Programmed Death-1 Pathway Research Articles

PD1 inhibits PKCθ-dependent phosphorylation of cytoskeleton-related proteins and immune synapse formation

AbstractThe inhibitory surface receptor programmed cell death protein 1 (PD1) is a major target for antibody–based cancer immunotherapies. Nevertheless, a substantial number of patients fail to respond to the treatment or experience adverse effects. An improved understanding of intracellular pathways targeted by PD1 is thus needed to develop better predictive and prognostic biomarkers. Here, via unbiased phosphoproteome analysis of primary human T cells, we demonstrate that PD1 triggering inhibited the phosphorylation and physical association with protein kinase Cθ (PKCθ) of a variety of cytoskeleton-related proteins. PD1 blocked activation and recruitment of PKCθ to the forming immune synapse (IS) in a Src homology-2 domain–containing phosphatase-1/2 (SHP1/SHP2)-dependent manner. Consequently, PD1 engagement led to impaired synaptic phosphorylation of cytoskeleton-related proteins and formation of smaller IS. T-cell receptor induced phosphorylation of the PKCθ substrate and binding partner vimentin was long-lasting and it could be durably inhibited by PD1 triggering. Vimentin phosphorylation in intratumoral T cells also inversely correlated with the levels of the PD1 ligand, PDL1, in human lung carcinoma. Thus, PKCθ and its substrate vimentin represent important targets of PD1-mediated T-cell inhibition, and low levels of vimentin phosphorylation may serve as a biomarker for the activation of the PD1 pathway.

Silencing of PD-1 combined with EBV-specific killer T cells for the treatment of EBV-associated B lymphoma

Epstein-Barr Virus (EBV) infection is closely associated with the development of lymphoma, as it plays a significant role in the malignant transformation of lymphocytes. The expression of programmed death-1 (PD-1), which binds to PD-L1 in tumor cells, can lead to immune evasion by lymphoma cells and promote tumor progression. In this study, immortalized B lymphoblastoid cell lines (B-LCLs) positive for EBV-specific proteins were established from human peripheral mononuclear cells (PBMCs) using EBV induction along with CpG-ODN 2006 and cyclosporin A. EBV-specific T cells (EBVST) were generated by multiple immunizations of CD3+ T lymphocytes using irradiated B-LCLs. Flow cytometry analysis confirmed the activation of EBVST through the detection of CD3+, CD4+, and CD8+ markers. Co-incubation of EBVST with EBV-positive B lymphocyte cell lines resulted in the secretion of perforin by EBVST, leading to granzyme B-mediated cell death and an increase in LDH levels. Silencing PD-1 in EBVST cells enhanced perforin production, increased granzyme B release, and upregulated cell death in co-incubated B lymphocytes. In a nude mice tumor transplantation model, silencing PD-1 in combination with EBV-specific killer T cells exhibited the maximum inhibition of B-lymphoblastoma. This treatment upregulated the expression of proteins associated with apoptosis and immune response, while inhibiting anti-apoptotic protein expression in tumor tissues. Silencing PD-1 also increased the infiltration of EBV-specific killer T cells in the tumor tissues. Overall, PD-1 silencing enhanced the tumor targeting effect of EBV-specific killer T cells on EBV-infected B lymphocytes and attenuated the immune escape effect mediated by the PD-1 pathway.

Regulatory Rheumatoid Factor is Specific to PD-1 and Uses PD-1 Pathway to Control CD4 T Lymphocytes

ABSTRACT Background We have earlier discovered a new factor of autoimmunity downregulation, called regulatory rheumatoid factor (regRF). Being anti-idiotypic antibodies, regRF restricts the expansion of CD4+ T lymphocytes to the idiotype of which it is specific, according to the negative feedback principle. It has been shown that only activated CD4+ T lymphocytes are the target of regRF. However, it is still not clear the way regRF distinguishes activated cells from naive ones. RegRF molecules, apart from individual paratopes specific to unique sequences of B- and T-cell receptors, have a shared paratope. We assume that regRF by means of a shared paratope recognizes one of the surface activation molecules of CD4+ T lymphocytes and initiates the cell death. Programmed death-1 (PD-1) has been tested as a potential receptor of the shared regRF paratope and transmitter of the negative regRF signal into activated CD4+ T lymphocytes. Methods The specificity of the shared regRF paratope to PD-1 was determined by ELISA. T cell activation was performed with immobilized anti-CD3ε antibodies. Flow cytometry was used to study the effect of regRF on PD-1+CD4+ lymphocytes. Results We found that regRF binds to PD-1. IgG Fc fragments carrying epitopes specific to the shared paratope of regRF compete with PD-1 for binding to regRF. It follows that regRF recognizes specifically PD-1 by means of a shared paratope. RegRF-containing serum reduced the number of PD-1+CD4+ lymphocytes in proportion to their increase resulting from the action of anti-CD3ε antibodies. Conclusion RegRF uses PD-1 pathway to control activated CD4+ T lymphocytes.

Association between programmed death-1 pathway and major depression

Objectives Major depression (MD) may be associated with inflammation and immunity. PD-1 (programmed death-1), PD-L1 (programmed death-ligand 1) and PD-L2 (programmed death-ligand 2) are among the inhibitory immune mediators on the PD-1 pathway. However, previous data regarding the association between MD and PD-1 pathway were still scarce; therefore, we investigated the association of PD-1 pathway with MD. Methods During a period of 2 years, patients with MD and healthy controls were recruited from a medical centre in this study. The diagnosis of MD was established according to the DSM-5 criteria. The severity of MD was assessed with 17-item Hamilton Depression Rating Scale. PD-1, PD-L1 and PD-L2 were detected in peripheral blood from MD patients after 4 weeks of treatment with antidepressant drugs. Results A total of 54 patients with MD and 38 healthy controls were recruited. According to the analyses, there is a significantly higher PD-L2 level in MD than in healthy controls and lower PD-1 level after age and BMI adjustment. Besides, moderately positive correlation between HAM-D scores and PD-L2 level was found. Conclusions It was found that PD-1 pathway might play an important role in MD. We need a large sample to prove these results in the future.

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)

RELATIVITY-106: A phase 1/2 trial of nivolumab (NIVO) + relatlimab (RELA) in combination with bevacizumab (BEV) in first-line (1L) hepatocellular carcinoma (HCC).

TPS636 Background: The current standard of care for 1L treatment of patients with advanced/metastatic HCC is atezolizumab + BEV, which demonstrated significantly prolonged progression-free survival (PFS) and overall survival (OS) compared to sorafenib in treatment-naïve patients. However, only 29.8% of patients show objective responses and additional therapy options are needed in the 1L setting. Programmed death-1 (PD-1) and lymphocyte-activation gene 3 (LAG-3) are distinct inhibitory immune checkpoint pathways that synergistically reduce T-cell function. RELA is a first-in-class human immunoglobulin G4 LAG-3-blocking antibody that binds to LAG-3 and restores the effector function of T cells. Dual checkpoint inhibition of the PD-1 and LAG-3 pathways with NIVO + RELA has the potential to boost immune surveillance in HCC. Preclinical data presume that BEV, a human vascular endothelial growth factor inhibitor, reverses abnormal vascularization to allow NIVO + RELA to inhibit hypoxia-induced programmed cell death ligand 1 and LAG-3 expression and enhance depth of response and OS in HCC. Here we describe the RELATIVITY-106 study investigating the triplet therapy of NIVO + RELA + BEV in the 1L treatment of advanced/metastatic HCC. Methods: RELATIVITY-106 (NCT05337137) is a phase 1/2, randomized, double-blind, placebo-controlled trial to assess the safety and efficacy of NIVO + RELA + BEV compared with NIVO + BEV in treatment-naïve patients with advanced/metastatic HCC. Key inclusion criteria include age ≥ 18 years; histologic confirmation of advanced/metastatic HCC in patients naïve to systemic therapy for advanced/metastatic HCC (prior neoadjuvant or adjuvant immunotherapy permitted if recurrence occurs ≥ 6 months after treatment completion); Child-Pugh A; and ECOG performance status 0 or 1. Key exclusion criteria include known fibrolamellar HCC, sarcomatoid HCC, or mixed hepatocellular cholangiocarcinoma; prior allogenic stem cell or solid organ transplantation; untreated symptomatic central nervous system metastases; clinically significant ascites; increased risk of bleeding; significant vascular disease or inadequately controlled hypertension; and major surgical procedure within 4 weeks prior to study treatment. Primary endpoints include incidence of dose-limiting toxicities assessed for up to 6 weeks and PFS by blinded independent central review (BICR) per RECIST v1.1 in all randomized patients in phase 1 and phase 2, respectively. Secondary endpoints include overall response rate (ORR) by BICR and OS in all randomized patients; ORR and PFS by BICR and OS in all randomized LAG-3-positive patients (≥ 1% by immunohistochemistry); and safety. Key exploratory endpoints include pharmacokinetics and immunogenicity assessed by antidrug antibody positivity. The study, initiated in May 2022, is currently enrolling globally. Clinical trial information: NCT05337137 .

Combined Immune Checkpoint Blockade Enhances Antiviral Immunity against Bovine Leukemia Virus.

Bovine leukemia virus (BLV) is a retrovirus that causes enzootic bovine leukosis (EBL) in cattle and is widespread in many countries, including Japan. Recent studies have revealed that the expression of immunoinhibitory molecules, such as programmed death-1 (PD-1) and PD-ligand 1, plays a critical role in immunosuppression and disease progression during BLV infection. In addition, a preliminary study has suggested that another immunoinhibitory molecule, T-cell immunoglobulin domain and mucin domain-3 (TIM-3), is involved in immunosuppression during BLV infection. Therefore, this study was designed to further elucidate the immunoinhibitory role of immune checkpoint molecules in BLV infection. TIM-3 expression was upregulated on peripheral CD4+ and CD8+ T cells in BLV-infected cattle. Interestingly, in EBL cattle, CD4+ and CD8+ T cells infiltrating lymphomas expressed TIM-3. TIM-3 and PD-1 were upregulated and coexpressed in peripheral CD4+ and CD8+ T cells from BLV-infected cattle. Blockade by anti-bovine TIM-3 monoclonal antibody increased CD69 expression on T cells and gamma interferon (IFN-γ) production from peripheral blood mononuclear cells from BLV-infected cattle. A syncytium formation assay also demonstrated the antiviral effects of TIM-3 blockade against BLV infection. The combined inhibition of TIM-3 and PD-1 pathways significantly enhanced IFN-γ production and antiviral efficacy compared to inhibition alone. In conclusion, the combined blockade of TIM-3 and PD-1 pathways shows strong immune activation and antiviral effects and has potential as a novel therapeutic method for BLV infection. IMPORTANCE Enzootic bovine leukosis caused by bovine leukemia virus (BLV) is an important viral disease in cattle, causing severe economic losses to the cattle industry worldwide. The molecular mechanisms of BLV-host interactions are complex. Previously, it was found that immune checkpoint molecules, such as PD-1, suppress BLV-specific Th1 responses as the disease progresses. To date, most studies have focused only on how PD-1 facilitates escape from host immunity in BLV-infected cattle and the antiviral effects of the PD-1 blockade. In contrast, how T-cell immunoglobulin domain and mucin domain-3 (TIM-3), another immune checkpoint molecule, regulates anti-BLV immune responses is rarely reported. It is also unclear why PD-1 inhibition alone was insufficient to exert anti-BLV effects in previous clinical studies. In this study, the expression profile of TIM-3 in T cells derived from BLV-infected cattle suggested that TIM-3 upregulation is a cause of immunosuppression in infected cattle. Based on these results, anti-TIM-3 antibody was used to experimentally evaluate its function in influencing immunity against BLV. Results indicated that TIM-3 upregulation induced by BLV infection suppressed T-cell activation and antiviral cytokine production. Some T cells coexpressed PD-1 and TIM-3, indicating that simultaneous inhibition of PD-1 and TIM-3 with their respective antibodies synergistically restored antiviral immunity. This study could open new avenues for treating bovine chronic infections.

The Programmed Death-1 Pathway Counter-Regulates Inflammation-Induced Osteoclast Activity in Clinical and Experimental Settings.

ObjectiveThe programmed death-1 (PD-1) pathway is essential for maintaining self-tolerance and plays an important role in autoimmunity, including rheumatoid arthritis (RA). Here, we investigated how membrane-bound and soluble (s)PD-1 influence bone homeostasis during chronic inflammation, exemplified in RA.MethodsBone mineral density and bone microstructure were examined in PD-1 and PD-L1 knockout (KO) mice and compared with wild-type (WT) mice. Receptor activator of nuclear factor kappa-B ligand (RANKL) was measured in serum, and the expression examined on activated bone marrow cells. Osteoclast formation was examined in cells from murine spleen and bone marrow and from human synovial fluid cells. sPD-1 was measured in chronic and early (e)RA patients and correlated to markers of disease activity and radiographic scores.ResultsPD-1 and PD-L1 KO mice showed signs of osteoporosis. This was supported by a significantly reduced trabecular bone volume fraction and deteriorated microstructure, as well as increased osteoclast formation and an increased RANKL/OPG ratio. The recombinant form of sPD-1 decreased osteoclast formation in vitro, but was closely associated with disease activity markers in eRA patients. Sustained elevated sPD-1 levels indicated ongoing inflammation and were associated with increased radiographic progression.ConclusionThe PD-1 pathway is closely associated with bone homeostasis, and lacking members of this pathway causes a deteriorated bone structure. The immunological balance in the microenvironment determines how the PD-1 pathway regulates osteoclast formation. In eRA patients, sPD-1 may serve as a biomarker, reflecting residual but clinically silent disease activity and radiographic progression.

Combined BRAF-Targeted Therapy with Immunotherapy in BRAF-Mutated Advanced Melanoma Patients.

To review evidence on the efficacy and safety of combined BRAF-targeted therapy and immune checkpoint inhibitors in patients with BRAF-mutated metastatic melanoma. Programmed death-1 pathway inhibitors administered with BRAF/MEK inhibitors showed promising anti-tumour activity in BRAF-mutated advanced melanoma and were investigated for safety and efficacy in three large international clinical trials. Although, in two out of those three randomized phase III studies, progression-free survival (PFS) did not reach statistical significance, results showed that duration of response (DOR) and overall survival (OS) were improved using combined therapy, sustaining the scientific rationale for its use at least in a subset of metastatic melanomas. However, the frequent occurrence of autoimmunity-induced toxicities should be considered since it is limiting the continuity and the wide application of these regimens. Novel treatment modalities combining targeted therapy with checkpoint inhibitors require further clinical investigation and elucidation of their effect on the immune system and cancer cell modulation.

Chromogranin A Deficiency Confers Protection From Autoimmune Diabetes via Multiple Mechanisms.

Recognition of β-cell antigens by autoreactive T cells is a critical step in the initiation of autoimmune type1 diabetes. A complete protection from diabetes development in NOD mice harboring a point mutation in the insulin B-chain 9–23 epitope points to a dominant role of insulin in diabetogenesis. Generation of NOD mice lacking the chromogranin A protein (NOD.ChgA−/−) completely nullified the autoreactivity of the BDC2.5 T cell and conferred protection from diabetes onset. These results raised the issue concerning the dominant antigen that drives the autoimmune process. Here we revisited the NOD.ChgA−/− mice and found that their lack of diabetes development may not be solely explained by the absence of chromogranin A reactivity. NOD.ChgA−/− mice displayed reduced presentation of insulin peptides in the islets and periphery, which corresponded to impaired T-cell priming. Diabetes development in these mice was restored by antibody treatment targeting regulatory T cells or inhibiting transforming growth factor-β and programmed death-1 pathways. Therefore, the global deficiency of chromogranin A impairs recognition of the major diabetogenic antigen insulin, leading to broadly impaired autoimmune responses controlled by multiple regulatory mechanisms.

Downregulation of Programmed Death-1 Pathway Promoting CD8 + T Cell Cytotoxicity in Primary Biliary Cholangitis

Primary biliary cholangitis (PBC) is an autoimmune disease. CD8 + T cell (CTLs) cytotoxicity played a crucial rule in of PBC with unclear detailed pathogenesis. The role of the programmed death-1 (PD-1) pathway in CD8 + T cell cytotoxicity in patients with PBC was determined. We recruited 69 patients with PBC and 57 healthy controls (HCs). PD-1 pathway in peripheral CD8 + T cells and related cytokines were detected, and gene expression levels were detected. Immunofluorescence staining of PD-1/PD-L1 was performed on liver tissue. PD-1 ± CTLs were cocultured with human intrahepatic biliary epithelial cells (HiBECs) to measure CTL cytotoxicity, proliferation and cytokine levels and HiBEC apoptosis. The upstream signaling pathway of PD-1 was detected. PBC patients exhibited Tbet gene upregulation and PD-1 downregulation in CTLs, with PD-1 expression reduced in CTLs and PD-L1 reduced in the liver portal region relative to HCs. Higher plasma IL-10, interferon-γ and transforming growth factor-β concentrations were observed in the PBC group than the HC group. In CTL and HiBEC coculture experiment, compared with PD-1- CTLs, PD-1 + CTLs exhibited weaker cytotoxicity, less proliferation and lower cytokine production. When the system was blocked by anti-PD-1 antibodies, these effects were antagonized. PD-1 expression in CD8 + T cells decreased, and PD-1 pathway-related cytokines changed in patients with PBC. PD-1/PD-L1 pathway silencing increased CD8 + T cell proliferation, related cytokine production and CTL cytotoxic effects on HiBECs in coculture experiment. The PD-1/PD-L1 pathway might represent an important pathway in the immunological mechanism underlying PBC.

P-61 Relatlimab + nivolumab in patients with advanced hepatocellular carcinoma who are naive to immuno-oncology therapy but progressed on tyrosine kinase inhibitors, a phase 2, randomized, open-label study: RELATIVITY-073

Immuno-oncology (IO) therapies, used alone or in combination, have improved outcomes in advanced hepatocellular carcinoma (HCC); however, not all patients benefit and novel IO combinations are needed to further enhance the benefit-risk profile. Lymphocyte-activation gene 3 (LAG-3) regulates an inhibitory immune checkpoint pathway, limiting T-cell activity, and is a marker for T-cell exhaustion and resistance to IO therapies. LAG-3 is upregulated in many tumors including HCC. Relatlimab (RELA), a LAG-3-blocking antibody, restores effector function of exhausted T cells. Nivolumab (NIVO) is a programmed death-1 (PD-1)-blocking antibody approved for second-line treatment of advanced HCC. Preclinical data indicate that dual inhibition of LAG-3 and PD-1 pathways with RELA + NIVO, respectively, may leverage potentially synergistic pathways to enable T-cell activation and improve immune response. RELATIVITY-073 will evaluate preliminary efficacy and safety of RELA + NIVO in previously treated IO-naive HCC. RELATIVITY-073 is an open-label, randomized, phase 2 study in IO-naive patients with advanced HCC who progressed on prior tyrosine kinase inhibitor therapy in the advanced/metastatic setting. Patients aged ≥ 18 years must have histologically confirmed, advanced HCC of any etiology, not eligible for curative surgical and/or locoregional therapy, Child-Pugh score of 5 or 6, and ECOG performance status 0 or 1. Tumor tissue must be provided for biomarker analysis and patients must have evaluable tumor LAG-3 expression status prior to randomization. Patients with active brain metastases or leptomeningeal metastases, uncontrolled or significant cardiovascular disease, and clinically significant ascites are excluded. Approximately 250 patients will be randomized 2:1:2 to NIVO (treatment arm A) or one of two regimens of RELA + NIVO (treatment arms B and C) to assess different RELA dose levels. The primary endpoint is objective response rate (ORR) per RECIST v1.1 as assessed by blinded independent central review. Other endpoints include safety and tolerability, disease control rate, duration of response, progression-free survival, overall survival, dose-response relationship using best overall response, and ORR by LAG-3 expression status. This study is enrolling patients globally. NCT04567615. All authors contributed to and approved the abstract; writing and editorial assistance was provided by Kathryn Woods and Adam Paton of Complete HealthVizion, funded by Bristol Myers Squibb. Bristol Myers Squibb.

Abstract 1625: Distribution and prevalence of LAG-3 expression in samples of melanoma and gastric/gastroesophageal junction cancer

Abstract Background: Immune checkpoint inhibitors (ICIs) have become the standard of care in a range of tumors. However, a large proportion of patients treated with ICIs have treatment-refractory/resistant tumors requiring innovative approaches. Lymphocyte-activation gene 3 (LAG-3) is an immune checkpoint receptor that inhibits effector T-cell function. Preclinical data suggest that simultaneous activation of the LAG-3 and programmed death-1 pathways results in greater T-cell exhaustion than either pathway alone, leading to impaired antitumor immune response and increased tumor growth. Therefore, LAG-3 may be a promising new target for cancer immunotherapy. The objective of this study was to assess the distribution of LAG-3 expression on lymphocytes in melanoma, gastric cancer (GC), and gastroesophageal junction cancer (GEJC) samples using an immunohistochemistry (IHC) assay. Methods: Formalin-fixed, paraffin-embedded samples from resected melanoma, GC, or GEJC ≤ 5 years old were procured for analysis. The LAG-3 IHC assay was developed and performed by LabCorp (Research Triangle Park, NC) using the anti-LAG-3 17B4 mouse antibody clone on a Leica Bond III platform. LAG-3 expression was assessed as the percentage of positive immune cells (ICs) within the invasive margin, tumor, and stroma. Programmed death ligand 1 (PD-L1) expression on tumor cells (TCs) and ICs was evaluated on the same samples using the Dako PD-L1 IHC 28-8 pharmDx assay to determine the percentage of PD-L1-expressing TCs and the combined positive score (CPS). Results: A total of 102 melanoma samples, 256 GC samples, and 84 GEJC samples were included in this study. LAG-3 expression was detected in the majority of samples across all tumor types, with higher expression in GEJC than in melanoma or GC. LAG-3 prevalence was similar across subgroups based on disease stage (melanoma, GC) and grade (GC). LAG-3 prevalence was also consistent across subgroups based on age, sex, and ethnicity (all tumor types), as well as alcohol consumption and smoking history (melanoma and GC). LAG-3 expression was weakly correlated with PD-L1 expression on TCs (Spearman's ρ of 0.31 for GC, 0.41 for GEJC, and 0.46 for melanoma) and moderately correlated with PD-L1 CPS (Spearman's ρ of 0.63 for GC and 0.73 for GEJC). Similar correlations were observed after binning PD-L1 TC score and CPS. Conclusions: LAG-3-expressing lymphocytes were identified in all tumor types tested. LAG-3 expression did not differ with age, sex, ethnicity, alcohol consumption, and smoking history, and appeared to be associated, but not strongly correlated, with PD-L1 expression in all tumor types. PD-L1 and LAG-3 expression in procured samples may not be reflective of expression observed in clinical trials, and further studies are needed to characterize LAG-3 expression in a clinical setting. Citation Format: Lloye M. Dillon, John Wojcik, Keyur Desai, Ming Lei, Lori Johnson, Bryan McCune, Krystal Johnson, Jeffrey Shuster, Steven M. Anderson, Bin Li. Distribution and prevalence of LAG-3 expression in samples of melanoma and gastric/gastroesophageal junction cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1625.

Higher Proportion of a Novel PD-1+ Foxp3+ γδ Regulatory T Cell Subset in De Novo and Recurrent AML

T cell immunodeficiency may be a common characteristic in acute myeloid leukemia (AML) and adoptive T cellular immunotherapy has a broad prospect for clinical treatment. γδ T cells is essential to anti-leukemia function through perforin (Perf) / granzyme B (Grmb) pathway and may become a new strategy for leukemia immunotherapy. However, γδ T cells have different functional subsets like regulatory cell subsets and relevant regulatory mechanism is still unknown. Programmed death-1 (PD-1) which is one of T cell inhibitory receptors may mediate T cell suppression and dysfunction in leukemia and relate to general regulatory T cells which express transcription factor forkhead box P3 (Foxp3). It would be interesting whether PD-1 pathway might involve in regulation of regulatory Foxp3+ γδ T cells. In this study, we first investigated the regulatory cell subsets of γδ T cells in patients with AML. The flow cytometry (FCM) was used to detect the expression proportions of γδ T cells and their subfamilies in peripheral blood of AML patients (including 15 cases of de novo AML, 5 cases of recurrent AML and 4 cases in complete remission (CR)). Real-time quantitative PCR(RQ-PCR)was used to detect the expression levels of PD-1, Foxp3, Grmb and Perf genes in the γδ T cells sorting by MACS technique from peripheral blood of de novo AML patients. Fifteen health individuals (HI) served as controls. The results showed decreasing trend of γδ T cell counts in AML patients. Compared with HI groups, higher expression levels of PD-1 gene in γδ T cells were found in de novo AML patients, while Grmb and Perf gene presented lower expression levels by RQ-PCR (P <0.05) (Figure A). The expression levels of PD-1 gene had a positive correlation to the Foxp3 gene in de novo AML group. It was noteworthy that we firstly found a novel PD-1+ Foxp3+ γδ T subset in peripheral blood of AML patients and HI controls with different frequencies. The proportions of Foxp3+ γδ T cells, PD1+ γδ T cells, and PD-1+ Foxp3+ γδ T cells were significantly higher either in de novo or the recurrent AML patients, respectively (P <0.05). The PD1+ Foxp3+ γδ T cells counts in AML patients showed the pattern as the recurrent AML > the de novo AML > the AML-CR groups (Figure B and C). In conclusion, a novel regulatory PD1+ Foxp3+ γδ T subset with lower expression levels of Grmb and Perf genes was found in peripheral blood of AML patients. Higher proportion of PD1+ Foxp3+ γδ T cells might influence the occurrence and development of AML. [Display omitted] DisclosuresLi:Guangdong Provincial Applied Science and Technology Research & Development Program (No. 2016B020237006): Research Funding; Guangzhou Science and Technology Project (No. 2015100010211): Research Funding; National Natural Science Foundation of China (91642111): Research Funding; Guangdong Provincial Basic Research Program (No. 2015B020227003: Research Funding. Wu:the Fundamental Research Funds for the Central Universities(No. 21617465): Research Funding; National Natural Science Foundation of China(No.8120038): Research Funding; the Natural Science Foundation of Guangdong Province (No. 2014A030313380): Research Funding.

Tumor Microenvironment (TME) Regulation of Programmed Death-1 (PD1) Receptor and Its Ligands PDL1 and PDL2 in Chronic Lymphocytic Leukemia (CLL)

The PD1 pathway is involved in the inactivation of immune effectors and is potentially reactive against neoplastic cells in the TME. T cells from CLL patients exhibit defective immunity, decreased proliferative capacity, and impaired effector function leading to T cell exhaustion. These functional defects coincide with higher CD244, CD160 and PD1 expression on T cells. In the Eμ-TCL1 transgenic CLL model, PDL1 checkpoint blockade restores immune dysfunction and impairs leukemia growth. PD1/PDL1 ligation also affects BCR signaling, and because PD1 is also expressed on CLL cells, PD1 interference might directly influence tumor growth and proliferation directly.We studied the 1) expression the PD1 receptor and its ligands and their correlation with TFTT; 2) role of the TME in controlling PD1 axis and miRNA expression; 3) role of the BCRi ibrutinib (IB) in the expression of the receptor and ligands.Early-stage CLL patients were prospectively enrolled at diagnosis (n=211, O-CLL1: clinicaltrial.govID:NCT00917540). Gene expression (GE) profiling was performed using the GeneChipVR Gene1.0 STArray (Affymetrix) and validated by qPCR (Thermofisher) and flow-cytometry (FC, BD Biosciences) in an independent subset of patients. CLL lymph node samples underwent in situ immunolocalization analyses. Autologous T-cells (AAT) were obtained by in vitro exposure of patient T-cells with anti CD3/CD28 Dynabeads (Thermofisher) and IL2 in co-culture with CLL cells. Cultures were monitored until substantial clumping was observed and tested for PD1 axis expression. HS5 stromal cells were used in co-culture experiments with B-CLL (ratio 1:100). MiR-424 expression was evaluated by qPCR using validated TaqMan® MicroRNA Assays (Thermofisher). In selected experiments IB was added to cell culture. PD1 GEP of circulating B-CLL cells was significantly higher than of ligands. No differences in GEP of PD1 or its ligands were observed stratifying cases based on CD38, ZAP-70, or FISH markers. Significantly higher PDL1 GE was detected in IGHV-M cases. GEP results were validated by qPCR on 27 independent CLL samples. The impact of GE of the PD1 axis on clinical outcomes in CLL cases (median follow-up 39 months, range 6−82 months) indicated TTFT was significantly shorter in cases with higher levels of PDL2, while no significant differences were detected based on higher or lower PD1 or PDL1 levels. Multivariate analysis indicated higher PDL2 gene expression retained an independent prognostic power (HR=1.9, 95%CI 1.1-3.4, P=.022) in predicting TTFT together with IGHV-UM status, B-lymphocytosis≥5000/mm3 (P=.020), and CD38 expression (P=.021). In situ immunolocalization of CLL tissue infiltrates showed variable expression of PD1 that characterized small lymphoid elements, while PDL1 and PDL2 (PDL2>PDL1) characterized larger medium-sized elements within proliferation centers. Co-localization studies revealed PD1 co-expression of CD20+CLL cells and in scattered CD3+ cells. Both PDL1 and PDL2 were variably expressed in CD20+CLL cells; few T-cells and macrophages showed expression of both ligands within CLL infiltrates. FC analysis of CLL cells after AAT co-culture showed a higher percentage expression of circulating PD1 axis members (n=27, PDL1>>PDL2>PD1) compared to baseline indicating that the changes in PD1 axis expression may be due to events triggered by activation of the TME. In contrast, T cell populations had higher expression of CD3+, CD4+ and CD8+ cells bearing both PD1 and PDL1, while no substantial increment for either CD3+, CD4+ or CD8+/PDL2+ cells following AAT co-culture. GE analysis also showed a similar increase in PDLs (n=15, PDL2>>PDL1>PD1) expression with respect to non-activated B-CLL cells. Interestingly, a 48h co-culture of B-CLL with HS5 stromal cells (n=6) showed a similar increase in PD1 axis GE pattern as AAT cultures. Expression of miR-424 also decreased significantly (<80%) after AAT co-cultures (n=6), indicating its potential involvement in PDL1 regulation. IB reduced PD1/PDL1 gene and protein expression on CLL B-cells; the IB inhibitory effect was also observed only on CD8+ cells.We indicate that PDL2 expression characterizes a subset of high-risk early stage CLL patients. PD1 axis expression is characteristic of the CLL TME whereby modulatory co-stimulatory signals may be counteracted by IB. Further studies will examine the role of miR-424 regulation with this cellular context. DisclosuresNo relevant conflicts of interest to declare.

The PD-1 Interactome.

T cell activation is a fine-tuned process that involves T cell receptor and costimulation signals. To prevent undue activation of T cells, inhibitory molecules including PD-1 (programmed death 1) are induced and function as brakes for T cell signaling. In a steady state, the interaction of PD-1 with its ligands PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC, CD273) maintains peripheral immune tolerance. However, the expression of PD-L1 on tumor cells and interaction with PD-1 on T cells dampen anti-tumor immunity. Therapeutic inhibitors of the PD-1 pathway have revolutionized tumor immunotherapy. Unfortunately, the majority of patients do not develop sustained anti-tumor responses. However, the knowledge about unique PD-1 interactions and their role in mediating PD-1 inhibitory signals is currently limited. Advances in the mechanistic understanding of the molecular and signaling integration of the PD-1 pathway could unleash the great potential in tumor immunotherapy by allowing the development of combinatorial approaches that target not only PD-1 and its ligands but also its unique downstream signal mediators. In this review, the current advances in understanding the mechanisms of extracellular and intracellular PD-1 interactions and their significance in potential future therapeutic approaches are discussed.

Programmed death-1 mediates venous neointimal hyperplasia in humans and rats

Venous neointimal hyperplasia can be a problem after vein interventions. We hypothesized that inhibiting programmed death-1 (PD-1) can decrease venous neointimal hyperplasia in a rat inferior vena cava (IVC) patch venoplasty model. The rats were divided into four groups: the control group was only decellularized without other special treatment; the PD-1 group was injected with a single dose of humanized PD-1 antibody (4 mg/kg); the PD-1 antibody coated patches group; the BMS-1 (a PD-1 small molecular inhibitor) coated patches group (PD-1 inhibitor-1). Patches were implanted to the rat IVC and harvested on day 14 and analyzed. Immunohistochemical analysis showed PD-1-positive cells in the neointima in the human samples. There was high protein expression of PD-1 in the neointima in the rat IVC venoplasty model. PD-1 antibody injection can significantly decrease neointimal thickness (p < 0.0001). PD-1 antibody or BMS-1 was successfully conjugated to the decellularized rat thoracic artery patch by hyaluronic acid with altered morphology and reduced the water contact angle (WCA). Patches coated with humanized PD-1 antibody or BMS-1 both can also decrease neointimal hyperplasia and inflammatory cells infiltration. PD-1-positive cells are present in venous neointima in both human and rat samples. Inhibition of the PD-1 pathway may be a promising therapeutic strategy to inhibit venous neointimal hyperplasia.

Comparative Efficacy and Safety of Programmed Death-1 Pathway Inhibitors in Advanced Gastroesophageal Cancers: A Systematic Review and Network Meta-Analysis of Phase III Clinical Trials.

Simple SummaryThe use of checkpoint inhibitors has changed the treatment landscape for gastroesophageal cancer in the third-line setting. However, success rates in earlier treatment lines are highly variable across trials. Herein, we compare the efficacy and safety of the different anti-PD-1/PD-L1 regimens with or without chemotherapy.Background: The use of checkpoint inhibitors has changed the treatment landscape for gastroesophageal cancer in the third-line setting. However, success rates in earlier treatment lines are highly variable across trials. Herein, we compare the efficacy and safety of the different anti-PD-1/PD-L1 regimens with or without chemotherapy; Methods: We performed a network meta-analysis (NMA) of anti-PD-1/PD-L1 monotherapy or combined with chemotherapy (chemoimmunotherapy) for gastroesophageal cancers without ERBB2 overexpression; Results: The first-line NMA included four trials (N = 3817), showing that chemoimmunotherapy improved OS and PFS without significant safety difference: Nivolumab-chemotherapy, OS (HR: 0.83 [95% CI, 0.75–0.92]), PFS (HR 0.68 [95% CI, 0.57–0.81]), Pembrolizumab-chemotherapy: OS (HR 0.77 [95% CI, 0.67–0.88]), PFS (HR: 0.72 [95% CI, 0.60–0.85]. Pembrolizumab monotherapy was the safest first-line treatment, SAE (OR 0.02 [95% CI, 0.00–0.2]) but showed no survival benefit. The second-line NMA encompassed four trials (N = 2087), showing that anti-PD-1 significantly improved safety but not survival: camrelizumab, SAE (OR 0.37; [95% CI, 0.24–0.56]); nivolumab, SAE (OR 0.13, [95% CI, 0.08–0.2]) pembrolizumab, SAE (OR 0.4; [95% CI, 0.30–0.53]); Conclusions: chemoimmunotherapy improves OS and PFS in previously untreated gastroesophageal cancers. Anti-PD-1 monotherapies improve safety in refractory disease, with no significant survival benefit.

Programmed death 1 (PD-1) serum level and gene expression in recent onset systemic lupus erythematosus patients

Aim of the workTo investigate the potential association of protein programmed death 1 (PD-1) serum level and its gene expression inrecent onset systemic lupus erythematosus (SLE) patients and study its association with the disease activity. Patients and methodsThe study included 80 recently diagnosed SLE patients and 80 healthy controls. SLE disease activity index (SLEDAI) was assessed. The serum level of soluble (sPD-1) was assessed by enzyme linked immunosorbent assay (ELISA) and its gene expression level was evaluated by real time-polymerase chain reaction (RT-PCR). ResultsThey were 68 females and 12 males (F: M 5.7:1) with age 30.8 ± 8.7 years and disease duration of 3.2 ± 1.7 months. The sPD-1 and PD-1 gene expression level (folds) were significantly elevated in patients (1280.6 ± 1448.1 pg/ml and 0.3 ± 0.06 folds) than controls (109.1 ± 11.9 pg/ml and 0.03 ± 0.008 folds) (p < 0.001). A significant correlation was found between sPD-1 and hematuria, pyuria, fever and C3 level (p = 0.01, p = 0.001, p = 0.02, and p = 0.03 respectively), and between PD-1gene expression and psychosis and fever (p = 0.03, p = 0.014). No significant correlation was found between SLEDAI and PD-1 gene expression or sPD-1 level (p = 0.1, p = 0.23 respectively). No significant correlation was found between sPD-1 and PD-1 gene expression levels and the autoantibodies. ConclusionPD-1 gene expression as well as the serum level of sPD-1 are elevated significantly in recent onset SLE patients denoting that they may have a role in the pathogenesis of the disease while there was no relation to the disease activity. This biomarker may be potentially promising for the development of a novel lupus immunotherapy by targeting the PD-1 pathway.

Mechanisms of immunogenic cell death and immune checkpoint blockade therapy.

Immunogenic cell death (ICD) refers to a form of regulated cell death that activates adaptive immunity, forming long-term immunological memory. Using chemotherapeutic drugs to induce ICD in cancer cells can help create an inflamed, immunogenic tumor environment, key for optimal immune checkpoint blockade (ICB) therapy response. ICB targets immune checkpoints such as cytotoxic T-lymphocyte-associated antigen 4 (CTLA4) and programmed death 1 (PD-1). Durable responses and better quality of life in ICB patients compared with many other treatments has prompted additional investigation into its therapeutic potential and possible approaches, in an effort to further understand the functions of the costimulatory molecules and how new treatments may be designed. In this review, we will summarize ICD induction, including stress responses, damage-associated molecular patterns, and various assays by which immunogenicity is evaluated in dying cells. In addition, the mechanisms and biomarkers underlying the CTLA4 and PD-1 pathways of checkpoint blockade will be covered. Finally, we will review the synergistic effects of ICD induction combined with ICB therapy, as well as combination blockade therapies involving the use of multiple drugs.