PD-L1 Blockade Differentially Impacts Regulatory T Cells from HIV-Infected Individuals Depending on Plasma Viremia

Blocking the PD-1/PD-L1 pathway has emerged as a potential therapy to restore impaired immune responses in human immunodeficiency virus (HIV)-infected individuals. Most reports have studied the impact of the PD-L1 blockade on effector cells and neglected possible effects on regulatory T cells (Treg cells), which play an essential role in balancing immunopathology and antiviral effector responses. The aim of this study was to define the consequences of ex vivo PD-L1 blockade on Treg cells from HIV-infected individuals. We observed that HIV infection led to an increase in PD-1+ and PD-L1+ Treg cells. This upregulation correlated with disease progression and decreased under antiretroviral treatment. Treg cells from viremic individuals had a particularly high PD-1 expression and impaired proliferative capacity in comparison with Treg cells from individuals under antiretroviral treatment. PD-L1 blockade restored the proliferative capacity of Treg cells from viremic individuals but had no effect on its suppressive capacity. Moreover, it increased the viral production in cell cultures from viremic individuals. This increase in viral production correlated with an increase in Treg cell percentage and a reduction in the CD4/Treg and CD8/Treg cell ratios. In contrast to the effect of the PD-L1 blockade on Treg cells from viremic individuals, we did not observe a significant effect on the proliferative capacity of Treg cells from individuals in whom viremia was controlled (either spontaneously or by antiretroviral treatment). However, PD-L1 blockade resulted in an increased proliferative capacity of HIV-specific-CD8 T cells in all subjects. Taken together, our findings suggest that manipulating PD-L1 in vivo can be expected to influence the net gain of effector function depending on the subject’s plasma viremia.


Introduction
Inhibiting programmed cell death 1 (PD-1) signalling has a potential therapeutic value for treating cancers and persistent viral infections (reviewed in [1][2][3][4][5]). PD-1 is a co-inhibitory receptor that plays a major role in exhaustion, a dysfunctional state of effector cells caused by antigen persistence [6]. Exhausted T cells present defects in effector function including impaired proliferation, cytotoxic capacity and cytokine production. These defects can be partially restored by blocking the interaction between PD-1 and its ligand programmed death ligand-1 (PD-L1), which notably reduces viral loads in several animal infection models [7][8][9][10]. This observation has also been extended to important persistent human infections such as the human immunodeficiency virus (HIV) infection, both in vitro [11][12][13][14] and in vivo in HIVinfected humanized mice [15,16]. Since the HIV load is directly correlated with disease progression [17], an augmentation of antiviral immune responses by blocking the PD-1/PD-L1 pathway might help to control viral replication and slow down pathogenesis. Furthermore, it may facilitate clearance of latently infected cells, and thus may represent a promising strategy to reach a functional cure of HIV infection [18,19].
PD-1 and PD-L1 are expressed on several cell types including regulatory T cells (Treg cells) [20]. Treg cells are a suppressive T cell subset mediating self-tolerance and immune homeostasis (reviewed in [21,22]). During HIV-infection, Treg cells have both, beneficial and detrimental roles (reviewed in [23][24][25]). For example, Treg cells control excessive immune activation that limits immunopathology and the availability of HIV target cells. On the contrary, Treg cells contribute to the destruction of the lymphatic tissue architecture, and inhibit HIV-specific immune responses promoting virus persistence. Therefore, any therapeutic alteration of Treg cell numbers and function may directly influence the balance between immunopathology and viral control.
PD-L1 blockade therapy in HIV-infected individuals is expected to affect their Treg cells. Indeed, several roles of the PD-1/PD-L1 pathway are already described for this cell subset. For example, PD-1/PD-L1 pathway is essential in the induction of Treg cells in the periphery [26][27][28] and the maintenance of their suppressive capacity [28][29][30][31][32][33]. PD-1 is also described as a negative regulator of Treg cells in hepatitis C virus infection [34]. Likewise, in vivo blockade of PD-L1 increased the numbers of Treg cells in the Friend virus mice model [35]. In the context of a HIV infection, PD-1 was found up-regulated in Treg cells compared with healthy controls [36][37][38]. Nonetheless, as most reports have focused on effector cells, possible effects from PD-L1 blockade on Treg cells have been neglected. In light of the upcoming therapeutic trials blocking PD-L1 in HIV-infected patients, it is important to understand its consequences for Treg cells, as they are essential players balancing immunopathology and antiviral effector responses.
The purpose of this report was to investigate the impact of ex vivo PD-L1 blockade on Treg cells from HIV-infected individuals. We found that PD-L1 blockade had no effect on Treg cells' suppressive capacity. However, PD-L1 blockade increased the proliferative capacity of Treg cells from viremic individuals but had no significant effect on Treg cells from individuals that control viremia. Interestingly, we found that PD-L1 blockade in peripheral blood mononuclear cells (PBMC) from viremic individuals increased virus production. This increase was related with increased Treg cell frequencies, suggesting that the inhibitory function of Treg cells may play a role in virus expansion upon PD-L1 blockade. In contrast to the differential effect from PD-L1 blockade on Treg cell proliferation, we observed an increase in the proliferation of effector T cells in all groups of HIV-infected individuals. Therefore, manipulating PD-L1 in vivo is expected to influence the net gain of effector function depending on the subject's plasma viremia.

PD-1 and PD-L1 are increased on Treg cells from HIV-infected individuals
Previous studies have shown that (i) PD-1 is overexpressed on CD4-and CD8-T cells in several persistent infections and cancers, and (ii) that this overexpression plays a key role in the exhausted phenotype of these cells (reviewed in [39]). To first evaluate the expression of PD-1 and its ligand PD-L1 on Treg cells from HIV-infected individuals, we used the gating strategy of Miyara and colleagues [40]. It distinguishes between effector Treg cells (eTreg, CD4+-CD45RA-FOXP3 hi ) and resting Treg cells (rTreg, CD4+CD45RA+FOXP3 lo ) (Figs 1, S1 and S2). The advantage over the traditional Treg cell characterization by CD4+-CD25 hi CD127 lo FOXP3+ is that conventional CD4 T cells with an up-regulated CD25 and FOXP3 expression due to the generalized, infection-related immune activation are excluded from the analysis [41]. PBMC from HIV-infected individuals and healthy controls were isolated, stained with fluorescence-labelled antibodies and characterized by flow cytometry. A significantly higher percentage of PD-1+ Treg cells was observed for HIV-infected individuals (8.2% ± 0.8 SEM) compared with controls (3.0% ± 0.4 SEM) (Fig 1B). This difference in PD-1 expression was due to PD-1 on effector Treg cells (13.6% ± 1.2 SEM) since very little PD-1 was expressed on resting Treg cells (1.5% ± 0.2 SEM). These observations are concordant with previous data [36][37][38] and fit to the current understanding of PD-1 upregulation induced by T cell stimulation [42]. We also found a higher percentage of PD-L1+ Treg cells for HIV-infected individuals (6.59% ± 1.1 SEM) compared with controls (1.75% ± 0.33 SEM) ( Fig 1B). However, in contrast to the expression pattern of PD-1, an increased percentage of PD-L1+ cells was observed for both, effector and resting Treg cells. Furthermore, the expression of PD-1 and PD-L1 on Treg cells from HIV-infected individuals correlated positively (Fig 1C).
The differential distribution of PD-1 and PD-L1 on resting and effector Treg cells from HIV-infected individuals and healthy controls suggested that the virus itself could induce PD-L1 upregulation on Treg cells. To test this hypothesis, PBMC from healthy controls were isolated and exposed to HIV-1Bal containing supernatants without additional stimuli or additional interleukin-2. As controls, we used supernatants from non-infected PBMC that have been cultured under similar conditions as the virus-exposed cells. While culture supernatants from non-infected PBMC increased the frequency of PD-L1+ effector and resting Treg cells, virus exposure dramatically augmented this effect in a dose-dependent manner (Figs 1D and S3A). In contrast, virus exposure had no effect on PD-1 expression (Fig 1D).
To evaluate whether PD-L1 upregulation occurred without infection, we cultured PBMC with infectious HIV-1Bal in the presence or absence of the HIV entry inhibitor T20. PD-L1 upregulation on Treg cells occurred after virus exposure even in the presence of T20 (S3B Fig). These data are in line with previous studies that demonstrate a PD-L1 upregulation upon HIV exposure in different cell populations including monocytes, macrophages, dendritic cells, neutrophils and CCR5+T cells [43][44][45][46]. In addition, we observed that the HIV-envelope protein gp120 induced PD-L1 upregulation in Treg cells providing an extra mechanistic insight into how HIV can induce PD-L1 (S3C Fig). When taken together, our results show an upregulation of PD-1 and PD-L1 on Treg cells of HIV-infected individuals that may be mediated by different routes and suggest that the Treg cell compartment is likely to be influenced by immunotherapy targeting the PD-1/PD-L1 pathway.

PD-1 expression on Treg cells is associated with disease progression
PD-1 expression on CD4-and CD8-T cells correlates with HIV disease progression [11,13,47]. To test whether the same is true for Treg cells, we analysed PD-1 expression on these cells from HIV-infected individuals categorized into 4 groups according to CD4 T cell counts and viral load (S2 Table). The highest percentage of PD-1-expressing Treg cells was found in the HIV study group with the lowest CD4 T cell counts and highest viral loads ( Fig 2B). As many as 13.6% ± 2.3 SEM of Treg cells where PD-1+ in this group whereas only 4.7% ± 0.4 SEM of Treg cells where PD-1+ in the group of individuals under combination antiretroviral therapy (cART). PD-1 expression on Treg cells paralleled PD-1 on total CD4-T cells but differed from CD8-T cells (S4A Fig). Within viremic individuals, the percentage of PD-1+ CD8 T cells was high irrespective of the CD4 T cell counts, whereas the percentage of PD-1+ Treg and PD-1 + total CD4 T cells was higher in individuals with low CD4 T cell counts (<500 CD4/μL).
Consistently, PD-1 on Treg cells correlated positively with viral load and negatively with CD4 T cell counts (Fig 2C). This is concordant with previous observations made for CD4 T cells and CD8 T cells from HIV-infected patients [11,13,47] and reproduced here with individuals of our study groups (S4B Fig). To further substantiate the relation between PD-1 expression on Treg cells and antigen exposure, we followed 5 patients before and after antiretroviral treatment interruptions. Samples were collected from the same patient at 4 time-points, (1) before starting treatment, (2) during treatment, (3) upon interruption of treatment, and (4) after restarting treatment. As can be seen in Fig 3, the percentage of PD-1 expressing Treg cells followed viremia and cART reduced PD-1 expression on Treg cells.
PD-1 is a negative regulator of the proliferative capacity in effector T cells. To characterize the relationship between PD-1 expression and the proliferative capacity of Treg cells, PBMC from individuals of the different HIV study groups were labelled with Carboxyfluorescein succinimidyl ester (CFSE), stimulated with HIV Gag peptides and analysed for proliferation by CFSE dilution via flow cytometry. As shown in Fig 4B, the proliferative capacity of Treg cells was strikingly impaired in non-treated individuals. It correlated positively with CD4 T cell counts and negatively with viral loads (Fig 4C and 4D) and PD-1 expression on Treg cells prior to stimulation (Fig 4E). These observations parallel those reported for effector T cells [11] and suggest a negative role of PD-1 for Treg cell proliferation.

PD-L1 blockade increases the proliferative capacity of Treg cells but not their suppressive capacity per cell
To analyse the impact of a PD-L1 blockade on the proliferative capacity of Treg cells from HIV-infected individuals, CFSE-labelled PBMC were cultured in the presence of HIV Gag peptides and PD-L1 blocking antibody or an isotype control antibody. Cell proliferation was quantified by CFSE dilution via flow cytometry. A significant gain on the proliferative capacity of Treg cells, as well as that of effector CD4-and CD8-T cells, is shown in Fig 5 as fold change in proliferation relative to the isotype antibody control stimulations (p <0.0001). PD-L1 blockade led to a roughly 2 fold mean increase in the percentage of proliferating Treg cells, comparable to that of effector CD4-and CD8-T cells ( Fig 5B) (p = 0.668). The range of responses was broad. The increase in Treg cell proliferation positively correlated with the viral load of the analysed individuals (Fig 5C).
To further analyse the functional consequences that a PD-L1 blockade may have on Treg cell function after an antigenic stimulation, we analysed the increase of Treg cells expressing effector molecules such as CD39 and CTLA4 as well as their suppressive capacity. Upon PD-L1 blockade a slight but significant increase in the frequency of CD39-and CTLA4-expressing Treg cells was observed relative to the control condition (p = 0.026 and 0.039, respectively) as well as to the fold change in the percentage of Helios-expressing Treg cells (p = 0.027) (Figs 5E and S6A). The latter is a transcription factor suggested to identify thymic Treg cells, and used as a control. As expected, the frequency of Helios-expressing Treg cells did not increase upon PD-L1 blockade (p = 0.862). To test the capacity of expanded Treg cells to suppress CD8 T cell proliferation, Treg cells were isolated from PBMCs after a 6-day-culture in the presence of PD-L1 blocking antibody or an isotype control antibody, and co-cultured with CFSE-labelled PBMCs in the presence of anti-CD3/anti-CD28 and interleukin-2. Proliferation of CD8 T cells was quantified by analysing CFSE profiles by flow cytometry. A dose-dependent inhibition of CD8 T cell proliferation was observed that was not significantly different from that of isolated Treg cells expanded under control conditions (Fig 5F). Although the in vitro study of the suppressive capacity of Treg cells might not always be predictive of in vivo function, the presented data suggest that the relief of the PD-1/PD-L1 interaction during expansion does not alter the suppressive capacity of Treg cells on a per cell basis.

The proliferative capacity of Treg cells and CD8 T cells from HIVinfected individuals is differentially restored by PD-L1 blockade and depends on the plasma viremia of the host
To analyse whether the PD-L1 blockade-mediated restoration of the proliferative capacity of Treg, CD4-and CD8-T cells as shown in Fig 5B was dependent on the HIV infection stage of the host, the respective data points were grouped according to viremia, CD4 T cells counts and antiretroviral treatment (patient grouping as of S2 Table). The PD-L1 blockade significantly increased the proliferation of Treg cells from patients with high viremia irrespective of their CD4 T cell counts (Figs 6A and S6B). Treg cells from patients that controlled viremia (either spontaneously or by cART) showed no significant proliferation increase compared with their isotype antibody control stimulation. In contrast, the increase of CD8 T cell proliferation mediated by the PD-L1 blockade was significant with respect to the isotype antibody control for all 4 patient groups (Figs 6A and S6B). Importantly, the PD-L1 blockade affected the Treg cells  The mean ± SEM (standard error of the mean) is shown. Significant differences were determined by a Mann-Whitney U test, corrected for multiple comparisons using Bonferroni method, and indicated by asterisks (**p <0.01; ***p <0.001). Panels C to E show correlations between the percentage of from the high viremic groups more than the respective CD8 T cells. The inverse was true in the groups with controlled viremia.
To confirm that PD-L1 blockade differentially impacts Treg cells and CD8 T cells depending on the plasma viremia of the host, fold changes in proliferation upon PD-L1 blockade were measured longitudinally in 7 additional individuals before and after antiretroviral treatment (pre-cART and on-cART, respectively). As can be seen in Fig 6B, in samples from pre-cART treatment, PD-L1 blockade increased the proliferative capacity of Treg cells by approximately 2 fold, which is comparable to that of effector CD4-and CD8-T cells. However, in samples from the same patients after a cART period, PD-L1 blockade had no significant effect on Treg cell proliferation. In contrast, the proliferative capacity of effector T cells was increased 2 fold upon PD-L1 blockade comparable to that of pre-cART samples ( Fig 6B). As shown in Fig 6C, PD-L1 blockade preferentially increased the proliferative capacity of effector T cells over regulatory T cells in samples from individuals under cART treatment. Thus the net gain of T cell effector function after PD-L1 blockade may critically depend on plasma viremia.
To analyse the consequences of the PD-L1 blockade for ex vivo HIV reactivation, supernatants of the above-described PBMC cultures were collected and tested for viral production by HIV p24 antigen determination. HIV production was readily detectable in most PBMC cultures from the viremic patient groups (16 from 19 samples) (Fig 7A). The blockade of PD-L1 consistently increased viral production relative to the isotype antibody control stimulations. This increase of HIV production correlated positively with the increase in CD4 T cell proliferation ( Fig 7B) and the fraction of Treg cells in the lymphocyte population ( Fig 7C) but not with the increase in Treg cell proliferation (S7 Fig). Furthermore, the increase in HIV production correlated negatively with the fold changes of CD4 T cell/Treg cell as well as CD8 T cell/Treg cell ratios respectively (Fig 7D and 7E). Together this suggests that the inhibitory function of the Treg cells rather than their capacity of being an HIV target cell may play a role in virus expansion under these conditions.

Discussion
Perhaps the most striking finding of this work is the observation that PD-L1 blockade restores the proliferative capacity of regulatory T cells (Treg cells) from HIV-infected individuals differentially depending on plasma viremia. Treg cells from viremic patients show the largest fold increase in proliferation while the cell´s suppressive capacity is maintained. As Treg cells contribute to maintain exhaustion [48,49], therapeutic interventions aiming to disrupt T cell exhaustion by means of blocking the PD-1 signalling pathway should first reduce the HIV load by antiretroviral drugs. Only this may guarantee the biggest possible net gain of effector T cell function and subsequent better immunological control over HIV.
Relative to healthy controls, the frequency of PD-1-expressing Treg cells was significantly increased in all four groups of HIV-infected individuals and correlated positively with markers of disease progression such as virus load and reduction of CD4 T cells. With this, Treg cells follow the same trend as total CD4-and CD8-T cells (S4 Fig) as previously described [11,13,47]. This is intriguing, as a coordinated up-regulation of a negative signalling receptor on both effector T cells and suppressor T cells seems counterintuitive. However, it is consistent with the model of dynamic co-evolution of memory and regulatory T cells at sites of infection [50,51] and data from subsequent studies of Treg cells in chronic hepatitis C virus infection [34,52]. Accordingly, an expanding virus triggers an effector T cell response with concomitant Treg cell

(C) Correlation between the fold change in proliferating Treg cells and HIV viral load. (D) Representative flow cytometry dot plots showing gating of CD39, CTLA4 and Helios on Treg cells for one individual (eTreg cells in red and rTreg cells in black). Numbers indicate the frequencies of CD39, CTLA4 or Helios expressing Treg cells (including eTreg and rTreg). (E) Fold change in the frequencies of CD39, CTLA4 or Helios expressing Treg cells. (F) PBMC from HIV-infected individuals were stimulated with Gag peptides in the
presence of a PD-L1 blocking antibody or an isotype control antibody, after 6 days Treg cells were isolated and co-cultured with CFSE-labelled-PBMC at different PBMC-to-Treg cell ratios. The percentage of inhibition of CD8 proliferation after anti-CD3/anti-CD28 T cell stimulation is shown as a function of different PBMC-to-Treg cell ratios. Black dots connected with a solid line correspond to suppression assays performed with Treg cells from PD-L1 blockade conditions whereas empty dots connected with a dashed line correspond to suppression assays performed with Treg cells from isotype control conditions. Mean and SEM from 4 independent experiments are indicated.  generation. The killing of infected cells by effector T cells then promotes tissue injury. This is dampened via PD-1 signalling on effector cells as well as expanding Treg cells. To limit exaggerated suppression and maintain homeostasis, Treg cell expansion is also controlled by PD-1 on Treg cells. While the data from our cross-sectional study do not enable the analysis of the temporal appearance of the PD-1-expressing T cell subsets in the HIV-infected individuals studied, they are concordant with this model of an antigen-driven coordinated response in order to balance (i) virus reduction by effector T cells and (ii) reduction of immunopathology by Treg cells with (iii) maintenance of the adaptability of T cell responses to subsequent viral bursts.
The direct correlation of PD-1 on Treg cells with patient's viral load is consistent with the idea that persistent antigen exposure is a main trigger of PD-1 expression. However, exposure of PBMCs to HIV under non-stimulating conditions did not induce PD-1 on effector or on resting Treg cells significantly thus suggesting that additional activation signals are required. Interestingly, this exposure to HIV was sufficient to massively up-regulate PD-L1 on both eTreg and rTreg cells in a virus dose-dependent manner. As PD-L1 can participate in Treg cell induction [26][27][28] as well as PD-1-mediated suppression [29,30,32], the role of both Treg cell states in virus sensing and subsequent signal conversion merits further investigation.
Upon PD-L1 blockade, the fold change of Treg cell proliferation was the highest in viremic individuals and correlated with viral loads. However the fold change of Treg cell proliferation was not significantly correlated with PD-1 expression itself although PD-1 expression was correlated to viral loads. Thus there might be additional factors that have participated in the observed gain in proliferation. One good candidate for this is IL-2 that is produced upon T cell PD-L1 Blockade and Regulatory T Cells in HIV stimulation but may have been limiting with increasing PD-1 expression and negative signalling on CD4 T cells, the main IL-2 producers. Consistent with this are previous observations that (i) the PD-1/PD-L1 pathway negatively regulates Treg cell proliferation by inhibiting the IL-2 signalling cascade [34] and (ii) exogenous IL-2 can overcome PD-1/PD-L1-mediated inhibition of proliferation [53].
The PD-L1 blockade of PBMC from viremic HIV-infected individuals under stimulating conditions commonly led to increased reactivation of HIV ex vivo. The observed correlations between the fold change in virus p24 with the fold change in proliferating CD4 T cells, the percentage of Treg cells as well as the negative correlations with the fold changes of CD4 and CD8 to Treg cell ratios respectively, may indicate that an increase of Treg cells in relation to CD8 T cells promotes virus expansion. These observations are compatible with the characteristics of HIV biology and stress the importance of applying antiviral treatment in addition to the PD-L1 blockade therapy.
In summary, this ex-vivo study of Treg cell behaviour from different HIV-infected patient groups demonstrates (i) an up-regulation of PD-1 and PD-L1 that correlates with markers of disease progression and (ii) a differential and plasma viremia-dependent gain of Treg cell proliferation and overall suppressive function upon PD-L1 blockade. This has direct consequences for patient selection to enter clinical trials targeting the PD-1/PD-L1 signalling pathway and treatment modalities. While the translation of our results to HIV infection in vivo is complex, and many aspects about the weight of the possible PD-1/PD-L1 roles for HIV expansion at different anatomical sites are not completely defined, the upcoming clinical trials will definitely increase our knowledge on Treg cell biology and provide a clear picture on the pros and cons of immune checkpoint modification in HIV infection.

Human subjects and PBMC isolation
Blood was obtained from healthy, HIV-uninfected volunteers and HIV-infected individuals at the Hospital Clinic and the Hospital del Mar, both in Barcelona, Spain. HIV-infected individuals were categorized into 4 groups: (1) fewer than 500 CD4/μL and more than 2000 RNA copies/mL blood; (2) more than 500 CD4/μL and more than 2000 RNA copies/mL blood; (3) more than 500 CD4/μL and fewer than 2000 RNA copies/mL blood; (4) HIV-infected patients under successful antiretroviral treatment (cART) for at least 2 years with more than 500 CD4/μL blood and viral loads below the limit of detection (40 RNA copies/mL blood). HIV-infected individuals from the cross-sectional study with the exception of the cART group were naïve to antiretroviral therapy at the time of testing and were not in the primary infection phase (S1 and S2 Tables). In addition, we studied two subgroups of patients longitudinally. A group of 5 individuals were followed before starting cART, during cART, upon interruption of cART, and after restarting cART (S3 Table, group A). A second group of 7 individuals were followed before cART and after 2 years receiving cART (S3 Table, group B). Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll density centrifugation (Invitrogen) and frozen for subsequent analyses.

Ethics statement
Ethical committee approval and written informed consent from all subjects, in accordance with the Declaration of Helsinki, were obtained prior to study initiation.
Flow cytometry data were collected on a LSR Fortessa (BD biosciences) and analysed with Flow Jo software (Tree Star). Panels containing the corresponding isotype controls were collected to set PD-1, PD-L1, CTLA4 and Ki-67 gates. Treg cells were identified as a joint population of effector Treg cells (CD4+CD45RA-FOXP3 hi ) (eTreg) and resting Treg cells (CD4+CD45RA+FOXP3lo) (rTreg), in which the cut offs for FOXP3 were set manually in relation to CD45RA expression as previously described [40] (Figs 1 and S1A).
To verify that the CD4+CD45RA+FOXP3lo & CD4+CD45RA-FOXP3hi cell populations define Treg cells after a 6-day culture, both rTreg cells (CD4+ CD127lo CD25+ CD45RA+) and conventional CD4 T cells (CD4+ CD127+ CD25-) were isolated from HIV-infected individuals by flow cytometry, labelled with CFSE and cultured in the presence of autologous, unlabelled PBMC and Gag peptides as described below. For this, PBMC samples were enriched for CD4 T cells using magnetic beads (Miltenyi Biotec) and sorted by an ARIA SORP (BD biosciences) (S1B Fig).

PBMC exposure to HIV
HIV-1Bal was obtained from the Centre for AIDS Reagents NIBSC (repository reference: ARP118) and propagated in PHA-stimulated PBMC in RPMI-1640 media (Gibco) supplemented with 20% FBS (Sigma), 1% penicillin/ streptomycin (Gibco) and 10U/mL rhIL-2 (R&D Systems) for 7 days. Supernatants were collected, titrated on TZM-bl cells and frozen at -80°C until use. Supernatants from non-infected PBMC cultured under identical conditions were collected as mock control.
PBMC from healthy controls were cultured in the presence of HIV-1Bal (or mock) at a multiplicity of infection (MOI) of 0.3 and 0.03. To discard that PD-L1 upregulation requires HIV infection, PBMC were infected at MOI 0.3 in the presence or absence of 5 μM T20 HIV entry inhibitor. In parallel, PBMC were also cultured with HIV-1 Bal gp120 (NIH reagent program catalogue number 4961) at 0.01ng/mL and 1ng/mL. After 4 hours, HIV-exposed cells were washed twice while gp120-exposed cells were left as such. 0.5Á10 6 PBMC/well were cultured in 48-well plates in RPMI-1640 media (Gibco) supplemented with 10% FBS (Sigma), 1% penicillin/ streptomycin (Gibco). After 3 days, cells were harvested and stained to analyse PD-1 and PD-L1 expression on Treg cells by flow cytometry.
The efficacy of HIV-1Bal inhibition by T20 treatment was controlled by stimulating the virus-exposed cells from above with 5μg/ml PHA at day 3 and culturing them for further 7 days. The presence or absence of virus production was determined by a p24 HIV core antigen ELISA kit (Innogenetics). T20 treatment completely blocked HIV-1Bal infection under these conditions. Cell culture and proliferation assay 2Á10 6 PBMC/well were cultured in 24-well plates in RPMI-1640 media (Gibco) supplemented with 10% FBS (Sigma), 1% penicillin/ streptomycin (Gibco) and 1μg/mL anti-CD28 and anti-CD49d antibodies. Cells were either left unstimulated or incubated with 1μg/mL Gag pool of overlapping peptides (Gag peptides; NIH reagent program catalogue number 8117 and 8118, in part kindly provided by Anja Germann and Hagen von Briesen, Fraunhofer IBMT, Germany) plus 5μg/mL anti-PD-L1 blocking or isotype control antibodies (eBioscience).
For proliferation assays, PBMC were stained with Carboxyfluorescein succinimidyl ester (CFSE) (Invitrogen) as described in (Quah et al., Nature Protocols, 2007). After 6-day-culture cells were harvested and stained to analyse proliferation of Treg, CD4-and CD8-T cell subsets. Alternatively, to analyse proliferation after a 6-day culture in longitudinal samples for which cell numbers were limited, non-CFSE stained PBMC were cultured as previously described, and stained with Ki67 or isotype control antibodies. Fold change in proliferation (FC proliferation) was calculated as a ratio of proliferation under PD-L1 blockade condition divided by proliferation under control condition.
In vitro suppression assay for functional assessment of regulatory T cells PBMC were stimulated with Gag peptides in the presence of anti-PD-L1 blocking antibody or isotype control antibody as described above. After 6-day culture, Treg cells where isolated by magnetic beads using the CD4+CD25+CD127dim/-regulatory T cell isolation kit II (Mitenyi Biotec; Treg cell purity shown in S5 Fig). Purified Treg cells were co-cultured with 50.000 CFSE-labelled PBMC at different ratios and stimulated with 0.5μg/mL anti-CD3 and 1μg/mL anti-CD28 antibodies. Cells were cultured in 96-U bottom well plates (Greiner bio-one) in RPMI-1640 media (Gibco) supplemented with 10% FBS (Sigma), 1% penicillin/ streptomycin (Gibco), 50U/mL rhIL-2 (R&D Systems) and 1mM sodium pyruvate (Sigma). After a 3-dayculture, cells were harvested and stained to analyse proliferation of CD8 T cells. The Treg cell suppressive capacity was determined by the percentage of inhibition of CD8 proliferation, calculated as: [(CD8 proliferation − CD8 proliferation in presence of Treg cells) / CD8 proliferation] x 100.

ELISA
Culture supernatants after a 4-day culture in the presence of Gag peptides and anti-PD-L1 blocking or isotype control antibodies (as described in previous section "Cell culture and proliferation assay") were collected for p24 HIV core antigen quantification by an ELISA kit (Innogenetics).

Statistical analysis
Comparisons between two groups were performed using Mann-Whitney U test, between more than two groups using Kruskal-Wallis test and within the same patient using Wilcoxon matched pairs test. Bonferroni correction was applied to adjust significance for multiple comparisons. Correlation coefficients (r) were calculated using the Spearman rank correlation test. Categorical variables between study groups were compared using Chi-squared and Fisher's exact test.
Statistical analyses were performed using GraphPad Prism 5.0 (San Diego, CA, USA) and SPSS 15.0 statistical software (Chicago, IL, USA). p-values (P) below 0.05 were considered significant and were indicated by asterisks: Ã p<0.05; ÃÃ p<0.01; ÃÃÃ p<0.001. Non-significant differences were indicated as "ns". Supporting Information S1 Table. Comparison of demographic characteristics of HIV-infected individuals and healthy controls used in Fig 1. (PDF) S2