PD-L1 Inhibitory Pathway Research Articles

STAT5 activation enhances adoptive therapy combined with peptide vaccination by preventing PD-1 inhibition.

Adoptive cell therapy (ACT) using retrovirally transduced T cells represents a promising strategy for enhancing antitumor responses. When used with TriVax, a peptide vaccination strategy, this approach synergistically expands antigen-specific cell populations. STAT5 plays a vital role as a transcription factor in regulating T cell proliferation and their differentiation into effector and memory T cells. We aimed to explore the combination therapy using CD8 T cells engineered to express constitutively active STAT5 (CA-STAT5) with vaccines. CD8 T cells were transduced with a retrovirus (RV) encoding the mouse gp100 T cell receptor (TCR). In certain treatment groups, cells were also co-transduced with RV encoding CA-STAT5. We assessed transduction efficiency and functional activity through flow cytometry and various functional assays. B16F10 tumor-bearing mice were treated with ACT using RV-transduced CD8 T cells and subsequently vaccinated with TriVax. We demonstrate that TriVax selectively enhanced the expansion of ACT cell populations bearing gp100-specific TCRs. T cells engineered to express CA-STAT5 showed not only increased expansion and polyfunctionality but also reduced PD-1 expression, leading to decreased cellular exhaustion. In a B16F10 melanoma mouse model, our approach yielded a potent antitumor effect, with CA-STAT5 further amplifying this response. We found that CA-STAT5 improved antitumor activities, in part, by attenuating the PD-1/PD-L1 inhibitory pathway. These findings indicate that TCR-transduced CD8 T cells can undergo antigen-dependent expansion when exposed to TriVax. Additionally, the expression of CA-STAT5 enhances T cell proliferation and persistence, partly by promoting resistance to PD-1/PD-L1-mediated inhibition in antitumor T cells.

Plant-derived Durvalumab variants show efficient PD-1/PD-L1 blockade and therapeutically favourable FcR binding.

Immune checkpoint blocking therapy targeting the PD-1/PD-L1 inhibitory signalling pathway has produced encouraging results in the treatment of a variety of cancers. Durvalumab (Imfinzi®) targeting PD-L1 is currently used for immunotherapy of several tumour malignancies. The Fc region of this IgG1 antibody has been engineered to reduce FcγR interactions with the aim of enhancing blockade of PD-1/PD-L1 interactions without the depletion of PD-L1-expressing immune cells. Here, we used Nicotiana benthamiana to produce four variants of Durvalumab (DL): wild-type IgG1 and its 'Fc-effector-silent' variant (LALAPG) carrying further modifications to increase antibody half-life (YTE); IgG4S228P and its variant (PVA) with Fc mutations to decrease binding to FcγRI. In addition, DL variants were produced with two distinct glycosylation profiles: afucosylated and decorated with α1,6-core fucose. Plant-derived DL variants were compared to the therapeutic antibody regarding their ability to (i) bind to PD-L1, (ii) block PD-1/PD-L1 inhibitory signalling and (iii) engage with the neonatal Fc receptor (FcRn) and various Fcγ receptors. It was found that plant-derived DL variants bind to recombinant PD-L1 and to PD-L1 expressed in gastrointestinal cancer cells and are able to effectively block its interaction with PD-1 on T cells, thereby enhancing their activation. Furthermore, we show a positive impact of Fc amino acid mutations and core fucosylation on DL's therapeutic potential. Compared to Imfinzi®, DL-IgG1 (LALAPG) and DL-IgG4 (PVA)S228P show lower affinity to CD32B inhibitory receptor which can be therapeutically favourable. Importantly, DL-IgG1 (LALAPG) also shows enhanced binding to FcRn, a key determinant of serum half-life of IgGs.

Abstract LB100: Understanding how IL-2 cytokine synergizes with PD-1 therapy during chronic viral infection

Abstract In this study we have examined how the cytokine interleukin-2 (IL-2) synergizes with programmed cell death-1 (PD-1) directed immunotherapy during chronic lymphocytic choriomeningitis virus (LCMV) infection. PD-1 blockade in combination with IL-2 is one of the most effective combination therapies in this very stringent LCMV mouse model of life-long chronic infection with irreversible T-cell exhaustion. Our paper makes the following points: First, we show that the more effective viral control seen after PD-1 + IL-2 combination therapy compared to PD-1 monotherapy is mediated by the CD8+ T-cell response. Then we identify the virus-specific CD8+ T cells that proliferate and respond to the combination therapy and show that these are the same lymphoid resident PD-1+ TCF-1+ stem-like CD8+ T cells that act as resource cells to maintain the CD8+ T-cell response during chronic infection and also respond to PD-1 blockade. However, the combination therapy dramatically changes the differentiation program of these chronic resource CD8+ T cells and results in the generation of transcriptionally and epigenetically distinct effector CD8+ T cells that resemble highly functional effector CD8+ T cells seen after an acute viral infection. In contrast, PD-1 monotherapy does not modify the differentiation program and one gets more virus-specific CD8+ T cells but they are transcriptionally and epigenetically similar to what is seen in untreated chronically infected mice. This epigenetic inflexibility of exhausted CD8+ T cells is a potential barrier to PD-1 therapy and the ability of this combination therapy to modify the epigenetic signature of virus-specific CD8+ T cells during chronic infection could be an important determinant of the striking synergy seen between IL-2 therapy and PD-1 blockade. We also highlight the importance of blocking the PD-1/PD-L1 inhibitory pathway at the target site for effective viral control. Expanding the CD8+ T cell population and generating better effector cells is important but it is also critical to block PD-1 inhibitory signals at the target site for optimal immunotherapy. Finally, we show that CD25 engagement with IL-2 plays an important and essential role in the observed synergy between IL-2 cytokine and PD-1 blockade. Either blocking CD25 with an antibody or using a mutated version of IL-2 that does not bind CD25 but still binds CD122/132 almost completely abrogated the synergistic effects seen after PD-1/IL-2 combination therapy. There is currently considerable interest in PD-1 + IL-2 combination therapy for cancer patients and our fundamental studies defining the underlying mechanisms of how IL-2 synergizes with PD-1 blockade should inform these human translational studies. Citation Format: Masao Hashimoto. Understanding how IL-2 cytokine synergizes with PD-1 therapy during chronic viral infection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB100.

Abstract 6372: In situ detection of various activated immune checkpoints via next-generation proximity ligation assays in tumor tissue

Abstract Immune checkpoints (ICs), which are inhibitory signaling pathways that can down-modulate the immune responses of T cells, are pivotal in peripheral tissues and for maintaining immune self-tolerance. Among the many molecularly defined IC proteins, some of the most studied ones are CTLA-4 and its binding partners important for early T-cell co-inhibition, along with the PD-1/PD-L1 axis proteins that carry out late co-inhibitory signals. Notably, in addition to antigen presenting and other immune cells, many tumors also express CTLA-4 and PD-L1, which facilitate tumor evasion from the immune system. The inhibition of these IC proteins has revolutionized the field of cancer therapy, but its efficiency has been limited to a poorly defined subset of patients. Patient outcomes are likely to have a stronger correlation to high levels of PD-1/PD-L1 interaction and other hallmarks of pathway activation, such as PD-1 phosphorylation and recruitment of SHP-2, than to the overexpression of a single IC protein alone. To improve the predictive value of tissue staining, we created an immuno-oncology line of ultrasensitive kits based on proximity ligation, which detect the activation of multiple ICs. Naveni™ PD1/PD-L1 is a tool for direct visualization of the interaction between PD-1 and PD-L1, and has been verified in various FFPE tumor tissues. Naveni™ pY PD1 sensitively detects PD1 phosphorylation, which is the first step in the PD-1/PD-L1 inhibitory pathway activation. To study the subsequent recruitment of SHP-2 to the activated PD-1 receptor, which is crucial in immune cells but dispensable when the pathway is activated between two tumor cells, one can use the upcoming Naveni™ PD1/SHP-2 kit. Furthermore, we are developing assays to observe the competing interactions of CTLA-4 and CD28 with their binding partners CD80 and CD86, the interplay of which is pivotal for the initiation of early co-inhibition. All kits can be used on consecutive tissue sections to obtain a comprehensive picture of tumor IC pathway activation, which may not always correspond to CTLA-4, PD-1 or PD-L1 overexpression. While these kits have a chromogenic readout applicable to brightfield microscopy, we also have preliminary data on a PD-1/PD-L1 assay with a fluorescent readout thanks to which it can also be multiplexed. The multiplex feature generates an immune profile which may help improve immunotherapeutic strategies. The Naveni™ assays can be applied in basic research to elucidate the interplay of IC axes and downstream molecules, in pre-clinical and clinical research to compare stainings with the existing IHC assays and evaluate the potential prognostic value of interaction detection, and in pharma, aiding the development of new drugs or bispecific antibodies. Citation Format: Desirée Edén, Ka I Au Ieong, Naomi Cook, Doroteya Raykova, Agata Zieba Wicher. In situ detection of various activated immune checkpoints via next-generation proximity ligation assays in tumor tissue. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6372.

Diacylglycerol kinase α inhibition cooperates with PD-1-targeted therapies to restore the T cell activation program.

Antibody-based therapies blocking the programmed cell death-1/ligand-1 (PD-1/PD-L1) axis have provided unprecedent clinical success in cancer treatment. Acquired resistance, however, frequently occurs, commonly associated with the upregulation of additional inhibitory molecules. Diacylglycerol kinase (DGK) α limits the extent of Ras activation in response to antigen recognition, and its upregulation facilitates hypofunctional, exhausted T cell states. Pharmacological DGKα targeting restores cytotoxic function of chimeric antigen receptor and CD8+ T cells isolated from solid tumors, suggesting a mechanism to reverse T cell exhausted phenotypes. Nevertheless, the contribution of DGKα downstream of the PD-1/PD-L1 inhibitory axis in human T cells and the consequences of combining DGKα and anti-PD-1/PD-L1 inhibitors are still unresolved relevant issues. We used a human triple parameter reporter cell line to investigate DGKα contribution to the PD-1/PD-L1 inhibitory pathway. We also addressed the impact of deleting DGKα expression in the growth dynamics and systemic tumor-derived effects of a PD-1-related tumor model, the MC38 colon adenocarcinoma. We identify DGKα as a contributor to the PD-1/PD-L1 axis that strongly limits the Ras/ERK/AP-1 pathway. DGKα function reinforces exhausted T cell phenotypes ultimately promoting tumor growth and generalized immunosuppression. Pharmacological DGKα inhibition selectively enhances AP-1 transcription and, importantly, cooperates with antibodies blocking the PD-1/PD-L1 interrelation. Our results indicate that DGKα inhibition could provide an important mechanism to revert exhausted T lymphocyte phenotypes and thus favor proper anti-tumor T cell responses. The cooperative effect observed after PD-1/PD-L1 and DGKα blockade offers a promising strategy to improve the efficacy of immunotherapy in the treatment of cancer.

Role of the PD-1/PD-L1 Dyad in the Maintenance of Pancreatic Immune Tolerance for Prevention of Type 1 Diabetes.

The human pancreas, like almost all organs in the human body, is immunologically tolerated despite the presence of innate and adaptive immune cells that promptly mediate protective immune responses against pathogens in situ. The PD-1/PD-L1 inhibitory pathway seems to play a key role in the maintenance of immune tolerance systemically and within the pancreatic tissue. Tissue resident memory T cells (TRM), T regulatory cells (Treg), macrophages and even β cells exhibit PD-1 or PD-L1 expression that contributes in controlling pancreatic immune homeostasis and tolerance. Dysregulation of the PD-1/PD-L1 axis as shown by animal studies and our recent experience with checkpoint inhibitory blockade in humans can lead to immune dysfunctions leading to chronic inflammatory disease and to type 1 diabetes (T1D) in genetically susceptible individuals. In this review, we discuss the role of the PD-1/PD-L1 axis in pancreatic tissue homeostasis and tolerance, speculate how genetic and environmental factors can regulate the PD-1/PD-L1 pathway, and discuss PD-1/PD-L1-based therapeutic approaches for pancreatic islet transplantation and T1D treatment.

Multifunctional Immunoliposomes Combining Catalase and PD-L1 Antibodies Overcome Tumor Hypoxia and Enhance Immunotherapeutic Effects Against Melanoma.

BackgroundImmune checkpoint blockades (ICBs) are a promising treatment for cancers such as melanoma by blocking important inhibitory pathways that enable tumor cells to evade immune attack. Programmed death ligand 1 monoclonal antibodies (aPDL1s) can be used as an ICB to significantly enhance the effectiveness of tumor immunotherapy by blocking the PD-1/PD-L1 inhibitory pathway. However, the effectiveness of aPDL1s may be limited by low selectivity in vivo and immunosuppressed tumor microenvironment including hypoxia.PurposeTo overcome the limitations, we develop a multifunctional immunoliposome, called CAT@aPDL1-SSL, with catalase (CAT) encapsulated inside to overcome tumor hypoxia and aPDL1s modified on the surface to enhance immunotherapeutic effects against melanoma.MethodsThe multifunctional immunoliposomes (CAT@aPDL1-SSLs) are prepared using the film dispersion/post-insertion method. The efficacy of CAT@aPDL1-SSLs is verified by multiple experiments in vivo and in vitro.ResultsThe results of this study suggest that the multifunctional immunoliposomes preserve and protect the enzyme activity of CAT and ameliorate tumor hypoxia. Moreover, the enhanced cellular uptake of CAT@aPDL1-SSLs in vitro and their in vivo biodistribution suggest that CAT@aPDL1-SSLs have great targeting ability,resulting in improved delivery and accumulation of immunoliposomes in tumor tissue.Finally, by activating and increasing the infiltration of CD8+ T cells at the tumor site, CAT@aPDL1-SSLs inhibit the growth of tumor and prolong survival time of mice,with low systemic toxicity.ConclusionIn conclusion, the multifunctional immunoliposomes developed and proposed in this study are a promising candidate for melanoma immunotherapy, and could potentially be combined with other cancer therapies like radiotherapy and chemotherapy to produce positive outcomes.

Tissue-Resident Memory T Cells Mediate Immune Homeostasis in the Human Pancreas through the PD-1/PD-L1 Pathway

SUMMARYNon-recirculating tissue-resident memory T cells (TRMs) are the predominant T cell subset in diverse tissue sites, where they mediate protective immune responses in situ. Here, we reveal a role for TRM in maintaining immune homeostasis in the human pancreas through interactions with resident macrophages and the PD-1/PD-L1 inhibitory pathway. Using tissues obtained from organ donors, we identify that pancreas T cells comprise CD8+PD-1hi TRMs, which are phenotypically, functionally, and transcriptionally distinct compared to TRMs in neighboring jejunum and lymph node sites. Pancreas TRMs cluster with resident macrophages throughout the exocrine areas; TRM effector functions are enhanced bay macrophage-derived co-stimulation and attenuated by the PD-1/PD-L1 pathways. Conversely, in samples from chronic pancreatitis, TRMs exhibit reduced PD-1 expression and reduced interactions with macrophages. These findings suggest important roles for PD-1 and TRM-macrophage interactions in controlling tissue homeostasis and immune dysfunctions underlying inflammatory disease, with important implications for PD-1-based immunotherapies.

Tissue-Resident Memory T Cells Mediate Immune Homeostasis in the Human Pancreas Through the PD-1/PD-L1 Pathway

Non-recirculating tissue resident memory T-cells (TRM) are the predominant T cell subset in diverse tissue sites where they mediate protective immune responses in situ. Here, we reveal a role for TRM in maintaining immune homeostasis in the human pancreas, through interactions with resident macrophages and the PD-1/PD-L1 inhibitory pathway. Using tissues obtained from organ donors, we identify that pancreas T cells comprise CD8+PD-1hi TRM, which are phenotypically, functionally and transcriptionally distinct compared to TRM in neighboring jejunum and lymph node sites. Pancreas TRM cluster with resident macrophages throughout the exocrine areas; TRM effector functions are enhanced by macrophage-derived co-stimulation and attenuated by the PD-1/PD-L1 pathways. Conversely, in samples from chronic pancreatitis, TRM exhibit reduced PD-1 expression and reduced interactions with macrophages. These findings suggest important roles for PD-1 and TRM-macrophage interactions in controlling tissue homeostasis and immune dysfunctions underlying inflammatory disease, with important implications for PD-1-based immunotherapies.

Abstract 3193: Combining immunotherapies: A strategy for improving CD8 T cell-mediated antitumor responses

Abstract Adoptive cell therapy (ACT) of retrovirally transduced (RV) CD8 T cells is a powerful technique that has shown promise in tumor eradication in cancer patients. However, some major barriers to current methods are that ACT is expensive, time consuming, and requires harmful and toxic adjunct procedures. Our laboratory has demonstrated the use of TriVax, a potent peptide vaccination strategy that dramatically expands ACT cell populations and bypasses the necessity for adjunct procedures. The purpose of this project was to enhance current methods of ACT+TriVax by testing an antigen-specific antitumor response of RV CD8 T cells and if it could be improved with constitutively active STAT5 (CA-STAT5) expression, a protein activated downstream several cytokine pathways that have been shown to play a role in increasing CD8 T cell persistence and resistance to apoptosis. We tested the hypothesis that CA-STAT5 in CD8 T cells enhances an antitumor effect by increasing T cell persistence and efficacy. Our results show that TriVax administration selectively expanded frequencies of the ACT cell population expressing gp100-TCR in both blood and spleen. When co-transduced with CA-STAT5, an even higher fold expansion of antigen-specific cells was observed. +CA-STAT5 T cells were able to expand more robustly than -CA-STAT5 T cells upon repeated antigen stimulation (vaccine boost), demonstrating nearly 4000-fold increases in antigen-specific CD8 T cells. +CA-STAT5 T cells also seemed to persist longer in vivo over time, and they expressed lower levels of surface PD-1. Using B16F10 melanoma, ACT+TriVax of these cell populations into tumor-bearing mice demonstrated a powerful antitumor effect, leading to tumor regression in treated groups. CA-STAT5 seemed to recapitulate similar antitumor effects our laboratory observed previously with combinatorial anti-PD-L1 treatment or IL2/anti-IL2 mAb complexes (IL2Cx), suggesting a potential role for STAT5 in resisting the PD-1/PD-L1 inhibitory pathway. When compared to tumor-bearing mice treated with ACT+TriVax with anti-PD-L1 antibody, our approach using +CA-STAT5 T cells generated a comparable antitumor response. Altogether, these results demonstrate that RV CD8 T cells expressing gp100-TCR and CA-STAT5 are capable of antigen-dependent expansion in response to TriVax. CA-STAT5 plays a role in increasing T cell proliferation and persistence, as well as increasing efficacy through mechanisms that seem to indicate resistance to PD-1/PD-L1 inhibition. Citation Format: Aaron E. Fan, Hussein Sultan, Takumi Kumai, Esteban Celis. Combining immunotherapies: A strategy for improving CD8 T cell-mediated antitumor responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3193.

Effects and mechanism of recombinant oncolytic adenovirus expressing soluble programmed cell death protein 1 on proliferation and apoptosis of B16/F10 cell

Objective To construct a recombinant oncolytic adenovirus expressing soluble programmed cell death protein 1 (sPD1) and investigate its effects and mechanism on proliferation and inhibition of B16/F10 cells. Methods The recombinant oncolytic adenovirus Ad5-sPD1 was constructed, and infected into B16/f10 cells. The quantitative fluorescent polymerase chain reaction was used to detect the viral replication. MTT assay was applied to examine the inhibition of B16/F10 cells. The viruses were injected subcutaneously into the mouse B16/F10 tumor model to study the antitumor effect of Ad5-sPD1 in vivo. EliSpot technique was exployed to test the number of tumor proliferative lymph nodes. Results After viral treatment of B16/F10 cells for 6, 24, 36, 48, 60 and 72 h, the copies fold increases in the Ad5 treatment group were respectively 0.99±0.10, 1.12±0.05, 1.57±0.15, 3.59±1.03, 6.26±0.74, 9.69±1.46, and those in the Ad5-sPD1 treatment group were respectively 1.00±0.104, 0.99±0.08, 1.05±0.03, 3.56±1.04, 6.04±0.38, 11.05±1.12. There was no significant difference between the two groups (P=0.754). After treating B16/F10 cells for 72 h with multiplicity of infection (MOI) of 0, 5, 10, 15 and 20, the cell viability of Ad5 treatment group was respectively (100.00±6.39)%, (101.60±5.21)%, (98.66±7.31)%, (93.50±4.54)%, (73.16±2.45)%, and that in the Ad5-sPD1 treatment group was respectively (100.00±6.38)%, (97.10±6.37)%, (96.96±7.44)%, (95.67±7.46)%, (74.49±4.14)%. There was no significant difference between the two groups (P=0.779). After treating mice model of B16/F10 subcutaneous tumor with Ad5 and Ad5-sPD1 viruses, tumor volume was respectively 3 638±1.823, 2 603±336 and 1 097±532 at the termination of the experiments with the difference being significant (Mock and Ad5: P=0.044; Mock and Ad5-sPD1: P=0.000; Ad5 and Ad5-sPD1: P=0.000). After respectively injecting mice model of B16/F10 subcutaneous tumor with Mock, Ad5 and Ad5 -sPD1 viruses, the number of IFN-γ spots was respectively 19.4±7.75, 149.8±25.18 and 314±32.34 with the the difference being significant (Mock and Ad5: P=0.000; Mock and Ad5-sPD1: P=0.000; Ad5 and Ad5-sPD1: P=0.000). Conclusion Recombinant oncolytic adenovirus expressing PD1 gene can significantly improve the anti-tumor effect of the oncolytic adenovirus by blocking PD-1/PD-L1 inhibitory pathway. Key words: Oncolytic adenovirus; Tumor; Immune response; Programmed cell death protein 1

Mycoplasma bovis-Induced Inhibition of Bovine Peripheral Blood Mononuclear Cell Proliferation Is Ameliorated after Blocking the Immune-Inhibitory Programmed Death 1 Receptor.

Mycoplasma bovis-induced immune suppression is a major obstacle faced by the host for controlling infections. M. bovis impairment of antigen-specific T-cell responses is achieved through inhibiting the proliferation of peripheral blood mononuclear cells (PBMCs). This impairment may contribute to the persistence of M. bovis infection in various sites, including lungs, and its systemic spread to various organs such as joints, with the underlying mechanisms remaining elusive. Here, we elucidated the role of the immune-inhibitory receptor programmed death 1 (PD-1) and its ligand (PD-L1) in M. bovis infection. Flow cytometry (FCM) analyses revealed an upregulation of PD-L1 expression on tracheal and lung epithelial cell lines after M. bovis infection. In addition, we found increased PD-L1 expression on purified lung lavage macrophages following M. bovis infection by FCM and determined its localization by immunofluorescence analysis comparing infected and control lung tissue sections. Moreover, M. bovis infection increased the expression of the PD-1 receptor on total PBMCs and in gated CD4+ and CD8+ T-cell subpopulations. We demonstrated that M. bovis infection induced a significant decrease in CD4+ PD-1INT and CD8+ PD-1INT subsets with intermediate PD-1 expression, which functioned as progenitor pools giving rise to CD4+ PD-1HIGH and CD8+ PD-1HIGH subsets with high PD-1 expression levels. We blocked PD-1 receptors on PBMCs using anti-PD-1 antibody at the beginning of infection, leading to a significant restoration of the proliferation of PBMCs. Taken together, our data indicate a significant involvement of the PD-1/PD-L1 inhibitory pathway during M. bovis infection and its associated immune exhaustion, culminating in impaired host immune responses.

PD-L1 Is Not Constitutively Expressed on Tasmanian Devil Facial Tumor Cells but Is Strongly Upregulated in Response to IFN-γ and Can Be Expressed in the Tumor Microenvironment.

The devil facial tumor disease (DFTD) is caused by clonal transmissible cancers that have led to a catastrophic decline in the wild Tasmanian devil (Sarcophilus harrisii) population. The first transmissible tumor, now termed devil facial tumor 1 (DFT1), was first discovered in 1996 and has been continually transmitted to new hosts for at least 20 years. In 2015, a second transmissible cancer [devil facial tumor 2 (DFT2)] was discovered in wild devils, and the DFT2 is genetically distinct and independent from the DFT1. Despite the estimated 136,559 base pair substitutions and 14,647 insertions/deletions in the DFT1 genome as compared to two normal devil reference genomes, the allograft tumors are not rejected by the host immune system. Additionally, genome sequencing of two sub-strains of DFT1 detected greater than 15,000 single-base substitutions that were found in only one of the DFT1 sub-strains, demonstrating the transmissible tumors are evolving and that generation of neoantigens is likely ongoing. Recent evidence in human clinical trials suggests that blocking PD-1:PD-L1 interactions promotes antitumor immune responses and is most effective in cancers with a high number of mutations. We hypothesized that DFTD cells could exploit the PD-1:PD-L1 inhibitory pathway to evade antitumor immune responses. We developed recombinant proteins and monoclonal antibodies (mAbs) to provide the first demonstration that PD-1 binds to both PD-L1 and PD-L2 in a non-placental mammal and show that PD-L1 is upregulated in DFTD cells in response to IFN-γ. Immunohistochemistry showed that PD-L1 is rarely expressed in primary tumor masses, but low numbers of PD-L1+ non-tumor cells were detected in the microenvironment of several metastatic tumors. Importantly, in vitro testing suggests that PD-1 binding to PD-L1 and PD-L2 can be blocked by mAbs, which could be critical to understanding how the DFT allografts evade the immune system.

Abstract 903: IFN-ã induced PD-L1 on tumor and host cells co-operatively prevents tumor immune elimination after cancer immunoediting

Abstract Tumor cells can escape immune destruction via engaging the PD-1/PD-L1 inhibitory pathway. However since PD-L1 is induced on many different cell types by IFN-ã, the importance of PD-L1 on tumor versus host cells in mediating immune escape remains unclear. In this study, we assessed the role of PD-L1 expression on two closely-related but distinct clones derived from the same d42m1 methylcholanthrene (MCA)-induced sarcoma line. Edited d42m1-T3 sarcoma cells grow progressively in wild type (WT) mice but are rejected in mice treated with anti-PD-L1. Upregulation of PD-L1 was observed on both immune and tumor cells in vivo. To assess the role of PD-L1 specifically on tumor cells, we generated d42m1-T3 cells lacking PD-L1 (T3ÄPDL1) using CRISPR-Cas9. T3ÄPDL1 cells were spontaneously rejected in naïve syngeneic WT mice but formed progressively growing tumors in Rag2-/- mice. To explore the role of host-cell expressed PD-L1, we challenged WT mice with 3-fold higher numbers of T3ÄPDL1 cells and found that the tumor cells formed progressively growing tumors in WT mice. However, when tumor bearing mice injected with the high dose of T3ÄPDL1 cells were treated with blocking PD-L1 mAb, they all rejected their tumors. Thus, PD-L1 expression on both tumor and host cells plays a critical role in preventing immune elimination of edited MCA sarcoma cells. Alternatively highly immunogenic, unedited d42m1-T9 sarcoma cells were spontaneously rejected in WT mice due to the presence of the strong rejection antigen, mutant Spectrin-â2. Enforced expression of PD-L1 on unedited d42m1-T9 cells to levels similar to those induced under physiologic in vivo conditions did not alter the regressor phenotype of these cells. In contrast, enforced overexpression of PD-L1 on d42m-T9 cells to levels that were 20 fold higher than physiologic led to formation of progressively growing tumors in WT mice. Thus, physiologic levels of PD-L1 expression on highly immunogenic unedited tumor cells are not sufficient to counteract immune effector mechanisms driven by “strong” tumor antigens. This result shows that adaptive immune resistance occurs only on tumor cells that have undergone immunoediting. Citation Format: Takuro Noguchi, Jeffrey P. Ward, Matthew M. Gubin, Cora Arthur, Robert D. Schreiber. IFN-ã induced PD-L1 on tumor and host cells co-operatively prevents tumor immune elimination after cancer immunoediting. [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 903.

Lipidoid Nanoparticles Containing PD-L1 siRNA Delivered In Vivo Enter Kupffer Cells and Enhance NK and CD8+ T Cell-mediated Hepatic Antiviral Immunity

Effective clinical application of antiviral immunotherapies necessitates enhancing the functional state of natural killer (NK) and CD8+ T cells. An important mechanism for the establishment of viral persistence in the liver is the activation of the PD-1/PD-L1 inhibitory pathway. To examine the role of hepatic myeloid PD-L1 expression during viral infection, we determined the magnitude and quality of antiviral immune responses by administering PD-L1 short-interfering RNA (siRNA) encapsulated in lipidoid nanoparticles (LNP) in mice. Our studies indicate that Kupffer cells (KC) preferentially engulfed PD-L1 LNP within a short period of time and silenced Pdl1 during adenovirus and MCMV infection leading to enhanced NK and CD8+ T cell intrahepatic accumulation, effector function (interferon (IFN)-γ and granzyme B (GrB) production), CD8+ T cell–mediated viral clearance, and memory. Our results demonstrate that PD-L1 knockdown on KCs is central in determining the outcome of liver viral infections, and they represent a new class of gene therapy.

In vivo delivery of lipidoid nanoparticles containing PD-L1 siRNA enhances NK and CD8+ T cell-mediated hepatic antiviral immunity (168.6)

Abstract Effective clinical application of anti-viral immunotherapies necessitates enhancing the functional state of NK and CD8+ T cells. Persistence of viral infections in the liver is established by activation of the PD-1/PD-L1 inhibitory pathway. The expression of PD-1 ligand (PD-L1) is widespread and found on multiple cell types including Kupffer cells, liver sinusoidal endothelial cells, non-resident macrophages, dendritic cells, NK cells, T cells, and hepatocytes. To examine the role of PD-L1 expression by myeloid cells, we determined the magnitude and quality of antiviral immune responses by administering PD-L1 siRNA encapsulated in lipidoid nanoparticles (LNP) in mice prior to or after viral infection. Our studies indicate that Kupffer cells preferentially engulfed PD-L1 LNP leading to specific silencing of Pdl1 during adenovirus and MCMV infection. Treatment of PD-L1 LNP enhanced NK cell and CD8+ T cell intrahepatic accumulation, and depending upon timing of PD-L1 LNP treatment, either NK cell or CD8+ T cell effector function, as assessed by IFN-γ and granzyme B production, were enhanced. Importantly, CD8+ T cell-mediated viral clearance was enhanced through targeted blockade of the PD-1/PD-L1 inhibitory pathway. Our results demonstrate that PD-L1 knockdown on Kupffer cells is central in determining the outcome of liver viral infections. The implications of this research also extend into other diseases including cancer where reversal of CD8+ T cell tolerance is critical.

Correlation of Serum IL-6 Level with Exhaustion of Cytomegalovirus-specific T Cells After Hematopoietic Stem Cell Transplantation

Abstract 360Preemptive treatments employing antiviral drugs have decreased in cytomegalovirus (CMV) infection. However, CMV has remained one of the most common pathogens causing morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT). Chronic viral infections with several human viruses were shown to be associated with exhausted T cells with high expression of the inhibitory molecule programmed death 1 (PD-1). PD-1 expression on CMV-specific T cells after HSCT has not been well examined. We evaluated the involvement of exhausted CMV-specific T cells characterized by high PD-1 expression in persistent CMV infection after allogeneic HSCT. Although some cytokines, such as IL-10, reportedly upregulate PD-1 expression, what increases PD-1 expression has not been well known. We also aimed to identify soluble factors regulating PD-1 expression on exhausted T cells.Peripheral blood mononuclear cells (PBMC) and serum were obtained from an HLA-A*2402-positive patient who had received bone marrow transplantation from an HLA-A, B, C and DR matched unrelated donor. This patient failed to eliminate CMV for more than one year after transplantation despite intermittent administration of antiviral drugs. Control PBMC and serum were obtained from an HLA-A*2402-positive healthy volunteer. All blood was collected with written informed consent. We at first analyzed frequencies of CMV-specific CD8+ T cells in patient and control PBMC by flow cytometer using QYDPVAALF/A*2402-specific tetramer and CD8 antibodies. QYDPVAALF is derived from CMV pp65 protein and presented by the HLA-A*2402 molecule. Tetramer-stained cells were detected in patient PBMC but not control PBMC (0.11% versus undetectable). Patient and control PBMC were stimulated by a synthetic peptide QYDPVAALF in culture media containing IL-2 for 14 days, and stained with QYD/A*2402-specific tetramer. Remarkably, post-stimulated patient PBMC contained only 0.54% of tetramer-stained CD8+ T cells, whereas a more dramatic increase (14.1%) was found in control PBMC. We analyzed frequencies of IFN-g secreting CD8+ T cells in PBMC after 4-hour stimulation with a peptide pool covering the whole CMV pp65 protein. Less patient CD8+ T cells produced IFN-g, compared with the control CD8+ T cells (0.1% versus 0.5%). These data demonstrate dysfunction of CMV-specific CD8+ T cells in the patient with persistent CMV infection. To examine the involvement of PD-1 in dysfunction of CMV-specific CD8+ T cells, we analyzed the expression of PD-1 on CMV-specific CD8+ T cells 14 days after stimulation with QYDPVAALF peptide. CMV-specific CD8+ T cells generated from patient PBMC exhibited higher PD-1 expression, compared with CMV-specific CD8+T cells generated from control PBMC. Next, we stimulated patient PBMC with QYDPVAALF peptide for 14 days in the presence or absence of anti-PD-L1 antibody, which blocks PD-1/PD-L1 inhibitory pathway. Blockade of the PD-1/PD-L1 pathway resulted in a 3.9-fold increase in patient CMV-specific T cells. These findings demonstrate that PD-1 is associated with the exhaustion of CMV-specific CD8+ T cells during persistent CMV infection in this patient. Next, to examine the effect of soluble factors on CMV-specific CD8+ T cells, we analyzed the levels of cytokines in patient and control sera by multiplex bead assay. Patient serum exhibited a higher IL-6 level compared with control serum. We stimulated patient and control PBMC with QYDPVAALF peptide for 14 days in culture media with patient or control serum. Patient serum led to poorer growth and higher PD-1 expression of CMV-specific CD8+ T cells. In addition, as the growth of CMV-specific CD8+ T cells was worse, the IL-6 level in the supernatant was higher (Fig). Blockade of IL-6/IL-6 receptor interaction using anti-IL-6 receptor antibody resulted in a 5-fold increase in patient CMV-specific T cells. We analyzed the IL-6 concentration in sera from healthy donors (n=5) and patients with temporary (n=4) and persistent (n=3) CMV infection after allogeneic HSCT. Serum from patients with persistent CMV infection showed a significantly higher IL-6 level. These findings suggest that IL-6 was associated with exhaustion of CMV- specific T cells.Although further investigations to clarify how IL-6 induces T cell exhaustion are warranted, blockade of IL-6/IL-6 receptor interaction might improve exhausted T cell function and contribute to effective adoptive T cell therapy. [Display omitted] Disclosures:Naoe:Dainipponn-Sumitomo Pharma.: Research Funding; Chugai Pharma.: Research Funding; Novartis Pharma.: Honoraria, Speakers Bureau; Zenyaku-Kogyo: Research Funding; Otsuka Pharma.: Research Funding; Kyowa-Hakko Kirin.: Research Funding.

Exhaustion of CMV Specific T Cells with Enhanced PD-1 Expression In Persistent Cytomegalovirus Infection After Allogeneic Stem Cell Transplantation

AbstractAbstract 3912Cytomegalovirus (CMV) is one of the most common pathogens causing morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT), despite preemptive treatments employing antiviral drugs. Cytotoxic T cells are indispensable to control CMV infections. Chronic viral infections with human immunodeficiency virus or hepatitis C virus were shown to be associated with exhausted T cells with high expression of the inhibitory molecule programmed death 1 (PD-1). Recently, it has been reported that PD-1 up-regulation on CMV specific T cells was associated with CMV infection after renal and liver transplantation. PD-1 expression on CMV specific T cells after HSCT has not been well examined. We evaluated the involvements of exhausted CMV specific T cells characterized by high PD-1 expression in persistent CMV infection after allogeneic HSCT. Peripheral blood mononuclear cells (PBMC) and serum were obtained from an HLA-A*2402-positive patient who had received bone marrow transplantation from an HLA-A, B, C and DR matched unrelated donor. This patient failed to eliminate CMV for more than one year after transplantation despite intermittent administration of ganciclovir and foscarnet. Control PBMC and serum were obtained from an HLA-A*2402-positive healthy volunteer because the Japan Marrow Donor Program prohibits blood collection for research use from donor. All blood was collected with written informed consent. We at first analyzed frequencies of CMV-specific CD8+ T cells in patient and control PBMC by flow cytometer using QYDPVAALF/A*2402-specific tetramer and CD8 antibodies. QYDPVAALF is derived from CMV pp65 protein and presented by the HLA-A*2402 molecule. Tetramer stained cells were detected in the patient PBMC but control PBMC (0.11% versus undetectable). Patient and control PBMC were stimulated by a synthetic peptide QYDPVAALF in culture media containing IL-2 for 14 days, and stained with QYD/A*2402-specific tetramer. Remarkably, post-stimulated patient PBMC contained only 0.54% of tetramer stained CD8+ T cells, whereas a more dramatic increase (14.1%) in control PBMC. We analyzed frequencies of IFN-g secreting CD8+ T cells in PBMC after stimulation with a peptide pool covering the whole CMV pp65 protein for 4 hours. Less patient CD8+ T cells produced IFN-g, compared with the control CD8+ T cells (0.5% versus 1.1%) These data demonstrate dysfunction of CMV-specific CD8+ T cells in the patient with persistent CMV infection. To examine the mechanism of dysfunction of CMV-specific CD8+ T cells, we analyzed the expression of PD-1 on CMV-specific CD8+ T cells 14 days after stimulation with QYDPVAALF peptide. Multiparameter flow cytometry and tetramer assay exhibited higher expression of PD-1 on CMV-specific CD8+ T cells generated from patient PBMC, compared with CMV-specific CD8+ T cells generated from control PBMC. To find out whether the engagement of PD-1 to its ligand (PD-L1) leads to T cell exhaustion, we stimulated patient PBMC with QYDPVAALF peptide for 14 days in the presence or absence of anti PD-L1 antibody which blocks PD-1/PD-L1 inhibitory pathway. Blockade of PD-1/PD-L1 pathway resulted in 3.9-fold increase in patient CMV specific T cells. These findings demonstrate that PD-1 is associated with the exhaustion of CMV specific CD8+ T cells during persistent CMV infection in this patient. To examine the effect of patient serum on CMV specific CD8+ T cells, we stimulated patient PBMC with QYDPVAALF peptide for 14 days in culture media with patient or control serum. CMV specific CD8+ T cells increased 4-fold and 55-fold in the presence of patient and control serum, respectively. Patient serum led to higher PD-1 expression on CMV specific CD8+ T cells, compared with control serum (Fig). These findings suggest that patient serum may contain what regulates PD-1 expression level of exhausted T cells. Further investigations to identify factors regulating PD-1 expression in patient serum are in progress. The identification of the factors may provide new strategies to improve exhausted T cell function. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Intestinal Tolerance Is Converted to Autoimmune Enteritis upon PD-1 Ligand Blockade

The B7 family member programmed death-1 ligand (PD-L1) has been shown to play an inhibitory role in the regulation of T cell responses in several organs. However, the role of PD-L1 in regulating tolerance to self-Ags of the small intestine has not been previously addressed. In this study, we investigated the role of PD-L1 in CD8(+) T cell tolerance to an intestinal epithelium-specific Ag using the iFABP-tOVA transgenic mouse model, in which OVA is expressed as a self-Ag throughout the small intestine. Using adoptive transfer of naive OVA-specific CD8(+) T cells, we show that loss of PD-1:PD-L1 signaling, by either Ab-mediated PD-L1 blockade or transfer of PD-1(-/-) T cells, leads to considerable expansion of OVA-specific CD8(+) T cells and their differentiation into effector cells capable of producing proinflammatory cytokines. A fatal CD8(+) T cell-mediated inflammatory response develops rapidly against the small bowel causing destruction of the epithelial barrier, severe blunting of intestinal villi, and recruitment and activation of myeloid cells. This response is highly specific because immune destruction selectively targets the small intestine but not other organs. Collectively, these results indicate that loss of the PD-1:PD-L1 inhibitory pathway breaks CD8(+) T cell tolerance to intestinal self-Ag, thus leading to severe enteric autoimmunity.

Restoring function in exhausted CD8 T cells during chronic viral infection

Functional impairment of antigen-specific T cells is a defining characteristic of many chronic infections, but the underlying mechanisms of T-cell dysfunction are not well understood. To address this question, we analysed genes expressed in functionally impaired virus-specific CD8 T cells present in mice chronically infected with lymphocytic choriomeningitis virus (LCMV), and compared these with the gene profile of functional memory CD8 T cells. Here we report that PD-1 (programmed death 1; also known as Pdcd1) was selectively upregulated by the exhausted T cells, and that in vivo administration of antibodies that blocked the interaction of this inhibitory receptor with its ligand, PD-L1 (also known as B7-H1), enhanced T-cell responses. Notably, we found that even in persistently infected mice that were lacking CD4 T-cell help, blockade of the PD-1/PD-L1 inhibitory pathway had a beneficial effect on the 'helpless' CD8 T cells, restoring their ability to undergo proliferation, secrete cytokines, kill infected cells and decrease viral load. Blockade of the CTLA-4 (cytotoxic T-lymphocyte-associated protein 4) inhibitory pathway had no effect on either T-cell function or viral control. These studies identify a specific mechanism of T-cell exhaustion and define a potentially effective immunological strategy for the treatment of chronic viral infections.