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IL7RA single nucleotide polymorphisms are associated with the size and function of the MAIT cell population in treated HIV-1 infection

MAIT cells are persistently depleted and functionally exhausted in HIV-1-infected patients despite long-term combination antiretroviral therapy (cART). IL-7 treatment supports MAIT cell reconstitution in vivo HIV-1-infected individuals and rescues their functionality in vitro. Single-nucleotide polymorphisms (SNPs) of the IL-7RA gene modulate the levels of soluble(s)IL-7Rα (sCD127) levels and influence bioavailability of circulating IL-7. Here we evaluate the potential influence of IL-7RA polymorphisms on MAIT cell numbers and function in healthy control (HC) subjects and HIV-1-infected individuals on long-term cART. Our findings indicate that IL-7RA haplotype 2 (H2*T), defined as T-allele carriers at the tagging SNP rs6897932, affects the size of the peripheral blood MAIT cell pool, as well as their production of cytokines and cytolytic effector proteins in response to bacterial stimulation. H2*T carriers had lower sIL-7Rα levels and higher MAIT cell frequency with enhanced functionality linked to higher expression of MAIT cell-associated transcription factors. Despite an average of 7 years on suppressive cART, MAIT cell levels and function in HIV-1-infected individuals were still significantly lower than those of HC. Notably, we observed a significant correlation between MAIT cell levels and cART duration only in HIV-1-infected individuals carrying IL-7RA haplotype 2. Interestingly, treatment with sIL-7Rα in vitro suppressed IL-7-dependent MAIT cell proliferation and function following cognate stimulations. These observations suggest that sIL-7Rα levels may influence MAIT cell numbers and function in vivo by limiting IL-7 bioavailability to MAIT cells. Collectively, these observations suggest that IL-7RA polymorphisms may play a significant role in MAIT cell biology and influence MAIT cells recovery in HIV-1 infection. The potential links between IL7RA polymorphisms, MAIT cell immunobiology, and HIV-1 infection warrant further studies going forward.

MAIT cells express high levels of IL-7Ra (CD127) (31,32), and IL-7 promotes the activation of circulating MAIT cells, arms cytotoxic capacity, and enhances cytokine production (32). Furthermore, IL-7 amplifies the activation of MAIT cells via TCR or non-TCR signals (31,32). In addition, IL-7 induces high levels of IL-18Ra on MAIT cells and increases their expression of the transcription factors promyelocytic leukemia zinc finger (PLZF), retinoic acid receptor-related orphan receptor (ROR)gt, T-box transcription factor 21 (TBX21, also known as T-bet), and eomesodermin (Eomes) (32)(33)(34). In disease settings, plasma IL-7 levels positively correlate with MAIT cell frequencies in HIV-1infected patients (32,35). IL-7 treatment expands the MAIT cell compartment in HIV-1-infected patients in vivo and rescues the antimicrobial effector function of MAIT cells of HIV-1-infected patients in response to E. coli stimulation in vitro (32,36). In rhesus macaques with chronic SHIV infection, IL-7 alone enhances the proliferation but not degranulation of MAIT cells (33). In mice, pretreatment with IL-7 improves survival in Streptococcus pneumoniae infection (34). IL-7 is a homeostatic cytokine required for both the production and survival of naïve and memory T cells and is critical for immune recovery in lymphopenic states (37, 38). IL-7 exerts its biological effect by binding to its membrane-bound receptor, mIL-7Ra on T-cells (39). There is also a soluble form of this receptor (sIL-7Ra), which is thought to modulate IL-7 availability (40). In vitro studies found that levels of sIL-7Ra in culture affected IL-7-mediated functions including T cell survival and proliferation (41). This suggests that sIL-7Ra may influence IL-7 mediated function including its capacity to drive immune recovery in HIV-1-infected individuals.
Four common haplotypes of the IL7RA gene (haplotypes 1, 2, 3 and 4) have been previously defined and shown to be associated with sIL-7Ra levels (42, 43). Haplotype 2 (tagged by carriage of T allele in rs6897932) is associated with reduced sIL7Ra levels in plasma. This single nucleotide polymorphism (SNP) influences the amount of soluble and membrane bound IL-7Ra isoforms produced by disrupting an exon splicing silencer (44). Studies assessing the association between rs6897932 and immune reconstitution have reported both positive and negative associations. In a predominantly Caucasian Australian cohort of HIV-1-infected people, there was a significant association between the IL7RA haplotype 2 (H2*T) and faster CD4 + T cell recovery in those receiving suppressive ART (45). In a replicate study in Uganda, the IL7RA H2*T had an inverse association with CD4 + T cell recovery compared to that found in Caucasians, and was associated with poorer CD4 + T cell recovery (46). Subsequently, the differential influences of IL7RA haplotypes on HIV-1 disease progression in Caucasians and Africans have been confirmed (47,48). The reason for the different associations seen in the various ethnic populations despite a consistent influence of IL7RA haplotype 2 on low sIL-7Ra levels is currently unclear. T-allele carriers of the rs6897932 SNP have also been associated with reduced susceptibility to multiple autoimmune diseases including multiple sclerosis, systemic lupus erythematosus (49), chronic inflammatory arthropathies (50), sarcoidosis (51) and type 1 diabetes (52) in HIV-1uninfected individuals. Despite the wide ranging effects of this SNP in multiple immune-mediated diseases, there is a notable lack of studies systematically assessing its immunologic effects.
Although the effects of IL-7 on MAIT cells have been clear for some time, it is unknown whether IL7RA polymorphisms affect MAIT cell phenotype and function in health and during HIV-1 infection. In this study, we investigate the association of IL7RA haplotypes with MAIT cell levels and effector function in health and in chronic HIV-1 infection with long-term cARTmediated viral suppression. Our findings indicate that IL7RA H2*T affects the size of the peripheral MAIT cell pool, as well as their production of cytokines and cytolytic effector proteins in response to bacterial stimulation. Furthermore, presence of IL7RA H2*T positively associates with circulating MAIT cell levels following the long-term suppressive ART in chronically HIV-1-infected individuals.

Results
IL7RA haplotype 2 is associated with higher MAIT cell levels in healthy individuals IL-7 treatment supports increased MAIT cell numbers in vivo in humans and IL7RA SNPs influence IL-7 uptake and downstream biological activity (36,53). Thus, the relationship between IL7RA SNPs (Supplementary Table 1) and MAIT cell numbers in circulation of healthy individuals (Supplementary Table 2) was initially determined. Peripheral blood (PB) MAIT cells were identified by the combined staining of TCR Va7.2 with high expression of CD161 ( Figure 1A). The vast majority of Va7.2 + CD161 hi MAIT cells also stained positive with MR1-5-OP-RU tetramer and expressed the CD8a co-receptor ( Figure 1A), consistent with previously published studies (10,54). Healthy individuals carrying IL7RA H2*T, defined as Tallele carriers (heterozygous or homozygous) at the tagging SNP rs6897932 (Supplementary Table 1), had higher levels of MAIT cells in circulation than those with non-haplotype 2 (non-H2*T) polymorphisms ( Figure 1B). There was no significant age differences between the haplotype groups when stratified by sex (Supplementary Figure 1A). Furthermore, MAIT cell numbers were comparable between females and males when stratified by haplotypes (Supplementary Figure 1B). Correlative analyses also showed that there were no associations between age and MAIT cell numbers as a whole or stratified either by sex or haplotypes (Supplementary Table 3). Therefore, the difference in MAIT cell frequency between the IL7RA haplotypes was probably not influenced by differences in age or sex between the groups. There was no difference in the representation of CD8 + and DN MAIT cells between the IL7RA haplotypes as a whole or when stratified by sex ( Figures 1C, D). Similarly, no differences were observed in IL-7Ra surface expression (mIL-7Ra/CD127) by MAIT cells ( Figure 1E) and the plasma levels of IL-7 from individuals carrying different IL7RA haplotypes ( Figure 1F), but those with H2*T haplotypes had significantly lower sIL-7Ra in their plasma ( Figure 1G), consistent with previous studies (42, 44). Notably, there was a significant inverse correlation between MAIT cell frequency and plasma levels of sIL-7Ra in this cohort of healthy individuals ( Figure 1H). However, there was no correlation between age and plasma levels of sIL-7Ra as a whole or stratified by haplotype or sex (Supplementary  Table 3). Altogether, these results suggest that IL7RA polymorphisms associates with MAIT cell levels in healthy adults.
IL7RA SNPs are associated with MAIT cell expression of transcription factors and function in healthy individuals MAIT cell treatment with IL-7 in vitro leads to enhanced expression of transcription factors (TFs) and augmented MAIT cell antimicrobial function, such as increased production of IFNg and TNF (31,32). Therefore, whether IL7RA polymorphisms affect expression of critical TFs and MAIT cell function was next evaluated. In line with higher MAIT cell frequency in individuals carrying H2*T, MAIT cells in these individuals expressed higher levels of PLZF ( Figure 2A). RORgt expression also appeared to be higher in MAIT cells from H2*T individuals ( Figure 2B). IL-7 downstream biological activity is associated with increased cellular survival partly via the anti-apoptotic protein Bcl-2 (55). However, Bcl-2 expression levels in MAIT cells from individuals carrying the H2*T and non-H2*T haplotypes were comparable ( Figure 2C). We were unable to stratify these differences based on sex due to limited sample size. To assess for antimicrobial effector function, MAIT cells were activated using an assay that was previously developed by our group (56,57). Following stimulation with formaldehyde-fixed E. coli for 24 h, MAIT cells from healthy adults with the H2*T haplotype had increased levels of IFNg and TNF, and appeared to also be more bifunctional (Figures 2D-G). Such higher expression levels of cytokines were still evident in females after stratification by sex, whereas there was a trend of increasing cytokine production by MAIT cells from male H2*T IL7RA carriers. There was no correlation between age and cytokine expression by MAIT cells stratified by either sex or haplotype (Supplementary Table 3). These findings suggest that IL7RA polymorphism may affect MAIT cell functional activity in vivo through modulation of TF levels.

MAIT cell levels and function are significantly lower in HIV-1-infected individuals on long-term suppressive ART
The frequency and function of MAIT cells are impaired in chronic untreated HIV-1-infected patients and do not fully recover following long-term cART (19,22,25). In agreement with previous studies (19, 22, 58), circulating MAIT cell levels from HIV-1infected individuals with long-term cART (Supplementary Table 4) were still significantly lower than those from uninfected adult males from the same HC cohort ( Figure 3A). The persistent depletion of MAIT cells from the circulation of long-term treated HIV-1-infected individuals was still observed in IL-7RA haplotype 2 carriers but not in non-haplotype 2 carriers ( Figure 3A). Since there was no association between MAIT cell levels and age stratified by haplotype in uninfected and HIV-1-infected cohorts in the current study (Supplementary Tables 3, 5, respectively), the differences seen in MAIT cell levels may be less likely due to age differences between the HC and HIV-1-infected cohorts. Similarly, no differences in the proportion of CD8 + and DN MAIT cell subpopulations, as well as in the expression of IL-7Ra by MAIT cells, were noted in healthy controls as compared with those of HIV-1-infected patients ( Figures 3B-D). There was no difference in plasma levels of IL-7 and sIL-7Ra between healthy controls and HIV-1-infected patients bearing H2*T and non-H2*T haplotypes ( Figures 3E, F). In addition, HIV-1 infection was associated with poor IFNg and TNF production by MAIT cells following E. coli stimulation despite long-term cART (Figures 3G-J) in line with previous observations (19). This impairment was more obvious in HIV-1infected individuals carrying the non-H2*T haplotypes ( Figures 3G-J). The low sample size for HIV-1-infected individuals carrying the non-H2*T haplotype may explain why there was no significant difference in IFNg expression found when compared to healthy controls carrying the same IL7RA haplotype. Taken together, these results indicate that MAIT cell dysfunction in chronic HIV-1 infection persists despite long-term viral suppression when compared to healthy controls. This dysfunction may occur regardless of the IL7RA haplotypes.

IL7RA haplotypes associate with MAIT cell function and recovery in HIV-1infected patients on ART
Based on the findings that MAIT cell dysfunction persists in HIV-1-infected patients receiving long-term cART, we next revisited the dataset from the same HIV-1-infected cohort and evaluated the degree of impairment between patients carrying the H2*T and non-H2*T haplotypes. No significant differences in clinical parameters were noted between the HIV-1-infected patient groups carrying IL7RA haplotypes H2*T and non-H2*T, including age, pretreatment CD4 counts and viral loads, duration of ART ( Supplementary Table 4). Interestingly, there was comparable MAIT cell frequency in virally-suppressed chronic HIV-1-infected patients with H2*T and non-H2*T haplotypes ( Figure 4A). Additionally, haplotypes H2*T and H-J: HIV-1-uninfected N=10, N=5 for both haplotype 2 and non-haplotype 2; HIV-1-infected N=10, N=6 for haplotype 2 and N=4 for nonhaplotype 2), median, and the interquartile range. Statistical significance was determined using Mann-Whitney's test. *** p<0.001,** p<0.01,* p<0.05. ns, not significant. M, male; H2*T, IL7RA haplotype 2. Male HC dataset were retrieved from HC cohort depicted in Figure 1.
non-H2*T did not appear to influence MAIT cell major subset distribution within the HIV-1-infected patient group ( Figures 4B, C). Although comparable levels mIL-7Ra and plasma IL-7 were observed between the two groups ( Figures 4D, E), individuals carrying non-H2*T haplotype had a higher level of plasma sIL-7Ra ( Figure 4F), consistent with the observation in healthy individuals ( Figure 1G). Moreover, there was no association between age and plasma sIL-7Ra levels as a whole or stratified by haplotype in this HIV-1-infected cohort (Supplementary Table 5). In addition, HIV-1-infected individuals with H2*T haplotype had better MAIT cell functionality following bacterial stimulation when compared to those carrying other haplotypes ( Figures 4G-I).
There was also no correlation between age and cytokine expression by MAIT cells following E. coli stimulation as a whole or stratified by haplotype in the current HIV-1-infected cohort, although the sample size was small (Supplementary  Table 5). Interestingly, despite that there was no difference in MAIT cell levels between group with the different IL7RA haplotypes, duration of ART positively correlated with MAIT cell levels in individuals bearing the H2*T haplotype ( Figure 4J).

Soluble IL-7Ra inhibits IL-7-mediated MAIT cell proliferation and effector function in vitro
Levels of sIL-7Ra affects IL-7-mediated functions including T cell survival and proliferation (41). Given plasma sIL-7Ra levels negatively correlate with MAIT cell frequency ( Figure 1H), we next explored the impact of sIL-7Ra treatment in vitro on IL-7-mediated MAIT cell proliferation and function. Downregulation of surface expression of mIL-7Ra is a surrogate marker of MAIT cell activation following IL-7 treatment in vitro (32). By using this approach, initial e x p e r i m e n t s s h o w e d t h a t s I L -7 R a m i t i g a t e d t h e downregulation of mIL-7Ra on MAIT cells following IL-7 treatment in vitro ( Figure 5A), suggesting that sIL-7Ra reduces IL-7 binding to mIL-7Ra on MAIT cells. Indeed, sIL-7Ra treatment in vitro reduced MAIT cell proliferation following stimulation with low dose of the prototypical MR1 ligand 5-OP-RU supplemented with low dose IL-7 (Figs. 5B-5G). This was shown by lower levels of Ki67 expression, lower MAIT cell numbers, as well as reduced proliferation and expansion indices calculated from CellTrace Violet (CTV) dilution assay ( Figures 5B-G), performed as previously described (56,59). Such inhibition appeared to be dose-dependent and remained apparent at sIL-7Ra:IL-7 molar ratio > 100 ( Figure 5H). The inhibitory effect of sIL-7Ra on MAIT cell proliferation did not occur when IL-2 substituted IL-7 ( Supplementary Figures 2A-G), indicating that sIL-7Ramediated impairment is specific to IL-7-dependent activity on MAIT cells. Interestingly, sIL-7Ra also inhibited IL-7-mediated enhancement on resting MAIT cell IFNg and TNF production and Ki-67 expression following E. coli stimulation ( Figures 5I-K). This inhibition was also dose-dependent as MAIT cell cytokine production and Ki-67 expression remained reduced at sIL-7Ra: IL-7 molar ratio > 100 ( Figure 5L).

Discussion
In the present study, we report an association between IL7RA polymorphisms and MAIT cell levels and functional activity in healthy individuals and HIV-1-infected individuals with long-term viral suppression. Specifically, we show that IL7RA haplotype 2 positively impacts the frequency and functional activity of MAIT cells in healthy individuals. This IL7RA haplotype is correlated with decreased plasma sIL-7Ra concentration both in health and chronic HIV-1 infection, which is linked with higher MAIT cell levels in healthy controls. Importantly, MAIT cell levels correlate with duration on cART in HIV-1-infected individuals carrying IL7RA haplotype 2, suggesting favorable MAIT cell reconstitution in these individuals. Such MAIT cells also appear to display better functionality upon bacterial stimulation. This observation suggests that IL7RA haplotype 2 may be beneficial for MAIT cell reconstitution in vivo. Finally, in vitro experiments support the notion that the positive effects of IL7RA haplotype 2 on MAIT cell number and function is associated with lower sIL-7Ra concentration, as sIL-7Ra treatment in vitro effectively inhibits IL-7-dependent MAIT cell proliferation and effector function. Altogether, these observations suggest favorable effects of the IL7RA haplotype 2 on MAIT cell reconstitution in virally suppressed chronic HIV-1 infection.
How IL7RA polymorphisms may affect MAIT cell biology in health and during HIV-1-infection is unclear. It is possible that decreased production of sIL-7Ra isoform in IL7RA haplotype 2carrying healthy and HIV-1-infected individuals may diminish the interference with IL-7 binding to its membrane-bound receptors on T cells (42, 53). However, the effects of sIL-7Ra in shaping IL-7 bioavailability are complex. Several reports demonstrated that sIL-7Ra serves as a reservoir of IL-7 and hence increases its half-life due to relatively low affinity (53, 60). Lundström et al. provided a model for sIL-7Ra-mediated IL-7 regulation, proposing that sIL-7Ra/rhIL-7 with a very high molar ratio (>5000) may increase the bioactivity of IL-7 by stabilizing its reservoir (53). Furthermore, in vivo experiments showed that IL7 −/− mice had transiently increased IL-7 levels following administration of rhIL-7 with sIL-7Ra versus rhIL-7 alone (53). In the current study, we saw the plasma levels of sIL-7Ra were significantly lower in haplotype 2 individuals in both healthy and HIV-1-infected individuals, consistent with previous studies (42, 44). Moreover, we found that MAIT cell levels in IL7RA haplotype 2 carriers with chronic HIV-1 infection correlated with duration on cART, whereas non-haplotype 2 carriers did not show this correlation. This observation is in line with better recovery from lymphopenia for haplotype 2 carriers on cART (42). HIV-1-infected IL7RA haplotype 2 carriers also had partial recovery of the cytokine production following E. coli stimulation. Given the stimulatory effects of IL-7 on MAIT cells (31,32,61), all these together suggest that IL7RA haplotype 2 may influence MAIT cell homeostasis via enhanced bioactivity of IL-7 due to low plasma sIL-7Ra.
Indeed, our data indicate dose-dependent inhibition of IL-7mediated MAIT cell proliferation and effector function by sIL-7Ra in vitro. Firstly, sIL-7Ra mitigated the downregulation of mIL-7Ra following low dose IL-7, indicating reduced binding of IL-7 to mIL-7Ra on MAIT cells. Furthermore, sIL-7Ramediated inhibition was seen when MAIT cells were stimulated with low dose of the prototypical antigen 5-OP-RU in the presence of low dose of IL-7, but not when in the presence of low dose of a similar gc cytokine IL-2. This indicates the specificity of sIL-7Ra-mediated inhibition on IL-7. Finally, sIL-7Ra negates IL-7-dependent enhancement of MAIT cell effector function following E. coli stimulation. Particularly, IFNg and TNF levels by IL-7-primed MAIT cells were comparable to those of unprimed MAIT cells following E. coli stimulation when coadministered with sIL-7Ra. Collectively, our in vitro data support the notion that favorable effects of IL7RA haplotype 2 on MAIT cell numbers and function in both HC and HIV-1infected patients is at least partially a consequence of enhanced bioavailability of IL-7 to MAIT cells due to lower sIL-7Ra levels in vivo. Our in vitro study used much lower sIL-7Ra/rhIL-7 molar ratios (0.015 to 150) and showed inhibitory effect of sIL-7Ra on IL-7 bioactivity, whereas a previous study that used a very high sIL-7Ra/rhIL-7 molar ratio of 5000 showed an enhancement of IL-7 bioactivity (53). Therefore, because of the complexity of IL-7/IL-7Ra biology, it will be important to further examine the influence of these receptor polymorphisms on IL-7 utilization and MAIT cell biology and the mechanisms behind these effects in both HC and HIV-1-infected individuals going forward. The positive effects of haplotype 2 on MAIT cells may additionally occur through the modulation of critical TFs for MAIT cell functions. Our data indicate that the IL7RA haplotype 2 appears to support higher expression of PLZF and RORgt by peripheral MAIT cells in healthy individuals. These TFs are essential for both TCR-dependent and -independent MAIT cell development and cytokine production (3). Therefore, upregulation of PLZF and RORgt may potentially lead to higher frequency and function of MAIT cells in haplotype 2 individuals. Tang et al. observed upregulation of anti-apoptotic molecule Bcl-2 in intrasinusoidal MAIT cells upon IL-7 stimulation (31). Of note, sIL-7Ra inhibits IL-7-induced proliferation and Bcl-2 expression in peripheral CD8 + T cells (41,53). Thus, lower sIL-7Ra and increased IL-7 utilisation by MAIT cells in haplotype 2 individuals may potentially lead to enhanced survival of MAIT cells via increased Bcl-2 activity. However, in the current study we did not observe any changes in Bcl-2 expression in the setting of lower plasma sIL-7Ra levels in haplotype 2 individuals.
Our study has several important limitations. Firstly, our sample size was relatively small and there was some differences with regard to age and sex distribution between the healthy and HIV-1-infected cohorts. Nevertheless, our correlative analyses suggest that these differences do not seem to significantly associate with the MAIT cell levels in either HC or HIV-1infected individuals. Indeed, the differences in MAIT cell numbers and function in individuals carrying different IL7RA haplotypes hold when stratified by sex in HC cohort. Unfortunately, the low number of male donors from either haplotypes in HC cohort does not allow us to make firm conclusion on the influence of IL7RA haplotypes on MAIT cell biology in adult males. We also used only the dataset from male HC for the comparison with the all-male HIV-1-infected cohort, thereby removing biological sex as a confounding factor in these analyses. Secondly, the potential influence of other confounders cannot be ruled out due to the relatively small sample size. In future studies, it will be important to recruit a larger cohort with matching healthy controls, as well as longitudinal cohorts from the HIV-1-infected individuals to confirm better MAIT cell recovery of haplotype 2 carriers. Moreover, further in vitro studies are warranted to tease out the potential mechanisms by which IL-7 and IL-7RA haplotypes influence MAIT cell biology. Complex interaction between MAIT cell biology, IL-7RA polymorphisms, and HIV-1 infection should be explored in-depth going forward. Understanding intricate utilization of IL-7 in reconstituting MAIT cells and enhancing their functions may ultimately benefit the host defense during HIV-1 infection.

Resource availability Lead contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Edwin Leeansyah (https://edwin.leeansyah@sz.tsinghua.edu.cn)

Data and code availability
No data sets or code were generated or analysed in this study. All raw data associated with this study are available upon request from the Lead Contact. All software is commercially available.

HIV-1-infected and uninfected cohorts
HIV-1-infected (n=39) individuals previously genotyped for the IL-7Ra SNPs were identified from a previous cohort established to explore biological factors associated with immune recovery (64).
Briefly, all patients were recruited at the Infectious Diseases Unit (IDU) in University Malaya Medical Centre (UMMC) during their routine follow-up. Patients who fulfilled the following inclusion criteria were enrolled into the study: 1) Men or women aged at least 18 years, 2) first antiretroviral regimen composed of cART defined by at least three antiretroviral drugs, 3) CD4 T-cell count <500 cells/ µl at the start of cART and 4) patients achieving HIV-1 RNA levels ≤50 copies/ml within 12 months following commencement of cART. Demographic and clinical parameters including age at cART initiation, gender, ethnicity, history of AIDS defining illnesses, antiretroviral prescribed and CD4 T cell count and HIV RNA measurements were obtained from patients medical records. HIV-1-negative controls (n=35) were recruited amongst healthy laboratory staff volunteers. All samples were processed using identical protocols within 4 hours of venipuncture.

Blood processing
Peripheral blood mononuclear cells (PBMCs) were isolated by standard Ficoll-Histopaque density gradient separation (Axis-Shield Poc As ™ ). Isolated PBMCs were cryopreserved in liquid nitrogen until further required.

Preparation of MAIT cell antigen 5-OP-RU
The MAIT cell antigen 5-OP-RU was synthesized according to a previously published procedure (62,63). It is stable in DMSO solutions but converts rapidly to much less active lumazine in aqueous media, the exposure time to which should be minimized as much as possible to maximize activity. All 5-OP-RU working solutions were diluted from the DMSO stocks with appropriate culture medium immediately (within 5 min) prior to the proliferation assay.
Preparation of sIL-7Ra/IL-7 or sIL-7Ra/ IL-2 mixture Recombinant human IL-7 (R&D System) or IL-2 (PeproTech) at 2.5 ng/mL in MAIT cell Expansion Medium (see below) were incubated with sIL-7Ra (Biolegend) at varying amounts corresponding to the indicated molar ratios for 1 h at 37°C. PBMCs were then cultured in these media for further MAIT cell functional and proliferation assay as indicated.

MAIT cells activation and proliferation assays
MAIT cells within bulk PBMCs, identified as Va7.2 + CD161 hi CD3 + T cells, were activated using formaldehyde-fixed E. coli at the microbial dose of 1 for 24 h as previously described (65). Monensin (Golgi Stop, BD Biosciences) was added for the last 6 h of incubation. To determine the effect of sIL-7Ra on IL-7dependent MAIT cell activation, cryopreserved PBMCs were thawed and rested for 48 h in a 37°C/5%CO 2 incubator in ImmunoCult-XF T cell expansion medium supplemented with 8% (v/v) xeno-free CTS Immune Cell Serum Replacement (ThermoFisher Scientific), 50 mg/mL gentamicin (ThermoFisher Scientific), and 100 mg/mL normocin (Invivogen) (collectively: Expansion Medium). PBMCs were subsequently supplemented with IL-7 alone or in combination with sIL-7Ra as indicated for a further 24 h prior to analyses. In selected experiments, PBMCs were further stimulated with formaldehyde-fixed E. coli at the microbial dose of 0.1 for another 24 h.
For MAIT cell proliferation assay, MAIT cells were expanded as previously described (59, 66) with modifications. Briefly, cryopreserved PBMCs were thawed and stained using CTV cell proliferation dye as previously described (67), and rested for 48h in a 37°C/5%CO 2 incubator in Expansion Medium. Subsequently, PBMCs were cultured with 100 pM 5-OP-RU with or without 2.5 ng/mL IL-7 or IL-2 alone, or with varying amounts of sIL-7Ra as indicated. No further media changes and cytokine supplementations were performed. Expansion and proliferation indices of MAIT cells, identified as MR1-OP-RU tetramer + Va7.2 + CD3 + T cells, were determined based on CTV dilution at day 5 post-stimulation using the FlowJo v. 10 software. Absolute MAIT cell numbers were calculated using Precision Count Beads (BioLegend) according to the manufacturer's instruction.

Measurement of IL-7
IL-7 levels in plasma samples were measured using Quantikine HS ELISA kit following the manufacturer instruction (R&D Systems). This assay was performed on patient samples in the kinetics study. Briefly, plasma samples were diluted in 1 in 2 using the diluent provided. All samples were assayed in duplicate. Pre-and post-cART samples from the same patient were assayed in the same plate. The inter-assay and intra-assay CV for this assay were 16.3% (n=6) and 9.33% (n=6), respectively. The standard concentrations for this assay ranged from 0.25-16 pg/ml.

Measurement of soluble IL-7Ra (sIL-7Ra)
sIL-7Ra levels in plasma samples were measured using an in-house ELISA method as previously described (42). Human IL-7Ra/Fc chimera extracellular domain immunoglobulin (R&D Systems) was used as standards and the concentration ranged from 0.625-40 ng/ml. Samples, standards, detection antibody and streptavidin-HRP were diluted using 1% BSA in PBS. The inter-assay and intra-assay CV precision for this assay were 20% (n=6) and 2.4% (n=6), respectively. All samples were assayed in duplicate.

Statistical analysis
Statistical analyses were performed using Prism software v.9.4 (GraphPad). Data sets were first assessed for data normality distribution. Box and whisker plots show median, the 10 th to 90 th percentile, and the interquartile range. Statistically significant differences between samples were determined as appropriate using the unpaired t-test or Mann-Whitney's test for unpaired samples. The Friedman test, one-way analysis of variance (ANOVA), or mixed-effects analysis followed by the appropriate post-hoc test as indicated was used to detect differences across multiple samples. Correlations were assessed using the Spearman rank correlation. Two-sided p-values < 0.05 were considered significant.

Data availability statement
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Ethics statement
The studies involving human participants were reviewed and approved by The University Malaya Medical Centre's ethics review board (MEC Ref: 896.32). The patients/participants provided their written informed consent to participate in this study.