Targeting the programmed cell death 1: programmed cell death ligand 1 pathway reverses T cell exhaustion in patients with sepsis

Introduction

A major pathophysiologic mechanism in sepsis is impaired host immunity which results in failure to eradicate invading pathogens and increased susceptibility to secondary infections. Although many immunosuppressive mechanisms exist, increased expression of the inhibitory receptor programmed cell death 1 (PD-1) and its ligand (PD-L1) are thought to play key roles. The newly recognized phenomenon of T cell exhaustion is mediated in part by PD-1 effects on T cells. This study tested the ability of anti-PD-1 and anti-PD-L1 antibodies to prevent apoptosis and improve lymphocyte function in septic patients.

Methods

Blood was obtained from 43 septic and 15 non-septic critically-ill patients. Effects of anti-PD-1, anti-PD-L1, or isotype-control antibody on lymphocyte apoptosis and interferon gamma (IFN-γ) and interleukin-2 (IL-2) production were quantitated by flow cytometry.

Results

Lymphocytes from septic patients produced decreased IFN-γ and IL-2 and had increased CD8 T cell expression of PD-1 and decreased PD-L1 expression compared to non-septic patients (P<0.05). Monocytes from septic patients had increased PD-L1 and decreased HLA-DR expression compared to non-septic patients (P<0.01). CD8 T cell expression of PD-1 increased over time in ICU as PD-L1, IFN-γ, and IL2 decreased. In addition, donors with the highest CD8 PD-1 expression together with the lowest CD8 PD-L1 expression also had lower levels of HLA-DR expression in monocytes, and an increased rate of secondary infections, suggestive of a more immune exhausted phenotype. Treatment of cells from septic patients with anti-PD-1 or anti-PD-L1 antibody decreased apoptosis and increased IFN-γ and IL-2 production in septic patients; (P<0.01). The percentage of CD4 T cells that were PD-1 positive correlated with the degree of cellular apoptosis (P<0.01).

Conclusions

In vitro blockade of the PD-1:PD-L1 pathway decreases apoptosis and improves immune cell function in septic patients. The current results together with multiple positive studies of anti-PD-1 and anti-PD-L1 in animal models of bacterial and fungal infections and the relative safety profile of anti-PD-1/anti-PD-L1 in human oncology trials to date strongly support the initiation of clinical trials testing these antibodies in sepsis, a disorder with a high mortality.     

PD-L1 blockade synergizes with IL-2 therapy in reinvigorating exhausted T cells

The inhibitory receptor programmed cell death 1 (PD-1) plays a major role in functional exhaustion of T cells during chronic infections and cancer, and recent clinical data suggest that blockade of the PD-1 pathway is an effective immunotherapy in treating certain cancers. Thus, it is important to define combinatorial approaches that increase the efficacy of PD-1 blockade. To address this issue, we examined the effect of IL-2 and PD-1 ligand 1 (PD-L1) blockade in the mouse model of chronic lymphocytic choriomeningitis virus (LCMV) infection. We found that low-dose IL-2 administration alone enhanced CD8⁺ T cell responses in chronically infected mice. IL-2 treatment also decreased inhibitory receptor levels on virus-specific CD8⁺ T cells and increased expression of CD127 and CD44, resulting in a phenotype resembling that of memory T cells. Surprisingly, IL-2 therapy had only a minimal effect on reducing viral load. However, combining IL-2 treatment with blockade of the PD-1 inhibitory pathway had striking synergistic effects in enhancing virus-specific CD8⁺ T cell responses and decreasing viral load. Interestingly, this reduction in viral load occurred despite increased numbers of Tregs. These results suggest that combined IL-2 therapy and PD-L1 blockade merits consideration as a regimen for treating human chronic infections and cancer.     

PD-L1 has distinct functions in hematopoietic and nonhematopoietic cells in regulating T cell responses during chronic infection in mice

The inhibitory receptor programmed death 1 (PD-1) is upregulated on antigen-specific CD8⁺ T cells during persistent viral infections. Interaction with PD-1 ligand 1 (PD-L1) contributes to functional exhaustion of responding T cells and may limit immunopathology during infection. PD-L1 is expressed on both hematopoietic and nonhematopoietic cells in tissues. However, the exact roles of PD-L1 on hematopoietic versus nonhematopoietic cells in modulating immune responses are unclear. Here we used bone marrow chimeric mice to examine the effects of PD-L1 deficiency in hematopoietic or nonhematopoietic cells during lymphocytic choriomeningitis virus clone 13 (LCMV CL-13) infection. We found that PD-L1 expression on hematopoietic cells inhibited CD8⁺ T cell numbers and function after LCMV CL-13 infection. In contrast, PD-L1 expression on nonhematopoietic cells limited viral clearance and immunopathology in infected tissues. Together, these data demonstrate that there are distinct roles for PD-L1 on hematopoietic and nonhematopoietic cells in regulating CD8⁺ T cell responses and viral clearance during chronic viral infection.     

Intravaginal immunization with HPV vectors induces tissue-resident CD8+ T cell responses

The induction of persistent intraepithelial CD8⁺ T cell responses may be key to the development of vaccines against mucosally transmitted pathogens, particularly for sexually transmitted diseases. Here we investigated CD8⁺ T cell responses in the female mouse cervicovaginal mucosa after intravaginal immunization with human papillomavirus vectors (HPV pseudoviruses) that transiently expressed a model antigen, respiratory syncytial virus (RSV) M/M2, in cervicovaginal keratinocytes. An HPV intravaginal prime/boost with different HPV serotypes induced 10-fold more cervicovaginal antigen-specific CD8⁺ T cells than priming alone. Antigen-specific T cell numbers decreased only 2-fold after 6 months. Most genital antigen-specific CD8⁺ T cells were intra- or subepithelial, expressed α_E-integrin CD103, produced IFN-γ and TNF-α, and displayed in vivo cytotoxicity. Using a sphingosine-1-phosphate analog (FTY720), we found that the primed CD8⁺ T cells proliferated in the cervicovaginal mucosa upon HPV intravaginal boost. Intravaginal HPV prime/boost reduced cervicovaginal viral titers 1,000-fold after intravaginal challenge with vaccinia virus expressing the CD8 epitope M2. In contrast, intramuscular prime/boost with an adenovirus type 5 vector induced a higher level of systemic CD8⁺ T cells but failed to induce intraepithelial CD103⁺CD8⁺ T cells or protect against recombinant vaccinia vaginal challenge. Thus, HPV vectors are attractive gene-delivery platforms for inducing durable intraepithelial cervicovaginal CD8⁺ T cell responses by promoting local proliferation and retention of primed antigen-specific CD8⁺ T cells.     

Critical role of the programmed death-1 (PD-1) pathway in regulation of experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is mediated by autoantigen-specific T cells dependent on critical costimulatory signals for their full activation and regulation. We report that the programmed death-1 (PD-1) costimulatory pathway plays a critical role in regulating peripheral tolerance in murine EAE and appears to be a major contributor to the resistance of disease induction in CD28-deficient mice. After immunization with myelin oligodendrocyte glycoprotein (MOG) there was a progressive increase in expression of PD-1 and its ligand PD-L1 but not PD-L2 within the central nervous system (CNS) of mice with EAE, peaking after 3 wk. In both wild-type (WT) and CD28-deficient mice, PD-1 blockade resulted in accelerated and more severe disease with increased CNS lymphocyte infiltration. Worsening of disease after PD-1 blockade was associated with a heightened autoimmune response to MOG, manifested by increased frequency of interferon gamma-producing T cells, increased delayed-type hypersensitivity responses, and higher serum levels of anti-MOG antibody. In vivo blockade of PD-1 resulted in increased antigen-specific T cell expansion, activation, and cytokine production. Interestingly, PD-L2 but not PD-L1 blockade in WT animals also resulted in disease augmentation. Our data are the first demonstration that the PD-1 pathway plays a critical role in regulating EAE.     

Immunoregulatory mechanisms triggered by viral infections protect from type 1 diabetes in mice

Type 1 diabetes (T1D) is an autoimmune disease that is caused by the destruction of insulin-producing β cells. Viral infections induce immune responses that can damage β cells and promote T1D or on the other hand prevent the development of the disease. However, the opposing roles of viral infections in T1D are not understood mechanistically. We report here that viruses that do not inflict damage on β cells provided protection from T1D by triggering immunoregulatory mechanisms. Infection of prediabetic NOD mice with Coxsackie virus B3 or lymphocytic choriomeningitis virus (LCMV) delayed diabetes onset and reduced disease incidence. Delayed T1D onset was due to transient upregulation of programmed cell death–1 ligand 1 (PD-L1) on lymphoid cells, which prevented the expansion of diabetogenic CD8⁺ T cells expressing programmed cell death–1 (PD-1). Reduced T1D incidence was caused by increased numbers of invigorated CD4⁺CD25⁺ Tregs, which produced TGF-β and maintained long-term tolerance. Full protection from T1D resulted from synergy between PD-L1 and CD4⁺CD25⁺ Tregs. Our results provide what we believe to be novel mechanistic insight into the role of viruses in T1D and should be valuable for prospective studies in humans.     

Active Immunotherapy Combined With Blockade of a Coinhibitory Pathway Achieves Regression of Large Tumor Masses in Cancer-prone Mice

Vaccines that aim to expand tumor-specific CD8⁺ T cells have yielded disappointing results in cancer patients although they showed efficacy in transplantable tumor mouse models. Using a system that more faithfully mimics a progressing cancer and its immunoinhibitory microenvironment, we here show that in transgenic mice, which gradually develop adenocarcinomas due to expression of HPV-16 E7 within their thyroid, a highly immunogenic vaccine expressing E7 only induces low E7-specific CD8⁺ T-cell responses, which fail to affect the size of the tumors. In contrast, the same type of vaccine expressing E7 fused to herpes simplex virus (HSV)-1 glycoprotein D (gD), an antagonist of the coinhibitory B- and T-lymphocyte attenuator (BTLA)/CD160-herpes virus entry mediator (HVEM) pathways, stimulates potent E7-specific CD8⁺ T-cell responses, which can be augmented by repeated vaccination, resulting in initial regression of even large tumor masses in all mice with sustained regression in more than half of them. These results indicate that active immunization concomitantly with blockade of the immunoinhibitory HVEM-BTLA/CD160 pathways through HSV-1 gD may result in sustained tumor regression.     

Nonviral Delivery of Small Interfering RNA Into Pancreas-associated Immune Cells Prevents Autoimmune Diabetes

The development of small interfering RNA (siRNA) for the treatment of human disorders has been often hampered by their low transfection efficiency in vivo. In order to overcome this major drawback, various in vivo siRNA transfection methods have been developed. However, their capacity to transfect immune or insulin-producing β-cells within the pancreas for the treatment of autoimmune diabetes remains undetermined. We found that lipid- or polyethylenimine-based delivery agents were efficient to address siRNA molecules within pancreas-associated antigen-presenting cells (APCs) (but not β-cells) and particularly a CD11b⁺ cell population comprising both CD11b⁺CD11c^neg macrophages and CD11b⁺CD11c⁺ dendritic cells. However, the route of administration and the carrier composition greatly affected the transfection efficacy. Therapeutically, we showed that early (starting at 6-week-old) short-course treatment with lipid/Alox15-specific siRNA complex promoted long-term protection from type 1 diabetes (T1D) in wild-type (WT) nonobese diabetic (NOD) mice. Alox15 downregulation in pancreas-associated CD11b⁺ cells significantly upregulated a variety of costimulatory molecules and particularly the programmed death 1 ligand 1 (PD-L1) pathway involved in tolerance induction. Concomitantly, we found that regulatory T cells were increased in the pancreas of lipid/Alox15 siRNA-treated NOD mice. Collectively, our data provide new insights into the development of siRNA-based therapeutics for T1D.     

Programmed cell death 1 forms negative costimulatory microclusters that directly inhibit T cell receptor signaling by recruiting phosphatase SHP2

Programmed cell death 1 (PD-1) is a negative costimulatory receptor critical for the suppression of T cell activation in vitro and in vivo. Single cell imaging elucidated a molecular mechanism of PD-1-mediated suppression. PD-1 becomes clustered with T cell receptors (TCRs) upon binding to its ligand PD-L1 and is transiently associated with the phosphatase SHP2 (Src homology 2 domain-containing tyrosine phosphatase 2). These negative costimulatory microclusters induce the dephosphorylation of the proximal TCR signaling molecules. This results in the suppression of T cell activation and blockade of the TCR-induced stop signal. In addition to PD-1 clustering, PD-1-TCR colocalization within microclusters is required for efficient PD-1-mediated suppression. This inhibitory mechanism also functions in PD-1(hi) T cells generated in vivo and can be overridden by a neutralizing anti-PD-L1 antibody. Therefore, PD-1 microcluster formation is important for regulation of T cell activation.     

Overexpression of CD155 is associated with PD-1 and PD-L1 expression on immune cells,rather than tumor cells in the breast cancer microenvironment

BACKGROUNDCD155 is an immune checkpoint protein in cancers and interacts with ligands to regulate the immune microenvironment. The expression of CD155 is correlated with the prognosis and pathological features of breast cancer.AIMTo investigate the expression status of CD155 and the association with exhausted CD4⁺ helper and CD8⁺ cytotoxic tumor infiltrating lymphocytes (TILs) and PD-L1 in the breast cancer microenvironment.METHODSOne hundred and twenty-six breast cancer patients with invasive ductal breast cancer were consecutively recruited into this study. Immunohistochemistry was used to detect the expression CD155, PD-L1 and PD-1 on tumor-infiltrating immune cells and tumor cells in the microenvironment.RESULTSThe proportion of patients with CD155 expression was higher in triple negative breast cancer (72.7%) than in Luminal A patients (22.2%, P < 0.05). Patients with positive CD155 expression had a higher percentage of CD4⁺/PD-1⁺ helper TILs (30%) than patients with negative CD155 expression (21%, P < 0.05). Patients with positive CD155 expression also had higher cell counts of exhausted CD4⁺ TILs [47 vs 20/high-power fields (HPF)] and unexhausted CD8⁺ TILs (30 vs 17/HPF) than patients with negative expression (P < 0.05). CD155 expression was correlated with increased PD-L1 expression in immune cells, 0.8% and 0.02% immune cells expressed PD-L1 in patients with positive and negative CD155 expression, respectively (P < 0.05).CONCLUSIONCD155 was related to an inhibitory immune breast cancer microenvironment. CD155 was associated with a high proportion of exhausted CD4⁺ and unexhausted CD8⁺ TILs and high PD-L1 expression in immune cells.     

Comparative Analysis of Immune Responses Induced by Vaccination With SIV Antigens by Recombinant Ad5 Vector or Plasmid DNA in Rhesus Macaques

DNA vaccines have undergone important enhancements in their design, formulation, and delivery process. Past literature supports that DNA vaccines are not as immunogenic in nonhuman primates as live vector systems. The most potent recombinant vector system for induction of cellular immune responses in macaques and humans is adenovirus serotype 5 (Ad5), an important benchmark for new vaccine development. Here, we performed a head-to-head evaluation of the Merck Ad5 SIV vaccine and an optimized electroporation (EP) delivered SIV DNA vaccine in macaques. Animals receiving the Ad5 vaccine were immunized three times, whereas the DNA-vaccinated animals were immunized up to four times based on optimized protocols. We observed significant differences in the quantity of IFNγ responses by enzyme-linked immunosorbent spot (ELISpot), greater proliferative capacity of CD8⁺ T cells, and increased polyfunctionality of both CD4⁺ and CD8⁺ T cells in the DNA-vaccinated group. Importantly, Ad5 immunizations failed to boost following the first immunization, whereas DNA responses were continually boosted with all four immunizations demonstrating a major advantage of these improved DNA vaccines. These optimized DNA vaccines induce very different immune phenotypes than traditional Ad5 vaccines, suggesting that they could play an important role in vaccine research and development.     

Development of cancer immunotherapy based on PD-1/PD-L1 pathway blockade

Programmed death receptor 1 (PD-1)/programmed death ligand 1 (PD-L1) blockade therapy has achieved considerable success in various tumours. However, only a fraction of patients benefit from its clinical application, and some patients might be suffer from tumour resistance against PD-1/PD-L1 blockade therapy after the original response. In this review, we summarized the main reasons that caused the low response rate of PD-/PD-L1 blockade therapy: firstly, the off-target of PD-1/PD-L1 blocking agents, which is also the main factor of the side effect of autoimmune disorders; secondly, the insufficient infiltration of T cells in a tumour microenvironment; thirdly, the low immunogenicity of tumor cells; fourth, other immunosuppressive components impairing the therapeutic efficacy of the immunotherapy based on the PD-/PD-L1 blockade, and introducing some updated the delivery system of PD-1/PD-L1 blocking agents and the combination therapy based on PD-1/PD-L1 inhibitors and other therapeutics that can complement and promote each other to achieve improved immune response.

The possible reasons that caused low response rate and severe side effects of programmed death receptor 1 (PD-1)/programmed death ligand 1 (PD-L1) blockade therapy and corresponding strategies.     

Adjuvanting a DNA vaccine with a TLR9 ligand plus Flt3 ligand results in enhanced cellular immunity against the simian immunodeficiency virus

DNA vaccines offer promising strategies for immunization against infections. However, their clinical use requires improvements in immunogenicity. We explored the efficacy of Toll-like receptor (TLR) ligands (TLR-Ls) on augmenting the immunogenicity of a DNA prime–modified vaccinia virus Ankara (MVA) boost vaccine against SIV. Rhesus macaques were injected with Fms-like tyrosine kinase 3 (Flt3)–ligand (FL) to expand dendritic cells (DCs) and were primed with a DNA vaccine encoding immunodeficiency virus antigens mixed with ligands for TLR9 or TLR7/8. Subsequently, the animals were boosted with DNA and twice with recombinant MVA expressing the same antigens. TLR9-L (CpG DNA) mediated activation of DCs in vivo and enhanced the magnitude of antigen-specific CD8⁺ interferon (IFN) γ⁺ T cells and polyfunctional CD8⁺ T cells producing IFN-γ, tumor necrosis factor α, and interleukin 2. Although this trial was designed primarily as an immunogenicity study, we challenged the animals with pathogenic SIVmac₂₅₁ and observed a reduction in peak viremia and cumulative viral loads in the TLR9-L plus FL-adjuvanted group relative to the unvaccinated group; however, the study design precluded comparisons between the adjuvanted groups and the group vaccinated with DNA/MVA alone. Viral loads were inversely correlated with the magnitude and quality of the immune response. Thus, the immunogenicity of DNA vaccines can be augmented with TLR9-L plus FL.     

Nonredundant Roles of IL-10 and TGF-�� in Suppression of Immune Responses to Hepatic AAV-Factor IX Gene Transfer

Hepatic gene transfer using adeno-associated viral (AAV) vectors has been shown to efficiently induce immunological tolerance to a variety of proteins. Regulatory T-cells (Treg) induced by this route suppress humoral and cellular immune responses against the transgene product. In this study, we examined the roles of immune suppressive cytokines interleukin-10 (IL-10) and transforming growth factor-β (TGF-β) in the development of tolerance to human coagulation factor IX (hF.IX). Interestingly, IL-10 deficient C57BL/6 mice receiving gene transfer remained tolerant to hF.IX and generated Treg that suppressed anti-hF.IX formation. Effects of TGF-β blockade were also minor in this strain. In contrast, in C3H/HeJ mice, a strain known to have stronger T-cell responses against hF.IX, IL-10 was specifically required for the suppression of CD8⁺ T-cell infiltration of the liver. Furthermore, TGF-β was critical for tipping the balance toward an regulatory immune response. TGF-β was required for CD4⁺CD25⁺FoxP3⁺ Treg induction, which was necessary for suppression of effector CD4⁺ and CD8⁺ T-cell responses as well as antibody formation. These results demonstrate the crucial, nonredundant roles of IL-10 and TGF-β in prevention of immune responses against AAV-F.IX-transduced hepatocytes.     

Enhancement of vaccine-induced primary and memory CD8(+) T-cell responses by soluble PD-1

Programmed death 1 (PD-1) signaling through its ligands, PD-L1 and PD-L2, has been known to negatively regulate T-cell responses. In addition, PD-L1 has been shown to interact with B7-1 costimulatory molecule to inhibit T-cell responses. Extensive studies have shown that PD-1/PD-L blockade restores exhausted T cells during chronic viral infections and tumors. In this study, we evaluated the effects of soluble PD-1 (sPD-1) as a blockade of PD-1 and PD-L1 on vaccine-elicited antigen-specific T-cell responses in mice. Coadministration of sPD-1 DNA with human papilloma virus-16 E7 DNA vaccine significantly enhanced E7-specific CD8(+) T-cell responses, resulting in potent antitumor effects against E7-expressing tumors. We also found that sPD-1, codelivered with adenovirus-based vaccine, could increase antigen-specific CD8(+) T-cell responses, indicating vaccine type-independent adjuvant effect of sPD-1. In addition, the frequency and functional activity of adoptively transferred OT-I cells, particularly memory CD8(+) T cells, were augmented by coadministration of sPD-1 DNA, which was closely associated with increased T-cell proliferation and reduced T-cell apoptosis through upregulation of Bcl-xL expression during T-cell activation. Codelivery of sPD-1 DNA also enhanced maturation of dendritic cells (DCs) in vivo which was accompanied by upregulation of DC maturation markers such as major histocompatibility complex class II. Taken together, our findings show that sPD-1 potently enhances codelivered antigen-specific CD8(+) T-cell responses and in vivo maturation of DCs during activation of naive CD8(+) T cells, suggesting that an immunization strategy with sPD-1 as an adjuvant can be used to increase antigen-specific T-cell immunity elicited by vaccination.     

Targeting Tim-3 and PD-1 pathways to reverse T cell exhaustion and restore anti-tumor immunity

The immune response plays an important role in staving off cancer; however, mechanisms of immunosuppression hinder productive anti-tumor immunity. T cell dysfunction or exhaustion in tumor-bearing hosts is one such mechanism. PD-1 has been identified as a marker of exhausted T cells in chronic disease states, and blockade of PD-1–PD-1L interactions has been shown to partially restore T cell function. We have found that T cell immunoglobulin mucin (Tim) 3 is expressed on CD8⁺ tumor-infiltrating lymphocytes (TILs) in mice bearing solid tumors. All Tim-3⁺ TILs coexpress PD-1, and Tim-3⁺PD-1⁺ TILs represent the predominant fraction of T cells infiltrating tumors. Tim-3⁺PD-1⁺ TILs exhibit the most severe exhausted phenotype as defined by failure to proliferate and produce IL-2, TNF, and IFN-γ. We further find that combined targeting of the Tim-3 and PD-1 pathways is more effective in controlling tumor growth than targeting either pathway alone.The importance of the immune system in protection against cancer was originally proposed in the theory of cancer immunosurveillance. This theory holds that the immune system can recognize cancerous cells as they arise and can mount both innate and adaptive immune responses to eliminate them. In support of cancer immunosurveillance is the fact that both immunodeficient or immunosuppressed patients and experimental animals are more susceptible to tumor development (for reviews see Dunn et al., 2004; Zitvogel et al., 2006; Swann and Smyth, 2007). Counter to the role of the immune system in staving off cancer is the ability of tumors to escape the immune system by engendering a state of immunosuppression (for review see Zitvogel et al., 2006). One example of a mechanism of immunosuppression present in tumor-bearing hosts is the promotion of T cell dysfunction or exhaustion.T cell exhaustion describes a state of T cell dysfunction that was initially observed during chronic lymphocytic choriomeningitis virus (LCMV) infection in mice (Zajac et al., 1998). Exhausted T cells fail to proliferate and exert effector functions such as cytotoxicity and cytokine secretion in response to antigen stimulation. Further studies identified that exhausted T cells are characterized by sustained expression of the inhibitory molecule PD-1 (programmed cell death 1) and that blockade of PD-1 and PD-L1 (PD-1 ligand) interactions can reverse T cell exhaustion and restore antigen-specific T cell responses in LCMV-infected mice (Barber et al., 2006). T cell exhaustion also occurs during chronic infections in humans (for review see Klenerman and Hill, 2005). CD8⁺ T cells in humans chronically infected with HIV (Day et al., 2006; Petrovas et al., 2006; Trautmann et al., 2006), hepatitis B virus (Boettler et al., 2006), and hepatitis C virus (HCV; Urbani et al., 2006) express high levels of PD-1, and blocking of PD-1–PD-L interactions can restore T cell function in vitro.Several lines of evidence also implicate the PD-1–PD-L pathway in T cell exhaustion in cancer. First, PD-1 expression is found on tumor-infiltrating CD8⁺ T cells in multiple solid tumors (Blank et al., 2006; Ahmadzadeh et al., 2009; Gehring et al., 2009) and on antigen-specific CD8⁺ T cells in hosts with nonsolid tumors (Yamamoto et al., 2008; Mumprecht et al., 2009). Second, these PD-1⁺ T cells are dysfunctional. Third, PD-L1 is expressed at high levels in several different cancers (Latchman et al., 2001; Dong et al., 2002; Brown et al., 2003), and high expression of PD-L1 on tumors is strongly associated with poor prognosis (Thompson et al., 2006). Fourth, interference with PD-1–PD-L signaling, either through antibody blockade or PD-1 deficiency, has been shown to improve clinical outcome and restore functional T cell responses in several cancers (Blank et al., 2006; Yamamoto et al., 2008; Mumprecht et al., 2009; Zhang et al., 2009). However, targeting the PD-1–PD-L1 pathway does not always result in reversal of T cell exhaustion (Blackburn et al., 2008; Gehring et al., 2009) and PD-1 expression is not always associated with exhausted phenotype (Petrovas et al., 2006; Fourcade et al., 2009), indicating that other molecules are likely involved in T cell exhaustion.A recent study in patients with HIV has shown that the immune regulator T cell immunoglobulin mucin (TIM) 3 is up-regulated on exhausted CD8⁺ T cells (Jones et al., 2008). Tim-3 is a molecule originally identified as being selectively expressed on IFN-γ–secreting Th1 and Tc1 cells (Monney et al., 2002). Interaction of Tim-3 with its ligand, galectin-9, triggers cell death in Tim-3⁺ T cells. Thus, both Tim-3 and PD-1 can function as negative regulators of T cell responses. In HIV patients, TIM-3 and PD-1 mark distinct populations of exhausted cells, with cells positive for both PD-1 and TIM-3 comprising the smallest fraction (Jones et al., 2008) of CD8⁺ T cells. Similarly, another group has shown that TIM-3 is up-regulated on exhausted T cells in patients with HCV (Golden-Mason et al., 2009). In this case, cells that coexpress TIM-3 and PD-1 are the most abundant fraction among HCV-specific CD8⁺ T cells. In both studies, blocking TIM-3 restored T cell proliferation and enhanced cytokine production.Because targeting the PD-1–PD-L pathway alone does not result in complete restoration of T cell function (Blackburn et al., 2008), and in some cancers targeting the PD-1–PD-L pathway does not restore T cell function at all (Gehring et al., 2009), there is a need to identify other molecules and inhibitory pathways that are involved in T cell exhaustion. Indeed, one study has identified LAG-3 as being expressed on exhausted T cells, and although treatment with anti–LAG-3 alone did not restore T cell function in LCMV-infected mice, it synergized with PD-1 blockade to improve T cell responses and reduce viral load (Blackburn et al., 2009). Unfortunately, this study did not identify whether LAG-3 and PD-1 are expressed on distinct or overlapping populations of exhausted T cells. Given these observations, it appears that targeting multiple pathways may prove most effective in reversing T cell exhaustion.We report in this paper the coexpression of Tim-3 and PD-1 on a large fraction of tumor-infiltrating lymphocytes (TILs) in mice bearing solid tumors. TILs that coexpress Tim-3 and PD-1 predominate among CD8⁺ TILs and exhibit the most profound defects in T cell effector function. We further show that combined targeting of the Tim-3 and PD-1 pathways is highly effective in controlling tumor growth.     

Enforced IL-10 Expression Confers Type 1 Regulatory T Cell (Tr1) Phenotype and Function to Human CD4+ T Cells

Type 1 regulatory T (Tr1) cells are an inducible subset of CD4⁺ Tr cells characterized by high levels of interleukin (IL)-10 production and regulatory properties. Several protocols to generate human Tr1 cells have been developed in vitro. However, the resulting population includes a significant fraction of contaminating non-Tr1 cells, representing a major bottleneck for clinical application of Tr1 cell therapy. We generated an homogeneous IL-10–producing Tr1 cell population by transducing human CD4⁺ T cells with a bidirectional lentiviral vector (LV) encoding for human IL-10 and the marker gene, green fluorescent protein (GFP), which are independently coexpressed. The resulting GFP⁺ LV-IL-10–transduced human CD4⁺ T (CD4^LV-IL-10) cells expressed, upon T-cell receptor (TCR) activation, high levels of IL-10 and concomitant low levels of IL-4, and markers associated with IL-10. Moreover, CD4^LV-IL-10 T cells displayed typical Tr1 features: the anergic phenotype, the IL-10, and transforming growth factor (TGF)-β dependent suppression of allogeneic T-cell responses, and the ability to suppress in a cell-to-cell contact independent manner in vitro. CD4^LV-IL-10 T cells were able to control xeno graft-versus-host disease (GvHD), demonstrating their suppressive function in vivo. These results show that constitutive over-expression of IL-10 in human CD4⁺ T cells leads to a stable cell population that recapitulates the phenotype and function of Tr1 cells.     

Immunomodulators targeting the PD-1/PD-L1 protein-protein interaction: From antibodies to small molecules

Cancer immunotherapy has made great strides in the recent decade, especially in the area of immune checkpoint blockade. The outstanding efficacy, prolonged durability of effect, and rapid assimilation of anti-PD-1 and anti-PD-L1 monoclonal antibodies in clinical practice have been nothing short of a medical breakthrough in the treatment of numerous malignancies. The major advantages of these therapeutic antibodies over their small molecule counterparts have been their high binding affinity and target specificity. However, antibodies do have their flaws including immune-related toxicities, inadequate pharmacokinetics and tumor penetration, and high cost burden to manufacturers and consumers. These limitations hinder broader clinical applications of the antibodies and have heightened interests in developing the alternative small molecule platform that includes peptidomimetics and peptides to target the PD-1/PD-L1 immune checkpoint system. The progress on these small molecule alternatives has been relatively slow compared to that of the antibodies. Fortunately, recent structural studies of the interactions among PD-1, PD-L1, and their respective antibodies have revealed key hotspots on PD-1 and PD-L1 that may facilitate drug discovery efforts for small molecule immunotherapeutics. This review is intended to discuss key concepts in immuno-oncology, describe the successes and shortcomings of PD-1/PD-L1 antibody-based therapies, and to highlight the recent development of small molecule inhibitors of the PD-1/PD-L1 protein-protein interaction.     

Inhibiting the MNK1/2-eIF4E axis impairs melanoma phenotype switching and potentiates antitumor immune responses

Melanomas commonly undergo a phenotype switch, from a proliferative to an invasive state. Such tumor cell plasticity contributes to immunotherapy resistance; however, the mechanisms are not completely understood and thus are therapeutically unexploited. Using melanoma mouse models, we demonstrated that blocking the MNK1/2-eIF4E axis inhibited melanoma phenotype switching and sensitized melanoma to anti–PD-1 immunotherapy. We showed that phospho-eIF4E–deficient murine melanomas expressed high levels of melanocytic antigens, with similar results verified in patient melanomas. Mechanistically, we identified phospho-eIF4E–mediated translational control of NGFR, a critical effector of phenotype switching. Genetic ablation of phospho-eIF4E reprogrammed the immunosuppressive microenvironment, exemplified by lowered production of inflammatory factors, decreased PD-L1 expression on dendritic cells and myeloid-derived suppressor cells, and increased CD8⁺ T cell infiltrates. Finally, dual blockade of the MNK1/2-eIF4E axis and the PD-1/PD-L1 immune checkpoint demonstrated efficacy in multiple melanoma models regardless of their genomic classification. An increase in the presence of intratumoral stem-like TCF1⁺PD-1⁺CD8⁺ T cells, a characteristic essential for durable antitumor immunity, was detected in mice given a MNK1/2 inhibitor and anti–PD-1 therapy. Using MNK1/2 inhibitors to repress phospho-eIF4E thus offers a strategy to inhibit melanoma plasticity and improve response to anti–PD-1 immunotherapy.