Blockade ofthe negative co-stimulatory molecules PD-1 and CTLA-4 improves survival in primary and secondary fungal sepsis

Sepsis, the host response to severe infection, is the 10th leading cause of death in the United States and the mostcommon cause of mortality in most intensive care units [1, 2].Improved treatment protocols have resulted in the majority of patients surviving the initial 72 hours of sepsis onset only to succumb later in the time course of the disease [3].There is increasing recognition that a state of impaired immunity follows the initial hyper-inflammatory phase of sepsis [4‐8].During this phase of impaired immunity, patients are more susceptible to secondary nosocomial infections, often with opportunistic organisms that typically infect immunocompromised individuals.One of the most important opportunistic infections in patients in the ICU is Candida albicans [9‐12].Candida infections are currently the third or fourth most common cause of bloodstream infections in many intensive care units.Although excellent antimicrobial therapy against most Candida species exists, mortality remains high at approximately 30 to 40% for fungal sepsis [10‐12].

The fact that mortality from fungal infections remains high despite the use of antimicrobial agents that are highly active against fungal organisms, suggests that defects in host immunity may contribute to the persistent high mortality.Therefore, methods that improve host immune function may be fundamental to improving survival. In this regard, recent studies suggest that immuno-adjuvant therapy in invasive fungal infections may be a viable strategy [13‐15].IL-7, a pleuripotent cytokine that enhances adaptive immunity throughimmunostimulatory effects on CD4 and CD8 T cells, caused an approximately1.7-fold improvement in survival in a murine fungal sepsis model [13].In addition to animal studies, a few clinical studies support the use of immuno-adjuvant therapy in invasive fungal infections [14, 15].A randomized trial of interferon gamma (IFN-γ), a potent activator of macrophages and monocytes in HIV patients with cryptococcal meningitis, showed that treatment led to a significantly faster rate of clearing of cerebrospinal fluid, a finding that has been shown to correlate with survival [14].IFN-γ is currently approved for use in patients with chronic granulomatous disease who have invasive fungal infections [15].

Another potential strategy for improving host immunologic defenses that has shown efficacy in various infectious models is the use of agents which up-regulateadaptive immunity by blocking inhibitory receptors expressed on T lymphocytes [16‐19].T cell activation is carefully controlled by expression of positive and negative co-stimulatory molecules that prevent excessive T cell function.CD28 is the classic positive co-stimulatory receptor that, acting in conjunction with the T cell receptor (TCR), induces T cells to proliferate and produce cytokines including, for example, IL-2 and IFN-γ that have extensive effects on other cells.To prevent excessive T cell activation, lymphocytes express a number of negative co-stimulatory molecules that suppress and down-regulate their function [18, 19].

Programmed cell death-1 (PD-1) is a member of the B7-CD28 superfamily that functions in an inhibitory role.During T cell activation, PD-1 is rapidly induced and expressed on the surface of CD4 and CD8 T cells where it interacts with its ligands PD-L1 and PD-L2 [20‐22].PD-L1 is expressed on both hematopoietic and non-hematopoietic cells and its expression is highly up-regulated during inflammatory states [20].Activation of PD-1 by its ligands causes inhibition ofmanyT cell functions, including proliferation, cytotoxic activity and cytokine production.The essential role of PD-1 in regulating immunity is demonstrated in studies showing that PD-1 knockout mice develop autoimmune diseases, including cardiomyopathy and a lupus-like syndrome [16, 20, 21].Increased T cell expression of PD-1 is known to occur under conditions of chronic antigenic stimulation, such as persistent viral infections, and lead to T cell "exhaustion" [16, 17, 20].These "exhausted" T cells are non-functional, prone to undergo apoptosis, and are unable to participate in an effective immune response, thereby contributing to the chronic nature of the viral infections.Blockade of PD-1using inhibitory antibodies has been shown to restore T cell function, increase antiviral T cell responses, and reduce viral load in certain infections [18, 19].In addition to viral infections, blockade of the PD-1 pathway has improved survival in bacterial infections.Three independent groups demonstrated that blockade of the PD-1 pathway decreased mortality in clinically-relevant animal models of bacterial sepsis [23‐25].The potential clinical relevance of these animal studies is highlighted by recent studies showing that PD-1 over-expression on circulating T cells from patients with sepsis correlated with decreased T cell proliferative capacity, increased secondary nosocomial infections and mortality [26].

The purpose of this study was to determine if inhibition of the T cell negative co-stimulatory PD-1:PD-L1 pathwaycould reverse immune dysfunction and improve survival in primary fungal infections and in secondary fungal sepsis occurring after bacterial infection.Both the anti-PD-1 and the anti-PD-L1 antibodysignificantly improved survival in the two different models of fungal sepsis.This improvement in survival was associated with increased production of IFN-γ, a cytokine which is critical in host defenses against fungal organisms, and reversed the fungal-induced depression of HLA-DR expression in monocytes and dendritic cells.Blockade of cytotoxic T-lymphocyte antigen 4 (CTLA-4), a second negative co-stimulatory molecule that suppresses T cell function, also improved survival in primary and secondary fungal sepsis thereby supporting the concept that immuno-adjuvant therapy offers a rational, new approach to treatment of this highly lethal infection.

The present results demonstrating that blockade ofPD-1, PD-L1 or CTLA-4 leads to improved survival in both primary and secondary fungal sepsis strongly support the concept that inhibition ofT cell negative co-stimulatory pathways represents an effective therapeutic approach to disseminated fungal sepsis.Previous reports by three independent groups showed that inhibition of the PD-1:PD-L1 pathway improved survival in the clinically relevant CLP model of bacterial sepsis [23‐25].Recent studies have also reported that inhibition of PD-1 restores T cell function and decreases viral load in murine models of hepatitis B and HIV-1 [29, 30].In a related fashion, our group has reported that an antagonistic antibody to CTLA-4 improved survival in a clinically-relevant animal model of bacterial peritonitis [28].Additional studies havedemonstrated that increased CTLA-4 expression adversely affected pathogen clearance in murine models of Helicobacterpylori, Leishmania and Trypanosoma [31‐33].Considered together, these studies make a compelling case that modulation of the negative co-stimulatory pathway mediated by PD-1/PD-L1 and CTLA-4 represents a novel and potentially highly effective approach to a broad range of infectious agents.

The findings from the current studies showing that blockade of negative co-stimulatory pathways improves survival in disseminated fungal sepsisprovide additional support for the hypothesis that immunosuppression is a major pathophysiologic phenomenon in sepsis.Although there are multiple interacting mechanisms responsible for immunosuppression in sepsis, including, for example, increased T regulatory cells, increased myeloid derived suppressor cells, and apoptosis-induced depletion of immune effector cells, recent studies highlight the likely role of PD-1 mediated T cell "exhaustion" in impaired immunity [23‐25, 34, 35].Guignantet al. showed that PD-1 over-expression occurred on circulating T cells from patients with sepsis and correlated with decreased T cell proliferative capacity, increased secondary nosocomial infections and mortality [26].Studies from our group documented an increase in the percentage of PD-L1 expressing monocytes in patients who die of sepsis versus sepsis survivors (data not shown).A recent postmortem study from our group also demonstrated significantly increased expression of PD-1 and PD-L1 on splenic T cells and antigen presenting cells respectively in septic versus non-septic patients [34].This increased expression of PD-1 and PD-L1 was associated with marked suppression of stimulated cytokine production by splenocytes from septic patients; splenocytes from septic patients produced less than approximately 10% of the amount of cytokines produced by splenocytes from critically-ill non-septic patients.Collectively, these studies are consistent with a major role for PD-1:PD-L1-mediated immune suppression in patients with sepsis.

Blockade of PD-1, PD-L1 and CTLA-4 not only offers new hope in infectious disease but also appears especially promising in cancer therapy [21, 22, 36‐38].Anti-CTLA-4 antibody reinvigorated the concept of immunotherapy as a valid approach to cancer with its success in inducing significant remissions in patients with widely metastatic malignant melanoma, many of whom had failed other therapies [38].Anti-PD-1 antibody, which has a better safety profile compared to anti-CTLA-4, produced clinical responses in approximately 25% of patients with a variety of diverse tumors [36, 37].Anti-CTLA-4 and anti-PD-1 restore T cell function thereby reactivating host immunity allowing it to eliminate tumor cells.Remarkably, many remissions with anti-CTLA-4 and anti-PD-1 appear to be durable, that is, they have lasted for over a year, and may be permanent [39].The encouraging results of cancer immunotherapy with anti-CTLA-4 and anti-PD-1 are highly relevant to sepsis patients because cancer and sepsis share many of the same immunosuppressive mechanisms,including increased T cell CTLA-4 expression and increased PD-1 and PD-L1 expression in T cells and monocytes, respectively, with T cell "exhaustion" [4‐8, 34, 35].Sepsis, like cancer, is an ideal condition for persistent antigenic exposure which is thought to be one mechanism driving induction of PD-1/PD-L1 and T cell dysfunction.

Although there are likely a number of mechanisms that are responsible for the protective effect of anti-PD-1, anti-PD-L1 and anti-CTLA-4 in sepsis, one likely mechanism is their ability to reverse the sepsis-induced suppression in IFN-γ production.All three antibodies had demonstrable efficacy in increasing either splenocyte IFN-γ production or the percentage of IFN-γ positive CD4 and/or CD8 T cells (Figures 4, 5, 6, 7).(Note that differing effects on host immunity have recently been observed in animals treated with different isotypeantibodies to CTLA-4 [40].)T cells are major producers of IFN-γ, which is essential for optimal function of monocytes and macrophages.Sepsis-induced suppression of IFN-γ production is likely a major driving force for the immune suppression in the disorder [41, 42].Administration of IFN-γ during the immunosuppressive phase of sepsis has been reported to be beneficial in several small clinical studies [41, 42].The ability of anti-PD-1, anti-PD-L1 and anti-CTLA-4 antibodies to improve IFN-γ production may be particularly germane to fungal sepsis.Administration of IFN-γ improves outcome in patients with chronic granulomatous disease who have fungal sepsis.Recently, a trial of IFN-γ in a small number of patients with fungal meningitis showed that IFN-γ leads to more rapid clearing of fungal organisms from the cerebrospinal fluid, an important clinical finding that correlates with survival [14].

Another interesting finding regarding effects of the anti-PD-1 antibody on cytokines was its action to increase production of the pro-inflammatory cytokine IL-6 and the immunosuppressive cytokine IL-10 (Figure 4).The effect of anti-PD-1 to increase IL-10 in the present study is somewhat surprising given findings by Said et al. who reported that PD-1 induces IL-10 production in monocytes in patients with HIV, thereby resulting in impaired T cell activation [43].Our findings, showing an effect of anti-PD-1 antibody to increase IL-10 production, are similar to the report by Wong et al. who noted that anti-PD-L-1 antibody caused an increase in both IFN-γ and IL-10 in a lymphocyte incubation study [44].The increase in IL-10 splenocyte production in the present study was relatively small compared to the larger effect of the anti-PD-1 antibody on IFN-γ production (Figure 4).Thus, we believe that the predominant effect of the anti-PD-1 antibody is to enhance activation of T cells, a highly beneficial effect in sepsis.

Similar to our previous findings in the CLP model of sepsis [28], the anti-CTLA-4 antibody worsened outcome in fungal sepsis if administered at higher doses or too early during the course of the disease (data not shown).No such adverse effects were observed with anti-PD-1 or anti-PD-L1 antibody at any dose or time of administration (data not shown).Thus, anti-PD-1 and anti-PD-L1 appear to have a better safety profile compared to anti-CTLA-4 antibody and this finding mirrors clinical studies in cancer patients which show a lower incidence of autoimmune effects in patients treated with anti-PD-1 compared to anti-CTLA-4 [36‐39].

Additional evidence for beneficial effects of anti-PD-1 and anti-PD-L1 on host immunity is provided by their ability to increase MHC II expression on macrophages and dendritic cells (Figure 8).Decreased MHC II expression is a hallmark of sepsis and is used as a marker of immune suppression during the disorder [7].MHC II molecules function to present microbial antigens to CD4 T cells thereby activating these cells for optimal response to the infectious challenge.Although both anti-PD-1 and anti-PD-L1 improved dendritic cell and macrophage MHC II expression, anti-PD-L1 had a greater effect than anti-PD-1 (Figure 8).This more robust effect of anti-PD-L1 antibody compared to anti-PD-1 antibody to increase MHC II expression may be related to the fact that PD-L1 is expressed on macrophages and dendritic cells and has direct suppressive effects on these cells.

The present experimental findings providepreclinical hypothesis-generating data which support the contention that immuno-adjuvant therapy with anti-PD-1/PD-L1 and anti-CTLA-4 offers a new approach to treatment of fungal infections and likely infectious disease in general.The remarkable efficacy of anti-PD-1 and anti-CTLA-4 antibody therapy to induce remissions in a relatively high percentage of cancer patients [36‐38] is evidence that this immunologic approach has profound effects to enhance host immunity.Sepsis, another life threatening failure of host immunity that shares many immune defects with cancer, is likely amenable to this immuno-stimulatory approach as well.Although serious autoimmune side effects of anti-PD-1 and anti-CTLA-4 have occurred in a small percentage of cancer patients treated with these agents [36‐38], these adverse effects would likely be less problematic in patients with sepsis because of two factors.First, compared to cancer patients, most septic patients have more severe impairment in host immunity.Secondly,most patients with sepsis would need only short-term therapy with anti-PD-1 or anti-CTLA-4 and, therefore, would be less likely to develop autoimmunity.Additionally, anti-PD-1 antibody could be tailored to septic patients whose peripheral circulating T cells or monocytes have persistently increased expression of PD-1 or PD-L1, respectively, evidence that the patients have entered an immunosuppressive phase of the disorder.Thus, a readily available biomarker could be used to select ideal candidate patients for immune therapy using these agents.

Blockade of the negative co-stimulatory molecules PD-1, PD-L1 and CTLA-4 improved survival in two models of fungal sepsis.Potential mechanisms of action for the beneficial effects of blocking negative co-stimulatory molecules include increased IFN-γ production and increased expression of MHC II on antigen presenting cells.If ongoing, large phase 3 studies of anti-PD-1 antibody in cancer patientscontinue to show its relative safety, carefully conducted clinical trials of anti-PD-1 antibody should be conducted in patients with sepsis.Such a novel immunologic therapy could represent a major advance against this highly lethal disease.