamine+ Penicillin Streptomycin + 10% FCS) and subsequently stained with antibodies corresponding to either the chemokine/cytokine receptor, cytotoxic, or transcription factor panels (see below). A FoxP3 permeabilization kit (BD Pharmingen) was used for intracellular/intranuclear staining. Staining of the chemokine panel was carried out at 37 °C. Samples were acquired on an LSRII flow cytometer (BD Biosciences) using the FACSDiva software package (BD Biosciences). Prior to each run, all samples were fixed in 2% PFA. Samples were then analyzed using the Flowjo software package (FlowJo, LLC). Gating strategies were developed based on florescence-minus-one (FMO) controls.

Base panel

Live/Dead dye (Invitrogen), CD4 (RPA-T4, BD Biosciences), CD3 (UCHT1), CD8 (5K1), Vα7.2 (3C10), and CD161 (191B8) [all Biolegend].

Activation/inhibitory receptor panel

GzmB (GB11), PD-1 (EH12.1) and TIM-3(7D3) all Biolegend.

CD8 MACS enrichment

CD8 T cells where enriched from whole PBMCs using a CD8 microbead-positive selection kit (Miltenyi) according to the manufacturer’s instructions. Briefly, whole PBMCs were isolated, washed in MACS buffer and incubated with CD8 microbeads for 15 min on ice. Cells were washed again and run over magnetic LS selection columns placed in a MACS magnet. After three rinsing steps, the columns were removed from the magnet and the enriched CD8 T cells were eluted from the columns. Enriched CD8 T cells were washed, counted and subjected to downstream experiments. A fraction of the enriched CD8 T cells were stained with Live/dead dye and CD8 antibodies to determine purity by FACS staining. On average enriched CD8 T cells had a viability >95% and a purity >96%.

MAIT cell sorting

To obtain pure MAIT cells for functional analyses, MACS-enriched CD8s were isolated and stained for FACS-sorting with the following antibodies and dyes: CCR6 (Biolegend, G034E3), CD161 (Miltentyi, 191B8), CD8 (Biolegend, SK1) and near-infrared Live/Dead dye (Invitrogen). To avoid TCR-triggering by antibodies, no antibodies against Vα7.2 and CD3 were included in the sorting panel and MAIT cells were instead identified by surrogate markers as CD161hi CCR6+ cells. Sorting was performed on a MA-900 sorter (SONY) using a 100 µm sorting chip. To validate the sorting strategy and to determine the purity of the cells a small fraction of each sorted sample was stained with antibodies against CD3 and Vα7.2 as well as Live/Dead dye. Viability of sorted MAIT cells was on average >93% and > 98% of the sorted cells stained positive for Vα7.2. Samples, which contained considerable populations of dead ( > 15%) or Va7.2 negative ( > 10%) cells were excluded from downstream experiments.

Cell lines

THP1 (ATCC), CEM-GXR (NIH AIDS Reagent Program), CEM-RR5 and Jurkat-Tat-R5 (both kindly provided by Quentin Sattentau) were maintained in RPMI 1640 media containing 10% FCS, L-glutamine and penicillin/streptomycin (Sigma-Aldrich). Cells were cultured every 3–4 days and incubated at 37 °C and 5% CO2. All cell lines used were mycoplasma negative.

In vitro stimulations of MAIT cells

For the TCR-specific triggering of MAIT cells, PBMC-derived CD8 T cells were enriched over MACS columns and 2 × 105 cells were co-cultured with 1 × 105 THP1 (ATCC) cells which had been pulsed with 10 nM 5-OP-RU for 2 hr. Unpulsed THP1s were used as controls. CD8s and THP1s were cultured together for three or 6 days and the expression of CXCL12 in MAIT cells was assessed by flow cytometry. To ensure that the observed effects were TCR-mediated, an anti-MR1 blocking antibody (5 µg/mL, clone 26.5, Biolegend) was added to control wells. For cytokine-induced MAIT cell activation, 2 × 105 MACS-enriched CD8s or FACS-sorted MAIT cells were stimulated overnight with IL-12 and IL-18 (both at 50 ng/mL). The supernatants of these cultures were harvested and stored at –80 °C before being used to treat HIV-1-BAL infected cells or to assess the expression of viral restriction factors. Supernatant from unstimulated cultures was used as controls.

Intracellular cytokine staining assay

2 × 105 MACS-enriched CD8 cells were incubated overnight with or without IL-12 and IL –18 (both at 50 ng/mL) at 37 °C. For the last 4 hr of incubation, Brefeldin A and monensin (Biolegend) were added to block the release of cytokines. Cells were harvested and permeabilized with Cytofix/perm (Becton Dickinson) and stained with intracellular antibodies to base panel as described above, and IFNỿ (B27, Biolegend), CCL4 (MIP-ẞ, FL3423L. BD or REA511, Miltentyi), Caspase-3 (C92-605, BD), CXCL12/SDF-1 (79018, R&Dsystems). MAIT cells were identified as CD161HIV-1α7.2+ cells.

Cytometric bead array and ELISA

A total of 2 × 105 FACS-sorted MAIT cells were incubated overnight with or without IL-12 and IL –18 (both at 50 ng/mL) at 37 °C. Cells were pelleted by centrifugation and the supernatants of the cultures was harvested to assess MAIT-specific production of CCL3,4 and 5. CCL3 and 5 production was analyzed by a Cytometric bead array (BD) following the manufacturer’s instructions. Briefly, 50 µl of undiluted supernatant was iteratively mixed and incubated with beads and detection reagents specific for CCL3 or CCL5 and the resulting fluorescent signals were recorded on a LSR II flow cytometer (BD). Protein concentrations were calculated from these fluorescent values based on standard curves generated in each experiment from serially diluted CCL3 and CCL5-stocks with defined concentrations. The concentration of CCL4 in each supernatant was determined using a DuoSet ELISA kit (R&D) following the manufacturer’s instructions. The supernatant was serially diluted and a one in two dilution was used for the final analysis. The concentration was determined from a standard curve which was run in parallel.

In vitro virus experiments

The THP1 monocytic cell line was incubated with the HIV-1 lab strain HIVBAL or aldrithiol (AT-2) inactivated HIVJRFL virus at an MOI of 0.2 for 6 hr at 37 °C. Positively bead (Thermo Scientific) selected CD8+ T cells were then added to the culture overnight at a ratio of 1:1 with THP1 cells. Anti-IL-12 and anti-IL-18 blocking antibodies (R&D) were used at 10 µg/mL and added to the culture prior to the addition of CD8+ cells. Formaldehyde-treated TOP10 E. coli cells (Thermofisher) were added to THP1 cells as a positive control. IFNỿ (B27, Biolegend) expression was then measured on an LSRII cytometer (BD biosciences).

HIV-1 inhibition assay- CEM-GXR cells, which express GFP under the control of the HIV-1 promoter, CEM-CCR5 cells or Jurkat-Tat-R5 cells were used for the inhibition assays as they express endogenous levels of CXCR4 and have been engineered to overexpress CCR5. Cells were incubated with unstimulated, or IL-12/18 stimulated supernatant for 6 hr at 37 °C. HIV-1BAL, HIVJRFL-GFP or HIVLAI was added to the culture at an MOI of 0.2. Where indicated, anti-CCL3/4/5 were added at a concentration of 10 µg/mL and anti-IFNγ (clone B27, Biolegend) at a concentration of 50 µg/mL. Expression of GFP or p24 (KC57, Beckman Coulter) was measured 4 days post infection by FACS.

Healthy donor PBMCs were incubated with unstimulated or IL-12/18-stimulated supernatant for 6 hr at 37 °C and HIVJRFL-GFP primary viral isolate was added to the culture. GFP expression was analyzed at day 7 on a BD LSRII.

Transwell - For cell contact experiments, MAIT cells were either co-cultured with CEM-GXR cells or separated using Transwell plates (Corning). HIVBAL was then added and GFP was measured at day 4.

Statistical analysis

Ex vivo data from six or more healthy donors and HIV-1+ donors (PBMC and Tissue), and four or more from in vitro stimulated samples were used for statistical calculations. All column graphs are presented as medians with inter-quartile ranges. Wilcoxon paired t test was used to analyze statistical data employing Prism 7.0 (GraphicPad, La Jolla, CA, USA) software. For unpaired samples the Mann-Whitney U test was used. p-Values < 0.05 were considered significant (* < 0.05, ** < 0.01, *** < 0.001, and **** < 0.0001).

Acknowledgements

We thank Dr Vera Klemm for proofreading our manuscript. PK and CP are supported by the Wellcome Trust (WT109965MA), NIHR Senior Fellowship (PK), and the NIHR Biomedical Research Centre, Oxford. JM and DF are supported by the ARC (CE140100011) and NHMRC (1117017). We thank all healthy donors and LTNP participants. We thank Jeff Lifson for the AT-2 inactivated virus stocks. We thank the participants of SPARTAC and HEATHER. The HEATHER study is conducted as part of the CHERUB (Collaborative HIV-1 Eradication of Reservoirs): (UK BRC) collaboration. (CHERUB Steering Committee): Andrew Lever (University of Cambridge), Mark Wills (University of Cambridge), Jonathan Weber (Imperial College, London), Sarah Fidler (Imperial College, London), John Frater (University of Oxford), Lucy Dorrell (University of Oxford), Mike Malim (King’s College, London), Julie Fox (King’s College London), Ravi Gupta (University College London), Clare Jolly (University College London).

Funding Statement

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Contributor Information

Chansavath Phetsouphanh, Email: c.phetsouphanh@gmail.com.

Paul Klenerman, Email: paul.klenerman@ndm.ox.ac.uk.

Stipan Jonjic, University of Rijeka, Croatia.

Tadatsugu Taniguchi, Institute of Industrial Science, The University of Tokyo, Japan.

Funding Information

This paper was supported by the following grant:

  • Wellcome Trust WT109965MA to Paul Klenerman.

Additional information

Competing interests

No competing interests declared.

No competing interests declared.

Author contributions

Conceptualization, Data curation, Funding acquisition, Investigation, Methodology, Project administration, Validation, Writing – original draft, Writing – review and editing.

Data curation, Investigation, Methodology, Writing – review and editing.

Data curation, Investigation, Methodology, Writing – review and editing.

Methodology, Resources.

Methodology, Resources.

Methodology, Resources.

Methodology, Resources.

Methodology, Resources.

Methodology, Resources.

Methodology.

Methodology, Resources.

Methodology, Resources.

Methodology, Resources.

Methodology, Resources, Writing – review and editing.

Methodology, Resources.

Methodology, Resources, Writing – review and editing.

Conceptualization, Supervision.

Conceptualization, Resources, Supervision, Writing – original draft, Writing – review and editing.

Ethics

Clinical trial registration 2004-000446-20.

Human subjects: The SPARTAC trial (EudraCT Number: 2004-000446-20) was approved by the following authorities: the Medicines and Healthcare products Regulatory Agency (UK), the Ministry of Health (Brazil), the Irish Medicines Board (Ireland), the Medicines Control Council (South Africa) and the Uganda National Council for Science and Technology (Uganda). It was also approved by the following ethics committees in the participating countries: the Central London Research Ethics Committee (UK), Hospital Universitário Clementino Fraga Filho Ethics in Research Committee (Brazil), the Clinical Research and Ethics Committee of Hospital Clinic in the province of Barcelona (Spain), the Adelaide and Meath Hospital Research Ethics Committee (Ireland), the University of Witwatersrand Human Research Ethics Committee, the University of Kwazulu-Natal Research Ethics Committee and the University of Cape Town Research Ethics Committee (South Africa), Uganda Virus Research Institute Science and ethics committee (Uganda), the Prince Charles Hospital Human Research Ethics Committee and St Vincent's Hospital Human Research Ethics Committee (Australia) and the National Institute for Infectious Diseases Lazzaro Spallanzani, Institute Hospital and the Medical Research Ethics Committee, and the ethical committee of the Central Foundation of San Raffaele, MonteTabor (Italy). Recruitment for and studies within the HEATHER cohort were approved by the West Midlands-South Birmingham Research Ethics Committee (reference 14/WM/1104). Recruitment of CHI participants were approved by The University of the Free State Ethics Committee (ETOVS 171/08). LTNPs recruitment was approved by The St Vincent's Human Research Ethics Committee (EC00140) approval number: HREC/12/SVH/298, SVH 12/217. PBMCs obtained from healthy donors were approved by the Sheffield Research Ethics Committee (reference 16/YH/0247). Participants were aged 18 years or older, all participants from each of the above mentioned cohorts gave informed and written consent for their participation in these studies.

Additional files

Transparent reporting form

Data availability

Raw data from main figures are provided as source data files.

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Decision letter

Editor: Stipan Jonjic1

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Decision letter after peer review:

Thank you for sending your article entitled "Human MAIT cells respond to and suppress HIV" for peer review at eLife. Your article is being evaluated by 3 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation is being overseen by Tadatsugu Taniguchi as the Senior Editor.

Summary:

The study by Phetsouphanh et al. deals with the role of MAIT cells in HIV infection. As shown previously, the authors found that MAIT cells are activated/exhausted in primary and chronic HIV infection, but, at the same time, their numbers are dramatically reduced. in vitro stimulation of MAIT cells with HIV infected THP-1 monocytes was used to characterize the mechanism of MAIT cell activation. Infected cells secrete IL-12 and IL-18 which then stimulate MAIT cells for production of effector molecules which inhibit HIV. Namely, MAIT cell secretion of chemokines is sufficient to inhibit HIV infection of a CCR5-overexpressing CEM cell line. In general, the study is relevant and interesting. However, too many open issues need serious additional work. Having in mind eLife's policy not to accept the papers that require substantial interventions, we need the authors to let us know a realistic expectation that requested experiments will be completed in 2 months. If the authors find unrealistic that they can revise the paper within two months, we advise the authors to resubmit the manuscript after finishing all the requested experiments. Please note that in that case the resubmitted manuscript will be considered as a new submission.

Essential revisions:

1. It is well established through multiple studies that MAIT cells (defined by surface marker expression) are depleted during HIV infection and poorly reconstituted during ART. Additionally, changes in GzmB and Tim-3 expression, as well as general MAIT cell activation, during HIV infection have previously been reported (Leeansyah 2013, Leeansyah 2015, and others). It is surprising that the current study does not take advantage of MR1 tetramers to identify the MAIT cell population, as this would present the opportunity to characterize the MAIT population in HIV-infected cohorts in a manner which would be more novel. The data on MAIT cells in the GALT would be stronger if representative staining were shown, but would also benefit immensely from use of the MR1 tetramer to characterize GALT MAIT cells, as this has not previously been done.

2. Infection of THP-1 monocytes in vitro was used to show that HIV can stimulate MAIT cells via IL-12 and IL-18. The use of THP-1 as the target cells for HIV-1 infections was somewhat intriguing since THP-1 cells usually don't support significant productive HIV-1 infection. With this in mind, it would be better to use cells that support productive HIV-1 BaL infection eg. monocyte-derived macrophages (MDM). It remained unclear are IL-12 and IL-18 produced by infected or bystander THP-1 monocytes? Can inactivated HIV induce IL-12 and IL-18? IL-12/18 produced by THP-1 cells themselves might also have some antiviral effects through various MAIT cell-independent mechanism. Have the authors ruled out this potential effect? Are the levels of IL-12 and IL-18 elevated in HIV+ patients in blood and in tissues?

3. The data shown in Figure 3 does clearly demonstrate that exposure of THP-1 cells to HIV results in IL-12/IL-18-driven MAIT cell activation in vitro. It is important to note, however, that this assay recapitulates only a very specific aspect of HIV infection, and does not include any assessment of MAIT cell activation by HIV-infected CD4+ T cells or other mechanisms such as microbial translocation. It is surprising that MAIT cell production of IFNγ was not attributed any anti-viral role, considering that similar experiments with iNKT cells have shown that iNKT-derived IFNγ limits HIV infection of primary T cells in vitro (Vasan 2007). Considering recently published data indicating the CCL3 and CCL4 production by MAIT cells is significantly greater following TCR-mediated stimulation than cytokine stimulation (Lamichhane 2019), and the impact that microbial translocation could have on MAIT cell activation, it is unclear what the relative contribution of cytokine-based activation would be on MAIT cells in vivo during HIV infection.

4. Did the authors also see contact-dependent MAIT cell-mediated inhibition of HIV replication? Similarly, would TCR-activated MAIT cells mediate antiviral activity through CCL3-5 production and/or contact-dependent mechanisms?

5. Technically, there is no indication of the use of uninfected THP-1 cultures as a control in Figure 3A or B, and it is unclear as to whether the data presented in Figure 3B is background subtracted. Sorting on CD8+ T cells prior to culture with the THP-1 cells indicates that only CD8+ MAIT cells were studied in this experiment, which is a caveat that should be noted, as MAIT cells can include a substantial CD4-CD8- population as well.

6. The assay in Figure 4 using the CEM-GXR cells is likely biased to detect an impact of β-chemokines on HIV entry, given the cell line overexpresses CCR5. However, it would be important to confirm MAIT cell-mediated anti-HIV activity using primary human immune cells that productively support HIV-1 BaL replication, such as MDM and/or PHA/IL-2-activated PBMCs, plus measurements of productive viral replication (eg. p24, RT activity, etc).

7. Have the authors tested other R5 strains apart from BaL? It would be important to see that this antiviral effect is not just restricted to the BaL strain of R5 HIV-1. Along this line, there were only n=4 of presumably independent MAIT cell donors in Figure 4B (or was it 4 different CEM-GXR expts but using a single or pooled MAIT cell donors?). It would be good to increase the sample size to strengthen the manuscript. Similarly, have the authors tested X4 strains, lab-adapted or primary isolates? It would be an important control and interesting to see if MAIT cells can also/cannot inhibit X4 viruses. Along this line, did MAIT cells produce CXCR4-binding chemokines (eg. CXCL12/SDF-1)?

8. The authors show that IL-12 + IL-18 stimulation induces apoptosis in MAIT cells. While this is interesting observation, it does not explain why there is no increase in MAIT cell numbers following years of ART treatment. One can hypothesize that due to lower HIV load there will be less of IL-12 and IL-18, and subsequently less of MAIT cell stimulation. This is the case with the expression of GzmB, PD-1 and Tim-3. However, the numbers of MAIT cells remain the same. This discrepancy should be commented.

9. The methods section lacks important details for several experiments. The E. coli stimulations do not appear to be described at all. There is no indication of the number or ratio of T cells used in the THP-1 co-culture experiments.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Thank you for submitting your article "Human MAIT cells respond to and suppress HIV" for consideration by eLife. Your article has been reviewed by 2 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Tadatsugu Taniguchi as the Senior Editor. The reviewers have opted to remain anonymous.

Although several of the requested data have been added, thus improving the quality of the manuscript, the reviewers pointed out that major concerns raised were not fully addressed to warrant sufficient novelty for publication in eLife. Additionally, on several occasions in response to requests from reviewers the authors stated what they could do, instead of dealing with the specific requests raised by reviewers. Since we still believe that your data showing that MAIT cells can inhibit HIV infection via secretion of R5 chemokines is worth considering for eLife, we decided to offer you another chance to fully deal with all reviewer's concerns, providing that you can assure us that all requested experiments will be conducted and re-revised manuscript submitted in frame of 2 months.

Below please find the summary of major concerns raised by reviewers.

1) The data in Figures 1 and 2 remain to be only confirmatory of multiple previously published studies. Without the data on GALT cells studied with MR1 tetramer, this portion of the manuscript does not add new information to the field. The authors argument that "this study commenced prior to these tetramers becoming available for staining, so for consistency we pursued this throughout" is somewhat in contradiction with their sentence stating that they have recently published the paper using MR1 tetramers to demonstrate MAIT cells in a GI tract. Some attempt at providing a confirmation of data should be done.

2) The response to reviewer's suggestion to assess the cells that support productive HIV-1 infection to stimulate MAIT cells instead of THP-1 cells, was also not clear. The authors state: "We can clarify the background to this experiment further" or "We can however readily test further whether combinations of monocyte-derived macrophages and BAL also work to activate MAIT cells in vitro, as previously tested for the other viruses …". Still, it remained unclear whether the authors performed the requested experiment and if so, it should be included in the manuscript. Since inactivated HIV did not result in THP-1 activation of MAIT cells, the question is what mechanism is behind this finding. This was not further discussed in the manuscript.

3) Potential anti-viral role of IFNγ was not explored. The authors state: "Regarding the IFNγ, we can certainly test this using blockade as we have done in other settings (eg HCV in vitro and influenza in vivo)."; Again, it is unclear whether or not these experiments have been performed. Although we agree that it is unlikely that IFNγ would result in a block of viral entry, it would still be relevant to test the impact of other MAIT cell-secreted cytokines and chemokines on viral replication, as the presence of MAIT cells at mucosal surfaces is clearly insufficient to block HIV infection. If the authors believe that mechanisms are the same in case of other viruses, we do not see the point of this study.

4) Control experiments using X4 viruses will further strengthen the manuscript, given the inhibition by MAIT cells is acting on R5 viruses (and thus no inhibition should be seen on X4 viruses).

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Thank you for submitting your revised manuscript '"Human MAIT cells respond to and suppress HIV' to eLife. Your manuscript was evaluated by three reviewers and the evaluation has been overseen by a Reviewing Editor and a Senior Editor. Although the two original reviewers were satisfied with the new results built into the revised manuscript, a third reviewer pointed to several weaknesses of the study.

The main objective of this reviewer is that the data provided so far do not fully support the conclusions made by the authors. Specifically, the reviewer emphasized the experiments involving stimulation of MAIT cells – under the experimental setting described only a portion of these cells belong to MAIT, while the vast majority are classical cytotoxic T lymphocytes. According to the reviewer, this fact calls into question the conclusions about the specific role of MAIT cells in HIV infection. The reviewer, therefore, requests the experiment on sorted MAIT cells instead of total CD8 T cells to exclude the possibility that CCL3/4/5 chemokines are produced by non-MAIT CD8 cells in response to IL-12/18.

Since during the consultations between the reviewers a unanimous opinion was not reached, the senior editor and the reviewing editor concluded that the above-specified request by the third reviewer was not entirely unfounded. In other words, although we are in favor of the final acceptance of the manuscript for publication in eLife, we request the authors to provide evidence that stimulated IL-12/18 sorted MAIT cells secrete CCL3/4/5 chemokines and demonstrate their function in functional assays.

Apart from above, one of the reviewers requested following corrections: Figure 3—figure supplement 3 – The units for CCL3-5 are in pg/mL whereas for CXCL12 is in gMFI. The authors should paraphrase the related sentence as these two units cannot be directly compared to each other. Otherwise, show the CXCL12 unit in pg/mL also.

Reviewer #2:

The authors have addressed my concerns.

Reviewer #3:

The authors have responded to the previous reviewer concerns and substantially improved the manuscript.

Reviewer 4:

– Experiments done in one figure should support the findings in the other figures. Now all 4 figures are completely separate.

– If you want to draw conclusions about the specific effects of MAIT cells, you need to purify MAIT cells and not total CD8 T cells. i.e. FACS sort CD161+ Va7.2+ cells

– Why study ART at all if you are not going to se this data on a functional level? Remove the data or include it in functional studies.

– Why study PD1 and TIM3 at all if you are not going to look at these markers in the rest of the study? Remove the data or include it in functional studies.

– Why not investigate CCL3/4/5 production by MAIT cells?

eLife. 2021 Dec 24;10:e50324. doi: 10.7554/eLife.50324.sa2

Author response

Essential revisions:

1. It is well established through multiple studies that MAIT cells (defined by surface marker expression) are depleted during HIV infection and poorly reconstituted during ART. Additionally, changes in GzmB and Tim-3 expression, as well as general MAIT cell activation, during HIV infection have previously been reported (Leeansyah 2013, Leeansyah 2015, and others). It is surprising that the current study does not take advantage of MR1 tetramers to identify the MAIT cell population, as this would present the opportunity to characterize the MAIT population in HIV-infected cohorts in a manner which would be more novel. The data on MAIT cells in the GALT would be stronger if representative staining were shown, but would also benefit immensely from use of the MR1 tetramer to characterize GALT MAIT cells, as this has not previously been done.

We agree that usage of MR1 tetramers would be novel in identifying MAIT cells in GI derived tissue. However, this study commenced prior to these tetramers becoming available for staining, so for consistency we pursued this throughout. We have however recently published an example in the GI tract (Leng et al. Cell Reports, Sept 2019; Supplementary Figure 3C). We have also shown that tetramer positive MAIT cells maintain the CD161++Va7.2+ phenotype in the GALT (please see Author response image 1).

Author response image 1. Double Tetramer positive MAIT cells are CD161++Va7.

Author response image 1.

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2+.

2. Infection of THP-1 monocytes in vitro was used to show that HIV can stimulate MAIT cells via IL-12 and IL-18. The use of THP-1 as the target cells for HIV-1 infections was somewhat intriguing since THP-1 cells usually don't support significant productive HIV-1 infection. With this in mind, it would be better to use cells that support productive HIV-1 BaL infection eg. monocyte-derived macrophages (MDM). It remained unclear are IL-12 and IL-18 produced by infected or bystander THP-1 monocytes? Can inactivated HIV induce IL-12 and IL-18? IL-12/18 produced by THP-1 cells themselves might also have some antiviral effects through various MAIT cell-independent mechanism. Have the authors ruled out this potential effect? Are the levels of IL-12 and IL-18 elevated in HIV+ patients in blood and in tissues?

We used THP1 cells as they are a robust surrogate for monocyte/macrophages and where we have found they respond well to viruses (and bacteria) to drive MAIT cell activation (Van Wilgenburg et al., Nature Comms 2016). We can clarify the background to this experiment further. We turned to this approach, which has been used by many labs now in the MAIT cell field, as the simpler experiment of using ex vivo PBMCs had failed to activate MAIT cells (Ussher et al. Blood 2012). We can however readily test further whether combinations of monocyte-derived macrophages and BAL also work to activate MAIT cells in vitro, as previously tested for the other viruses.

IL-12 and IL-18 production by THP-1 cells is most likely due to activation of these cells by HIV through innate sensing. We have encountered this in previous studies (eg Van Wilgenburg et al., above), where influenza drives MAIT cell activation by the same route, but influenza does not replicate in the cells used. A similar finding using a replication deficient adenovirus in monocytes is also seen and the mechanism has been further explored (Provine et al., BioRxiv 2019). These papers have been referenced (line 290) in order to explain this – overall it seems very plausible that such presenting cells can drive activation even if a full viral life cycle is not completed.

Inactivated HIV did not activate THp1 cells that in-turn did not stimulate MAIT cells to produce IFNγ (Figure 3—figure supplement 1).

This manuscript is focused on the role of IL-12/18 on inducing MAIT cells’ anti-viral function. We show that was no viral inhibition following blockade of CCL3/4/5 with antibodies. IL-12/18 should be in the supernatant, but does not seem to have antiviral effects in this setting.

There are studies where these inflammatory cytokines (and others which may impact on MAIT cell function that have been described) have been studied in acute and chronic HIV infection. We can readily include references to such data in the revised manuscript.

3. The data shown in Figure 3 does clearly demonstrate that exposure of THP-1 cells to HIV results in IL-12/IL-18-driven MAIT cell activation in vitro. It is important to note, however, that this assay recapitulates only a very specific aspect of HIV infection, and does not include any assessment of MAIT cell activation by HIV-infected CD4+ T cells or other mechanisms such as microbial translocation. It is surprising that MAIT cell production of IFNγ was not attributed any anti-viral role, considering that similar experiments with iNKT cells have shown that iNKT-derived IFNγ limits HIV infection of primary T cells in vitro (Vasan 2007). Considering recently published data indicating the CCL3 and CCL4 production by MAIT cells is significantly greater following TCR-mediated stimulation than cytokine stimulation (Lamichhane 2019), and the impact that microbial translocation could have on MAIT cell activation, it is unclear what the relative contribution of cytokine-based activation would be on MAIT cells in vivo during HIV infection.

Thank you for the detailed comments. Here we have tried to focus here on the TCR-independent activation of MAIT cells by HIV as this seemed most relevant to their role as antiviral cells – this was prompted by our studies showing such activity is relevant in influenza in vivo. We agree that in the presence of ligand, MAIT cells will be activated via their TCR, and this could certainly affect their overall behaviour in vivo – but this would be a very diffuse effect since the bacteria or ligand would be distributed on non-HIV infected cells. Although this in theory could contribute to MAIT cell activation (in concert with cytokines) and behaviours such as activation induced cell death, this was not what we were originally trying to address in this study. We do have additional data showing the interdependency of TCR stimulation with cytokines, suggesting that even low levels of TCR triggering enhances the response to cytokines. We could include such data to further address that point i.e. that gut translocation could sensitise MAIT cells for antiviral functions.

We would politely disagree with the reviewer about the relative degree of activation with cytokines and the induction of chemokines. 3 papers were published in parallel recently which included the Lamichhane paper. In our own paper where we compare cytokine induced vs TCR triggered MAIT cell activation and we see a much larger response with cytokines and where clear activation of the relevant chemokines is seen (Leng et al., Cell Reports, Sept 2019; Figure 4F and G). The differences in the papers are due to slightly different timings and stimulation conditions – overall they are relatively congruent as we tested in our study through data integration, as well as similar to in vitro and in vivo mouse data. Indeed, generally all groups have found that cytokine induced activation is slower but more sustained. Certain functions of MAIT cells are highly TCR dependent (even if amplified by cytokines), but we do have good data that the antiviral chemokines are readily induced by cytokines.

Regarding the IFNγ, we can certainly test this using blockade as we have done in other settings (e.g. HCV in vitro and influenza in vivo). However, we note that blockade of chemokines neutralises the antiviral effect completely, so unless both are required, we think the impact in our assay is likely to be small. We have added this in the Discussion section (Line 323-328)

4. Did the authors also see contact-dependent MAIT cell-mediated inhibition of HIV replication? Similarly, would TCR-activated MAIT cells mediate antiviral activity through CCL3-5 production and/or contact-dependent mechanisms?

We did not observe any contact-dependent inhibition of HIV (Figure 4—figure supplement 2)

5. Technically, there is no indication of the use of uninfected THP-1 cultures as a control in Figure 3A or B, and it is unclear as to whether the data presented in Figure 3B is background subtracted. Sorting on CD8+ T cells prior to culture with the THP-1 cells indicates that only CD8+ MAIT cells were studied in this experiment, which is a caveat that should be noted, as MAIT cells can include a substantial CD4-CD8- population as well.

The figure legend has been amended to indicate the use of uninfected THP-1 cells as controls (Figure 3B). A caveat has been included in the Results section to explain that MAIT cells can also be found in the CD4-8- subset (Line 157-159).

6. The assay in Figure 4 using the CEM-GXR cells is likely biased to detect an impact of β-chemokines on HIV entry, given the cell line overexpresses CCR5. However, it would be important to confirm MAIT cell-mediated anti-HIV activity using primary human immune cells that productively support HIV-1 BaL replication, such as MDM and/or PHA/IL-2-activated PBMCs, plus measurements of productive viral replication (eg. p24, RT activity, etc).

HIV inhibition by MAIT cells was also observed in Primary PBMCs (Figure 4B). Here we used HIV-iGFP (JR-FL) R5 tropic virus.

7. Have the authors tested other R5 strains apart from BaL? It would be important to see that this antiviral effect is not just restricted to the BaL strain of R5 HIV-1. Along this line, there were only n=4 of presumably independent MAIT cell donors in Figure 4B (or was it 4 different CEM-GXR expts but using a single or pooled MAIT cell donors?). It would be good to increase the sample size to strengthen the manuscript. Similarly, have the authors tested X4 strains, lab-adapted or primary isolates? It would be an important control and interesting to see if MAIT cells can also/cannot inhibit X4 viruses. Along this line, did MAIT cells produce CXCR4-binding chemokines (eg. CXCL12/SDF-1)?

We have also used HIV-iGFP (JR-FL) R5 tropic virus. The total n for donors in these experiments = 8. These were 8 individual donor supernatants and not pooled. We have not tested X4 strains, as expression CXCL12 from MAIT cells were very low (Figure 3—figure supplement 3).

8. The authors show that IL-12 + IL-18 stimulation induces apoptosis in MAIT cells. While this is interesting observation, it does not explain why there is no increase in MAIT cell numbers following years of ART treatment. One can hypothesize that due to lower HIV load there will be less of IL-12 and IL-18, and subsequently less of MAIT cell stimulation. This is the case with the expression of GzmB, PD-1 and Tim-3. However, the numbers of MAIT cells remain the same. This discrepancy should be commented.

This is an interesting point for discussion. There are many unknown issues regarding MAIT cell (re)generation in adults. It takes around a decade to expand the MAIT cell population in early life, and the cells start to decline with age in a very reproducible way. These features suggest that the turnover of this subset is different to regular CD8+ T cell memory cells and so they may overall recover slowly following a depletion. A major caveat to this is that such studies have been largely performed in blood. Thus the overall recovery may not be solely viral load dependent but depends on features such as age, cell turnover and cell redistribution. We have submitted a proposal to study MAIT cell turnover (in heath and HIV infection) using heavy water but this is out with the scope of this project. Discussed in lines (302-307).

9. The methods section lacks important details for several experiments. The E. coli stimulations do not appear to be described at all. There is no indication of the number or ratio of T cells used in the THP-1 co-culture experiments.

The methods section has been amended to be more detailed (line 460-480)

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Below please find the summary of major concerns raised by reviewers.

1) The data in Figures 1 and 2 remain to be only confirmatory of multiple previously published studies. Without the data on GALT cells studied with MR1 tetramer, this portion of the manuscript does not add new information to the field. The authors argument that "this study commenced prior to these tetramers becoming available for staining, so for consistency we pursued this throughout" is somewhat in contradiction with their sentence stating that they have recently published the paper using MR1 tetramers to demonstrate MAIT cells in a GI tract. Some attempt at providing a confirmation of data should be done.

Figures 1 and 2 were used to show the depletion of MAIT cells in blood and tissue during HIV infection. Although this does initially confirm other studies, here we use patient samples form primary and chronic infection, as well as, in particular, a set of very difficult to obtain long-term non-progressor/Elite controller samples. We have now in Figure 2—figure supplement 1- added a dataset to clearly show that expression of CD161 and Vα7.2 is comparable to MR1-5OPRU tetramer staining in gut tissue for the identification of MAIT cells. This data is quite robust as we used a specific dual-staining approach and a good negative control with careful gating.

Tetramers were not available at the time for staining of HIV+ patient tissue samples – these samples were obtained from the HEATHER cohort and samples were stained real-time during collection. The data on MAIT cells in the GI tract – taken at the time of an operation for cancer – were obtained after the HIV study. The data obtained here showing a side by side comparison are very useful for the field and hopefully displayed in a clear format that allows for evaluation by the reviewers and ultimately the readers.

This paragraph has been added to the Results section.

“While MR1-Tetramers loaded with the MAIT cell ligand 5OPRU represent the most reliable way to identify MAIT cells in the blood, MAITs can also be identified as CD161++ Vα7.2+ cells, especially within the CD8+ population (Kurioka et al., 2017). In order to test whether this would also be the case in the gut, we co-stained CD161 and Vα7.2 with 5-OPRU-loaded or control MR1-tetramers and assessed whether CD161++ Vα7.2+ cells stained positive for the tetramers (Figure 2—figure supplement 1). Within the CD8+ and double-negative (DN) populations, almost all CD161++ Vα7.2+ cells reacted with both 5-OPRU-loaded but not the 6FP-loaded control tetramers, suggesting that the combination of high CD161 expression with Vα7.2 can be used to identify MAIT cells within the CD8 and DN T-cell populations in the gut. CD161++ Vα7.2+ expression on CD8+ T-cells was comparable with MR1-5OPRU- tetramer+ cells and could identify the majority of MAIT cells within this compartment.”

2) The response to reviewer’s suggestion to assess the cells that support productive HIV-1 infection to stimulate MAIT cells instead of THP-1 cells, was also not clear. The authors state: “We can clarify the background to this experiment further” or “We can however readily test further whether combinations of monocyte-derived macrophages and BAL also work to activate MAIT cells in vitro, as previously tested for the other viruses …”. Still, it remained unclear whether the authors performed the requested experiment and if so, it should be included in the manuscript. Since inactivated HIV did not result in THP-1 activation of MAIT cells, the question is what mechanism is behind this finding. This was not further discussed in the manuscript.

We have now had the chance to address this directly by using unmanipulated human PBMCs as targets and infecting with a GFP expressing JRFL virus. This is now shown in figure 4B. We were able to reproduce the data previously obtained using THP1 cells. IL-12/18 stimulated MAIT cell supernatant was able to inhibit infection of PBMCs in vitro. We hope this use of primary cells explores the area suitably for the reviewers and addresses the question posed.

We have included more data on HIV inactivation. This is now discussed in a section (line 279-289) on inactivated HIV as outline below.

“The THP1 monocytic cell line was used as it has been extensively trialed by our group and many others as an effective APC for MAIT cells in microbial and viral infections (Ussher et al., 2018, van Wilgenburg et al., 2016). MAIT cell activation was dependent on IL-12 and IL-18 produced by THP1 cells via innate sensing (van Wilgenburg et al., 2016a, Provine N, 2019). THP1 sensing of HIV-BaL may be through TLR7/8 or cytosolic RIG-like receptors (Diget et al., 2013, Guo et al., 2014), which in-turn activates THP1 cells to secrete inflammatory cytokines such as IL-12 and IL-18 (Bandera et al., 2018). Inactivated virus was incapable of activating THP1 cells. This may be due to cross-linking of nucleocapsid P7 protein by aldrithiol-2, which may not allow single stranded HIV RNA within virions to bind to TLR7/8 (Rosio et al, 1998).We note that inactivation of influenza and HCV also impacted on MAIT cell recognition in vitro (van Wilgenburg et al 2018), even in a macrophage culture where true infection and replication does not occur. ”

3) Potential anti-viral role of IFNγ was not explored. The authors state: "Regarding the IFNγ, we can certainly test this using blockade as we have done in other settings (eg HCV in vitro and influenza in vivo)."; Again, it is unclear whether or not these experiments have been performed. Although we agree that it is unlikely that IFNγ would result in a block of viral entry, it would still be relevant to test the impact of other MAIT cell-secreted cytokines and chemokines on viral replication, as the presence of MAIT cells at mucosal surfaces is clearly insufficient to block HIV infection. If the authors believe that mechanisms are the same in case of other viruses, we do not see the point of this study.

Apologies for this confusion but the original statement was from the first response to the editor and carried over. We have now had the chance to address this experimentally. We found that IFNg did not have anti-HIV effects when blocking antibodies were used. This is now Fig.4-figure supplement 4. Low antiviral effect of IFNg has been documented by other groups. This has now been added to results section (line 221-225) and the discussion section (line 277-279 and 314-324).

“IFNỿ expression in MAIT cells was observed when HIV-BaL was used to stimulate the antigen presenting cell (THP1), but had no anti-HIV effect.”

“Both MAIT cells and THP-1 cells will produce a broad range of factors following stimulation as recently described (Hinks et al., 2019, Lamichhane et al., 2019, Leng et al., 2019). However, even though factors such as IFNỿ could potentially possess anti-HIV activity, as has been described for iNKT cells (Paquin-Proulx et al., 2016), we were able to block the suppressive activity very effectively with anti-chemokine antibodies, suggesting that at least in our models, these are the most potent effectors. There has been no significant difference in HIV specific IFNỿ response reported in both progressors and long-term non-progressor patients with chronic HIV infection (Roff et al., 2013, Zanussi et al., 1996). This is consistent with original in vitro studies which revealed no antiviral effect of IFNg and even enhancement of infection in primary cells and several clinical trials which revealed no impact of this cytokine in vivo.”

4) Control experiments using X4 viruses will further strengthen the manuscript, given the inhibition by MAIT cells is acting on R5 viruses (and thus no inhibition should be seen on X4 viruses).

We have shown MAIT cells to express low levels of the CXCR4 ligand (Fig. 3-figure supplement 3), much lower than beta chemokines in our assays. We have now had the opportunity to address the experiment suggested and show that anti-HIV effect of MAIT cells is confined to R5 tropic viruses. We used HIV-LAI (X4 tropic) virus and saw no inhibition of infection when stimulated supernatant from MAIT cells were added (Fig.4 figure supplement 2), highlighted in lines 212-214.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Reviewer 4:

– Experiments done in one figure should support the findings in the other figures. Now all 4 figures are completely separate.

Thank you for the comment. We think this is a critique of the flow of the paper. The idea behind it is as follows: 1. Show MAITs are activated in vivo (here the addition of ART makes sense as it shows the role of viral replication and acts an in vivo control). 2. Address the mechanism in vitro. 3. Show the activation leads to relevant functional consequences for the MAIT cell. 4. Show that this responsiveness is meaningful in terms of antiviral responses against HIV. We think this combination of data, presented in this order – and adjusted to incorporate all the previous reviewers’ comments – hangs together reasonably. If we just went for the in vitro mechanisms we would not really be able to link this to relevant in vivo findings and vice versa, so the approach demands a bit of both. It follows a similar flow to a previous paper where we studied HCV, and which had a different mechanism (van Wilgenburg et al., Nat Comms 2016). We did need to address the issue of cell redistribution as this is unclear currently and actually highly relevant to the antiviral role.

We have tried to smooth all of this out throughout, so it is a bit easier to follow. We also reordered the last part of the paper (Figure 3 and 4; lines 201-258) to help with this. New amendments are highlighted within the revised manuscript (related manuscript file).

– If you want to draw conclusions about the specific effects of MAIT cells, you need to purify MAIT cells and not total CD8 T cells. i.e. FACS sort CD161+ Va7.2+ cells

We have now added new experimental data that was performed on FACS sorted MAIT cells and our original data on bulk CD8 T cells were reproducible (Figure 3C). We show that the major producer of CCL4 are indeed CD161+ Va7.2+ MAIT cells (Figure 4C and D). This is pretty much consistent with all our prior data (and that of other groups) showing the very high responsiveness compared to other T cells of human MAIT cells in the presence of such cytokine combinations.

– Why study ART at all if you are not going to se this data on a functional level? Remove the data or include it in functional studies.

Patient data following ART was to show the impact on activation and exhaustion of MAIT cells that is highly evident during HIV infection. We showed on figure 2b that even during acute HIV infection MAIT cell frequencies are dramatically reduced compared to healthy controls. There is also very high expression of PD-1, Tim-3 and GzmB in these cells ex vivo. This response is modified by ART at the level of phenotype as clearly shown here. Any functional work on these cells is technically very difficult as they die in culture. Hence, we sorted MAITs from healthy donors and show anti-HIV function when they are activated by the virus, or with IL12 and IL-18 as a reduced surrogate. This is for proof of principle. Thus, we were not trying to address specifically the impact of chronic stimulation or ART on MAIT cell function – we needed to prove first whether this is of any impact for HIV. If MAIT cells are diminished for any reason (by deletion is the most obvious) then this effect will be reduced.

– Why study PD1 and TIM3 at all if you are not going to look at these markers in the rest of the study? Remove the data or include it in functional studies.

The addition of these markers was to assess the impact of HIV on MAIT cell activation. We could have used a range of alternatives – for example CD69 as we have used elsewhere but these markers show a good dynamic range and staining is well established. We also used GzmB which we know is highly responsive (see eg van Wilgenburg paper as quoted earlier). Having shown this responsiveness in a robust way, we don’t think using these markers in later studies is needed to address the point we were trying to understand, which is whether this activation is functionally relevant. For this reason, we have left it in but described the meaning in more detail.

– Why not investigate CCL3/4/5 production by MAIT cells?

This is now included in Figure 4A, 4B, 4C and 4D.

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    Figure 1—source data 1. Marker expression on MAIT cells during HIV-1 infection.

    (B) GzmB, PD-1, and TIM-3 expression levels on MAIT cells during PHI and CHI HIV-1 infection. Fig. Suppl.(1) TIM-3 expression levels on MAIT cells in EC and VC.

    elife-50324-fig1-data1.xlsx (10.3KB, xlsx)
    Figure 2—source data 1. Frequency of MAITs cells in blood and tissue during HIV-1 infection.

    (B) MAIT cell frequencies in HC compared to PHI, CHI, EC, and VCs at baseline. (C) MAIT cell frequencies during CHI at baseline and 1 year post-ART. (D) MAIT cell frequencies during PHI at baseline and 1 year post-ART. (E) longitudinal MAIT cell frequencies during CHI at 1, 3, and 5 years post-ART. (F) Comparison of MAIT cell frequencies between GALT and PBMC. (G) Presence of MAIT cells in rectum and terminal ileum of PHI ART-treated subjects.

    elife-50324-fig2-data1.xlsx (10.4KB, xlsx)
    Figure 3—source data 1. MAIT cells are activated by HIV-1 in an IL-12 and IL-18-dependent manner.

    (A) percentage of MAIT cells expressing IFN-γ upon in vitro stimulation with fixed E. coli or HIVBAL in the presence or absence of blocking antibodies directed against IL-12 and IL-18. (B) frequency of GFP positive CEM-GXR cells following infection with HIVBAL (MOI = 0.2) and pre-treatment with stimulated supernatant from MAIT cells. (C) HIVJRFL-GFP infection in primary human PBMCs or CEM-CCR5 cells by addition of control or IL-12/18-treated supernatants obtained from MACS-enriched CD8s (top) or FACS-sorted MAIT cells (bottom).

    elife-50324-fig3-data1.xlsx (10.5KB, xlsx)
    Figure 4—source data 1. MAIT-cell-derived antiviral restriction factors are essential for suppressing HIV-1.

    CCL4 (MIP-1β) concentration was measured in the supernatants by ELISA after 20 hr post stimulation with IL-12/18. (B) Concentrations of CCL3 (MIP1α) and CCL5 (RANTES) were measured in the supernatants by cytometric bead array (CBA) after 20 hr post stimulation with IL-12/18. (C) Expression of CCL4 by MAITs after incubation with IL-12/18 for 20 hr. MAIT cells were identified as CD161++ Vα7.2+ cells within MACS-enriched CD8s. (D) Percentage of MAIT cells as identified by co-expression of Vα7.2 with high levels of CD161 within MACS-enriched CD8s and within all CCL4-expressing CD8 T cells from the same culture. CD8 T cells were stimulated with IL-12/18 for 20 hr. (E) GFP-positive CEM-GXR cells following blocking of restriction factors (CCL3/4/5), after treatment with IL12/18 stimulated supernatant from CD8 cells and infection with HIVBAL.

    elife-50324-fig4-data1.xlsx (10.4KB, xlsx)
    Transparent reporting form
    elife-50324-transrepform1.docx (66.8KB, docx)

    Data Availability Statement

    Raw data from main figures are provided as source data files.

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