Lymphocyte Effector Functions Research Articles

Abstract 4861: Oral immune checkpoint antagonists targeting PD-L1/VISTA or PD-L1/Tim3 for cancer therapy

Abstract Recent successes in achieving highly durable clinical responses with antibodies to immune checkpoint receptors such as CTLA4 and PD1 have transformed the outlook for cancer therapy. While these antibody-based therapies show impressive clinical activity, they suffer from the shortcomings including the need to administer by intravenous injection, failure to show response in majority of patients and immune-related adverse events (irAEs) due to the breaking of immune self-tolerance. Sustained target inhibition as a result of a long half-life (>15-20 days) and >70% target occupancy for months may be factors contributing to irAEs observed. We sought to discover and develop small molecule immune checkpoint antagonists capable of targeting PD-L1 and another immune checkpoint pathway. We reasoned that such therapeutic agents will be amenable for oral dosing, likely show greater response rate due to dual antagonism and allow better management of irAEs due a shorter pharmacokinetic profile. A focused library of compounds mimicking the interaction of checkpoint proteins was designed and synthesized. Screening and analysis of the resulting library led to the identification of hits capable of functional disruption of the checkpoint protein(s) signaling depending upon the pockets of sequence similarity of interacting proteins. Further optimization resulted in compounds targeting PD-L1/VISTA or PD-L1/TIM-3 with desirable physico-chemical properties and exposure upon oral administration.. The ability of compounds to disrupt specific immune checkpoint pathways was confirmed though functional studies. Identified lead compounds exhibit potent activity when tested in assays to rescue lymphocyte proliferation and effector functions inhibited by respective ligands/proteins. In a panel of functional assays, the selected lead compounds showed selectivity against other immune checkpoint pathways including CTLA4, LAG3 and BTLA. Lead compounds exhibited sustained immune PD in vitro and in vivo suggesting that drug efficacy may extend beyond drug clearance. Lead compounds exhibited significant efficacy in syngeneic pre-clinical tumor models of melanoma, breast carcinoma and colon cancers upon once a day oral dosing. In repeated dose toxicity studies, the most advanced compound, AUPM-170, a dual antagonist of PD-L1 and VISTA, was well tolerated at >100x of the efficacious doses. The data demonstrating the inhibition of PD-L1 and another immune checkpoint pathway (VISTA or Tim3) resulting in activation of T cells and anti-tumor activities support further development of these orally bioavailable agents. IND-enabling studies with one of the lead compounds, AUPM-170, are underway towards advancing it to the clinic. Citation Format: Pottayil Sasikumar, N S Sudarshan, Nagaraj Gowda, D S Samiulla, Raghuveer Ramachandra, T Chandrasekhar, Sreenivas Adurthi, Jiju Mani, Rashmi Nair, Sharad Singh, Amit Dhudashia, Nagesh Gowda, Murali Ramachandra. Oral immune checkpoint antagonists targeting PD-L1/VISTA or PD-L1/Tim3 for cancer therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4861.

Gut bacteria modulate dendritic cell priming of diabetogenic T cell effector functions in a mouse model of autoimmune diabetes

Abstract Increasing evidence implicates a contribution of the microbiome, within the gastrointestinal tract, in the initiation and progression of type 1 diabetes (T1D). Notably, our understanding of mechanisms whereby gut flora interaction with the host immune system can influence T1D pathogenesis remain poorly understood. Comparison of stool samples from the non-obese diabetic (NOD) mouse model of spontaneous T1D and non-obese resistant (NOR) mice revealed distinct bacterial flora communities present within the two strains correlated to a Th17 bias. Given that dendritic cells (DC) are critical in sampling luminal antigens and priming naïve T lymphocyte effector functions, we next interrogated the populations of CD11c+ DC within the lamina propria (LP) of NOD and NOR mice. Phenotypic characterization of LP DC revealed that NOR mice possessed more mature DC denoted by a higher frequency of CD11c+ MHCII+ cells and CD103+ CD86+ cells. We have previously shown that the oral administration of a gut bacterium Lactobaccillus johnsonii (LjN6.2), present in diabetes resistant rodents, was capable of conferring diabetes resistance to diabetes prone rodents while another strain Lactobacillus reuterii (LrTD1) had no effect. Notably, T1D resistance was correlated to a Th17 bias. Hence, we sought to investigate the influence of LjN6.2 on NOD and NOR DC in comparison to LrTD1 in vitro. We found that LjN6.2, but not LrTD1 mediated an increase in IL6 and IL10 cytokine production by DC. Together, these data suggest that gut bacteria have the important ability to shape the maturation and priming abilities of DC. These results could have critical implications regarding the ability of bacterial flora to regulate the generation of a diabetogenic immune repertoire.

Integrin‐mediated Signaling Regulates Efficiency of Lymphocyte Effector Functions

Integrin‐mediated Signaling Regulates Efficiency of Lymphocyte Effector Functions

Regulatory T Cells in Post-stroke Immune Homeostasis

The secondary neuroinflammatory response has come into focus of experimental stroke research. Immunological mechanisms after acute stroke are being investigated in the hope to identify novel and druggable pathways that contribute to secondary infarct growth after stroke. Among a variety of neuroimmunological events after acute brain ischemia, including microglial activation, brain leukocyte invasion, and secretion of pro-inflammatory factors, lymphocytes have been identified as the key leukocyte subpopulation driving the neuroinflammatory response and contributing to stroke outcome. Several studies have shown that pro-inflammatory lymphocyte subpopulations worsen stroke outcome and that inhibiting their invasion to the injured brain is neuroprotective. In contrast to the effector functions of pro-inflammatory lymphocytes, regulatory T cells (Treg) are critically involved in maintaining immune homeostasis and have been characterized as disease-limiting protective cells in several inflammatory conditions, particularly in primary inflammatory diseases of the central nervous system (CNS). However, due to the complex function of regulatory cells in immune homeostasis and disease, divergent findings have been described for the role of Treg in stroke models. Emerging evidence suggests that this discrepancy arises from potentially differing functions of Treg depending on the predominant site of action within the neurovascular unit and the surrounding inflammatory milieu. This article will provide a comprehensive review of current findings on Treg in brain ischemia models and discuss potential reasons for the observed discrepancies.

Expression and Function of NKG2D Is Impaired in CD8+ T Cells of Chronically HIV-1-Infected Patients Without ART.

Increasing line of evidence indicates that the NKG2D-activating receptor plays a relevant role in the effector functions of cytotoxic lymphocytes. In this study, we investigated the expression and function of NKG2D in CD8⁺ T cells from chronically HIV-1-infected patients with or without antiretroviral therapy (ART). We measured by flow cytometry the expression of NKG2D on CD8⁺ T-cell subsets of ART-naive and ART patients as well as seronegative healthy subjects (HIV-neg). An intrapatient analysis before and after ART initiation was also performed. Results were correlated with viral load, CD4⁺ T-cell counts, markers of immune activation (CD38, sCD14), and soluble NKG2D ligands (sMICA and sULBP2). The function of NKG2D on CD8⁺ T cell cytotoxicity was tested by ex vivo degranulation assays. We showed that NKG2D was downregulated on all CD8⁺ T-cell subsets of ART-naive patients. The expression of NKG2D on CD8⁺ T cells inversely correlated with viral load and CD38 expression but not with plasma levels of sMICA and sULBP2. Importantly, we found that NKG2D-mediated costimulation of CD8⁺ T-cell lytic activity was strongly reduced in ART-naive patients if compared with HIV-neg and ART subjects. Finally, intrapatient analysis demonstrated that effective anti-HIV-1 therapy restores NKG2D expression and NKG2D-induced cytotoxicity by CD8⁺ T cells. These data underscore that NKG2D downregulation contributes to impaired CD8⁺ T-cell responses in untreated HIV-1 infection and have implications for monitoring immune functions and response to treatments, and for the development of novel anti-HIV-1 strategies combining ART with drugs that stimulate NKG2D expression and function.

Abstract A96: First-in-class orally available immune checkpoint antagonists for cancer therapy

Abstract Highly durable clinical responses observed with antibodies to immune checkpoint receptors such as CTLA4 and PD1 have revolutionized the outlook of cancer therapy. However, while these antibodies show impressive clinical activity, they suffer from the shortcomings including the need to administer by intravenous injection and immune-related adverse events due to the breaking of immune self-tolerance. Sustained target inhibition as a result of a long half-life (>15-20 days) and >70% target occupancy for months are likely contributing to irAEs observed. Herein we report the discovery of the first-in-class small molecule PD-L1 antagonists that are amenable for oral dosing to achieve potent anti-tumor activity but with a shorter pharmacokinetic profile as a strategy to better manage irAEs. A focused library of compounds mimicking the interaction of PD1 with PD-L1 was designed and synthesized. Screening and analysis of the resulting library led to the identification of compounds capable of functional disruption of the PD-L1 signaling. Further optimization of the initial hits resulted in compounds with desirable physico-chemical properties and exposure upon oral administration. Disruption of the PD1-PDL1 interaction by lead compounds was confirmed in binding assays. Potent activity comparable to that obtained with an anti-PD1 antibody in rescuing lymphocyte proliferation and effector functions inhibited by PD-L1 was observed with lead compounds. In a panel of functional assays, the lead compounds showed selectivity against other immune checkpoint pathways including CTLA4, TIM3, LAG3 and BTLA. In syngeneic pre-clinical models of melanoma and colon cancers, significant efficacy comparable to that observed with an anti-PD1 antibody in inhibition of both primary tumor growth and metastasis was noted upon once a day oral dosing. In a 14-day repeated dose toxicity studies, the lead compound was well tolerated at >100x of the efficacious doses. The findings demonstrating the inhibition of PD-L1 pathway resulting in activation of T cells and anti-tumor activities support further development of these orally bioavailable agents. IND-enabling studies with one of the lead compounds are underway towards advancing it to the clinic. Citation Format: Pottayil G. Sasikumar, Sudarshan S. Naremaddepalli, Nagaraj Gowda, Sreenivas Adurthi, Samiulla Dhodheri, Amit Dhudashia, Raghuveer Ramachandra, Murali Ramachandra. First-in-class orally available immune checkpoint antagonists for cancer therapy. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A96.

Progression of Lung Cancer Is Associated with Increased Dysfunction of T Cells Defined by Coexpression of Multiple Inhibitory Receptors.

Dysfunctional T cells present in malignant lesions are characterized by a sustained and highly diverse expression of inhibitory receptors, also referred to as immune checkpoints. Yet, their relative functional significance in different cancer types remains incompletely understood. In this study, we provide a comprehensive characterization of the diversity and expression patterns of inhibitory receptors on tumor-infiltrating T cells from patients with non-small cell lung cancer. In spite of the large heterogeneity observed in the amount of PD-1, Tim-3, CTLA-4, LAG-3, and BTLA expressed on intratumoral CD8(+) T cells from 32 patients, a clear correlation was established between increased expression of these inhibitory coreceptors and progression of the disease. Notably, the latter was accompanied by a progressively impaired capacity of T cells to respond to polyclonal activation. Coexpression of several inhibitory receptors was gradually acquired, with early PD-1 and late LAG-3/BTLA expression. PD-1 blockade was able to restore T-cell function only in a subset of patients. A high percentage of PD-1(hi) T cells was correlated with poor restoration of T-cell function upon PD-1 blockade. Of note, PD-1(hi) expression marked a particularly dysfunctional T-cell subset characterized by coexpression of multiple inhibitory receptors and thus may assist in identifying patients likely to respond to inhibitory receptor-specific antibodies. Overall, these data may provide a framework for future personalized T-cell-based therapies aiming at restoration of tumor-infiltrating lymphocyte effector functions.

NK Cell Functional Impairment after Allogeneic Hematopoietic Stem Cell Transplantation Is Associated with Reduced Levels of T-bet and Eomesodermin.

NK cells play a major role in protection against tumor recurrence and infection after allogeneic hematopoietic stem cell transplantation (HSCT). It has been shown that NK cell function after HSCT is impaired, but underlying molecular mechanisms are not well-known. In this report we show that the level of T-bet and Eomesodermin (Eomes), two T-box transcription factors regulating lymphocyte effector functions, is strongly reduced in NK cells from HSCT recipients compared with healthy control subjects. Reduction of T-bet and Eomes expression appeared early and persisted for years after HSCT, affecting all peripheral blood NK cells independently of their differentiation status. Reduced T-bet levels in NK cells from allogeneic HSCT recipients significantly correlated with reduced perforin expression. Acute, but not chronic, graft-versus-host disease, as well as CMV reactivation, was associated with further downregulation of T-bet expression in NK cells. Lower levels of T-bet expression in NK cells were associated with less favorable outcome after HSCT as a result of increased nonrelapse mortality. Collectively, our results provide a possible molecular explanation for the previously reported functional exhaustion of NK cells after allogeneic HSCT and suggest an impact of the NK transcriptional machinery status on HSCT outcome.

Decreased expression of pSTAT, IRF-1 and DAP10 signalling molecules in peripheral blood lymphocytes of patients with metastatic melanoma

AimsAs numerous signalling molecules regulate effector functions of peripheral blood lymphocytes (PBLs) that have an important anti-tumour activity, the aim of this study was to analyse their level in patients...

The Fyn-ADAP Axis: Cytotoxicity Versus Cytokine Production in Killer Cells.

Lymphocyte signaling cascades responsible for anti-tumor cytotoxicity and inflammatory cytokine production must be tightly regulated in order to control an immune response. Disruption of these cascades can cause immune suppression as seen in a tumor microenvironment, and loss of signaling integrity can lead to autoimmunity and other forms of host-tissue damage. Therefore, understanding the distinct signaling events that exclusively control specific effector functions of “killer” lymphocytes (T and NK cells) is critical for understanding disease progression and formulating successful immunotherapy. Elucidation of divergent signaling pathways involved in receptor-mediated activation has provided insights into the independent regulation of cytotoxicity and cytokine production in lymphocytes. Specifically, the Fyn signaling axis represents a branch point for killer cell effector functions and provides a model for how cytotoxicity and cytokine production are differentially regulated. While the Fyn–PI(3)K pathway controls multiple functions, including cytotoxicity, cell development, and cytokine production, the Fyn–ADAP pathway preferentially regulates cytokine production in NK and T cells. In this review, we discuss how the structure of Fyn controls its function in lymphocytes and the role this plays in mediating two facets of lymphocyte effector function, cytotoxicity and production of inflammatory cytokines. This offers a model for using mechanistic and structural approaches to understand clinically relevant lymphocyte signaling.

Inhibition of the lymphocyte metabolic switch by the oxidative burst of human neutrophils.

Activation of the phagocytic NADPH oxidase-2 (NOX-2) in neutrophils is a critical process in the innate immune system and is associated with elevated local concentrations of superoxide, hydrogen peroxide (H2O2) and hypochlorous acid. Under pathological conditions, NOX-2 activity has been implicated in the development of autoimmunity, indicating a role in modulating lymphocyte effector function. Notably, T-cell clonal expansion and subsequent cytokine production requires a metabolic switch from mitochondrial respiration to aerobic glycolysis. Previous studies demonstrate that H2O2 generated from activated neutrophils suppresses lymphocyte activation but the mechanism is unknown. We hypothesized that activated neutrophils would prevent the metabolic switch and suppress the effector functions of T-cells through a H2O2-dependent mechanism. To test this, we developed a model co-culture system using freshly isolated neutrophils and lymphocytes from healthy human donors. Extracellular flux analysis was used to assess mitochondrial and glycolytic activity and FACS analysis to assess immune function. The neutrophil oxidative burst significantly inhibited the induction of lymphocyte aerobic glycolysis, caused inhibition of oxidative phosphorylation and suppressed lymphocyte activation through a H2O2-dependent mechanism. Hydrogen peroxide and a redox cycling agent, DMNQ, were used to confirm the impact of H2O2 on lymphocyte bioenergetics. In summary, we have shown that the lymphocyte metabolic switch from mitochondrial respiration to glycolysis is prevented by the oxidative burst of neutrophils. This direct inhibition of the metabolic switch is then a likely mechanism underlying the neutrophil-dependent suppression of T-cell effector function.

Control of lymphocyte homeostasis and effector function by the aryl hydrocarbon receptor

The adaptive immune system, composed of lymphocytes, recognizes diversified antigens and generates immunological memory. According to the canonical model, it is the innate immune system that captures pathogens and senses environment to activate adaptive lymphocytes through antigen presentation, costimulatory signals and cytokine milieu. Emerging evidence indicates that environmental cues can be directly conveyed to lymphocytes by the aryl hydrocarbon receptor (AhR). AhR is a ligand-activated transcription factor that widely expresses in many immune cell lineages and recognizes a broad range of ligands including endogenous and dietary metabolites, microbial derivatives and xenobiotics. This review will focus on the regulatory role of AhR in not only adaptive but also innate lymphocytes including recently discovered innate lymphoid cells.

CD200 receptor restriction of myeloid cell responses antagonizes antiviral immunity and facilitates cytomegalovirus persistence within mucosal tissue.

CD200 receptor (CD200R) negatively regulates peripheral and mucosal innate immune responses. Viruses, including herpesviruses, have acquired functional CD200 orthologs, implying that viral exploitation of this pathway is evolutionary advantageous. However, the role that CD200R signaling plays during herpesvirus infection in vivo requires clarification. Utilizing the murine cytomegalovirus (MCMV) model, we demonstrate that CD200R facilitates virus persistence within mucosal tissue. Specifically, MCMV infection of CD200R-deficient mice (CD200R-/-) elicited heightened mucosal virus-specific CD4 T cell responses that restricted virus persistence in the salivary glands. CD200R did not directly inhibit lymphocyte effector function. Instead, CD200R-/- mice exhibited enhanced APC accumulation that in the mucosa was a consequence of elevated cellular proliferation. Although MCMV does not encode an obvious CD200 homolog, productive replication in macrophages induced expression of cellular CD200. CD200 from hematopoietic and non-hematopoietic cells contributed independently to suppression of antiviral control in vivo. These results highlight the CD200-CD200R pathway as an important regulator of antiviral immunity during cytomegalovirus infection that is exploited by MCMV to establish chronicity within mucosal tissue.

Tryptophan-degrading enzymes in tumoral immune resistance.

Tryptophan is required for T lymphocyte effector functions. Its degradation is one of the mechanisms selected by tumors to resist immune destruction. Two enzymes, tryptophan-2,3-dioxygenase and indoleamine 2,3-dioxygenase 1, control tryptophan degradation through the kynurenine pathway. A third protein, indoleamine 2,3-dioxygenase 2, was identified more recently. All three enzymes were reported to be expressed in tumors, and are candidate targets for pharmacological inhibition aimed at restoring effective anti-tumoral immunity. In this review, we compare these three enzymes in terms of structure, activity, regulation, and expression in healthy and cancerous tissues, in order to appreciate their relevance to tumoral immune resistance.

Abstract 15489: Hematopoietic PI3-Kinase δ-Deficiency Exacerbates Lesional Macrophage Infiltration and Atherosclerosis in LDLR-/- Mice

Atherosclerosis and its consequences such as myocardial infarction and ischemic stroke remains the leading cause of death in western countries. Atherosclerosis is a chronic inflammatory disease of arterial blood vessels, critically involving macrophages, dendritic cells and T lymphocytes. Leukocytes highly express the catalytic phosphoinositide 3-kinase isoform p110δ (PI3Kδ), exerting a key role in the regulation of immune responses including activation, differentiation and effector function of lymphocytes. Therefore, PI3Kδ represents a promising target for the modulation of inflammatory processes during atherogenesis. To investigate the role of leukocytic PI3Kδ during atherogenesis, lethally irradiated LDLR -/- mice were either transplanted with bone marrow from PI3Kδ -/- or PI3Kδ +/+ mice. After recovery, recipient mice were fed an atherogenic diet for 6 weeks. Hypercholesterolemic PI3Kδ -/- recipient LDLR -/- mice displayed a profound reduction of peripheral B and T cells as well as strongly impaired CD4 + T-cell activation, T-helper 1 response and regulatory T-cell numbers in paraaortic lymph nodes and spleen compared with PI3Kδ +/+ transplanted controls. Surprisingly, the profound impairment of the adaptive immune system by PI3Kδ-deficiency caused a considerable aggravation of atherosclerosis in LDLR -/- mice. Atherosclerotic lesion area at the aortic root and abdominal aorta of PI3Kδ -/- recipient LDLR -/- mice was significantly increased by 72% and 218% compared with PI3Kδ +/+ recipients, respectively (n = 10[[Unable to Display Character: –]]20; P < 0.001). Importantly, atherosclerotic lesions of PI3Kδ-deficient LDLR -/- mice were characterized by a lower fraction of CD4 + T cells and a higher proportion of MOMA-2 + monocytes/macrophages compared with controls despite unaltered circulating monocyte subsets. Thus, PI3Kδ-deficiency in mononuclear phagocytes may contribute to enhanced plaque growth. In summary, we demonstrate that hematopoietic PI3Kδ plays a crucial role in regulating innate and adaptive immune responses within the arterial wall by exerting protective functions during atherogenesis. Current studies aim to dissect PI3Kδ-dependent mechanisms that modulate inflammatory and regulatory processes in multiple stages of atherosclerosis.

Control of lymphocyte egress from lymph nodes through β2-adrenergic receptors.

Lymphocyte recirculation through secondary lymphoid organs is essential for immunosurveillance and lymphocyte effector functions. Here, we show that signals through β2-adrenergic receptors (β2ARs) expressed on lymphocytes are involved in the control of lymphocyte dynamics by altering the responsiveness of chemoattractant receptors. Agonist stimulation of lymphocyte β2ARs inhibited egress of lymphocytes from lymph nodes (LNs) and rapidly produced lymphopenia in mice. Physiological inputs from adrenergic nerves contributed to retention of lymphocytes within LNs and homeostasis of their distribution among lymphoid tissues. β2ARs physically interacted with CCR7 and CXCR4, chemokine receptors promoting lymphocyte retention in LNs. Activation of β2ARs enhanced retention-promoting signals through CCR7 and CXCR4, and consequently inhibited lymphocyte egress from LNs. In models of T cell-mediated inflammatory diseases, β2AR-mediated signals inhibited LN egress of antigen-primed T cells and reduced their recruitment into peripheral tissues. Thus, this study reveals a novel mechanism for controlling lymphocyte trafficking and provides additional insights into immune regulation by the nervous system.

Hypoxia-inducible factors in regulation of immune responses in tumour microenvironment.

Hypoxia is one of the hallmarks of the tumour microenvironment. It is the result of insufficient blood supply to support proliferating tumour cells. In response to hypoxia, the cellular machinery uses mechanisms whereby the low level of oxygen is sensed and counterbalanced by changing the transcription of numerous genes. Hypoxia-inducible factors (HIF) play a critical role in the regulation of cellular responses to hypoxia. In recent years ample evidence has indicated that HIF play a prominent role in tumour immune responses. Up-regulation of HIF1αpromotes immune suppressive activity of myeloid-derived suppressive cells (MDSC) and tumour-associated macrophages (TAM) and rapid differentiation of MDSC to TAM. HIF1α does not affect MDSC differentiation to dendritic cells (DC) but instead causes DC activation. HIF inhibit effector functions of tumour-infiltrating lymphocytes. HIF1α inhibits regulatory T (Treg) cell development by switching the balance towards T helper type 17 cells. However, as a major part of Treg cell differentiation does not take place in the tumour site, a functionally more important role of HIF1α is in the promotion of Treg cell recruitment to the tumour site in response to chemokines. As a result, the presence of Treg cells inside tumours is increased. Hence, HIF play a largely negative role in the regulation of immune responses inside tumours. It appears that therapeutic strategies targeting HIF in the immune system could be beneficial for anti-tumour immune responses.

Abstract 5022: Peptide therapeutic inhibiting PD1 immune checkpoint ligands: Potential for greater anti-tumor immune response and better management of immune-related adverse events

Abstract Antibodies to immune checkpoint receptors have revolutionized the outlook of cancer therapy by achieving highly durable clinical responses. However, along with impressive clinical activity (response rate of ∼25% with either anti-CTLA4 or anti-PD1 as single agent, but > 50% with a combination), severe immune-related adverse events (irAEs) due to the breaking of immune self- tolerance (25-30% with anti-CTLA4 and up to 15-17% with anti-PD1) are becoming increasingly evident. Sustained target inhibition as a result of a long half-life (>15-20 days) and >70% target occupancy for months are likely contributing to severe irAEs observed in the clinic with antibodies targeting immune checkpoint proteins. The antibodies targeting PD-1 signaling pathway are either based on receptor (PD-1) or its ligands (PD-L1 & PDL2). It has also been reported that PDL1 also interacts with B7-1(CD80) resulting in negative T-cell regulation. Our efforts are therefore focused on (a) developing a first in class peptide therapeutic inhibiting all three reported interaction of PD-1 pathway to improve the anti-tumor immune response (b) developing immune checkpoint blockers with potent anti-tumor activity but with a shorter pharmacokinetic profile as a strategy to better manage severe irAEs. Peptide antagonist AUR-012, constructed with elements from human PD-1 receptor, displayed equipotent antagonism towards PD-L1 and PD-L2 with potent activity in rescue of lymphocyte proliferation and effector functions. Additionally crosslinking study to evaluate the effect of AUR-12 on B7.1 - PDL-1 interaction supports the finding that AUR-012 prevents the interaction of B7.1 with PD-L1. Rescue of proliferation of immune cells analyzed upon stimulation with anti-CD3/anti-CD-28 indicated a complete rescue of CD4+ and CD8+ T cells and a complete suppression of regulatory T cells. Sustained activation of circulatory immune cells and their ability to secrete IFN-γ up to 72 h indicate that pharmacodynamic effects persist even after the clearance of AUR-012 in animal models, thus supporting a dosing interval of up to 3 days. In models of melanoma, breast, kidney and colon cancers, AUR-012 showed efficacy in inhibition of both primary tumor growth and metastasis. Additionally, anti-tumor activity of AUR-012 in a pre-established CT26 model correlated well with pharmacodynamic effects as indicated by intratumoral recruitment of CD4+ and CD8+ T cells, and a reduction in PD1+ T cells (both CD4+ & CD8+) in tumor and blood. In 14-day repeated dose toxicity studies, AUR -012 was well tolerated at 100x of the efficacious doses. These findings demonstrating inhibition of all PD-1/ PD-1 ligands mediated co-inhibitory signaling pathway and the observed correlation between anti-tumor activities with the modulation of specific T-cell populations support further development of AUR-012 in the clinic. Citation Format: Murali Ramachandra, Pottayil G. Sasikumar, Rajeev K. Shrimali, Sreenivas Adurthi, Raghuvir Ramachandra, Leena K. Satyam, Amit A. Dhudashia, Samiulla Dhodheri, K B. Sunilkumar. Peptide therapeutic inhibiting PD1 immune checkpoint ligands: Potential for greater anti-tumor immune response and better management of immune-related adverse events. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5022. doi:10.1158/1538-7445.AM2014-5022

NF-κB factors control the induction of NFATc1 in B lymphocytes.

In peripheral lymphocytes, the transcription factors (TFs) NF-κB, NFAT, and AP-1 are the prime targets of signals that emerge from immune receptors. Upon activation, these TFs induce gene networks that orchestrate the growth, expansion, and effector function of peripheral lymphocytes. NFAT and NF-κB factors share several properties, such as a similar mode of induction and architecture in their DNA-binding domain, and there is a subgroup of κB-like DNA promoter motifs that are bound by both types of TFs. However, unlike NFAT and AP-1 factors that interact and collaborate in binding to DNA, NFAT, and NF-κB seem neither to interact nor to collaborate. We show here that NF-κB1/p50 and c-Rel, the most prominent NF-κB proteins in BCR-induced splenic B cells, control the induction of NFATc1/αA, a prominent short NFATc1 isoform. In part, this is mediated through two composite κB/NFAT-binding sites in the inducible Nfatc1 P1 promoter that directs the induction of NFATc1/αA by BCR signals. In concert with coreceptor signals that induce NF-κB factors, BCR signaling induces a persistent generation of NFATc1/αA. These data suggest a tight connection between NFATc1 and NF-κB induction in B lymphocytes contributing to the effector function of peripheral B cells.

Targeting of TLRs inhibits CD4+ regulatory T cell function and activates lymphocytes in human peripheral blood mononuclear cells.

Accumulating evidence suggests elements within tumors induce exhaustion of effector T cells and infiltration of immunosuppressive regulatory T cells (Tregs), thus preventing the development of durable antitumor immunity. Therefore, the discovery of agents that simultaneously block Treg suppressive function and reinvigorate effector function of lymphocytes is key to the development of effective cancer immunotherapy. Previous studies have shown that TLR ligands (TLRLs) could modulate the function of these T cell targets; however, those studies relied on cell-free or accessory cell-based assay systems that do not accurately reflect in vivo responses. In contrast, we used a human PBMC-based proliferation assay system to simultaneously monitor the effect of TLRLs on T cells (CD4(+), CD8(+), Tregs), B cells, and NK cells, which gave different and even conflicting results. We found that the TLR7/8L:CL097 could simultaneously activate CD8(+) T cells, B cells, and NK cells plus block Treg suppression of T cells and B cells. The TLRLs TLR1/2L:Pam3CSK4, TLR5L:flagellin, TLR4L:LPS, and TLR8/7L:CL075 also blocked Treg suppression of CD4(+) or CD8(+) T cell proliferation, but not B cell proliferation. Besides CL097, TLR2L:PGN, CL075, and TLR9L:CpG-A, CpG-B, and CpG-C) were strong activators of NK cells. Importantly, we found that Pam3CSK4 could: 1) activate CD4(+) T cell proliferation, 2) inhibit the expansion of IL-10(+) naturally occurring FOXP3(+) Tregs and induction of IL-10(+) CD4(+) Tregs (IL-10-producing type 1 Treg), and 3) block naturally occurring FOXP3(+) Tregs suppressive function. Our results suggest these agents could serve as adjuvants to enhance the efficacy of current immunotherapeutic strategies in cancer patients.