Programmed Death-1 Pathway Blockade Research Articles

MTOR signals regulate T cell exhaustion and T cell responses during PD-1 targeted immunotherapy in chronic viral infection.

Abstract Antigen-specific CD8 T cells gradually have impaired functions in chronic infection and cancers, which is termed T cell exhaustion. Since T cell exhaustion is associated with programmed death 1 (PD-1), the blockade of PD-1 pathway reinvigorates exhausted CD8 T cells, resulting in better control of infection and cancers. Stem-like CD8 T cells are important for maintenance of antigen-specific CD8 T cells during chronic infection and cancers because they have self-renewal ability and provide effector-like CD8 T cells which possess transiently cytotoxicity. PD-1 targeted therapies enhance functional T cells response by promoting differentiation of stem-like CD8 T cells toward effector-like CD8 T cells. mTOR is a serine/threonine kinase that regulates cellular functions and proliferation. However, the role of mTOR during T cell exhaustion and PD-1 targeted therapies remains elusive. Here, we found that mTOR inhibition enhanced accumulation of stem-like CD8 T cells at the early phase of infection, leading to improved efficacy of PD-1 blockade. In contrast, after the establishment of chronic LCMV infection, mTOR inhibition suppressed the transition to effector-like CD8 T cells from stem-like CD8 T cells. Mechanistically, a cell intrinsic mTOR signal was critical for differentiation of stem-like CD8 T cells into effector-like CD8 T cells during chronic infection as well as PD-1 targeted therapy. Thus, simultaneous treatments of PD-1 blockade with mTOR inhibition failed to induce effector-like CD8 T cells, resulting in reduced efficacy of PD-1 targeted therapy. Our data demonstrate that mTOR signals regulate T cell exhaustion and PD-1 targeted therapy and have important implications for combination cancer therapies with PD-1 blockade. Supported by grants from NIH (R01 AI139675)

The Landscape of Checkpoint Inhibition in the Management of Hematological Malignancies

The programmed-death 1 (PD-1) and cytotoxic T-lymphocyte associated protein 4 (CTLA-4) pathways are inhibitory immune checkpoints involved in the escape of cancer cells from the immune system. Inhibition of these immune checkpoints can lead to the induction of body’s immune response to these cancer cells. To activate the immune system against the tumor cells, various monoclonal antibodies targeting these pathways have been developed. Many of such antibodies have been approved for therapy in solid tumor malignancies and now some hematological malignancies. Here, we review the available data regarding the response to PD-1 and CTLA-4 pathway blockade in hematological malignancies including Hodgin lymphoma, non-Hodgkin lymhoma, multiple myeloma and myeloid neoplasms as well as before and after hematopoietic cell transplantation. We also discuss the specific concerns and differences related to their use in hematological malignancies (HMs) as compared to solid tumors.

Abstract 1727: Antitumor activity associated with dual targeting of CD38 and programmed death-1 (PD-1) pathways in preclinical models

Abstract Background: Blockade of the programmed death-1 (PD-1) pathway has shown efficacy across a range of tumor types. However, some tumors do not respond and a combination strategy with other treatments may be required. CD38, a leukocyte receptor and ectoenzyme, was recently shown to be upregulated following PD-1 pathway blockade in mouse tumor models (Chen et al. ASCO-SITC 2017). Daratumumab is a fully human monoclonal antibody (mAb) that binds to CD38. It has been shown to exhibit immunomodulatory properties in patients with multiple myeloma (Krejcik et al. Blood. 2016). Studies in syngeneic mouse models and in vitro assays were conducted to investigate the potential of targeting CD38 in combination with anti-PD-1 to increase antitumor response. Methods: mAbs targeting mouse CD38 (mCD38-mg2a; mCD38) and mouse PD-1 (mPD1-mIgG1-D265A; mPD-1) were tested alone or in combination in several syngeneic models: colon adenocarcinoma (MC38), plasmacytoma (J558), and lung carcinoma (M109). The effect of these mAbs on the phenotype and functionality of tumor-infiltrating and circulating lymphocytes was evaluated using flow cytometry and immunohistochemistry. In addition, the effect of daratumumab on human regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) was assessed. Results: Combined anti-mCD38/anti-mPD-1 mAb treatment enhanced tumor regression and extended survival compared with single-mAb treatment in mouse tumor models. In the MC38 model (n=10/group), 55% of mice were tumor free (TF) following combined treatment, whereas 0% and 25% were TF following individual administration of anti-mCD38 or anti-mPD-1 mAbs, respectively. Similar effects were observed in J558- and M109-bearing mice (n=10/group for both models): with combined treatment, 85% of J558 and 20% of M109 mice were TF, compared with 50% (J558) and 0% (M109) for anti-mCD38 and 25% (J558) and 0% (M109) for anti-mPD-1 mAbs, respectively. Enhanced antitumor activity with combination treatment was associated with increased infiltration and activation of intratumoral CD4+ and CD8+ T cells, as well as increased levels of Ki67 on circulating T cells in MC38 and J558 models. The antitumor activity of anti-mCD38 mAb was dependent on active Fc function, suggesting that CD38+ cells may limit antitumor response. Consistent with this hypothesis, anti-mCD38 mAb led to a reduction in the frequency of CD38+ immunosuppressive Tregs and MDSC populations in mice. In vitro, daratumumab mediated antibody-dependent cellular cytotoxicity of human Tregs and led to the depletion of human monocytic MDSCs in blood of patients with lung cancer (n=17). Conclusion: These results demonstrate the combined effect of targeting both CD38 and PD-1 pathways in regulating antitumor immunity, and suggest that dual targeting of CD38 with daratumumab and PD-1 with nivolumab may represent a promising combination strategy for treating cancer. Citation Format: Natalie A. Bezman, Michelle Kinder, Amy D. Jhatakia, Bethany K. Mattson, Darlene Pizutti, Edward W. Thompson, Dorie A. Capaldi, Mark W. Mendonca, Aravind Anandam, Gopal Dhar, Lavanya Kovvuri, Archana Devi, Swagatam Ray, Vivek Surse, Elizabeth Saravia, Robert F. Graziano, Natalie A. Hutnick, Alan Korman. Antitumor activity associated with dual targeting of CD38 and programmed death-1 (PD-1) pathways in preclinical models [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 1727.

Inhibitors of the PD-1 Pathway in Tumor Therapy.

The programmed death 1 (PD-1) pathway delivers inhibitory signals that function as a brake for immune responses. This pathway limits the initiation and duration of immune responses, thereby protecting tissues from immune-mediated damage and autoimmune diseases. However, the PD-1 pathway also inhibits immune responses to tumors. The critical role of PD-1 in preventing antitumor immunity is demonstrated by the transformative effects of PD-1 pathway blockade in a broad range of cancers with the hallmark of durability of response. Despite this success, most patients do not respond to PD-1 monotherapy, and some patients experience adverse events. In this review, we discuss the functions of the PD-1 pathway and its translation to cancer immunotherapy. We also consider current challenges and opportunities for PD-1 cancer immunotherapy, including mechanisms of response and resistance, identification of biomarkers of response to PD-1 therapy, characterization and treatment of PD-1 therapy-related adverse events, and development of safe and effective combination therapies.

Programmed Death-1 Inhibition of Phosphatidylinositol 3-Kinase/AKT/Mechanistic Target of Rapamycin Signaling Impairs Sarcoidosis CD4+ T Cell Proliferation.

Patients with progressive sarcoidosis exhibit increased expression of programmed death-1 (PD-1) receptor on their CD4+ T cells. Up-regulation of this marker of T cell exhaustion is associated with a reduction in the proliferative response to T cell receptor (TCR) stimulation, a defect that is reversed by PD-1 pathway blockade. Genome-wide association studies and microarray analyses have correlated signaling downstream from the TCR with sarcoidosis disease severity, but the mechanism is not yet known. Reduced phosphatidylinositol 3-kinase (PI3K)/AKT expression inhibits proliferation by inhibiting cell cycle progression. To test the hypothesis that PD-1 expression attenuates TCR-dependent activation of PI3K/AKT activity in progressive systemic sarcoidosis, we analyzed PI3K/AKT/mechanistic target of rapamycin (mTOR) expression at baseline and after PD-1 pathway blockade in CD4+ T cells isolated from patients with sarcoidosis and healthy control subjects. We confirmed an increased percentage of PD-1+ CD4+ T cells and reduced proliferative capacity in patients with sarcoidosis compared with healthy control subjects (P < 0.001). There was a negative correlation with PD-1 expression and proliferative capacity (r = -0.70, P < 0.001). Expression of key mediators of cell cycle progression, including PI3K and AKT, were significantly decreased. Gene and protein expression levels reverted to healthy control levels after PD-1 pathway blockade. Reduction in sarcoidosis CD4+ T cell proliferative capacity is secondary to altered expression of key mediators of cell cycle progression, including the PI3K/AKT/mTOR pathway, via PD-1 up-regulation. This supports the concept that PD-1 up-regulation drives the immunologic deficits associated with sarcoidosis severity by inducing signaling aberrancies in key mediators of cell cycle progression.

A first-in-human study of REGN2810, a monoclonal, fully human antibody to programmed death-1 (PD-1), in combination with immunomodulators including hypofractionated radiotherapy (hfRT).

3024Background: Monotherapy with PD-1 pathway blockade is effective in a significant minority of patients with various malignancies. Combination therapy may further augment immune and clinical responses. This phase 1 multicenter study assessed the safety and preliminary anti-tumor activity of REGN2810, an anti-PD-1 antibody, as monotherapy and in combination with other anti-tumor treatments in patients with advanced solid tumors. Methods: REGN2810 was evaluated at 1, 3, or 10 mg/kg IV q 2 weeks (W) as monotherapy, and at 1 and 3mg/kg IV q 2W in combination with hfRT (either 9 Gy X 3 days or 6 Gy X 5 days in cycle 1 week 2), or with (CTX) or CTX + hfRT. A standard 3 + 3 dose escalation design was used, with additional patients (up to 6 total) to confirm safety. DLT monitoring period was 28 days. Tumor response (RECIST 1.1; in non-irradiated lesions) was evaluated every 8 weeks. Results: As of Feb 2016, 58 pts (median age 57.5 yrs, 57% male) have been treated in the dose escalation/safety confirmation porti...

Tumor control with PD-1 inhibition in a patient with concurrent metastatic melanoma and renal cell carcinoma.

Blockade of the immunological checkpoint programmed death 1 (PD-1) using monoclonal antibodies has shown robust anti-tumor activity across a broad range of solid and hematological malignancies including melanoma and renal cell carcinoma (RCC). Characteristic markers such as the presence of tumor infiltrating lymphocytes, PD-L1 status, and mutational load may be equally or even more important in predicting clinical benefit from PD-1 pathway blockade than tumor histology. This case of a patient with concurrent metastatic melanoma and metastatic RCC, both of which were controlled for more than a year after a single dose of the anti-PD-1 antibody pembrolizumab, illustrates the potential to simultaneously treat distinct immunogenic tumors with anti-PD-1 agents.

Fractionated radiotherapy combined with PD-1 pathway blockade promotes CD8 T cell-mediated tumor clearance for the treatment of advanced malignancies.

Programmed death-1 (PD-1) is a T cell inhibitory receptor, expressed on recently activated and chronically stimulated CD4 and CD8 T cells (1,2). Through interacting with programmed death ligand-1 (PD-L1), PD-1 limits T cell receptor signaling, and maintains peripheral tolerance (1,2). PD-1 pathway blockade has the potential to restore effector function to exhausted T cells, thus boosting their antiviral and antitumor activity (2). This has prompted the development of PD-1/PD-L1 antibodies for treating cancer. Success in numerous preclinical studies (3-5) led to multicenter clinical trials, and FDA approval of anti- PD-1 agents (nivolumab or Opdivo ® and pembrolizumab or Keytruda ® ) for the treatment of metastatic melanoma and non-small cell lung cancer (6-8). With as many as 31% of patients benefiting from treatment and median response duration lasting 2 years (8), it is not surprising that PD-1 pathway blockade, and interference with other T cell signaling checkpoints such as cytotoxic T lymphocyteassociated antigen 4 (CTLA-4), continues to generate excitement for cancer immunotherapy. Given that PD-1 and CTLA-4 blockade exert distinct effects on tumor control, combined blockade has been successful in mouse models of melanoma and clinical trials with 53% response rate (9). In order to further increase efficacy, it is necessary to understand how immunotherapy could complement already approved treatment modalities, such as chemotherapy and radiation.

PD-1 pathway-mediated regulation of islet-specific CD4+ T cell subsets in autoimmune diabetes.

Type 1 diabetes (T1D) is a CD4+ T cell-driven autoimmune disease resulting from the destruction of insulin-producing pancreatic beta cells. Clinical evidence and studies in non-obese diabetic (NOD) mice suggest that insulin is a major autoantigen. With this in mind, we developed insulin B10-23:IAg7 tetramer reagents to track insulin-specific CD4+ T cells in mice and interrogated the role of Programmed death-1 (PD-1) for peripheral tolerance. PD-1 is a T cell inhibitory receptor necessary to maintain tolerance and prevent T1D in NOD mice. PD-1 pathway inhibitors are increasingly used in the clinic for treating malignancies, and while many patients benefit, some develop adverse autoimmune events, including T1D. We therefore sought to understand the role of PD-1 in maintaining islet-specific tolerance in diabetes-resistant strains. B6.g7 mice express the same MHC Class II allele as NOD mice, have predominantly naïve insulin-specific CD4+ T cells in the periphery, and remain diabetes-free even after PD-1 pathway blockade. Here, we examined the trafficking potential of insulin-specific CD4+ T cells in NOD and B6.g7 mice with or without anti-PD-L1 treatment, and found that PD-L1 blockade preferentially increased the number of CD44highCXCR3+ insulin-specific cells in NOD but not B6.g7 mice. Additionally, we investigated whether pancreatic islets in NOD and B6.g7 mice expressed CXCL10, a lymphocyte homing chemokine and ligand for CXCR3. Anti-PD-L1 treated and control NOD mice had detectable CXCL10 expression in the islets, while B6.g7 islets did not. These data suggest that islet tolerance may be in part attributed to the pancreatic environment and in the absence of pancreas inflammation, chemotactic cytokines may be missing. This, together with our previous data showing that PD-1 pathway blockade preferentially affects effector but not anergic self-specific T cells has implications for the use of checkpoint blockade in treating tumor patients. Our work suggests that determining tumor- and self-specific CD4+ T cell activation status (naïve, effector or anergic) prior to initiation of immunotherapy would likely help to stratify individuals who would benefit from this therapy versus those who might have adverse effects or incomplete tumor control.

Role of PD-1/PD-L1 in Acute and Chronic Graft Versus Host Disease

Programmed death 1 (PD-1) and its ligands, PD-L1 and PD-L2, play an important role in the maintenance of peripheral tolerance. PD-1 is an inhibitory receptor that attenuates TCR signaling. Its expression is inducible on T-cells, B-cells, NKT-cells, and activated monoytes. Interactions between PD-1 and its ligands deliver inhibitory signals that regulate T-cell activation, tolerance, and immune-mediated tissue damage.A blocking anti-PD-1 mAb given at the time of transplant markedly accelerated acute GVHD lethality in preclinical models via an interferon-gamma dependent mechanism. Both PD-L1 and PD-L2 expression were upregulated in the spleen, liver, colon, and ileum of GVHD mice. PD-L2 expression was limited to hematopoietic cells, but hematopoietic and endothelial cells expressed PD-L1. PD-1/PD-L1, but not PD-1/PD-L2, blockade markedly accelerated GVHD-induced lethality. PD-L1-deficient hosts exhibit rapid mortality associated with increased gut T-cell homing and loss of intestinal epithelial integrity, increased donor T-cell proliferation, activation, Th1 cytokine production, and reduced apoptosis. Bioenergetics profile analysis of proliferating alloreactive donor T-cells demonstrated increased aerobic glycolysis and oxidative phosphorylation, hyperpolarized mitochondrial membrane potential, increased superoxide production, and increased expression of a glucose transporter. During acute GVHD, PD-L1 was up-regulated on donor T-cells. Surprisingly, GVHD-induced lethality was significantly reduced in recipients of donor T cells devoid of PD-L1 and associated with reduced PD-L1-/- donor T-cell infiltration into lymphoid organs and gut, a retention of intestinal epithelial integrity, and a lower production of inflammatory cytokines. During GVHD, PD-L1-/- donor T cells showed increased apoptosis and reduced proliferation, as well as reduced glycolysis, glutaminolysis, and fatty acid metabolism. A role for PD-L1 in glucose-mediated acetyl-CoA production was seen, highlighting the important of glucose as an important carbon source in in alloreactive T cells undergoing clonal expansion. Further data support the hypothesis that the PD-1/PD-L1 pathway regulates T-T interaction. Together our studies indicate that PD-L1 expression that is upregulated on alloreactive donor T cells increases their survival and alters their metabolic pathway utilization in GVHD mice. In contrast to acute GVHD models, we have found that PD-1 pathway blockade can reduce chronic GVHD in a mouse model of multi-organ system disease in which one prominent component is bronchiolitis obliterans. This may occur via effects on T follicular regulatory or germinal center B cells. In summary, we have identified distinct consequences of PD-1/PD-L1 engagement in preclinical acute and chronic GVHD models: PD-1/PD-L1 interactions restrain acute GVHD but increase chronic GVHD. These findings illustrate the important but complex regulatory features of this pathway on a wide array of cell types. Our finding suggests PD-1 pathway modulation may provide unique opportunities for altering immune regulation post-transplant. DisclosuresMilone:Novartis: Patents & Royalties, Research Funding. Sharpe:Costim Pharmaceuticals: Patents & Royalties.

Abstract IA19: Response and resistance PD-1 pathway blockade

Abstract Melanoma is a genetically complex cancer harboring a multitude of somatic mutations for potential targeting by the immune system. However, melanoma cells can co-opt normal immune tolerance mechanisms to evade elimination. The immunoglobulin-like molecule PD-L1 (B7-H1), expressed by many melanomas in situ, creates a protective shield against immune attack. PD-L1 is a ligand for the programmed death-1 (PD-1) receptor expressed on activated T and B cells, and it delivers an inhibitory signal to suppress antitumor immunity. Blocking the PD-1:PD-L1 interaction can rejuvenate anti-melanoma immunity and mediate tumor regression. Multicenter clinical trials of blocking antibodies against PD-1 (nivolumab, pembrolizumab) or PD-L1 (BMS-936559, MPDL3280A, MEDI4736) have been undertaken. In an early phase trial of biweekly nivolumab administration, durable objective tumor regressions and disease stabilization were observed among 31% and 7%, respectively, of 107 patients with advanced treatment-refractory melanoma. Median overall survival in this trial was 17 months. Partial tumor regressions that are durable and persist after drug discontinuation suggest that PD-1 blockade can reset the balance between the immune system and melanoma. In some patients, “mixed” tumor regressions or initial disease progression followed by regression suggest that tumor lesions separated anatomically or chronologically may be immunologically heterogeneous. Durable objective tumor regressions reported in clinical trials of various antibodies blocking the PD-1 and PD-L1 immune checkpoints validate the importance of targeting this pathway in melanoma therapy. Ongoing investigations into pharmacodynamics and potential biomarkers of response are designed to reveal the basis for immunological heterogeneity among different patients, and among diverse tumor sites in individual patients, to enable the optimal clinical development of these drugs. Supported by research grants from Bristol-Myers Squibb, the National Institutes of Health, Stand Up 2 Cancer, the Barney Family Foundation, the Laverna Hahn Charitable Trust, Moving for Melanoma of Delaware, and the Melanoma Research Alliance. Citation Format: Suzanne L. Topalian. Response and resistance PD-1 pathway blockade. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr IA19.

Abstract PL02-03: Immune modulation for cancer therapy: Assessing antagonists and agonists

Abstract PL02-03: Immune modulation for cancer therapy: Assessing antagonists and agonists

Blockade of the programmed death-1 pathway restores sarcoidosis CD4(+) T-cell proliferative capacity.

Effective therapeutic interventions for chronic, idiopathic lung diseases remain elusive. Normalized T-cell function is an important contributor to spontaneous resolution of pulmonary sarcoidosis. Up-regulation of inhibitor receptors, such as programmed death-1 (PD-1) and its ligand, PD-L1, are important inhibitors of T-cell function. To determine the effects of PD-1 pathway blockade on sarcoidosis CD4(+) T-cell proliferative capacity. Gene expression profiles of sarcoidosis and healthy control peripheral blood mononuclear cells were analyzed at baseline and follow-up. Flow cytometry was used to measure ex vivo expression of PD-1 and PD-L1 on systemic and bronchoalveolar lavage-derived cells of subjects with sarcoidosis and control subjects, as well as the effects of PD-1 pathway blockade on cellular proliferation after T-cell receptor stimulation. Immunohistochemistry analysis for PD-1/PD-L1 expression was conducted on sarcoidosis, malignant, and healthy control lung specimens. Microarray analysis demonstrates longitudinal increase in PDCD1 gene expression in sarcoidosis peripheral blood mononuclear cells. Immunohistochemistry analysis revealed increased PD-L1 expression within sarcoidosis granulomas and lung malignancy, but this was absent in healthy lungs. Increased numbers of sarcoidosis PD-1(+) CD4(+) T cells are present systemically, compared with healthy control subjects (P < 0.0001). Lymphocytes with reduced proliferative capacity exhibited increased proliferation with PD-1 pathway blockade. Longitudinal analysis of subjects with sarcoidosis revealed reduced PD-1(+) CD4(+) T cells with spontaneous clinical resolution but not with disease progression. Analogous to the effects in other chronic lung diseases, these findings demonstrate that the PD-1 pathway is an important contributor to sarcoidosis CD4(+) T-cell proliferative capacity and clinical outcome. Blockade of the PD-1 pathway may be a viable therapeutic target to optimize clinical outcomes.

Profound Immune Exhaustion and Downregulation Of Interleukin-7 Alpha Receptor (CD127) In CD8 and CD4 T-Cell Subsets In Jamaican Patients Co-Infected With Human T-Cell Lymphotropic Virus Type-1 and Human Immunodeficiency Virus Type-1 Despite Normal Or Above Normal CD4 Counts

BackgroundNext to Sub-Saharan Africa, the Caribbean has the second highest prevalence of human immunodeficiency virus type 1 (HIV-1) infection; and human T-cell lymphotropic virus type 1 (HTLV-1), another human retrovirus, is also endemic. Fewer than five percent of persons with HTLV-1, predominantly transmitted through breast feeding, progress to any of the myriad associated diseases, including adult T-cell leukemia, HTLV-1 associated myelopathy/Tropical Spastic Paraparesis (HAM/TSP), or polymyositis. HTLV-1, through its oncoprotein-like transactivating (Tax) protein, results in aberrant proliferation of CD4 cells through interaction with cell cycle regulators, and activation of nuclear factor kappa B and the interleukin-2 pathway. Elevated CD4 counts, often above normal range, observed in patients with HTLV-1/HIV-1 co-infection, may be spurious, posing a challenge for laboratory monitoring of lymphoproliferative disorders or HIV-1 progression. MethodsWe compared CD4 counts, HIV-1 viral load, and clinical parameters in a cohort of patients co-infected with HTLV-1 and HIV-1 (syphilis, hepatitis B and C non-reactive), with controls with HIV-1 infection only matched for age, sex, and duration of antiretroviral therapy, as well as donors without HTLV-1 or HIV-1 infection. We used flow cytometry to characterize CD4 and CD8 naïve and memory T-cell subsets, expression of T-cell survival and homeostatic cytokine interleukin-7 alpha receptor (CD127), and examined the role of co-inhibitory programmed death-1 in HTLV-1 infected (intracellular HTLV-1 Tax protein-expressing) and HIV-1 infected (intracellular HIV-1 Gag protein-expressing) CD4 cells in immune evasion. Additionally, we assessed the effects of exogenous interleukin-7 (IL-7) and programmed death-1 pathway blockade on the function of responding CD8 cytotoxic T-lymphocyte (CTL) subsets ex vivo. ResultsAll patients with HTLV-1 and HIV-1 co-infection (n = 5) were female and of median age 42 years (range, 22 to 49 years), with normal or above normal CD4 counts. Four of five patients with HTLV-1/HIV-1 co-infection were WHO Stage 4 HIV/AIDS at diagnosis (e.g., oesophageal candidiasis, HIV nephropathy). Median duration since diagnosis with HIV-1 infection was six years (range, 1 to 18 years), and unexpectedly high CD4 count was the reason for HTLV-1/2 testing in all cases. Nadir CD4 count among patients with HTLV-1/HIV-1 co-infection was significantly higher than controls with HIV-1 infection only (n = 12) matched for age, sex, and duration of antiretroviral therapy (median 684, range 467 to 1474; and median 36, range, 8 to 352 cells per microliter, respectively; p = 0.007, Mann-Whitney U test). Flow cytometric analyses of CD8 and CD4 T-cell memory subsets revealed more profound loss of CD127 in the CD8 compared to the CD4 compartment; and CD8(+) effector memory T-cells showed the most significant downregulation of CD127 when patients with HTLV-1/HIV-1 co-infection were compared with seronegative donors (40.3±17.0%, n =5; and 73.1±7.48%, n = 10, respectively; p = 0.032, Mann-Whitney U). In ex vivo experiments in the context of peripheral blood mononuclear cells, preliminary data suggest that patients with HTLV-1/HIV-1 co-infection have impaired CD8(+) CTL function in response to HLA-restricted HTLV-1 Tax11-19 and cytomegalovirus (CMV) pp65 peptide stimulation. Further, we sought to describe expression of co-inhibitory marker programmed death-1 ligand (PDL1) on intracellular HTLV-1 Tax protein- as well as intracellular HIV-1 Gag protein-expressing CD4 cells. Blockade of PDL1 resulted in partial recovery of CTL function in patients with HTLV-1/HIV co-infection, and enhanced killing of target cells infected with either HTLV-1 or HIV-1. ConclusionsPatients with HTLV-1/HIV-1 co-infection show profound CTL exhaustion; and CD4 cells, although within or above normal CD4 count range, show aberrant naïve and memory T-cell distribution and possible immune evasion by upregulation of the programmed death-1 pathway, blockade of which showed a tendency towards enhanced virus-specific CTL function and killing of target cells infected with HTLV-1 or HIV-1. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

PD-1 Expression On Total and CMV-Specific T Cells In Early Post Transplant Is Associated With Donor Source, T Cell Maturation Profile, and Effectiveness Of CMV Control

T cell inhibitory receptors upregulate in parallel with activating receptors to prevent overwhelming immune response. Excessive or persistent inhibitory receptor expression on T cells, however, has been associated with several pathologic states, including chronic viral infection and malignancy. Programmed death-1 (PD-1) is among the most well studied inhibitory receptors, and PD-1 inhibition has resulted in regression of solid tumor malignancies and improved control of HIV infection.To examine the role of PD-1 in T cell function post allotransplant we examined PD-1 expression on total and CMV antigen specific T cells on samples collected from 43 patients between 55 and 85 days post cord blood transplant (CBT) (n=13),matched unrelated donor transplant (MURD) (n=12), matched related donor transplant (MRD) (n=18) and compared results to normal adult peripheral blood controls (PBMCs) (n=22), and cord blood controls (CB) (n=11). PD-1 expression was significantly upregulated (p<0.05) on CD4+ T cells following CBT and MURD transplantation as compared to PBMCs and on all donor sources as compared to normal CB. On CD8+ T cells, PD-1 expression was significantly upregulated (p<0.05) following CB and MURD transplants as compared to PBMC. For each donor source, there was no difference in PD1 expression on CD4+ or CD8+ T cells based on conditioning intensity, GVHD prophylaxis, use of prednisone at the time the sample was drawn, or degree of T cell chimerism. Naïve CD4+ T cells were a statistically smaller proportion of total T cells following CB transplants as compared to PBMC and central memory (CM) cells were a statistically greater proportion of total T cells following CB and MURD transplant compared to PBMC. Effector memory terminally differentiated (EMTD)s were a statistically greater portion of total T cells on MRD transplants as compared to CB.We next assessed PD-1 expression on CMV specific CD8+ T cells from HLA A2 patients using a CMVpp65 peptide (NLVPMVATV) specific A2 tetramer. Among 15 CMV positive patients assessed between 55 and 85 days post-transplant, tetramer+, CD8+ T cells were detectable in 10 patients. In patients who effectively controlled CMV clinically, defined as no reactivation (n=12) or reactivation controlled within 1 week of preemptive therapy (n=2), tetramer+, CD8+ T cell populations consisted primarily of EM/EMTD cells (28.9-98.2% of cells, median 75%, n=8) and PD-1 expression was comparable, though modestly increased, compared to total CD8+ T cell populations. In 2 patients not controlling CMV, tetramer+, CD8+ T cells had a greater proportion of naïve/CM phenotype (45.7-54.6% of cells, median 50%, n=2) and expressed markedly higher PD-1 levels than total CD8+T cells.To investigate the potential for PD-1 pathway blockade to enhance CMV responses, we performed stimulations with and without PD-L1 antibody blockade on 17 patient samples collected between 55 and 85 days post-transplant. Eleven patients demonstrated T cell proliferation in response to CMV. For nine of 11 patients, proliferative responses in CD4+ T cells were enhanced by PD-L1 antibody blockade and for eight of 11 patients proliferative responses in CD8+ T cells were enhanced by PD-L1 antibody blockade.Our observations suggest different patterns of T cell immune reconstitution depending on donor source. Similar patterns of immune reconstitution were demonstrated in the case of CMV specific responses. Our data suggests that PD-1 interactions might be manipulated to affect immune responses following transplantation. Further investigation of PD-1 and its interactions with other inhibitory receptors as well as the potential to manipulate PD-1 to enhance GVT and abrogate GVHD are ongoing. Disclosures:No relevant conflicts of interest to declare.

A Randomized, Double-Blind, Placebo-Controlled Assessment of BMS-936558, a Fully Human Monoclonal Antibody to Programmed Death-1 (PD-1), in Patients with Chronic Hepatitis C Virus Infection

Expression of the programmed death 1 (PD-1) receptor and its ligands are implicated in the T cell exhaustion phenotype which contributes to the persistence of several chronic viral infections, including human hepatitis C virus (HCV). The antiviral potential of BMS-936558 (MDX-1106) – a fully human anti-PD-1 monoclonal immunoglobulin-G4 that blocks ligand binding – was explored in a proof-of-concept, placebo-controlled single-ascending-dose study in patients (N = 54) with chronic HCV infection. Interferon-alfa treatment-experienced patients (n = 42) were randomized 5∶1 to receive a single infusion of BMS-936558 (0.03, 0.1, 0.3, 1.0, 3.0 mg/kg [n = 5 each] or 10 mg/kg [n = 10]) or of placebo (n = 7). An additional 12 HCV treatment-naïve patients were randomized to receive 10 mg/kg BMS-936558 (n = 10) or placebo (n = 2). Patients were followed for 85 days post-dose. Five patients who received BMS-936558 (0.1 [n = 1] or 10 mg/kg) and one placebo patient achieved the primary study endpoint of a reduction in HCV RNA ≥0.5 log10 IU/mL on at least 2 consecutive visits; 3 (10 mg/kg) achieved a >4 log10 reduction. Two patients (10 mg/kg) achieved HCV RNA below the lower limit of quantitation (25 IU/mL), one of whom (a prior null-responder) remained RNA-undetectable 1 year post-study. Transient reductions in CD4+, CD8+ and CD19+ cells, including both naïve and memory CD4+ and CD8+ subsets, were observed at Day 2 without evidence of immune deficit. No clinically relevant changes in immunoglobulin subsets or treatment-related trends in circulating cytokines were noted. BMS-936558 exhibited dose-related exposure increases, with a half-life of 20–24 days. BMS-936558 was mostly well tolerated. One patient (10 mg/kg) experienced an asymptomatic grade 4 ALT elevation coincident with the onset of a 4-log viral load reduction. Six patients exhibited immune-related adverse events of mild-to-moderate intensity, including two cases of hyperthyroidism consistent with autoimmune thyroiditis. Further investigation of PD-1 pathway blockade in chronic viral disease is warranted.Trial RegistrationClinicalTrials.gov NCT00703469 NCT00703469

B and T lymphocyte attenuator is highly expressed on intrahepatic T cells during chronic HBV infection and regulates their function

T cell antiviral function is impaired during chronic hepatitis B (CHB). Programmed death-1 (PD-1) impairs antiviral T cell responses, but dysfunction is not always reversed by blockade of PD-1 pathway. Whether distinct T cell populations expressing different sets of inhibitory molecules exist has not been determined. We studied the expression of the B and T lymphocyte attenuator (BTLA) on both peripheral blood mononuclear cells (PBMC) and intrahepatic lymphocytes, and the effects of blocking BTLA on circulating and intrahepatic T cells in CHB patients. Sixty-three CHB patients who underwent liver biopsy were enrolled. The expression of BTLA and PD-1 on PBMC and intrahepatic T cells was assessed by flow cytometry with antibodies to T cell differentiation molecules. Functional recovery was evaluated by analyzing production of interferon (IFN)-γ and interleukin (IL)-2 after incubation of T cells with anti-CD3 and irradiated mature dendritic cells in the presence of anti-BTLA, anti-PD-1, or both. Intrahepatic T cells expressed higher levels of BTLA than their peripheral counterparts. A significant fraction of intrahepatic T cells coexpressed BTLA and PD-1 and showed deep exhaustion of T cell responses. Blockade of the BTLA pathway enhanced both intrahepatic and PBMC T cell proliferation and cytokine secretion, and exhibited an additive effect upon blockage of PD-1. Upregulation of inhibitory receptor BTLA restricts T cell responses in CHB. T cell exhaustion by high antigen concentrations exacerbates dysfunction of peripheral and intrahepatic T cells. Blockage of BTLA is a potential therapeutic approach for chronic HBV infection that may act by restoring antiviral T cell responses.

CO-INHIBITORY MOLECULE PROGRAMMED DEATH-1 AND ITS LIGANDS: A NEW ALTERNATIVE THERAPY FOR HUMAN IMMUNODEFICIENCY VIRUS INFECTION?

Chronic viral infections are characterized by the up-regulation of a set of immunomodulatory receptors. The over-expression of co-inhibitory molecules on T cells leads to a dysfunctional T cell response with an &#34exhausted&#34 phenotype. Programmed Death-1 (PD-1) is a molecule that exerts an inhibitory signal on the T cell receptor when it binds to the PD-L1 or PD-L2 ligands present on antigen-presenting cells. Also, the expression of these molecules has been associated to the loss of T cell functions as well as clinical markers of the progression of HIV infection. The study of these molecules has gained attention due to reports indicating that blockade of PD-1 pathway could partially reconstitute T cell functions. In fact, this mechanism has been proposed as an alternative treatment for some chronic viral infections such as HIV infection. This review is focused on those mechanisms that might be favouring the over-expression of PD-1 and its ligands during HIV infection and on the possible new approaches that, by reducing its expression, might represent new strategies for the treatment of HIV infection. Knowing the exact mechanism leading to PD-1, PD-L1 and PDL2 expression in physiologic and pathological conditions is essential for the development of successful treatments. Novel molecular mechanisms inhibiting PD-1 activation might have potential therapeutic use not only in HIV infection but also in other diseases.

PD-1 localization in the immunological synapse is different in exhausted as compared to effector LCMV specific CD8 T cells (90.12)

Abstract Understanding the molecular basis of ineffective T cell response during chronic viral infections may provide new therapeutic targets. Our studies in lymphocytic choriomeningitis virus (LCMV) infection provided the first indication that programmed death-1 (PD-1) pathway regulate T cell dysfunction during chronic infection. These findings were extended to HIV, SIV, HBV and HCV infections and suggest that in vivo blockade of PD-1 pathway may provide a therapeutic approach for human chronic infections. However, further studies are needed to determine how best to modulate PD-1 signaling to activate anti-viral T cells while minimizing immunopathology. Understanding these signaling effects would be greatly advanced by direct imaging of PD-1 in relation to signaling structures in the immunological synapse. Using LCMV model, we investigated PD-1 localization at the T:DC synapse by direct imaging of PD-1 in relation to T cell receptor by confocal microscopy. We compared P14 (LCMV gp33-specific) effector and exhausted cells from mice infected with LCMV Armstrong and clone 13 strains, respectively. Here we show that PD-1:TCR co-localization at the T:DC synapse is different between exhausted and effector CD8 T cells. Our study provides important informations on molecular mechanisms behind T cell exhaustion and may help to identify novel targets to modify PD-1 signaling for the treatment of human chronic infections.

PD-1 upregulation is associated with HBV-specific T cell dysfunction in chronic hepatitis B patients

Programmed death-1 (PD-1) is demonstrated to have an increased expression on antigen-specific T cells during chronic virus infections, and the blockage of PD-1/PD-ligand (PD-L1) pathway could restore the function of exhausted T cells. We measured the PD-1 expression levels on HBV-specific CD8 T cells and investigated the role of PD-1/PD-L1 pathway in T-cell responses of patients with different HBV infection statuses. Compared to the patients with convalescent acute hepatitis B, PD-1 expression on total CD8 T cells from chronic hepatitis B (CHB) patients was significantly upregulated, especially on the HBV pentamer-positive CD8 T cells. And PD-L1, but not PD-L2, was also significantly upregulated on PBMC from CHB patients. In CHB patients, HBV-specific T cells and cellular proliferation could be observed under the recombinant HBV-Ag stimulation in vitro, and blockade of PD-1 pathway significantly enhanced the IFN-gamma production and cellular proliferation of PBMC. Furthermore, PD-1 expression level on HBV-pentamers positive CD8 T cells was positively associated with plasma viral load in CHB patients. Thus, PD-1 upregulation on HBV-specific CD8 T cells is engaged in the dysfunction of T cells and high viraemia in CHB patients, and the antiviral T-cell responses could be improved by the blockade of this inhibitory PD-1/PD-L1 pathway.