1. Emory Vaccine Center, Emory University School of Medicine, Atlanta, Georgia 30322, USA
2. Division of Microbiology and Immunology, Yerkes National Primate Research Center, Emory University, Atlanta, Georgia 30329, USA
3. Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts 02115, USA
4. Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115, USA
5. University of Pennsylvania School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA
6. These authors contributed equally to this work.

Correspondence to: Rama Rao Amara^1,2 Correspondence and requests for materials should be addressed to R.R.A. (Email: ramara@emory.edu).

Virus-specific T cells show varying degrees of functional impairment during chronic infections^{6, 7}. Although these T cells retain some antiviral functions, they are less polyfunctional compared with antiviral T cells seen in acute infections. This defect in T-cell function greatly contributes to the inability of the host to eliminate the persisting pathogen. The exhaustion of virus-specific T cells was first shown during persistent lymphocytic choriomeningitis virus (LCMV) infection of mice^{8, 9} and was quickly extended to other model systems including human immunodeficiency virus (HIV), hepatitis B virus (HBV) and hepatitis C virus (HCV) infections in humans^{1, 2, 10}. The co-inhibitory receptor PD-1 has been shown to be highly expressed by exhausted LCMV-specific CD8 T cells and in vivo blockade of this pathway restores function in these cells¹¹. PD-1 is also upregulated on HIV-1-specific^{12, 13, 14} and SIV-specific^{15, 16} CD8 T cells and in vitro blockade of PD-1 enhances cytokine production and proliferative capacity of these cells. However, the importance of this PD-1 inhibitory pathway in regulating T-cell function during immunodeficiency virus infection in vivo is not known. Here, we use a SIV/macaque model to evaluate the effects of in vivo blockade of PD-1 on the safety and restoration of virus-specific cellular and humoral immunity during chronic immunodeficiency virus infections.

PD-1 blockade was performed using an antibody specific to human PD-1 that blocks the interaction between macaque PD-1 and its ligands (PDLs) in vitro¹⁵. Blockade was performed during the early (10 weeks) as well as late (90 weeks) phases of chronic SIV infection. Nine macaques (five during the early phase and four during the late phase) received the anti-PD-1 antibody and five macaques (three during the early phase and two during the late phase) received an isotype control antibody (Synagis, anti-respiratory syncytial virus (RSV)-specific)¹⁷.

PD-1 blockade during chronic SIV infection resulted in a rapid expansion of SIV-specific CD8 T cells in the blood of all macaques (Fig. 1a, b). We were able to study the CD8 T-cell responses to two immunodominant epitopes, Gag CM9 (ref. 18) and Tat SL8/TL8 (ref. 19), using major histocompatibility complex (MHC) I tetrameric complexes in seven of the anti-PD-1-antibody-treated and three of the control-antibody-treated macaques that expressed the Mamu A*01 histocompatibility molecule. Consistent with previous data¹⁵, most (>98%) of the Gag-CM9 tetramer-specific CD8 T cells expressed PD-1 before blockade (data not shown). After PD-1 blockade, the Gag-CM9 tetramer-specific CD8 T cells expanded rapidly and peaked by 7–21 days. At the peak response, these levels were about 2.5–11-fold higher than their respective levels on day 0 (P = 0.007) and remained elevated until 28–45 days (Fig. 1b). Similar results were observed with blockade during the early as well as late phases of chronic SIV infection. A 3–4-fold increase in the frequency of Gag-specific interferon (IFN)--positive CD8 T cells was also observed by day 14 after blockade in the two Mamu A*01-negative animals (RTd11 and RDb11), demonstrating that PD-1 blockade can enhance the frequency of virus-specific CD8 T cells that are restricted by non-Mamu A*01 alleles (data not shown). As expected, expansion of SIV-specific CD8 T cells was not observed in the control-antibody-treated macaques (Fig. 1).

Figure 1: In vivo PD-1 blockade during chronic SIV infection increases the Gag-CM9-specific CD8 T cells with improved functional quality in both blood and gut.

a, Representative FACS plots for macaque RRk10. b, c, The magnitude and phenotype of Gag-CM9-tetramer-positive CD8 T cells in blood (b) and gut (colorectal mucosal tissue) (c). Representative FACS plots are shown on the left and summary for all Mamu A*01-positive animals is shown on the right. Numbers on the FACS plots represent the frequency of tetramer-positive cells as a per cent of total CD8 T cells. Arrows and vertical lines indicate anti-PD-1 antibody or control antibody treatment.

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PD-1 blockade was also associated with a significant increase in the frequency of virus-specific CD8 T cells that were undergoing active cell division in vivo with improved functional quality (Fig. 1b). Consistent with the rapid expansion of SIV-specific CD8 T cells, the frequency of Gag-CM9 tetramer-specific CD8 cells that co-expressed Ki67 (marker for proliferating cells) also increased as early as by day 7 after blockade (P = 0.01). Similarly, we observed an increase in the frequencies of Gag-CM9 tetramer-specific CD8 T cells co-expressing perforin and granzyme B (cytolytic potential; P = 0.001 and P = 0.03, respectively), CD28 (co-stimulation potential; P = 0.001), CD127 (proliferative potential; P = 0.0003)²⁰ and CCR7 (lymph-node homing potential; P = 0.001)²¹. We also observed a transient 1.5–2-fold increase in the frequency of tetramer-negative and Ki67-positive CD8 T cells after blockade (data not shown). This could be due to expansion of CD8 T cells specific to other epitopes in Gag as well as other proteins of SIV, and other chronic viral infections in these animals. No significant enhancement was observed for these markers in the three control-antibody-treated macaques.

Notably, no expansion was observed for Tat-TL8-specific CD8 T cells after blockade (Supplementary Fig. 1a). This could be due to viral escape from recognition by Tat-TL8-specific CD8 T cells, as PD-1 blockade is known to result in expansion of T cells only when they simultaneously receive signals through T-cell receptor. To test this possibility, we sequenced the viral genomes present in the plasma just before the initiation of blockade from all three Mamu A*01-positive macaques that were infected with SIV251 and received the blocking antibody during the early phase of infection. Indeed, we found mutations in the viral genome corresponding to the Tat TL8 epitope region (Supplementary Fig. 1b). All these mutations either have been shown or predicted to reduce the binding of Tat SL8/TL8 peptide to Mamu A*01 MHC molecule and result in escape from recognition by the Tat-SL8/TL8-specific CD8 T cells^{18, 19}. These results suggest that in vivo blockade of PD-1 may not result in expansion of T cells that are specific to escape mutants of viral epitopes.

PD-1 blockade also resulted in expansion of Gag-CM9-specific CD8 T cells at the colorectal mucosal tissue (gut), a preferential site of SIV/HIV replication²² (Fig. 1c). Expansion was not observed for two of the seven macaques, although expansion was evident for one of them in blood. In contrast to blood, the expansion in gut peaked much later by day 42 and ranged from 2- to 3-fold compared with their respective day 0 levels (P = 0.003). Similar to blood, the Gag-CM9 tetramer-specific cells that co-expressed Ki67 (P = 0.01), perforin (P = 0.03), granzyme B (P = 0.01) and CD28 (P = 0.01) also increased in the gut after blockade.

More importantly, PD-1 blockade also enhanced the functional quality of anti-viral CD8 T cells and resulted in the generation of polyfunctional cells capable of co-producing the cytokines IFN-, tumour-necrosis factor (TNF)- and interleukin (IL)-2 (Fig. 2). On the day of initiation of PD-1 blockade during the late chronic phase of infection, the frequency of Gag-specific IFN--positive cells was low and they failed to co-express TNF- and IL-2 (Fig. 2a). However, after the blockade, the frequency of IFN--positive cells increased in all four PD-1 antibody-treated macaques (P = 0.03) and they acquired the ability to co-express TNF- and IL-2. The expansion of IFN--positive cells peaked by 14–21 days and the peak levels were 2–10-fold higher than the respective day 0 levels. On day 21, about 16% of the total Gag-specific cells co-expressed all three cytokines, and about 30% co-expressed IFN- and TNF- (Fig. 2b). This is in contrast to <1% of the total Gag-specific cells co-expressing all three cytokines (P = 0.01), and about 14% co-expressing IFN- and TNF- on day 0 (P = 0.04). Similar results were also observed after blockade during the early chronic phase of infection (data not shown).

Figure 2: In vivo PD-1 blockade during chronic SIV infection increases the polyfunctional virus-specific CD8 T cells.

a, Frequency of Gag-specific cytokine-secreting CD8 T cells as a percentage of total CD8 T cells. Representative FACS plots are shown on the left and summary for the group is shown on the right. Arrows and vertical lines indicate anti-PD-1 antibody or control antibody treatment. Green lines represent anti-PD-1-antibody-treated macaques and red lines represent control-antibody-treated macaques. NS, no stimulation. b, Cytokine co-expression subsets expressed as a percentage of total cytokine-positive cells. Mean for the group is shown. Single producers, IFN-⁺, TNF-⁺ or IL-2⁺; double producers, IFN-⁺TNF-⁺, IFN-⁺IL-2⁺ or IL-2⁺TNF-⁺; triple producers, IFN-⁺IL-2⁺TNF-⁺.

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Recent studies have shown that chronic immunodeficiency virus infections are also associated with B-cell dysfunction³ and very little is known about the role of PD-1 in regulating B-cell function/exhaustion. To understand the role of PD-1 in regulating B-cell function during chronic immunodeficiency virus infections, we characterized the B-cell responses after PD-1 blockade in SIV-infected macaques (Fig. 3). Analysis of PD-1 expression on different B-cell subsets before PD-1 blockade revealed preferential expression of PD-1 by memory B cells (CD20⁺CD27⁺CD21^-) compared to naive B cells (CD20⁺CD27^-CD21⁺; Fig. 3a, P < 0.001). In vivo blockade of PD-1 resulted in a 2–8-fold increase in the titre of SIV-specific binding antibody by day 28 after blockade (P < 0.001; Fig. 3b). To understand this further, we studied the proliferation of memory B cells in SIV-infected macaques that were treated simultaneously with anti-PD-1 antibody and anti-retroviral therapy and observed a significant increase in Ki67⁺ (proliferating) memory, but not naive, B cells as early as day 3 (Fig. 3c). These results demonstrate that the PD-1–PDL pathway could have a role in regulating B-cell dysfunction during chronic SIV infection. Neutralization assays revealed a twofold increase in titres against the easily neutralizable laboratory-adapted SIV251 and no increase in titres against hard-to-neutralize wild-type SIV251 or SIV239 (data not shown). In two of the nine animals treated with anti-PD-1 antibody, we observed only a minimal (<2-fold) expansion of SIV-specific antibody after blockade. Notably, the frequency of total memory B cells in these two animals was lower (40% of total B cells) compared with the remaining seven animals (60–90% of total B cells) before blockade (data not shown), indicating that the level of SIV-specific memory B cells before blockade may determine the level of expansion of SIV-specific antibody after blockade.

Figure 3: In vivo PD-1 blockade during chronic SIV infection enhances SIV-specific humoral immunity.

a, Expression of PD-1 on memory (CD20⁺CD27⁺CD21^-) and naive (CD20⁺CD27^-CD21⁺) B cells in blood after SIV infection and before in vivo PD-1 blockade. b, Titres of anti-SIV Env-binding antibody in serum after blockade. c, Ki67 expression (marker for proliferation) on memory and naive B cells after blockade. Numbers on the FACS plots represent Ki67-positive cells as a percentage of respective total naive/memory B cells. Macaques RAf11 and RJd11 were treated simultaneously with anti-PD-1 antibody and anti-retroviral therapy at 22 weeks after SIV infection.

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PD-1 blockade resulted in significant reductions in plasma viraemia (P = 0.03) and also prolonged the survival of SIV-infected macaques (P = 0.001; Fig. 4). In two of the five macaques treated with anti-PD-1 antibody during the early chronic phase, viral load declined by day 10 and persisted at or below this level until day 90 (Fig. 4a). In one macaque viral load declined transiently and in the remaining two macaques increased transiently and returned to pre-blockade levels. In contrast to the early chronic phase, all four macaques treated with the anti-PD-1 antibody during the late chronic phase showed a transient increase in viraemia by day 7, but rapidly reduced the virus load by day 21 to levels that were below their respective day 0 levels (Fig. 4b). However, the viral RNA levels returned to pre-blockade levels by day 43. As expected, no significant reductions in the plasma viral loads were observed in any of the five macaques treated with the control antibody (Fig. 4c). By 21–28 days after blockade, the viral RNA levels in the anti-PD-1-antibody-treated animals were 2–10-fold lower than their respective day 0 levels (P = 0.03; Fig. 4d). By day 150 after the blockade, four of the five macaques in the control group were killed owing to AIDS-related symptoms (for example loss of appetite, diarrhoea, weight loss), whereas all nine animals in the anti-PD-1-antibody-treated group had survived (P = 0.001; Fig. 4e).

Figure 4: In vivo PD-1 blockade reduces plasma viraemia and prolongs survival of SIV-infected macaques.

a–c, Plasma viral load in macaques treated with anti-PD-1 antibody during the early chronic phase of infection (a), macaques treated with anti-PD-1 antibody during the late chronic phase of infection (b), and macaques treated with control antibody during the early/late chronic phase of SIV infection (c). An asterisk indicates death of animal. d, Fold reduction in plasma viral load between day 0 and day 28 (early chronic study) or day 0 and day 21 (late chronic study). The colour scheme is the same as that used in a–c. e, Survival of SIV-infected macaques after PD-1 blockade.

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The observed initial rise in plasma viraemia levels in all of the late-phase-treated and some of the early-phase-treated animals could be due to an increase in the frequency of activated CD4 T cells. To determine this, we measured the percentage of Ki67-positive total CD4 T cells as well as the frequency of SIV Gag-specific IFN--producing CD4 T cells (preferential targets for virus replication²³) after blockade (Supplementary Fig. 2). These analyses revealed a transient increase in the percentage of Ki67-positive CD4 T cells by day 7–14 after blockade (P = 0.002) and this increase was higher in animals treated during the late phase than early phase of infection (P = 0.015; Supplementary Fig. 2a). Similarly, an increase in the frequency of Gag-specific CD4 T cells was also observed, but only in animals treated during the late phase of infection (Supplementary Fig. 2b). No significant increases were observed for these activated CD4 T cells in the control-antibody-treated macaques. These results suggest that the activated CD4 T cells could have contributed to the observed initial rise in plasma viraemia levels after blockade.

It is to be noted that, before initiation of PD-1 blockade, the set point viral load in plasma and total CD4 T cells in blood and gut were similar between the anti-PD-1-antibody-treated and control-antibody-treated groups (Supplementary Fig. 3). However, the frequencies of Gag CM9⁺ cells and Gag CM9⁺ cells co-expressing perforin, granzyme B or CD28 were not similar between the two treatment groups before in vivo blockade (Fig. 1b). This raises the possibility that these differences could have contributed to the expansion of Gag CM9⁺ cells after PD-1 blockade. To study the influence of the frequency of Gag CM9⁺ cells before blockade on their expansion after blockade, we divided the anti-PD-1-antibody-treated group into two subgroups based on the frequency of Gag CM9⁺ cells before initiation of blockade such that one group has similar levels and the other group has higher levels of Gag CM9⁺ cells compared with the control-antibody-treated group. These subgroups were then analysed for expansion of Gag CM9⁺ cells after blockade. Expansion of Gag CM9⁺ cells was evident in both subgroups of animals after blockade of PD-1, irrespective of whether they were at low or high levels before blockade (Supplementary Fig. 4). Similar results were also observed with subgroup analyses based on the frequency of Gag CM9⁺ cells co-expressing molecules associated with better T-cell function such as perforin, granzyme B, CCR7, CD127 or CD28 (Supplementary Fig. 4). However, we observed a trend towards better expansion of Gag CM9⁺CD28⁺ cells in animals with higher levels of Gag CM9⁺CD28⁺ cells before blockade, suggesting that CD28 expression may serve as a biomarker for predicting the outcome of in vivo PD-1 blockade.

To evaluate the safety of PD-1 blockade, we performed an extensive analysis of serum proteins, ions, lipids, liver and kidney enzymes, and complete blood count after blockade (Supplementary Tables 1 and 2). These analyses revealed no significant changes for all parameters tested between the anti-PD-1-antibody-treated and control-antibody-treated macaques (data not shown). Similarly, the levels of anti-nuclear antibodies (ANA) in serum (measure of autoimmunity) did not change significantly after treatment with anti-PD-1 antibody (Supplementary Fig. 5). In one macaque, the levels of ANA increased about 3-fold by day 10 after blockade, but returned to day 0 levels by day 56. These results demonstrate that anti-PD-1 antibody treatment during chronic SIV infection results in no observable toxicity. This is consistent with a recent study that demonstrated the safety of PD-1 blockade in patients with advanced haematological malignancies²⁴.

We studied the pharmacokinetics of the partially humanized anti-PD-1 antibody in serum after in vivo blockade. The titre of anti-PD-1 antibody rapidly declined between days 14 and 28 after blockade (Supplementary Fig. 6a) and coincided with macaques generating antibody response against the mouse immunoglobulin variable domains of anti-PD-1 antibody (Supplementary Fig. 6b). These results suggest that the use of completely humanized anti-PD-1 antibody may allow longer periods of treatment that may further enhance the efficacy of in vivo blockade.

Our results demonstrate that in vivo blockade of PD-1 during chronic SIV infection is safe and results in rapid expansion and restoration of SIV-specific polyfunctional CD8 T cells and enhanced B-cell responses. Expansion was observed with blockade performed during the early as well as late phases of chronic infection even under conditions of high levels of persisting viraemia and AIDS. Expansion was also observed at the colorectal mucosal tissue, a preferential site of SIV/HIV replication²². Importantly, PD-1 blockade resulted in a significant reduction of plasma viral load and also prolonged the survival of SIV-infected macaques. These results are highly significant considering the failure of blockade of a related co-inhibitory molecule CTLA-4 to expand virus-specific CD8 T cells and to reduce plasma viral load in SIV-infected macaques²⁵. The therapeutic benefits of PD-1 blockade may be improved further by using combination therapy with anti-retrovirals and/or therapeutic vaccination.

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Supplementary Information

Supplementary information accompanies this paper.

Acknowledgements

The authors thank J. D. Altman for provision of Gag CM9 and Tat SL8 tetramers, H. Drake-Perrow for administrative support, and D. Watkins and Wisconsin National Primate Research Center Genotyping Service for Mamu typing of animals. The authors also thank the Yerkes Division of Research Resources for pathology support, Emory CFAR virology core for viral load assays and the NIH AIDS Research and Reference Reagent Program for the provision of peptides. This work was supported by the National Institutes of Health/National Institute of Allergy and Infectious Diseases grants R01 AI057029, R01 AI071852, R01 AI074417 to R.R.A.; the Foundation for the NIH through the Grand Challenges in Global Health initiative P51 RR00165 to R.A., G.J.F. and R.R.A.; Yerkes National Primate Research Center base grant P51 RR00165; Emory CFAR grant P30 AI050409; and R24 RR16038 to David I. Watkins.

Author Contributions V.V. and K.T. contributed to the design of experiments, conducted analyses on T-cell responses, and contributed to manuscript preparation; S.H. performed analyses on T-cell phenotyping. T.H.V. performed analyses on viral escape. L.L. and A.P. performed analyses on humoral responses; L.C. performed the statistical analysis; G.S. supervised the analyses on viral escape and contributed to manuscript preparation; B.Z. and G.J.F. developed and provided the anti-human PD-1 blocking antibody, and contributed to the design of experiments and manuscript preparation. R.A. contributed to the concept, design of experiments and manuscript preparation. R.R.A. supervised the entire project, designed and coordinated the experiments, and contributed to manuscript preparation.

Author Information

Sequencing data related to the Tat SL8/TL8 epitope region have been deposited in GenBank (accession numbers FJ268664–FJ268704).

Competing interests statement

The authors declare  competing financial interests.

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