Surface Expression Of PD-1 Research Articles

Correlation between PD-1 and sPD-L1 expression levels in peripheral blood of DLBCL patients and their clinicopathological characteristics.

Molecular pathology and clinical characteristics play a crucial role in guiding treatment selection and predicting the prognosis of diffuse large B-cell lymphoma (DLBCL). The programmed cell death protein 1 (PD-1) and its ligand (PD-L1), have emerged as pivotal regulators of immune checkpoints in cancer. The objectives of this study are to investigate the correlation between the expression levels of PD-1 and soluble PD-L1 (sPD-L1) in the peripheral blood of DLBCL patients, analyze their clinicopathological characteristics, and identify the optimal beneficiary group for PD-1/PD-L1 blockade. Peripheral blood samples were collected from 36 DLBCL patients before their initial treatment at Shandong Cancer Hospital between December 2018 and July 2019. The expression levels of PD-1 and sPD-L1 were measured using flow cytometry and enzyme-linked immunosorbent assay (ELISA), respectively. The clinicopathological characteristics, including age, sex, Ann Arbor stage, International Prognostic Index (IPI) score, response to treatment, etc., were recorded for each patient. The surface expression of PD-1 on peripheral blood T cells was significantly higher in DLBCL patients compared to healthy controls. There was a significant association between elevated PD-1 expression levels and the advanced Ann Arbor stage (P=0.0153) as well as the B group (P=0.0184). Higher sPD-L1 levels were associated with the GCB subtype according to Hans's classification (P=0.0435). The expression levels of PD-1 and sPD-L1 in the peripheral blood of DLBCL patients are significantly correlated with advanced disease stage, B group, and GCB subtype according to Hans's classification. This suggests that the PD-1/PD-L1 axis play a critical role in specific subgroups of DLBCL. Targeting this axis could serve as a potential therapeutic strategy to enhance the clinical outcomes of DLBCL patients. Further studies are necessary to explore the prognostic implications of PD-1 and sPD-L1 expression levels in DLBCL patients.

Anti-PD-L1 therapy altered inflammation but not survival in a lethal murine hepatitis virus-1 pneumonia model.

Because prior immune checkpoint inhibitor (ICI) therapy in cancer patients presenting with COVID-19 may affect outcomes, we investigated the beta-coronavirus, murine hepatitis virus (MHV)-1, in a lethal pneumonia model in the absence (Study 1) or presence of prior programmed cell death ligand-1 (PD-L1) antibody (PD-L1mAb) treatment (Study 2). In Study 1, animals were inoculated intratracheally with MHV-1 or vehicle and evaluated at day 2, 5, and 10 after infection. In Study 2, uninfected or MHV-1-infected animals were pretreated intraperitoneally with control or PD-L1-blocking antibodies (PD-L1mAb) and evaluated at day 2 and 5 after infection. Each study examined survival, physiologic and histologic parameters, viral titers, lung immunophenotypes, and mediator production. Study 1 results recapitulated the pathogenesis of COVID-19 and revealed increased cell surface expression of checkpoint molecules (PD-L1, PD-1), higher expression of the immune activation marker angiotensin converting enzyme (ACE), but reduced detection of the MHV-1 receptor CD66a on immune cells in the lung, liver, and spleen. In addition to reduced detection of PD-L1 on all immune cells assayed, PD-L1 blockade was associated with increased cell surface expression of PD-1 and ACE, decreased cell surface detection of CD66a, and improved oxygen saturation despite reduced blood glucose levels and increased signs of tissue hypoxia. In the lung, PD-L1mAb promoted S100A9 but inhibited ACE2 production concomitantly with pAKT activation and reduced FOXO1 levels. PD-L1mAb promoted interferon-γ but inhibited IL-5 and granulocyte-macrophagecolony-stimulating factor (GM-CSF) production, contributing to reduced bronchoalveolar lavage levels of eosinophils and neutrophils. In the liver, PD-L1mAb increased viral clearance in association with increased macrophage and lymphocyte recruitment and liver injury. PD-L1mAb increased the production of virally induced mediators of injury, angiogenesis, and neuronal activity that may play role in COVID-19 and ICI-related neurotoxicity. PD-L1mAb did not affect survival in this murine model. In Study 1 and Study 2, ACE was upregulated and CD66a and ACE2 were downregulated by either MHV-1 or PD-L1mAb. CD66a is not only the MHV-1 receptor but also an identified immune checkpoint and a negative regulator of ACE. Crosstalk between CD66a and PD-L1 or ACE/ACE2 may provide insight into ICI therapies. These networks may also play role in the increased production of S100A9 and neurological mediators in response to MHV-1 and/or PD-L1mAb, which warrant further study. Overall, these findings support observational data suggesting that prior ICI treatment does not alter survival in patients presenting with COVID-19.

Engineering Cell-Derived Nanovesicles for Targeted Immunomodulation.

Extracellular vesicles (EVs) show promise for targeted drug delivery but face production challenges with low yields. Cell-derived nanovesicles (CDNVs) made by reconstituting cell membranes could serve as EV substitutes. In this study, CDNVs were generated from mesenchymal stem cells by extrusion. Their proteomic composition, in vitro and in vivo toxicity, and capacity for loading RNA or proteins were assessed. Compared with EVs, CDNVs were produced at higher yields, were comprised of a broader range of proteins, and showed no detrimental effects on cell proliferation, DNA damage, or nitric oxide production in vitro or on developmental toxicity in vivo. CDNVs could be efficiently loaded with RNA and engineered to modify surface proteins. The feasibility of generating immunomodulatory CDNVs was demonstrated by preparing CDNVs with enhanced surface expression of PD1, which could bind to PD-L1 expressing tumor cells, enhance NK and T cell degranulation, and increase immune-mediated tumor cell death. These findings demonstrate the adaptability and therapeutic promise of CDNVs as promising substitutes for natural EVs that can be engineered to enhance immunomodulation.

A CD64/FcγRI-mediated mechanism hijacks PD-1 from PD-L1/2 interaction and enhances anti-PD-1 functional recovery of exhausted T cells.

Therapeutic monoclonal antibodies (mAb) targeting the immune checkpoint inhibitor programmed cell death protein 1 (PD-1) have achieved considerable clinical success in anti-cancer therapy through relieving T cell exhaustion. Blockade of PD-1 interaction with its ligands PD-L1 and PD-L2 is an important determinant in promoting the functional recovery of exhausted T cells. Here, we show that anti-PD-1 mAbs act through an alternative mechanism leading to the downregulation of PD-1 surface expression on memory CD4+ and CD8+ T cells. PD-1 receptor downregulation is a distinct process from receptor endocytosis and occurs in a CD14+ monocyte dependent manner with the CD64/Fcγ receptor I acting as the primary factor for this T cell extrinsic process. Importantly, downregulation of surface PD-1 strongly enhances antigen-specific functional recovery of exhausted PD-1+CD8+ T cells. Our study demonstrates a novel mechanism for reducing cell surface levels of PD-1 and limiting the inhibitory targeting by PD-L1/2 and thereby enhancing the efficacy of anti-PD-1 Ab in restoring T cell functionality.

Tumor Cell-Derived Microparticles Induced by Methotrexate Augment T-cell Antitumor Responses by Downregulating Expression of PD-1 in Neutrophils.

Neutrophils act as a "double-edged sword" in the tumor microenvironment by either supporting or suppressing tumor progression. Thus, eliciting a neutrophil antitumor response remains challenging. Here, we showed that tumor cell-derived microparticles induced by methotrexate (MTX-MP) acts as an immunotherapeutic agent to activate neutrophils, increasing the tumor-killing effect of the cells and augmenting T-cell antitumor responses. We found that lactate induced tumor-associated neutrophils to elevate expression of programmed cell death protein 1 (PD-1) and that PD-1+ neutrophils had the properties of N2 neutrophils and suppressed T-cell activation through PD-1/programmed death-ligand 1 (PD-L1) signaling. By performing ex vivo experiments, we found that MTX-MPs-activated neutrophils had reduced surface expression of PD-1 as a result of PD-1 internalization and degradation in the lysosomes, leading to the cells showing a decreased capacity to suppress T-cell responses. In addition, we also found that MTX-MP-activated neutrophils released neutrophil elastase which could kill tumor cells and disrupt tumor stroma, leading to increased T-cell infiltration. Furthermore, using a combination of anti-PD-L1 and MTX-MPs, we observed that long-term survival increased in a mouse model of lung cancer. Collectively, these findings highlight the potential use of a combination of anti-PD-L1 and MTX-MPs to enhance the therapeutic effect of anti-PD-L1 alone.

Large Scale Ex Vivo Expansion of γδ T cells Using Artificial Antigen-presenting Cells.

Higher γδ T cell counts in patients with malignancies are associated with better survival. However, γδ T cells are rare in the blood and functionally impaired in patients with malignancies. Promising results are reported on the treatment of various malignancies with in vivo expansion of autologous γδ T cells using zoledronic acid (zol) and interleukin-2 (IL-2). Here we demonstrated that zol and IL-2, in combination with a novel genetically engineered K-562 CD3scFv/CD137L/CD28scFv/IL15RA quadruplet artificial antigen-presenting cell (aAPC), efficiently expand allogeneic donor-derived γδ T cells using a Good Manufacturing Practice (GMP) compliant protocol sufficient to achieve cell doses for future clinical use. We achieved a 633-fold expansion of γδ T cells after day 10 of coculture with aAPC, which exhibited central (47%) and effector (43%) memory phenotypes. In addition, >90% of the expanded γδ T cells expressed NKG2D, although they have low cell surface expression of PD1 and LAG3 inhibitory checkpoint receptors. In vitro real-time cytotoxicity analysis showed that expanded γδ T cells were effective in killing target cells. Our results demonstrate that large-scale ex vivo expansion of donor-derived γδ T cells in a GMP-like setting can be achieved with the use of quadruplet aAPC and zol/IL-2 for clinical application.

High ORM1 Expression Marks a Subset of Genetically Adverse-Risk B-ALL Characterized By MDSC Enrichment, T-Cell Dysfunction, and Inferior Overall Survival

Alterations in the transcriptional activity of certain bone marrow microenvironment populations have been shown to contribute to the pathogenesis of B-lymphoblastic leukemia (B-ALL); however, the precise mechanisms by which neoplastic lymphoblasts shape the gene expression programs of reactive immune and stromal cell populations to support a leukemogenic niche remain incompletely characterized. ORM1 is an acute phase protein with immunomodulatory properties that is overexpressed in a subset of high-risk B-ALL. In an initial exploratory analysis of 1,104 adult and pediatric B-ALL RNA-seq transcriptomes annotated with mature survival outcomes (PMID 30643249), high ORM1 expression was significantly associated with inferior event-free and overall survival and predominantly associated with Ph+, Ph-like, and KMT2A-r B-ALL subtypes. We hypothesized that B-ALL with high ORM1 expression is associated with transcriptionally distinct bone marrow microenvironment immune cell subsets that contribute to its aggressive clinical behavior and sought to delineate these alterations through digital cytometry and analysis of single cell proteogenomic sequencing (CITE-seq) data. To delineate the tumor microenvironment (TME) features of ORM1 high B-ALL through digital cytometry, we constructed a novel bone marrow signature matrix that recapitulates the native BM cellular landscape, including hematopoietic progenitors and mesenchymal stem cells, and performed de novo transcriptional subtype discovery agnostic to clinical outcomes using the Ecotyper platform on a cohort of 577 B-ALL microarray transcriptomes from the MILE study (GSE13159). Discovery analysis identified 51 transcriptionally defined TME cell states across 17 cell types. ORM1 overexpression significantly correlated with transcriptionally-distinct neutrophil, promyelocyte, monocyte, and macrophage subsets enriched for immunosuppressive gene expression programs as well as CD8+ and γδ T-cell subsets with hallmark transcriptional features of effector dysfunction and exhaustion. To validate the imputed cell states at single-cell resolution and define their immunophenotypic features, we drew upon several B-ALL CITE-seq datasets. Recovery and assignment of discovery cohort transcriptional states to single-cell transcriptomes in KMT2A-r and Ph+ cases confirmed enrichment for multiple myeloid-derived suppressor cell (MDSC) and dysfunctional T-cell transcriptional subsets. MDSCs were predominantly monocytic but also included neutrophilic promyelocytes with surface expression of CD123 and CD155/PVR, which interacts with TIGIT to suppress immune cell activation. Indeed, enrichment for CD8+ cytotoxic lymphocytes with surface expression of PD-1 and TIGIT was also present. Neutrophilic MDSCs were present at diagnosis, in pre-relapse remission samples and at the time of relapse, suggesting that their persistence following frontline therapy may facilitate the immune evasion and expansion of treatment-resistance leukemic clones that drive eventual relapse. Taken together, high ORM1 expression marks a subset of high-risk B-ALL characterized by enrichment for transcriptionally-distinct immunosuppressive TME cell states which may be targetable with emerging therapeutic strategies aimed at restoring host anti-tumor immunity.

Lyt-200, a Humanized Anti-Galectin-9 Antibody, Exhibits Preclinical Efficacy in Models of Hematological Malignancies

Patients with relapse and refractory (R/R) hematological malignancies typically have 5-year overall survival rates of less than 50% when receiving chimeric antigen receptor (CAR) T-cell therapy and less than 30% after receiving hematopoietic stem cell transplantation (HSCT). Human leukemia cells utilize a variety of biochemical mechanisms, which allow them to escape host immune surveillance. These molecular pathways cause impairment of the anti-cancer activities of immune cells, which could attack and kill leukemia cells. These dismal outcomes for patients with R/R disease highlight the need for novel treatment regimens when current therapeutic options are exhausted. Galectins are s-type lectins that promote diverse biological processes including adhesion, signaling, and immunosuppression. In acute myeloid leukemia (AML), galectin-9 (GAL-9) maintains the leukemia initiating cell (LIC) population and plays an important role in treatment resistance. We recently tested LYT-200, a fully humanized IgG4 αGAL-9 monoclonal antibody, developed by PureTech Health, which has so far been well tolerated with no dose limiting toxicities in a Phase I clinical trial for solid tumors (NCT04666688). We tested LYT-200 in vitro and in vivo across multiple hematological malignancies. Flow cytometry was used to assess the surface expression of GAL-9 on human B-ALL, AML, T-cell acute lymphoblastic leukemia (T-ALL), and diffuse large B-cell lymphoma (DLBCL) cell lines relative to receptor T-cell immunoglobulin and mucin domain-containing protein-3 (TIM-3) and Programmed cell death protein-1 (PD-1) expression. Compared to healthy human peripheral blood mononuclear cells (PBMCs), where GAL-9 surface expression was low or absent depending on the immune cell, GAL-9 was highly expressed on the surface of all human blood cancer cell lines tested (>100-fold increase). Notably, GAL-9 expression was comparable to PD-1 surface expression and higher than that of TIM-3 on AML, B-ALL, T-ALL, and DLBCL cells. Treating all cell lines with LYT-200 relative to control immunoglobulin resulted in extensive cell death, which occurred after 48 and 72 hours of treatment. Furthermore, a side-by-side comparison with αTIM-3 antibody treatment at equivalent concentrations, revealed that LYT-200 treatment was significantly more effective at killing blood cancer cells at low doses of 10-100 ng/mL in vitro. The in vitro efficacy of LYT-200 against multiple blood cancer subtypes extended to in vivo protection and survival benefit in murine models of T-ALL and AML, both in immunocompromised and immunocompetent mice. When immunocompromised mice were transplanted with human T-ALL cell lines or patient-derived samples, single-agent treatment with LYT-200 (1.5 mg/kg/weekly) was comparable to survival results obtained with methotrexate (0.75 mg/kg/weekly) administration. Notably, the combination of LYT-200 and methotrexate treatments led to the best survival outcomes in both contexts. Similar results were obtained in xenograft studies of AML, where single-agent LYT-200 (1.5 mg/kg/weekly) treatment was more effective at protecting mice than cytarabine (100 mg/kg/weekly). However, combining both treatments resulted in the best survival outcomes with greater than 80% of mice surviving over 2 months after disease appearance. In all, our results demonstrate that GAL-9 is an important driver of multiple blood cancer subtypes and that targeting GAL-9 with LYT-200 represents a potential novel treatment strategy which warrants additional pre-clinical and clinical testing.

Signatures of recent activation identify a circulating T cell compartment containing tumor-specific antigen receptors with high avidity.

T cell receptor (TCR) avidity is assumed to be a major determinant of the spatiotemporal fate and protective capacity of tumor-specific T cells. However, monitoring polyclonal T cell responses with known TCR avidities in vivo over space and time remains challenging. Here, we investigated the fate and functionality of tumor neoantigen-specific T cells with TCRs of distinct avidities in a well-established, reductionist preclinical tumor model and human patients with melanoma. To this end, we used polyclonal T cell transfers with in-depth characterized TCRs together with flow cytometric phenotyping in mice inoculated with MC38 OVA tumors. Transfer of T cells from retrogenic mice harboring TCRs with high avidity resulted in best tumor protection. Unexpectedly, we found that both high- and low-avidity T cells are similarly abundant within the tumor and adopt concordant phenotypic signs of exhaustion. Outside the tumor, high-avidity TCR T cells were not generally overrepresented but, instead, selectively enriched in T cell populations with intermediate PD-1 protein expression. Single-cell sequencing of neoantigen-specific T cells from two patients with melanoma-combined with transgenic reexpression of identified TCRs by CRISPR-Cas9-mediated orthotopic TCR replacement-revealed high-functionality TCRs to be enriched in T cells with RNA signatures of recent activation. Furthermore, of 130 surface protein candidates, PD-1 surface expression was most consistently enriched in functional TCRs. Together, our findings show that tumor-reactive TCRs with high protective capacity circulating in peripheral blood are characterized by a signature of recent activation.

The characteristics of antigenic specificity of memory regulatory t cells in women with unexplained recurrent pregnancy loss

Regulatory T cells (Tregs) proliferate after encountering the fetal antigen, which plays an important role in maintaining maternal-fetal tolerance. Activated Tregs increase number and function after antigen encounter and develop memory. Upon subsequent antigen exposure, Treg cells re-expand more rapidly. However, the characteristics of memory regulatory T cells (mTregs) during normal pregnancy and unexplained recurrent pregnancy loss (URPL) have not been elucidated well. In this study, we analyzed the proportion of Tregs and mTregs in the peripheral blood and their surface expression of PD-1, CCR6, and HLA-G in normal non-pregnant (n = 20) and pregnant (n = 20) women, and non-pregnant (n = 20) and pregnant URPL (n = 20) women. We found that the proportions of mTregs in lymphocytes, CD3+ T cells, CD4+ T cells, and Tregs were lower in pregnant URPL patients than in normal pregnant women. The proportions of CD4+CD45RO+ Th cells in lymphocytes, CD3+ T, and CD4+ T cells in the pregnant URPL group were the highest among the four groups (P < 0.05). There were no significant differences among the other three groups (P > 0.05). The proportions of CD4+/CCR6+/mTregs, CD4+/PD-1+/mTregs, CD4+/HLA-G+/mTregs were significantly lower in the non-pregnant normal group and non-pregnant URPL group than in normal pregnant group and pregnant URPL group (P < 0.05, respectively). The proportions of CD4+/CCR6+ mTregs, CD4+/PD-1+/mTregs, CD4+/HLA-G+/mTregs were lower in pregnant URPL group than in normal pregnant group (P < 0.05, respectively). These findings indicate that fetal antigen-specific mTregs play an important role in pregnancy maintenance, and the dysregulation of mTreg may contribute to URPL.

Interaction of immune checkpoint PD-1 and chemokine receptor 4 (CXCR4) promotes a malignant phenotype in pancreatic cancer cells.

Despite recent therapeutic advances, pancreatic ductal adenocarcinoma (PDAC) remains a devastating disease with limited therapeutic options. Immune checkpoint inhibitors (ICIs) have demonstrated promising results in many cancers, but thus far have yielded little clinical benefit in PDAC. Based on recent combined targeting of programmed cell death protein-1 (PD-1) and C-X-C chemokine receptor 4 (CXCR4) in patient-derived xenografts (PDXs) and a pilot clinical trial, we sought to elucidate potential interactions between PD-1 and CXCR4. We observed concomitant expression and direct interaction of PD-1 and CXCR4 in PDAC cells. This interaction was disrupted upon CXCR4 antagonism with AMD3100 and led to increased cell surface expression of PD-1. Importantly, CXCR4-mediated PDAC cell migration was also blocked by PD-1 inhibition. Our work provides a possible mechanism by which prior studies have demonstrated that combined CXCR4 and PD-1 inhibition leads to decreased tumor growth. This is the first report investigating PD-1 and CXCR4 interactions in PDAC cells and our results can serve as the basis for further investigation of combined therapeutic targeting of CXCR4 and PD-1.

A Combination of Cytokine-Induced Killer Cells With PD-1 Blockade and ALK Inhibitor Showed Substantial Intrinsic Variability Across Non-Small Cell Lung Cancer Cell Lines.

BackgroundCancer heterogeneity poses a serious challenge concerning the toxicity and adverse effects of therapeutic inhibitors, especially when it comes to combinatorial therapies that involve multiple targeted inhibitors. In particular, in non-small cell lung cancer (NSCLC), a number of studies have reported synergistic effects of drug combinations in the preclinical models, while they were only partially successful in the clinical setup, suggesting those alternative clinical strategies (with genetic background and immune response) should be considered. Herein, we investigated the antitumor effect of cytokine-induced killer (CIK) cells in combination with ALK and PD-1 inhibitors in vitro on genetically variable NSCLC cell lines.MethodsWe co-cultured the three genetically different NSCLC cell lines NCI-H2228 (EML4-ALK), A549 (KRAS mutation), and HCC-78 (ROS1 rearrangement) with and without nivolumab (PD-1 inhibitor) and crizotinib (ALK inhibitor). Additionally, we profiled the variability of surface expression multiple immune checkpoints, the concentration of absolute dead cells, intracellular granzyme B on CIK cells using flow cytometry as well as RT-qPCR. ELISA and Western blot were performed to verify the activation of CIK cells.ResultsOur analysis showed that (a) nivolumab significantly weakened PD-1 surface expression on CIK cells without impacting other immune checkpoints or PD-1 mRNA expression, (b) this combination strategy showed an effective response on cell viability, IFN-γ production, and intracellular release of granzyme B in CD3+ CD56+ CIK cells, but solely in NCI-H2228, (c) the intrinsic expression of Fas ligand (FasL) as a T-cell activation marker in CIK cells was upregulated by this additive effect, and (d) nivolumab induced Foxp3 expression in CD4+CD25+ subpopulation of CIK cells significantly increased. Taken together, we could show that CIK cells in combination with crizotinib and nivolumab can enhance the anti-tumor immune response through FasL activation, leading to increased IFN-γ and granzyme B, but only in NCI-H2228 cells with EML4-ALK rearrangement. Therefore, we hypothesize that CIK therapy may be a potential alternative in NSCLC patients harboring EML4-ALK rearrangement, in addition, we support the idea that combination therapies offer significant potential when they are optimized on a patient-by-patient basis.

Dynamic changes in regulatory T cells during normal pregnancy, recurrent pregnancy loss, and gestational diabetes

Regulatory T cells (Tregs) are critical to regulating maternal T-cell activation against trophoblast; however, the characteristics of maternal Tregs during pregnancy have not been elucidated well. In this study, we analyzed the proportion of CD4+ and CD8+ Tregs in the peripheral blood and their surface expression of PD-1, GITR, HLA-G, and CTLA-4 in normal pregnant women during the first (n = 28), second (n = 43), and the third trimester (n = 33), non-pregnant women (n = 57), pregnant women with a history of recurrent pregnancy loss (RPL) during the first trimester (n = 21), and pregnant women with gestational diabetes mellitus (GDM) during the second (n = 17) and third trimester (n = 28). The proportions of CD4+ and CD8+ Tregs were higher in normal pregnant women than that of non-pregnant women (P < 0.01 respectively). The proportion of CD4+ Tregs was peaked during the second trimester and then decreased. Contrarily, the proportion of CD8+ Tregs was increased throughout gestation and peaked during the third trimester. Proportions of CD4+/PD-1+ Tregs, CD4+/GITR+ Tregs, CD8+/PD-1+ Tregs, and CD8+/CTLA-4+ Tregs peak during the third trimester of normal pregnancy. Pregnant women with RPL and GDM had lower proportions of CD4+ Tregs (P < 0.05 and P < 0.01 respectively) and higher proportions of CD8+ Tregs (P < 0.01 and P < 0.01 respectively) than those of normal pregnancies.Together, our findings indicate that CD4+ and CD8+ Tregs play different roles in pregnancy maintenance, and the dysregulation may contribute to obstetrical complications.

Elevated Levels of PD-L1 on MDSCs in Patients with Ph(-) Myeloproliferative Neoplasm

Introduction. We previously reported that PD-1 and PD-L1 were increased in patients with Ph(-) myeloproliferative neoplasm (MPN) ( Wang et al., Leuk Res 2019). The PD-1 inhibitor therapy or immune checkpoint inhibitor therapy (ICI) trial in MPN by Hobbs et al. reported a negative result (Blood, 2020 (supplement )). Resistance to ICI therapy has been reported to be related to myeloid-derived suppressor cells (MDSCs) in melanoma and breast carcinoma. We also previously reported that MDSCs were increased in patients with Ph(-) MPN (Wang et al., Leu Res 2016). Regulation of immune suppression by MDSCs has been reported to be related to PD-1and PD-L1 expression on the MDSC. Therefore, the current study measured PD-1 and PD-L1 expression in MDSCs in patients with Ph(-) MPN.Materials and Methods. Twenty-six patients, including 11 essential thrombocythemias (ETs), six polycythemia vera (PV), and nine myelofibrosis (six primary MF, one post-ET-MF, two post-PVMF) were compared with ten normal volunteer controls. MDSCs were measured as follows: the pelleted PBMNCs were used for Magnetic-assisted cell separation (MACS, Miltenyi Biotech, Inc.). PBMNCs are sequentially selected for HLA-DR - cells, from which we further selected for CD14 + and CD14 - cells using Miltenyic magnetic microbeads kits, respectively, according to the manufacturer's protocol. The resulting HLA-DR -CD14 + and HLA-DR -CD14 - cells were stored at -80°C until use. Flow cytometric assay:HLA-DR -CD14 + and HLA-DR -CD14 - cells were stained with both anti-human CD274 (PD-L1) FITC and human CD279 (PD-1) PE (BD Biosceinces, Inc.), together with anti-human CD14 APC or anti-human CD33 APC (BD Biosceinces, Inc.), respectively. Flow cytometric assessment was done using the BD Accuri C6 flow cytometer. While HLA-DR -CD14 +Monocytic MDCS cells were gated and assayed for surface expression of PD-1 (CD274) and PDL-1 (CD279), G-MDSC (granulocytic MDSC), and M-NDSC (Monocytic MDSC) were assayed as HLA-DR -CD14 - , CD33 + and HLA-DR -CD14 +, respectively. These MDSCs were then assayed for surface expression of PD-1 (CD274) and PDL-1 (CD279). The flow data were analyzed using FlowJo V8 (Flowjo, LLC), and the mean fluorescent intensity (MFI) was calculated.Results. PD-L1 on the m-MDSCs was significantly elevated in patients with MPN (grouping patients with ET, PV, and MF) and MF than controls. PD-1 on the M-MDSCs was not different between ET, PV, MF, or MPN compared with normal controls. Compared to controls, PD-L1 on the G-MDSC was significantly elevated in patients when ET, PV, and MF were grouped as MPN. PD-1 on the G-MDSC were not different between ET, PV, MF, or MPN and controls.Conclusion. Ph(-) MPN was found to have significantly elevated PD-L1 on the M-MDSCs and G-MDSCs, but PD-1 levels were not significantly elevated. Further studies to employ drugs, such as a combination of IMiDs (e.g., lenalidomide) and anti-MDSC drugs in combination with ICI in vitro and clinical trials, may be warranted. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract 1805: ABTL0812, a Phase 2 clinical stage pro-autophagy anticancer drug exhibits immunomodulatory effects that modify tumor microenvironment in pancreatic cancer models

Abstract ABTL0812 induces cytotoxic autophagy in cancer cells through the combination of ER stress induction and Akt/mTOR blockade. ABTL0812 induces ER stress mediated activation of JNK-Jun pathway and inhibition of the STAT3-IL10 axis in cancer and immune cells in vitro and induces ER stress markers TRIB3 and CHOP in white blood cells of patients with no toxic effects and showing clinical efficacy in Phase 2 trial in combination with chemotherapy vs chemotherapy alone (historical data). In primary and immortalized monocytes-derived macrophages in vitro, ABTL0812 promoted M1 phenotypes potentiating IL-1β and TNFα expression and suppressed M2 phenotypes by decreasing IL-10 expression, as detected by RT-qPCR. Additionally, ABTL0812 inhibited the release of immunosuppressive chemokines (CXCL5, CCL5, CCL8) detected by protein array. In human primary T cells cultured in vitro, ABTL0812 decreased PD1 surface expression in non-activated and activated CD4 and CD8 cells detected by flow cytometry. In pancreatic cancer cells in vitro, ABTL0812 inhibited the release of immunosuppressive chemokines (CCL5, CXCL6, CXCL16), as detected by a protein array. ABTL0812 induced Immunogenic Cell Death (ICD) as indicated by a dose-dependent increase of ICD markers: extracellular Hmgb1 and ATP, surface calreticulin, and caspases 3 and 8 activation, as detected by ELISA, luciferase assay, flow cytometry and fluorescence based substrate assays, respectively. Cultured media from ABTL0812 treated pancreatic cancer cells transferred to differentiated macrophages potentiated the activation of immortalized THP1 macrophages (increased metabolic activity) and promoted M1 polarization potentiating IL-1β and TNFα expression measured by RT-qPCR. In a syngeneic murine model using MT5 murine pancreatic cancer cells (K-Ras and p53 mutated), ABTL0812 showed significant tumor volume reduction compared to vehicle treatment, and similar efficacy to anti-PD1 treatment. Flow cytometry analysis of tumors revealed that ABTL0812 increased intratumor myeloid and Natural Killer T-cells more efficiently than anti-PD1. Importantly, ABTL0812 also increased T helpers Th1/Th2 ratio in spleen. ABTL0812 shows immunomodulatory effects in vitro and in vivo in pancreatic cancer models through the inhibition of the secretion of immunosuppressive chemokines and the induction of ICD in cancer cells, while potentiates M1 phenotypes in macrophages and inhibits PD1 expression in T cells, ultimately promoting increased myeloid and NK cells within tumors and increased Th1/Th2 ratio. Overall, these multiple actions could potentially help to transform “cold” non-immunogenic tumors into “hot” immunogenic tumors and contribute to elicit anticancer immunosurveillance. These novel findings support further investigation of ABTL0812 in combination with immunotherapy to treat cancer Citation Format: Guillermo Yoldi, Pau Munoz-Guardiola, Elisabet Megias, Hector Perez-Montoyo, Marc Yeste-Velasco, Jose Alfon, Carles Domenech, Jose Miguel Lizcano. ABTL0812, a Phase 2 clinical stage pro-autophagy anticancer drug exhibits immunomodulatory effects that modify tumor microenvironment in pancreatic cancer models [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 1805.

Long-Term Treatment with Ibrutinib in Chronic Lymphocytic Leukemia Patients Normalizes CD3+ and CD8+ T Cells and Immune Checkpoints Expression but Gradually Leads to CD4+ T-Cell Depletion

In this study, we analyzed the changes in the peripheral blood mononuclear cells (PBMC) in symptomatic, relapsed or refractory chronic lymphocytic leukemia (CLL) patients (pts) during long-term treatment with the Bruton's tyrosine kinase inhibitor ibrutinib. Peripheral blood (PB) samples were collected before start of treatment with ibrutinib (420 mg/day p.o.) and at week (wk) 4, 10, 16, 22 and at 8, 12 and 24 months (mo) of treatment. Flow-cytometry analyses of PBMC included CLL cells, Natural Killer (NK) cells, T-cell memory subsets, helper subpopulations (Ths) and regulatory T cells (Tregs).Eleven pts were included in the study, 8 males and 3 females; median age was 73 years. Nine age- and sex-matched healthy individuals were included as controls. Eight pts were still on ibrutinib treatment after 24 mo; for the remaining 3 pts, the last follow-up time was 12 mo. Eight pts had high-risk and 3 intermediate disease according to the modified Rai staging system; 8/11 had 17p deletion. The median lymphocyte count at study entry was 45 x 109/L (range 3.1-109.5). Median number of previous treatment regimens was 1 (range 1-4). All but 2 pts stayed on full-dose ibrutinib during the whole treatment period. In the remaining 2 pts, the dose was decreased to 280 mg/day after approximately 1 year of treatment due to toxicity.In 9/11 pts, the number of CLL cells initially increased at wk 4 compared to baseline (p=0.03); then a gradual reduction occurred during treatment and became significant at wk 16 (p=0.005). At baseline, CLL pts had significantly higher absolute numbers of CD3+ and CD8+ cells compared to healthy controls. CD3+ normalized (i.e. reached values not significantly different from healthy individuals) by wk 10. In contrast to previously published data (Long M et al, J Clin Invest 2017), we observed that CD8+ T cell numbers started to decrease at wk 10 and were normalized by 12 mo. CD4+ T cells, which were at start of treatment non-significantly different from healthy, also gradually decreased in 9/11 pts from wk 16 reaching by 24 mo levels below normal (p=0.02) (Fig.1A). At 24 mo of follow-up, CD4+ cell numbers had decreased in 7/8 evaluable pts; the median reduction vs baseline was 59% (range 3-92%). Among the helper subsets, Th1 (CCR6-/ CXCR3+) cells, which at baseline were higher compared to healthy (p=0.0003), normalized by 12 mo, while Th2 (CCR6-/ CXCR3-) cells, at baseline non-significantly different from healthy, decreased below normal levels at 12 mo (p=0.006). Th17 (CCR6+/ CXCR3-) cells, lower than in healthy controls at baseline (p=0.04), remained unchanged (Fig. 1B). Tregs (CD4+/CD25+/ CCR4+/CD127low) and NK cells remained also unchanged.The surface expression of PD-1 and intracellular CTLA-4, which was significantly higher at baseline in pts compared to healthy controls in both CD4+ and CD8+ cells, decreased also gradually. In the CD4+ subset, PD-1 expression normalized by 24 mo, while in the CD8+ subset already at 12 mo (Fig.2A). The expression of CTLA-4 normalized in CD4+ cells only by wk 22 (Fig.2B). Proliferating (Ki67+) cells, which at baseline were higher in pts compared to controls both in CD4+ and CD8+ cells, also normalized at wk 10 and 12 mo, respectively (Fig.2C). The distribution of the CD4+ and CD8+ memory cell subsets, abnormal at baseline, normalized as well by 12 mo.In conclusion, these data indicate that ibrutinib treatment gradually led to normalization of T-cell counts in CLL patients. Down-regulation of immune checkpoints and cell proliferation markers also occurred, which paralleled the reduction of tumour burden. Moreover, the CD4+ T-cell counts gradually reached levels significantly lower than normal. This reduction affected both the Th1 and Th2 helper populations. The possible clinical implications remain to be shown during extended therapy. [Display omitted] DisclosuresMellstedt:Kancera AB: Equity Ownership; Sandoz: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees. Österborg:Gilead: Honoraria, Research Funding; Janssen-Cilag: Honoraria, Research Funding; Celgene: Research Funding; Pfizer: Honoraria; Abbvie: Honoraria.

Obese microenvironments attenuate the efficacy of endogenous and CD19-directed CAR T-cells

Abstract Background: Hematological malignancies represent the largest class of cancers in pediatric patients. Despite successes in treating primary diseases, patients with relapsed B-cell acute lymphoblastic leukemia (B-ALL) and diffuse large B-cell lymphoma (DLBCL) have significantly lower survival outcomes. This has led to the use of immunotherapies, notably chimeric antigen receptor (CAR) T-cells, to treat relapse. Unfortunately, only 50% respond to CAR-T therapy, with relapse common in less than a year. Objective: Given that obesity is associated with poorer survival outcomes in pediatric patients with B-cell malignancies and promotes a loss of immune homeostasis, we hypothesize that obesity compromises the function of endogenous and engineered T-cells in B-cell malignancies. Results: Using in vitro models of adiposity and diet-induced obesity mouse models to study the impact of obesity on primary T-cell function, we observe defects in proliferation, cytokine production, and the upregulation of cytolytic mediators when cultured in adipocyte but not in bone marrow cell or unconditioned media. The introduction of CD19-direct CARs did not overcome adipocyte-induced defects in T-cell function, which resulted in increased PD-1 surface expression, compromised cytokine production, and defective killing of human CD19-expressing B-ALL cells. Interestingly, human B-ALL cells cultured in adipocyte-secreted factors downregulated CD19, suggesting that adipocytes promote B-ALL pathogenesis by compromising T-cell function and reducing the surface expression of antigens on target cells. Conclusion: Our studies are the first to demonstrate the inhibitory impact of the adipocyte secretome on CAR T-cell function in models of B-ALL.

The Immune Checkpoint PD-1 in Natural Killer Cells: Expression, Function and Targeting in Tumour Immunotherapy.

Simple SummaryNatural killer cells are innate cytotoxic lymphocytes that play a key role in the anti-tumor immune response. In the tumor microenvironment, however, the effector functions of these cells are often impaired by the induction of inhibitory surface molecules, including PD-1. In the present review, we provide further insight into the expression and function of the immune checkpoint PD-1 in natural killer cells, together with the limitations and perspectives of immunotherapies aimed at blocking the interaction of this inhibitory receptor with its ligands.In the last years, immunotherapy with antibodies against programmed cell death protein 1 (PD-1) and programmed death-ligand 1 (PD-L1) has shown remarkable efficacy in the treatment of different types of tumours, representing a true revolution in oncology. While its efficacy has initially been attributed only to unleashing T cell responses, responsivity to PD-1/PD-L1 blockade was observed in some tumours with low Human Leukocyte Antigen (HLA) I expression and increasing evidence has revealed PD-1 surface expression and inhibitory function also in natural killer (NK) cells. Thus, the contribution of anti-PD-1/PD-L1 therapy to the recovery of NK cell anti-tumour response has recently been appreciated. Here, we summarize the studies investigating PD-1 expression and function in NK cells, together with the limitations and perspectives of immunotherapies. A better understanding of checkpoint biology is needed to design next-generation therapeutic strategies and to improve the clinical protocols of current therapies.

Large Scale Ex Vivo Expansion of Γδ T Cells Using Artificial Antigen Presenting Cells for the Treatment of Acute Myeloid Leukemia

Patients with relapsed or refractory acute myeloid leukemia (AML) are at increased risk of mortality. Higher γδ T cell count in a bone marrow or peripheral blood of patients with leukemia is associated with better survival. However, γδ T cells are rare in the blood and functionally impaired or exhausted in patients with malignancies. Promising results are reported on the treatment of various malignancies with in vivo expansion of autologous γδ T cells using zoledronic acid (zol) and IL-2. Here we demonstrated that zol and IL-2, in combination with a novel genetically engineered K562 CD3/CD137L/CD28/IL15RA quadruplet artificial antigen presenting cell (aAPC), efficiently expand allogeneic donor-derived γδ T cells using a GMP-compliant protocol sufficient to achieve cell doses for future clinical use. We achieved a 633-fold expansion of γδ T cells after day 10 of co-culture with aAPC, the majority of which exhibited central (47%) and effector (43%) memory phenotypes. Additionally, &amp;gt;90% of the expanded γδ T cells expressed NKG2D, while they have low cell surface expression of PD1 and LAG2 inhibitory checkpoint receptors. In vitro real-time cytotoxicity analysis showed that expanded, previously cryopreserved, γδ T cells were effective in killing target cells. Our results demonstrate that large scale ex vivo expansion of donor-derived γδ T cells can be achieved with the use of CD3/CD137L/CD28/IL15RA quadruplet aAPC and zol/IL-2 for clinical application as promising antineoplastic immunotherapy. Figure 1 Disclosures Lancet: Abbvie: Consultancy; Agios Pharmaceuticals: Consultancy, Honoraria; Astellas Pharma: Consultancy; Celgene: Consultancy, Research Funding; Daiichi Sankyo: Consultancy; ElevateBio Management: Consultancy; Jazz Pharmaceuticals: Consultancy; Pfizer: Consultancy. Sallman:Celgene, Jazz Pharma: Research Funding; Agios, Bristol Myers Squibb, Celyad Oncology, Incyte, Intellia Therapeutics, Kite Pharma, Novartis, Syndax: Consultancy. Bejanyan:Kiadis Pharma: Membership on an entity's Board of Directors or advisory committees.

Abstract 1066: PD-1 expressing ex-vivo cultured tumor infiltrating lymphocytes confers tumor reactivity in cervical cancer: Possible implication for adoptive cell therapy

Abstract Adoptive cell transfer (ACT) of ex-vivo tumor-infiltrating lymphocytes (TILs) has been shown to achieve durable clinical response in cervical cancer (CC). However, objective response is still limited. One of the ways to improve the efficacy of ACT is to enrich tumor reactive TILs for adoptive transfer. In this study, we aim to identify tumor-reactive CD8+ TILs by their ability to recognize antigen presenting cells loaded with pools of human papillomavirus (HPV) E6/E7 derived proteins, HPV E6/E7 overexpressing cells, or autologous tumors. TILs were generated from CC tumor specimens. Tissues were minced into 1-2 mm fragments and cultured in 6000 IU per mL of rhIL-2. Autologous dendritic cells (DCs) were generated from CD14+ cells isolated from patients' peripheral blood mononuclear cells (PBMCs). Autologous fibroblast cells were transfected with the E6/E7 genes. In some cases, autologous tumor cells were generated using monolayer or matrigel based 3D culture system. DCs loaded with pools of peptides derived from HPV16 or HPV18 E6/E7 oncoproteins, E6/E7 transfected autologous fibroblast cells, and autologous tumor cells were cocultured with TILs, and IFN-γ production was measured by enzyme-linked immunosorbent assay (ELISA). MHC class I or II restriction was evaluated using blocking antibodies. Knockdown of HPV gene was performed by using small interfering RNA (siRNA). Tumor-reactive and non-reactive T cells were then analyzed for the surface expression of PD-1, TIM-3, LAG-3, TIGIT, CD39, CD103, and CD69 by flow cytometry. In certain cases, T-cells were sorted using flow cytometry and cocultured with autologous tumor cells. We were able to generate TILs from 76% of the CC patients (n=101). The cultured TILs were reactive to DCs loaded with pools of peptides derived from HPV16 or HPV18 E6/E7 oncoproteins, autologous fibroblast cells transfected with HPV16 E6/E7 gene, and autologous tumor cells. IFN-γ was produced by TILs in either MHC class I or class II restricted manner. Production of IFN-γ was inhibited when HPV16 E6/E7 genes in the stimulator cells were silenced by siRNA. We found that only expression of PD-1 among tested molecules was significantly higher on tumor-reactive TILs than non-reactive TILs. As expected sorted PD-1 positive T cells produced significantly higher IFN-γ compared to PD-1 negative T cells. These data indicate that tumor-reactive CD8+ T-cells were enriched in the PD-1 positive T cells. Use of PD-1 positive tumor-reactive TILs may improve the efficacy of ACT for patients with CC compared with bulk culture TILs. Citation Format: Mohammad A. Sayem, Nuchsupha Sunthamala, Tomonori Yaguchi, Takashi Iwata, Tomonobu Fujita, Yutaka Kawakami. PD-1 expressing ex-vivo cultured tumor infiltrating lymphocytes confers tumor reactivity in cervical cancer: Possible implication for adoptive cell therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1066.