Lymphocyte Exhaustion Research Articles

Transgene IL-21-Engineered T Cell-Based Vaccine Potently Converts CTL Exhaustion via the Activation of the mTORC1 Pathway in Chronic Infection

CD8+ cytotoxic T lymphocyte (CTL) exhaustion is one of the major obstacles for the effectiveness of virus control in chronic infectious diseases. We previously generated novel ovalbumin (OVA)-specific 41BBL-expressing OVA-TEXO and human immunodeficiency virus (HIV-1) Gag-specific Gag-TEXO vaccines, inducing therapeutic immunity in wild-type C57BL/6 (B6) mice, and converting CTL exhaustion in recombinant OVA-specific adenovirus AdVOVA-infected B6 (AdVOVA-B6) mice with chronic infection. IL-21 cytokine plays an important role in controlling chronic infections. Therefore, in this study, we constructed recombinant transgene IL-21-expressing AdVIL-21, and generated IL-21-expressing OVA-TEXO/IL-21 and Gag-TEXO/IL21 vaccines, or control vaccines (OVA-TEXO/Null and Gag-TEXO/Null) by infecting OVA-TEXO and Gag-TEXO cells with AdVIL-21 or the control AdVNull, lacking transgene, and assessed their effects in B6 or AdVOVA-B6 mice. We demonstrate that both OVA-TEXO/IL-21 and control OVA-TEXO/Null vaccines are capable of converting CTL exhaustion in chronic infection. However, the OVA-TEXO/IL-21 vaccine more efficiently rescues exhausted CTLs by increasing stronger CTL proliferation and effector cytokine IFN-γ expression than the control OVA-TEXO/Null vaccine in AdVOVA-B6 mice with chronic infection, though both vaccines stimulated comparable OVA-specific CTL responses and protective immunity against OVA-expressing BL6-10OVA melanoma lung metastasis in wild-type B6 mice. In vivo, the OVA-TEXO/IL-21-stimulated CTLs more efficiently up-regulate phosphorylation of mTORC1-controlled EIF4E and expression of mTORC1- regulated T-bet molecule than the control OVA-TEXO/Null-stimulated ones. Importantly, the Gag-TEXO/IL21 vaccine induces stronger Gag-specific therapeutic immunity against established Gag-expressing BL6-10Gag melanoma lung metastases than the control Gag-TEXO/Null vaccine in chronic infection. Therefore, this study should have a strong impact on developing new therapeutic vaccines for patients with chronic infections.

Sepsis and Nosocomial Infection: Patient Characteristics, Mechanisms, and Modulation.

Sepsis is a leading cause of death worldwide. After initial trials modulating the hyperinflammatory phase of sepsis failed, generations of researchers have focused on evaluating hypo-inflammatory immune phenotypes. The main goal has been to develop prognostic biomarkers and therapies to reduce organ dysfunction, nosocomial infection, and death. The depressed host defense in sepsis has been characterized by broad cellular reprogramming including lymphocyte exhaustion, apoptosis, and depressed cytokine responses. Despite major advances in this field, our understanding of the dynamics of the septic host response and the balance of inflammatory and anti-inflammatory cellular programs remains limited. This review aims to summarize the epidemiology of nosocomial infections and characteristic immune responses associated with sepsis, as well as immunostimulatory therapies currently under clinical investigation.

PD-1 blockade inhibits lymphocyte apoptosis and restores proliferation and anti-viral immune functions of lymphocyte after CP and NCP BVDV infection in vitro

Bovine viral diarrhea virus (BVDV) is an important virus that can cause extensive economic losses in both dairy and beef industry worldwide. Acute infection with BVDV results in peripheral blood lymphopenia, apoptosis and immunosuppression. Up-regulated programmed death-1 (PD-1) expression induces functional exhaustion of lymphocytes, inhibition of proliferation and apoptosis of lymphocytes during acute and chronic viral infections, such as HIV and HCV. However, there are no reports showing the role of PD-1 in peripheral blood lymphopenia, apoptosis and immunosuppression after acute BVDV infection. Accordingly, we measured the mRNA and protein expression of PD-1 and programmed death-ligand 1 (PD-L1) in peripheral blood mononuclear cells (PBMCs) infected with BVDV, and analyzed the effects of PD-1 blockade on immune-associated function and activity in peripheral blood lymphocytes (PBLs). The results showed that both cytopathic (CP) BVDV (strain NADL) and non-cytopathic (NCP) BVDV (strain KD) infection stimulated the mRNA and protein expression of PD-1 and PD-L1 significantly. The upregulation of PD-1/PD-L1 was accompanied by the decreased PBLs proliferation and increased apoptosis. Additionally, PD-1 blockade restored proliferation, inhibited apoptosis, increased IFN-γ production and decreased BVDV load. Remarkably, the PD-1/PD-L1 interaction has a more substantial effect on the immunoregulation of inhibiting proliferation induced by CP BVDV infection. Our findings confirm that PD-1 plays a vital role in peripheral blood lymphopenia and apoptosis caused by acute BVDV infection, and provide new insights into exploring the immunopathological mechanisms of BVDV or other members of the Flaviviridae family, and a potential therapeutic strategy to control BVDV infection.

Abstract 4064: Spatial re-distribution of natural killer cells targets drug resistance and anti-tumor response, ex-vivo

Abstract Background: It is now clear that response or resistance to therapy is contingent on the tumor microenvironment, which is comprised of malignant cells, normal stroma, and immune landscape; attributes that are unique to each individual patient. This is particularly true for emerging anticancer drugs, such as immune checkpoint inhibitors, which recalibrate the body's own immune defense largely by modulating exhaustion of cytotoxic lymphocytes including T cells and natural killer (NK) cells. However, clinical response to therapy varies enormously. There is a critical gap in our understanding for the mechanisms that drive response or resistance to conventional drugs and immunotherapies at the individual patient level. Methods: Here, we used in-vitro and in-vivo experiments to study the role of NK cells in models of drug tolerance. Next, we employed (CANscriptTM), a clinically-validated ex-vivo tumor model that recreates and preserves the native, patient tumor microenvironment, which integrates an algorithm-driven method to predict clinical response to therapy (M-Score). Utilizing tissue from patients diagnosed with luminal, HER2 positive, and triple-negative (ER- PR- HER2-) breast cancers (N=10), we studied spatial heterogeneity of the tumor-immune contexture, M-Score, and phenotypic alterations under pressure of conventional standard-of-care regimens and immunotherapies including immune-checkpoint inhibitors. To do this, we used a comprehensive panel of immunological assays to evaluate changes in cytotoxic lymphocytes by flow cytometry, immunohistochemistry and cytokine profiling (i.e. CD56, MHC class 1A/B, NKG2D/C, CD8, CD3, PD-1, CTLA-4, TIM-3, LAG-3, 4-1BB, granzyme A/B). Results: We identified that drug resistance is associated to downregulation of activating proteins on NK cells including NKG2D/C, reducing their cytotoxic capability. Furthermore, tumor response, predicted by M-Score, correlates to a unique pattern of spatially-distributed NK cells, which associated a pro-inflammatory cytokine signature from the tumor microenvironment. Interestingly, we determined that therapy-induced expression of exhaustion biomarkers on NK cell exhaustion inversely correlated to the expression of cytotoxic granzyme B in the tumor microenvironment. Conclusions: Taken together, these data demonstrate an integral role that NK cells contribute to resistance and the antitumor response to therapy including both conventional and immuno-modulatory drugs. We further demonstrate that CANscriptTM can be harnessed to study response and resistance of the innate and adaptive immune system. Such an advance in our preclinical methods to study anticancer drugs at the individual patient level can help guide treatment decisions for clinicians while simultaneously functioning as a platform to study clinical efficacy of novel and emerging agents. Citation Format: Munisha Smalley, Basava U. Shanthappa, Hans Gertje, Mark Lawson, Manjusha Biswas, Saravanan Thiyagarajan, Biswanath Majumder, D. C. Doval, Anurag Mehta, Neyaz Alam, Nabendu Murmu, S. P. Somashekhar, Aaron Goldman. Spatial re-distribution of natural killer cells targets drug resistance and anti-tumor response, ex-vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4064.

Human Herpes Virus 8 in HIV-1 infected individuals receiving cancer chemotherapy and stem cell transplantation.

BackgroundHuman Herpes Virus 8 (HHV8) can cause Kaposi’s Sarcoma (KS) in immunosuppressed individuals. However, little is known about the association between chemotherapy or hematopoietic stem cell transplantation (HSCT), circulating HHV8 DNA levels, and clinical KS in HIV-1-infected individuals with various malignancies. Therefore, we examined the associations between various malignancies, systemic cancer chemotherapy, T cell phenotypes, and circulating HHV8 DNA in 29 HIV-1-infected participants with concomitant KS or other cancer diagnoses.MethodsWe quantified HHV8 plasma viral loads and cell-associated HHV8 DNA and determined the relationship between circulating HHV8 DNA and lymphocyte counts, and markers of early and late lymphocyte activation, proliferation and exhaustion.ResultsThere were no significant differences in plasma HHV8 DNA levels between baseline and post-chemotherapy time points or with the presence or absence of clinical KS. However, in two participants circulating HHV8 DNA increased following treatment for KS or HSCT for lymphoma,. We observed an approximately 2-log10 reduction in plasma HHV8 DNA in an individual with KS and multicentric Castleman disease following rituximab monotherapy. Although individuals with clinical KS had lower mean CD4+ T cell counts and percentages as expected, there were no significant associations with these factors and plasma HHV8 levels. We identified increased proportions of CD8+ and CD4+ T cells expressing CD69 (P = 0.01 & P = 0.04 respectively), and increased CD57 expression on CD4+ T cells (P = 0.003) in participants with detectable HHV8.ConclusionThese results suggest there is a complex relationship between circulating HHV8 DNA and tissue-based disease in HIV-1 and HHV8 co-infected individuals with various malignancies.

Lymphocyte subset expression and serum concentrations of PD-1/PD-L1 in sepsis - pilot study

BackgroundSepsis remains a major cause of mortality in critical care, for which specific treatments are lacking. The dysregulated response to infection seen in sepsis includes features of lymphocyte dysfunction and exhaustion, suggesting that immune-stimulatory therapy may improve outcomes in certain patient groups. Monoclonal antibodies targeting checkpoint molecules, such as programmed-death 1 protein (PD-1) and its ligand PD-L1, have shown success in stimulating the immune response in patients with cancer, and are being considered for future sepsis trials. The aims of this pilot study were to compare lymphocyte subset expression of PD-1 and its ligands between patients with sepsis and controls; to characterize serum levels of PD-1 and PD-L1 in patients with sepsis and controls, and determine if serum concentrations correlated with cell surface expression.MethodsExpression levels of PD-1, PD-L1 and PD-L2 on four lymphocyte subsets (CD27 + CD19+ B cells, CD27-CD19+ B cells, CD27 + CD4+ T cells and CD27-CD4+ T cells) were compared between 22 patients with sepsis (including 11 survivors and 11 non-survivors) and 11 healthy controls using flow cytometry. Levels of soluble PD-1 and PD-L1 were also compared using commercially available ELISA kits.ResultsExpression of PD-1 and PD-L1 was higher on all lymphocyte subsets in patients with sepsis compared to controls (p < 0.05). PD-L2 expression on CD27+ B cells was also higher in patients with sepsis (p = 0.0317). There was differential expression of PD-1 by CD27 status, with expression being higher in the B and T cell subsets associated with memory status (CD27+ and CD27-, respectively; p < 0.001). Higher PD-1 and PD-L1 expression was not associated with mortality or with a higher risk of nosocomial infection. There were no differences in levels of soluble PD-1 or PD-L1 between patients with sepsis and controls.ConclusionsHigher expression of PD-1 by memory subpopulations of B cells and CD4+ T cells, with normal soluble PD-1 and PD-L1 in patients with sepsis, are novel findings. This information may be useful to enrich sepsis populations for trials of PD-1/PD-L1 blockade.

Abstract P5-11-04: Therapy-induced priming of natural killer cells predicts patient-specific tumor rejection in multiple breast cancer indications

Abstract Background: Predicting patient-specific clinical response to anticancer therapy is the holy grail of treatment-selection. It is now clear that response or resistance to therapy depends on the heterogeneous tumor microenvironment, which is comprised of malignant cells, normal stroma, soluble ligands, and tumor-immune contexture; attributes that are unique to each individual patient. This is particularly true for emerging anticancer drugs, such as immune checkpoint inhibitors, which recalibrate the body's own immune defense largely by modulating exhaustion of cytotoxic lymphocytes including T cells and natural killer (NK) cells. However, clinical response to therapy varies enormously. There is a critical gap in our understanding for the mechanisms that drive response or resistance to conventional drugs and immunotherapies at the individual patient level. Methods: Here, we used a fully patient-autologous, clinically-validated ex-vivo tumor model that recreates and preserves the native, patient tumor microenvironment (CANscriptTM), which incorporates an algorithm-driven method to predict clinical response to therapy (M-Score). Utilizing tissue from patients diagnosed with luminal, HER2 positive, and triple-negative (ER- PR- HER2-) breast cancers (N=10), we studied phenotypic alterations to the tumor-immune contexture under pressure of conventional standard-of-care regimens and immunotherapies including immune-checkpoint inhibitors, ex-vivo. To do this, we used a comprehensive panel of immunological assays to evaluate changes in cytotoxic lymphocytes by flow cytometry and multiplex immunohistochemistry (i.e. CD56, MHC class 1A/B, NKG2D/C, CD8, CD3, PD-1, CTLA-4, TIM-3, LAG-3, 4-1BB, granzyme A/B). In addition, we used multiplex cytokine analysis to study the soluble components of the tumor microenvironment. Results: We identified that tumor response, predicted by M-Score, correlates to increased infiltration of NK cells, which associated a pro-inflammatory cytokine signature from the tumor microenvironment. Interestingly, these evidences were concordant with induction of the tumor-expressing biomarker MICA/B, which is known to attract and recruit active NK cells. Furthermore, we determined that therapy-induced expression of protein biomarkers associated with NK cell exhaustion inversely correlated to the expression of cytotoxic granzyme B in the tumor microenvironment. Conclusions: Taken together, these data demonstrate an integral role that NK cells contribute to the antitumor effect of therapy including conventional and immuno-modulatory drugs. It further demonstrates how a novel ex-vivo platform can be harnessed to study the mechanisms of response and resistance, which couldn't otherwise be known in a drug naïve state. Such an advance in our preclinical methods to study anticancer drugs at the individual patient level can help guide treatment decisions for clinicians while simultaneously functioning as a platform to study clinical efficacy of novel and emerging agents. Citation Format: Smalley M, Shanthappa BU, Gertje H, Lawson M, Ulaganathan B, Thayakumar A, Maciejko L, Radhakrishnan P, Biswas M, Thiyagarajan S, Majumder B, Gopinath KS, K GB, Goldman A. Therapy-induced priming of natural killer cells predicts patient-specific tumor rejection in multiple breast cancer indications [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-11-04.

Possible Critical Role of Latent Chronic Toxoplasma gondii Infection in Triggering, Development and Persistence of Autoimmune Diseases

T. gondii is globally distributed with a high proportion of the world population estimated to be seropositive, and in the U.S. the parasite is responsible for approximately million infections each year. T. gondii tachyzoites infect almost all nucleated cells and their intracellular multiplication and lifelong persistence in the host cells play an important role in triggering and development of autoimmune diseases (ADs). Latent chronic T. gondii infection may be associated with iron, iodine, and folic acid deficiencies that facilitate development and/or progression of ADs. The oral route is the natural portal of entry for the parasite and gastrointestinal manifestations are frequently reported in patients with ADs. Prolactin was found to bind to tachyzoites and this process impairs their adhesion and penetration into the host cells. Hyperprolactinemia (HPRL) demonstrated in patients with different ADs may therefore reflect host defense against T. gondii infection, and several antipsychotic drugs that induce HPRL also have antitoxoplasmatic activity. Leptin and obesity play an important role of in triggering and maintenance of inflammation and autoimmunity. T. gondii infection causes a significant increase in leptin levels and there is a significant positive association between the parasite seropositivity and obesity. Nitric oxide (NO) acts as a proapoptotic as well as an antiapoptotic biomodulator, and have a variety effects on autophagy. Overproduction of NO during T. gondii infection causes dysfunction of both these processes and therefore hinders cleaning service of the apoptotic/autophagic cell-derived antigenic remnants, finally leading to triggering and development of ADs. Damage of the olfactory system associated with chronic latent T. gondii infection may affect olfactory bulb volume and various olfactory functions, being therefore at least in part responsible for the smell impairment in ADs. The potent proinflammatory response of macrophages to infection with T. gondii type II may explain the ability of the strain to cause pathology after oral infection. The parasite also triggers the secretion of antiinflammatory cytokines, such as IL-10, TGF-b, and generation of reactive nitrogen intermediates, thus suppressing the development of the T H 1 immune responses and deactivating macrophages. Toxoplasma chronic infection-induced cytotoxic T lymphocyte exhaustion leads to development of ADs because of decreased polyfunctionality, cytotoxic capability, cytokine production, proliferative capacity, and metabolic deficiency. The process of CD4 + and CD8 + T-cell immune exhaustion inhibits the immune response, thus facilitating pathogen persistence. Systemic T. gondii infection triggers a rapid and persistent decrease in the size of naive CD4 + T lymphocyte pool, and a long-term thymic atrophy and output due to destruction of the thymic epithelium. Chronic parasite infections characterized by lower pathogen burden usually restricted to tissues, suggest alternative driving forces in the induction of T cell exhaustion, such as parasite encystations. A significantly lower occurrence of antibodies to persistent viral infections reported in patients with some ADs compared with controls may be due to suppressed (exhausted) function of host B cells. Both T. gondii- and viral-associated inflammatory processes may be mutually overlapping which lead to worsening or improving clinical course of ADs depending on final temporary or stable proinflammatory/antiinflammatory cytokine constellations. Dual-affinity T cell receptors may at least in part be responsible for frequently observed coinfections of T.gondii with some viruses and bacteria. Commonly reported comorbidities in ADs may at least in part be explained by liver damage caused by the pathogen. Vitamin D deficiency is often found in patients with ADs and there is vast evidence that the vitamin has an important beneficial impact on both innate and acquired immunity. Moreover, vitamin D exerts toxoplasmacidal effects and therefore should find a firm place in treatment regimens used in ADs.

Prostaglandin E2 and PD-1 mediated inhibition of antitumor CTL responses in the human tumor microenvironment.

Accumulating evidence indicates that inflammation plays a critical role in cancer development; however, mechanisms of immunosuppression hinder productive anti-tumor immunity to limit immunopathology. Tumor-specific cytotoxic T lymphocyte (CTL) dysfunction or exhaustion by upregulating inhibitory receptors such as programmed cell death 1 (PD-1) in tumor-bearing hosts is one such mechanism. Identification and blockade of the pathways that induce CTL dysfunction has been shown to partially restore CTL function in tumor-bearing hosts. Cyclooxygenase-2 (COX-2) is a rate-limiting enzyme for prostanoid biosynthesis, including prostaglandin E2 (PGE2), and plays a key role in both inflammation and cancer. The disruption of COX2/PGE2 signaling using COX2 inhibitors or PGE2 receptors EP2 and EP4 antagonists, combined with anti-PD-1 blockade was therapeutic in terms of improving eradication of tumors and augmenting the numbers of functional tumor-specific CTLs. Thus, COX2/PGE2 axis inhibition is a promising adjunct therapy to PD-1 blockade for immune-based therapies in cancer.

Abstract 3771: Concurrent GVAX and anti-OX40 therapy in murine intracranial glioma model

Abstract Introduction: Malignant glioma is the most aggressive brain tumor with a median survival time of less than 1.5 years post diagnosis despite standard treatments. However, recent advancements in immunotherapy hold significant promise. Irradiated whole tumor cell vaccination stimulates antitumor immunity and prolongs survival in mice with intracranial glioma tumors. We have shown that vaccination and immune checkpoint blockade is synergistic and these efforts are underway in the clinic. In contrast to immune checkpoint blockade, agonist stimulation of OX40, a member of the TNFR/TNF superfamily that is expressed on activated CD4 and CD8 T lymphocytes increases T cell proliferation, clonal memory differentiation, and cytokine release. We hypothesized that subcutaneous vaccination with irradiated GM-CSF expressing glioma cells (GVAX) would, therefore, synergize with systemic delivery of stimulating anti-OX40 antibody and provide effective immunity against intracranial gliomas. Methods: Gl261 cells were implanted in the right frontal lobes of syngeneic c57/bl6 mice using a stereotactic implantation technique. Mice received concurrent subcutaneous injection of irradiated GM-CSF expressing glioma cells and intraperitoneal injection of OX40 antibody, either monotherapy, or control treatment on days 3, 6 and 9 following tumor implantation. Results: Combination immunotherapy with irradiated GM-CSF expressing glioma cells and intraperitoneal delivery of agonist monoclonal antibody against OX40 was more effective than either therapy alone and prolonged survival in mice with established intracranial GL261 tumors. Elispot analyses of peripheral lymphocytes revealed that combination immunotherapy reverses the Th2 cytokine skew that is induced by the presence of intracranial glioma. OX40 signaling alone and in combination with vaccination drove high levels of interferon gamma expression by splenocytes harvested from glioma-bearing animals, while IL-10 expression was little changed. In the tumor microenvironment, vaccination improves the CD8+ /FoxP3+ T lymphocyte ratio, while OX40 ligation overcomes tumor-driven lymphocyte exhaustion, demonstrated by reduced expression of PD1 and LAG3, measured by flow cytometery 2 weeks after initiation of treatment. Conclusions: Combination treatment with GVAX and OX-40 agonist antibody increased survival in mice bearing intracranial gliomas and are mechanistically complementary. Citation Format: Nusrat Jahan, Hammad Talat, William T. Curry. Concurrent GVAX and anti-OX40 therapy in murine intracranial glioma model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3771. doi:10.1158/1538-7445.AM2017-3771

Interleukin-10/lymphocyte ratio predicts mortality in severe septic patients.

BackgroundImmunosuppression is common even in the early stage of severe sepsis. Interleukin-10 (IL-10) secretion and lymphocyte exhaustion are the main features of sepsis-induced immunosuppression. However, the relationship between IL-10 and the lymphocyte is still unclear. We investigated if IL-10/lymphocyte ratio (IL10LCR) were associated with mortality in severe septic patients.MethodsAdult patients with severe sepsis admitted to ICU of the First Affiliated Hospital of Guangzhou Medical University were identified from October 2012 to August 2013. Within 24 hours of ICU admission, peripheral whole blood was collected for the measurement of IL-10 using commercial multiplex bead-based assay kits and determination of lymphocyte count from laboratory data. The primary outcome was 28-day mortality.ResultsA total of 63 severe sepsis patients were identified. There were 20 (32%) patients died within 28 days. IL10LCR in non-survival patients was significantly higher than survival patients (median (IQR) 36.78 (12.34–79.63) ng/ml2 versus 11.01(5.41–27.50) ng/ml2, P = 0.002). Correlation analysis showed that IL10LCR was significantly correlated with APACHE II score (Spearman’s rho = 0.424, P<0.001). The receiver operating characteristic (ROC) curves showed the area under the curve was 0.749 for IL10LCR level to predict 28-day mortality with sensitivity and specificity at 70.0% and 74.4%, respectively. At an optimal cutoff of 23.39ng/ml2, Kaplan-Meier curve showed survival in patients with IL10LCR level above 23.39ng/ml2 was significantly lower than in patients with IL10LCR level less than 23.39ng/ml2 (P = 0.001 by log-rank test).ConclusionIL10LCR level is significantly associated with the severity and outcome of severe septic patients. It may serve as a biomarker for sepsis-induced immunosuppression.

T-Cell Immunoglobulin- and Mucin-Domain-Containing Molecule 3 Signaling Blockade Improves Cell-Mediated Immunity Against Malaria.

Cell-mediated immune responses play important roles in immune protection against Plasmodium infection. However, impaired immunity, such as lymphocyte exhaustion, is a common phenomenon in malaria. T-cell immunoglobulin- and mucin-domain-containing molecule 3 (Tim-3) is an important regulatory molecule in cell-mediated immunity and has been implicated in malaria. In this study, it was found that Tim-3 expression on key populations of lymphocytes was significantly increased in both Plasmodium falciparum-infected patients and Plasmodium berghei ANKA (PbANKA)-infected C57BL/6 mice. Upregulation of Tim-3 led to lymphocyte exhaustion, while blocking Tim-3 signaling with an anti-Tim-3 antibody restored lymphocyte activity in Plasmodium infections. Further, anti-Tim-3 treatment accelerated the parasite clearance and relieved the symptoms of cerebral malaria in PbANKA-infected mice. In conclusion, Tim-3 on immune cells negatively regulates cell-mediated immunity against Plasmodium infection, and blocking Tim-3 signaling enhances sterile immunity and may play a protective role during malarial parasite infections.

Reduced Immunocompetent B Cells and Increased Secondary Infection in Elderly Patients With Severe Sepsis.

Lymphocyte exhaustion was recently recognized as a mechanism of immunosuppression in sepsis. While B cells are known to play pivotal roles in bacterial infection and sepsis, changes in B-cell-mediated humoral immunity have not been evaluated in critically ill septic patients. We aimed to investigate changes in humoral immunity caused by defective B-cell function during severe sepsis. Thirty-three severe sepsis patients and 44 healthy subjects were prospectively enrolled. Blood was collected from patients within 72 h of and 8 to 11 h after sepsis onset to measure B-cell subtypes, serum immunoglobulin M concentration, and CpG-B oligodeoxynucleotide-induced immunoglobulin M (IgM) production ex vivo. Participants were divided into two age groups: adults (18-64 years) and elderly (≥65 years). The fraction of CD21 exhausted B cells in acute sepsis patients (3.18%) was higher than that observed in healthy donors (0.77%, respectively, P <0.01). Significantly, serum IgM in elderly septic patients (≥65 years) was negatively correlated with acute physiology and chronic health evaluation II score (r = -0.57, P <0.05). Consistently, in B cells stimulated ex vivo, both aging and sepsis induced significant reductions in supernatant IgM (P <0.01). This finding was clinically relevant, as elderly patients with decreased IgM production might be more susceptible to infection by Gram-negative bacteria and fungi. Reduced immunocompetent B cells may be related to increased secondary infection after sepsis, especially in the elderly. Finally, impaired humoral immunity with increased CD21 exhausted B cells and insufficient immunoglobulin M production may be a critical immunological change in sepsis.

Prognostic value of PDL1 expression in pancreatic cancer.

Pancreatic cancer is one of the most aggressive human cancers. PD1/PDL1-inhibitors recently showed promising results in different cancers with correlation between PDL1 tumor expression and responses. Expression of programmed cell death receptor ligand 1 (PDL1) has been scarcely studied in pancreatic cancer. In this retrospective study, we analyzed PDL1 mRNA expression in 453 clinical pancreatic cancer samples profiled using DNA microarrays and RNASeq. Compared to normal pancreatic samples, PDL1 expression was upregulated in 19% of cancer samples. Upregulation was not associated with clinicopathological features such as patients' age and sex, pathological type, tumor size, lymph node status, and grade, but was associated with shorter disease-free survival and overall survival in multivariate analyses. Analysis of correlations with biological parameters showed that PDL1 upregulation was associated with some degree of lymphocyte infiltration and signs of anti-tumor T-cell response, but to a lesser extent than what has been reported in breast cancer and GIST. PDL1-up pancreatic cancers displayed profiles of lymphocyte exhaustion, were more enriched in inhibitory molecules and pro-tumor populations (Tregs with upregulation of FOXP3 and IL10, myeloid-derived suppressor cells with upregulation of CD33 and S100A8/A9), and demonstrated a down-modulation of most MHC class I members (HLA-A/B/C, HLA-E/F/G) suggestive of a defect in antigen processing and presentation. In conclusion, our results suggest that PDL1 expression might refine the prediction of metastatic relapse in operated pancreatic cancer, and that PD1/PDL1 inhibitors might reactivate inhibited T-cells to increase the anti-tumor immune response in PDL1-upregulated tumors.

Sepsis in PD-1 light.

Increasing evidence suggests that after the first pro-inflammatory hours, sepsis is characterized by the occurrence of severe immunosuppression. Several mechanisms have been reported to participate in sepsis-induced immune alterations affecting both innate and adaptive immunity. Of these, the concept of ‘cell exhaustion’ has gained a lot of interest because some parallels can be drawn with the cancer field in which immunostimulation approaches through blocking immune checkpoints currently obtain remarkable success. Herein, perspectives regarding co-inhibitory receptors’ contribution to lymphocyte exhaustion in sepsis will be discussed in the context of a recently published study investigating the potential of PD-1 molecule expression (i.e. PD-1 on lymphocytes, PD-L1 on monocytes) to predict mortality in septic shock patients.

Novel exosome-targeted T-cell-based vaccine counteracts T-cell anergy and converts CTL exhaustion in chronic infection via CD40L signaling through the mTORC1 pathway.

CD8+ cytotoxic T lymphocyte (CTL) exhaustion is a chief issue for ineffective virus elimination in chronic infectious diseases. We generated novel ovalbumin (OVA)-specific OVA-Texo and HIV-specific Gag-Texo vaccines inducing therapeutic immunity. To assess their therapeutic effect in chronic infection, we developed a new chronic infection model by i.v. infecting C57BL/6 mice with the OVA-expressing adenovirus AdVova. During chronic AdVova infection, mouse CTLs were found to express the inhibitory molecules programmed cell-death protein-1 (PD-1) and lymphocyte-activation gene-3 (LAG-3) and to be functionally exhausted, showing a significant deficiency in T-cell proliferation, IFN-γ production and cytolytic effects. Naive CD8+ T cells upregulated inhibitory PD-ligand 1 (PD-L1), B- and T-lymphocyte attenuator and T-cell anergy-associated molecules (Grail and Itch) while down-regulating the proliferative response upon stimulation in mice with chronic infection. Remarkably, the OVA-Texo vaccine counteracted T-cell anergy and converted CTL exhaustion. The latter was associated with (i) the upregulation of a marker for CTL functionality, diacetylated histone-H3 (diAcH3), (ii) a fourfold increase in CTLs, occurring independent of host DCs or CD4+ T cells, and (iii) the restoration of CTL IFN-γ production and cytotoxicity. In vivo OVA-Texo-stimulated CTLs upregulated the activities of the mTORC1 pathway-related molecules Akt, S6, eIF4E and T-bet, and treatment of the CTLs with an mTORC1 inhibitor, rapamycin, significantly reduced the OVA-Texo-induced increase in CTLs. Interestingly, OVA-Texo-mediated CD40L signaling played a critical role in the observed immunological effects. Importantly, the Gag-Texo vaccine induced Gag-specific therapeutic immunity in chronic infection. Therefore, this study should have a serious impact on the development of new therapeutic vaccines for human immunodeficiency virus (HIV-1) infection.

Immune Exhaustion and Transplantation.

Exhaustion of lymphocyte function through chronic exposure to a high load of foreign antigen is well established for chronic viral infection and antitumor immunity and has been found to be associated with a distinct molecular program and characteristic cell surface phenotype. Although exhaustion has most commonly been studied in the context of CD8 viral responses, recent studies indicate that chronic antigen exposure may affect B cells, NK cells and CD4 T cells in a parallel manner. Limited information is available regarding the extent of lymphocyte exhaustion development in the transplant setting and its impact on anti-graft alloreactivity. By analogy to the persistence of a foreign virus, the large mass of alloantigen presented by an allograft in chronic residence could provide an ideal setting for exhausting donor-reactive T cells. The extent of T cell exhaustion occurring with various allografts, the kinetics of its development, whether exhaustion is influenced positively or negatively by different immunosuppressants, and the impact of exhaustion on graft survival and tolerance development remains a fertile area for investigation. Harnessing or encouraging the natural processes of exhaustion may provide a novel means to promote graft survival and transplantation tolerance.

Abstract B143: A phase 1a/1b study of FPA008 in combination with nivolumab in patients with selected advanced cancers

Abstract Introduction: Tumor-associated macrophages (TAMs) are a major cellular component of human tumors, and actively suppress anti-tumor immune responses. Colony stimulating factor 1 receptor (CSF1R) signaling plays a major role in the differentiation and survival of TAMs. Programmed cell death 1 (PD-1) is expressed on exhausted lymphocytes in the tumor, and delivers a negative signal to suppress lymphocyte activation. FPA008 is a humanized IgG4 monoclonal antibody that binds to human CSF1R, and blocks the ability of IL34 and CSF1 to activate CSF1R expressed on macrophages. Nivolumab is a fully-human lgG4 PD-1 immune checkpoint inhibitor that blocks the interaction of PD-1 with its ligands (PD-L1 and PD-L2), thereby inhibiting the negative signal and promoting the host immune response. This immune response can then recognize and eliminate tumor cells. Blocking CSF1R with FPA008 treatment could alleviate the immunosuppressive tumor environment that is generated by TAMs and create an environment that is more conducive to immune-based anti-cancer therapies such as nivolumab. Experimental Procedures: This open-label study is designed to evaluate the safety of FPA008 alone and in combination with nivolumab. Eligible patients (≥ 18 years) with advanced cancers will have an Eastern Cooperative Oncology Group performance status of 0 or 1. The Phase 1a dose escalation will include all advanced solid tumors and the Phase 1b expansion part of this study will focus on six tumor types: non-small cell lung cancer, melanoma, head and neck cancer, pancreatic cancer, colorectal cancer, and malignant glioma. For Phase 1a, the primary objectives are to assess the safety and tolerability of FPA008 alone and in combination with 3 mg/kg nivolumab, and to determine the maximum tolerated dose of FPA008 in this combination. Secondary objectives include an assessment of pharmacokinetics, objective response rate, overall survival, duration of response, and progression-free survival of FPA008 in combination with nivolumab. Pre- and on-treatment biopsies will be collected from patients to characterize the tumor microenvironment and examine pharmacodynamic responses to treatment. Additionally, post-progression biopsies in a subset of patients will be collected to study potential mechanisms of resistance. Exploratory objectives for Phase 1a and Phase 1b include an evaluation of biomarkers and their relationship to tumor response. Summary: This is a Phase 1 study designed in two parts to assess the safety and tolerability of FPA008 alone and in combination with nivolumab. This study will also assess efficacy and correlate response with pharmacodynamic markers of activity for both drugs. Conclusions: Emerging data elucidate the contribution TAMs make to immunosuppression in the tumor microenvironment. Inhibition of TAMs by FPA008 may reduce their immunosuppressive effects and increase T cell anti-tumor responses. This activity may be further enhanced by combining FPA008 with a PD-1 inhibitor, such as nivolumab. This study will evaluate the safety and preliminary efficacy of this novel combination in treating patients with advanced cancers. (NCT Number pending) Citation Format: Julie Brahmer, Drew Rasco, Minjia Chen, Emma Masteller, Ibrahim Qazi, Seema Rogers, Neil Sankar, Robert Sikorski, Julie Hambleton, F. Stephen Hodi. A phase 1a/1b study of FPA008 in combination with nivolumab in patients with selected advanced cancers. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B143.

Abstract 4064: Targeting the anti-tumor function of B cells in non-small cell lung cancer patient tumors

Abstract Despite improvements in surgical techniques and combined chemotherapies, the 5-year survival rate for all stages of non-small cell lung cancer (NSCLC) is only 18%. Understanding the function of tumor infiltrating lymphocytes (TILs) in NSCLC patient tumors will contribute to the development of rationally designed treatments and improved statistics. B cells in tumors (TIL-Bs) are detected in NSCLC and their frequency correlates with improved survival, however, they are understudied in human tumors. We hypothesize that TIL-Bs help generate potent, long-term immune responses against cancer. We used un-manipulated, primary human B cells from fresh tumor, tumor-adjacent, and normal (cancer-free) lung tissue. We performed a comprehensive flow cytometric analysis of all TILs in NSCLC: CD4 and CD8 TILs, T regulatory cells, TIL-Bs, NK and NKT cells. We further examined the phenotype of TIL-Bs by analyzing markers of lymphocyte activation (anti-tumor; CD69, CD86, HLA-DR, CD27) and exhaustion (pro-tumor; CD95, CD21, PD-1, PD-L1). We developed an in vitro antigen presentation assay that analyzed if TIL-Bs present tumor-specific antigens to CD4 TILs to help stimulate an anti-tumor response in NSCLC patient tumors. The total number of B cells at the site of the tumor versus the tumor-adjacent tissue was increased compared to other immune subsets. In analyzing the markers of activation and exhaustion, we observed a spectrum of activation of TIL-Bs. Further, we observed that TIL-Bs present autologous tumor antigens to CD4 TILs in a subset of NSCLC patients. These data suggest that some patients have functional TIL-Bs and some patients have nonfunctional TIL-Bs. XAGE-1b is an immunogenic, lung cancer-specific antigen that generates CD4 and CD8 T cell responses in lung cancer patients. It was overexpressed in the tumor cells of our NSCLC patients, and we demonstrated that some TIL-Bs could present recombinant XAGE-1b antigen to CD4 TILs. The TIL-Bs that could not present tumor-specific antigens had higher expression of B cell exhaustion markers on the cell surface. Further, because PD-1 expression is increased on CD4 TILs in our NSCLC patients, we are combining blockade of the immune inhibitory PD-1:PD-L1 pathway in our antigen presentation assays. In conclusion, the anti-tumor function of TIL-Bs can be stimulated in some NSCLC patients, and TIL-Bs that cannot be stimulated have increased immune exhaustion. Ultimately, results from this study will advance the understanding of the anti-tumor function of TIL-Bs in solid tumors and will help develop a model of B cell exhaustion in human cancer. It will also help predict which TIL-B functions to target in future TIL-B-specific immunotherapies or in combination with current successful immunotherapies for NSCLC patients like blockade of the immune inhibitory PD-1:PD-L1 pathway. Citation Format: Tullia Carmela Bruno, Brandon L. Moore, Daniel J. Munson, Peggy Ebner, Jeffrey Kern, Jill E. Slansky. Targeting the anti-tumor function of B cells in non-small cell lung cancer patient tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4064. doi:10.1158/1538-7445.AM2015-4064

Immunologic Aging in Adults with Congenital Heart Disease: Does Infant Sternotomy Matter?

Thymectomy is performed routinely in infants undergoing cardiothoracic surgery. Children post-sternotomy have decreased numbers of T lymphocytes, although the mechanisms involved and long-term consequences of this have not been defined. We hypothesized that lymphopenia in patients with adult congenital heart disease (ACHD) would be reflective of premature T cell maturation and exhaustion. Adults with ACHD who had sternotomy to repair congenital heart disease as infants (<1 year) and age-matched ACHD patients without prior sternotomy were studied using polychromatic flow cytometry interrogating markers of lymphocyte maturation, exhaustion and senescence. Group differences were analyzed using Mann-Whitney U and Fisher's exact tests. Eighteen ACHD patients aged 21-40 years participated: 10 cases and 8 controls. Median age at sternotomy for cases was 52 days. Cases and controls were matched for age (28.9 vs. 29.1 years; p = 0.83), gender (p = 0.15) and race (p = 0.62) and had similar case complexity. Cases had a lower mean percentage of cytotoxic CD8 lymphocytes compared to controls (26.8 vs. 33.9 %; p = 0.016), with fewer naive, undifferentiated CD8 T cells (31.0 vs. 53.6 %; p = 0.027). CD8 cells expressing PD1, a marker of immune exhaustion, trended higher in cases versus controls (25.6 vs. 19.0 %; p = 0.083). Mean percentage of CD4 cells was higher in cases versus controls (65.6 vs. 59.6 %; p = 0.027), without differences in CD4 T cell maturation subtype. In summary, ACHD patients who undergo sternotomy as infants exhibit differences in T lymphocyte composition compared to ACHD controls, suggesting accelerated immunologic exhaustion. Investigation is warranted to assess the progressive nature and clinical impact of this immune phenotypic change.