Tissue-Specific Differences in PD-1 and PD-L1 Expression during Chronic Viral Infection: Implications for CD8 T-Cell Exhaustion

The PD-1/PD-L pathway plays a major role in regulating T-cell exhaustion during chronic viral infections in animal models, as well as in humans, and blockade of this pathway can revive exhausted CD8⁺ T cells. We examined the expression of PD-1 and its ligands, PD-L1 and PD-L2, in multiple tissues during the course of chronic viral infection and determined how the amount of PD-1 expressed, as well as the anatomical location, influenced the function of exhausted CD8 T cells. The amount of PD-1 on exhausted CD8 T cells from different anatomical locations did not always correlate with infectious virus but did reflect viral antigen in some tissues. Moreover, lower expression of PD-L1 in some locations, such as the bone marrow, favored the survival of PD-1^Hi exhausted CD8 T cells, suggesting that some anatomical sites might provide a survival niche for subpopulations of exhausted CD8 T cells. Tissue-specific differences in the function of exhausted CD8 T cells were also observed. However, while cytokine production did not strictly correlate with the amount of PD-1 expressed by exhausted CD8 T cells from different tissues, the ability to degranulate and kill were tightly linked to PD-1 expression regardless of the anatomical location. These observations have implications for human chronic infections and for therapeutic interventions based on blockade of the PD-1 pathway.Chronic viral infections are often associated with CD8⁺ T-cell dysfunction (30). This dysfunction, termed exhaustion, includes defects in the ability to produce antiviral cytokines, poor cytotoxicity, a loss of antigen-independent self-renewal, and the inability to vigorously re-expand following antigen exposure (30). These functional deficiencies contrast with the highly functional memory CD8⁺ T cells that are generated after acute infection and maintained via interleukin-7 (IL-7)- and IL-15-mediated homeostatic proliferation (30). During chronic viral infections, T-cell exhaustion often correlates with poor control of viral replication (3, 8, 38, 39). Thus, there is considerable interest in developing strategies to reverse exhaustion and restore function in virus-specific CD8⁺ T cells during chronic infections.Recent studies have revealed an important role for the negative regulatory molecule PD-1 in CD8 T-cell exhaustion during chronic viral infections (29). PD-1, a member of the CD28/CTLA-4 family of costimulatory/coinhibitory receptors, contains both ITIM and ITSM motifs in the intracellular tail and can deliver negative signals, at least partly via recruitment of the phosphatase Shp-2 (29). A role for PD-1 in regulating T-cell responses to chronic viral infections was first observed using lymphocytic choriomeningitis virus (LCMV) infection of mice, where PD-1 was found to be highly expressed on exhausted CD8⁺ T cells from chronically infected animals but not on functional memory CD8⁺ T cells from mice that had cleared an acute strain of the virus (3). In vivo blockade of the PD-1 pathway led to a dramatic increase in the number of virus-specific CD8⁺ T cells, improved functionality of these cells, and enhanced control of viral replication (3). These observations were extended to human chronic viral infections, and a series of studies have demonstrated that human immunodeficiency virus (HIV)-, hepatitis C virus (HCV)-, and HBV-specific CD8⁺ T cells upregulate PD-1 in humans compared to CD8⁺ T cells specific for nonpersisting viruses such as influenza virus or vaccinia virus (6-8, 24, 26, 32, 33, 42). Increasing PD-1 expression also correlates with disease status during HIV infection (8, 42). In vitro blockade of PD-1-PD-L interactions can reinvigorate exhausted virus-specific T-cell responses in humans and appears to have a prominent impact on proliferative expansion and/or prevention of apoptosis in these cases (9, 24, 32). Finally, recent results from in vivo blockade in the macaque simian immunodeficiency virus (SIV) infection model demonstrated the effectiveness of blocking PD-1 in primates during chronic viral infection (36). In these studies, PD-1 blockade enhanced virus-specific T and B-cell responses, lowered viral load, and improved the survival of chronically infected animals. Thus, PD-1 has emerged as not only a major regulator of T-cell exhaustion and viral control during chronic infection but also as an important potential therapeutic target.Despite these important studies and the clear impact of PD-1 blockade on the reversal of T-cell exhaustion, important questions remain. For example, previous work has demonstrated that PD-1 expression is not uniform on subsets of exhausted CD8 T cells (4). However, the expression of PD-1 on exhausted CD8 T cells in multiple tissues, and the relationship between PD-1 expression in these tissues to viral load, the PD-1 ligands and function has not been examined. Given the nonlymphoid accumulation of virus-specific CD8 T cells during chronic viral infections (11, 39) and the predilection of many important chronic infections for replicating in anatomically restricted locations (e.g., HCV and the liver, HIV and mucosal tissues, etc.), the dynamics of PD-1 expression by exhausted CD8 T cells outside the blood and spleen could have important therapeutic implications.In the present study we examined these issues using the mouse model of LCMV infection. Our results demonstrate that exhausted CD8 T cells have a wide range of PD-1 expression in different tissues of chronically infected mice. Virus-specific CD8 T cells in some anatomical locations such as the liver, brain, and bone marrow (BM) expressed high PD-1 for substantially longer than virus-specific CD8⁺ T cells from the spleens or blood of the same mice. Although PD-1 expression in the spleen correlated well with reduced gamma interferon (IFN-γ) and tumor necrosis factor (TNF) production, the PD-1^Hi virus-specific CD8⁺ T cells from the BM remained capable of producing antiviral cytokines ex vivo. In contrast, a strong negative correlation between PD-1 expression and cytotoxicity existed for exhausted CD8 T cells from all tissues tested. PD-L1 expression was high in the spleen, whereas in the BM antigen-presenting cell (APC) populations expressed lower amounts of PD-L1. Survival of PD-1^Hi CD8⁺ T cells from the BM was decreased in the presence of splenic APCs, suggesting that different tissue microenvironments in vivo could selectively support the persistence of PD-1^Hi exhausted CD8 T cells. Since PD-1 expression differs by anatomical location, these observations suggest that PD-1 blockade in vivo will have varying impacts on exhausted CD8 T cells from different tissues or anatomical locations. These observations have implications for human chronic infections such as HBV, HCV, and HIV.     

Programmed death 1 expression on HIV-specific CD4+ T cells is driven by viral replication and associated with T cell dysfunction

Functional impairment of HIV-specific CD4(+) T cells during chronic HIV infection is closely linked to viral replication and thought to be due to T cell exhaustion. Programmed death 1 (PD-1) has been linked to T cell dysfunction in chronic viral infections, and blockade of the PD-1 pathway restores HIV-specific CD4(+) and CD8(+) T cell function in HIV infection. This study extends those findings by directly examining PD-1 expression on virus-specific CD4(+) T cells. To investigate the role of PD-1 in HIV-associated CD4(+) T cell dysfunction, we measured PD-1 expression on blood and lymph node T cells from HIV-infected subjects with chronic disease. PD-1 expression was significantly higher on IFN-gamma-producing HIV-specific CD4(+) T cells compared with total or CMV-specific CD4(+) T cells in untreated HIV-infected subjects (p = 0.0001 and p < 0.0001, respectively). PD-1 expression on HIV-specific CD4(+) T cells from subjects receiving antiretroviral therapy was significantly reduced (p = 0.007), and there was a direct correlation between PD-1 expression on HIV-specific CD4(+) T cells and plasma viral load (r = 0.71; p = 0.005). PD-1 expression was significantly higher on HIV-specific T cells in the lymph node, the main site of HIV replication, compared with those in the blood (p = 0.0078). Thus, PD-1 expression on HIV-specific CD4(+) T cells is driven by persistent HIV replication, providing a potential target for enhancing the functional capacity of HIV-specific CD4(+) T cells.     

Programmed Death-1 expression on Epstein Barr virus specific CD8+ T cells varies by stage of infection, epitope specificity, and T-cell receptor usage

Background

Programmed Death-1 (PD-1) is an inhibitory member of the CD28 family of molecules expressed on CD8+ T cells in response to antigenic stimulation. To better understand the role of PD-1 in antiviral immunity we examined the expression of PD-1 on Epstein-Barr virus (EBV) epitope-specific CD8+ T cells during acute infectious mononucleosis (AIM) and convalescence.

Methodology/Principal Findings

Using flow cytometry, we observed higher frequencies of EBV-specific CD8+ T cells and higher intensity of PD-1 expression on EBV-specific CD8+ T cells during AIM than during convalescence. PD-1 expression during AIM directly correlated with viral load and with the subsequent degree of CD8+ T cell contraction in convalescence. Consistent differences in PD-1 expression were observed between CD8+ T cells with specificity for two different EBV lytic antigen epitopes. Similar differences were observed in the degree to which PD-1 was upregulated on these epitope-specific CD8+ T cells following peptide stimulation in vitro. EBV epitope-specific CD8+ T cell proliferative responses to peptide stimulation were diminished during AIM regardless of PD-1 expression and were unaffected by blocking PD-1 interactions with PD-L1. Significant variability in PD-1 expression was observed on EBV epitope-specific CD8+ T cell subsets defined by V-beta usage.

Conclusions/Significance

These observations suggest that PD-1 expression is not only dependent on the degree of antigen presentation, but also on undefined characteristics of the responding cell that segregate with epitope specificity and V-beta usage.     

Liver-infiltrating lymphocytes in chronic human hepatitis C virus infection display an exhausted phenotype with high levels of PD-1 and low levels of CD127 expression

The majority of people infected with hepatitis C virus (HCV) fail to generate or maintain a T-cell response effective for viral clearance. Evidence from murine chronic viral infections shows that expression of the coinhibitory molecule PD-1 predicts CD8+ antiviral T-cell exhaustion and may contribute to inadequate pathogen control. To investigate whether human CD8+ T cells express PD-1 and demonstrate a dysfunctional phenotype during chronic HCV infection, peripheral and intrahepatic HCV-specific CD8+ T cells were examined. We found that in chronic HCV infection, peripheral HCV-specific T cells express high levels of PD-1 and that blockade of the PD-1/PD-L1 interaction led to an enhanced proliferative capacity. Importantly, intrahepatic HCV-specific T cells, in contrast to those in the periphery, express not only high levels of PD-1 but also decreased interleukin-7 receptor alpha (CD127), an exhausted phenotype that was HCV antigen specific and compartmentalized to the liver, the site of viral replication.     

T cells from Programmed Death-1 deficient mice respond poorly to Mycobacterium tuberculosis infection

Background

Programmed Death-1 (PD-1; CD279) receptor molecule is widely believed to be a negative regulator predominantly expressed by exhausted/activated mouse T cells. Upon interaction with its ligands, PD-L1 and PD-L2, PD-1 inhibits activation of T cells and cytokine production, which has been documented in various viral and fungal infections as well as in vitro studies. Therefore, inhibition of T cell responses by PD-1 resulted in disease resistance in a variety of mouse infection models studied heretofore.

Methodology/Principal Findings

Here, we report that PD-1 deficient (PD-1^−/−) mice infected with Mycobacterium tuberculosis (M. tb) H37Rv by the aerosol route have increased susceptibility as compared with their wild type littermates. Surprisingly, M. tb antigen-specific T cell proliferation was dramatically reduced in PD-1 deficient animals compared with wild-type littermates, and this was due to increased numbers of regulatory T cells (Tregs) and recruitment of mesenchymal stem cells. Furthermore, PD-1^−/− mice exhibited decreases in the autophagy-induced LC3-B marker protein in macrophages.

Conclusions/Significance

Our findings suggest that PD-1 does not play an inhibitory role during M. tb infection and instead promotes mycobacterial clearance in mice.     

Enhancing Virus-Specific Immunity In Vivo by Combining Therapeutic Vaccination and PD-L1 Blockade in Chronic Hepadnaviral Infection

Hepatitis B virus (HBV) persistence is facilitated by exhaustion of CD8 T cells that express the inhibitory receptor programmed cell death-1 (PD-1). Improvement of the HBV-specific T cell function has been obtained in vitro by inhibiting the PD-1/PD-ligand 1 (PD-L1) interaction. In this study, we examined whether in vivo blockade of the PD-1 pathway enhances virus-specific T cell immunity and leads to the resolution of chronic hepadnaviral infection in the woodchuck model. The woodchuck PD-1 was first cloned, characterized, and its expression patterns on T cells from woodchucks with acute or chronic woodchuck hepatitis virus (WHV) infection were investigated. Woodchucks chronically infected with WHV received a combination therapy with nucleoside analogue entecavir (ETV), therapeutic DNA vaccination and woodchuck PD-L1 antibody treatment. The gain of T cell function and the suppression of WHV replication by this therapy were evaluated. We could show that PD-1 expression on CD8 T cells was correlated with WHV viral loads during WHV infection. ETV treatment significantly decreased PD-1 expression on CD8 T cells in chronic carriers. In vivo blockade of PD-1/PD-L1 pathway on CD8 T cells, in combination with ETV treatment and DNA vaccination, potently enhanced the function of virus-specific T cells. Moreover, the combination therapy potently suppressed WHV replication, leading to sustained immunological control of viral infection, anti-WHs antibody development and complete viral clearance in some woodchucks. Our results provide a new approach to improve T cell function in chronic hepatitis B infection, which may be used to design new immunotherapeutic strategies in patients.     

Cutting edge: programmed death-1 up-regulation is involved in the attrition of cytomegalovirus-specific CD8+ T cells in acute self-limited hepatitis B virus infection

Attrition of heterologous virus-specific CD8(+) T cells has been demonstrated in murine viral infection; however, little is known regarding this phenomenon in human viral infections. In this study, we observed that CMV-specific CD8(+) T cells displayed numerical decline and functional impairment in the early phase of acute infection, whereas programmed death-1 (PD-1) expression was significantly up-regulated by these CMV-specific CD8(+) T cells. This early PD-1 up-regulation was found to be closely associated with the increased apoptotic sensitivity of CMV-specific CD8(+) T cells. The in vitro addition of anti-PD-1 further enhanced the spontaneous apoptosis of CMV-specific CD8(+) T cells; however, blockade of the PD-1-mediated pathway with anti-PD-L1 significantly restored the CMV-specific CD8(+) T cell proliferation and IFN-gamma production. Thus, PD-1 plays a crucial role in the attrition of CMV-specific CD8(+) T cells in acute hepatitis B virus infection, which in turn, influences the preexisting homeostatic virus-specific CD8(+) T cell pool.     

Synergistic Reversal of Intrahepatic HCV-Specific CD8 T Cell Exhaustion by Combined PD-1/CTLA-4 Blockade

Viral persistence is associated with hierarchical antiviral CD8 T cell exhaustion with increased programmed death-1 (PD-1) expression. In HCV persistence, HCV-specific CD8 T cells from the liver (the site of viral replication) display increased PD-1 expression and a profound functional impairment that is not reversed by PD-1 blockade alone. Here, we report that the inhibitory receptor cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) is preferentially upregulated in PD-1⁺ T cells from the liver but not blood of chronically HCV-infected patients. PD-1/CTLA-4 co-expression in intrahepatic T cells was associated with a profound HCV-specific effector dysfunction that was synergistically reversed by combined PD-1/CTLA-4 blockade in vitro, but not by blocking PD-1 or CTLA-4 alone. A similar effect was observed in circulating HCV-specific CD8 T cells with increased PD-1/CTLA-4 co-expression during acute hepatitis C. The functional response to combined blockade was directly associated with CTLA-4 expression, lost with CD28-depletion and CD4-independent (including CD4⁺FoxP3⁺ Tregs). We conclude that PD-1 and CTLA-4 pathways both contribute to virus-specific T cell exhaustion at the site of viral replication by a redundant mechanism that requires combined PD-1/CTLA-4 blockade to reverse. These findings provide new insights into the mechanisms of virus-specific T cell dysfunction, and suggest that the synergistic effect by combined inhibitory receptor blockade might have a therapeutic application against chronic viral infection in vivo, provided that it does not induce autoimmunity.     

Upregulation of PD-1 expression on HIV-specific CD8+ T cells leads to reversible immune dysfunction

The engagement of programmed death 1 (PD-1) to its ligands, PD-L1 and PD-L2, inhibits proliferation and cytokine production mediated by antibodies to CD3 (refs. 5,6,7). Blocking the PD-1-PD-L1 pathway in mice chronically infected with lymphocytic choriomeningitis virus restores the capacity of exhausted CD8(+) T cells to undergo proliferation, cytokine production and cytotoxic activity and, consequently, results in reduced viral load. During chronic HIV infection, HIV-specific CD8(+) T cells are functionally impaired, showing a reduced capacity to produce cytokines and effector molecules as well as an impaired capacity to proliferate. Here, we found that PD-1 was upregulated on HIV-specific CD8(+) T cells; PD-1 expression levels were significantly correlated both with viral load and with the reduced capacity for cytokine production and proliferation of HIV-specific CD8(+) T cells. Notably, cytomegalovirus (CMV)-specific CD8(+) T cells from the same donors did not upregulate PD-1 and maintained the production of high levels of cytokines. Blocking PD-1 engagement to its ligand (PD-L1) enhanced the capacity of HIV-specific CD8(+) T cells to survive and proliferate and led to an increased production of cytokines and cytotoxic molecules in response to cognate antigen. The accumulation of HIV-specific dysfunctional CD8(+) T cells in the infected host could prevent the renewal of a functionally competent HIV-specific CD8(+) repertoire.     

Enhanced PD-1 Expression by T Cells in Cerebrospinal Fluid Does Not Reflect Functional Exhaustion during Chronic Human Immunodeficiency Virus Type 1 Infection

During chronic viral infections, T cells are exhausted due to constant antigen exposure and are associated with enhanced programmed death 1 (PD-1) expression. Deficiencies in the PD-1/programmed death-ligand 1 (PD-L1) pathway are associated with autoimmune diseases, including those of the central nervous system (CNS). To understand the role of PD-1 expression in regulating T-cell immunity in the CNS during chronic infection, we characterized PD-1 expression in cerebrospinal fluid (CSF) and blood of individuals with chronic human immunodeficiency virus type 1 (HIV-1) infection. PD-1 expression was higher on HIV-specific CD8⁺ T cells than on total CD8⁺ T cells in both CSF and blood. PD-1 expression on CSF T cells correlated positively with CSF HIV-1 RNA and inversely with blood CD4⁺ T-cell counts, suggesting that HIV-1 infection drives higher PD-1 expression on CSF T cells. However, in every HIV-positive individual, PD-1 expression was higher on T cells in CSF than on those in blood, despite HIV-1 RNA levels being lower. Among healthy HIV-negative controls, PD-1 expression was higher in CSF than in blood. Furthermore, frequencies of the senescence marker CD57 were lower on CSF T cells than on blood T cells, consistent with our prior observation of enhanced ex vivo functional capacity of CSF T cells. The higher PD-1 expression level on CSF T cells therefore does not reflect cellular exhaustion but may be a mechanism to downregulate immune-mediated tissue damage in the CNS. As inhibition of the PD-1/PD-L1 pathway is pursued as a therapeutic option for viral infections, potential effects of such a blockade on development of autoimmune responses in the CNS should be considered.Programmed death 1 (PD-1; also called CD279) and its ligands, PD-L1 (also called B7-H1 or CD274) and PD-L2 (also known as B7-DC or CD-273), regulate T-cell activation, peripheral tolerance, and autoimmunity (22, 43). PD-1 can be expressed on CD8⁺ and CD4⁺ T cells, B cells, natural killer T cells, and activated monocytes. PD-L1 is expressed on various cells, including T and B cells, dendritic cells, macrophages, mast cells, nonhematopoietic cell types (including vascular endothelial cells, pancreatic islet cells, astrocytes, keratinocytes, and microglial cells), and cells in immune privileged sites, including the placenta and the eye (22). PD-L2 expression is inducible and is restricted to dendritic cells, monocytes, macrophages, and mast cells (22). During chronic infections, the PD-1/PD-L1 pathway inhibits antigen-specific T-cell responses (7, 8, 35, 46). In human immunodeficiency virus type 1 (HIV-1)-infected individuals, PD-1 expression on HIV-specific T cells in peripheral blood is upregulated and correlates positively with plasma viremia and inversely with CD4⁺ T-cell counts (7, 46). PD-1 expression on HIV-specific T cells is also associated with T-cell exhaustion, as defined by a reduced ability to proliferate and produce cytokines (7, 46). Inhibition of the PD-1/PD-L1 pathway augments HIV-specific CD8⁺ and CD4⁺ T-cell function, and antiretroviral therapy is associated with a significant reduction of PD-1 expression on HIV-specific T cells in peripheral blood (8).The PD-1/PD-L1 pathway also limits immune-mediated tissue damage that may be caused by overreactive peripheral T cells, especially in immune privileged sites such as the central nervous system (CNS). In 1999, the importance of PD-1 for peripheral tolerance was first suggested by studies which showed that PD1^−/− mice develop lupus-like autoimmune diseases (32). In humans, polymorphisms in the PDCD1 gene, which encodes PD-1, have been associated with autoimmune diseases, including lupus, diabetes, rheumatoid arthritis, and multiple sclerosis (20, 21, 25). Upregulation of PD-L1 in multiple sclerosis lesions from human brain tissue suggests a role for the PD-1/PD-L1 pathway in regulating T-cell activation and controlling immunopathological damage (33).The CNS is involved by HIV-1 early during primary infection (6, 13), and approximately 40% of patients who develop advanced AIDS without receiving antiretroviral therapy develop cognitive impairment (6, 13, 38). While HIV-1 proteins gp120 (3, 16) and Tat (30) are directly neurotoxic and may contribute to HIV-associated dementia, detrimental neuropathogenic effects have also been postulated for inflammatory and innate immune cells, especially monocytes/macrophages and T cells (11, 19, 49, 50). Immune responses cause neuropathogenesis during other viral infections, and cytotoxic T lymphocytes can worsen the disease through direct cytotoxicity or release of inflammatory cytokines such as gamma interferon (IFN-γ) (14). However, we recently described higher frequencies of functional HIV-specific CD8⁺ T cells in cerebrospinal fluid (CSF) than in blood among asymptomatic HIV-positive individuals with little or no HIV-1 RNA in CSF, suggesting that HIV-1-specific CD8⁺ T cells help to control intrathecal viral replication (40).To understand the role of the PD-1/PD-L1 pathway in regulating T-cell responses during viral infection of the CNS, we characterized PD-1 expression on T cells in CSF and peripheral blood among asymptomatic HIV-positive individuals. We hypothesized that T-cell PD1 expression would be lower in CSF than in blood, since HIV-1 RNA concentrations are lower in CSF than in plasma and the magnitude and breadth of IFN-γ-secreting HIV-specific T cells are greater in CSF than in blood (40). We show that, in CSF, HIV-1 RNA correlates directly with PD-1 expression on CD4⁺, CD8⁺, and HIV-specific CD8⁺ T cells. Unexpectedly, PD-1 expression on all T cells is higher in CSF than in blood in HIV-positive patients and healthy HIV-negative controls. In contrast, expression of the senescence marker CD57 is lower in CSF than in blood. These data suggest that higher PD-1 expression on T cells in CSF may be a mechanism to regulate T-cell immunity in the CNS, rather than indicating T-cell exhaustion, and that this regulation is increased by HIV-1 replication.     

PD-1 modulates regulatory T cells and suppresses T-cell responses in HCV-associated lymphoma

T regulatory (T(R)) cells suppress T-cell responses that are critical in the development of chronic viral infection and associated malignancies. Programmed death-1 (PD-1) also has a pivotal role in regulation of T-cell functions during chronic viral infection. To examine the role of PD-1 pathway in regulating T(R)-cell functions that inhibit T-cell responses during virus-associated malignancy, T(R) cells were investigated in the setting of hepatitis C virus-associated lymphoma (HCV-L), non-HCV-associated lymphoma (non-HCV-L), HCV infection alone and healthy subjects (HS). Relatively high numbers of CD4(+)CD25(+) and CD8(+)CD25(+) T(R) cells, as well as high levels of PD-1 expressions on these T(R) cells were found in the peripheral blood of subjects with HCV-L compared with those from non-HCV-L or HCV alone or HS. T(R) cells from the HCV-L subjects were capable of suppressing the autogeneic lymphocyte response, and depletion of T(R) cells in peripheral blood mononuclear cells from HCV-L improved T-cell proliferation. Additionally, the suppressed T-cell activation and proliferation in HCV-L was partially restored by blocking the PD-1 pathway ex vivo, resulting in both a reduction in T(R)-cell number and the ability of T(R) to suppress the activity of effector T cells. This study suggests that the PD-1 pathway is involved in regulating T(R) cells that suppress T-cell functions in the setting of HCV-associated B-cell lymphoma.     

PD-1, PD-L1 and PD-L2 Gene Expression on T-Cells and Natural Killer Cells Declines in Conjunction with a Reduction in PD-1 Protein during the Intensive Phase of Tuberculosis Treatment

Background

The PD-1 axis is a cell intrinsic immunoregulatory pathway that mediates T cell exhaustion in chronic infection particularly in some viral infections. We hypothesized that PD-1, PD-L1 and PD-L2 would be highly expressed in untreated tuberculosis patients compared to controls due to their chronic infection and would decrease with successful TB treatment.

Materials and Methods

Untreated tuberculosis patients (n = 26) were recruited at diagnosis and followed up during treatment. Household contacts (n = 24) were recruited to establish baseline differences. Blood gene expression ex vivo was investigated using qRT-PCR. Flow cytometry was performed to establish protein expression patterns.

Results

PD-L1 gene expression was found to be elevated in active TB disease; however, this was not observed for PD-1 or PD-L2. The intensive phase of TB treatment was associated with a significant decline in PD-1, PD-L1 and PD-L2 gene expression. PD-1 protein expression on the surface of NK cells, CD8⁺ and CD4⁺ T cells was similar in patients with active TB disease compared to controls but declined with successful TB treatment, with the greatest decline occurring on the NK cells followed by CD8⁺ T cells and then CD4⁺ T cells. Granzyme B/PD-1 co-expression declined with successful intensive phase treatment.

Conclusion

Modulation of PD-1/PD-L1 pathway through TB treatment indicates changes in the peripheral T cell response caused by live Mycobacterium tuberculosis (Mtb) followed by the response to dead bacilli, antigen-release and immuno-pathology resolution. The PD-1 axis could be a host drug target for immunomodulatory treatments in the future.     

Re-Evaluation of PD-1 Expression by T Cells as a Marker for Immune Exhaustion during SIV Infection

PD-1 expression is generally associated with exhaustion of T cells during chronic viral infections based on the finding that PD-1 expressing cells respond poorly to antigen activation and blockade of PD-1/PD-ligand interaction restores such antigen specific responses in vitro. We tested this hypothesis by examining PD-1 expression on virus-specific CD8 T cells and total T cells in vivo to determine whether PD-1 expression constitutes a reliable marker of immune exhaustion during SIV infection. The expression of PD-1 and Ki67 was monitored longitudinally on T cell subsets in peripheral blood, bone marrow, lymph node and rectal biopsy specimens from rhesus macaques prior to and post infection with pathogenic SIVmac239. During the course of infection, a progressive negative correlation was noted between PD-1 density and Ki67 expression in p11CM⁺ CD8⁺ T cells, as seen in other studies. However, for total and memory CD4 and CD8 T cells, a positive correlation was observed between PD-1 and Ki67 expression. Thus, while the levels of non-proliferating PD-1⁺ p11CM⁺ CD8 T cells were markedly elevated with progressing infection, such an increase was not seen on total T cells. In addition, total memory PD1⁺ T cells exhibited higher levels of CCR5 than PD-1⁻ T cells. Interestingly, few PD-1⁺ CD8⁺ T cells expressed CCR7 compared to PD-1⁺ CD4 T cells and PD-1⁻ T cells. In conclusion, overall PD1⁺ T cells likely represent a particular differentiation stage or trafficking ability rather than exhaustion and in the context of chronic SIV infection, the level of PD-1 expression by T cells does not by itself serve as a reliable marker for immune exhaustion.     

PD-1 blockage reverses immune dysfunction and hepatitis B viral persistence in a mouse animal model

Persistent hepatitis B viral (HBV) infection results in chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC). Recent studies in animal models of viral infection indicate that the interaction between the inhibitory receptor, programmed death (PD)-1, on lymphocytes and its ligand (PD-L1) play a critical role in T-cell exhaustion by inducing T-cell inactivation. High PD-1 expression levels by peripheral T-lymphocytes and the possibility of improving T-cell function by blocking PD-1-mediated signaling confirm the importance of this inhibitory pathway in inducing T-cell exhaustion. We studied T-cell exhaustion and the effects of PD-1 and PD-L1 blockade on intrahepatic infiltrating T-cells in our recently developed mouse model of HBV persistence. In this mouse animal model, we demonstrated that there were increased intrahepatic PD-1-expressing CD8+ and CD4+ T cells in mice with HBV persistence, but PD-1 upregulation was resolved in mice which had cleared HBV. The Intrahepatic CD8+ T-cells expressed higher levels of PD-1 and lower levels of CD127 in mice with HBV persistence. Blockade of PD-1/PD-L1 interactions increased HBcAg-specific interferon (IFN)-γ production in intrahepatic T lymphocytes. Furthermore, blocking the interaction of PD-1 with PD-L1 by an anti-PD-1 monoclonal antibody (mAb) reversed the exhausted phenotype in intrahepatic T lymphocytes and viral persistence to clearance of HBV in vivo. Our results indicated that PD-1 blockage reverses immune dysfunction and viral persistence of HBV infection in a mouse animal model, suggesting that the anti-PD-1 mAb might be a good therapeutic candidate for chronic HBV infection.     

Selective Loss of Early Differentiated,Highly Functional PD1high CD4 T Cells with HIV Progression

The role of PD-1 expression on CD4 T cells during HIV infection is not well understood. Here, we describe the differential expression of PD-1 in CD127^high CD4 T cells within the early/intermediate differentiated (EI) (CD27^highCD45RA^low) T cell population among uninfected and HIV-infected subjects, with higher expression associated with decreased viral replication (HIV-1 viral load). A significant loss of circulating PD-1^highCTLA-4^low CD4 T cells was found specifically in the CD127^highCD27^highCD45RA^low compartment, while initiation of antiretroviral treatment, particularly in subjects with advanced disease, reversed these dynamics. Increased HIV-1 Gag DNA was also found in PD-1^high compared to PD-1^low ED CD4 T cells. In line with an increased susceptibility to HIV infection, PD-1 expression in this CD4 T cell subset was associated with increased activation and expression of the HIV co-receptor, CCR5. Rather than exhaustion, this population produced more IFN-g, MIP1-a, IL-4, IL-10, and IL-17a compared to PD-1^low EI CD4 T cells. In line with our previous findings, PD-1^high EI CD4 T cells were also characterized by a high expression of CCR7, CXCR5 and CCR6, a phenotype associated with increased in vitro B cell help. Our data show that expression of PD-1 on early-differentiated CD4 T cells may represent a population that is highly functional, more susceptible to HIV infection and selectively lost in chronic HIV infection.