Comparison of transcriptional profiles between PD-1 antibody– (n
= 5) and control antibody–treated (n
= 3) SIV-infected RMs at 14 days following PD-1 blockade revealed significant changes (>1.5-fold change and P
< 0.05) in 236 genes in the gut and 312 genes in blood. We then categorized these significantly altered genes in terms of their role in biological pathways using ingenuity pathway analysis (IPA). Consistent with our prior findings (8
), IPA also revealed a significant upregulation of genes associated with TCR signaling (Figure A). However, we observed a significant downregulation of genes involved in type I IFN and IFN regulatory factor (IRF) activation signaling, which suggested a negative influence on these pathways and possible reduction of immune activation in PD-1 antibody–treated SIV-infected RMs.
PD-1 blockade downregulates type I IFN responses in SIV-infected RMs.
Sustained high levels of type I IFN responses in blood, lymphoid, and gut tissue of pathogenic SIV-infected RMs have been implicated in immune activation and faster disease progression (11
). Also, HIV-specific T cells from progressors compared with controllers express higher levels of type I IFN–responsive genes (14
). We further looked into the individual genes involved in IFN signaling that were significantly modulated at 14 days following PD-1 blockade in our microarray analyses and found a number of type I IFN stimulatory genes (ISGs) to be downmodulated by about 2- to 4-fold following PD-1 blockade, in both blood and gut (Figure B and Supplemental Figure 1; supplemental material available online with this article; doi:
). Temporal microarray analysis of ISG expression before and after PD-1 blockade in two SIV-infected late chronic RMs also revealed a similar downregulation of many ISGs (Supplemental Figure 2). It is important to note that consistent with previous reports (12
), expression of all of these ISGs was greatly increased following SIV infection (Figure B).
We next performed quantitative real-time PCR (qPCR) analysis to study the mRNA expression kinetics of one of the significantly modulated ISGs, MX1
, in the gut using longitudinal samples from the same macaque before and after infection, and following PD-1 blockade (Figure C). These analyses revealed a strong increase in MX1
transcripts following SIV infection and significant downregulation following PD-1 blockade that was evident even at 90 days (last point of analysis) following blockade. As expected, no significant reduction in MX1
levels was observed in control antibody–treated animals. This sustained reduction in immune activation in PD-1 antibody–treated animals was not due to decreased SIV levels, as the viral RNA levels were comparable in the gut between anti-PD-1 antibody–treated and control antibody–treated animals (Figure D) and remained relatively high in the plasma (Supplemental Figure 3). These results demonstrate that in vivo PD-1 blockade reduces expression of SIV-induced ISGs independent of virus levels, a condition that is normally seen in chronically SIV-infected sooty mangabeys (12
) and African green monkeys (13
Sustained proinflammatory responses could lead to changes in tight junction gene expression and alter the gut permeability barrier (15
). So, we next investigated whether in vivo PD-1 blockade influenced tight junction–associated gene expression and gut permeability barrier. First, we analyzed the expression of genes associated with tight junctions in our microarray analyses. We found that transcripts encoded by genes associated with tight junctions, such as claudin 5 (CLDN5
), junction adhesion molecule 2 (JAM2
), connexin-45 (Cx45
), and connexin-43 (Cx43
) increased following PD-1 blockade (Figure A). We then confirmed the upregulation of transcripts specific for JAM2
, and Cx45
using qPCR analysis, which showed enhanced expression of these genes following PD-1 blockade (Figure B). It is important to note that all of these genes have been shown to improve gut permeability barrier function (16
). These results demonstrate that in vivo PD-1 blockade induces expression of some of the important tight junction genes during chronic SIV infection and suggest an increase in gut permeability barrier function. The mechanisms by which PD-1 blockade induces the expression of tight junction genes are not clear. We speculate that the reduced proinflammatory responses could have contributed to enhanced survival of gut epithelial cells. Alternately, PD-1 blockade could directly influence gut epithelial cells, as these cells express one of the PD-1 ligands, PD-L1 (1
PD-1 blockade enhances gut junction–associated gene expression and reduces microbial translocation in SIV-infected RMs.
Previous studies demonstrated an association between decreased gut permeability barrier function and microbial translocation measured as increased LPS levels in plasma (19
); so, we measured LPS levels in plasma following SIV infection and PD-1 blockade. As expected, we observed a significant increase in plasma LPS levels following SIV infection, and we found a dramatic decrease in plasma LPS levels as early as 14 days following PD-1 blockade (Figure C). By 90 days following blockade, the plasma LPS in all animals reached preinfection levels. We could not follow LPS levels long-term in some of the control antibody–treated animals because they progressed to AIDS and were euthanized. However, in two of the control antibody–treated animals, we did not observe a significant decrease in LPS levels. These results suggest that in vivo PD-1 blockade during chronic SIV infection not only restores gut permeability barrier function but also reduces microbial translocation.
Reduced microbial translocation into the blood could also result from reduced microbial burden in the gut. Since PD-1 blockade can enhance the function of immunity against persistent antigens (1
), we investigated the effect of in vivo PD-1 blockade on cellular and humoral immunity in blood against the gut-resident pathogens. Specifically, we studied the antibody and CD8+
T cell responses against Campylobacter
, one of the common gut-resident bacteria in our macaque colony. The Campylobacter
-specific antibody titers increased significantly (>2-fold) in sera of 5 of the 9 PD-1 antibody–treated animals at 90 days following blockade (Figure A). Similarly, Campylobacter
T cell levels increased in 6 of the 9 PD-1 antibody–treated animals by 90 days following blockade (Figure B). A similar increase was also observed for Salmonella
-specific (another gut-resident bacterium) CD8+
T cells in the blood following PD-1 blockade (Figure B). Control RMs did not show any increase in CD8+
T cell responses against Campylobacter
, but rather we observed a gradual decrease in T cell responses in these animals. These results showed that the enhanced immune responses against gut-resident pathogens could have contributed to the reduced immune activation and microbial translocation following PD-1 blockade.
In vivo PD-1 blockade enhances immunity to pathogenic gut bacteria, decreases occurrence of opportunistic infections, and prolongs survival of SIV-infected RMs.
To further confirm the reduced microbial burden in the gut, we followed the occurrence of opportunistic microbial infections such as Campylobacter, Cryptosporidium, Shigella, and Trichuris in stool samples of the 5 early chronic PD-1 antibody–treated RMs following SIV infection and PD-1 blockade, compared with the 3 early chronic control antibody–treated and 8 early chronic “no antibody”–treated SIV-infected RMs with comparable set point viral load (Figure C, Supplemental Figure 3, and Supplemental Tables 1 and 2). For these analyses, we excluded the late chronic animals, because members of this group naturally had a low incidence of opportunistic infections, with lower set point viremia and better preservation of immune function as evidenced by longer survival. About 10%–20% of the animals in the control group developed one or more opportunistic infections by 3 months after SIV infection, with about 40% developing infections by 5 months. Similarly, about 20% of the animals in the PD-1 group were positive for opportunistic infection(s) at 2 months after SIV infection (2 weeks before blockade). However, following blockade, all animals in the PD-1 group remained negative for opportunistic infection(s) for about 5 months, demonstrating that in vivo PD-1 blockade enhanced the control of opportunistic infections in the gut.
Hyperimmune activation has been shown to be one of the strong predictors of disease progression, and the reduction of hyperimmune activation by the anti-PD-1 antibody treatment could contribute to enhanced survival. Consistent with this hypothesis, animals in the PD-1 group survived significantly longer than the animals in the control group following SIV infection (Figure D). About 55% (6 of 11) of the animals in the control group died by 270 days after SIV infection, whereas all 5 animals in the early chronic PD-1 group were alive at this time, demonstrating that in vivo PD-1 blockade enhanced the survival of SIV-infected RMs despite high viremia. This enhanced survival persisted even at 350 days after SIV infection (time of euthanasia to terminate the study), at which time 4 of 5 animals in the early chronic PD-1 group had survived. For the data presented in Figure D, we did not include data from the remaining 4 late chronic PD-1–treated animals for the reasons discussed above. However, none of these 4 late chronic PD-1–treated animals showed any evidence of opportunistic infections following blockade, and all survived for more than 200 days following PD-1 blockade (data not shown).
In conclusion, our results reveal an unanticipated but critical finding that a short treatment (10 days) with anti–PD-1 antibody during chronic SIV infection reduces hyperimmune activation and microbial translocation even under conditions of high viremia. More importantly, the reduced hyperimmune activation was associated with enhanced survival of SIV-infected RMs. These results are similar to what is seen in nonprogressive SIV infection in naturally infected hosts (12
) and HIV infection in viremic nonprogressors (21
). The effects of PD-1 blockade on reducing hyperimmune activation could be a result of a combination of enhanced immunity against gut-resident pathogenic bacteria and repair of gut permeability barrier function. These results could aid in the development of novel therapeutic strategies to treat HIV/AIDS. For example, it may be that combination antiretroviral therapy in humans reduces hyperimmune activation to just a limited extent (22
) because immunity against opportunistic infections is only partially restored. Our results suggest that combining PD-1 blockade with highly active antiretroviral therapy (HAART) may improve the benefits of HAART by enhancing immunity against opportunistic infections and reducing microbial translocation and hyperimmune activation.