Expression Of Co-stimulatory Receptors Research Articles

Pharmacologic HIF stabilization activates costimulatory receptor expression to increase antitumor efficacy of adoptive T cell therapy.

Adoptive cell transfer (ACT) is a therapeutic strategy to augment antitumor immunity. Here, we report that ex vivo treatment of mouse CD8+ T cells with dimethyloxalylglycine (DMOG), a stabilizer of hypoxia-inducible factors (HIFs), induced HIF binding to the genes encoding the costimulatory receptors CD81, GITR, OX40, and 4-1BB, leading to increased expression. DMOG treatment increased T cell killing of melanoma cells, which was further augmented by agonist antibodies targeting each costimulatory receptor. In tumor-bearing mice, ACT using T cells treated ex vivo with DMOG and agonist antibodies resulted in decreased tumor growth compared to ACT using control T cells and increased intratumoral markers of CD8+ T cells (CD7, CD8A, and CD8B1), natural killer cells (NCR1 and KLRK1), and cytolytic activity (perforin-1 and tumor necrosis factor-α). Costimulatory receptor gene expression was also induced when CD8+ T cells were treated with three highly selective HIF stabilizers that are currently in clinical use.

Uncovering the Expression Pattern of the Costimulatory Receptors ICOS, 4-1BB, and OX-40 in Exhausted Peripheral and Tumor-Infiltrating Natural Killer Cells from Patients with Cervical Cancer.

Cervical cancer (CC) poses a significant health burden, particularly in low- and middle-income countries. NK cells play a crucial role against CC; however, they can become exhausted and lose their cytotoxic capacity. This work explores the expression of costimulatory receptors (ICOS, 4-1BB, OX-40) in exhausted NK cells from CC patients. Peripheral blood and tumor biopsies were collected, and flow cytometry was used to evaluate the expression of costimulatory receptors in exhausted NK cells. There is an increase of peripheral exhausted NK cells (PD-1+TIGIT+) in CC patients; this subpopulation has a selectively increased expression of the costimulatory receptors ICOS and 4-1BB. An exhausted population is also highly increased in tumor-infiltrating NK cells, and it shows a dramatically increased expression of the costimulatory receptors ICOS (>15×) and 4-1BB (>10×) compared to peripheral NK cells. The exhausted cells, both in the periphery and in the tumor infiltrating lymphocytes (TILs), are also more likely than non-exhausted NK cell populations (PD-1-TIGIT-) to express these costimulatory receptors; increases ranging from 2.0× ICOS, 2.4× 4-1BB, and 2.6× OX-40 in CD56dim PBMCs to 1.5× ICOS, 5× 4-1BB, and 10× OX-40 in TILs were found. Our study demonstrates for the first time the increased expression of the costimulatory receptors ICOS, 4-1BB, and OX-40 in peripheral CD56dim, CD56bright, and tumor-infiltrating NK cells in CC. Targeting these receptors for stimulation could reverse exhaustion and be a promising immunotherapy strategy.

Immunomodulatory alpha neutrophils: The first-in-class neutrophil progenitor-based allogeneic immuno-cell therapy with cytotoxic and immunomodulatory functionality for the treatment of solid tumours.

e14523 Background: Adoptive cell therapy for the treatment of cancer has largely focused on the administration of autologous engineered αβ T cells. Although successful in some haematological malignancies, the solid tumour microenvironment (TME) poses significant challenges to the efficacy of CAR-T cells, including physical barriers, heterogeneous antigen expression or antigen escape and immunosuppressive mediators. Remodeling the immunosuppressive solid TME is pivotal to the success of immunotherapies. Recent studies have highlighted key roles for neutrophils in tumour control and in facilitating successful responses to cancer immunotherapy, with neutrophil functional heterogeneity within the TME becoming increasingly appreciated. Methods: At LIfT Biosciences we have developed the first-in-class, stem cell-derived immuno-cell therapy with anti-cancer neutrophil properties called Immunomodulatory Alpha Neutrophils (IMANs). IMANs are derived from CD34+ stem cells expanded and differentiated in proprietary media compositions in a simple and scalable GMP-compliant manufacturing process. IMANs are designed to recapitulate those neutrophil states that are both cytotoxic and capable of immunomodulating the solid TME for long-lasting tumour control. Off-the-shelf, allogeneic, innate IMANs have the potential to provide major clinical benefits in the treatment of multiple solid tumours. Results: IMANs multimodal mechanisms of action (MoA) have been demonstrated in different state-of-the-art solid tumour models, including co-culture with patient-derived xenograft organoids (PDX-O) and with 3D ex vivo patient tissues (tumour-on-a-chip). Direct antigen-independent cytotoxicity towards PDX-O from a range of solid tumour indications has been demonstrated. Additionally, and fundamental to IMANs MoA is their ability to migrate to the solid TME and recruit and activate recipients’ anti-tumour immune responses. We have demonstrated IMANs ability to rapidly migrate into the solid TME and promote recruitment of patients’ immune cells leading to enhanced tumour cell death using tumour-on-a-chip technology accompanied by daily live cell imaging. Mechanistically, co-culture with IMANs enhanced expression of co-stimulatory receptors on T and NK cells, such as OX40 and 4-1BB, as well as promoting secretion of TME-modulating factors, such as CXCL10. Furthermore, co-culture with IMANs enhanced secretion of IFN-γ by activated tumour-infiltrating lymphocytes. Conclusions: These cytotoxic and immunomodulatory functions enable IMANs to remodel the TME, leaving the solid tumour more susceptible to host immune responses.

Novel genetically glycoengineered human dendritic cell model reveals regulatory roles of α2,6-linked sialic acids in DC activation of CD4+ T cells and response to TNFα.

Dendritic cells (DCs) are central for the initiation and regulation of appropriate immune responses. While several studies suggest important regulatory roles of sialoglycans in DC biology, our understanding is still inadequate primarily due to a lack of appropriate models. Previous approaches based on enzymatic- or metabolic-glycoengineering and primary cell isolation from genetically modified mice have limitations related to specificity, stability, and species differences. This study addresses these challenges by introducing a workflow to genetically glycoengineer the human DC precursor cell line MUTZ-3, described to differentiate and maturate into fully functional dendritic cells, using CRISPR-Cas9, thereby providing and validating the first isogenic cell model for investigating glycan alteration on human DC differentiation, maturation, and activity. By knocking out (KO) the ST6GAL1 gene, we generated isogenic cells devoid of ST6GAL1-mediated α(2,6)-linked sialylation, allowing for a comprehensive investigation into its impact on DC function. Glycan profiling using lectin binding assay and functional studies revealed that ST6GAL1 KO increased the expression of important antigen presenting and co-stimulatory surface receptors and a specifically increased activation of allogenic human CD4 + T cells. Additionally, ST6GAL1 KO induces significant changes in surface marker expression and cytokine response to TNFα-induced maturation, and it affects migration and the endocytic capacity. These results indicate that genetic glycoengineering of the isogenic MUTZ-3 cellular model offers a valuable tool to study how specific glycan structures influence human DC biology, contributing to our understanding of glycoimmunology.

A Scalable, Off-the-Shelf, and Cost-Effective NK Cell Manufacturing Platform for Developing NK Cell-Based Immunotherapy

Natural killer (NK) cells are innate lymphocytes that play a key role in the control of infection and malignancy. NK cells are potent killers of target cells and act in an MHC-independent manner, enabling their application as allogenic therapy for cancer. Human iPSCs are pluripotent with the ability to self-renew, thus providing an inexhaustible source for deriving NK cells. With a two-tiered cell bank system, we can manufacture NK cells with more homogeneity between batches. In principle, one single iPSC clone-derived master cell bank and working cell banks can support the life cycle of an affordable, off-the-shelf NK cell product. We developed a robust stepwise protocol for large-scale generation of high-purity NK cells from human iPSCs without the cell isolation procedure. First, human iPSCs formed embryoid bodies and then were differentiated into highly purified (>98.0%) CD34 + CD43 + hematopoietic stem and progenitor cells (HSPCs) using a combination of cytokines and small molecles such as BMP-4, VEGF, BFGF, SCF, FLT-3 and ascorbic acid. During this period, we observed up to an 180-fold cell increase in the number of HSPCs compared with human iPSCs. Second, these HSPCs were differentiated into NK cells with FLT3, SCF, IL-3, IL-7, and IL-15 etc., with cell number increased approximately 9000-fold compared with iPSCs. In the final step, iPSC-derived NK cells were further expanded up to 8000-fold which enable us to produce 1E12 NK cells from less than 1 million iPSCs. After expansion, iNK cells became more homogenous and exhibited a more mature phenotype. More than 99.0% of cells expressed both CD45 and CD56, and more than 70% expressed CD16. The expression of activating and co-stimulatory receptors (NKG2D, NKp46,NKp44, NKp30, CD244 and CD226) of NK cells was also high in these iNK cells. Intrestingly, the cells exhibited robust anti-tumor efficacy against K562 (chronic myelogenous leukemia), RPMI 8226 (multiple myeloma). Furthermore, the cryopreserved iNK cells demonstrated dramatic cytotoxicity to U-87 MG cells (malignant gliomas) in 2D culture and the tumor organoid, indicating that iNK cells derived from our protocol can be used as off-the-shelf therapy for both hematological and solid tumors. We also observed that these iNK cells had variable cytotoxicity in other tumors. For example, our iNK were not obviously cytotoxic to the NCI-H929 cell line, derived from a plasmacytoma myeloma patient and expressing BCMA at a high level. To improve the specific cytotoxicity of NK cells to NCI-H929 cells, we transduced a anti-BCMA CAR to the donor iPSC. Following differentiation, the CAR-transduced iNK cells showed approximately 6-fold cytotoxicity compared with WT iNK. (Figure 1) Collectively, our serum-free and strom cell-free NK cell generation produre from human iPSCs provides a scalable, consistent, off-the-shelf and cost-effective NK/CAR-NK cell manufacturing platform for clinical application and NK cell-based immunotherapies.

Abstract 6378: An in vitro human CD8 T cell exhaustion model for the functional screening of immune checkpoint inhibitors

Abstract T cell exhaustion is a state of dysfunction that frequently occurs during chronic infections and cancer due to persistent antigen stimulation and inflammation. It is defined by poor effector function, sustained expression of inhibitory receptors, and a transcriptional state distinct from that of functional effector or memory T cells. In this state, cells remain in the local environment but lack effector functions necessary to combat the tumor or infection. In the current study, we developed an in vitro human antigen-specific CD8 T cell exhaustion model to evaluate the ability of immune checkpoint inhibitors to restore functionality to exhausted T cells. Using the fully human, autologous MIMIC (Modular IMmune In vitro Construct) effector CD8 T cell assay as a framework, we assessed the effect of antigen dose, timing, and frequency of repeated antigen stimulation with antiviral HLA Class I-restricted peptides to drive the development of Ag-specific CD8 T cells toward an exhausted state. Epitope-specific CD8 T cell responses were monitored by HLA class I-restricted pentamers and establishment of an exhausted profile was evaluated by increased expression of inhibitory co-stimulatory receptors and decreased intracellular expression of antiviral effector cytokine via flow cytometry relative to functional Ag-specific CD8 T cells from the same donor. Repeated antigen exposure led to decreased production of IFN-γ and TNF-α and increased expression of inhibitory co-stimulation markers on antigen-specific CD8 T cells. After establishment of an exhausted phenotype, treatment with various immune checkpoint inhibitor molecules restored functionality to Ag-specific CD8 T cells upon an additional antigen re-stimulation. Moreover, here using this model we characterized distinct transcriptomic signatures of functional and exhausted MIMIC CD8 T cells. Development of an in vitro human viral antigen-specific CD8 T cell exhaustion model with characteristic features of dysfunctional CD8 T cells as observed in vivo and the ability to restore effector function by treatment with immune checkpoint inhibitors, provides a platform to assess the effectiveness of therapeutic mono-specific or bi-specific “checkpoint blockade” antibody candidates and, in general, the potential to evaluate a myriad of immunotherapeutic approaches for cancer treatment. Citation Format: Roberto Carrio, Margot Cucchetti, Mikielia Devonish, Louis Poisson, Mihaela Babiceanu, Andrew Kettring, Yingchun Liu, Donald Jackson, Evan Gomes, Jeremy Baudhuin, Donald R. Drake, Valeria Fantin, Anthony Byers, Ingrid Sassoon. An in vitro human CD8 T cell exhaustion model for the functional screening of immune checkpoint inhibitors [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 6378.

CuMV VLPs Containing the RBM from SARS-CoV-2 Spike Protein Drive Dendritic Cell Activation and Th1 Polarization.

Dendritic cells (DCs) are the most specialized and proficient antigen-presenting cells. They bridge innate and adaptive immunity and display a powerful capacity to prime antigen-specific T cells. The interaction of DCs with the receptor-binding domain of the spike (S) protein from the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a pivotal step to induce effective immunity against the S protein-based vaccination protocols, as well as the SARS-CoV-2 virus. Herein, we describe the cellular and molecular events triggered by virus-like particles (VLPs) containing the receptor-binding motif from the SARS-CoV-2 spike protein in human monocyte-derived dendritic cells, or, as controls, in the presence of the Toll-like receptors (TLR)3 and TLR7/8 agonists, comprehending the events of dendritic cell maturation and their crosstalk with T cells. The results demonstrated that VLPs boosted the expression of major histocompatibility complex molecules and co-stimulatory receptors of DCs, indicating their maturation. Furthermore, DCs' interaction with VLPs promoted the activation of the NF-kB pathway, a very important intracellular signalling pathway responsible for triggering the expression and secretion of proinflammatory cytokines. Additionally, co-culture of DCs with T cells triggered CD4+ (mainly CD4+Tbet+) and CD8+ T cell proliferation. Our results suggested that VLPs increase cellular immunity, involving DC maturation and T cell polarization towards a type 1 T cells profile. By providing deeper insight into the mechanisms of activation and regulation of the immune system by DCs, these findings will enable the design of effective vaccines against SARS-CoV-2.

High Dimensional Mapping of Bone Marrow Immune Characteristics in Immune Thrombocytopenia

Introduction Immune thrombocytopenia (ITP) is an acquired autoimmune disease that is characterized by increased antibody-mediated platelet destruction, decreased megakaryopoiesis and impaired thrombopoiesis. An abnormal bone marrow microenvironment has been proposed to play an essential role in the progression of ITP [Stem Cells Dev, 2017]. Cytometry by Time of Flight (CyTOF) is a new analytic strategy that provides single-cell proteomics for enhanced cellular analysis, and describes the components of immune cell populations and the characteristics of new biomarkers. Here, we aim to provide a high-dimensional overview and summary of the changes in cell subsets and functions in the bone marrow of patients with ITP using CyTOF. Methods A total of 40 patients diagnosed with ITP (21 men and 19 women; 23-71 years of age, median 47 years) were enrolled between July and October 2021. Based on the onset and progression of the disease, the ITP patients were classified into 3 subgroups. Group ITP-N1 included 15 newly diagnosed treatment-naïve patients ≤3 months after diagnosis, Group ITP-N2 included 10 patients with an ITP diagnosis of > 3 months who had not received any standard treatment (>3-12 months after diagnosis), and Group ITP-R included 15 patients who had received standard treatment but relapsed following remission. The healthy control (HC) group consisted of 10 donors who underwent hematopoietic stem cell transplantation. CyTOF panels were designed using 42 parameters that were measured at the single-cell level in the bone marrow of the ITP patients and HCs. The plasma levels of immune checkpoint regulators, such as PD-1, PD-L1, CTLA-4, TIM-3, LAG-3 and Galectin-9, were measured using a multianalyte flow assay kit (BioLegend). Results The platelet counts of enrolled ITP patients ranged from 1 to 96×109/L, with a median count of 25×109/L. Using CyTOF, the immune cells in the bone marrow were classified into 7 major subsets, including B cells, γδT cells, CD4+ T cells, CD8+ T cells, natural killer (NK) cells, dendritic cells (DCs), and monocytes. The proportions of DCs and monocytes were significantly different between the ITP patients and HCs. The proportion of monocytes was observed to increase significantly in the bone marrow of the ITP patients. The proportion of the pDC subset of DCs showed a trend of being reduced in the ITP patients, while the proportion of mDCs was increased significantly in the ITP patients. NK cells can be divided into 2 subsets according to the expression of CD56, and these subsets exhibit different cytokine production patterns and cytotoxic functions. The proportion of CD56low NK cells was elevated and the proportion of CD56bright NK cells decreased significantly in ITP-R patients compared to HCs; similar results were not observed in the ITP-N1 or ITP-N2 groups. NK cells in bone marrow may play an important role in the relapse of ITP. We further investigated the functional status of T cells in the ITP patients using a CyTOF panel. The results showed that the proportions of both CD4+ naïve and CD8+ naïve T cells were decreased in the bone marrow of the ITP patients. In particular, the proportion of CD8+ effector T cells was increased significantly in the ITP patients. Next, we analyzed coinhibitory and costimulatory receptor expression in each T-cell subpopulation. CTLA4 expression was observed to be reduced in CD8+ effector T cells and CD4+ Tfh cells. In addition to the changes in coinhibitory receptor expression, costimulatory molecules such as CD28 and ICOS were found to be expressed at higher levels in CD4+ central memory T cells. Combined with the soluble regulators in plasma, we found that the B7.2 levels in plasma was positively correlated with the cell surface expression of CD28 and negatively correlated with the expression of CTLA4 in T cells. Conclusions This study provides a comprehensive overview of the human bone marrow immune characteristics of patients with ITP. Using CyTOF to conduct single-cell analyses, we mapped myeloid and T-cell subset phenotypes in particular and focused on the expression of coinhibitory molecules such as CTLA4. The different proportions of bone marrow immune cells and patterns of immune checkpoint molecule expression in different stages of ITP may provide novel insights into disease etiology and offer promising therapeutic targets for patients with ITP. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Single-Cell Transcriptomics Reveals Killing Mechanisms of Antitumor Cytotoxic CD4+ TCR-T Cells.

T cell receptor-engineered T cells (TCR-Ts) have emerged as potent cancer immunotherapies. While most research focused on classical cytotoxic CD8+ T cells, the application of CD4+ T cells in adoptive T cell therapy has gained much interest recently. However, the cytotoxic mechanisms of CD4+ TCR-Ts have not been fully revealed. In this study, we obtained an MHC class I-restricted MART-127-35-specific TCR sequence based on the single-cell V(D)J sequencing technology, and constructed MART-127-35-specific CD4+ TCR-Ts and CD8+ TCR-Ts. The antitumor effects of CD4+ TCR-Ts were comparable to those of CD8+ TCR-Ts in vitro and in vivo. To delineate the killing mechanisms of cytotoxic CD4+ TCR-Ts, we performed single-cell RNA sequencing and found that classical granule-dependent and independent cytolytic pathways were commonly used in CD4+ and CD8+ TCR-Ts, while high expression of LTA and various costimulatory receptors were unique features for cytotoxic CD4+ TCR-Ts. Further signaling pathway analysis revealed that transcription factors Runx3 and Blimp1/Tbx21 were crucial for the development and killing function of cytotoxic CD4+ T cells. Taken together, we report the antitumor effects and multifaceted killing mechanisms of CD4+ TCR-Ts, and also indicate that MHC class I-restricted CD4+ TCR-Ts could serve as potential adoptive T cell therapies.

Lurbinectedin shows clinical activity and immune-modulatory functions in patients with pre-treated small cell lung cancer and malignant pleural mesothelioma

PurposeLurbinectedin is a promising new drug being investigated in pre-treated patients with small cell lung cancer (SCLC) or malignant pleural mesothelioma (MPM). Its clinical activity in the real-world setting has not been investigated yet. Patients and methodsClinical data of patients with SCLC and MPM who were treated with lurbinectedin were prospectively collected. Comprehensive immune cell profiling by flow cytometry was performed on screening and treating peripheral blood samples. ResultsA total of 95 patients (43 SCLC and 52 MPM) were treated, mostly as ≥3-line of therapy. In the SCLC cohort, a median progression-free survival (mPFS) was 1.5 months (95% CI: 1.4–3.0), and median overall survival was 7.0 months (95% CI: 4.7–not reached). Objective radiological response and disease control rate after 12 weeks were 16% and 28%, respectively. In the MPM cohort, median progression-free survival was 2.8 months (95% CI: 1.4–4.2), and median overall survival was 7.2 months (95% CI: 5.9–not reached). Disease control rate after 12 weeks was 29%, whereas no partial responses were registered. No new safety signals were observed. Lurbinectedin treatment was significantly associated with the depletion of circulating classical monocytes, which correlated with a better PFS in patients with SCLC. Lurbinectedin increased the proliferation of CD4+ and CD8+ T cells (SCLC) and natural killer and natural killer T cells (SCLC and MPM) and altered co-stimulatory and co-inhibitory receptor expression on circulating lymphocytes. ConclusionLurbinectedin has a manageable safety profile and shows clinical activity in pre-treated patients with SCLC and MPM. Its immune-modulatory functions make lurbinectedin a potential platform for immunotherapy combinations.

Abstract 4228: Inhibition of B7-H3 by enoblituzumab elicits antitumor immune modulation in both innate and adaptive immunity

Abstract Introduction: B7-H3, a B7 family member, is widely expressed in multiple cancers and is associated with poor clinical prognosis. Enoblituzumab (also known as MGA271 or TJ271) is an investigational, Fc-optimized monoclonal antibody that targets B7-H3 and exhibits enhanced antibody-dependent cell-mediated killing of B7-H3 expressing cancer cells. Here we identified the immuno-modulatory function of TJ271 in different cell subsets and pathways as a new mechanism for cancer treatment. Experiments: The function of B7-H3 and TJ271 in regulating immune response was investigated in human PBMCs stimulated with anti-CD3 antibody with or without plate-coated B7-H3 in the presence or absence of TJ271. Gene expression profiles and immunophenotyping of PBMCs after treatments were evaluated by Nanostring nCounter platform and CyTOF mass cytometry, respectively. IFN-γ production from cell supernatant was measured by ELISA. In vitro tumor killing activity by TJ271 and urelumab, a 4-1BB agonist, was evaluated by co-culture of B7-H3 expressing tumor cells and PBMCs stimulated with anti-CD3. The tumor cell killing activity was evaluated by 7-AAD staining on the Incucyte platform. Results: Gene expression of cell lineage markers in antigen-presenting cells, including macrophages and neutrophils was induced, while those in natural killer (NK) cells was suppressed by B7-H3 stimulation, respectively. Furthermore, expression of activation markers and co-stimulatory receptors, including IFNγ, ULBP2, GITR, and 4-1BB, was down-regulated while expression of exhaustion markers in CD8+ T cells and NK cells, including PD-L1, PD-L2, and IDO1, and of genes associated with M2-like macrophages were up-regulated by B7-H3. All this B7-H3-induced differential gene expression pattern was reversed by TJ271 treatment. As revealed by CyTOF, TJ271 treatment abolished B7-H3-mediated increase in the frequency of M2-like monocytes (B7-H3+CD68+CD206+CD16lo or B7-H3-CD68+CD163+CD16+) and led to increased frequency of 4-1BB-expressing CD4, CD8, and NK cells. TJ271 exhibited strong tumor killing activity against ES-2, a B7-H3-expressing ovarian cancer cell line in vitro. Consistent with the TJ271-associated increased 4-1BB expression in T and NK cells, activation of 4-1BB by urelumab further enhanced TJ271-mediated tumor killing activity. Conclusion: Our data confirm the immunosuppressive function of B7H3 and demonstrate the immunoregulatory function of TJ271 as evidenced by the activation of cytolytic T cell and NK cells, reinvigoration of exhausted cells, and suppression of M2-like myeloid cells. These findings provide rationale for combination therapy with blockade of B7-H3 by TJ271 with other immunotherapies to achieve increased clinical efficacy against cancers. Citation Format: Yanni Zhang, Xuejun Liu, Xinyun Zhu, Zhen Sheng, Yan Liu, Zhengyi Wang, Taylor B. Guo. Inhibition of B7-H3 by enoblituzumab elicits antitumor immune modulation in both innate and adaptive immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4228.

Inhibitors of the Actin-Bundling Protein Fascin-1 Developed for Tumor Therapy Attenuate the T-Cell Stimulatory Properties of Dendritic Cells.

Simple SummaryExpression of the actin-bundling protein Fascin-1 (Fscn1) is largely restricted to neuronal cells and to activated dendritic cells (DCs). DCs are important inducers of (antitumor) immune responses. In tumor cells, de novo expression of Fscn-1 correlates with their invasive and metastatic activities. Pharmacological Fscn1 inhibitors, which are currently under clinical trials for tumor therapy, were demonstrated to counteract tumor metastasis. Within this study, we were interested in better understanding the effects of Fscn1 inhibitors on DCs and discovered that two distinct Fascin-1 inhibitors affect the immune-phenotype and T-cell stimulatory activity of DCs. Our results suggest that systemic application of Fscn1 inhibitors for tumor therapy may also modulate antitumor immune responses.Background: Stimulated dendritic cells (DCs), which constitute the most potent population of antigen-presenting cells (APCs), express the actin-bundling protein Fascin-1 (Fscn1). In tumor cells, de novo expression of Fscn1 correlates with their invasive and metastatic properties. Therefore, Fscn1 inhibitors have been developed to serve as antitumor agents. In this study, we were interested in better understanding the impact of Fscn1 inhibitors on DCs. Methods: In parallel settings, murine spleen cells and bone-marrow-derived DCs (BMDCs) were stimulated with lipopolysaccharide in the presence of Fscn1 inhibitors (NP-G2-044 and BDP-13176). An analysis of surface expression of costimulatory and coinhibitory receptors, as well as cytokine production, was performed by flow cytometry. Cytoskeletal alterations were assessed by confocal microscopy. The effects on the interactions of BMDCs with antigen-specific T cells were monitored by time lapse microscopy. The T-cell stimulatory and polarizing capacity of BMDCs were measured in proliferation assays and cytokine studies. Results: Administration of Fscn1 inhibitors diminished Fscn1 expression and the formation of dendritic processes by stimulated BMDCs and elevated CD273 (PD-L2) expression. Fscn1 inhibition attenuated the interaction of DCs with antigen-specific T cells and concomitant T-cell proliferation. Conclusions: Systemic administration of Fscn1 inhibitors for tumor therapy may also modulate DC-induced antitumor immune responses.

Diversity of Intratumoral Regulatory T Cells in Non-Hodgkin Lymphoma

Introduction: Regulatory T cells (Tregs), a highly immunosuppressive subset of CD4 + T cells, represent a key challenge in the tumor microenvironment by limiting potent antitumor immune responses. While high densities of tumor-infiltrating Tregs are associated with poor prognosis in patients with various types of solid cancers, their prognostic impact in B-cell non-Hodgkin lymphoma (NHL) remains unclear. Emerging studies suggest substantial heterogeneity in the phenotype and suppressive capacities of Tregs, emphasizing the importance of understanding Treg diversity and the need for additional markers to identify highly suppressive Tregs. Our in-depth characterization of Tregs in NHL tumors could open new paths for rational drug design, facilitating selective therapeutic manipulation of Tregs to reduce immunosuppression and improve anti-tumor immunity.Methods: Single-cell suspensions from NHL patients (diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL) and healthy donors (tonsils and peripheral blood)) were analyzed by fluorescent flow- and mass cytometry to characterize Tregs, focusing on their expression of co-stimulatory and co-inhibitory checkpoint receptors. The immunosuppressive capacity of Tregs was measured by in vitro co-culture of FACS-sorted subsets of Tregs together with autologous CellTrace Violet-labelled T effector cells as responder cells, using samples from FL and tonsils. Live CD4 + T cells were obtained by FACS sorting from DLBCL (n = 3), FL (n = 3) and healthy donor tonsils (n = 3) and subjected to single-cell RNA sequencing (scRNA-seq), Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) and scTCR-seq by the 10X Genomics platform. The computational framework of CIBERSORTx was used to generate unique signature matrices for the three Treg subsets identified by scRNA-seq, to facilitate validation in separate scRNA-seq cohorts (King, Sci Immunol 2021; Roider, Nat Cell Biol 2020), and to impute frequencies of the Treg subsets in cohorts with bulk RNA-seq data (Chapuy, Nat Med 2018; Schmitz, NEJM 2018; Pastore, Lancet Oncol 2015).Results: Immunophenotyping by mass cytometry revealed a subset of activated Tregs identified by co-expression of TIGIT, CTLA-4, PD-1, ICOS and OX40, and higher expression of FOXP3, CD25 and CD45RO, that was present in DLBCL and tonsils, but lacking in peripheral blood. This was validated by fluorescent flow cytometry, demonstrating significantly higher frequencies of activated Tregs in NHL tumors compared to PBMCs and tonsils from healthy donors. The phenotypic heterogeneity of intratumoral Tregs reflected different suppressive capacities as activated Tregs more potently suppressed the proliferation of autologous effector CD4 + and CD8 + T cells than naïve Tregs. For global transcriptomic profiling of CD4 + T cells from FL, DLBCL and tonsillar samples, we integrating scRNA-seq and CITE-seq data from 17,774 cells, revealing 13 distinct cellular states including three states of Tregs: naïve, activated and non-conventional LAG3 +FOXP3 - Tregs. Activated Tregs had higher expression of checkpoint receptors (TNFRSF4, TNFRSF18, ICOS), phosphatases (DUSP2, DUSP4), NF-κB pathway (NFKBIA, TNFAIP3, NFKBIZ, REL), chemokine receptors (CXCR4) and transcription factors (JUNB, IRF1, STAT3) as compared to naïve Tregs. We next used a computational approach to develop unique signature matrices for each Treg subset. This approach demonstrated strong concordance between CIBERSORTx estimated cell abundances of the three Treg subsets and the ground truth, and was validated in two external scRNA-seq cohorts. The development of unique signature matrices for Treg subsets facilitated imputation of their frequencies in bulk RNA-seq datasets. These analyses revealed that higher frequency of activated Tregs was enriched in the germinal B cell subtype of DLBCL and was associated with adverse outcome in FL.Conclusion: This study demonstrates that Tregs infiltrating NHL tumors are transcriptionally and functionally diverse and include highly immunosuppressive activated Tregs co-expressing several checkpoint receptors, which distinguish them from peripheral blood Tregs. Activated intratumoral Tregs could hamper clinical responses to checkpoint blockade, and identifying and targeting their vulnerabilities has the potential to improve anti-tumor immune responses. DisclosuresHolte: Gilead: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Nordic: Membership on an entity's Board of Directors or advisory committees; Nanovector: Membership on an entity's Board of Directors or advisory committees, Other: lectures honorarias; Novartis: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees. Alizadeh: Cibermed: Consultancy, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company; CAPP Medical: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company; Forty Seven: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company; Foresight Diagnostics: Consultancy, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company; Roche: Consultancy, Honoraria; Janssen Oncology: Honoraria; Celgene: Consultancy, Research Funding; Gilead: Consultancy; Bristol Myers Squibb: Research Funding.

Phosphoinositide 3-Kinase p110 Delta Differentially Restrains and Directs Naïve Versus Effector CD8+T Cell Transcriptional Programs.

Phosphoinositide 3-kinase p110 delta (PI3K p110δ) is pivotal for CD8+ T cell immune responses. The current study explores PI3K p110δ induction and repression of antigen receptor and cytokine regulated programs to inform how PI3K p110δ directs CD8+ T cell fate. The studies force a revision of the concept that PI3K p110δ controls metabolic pathways in T cells and reveal major differences in PI3K p110δ regulated transcriptional programs between naïve and effector cytotoxic T cells (CTL). These differences include differential control of the expression of cytolytic effector molecules and costimulatory receptors. Key insights from the work include that PI3K p110δ signalling pathways repress expression of the critical inhibitory receptors CTLA4 and SLAMF6 in CTL. Moreover, in both naïve and effector T cells the dominant role for PI3K p110δ is to restrain the production of the chemokines that orchestrate communication between adaptive and innate immune cells. The study provides a comprehensive resource for understanding how PI3K p110δ uses multiple processes mediated by Protein Kinase B/AKT, FOXO1 dependent and independent mechanisms and mitogen-activated protein kinases (MAPK) to direct CD8+ T cell fate.

165-OR: Antioxidant-Based Encapsulation Strategies to Enhance Islet Allotransplantation

The destruction of insulin-producing cells is a hallmark of type 1 diabetes (T1D) and results in a loss of glycemic control. Islet transplantation can restore glucose homeostasis and prevent hypoglycemic events; however, long-term graft survival is low. Oxidative stress mediates islet transplant rejection. Therefore, suppressing oxidative stress with nanothin encapsulation materials comprised of alternating layers of tannic acid (TA), an immunomodulatory antioxidant, and poly(N-vinylpyrrolidone) (PVPON), a scaffold polymer, may allow for improved graft survival. We hypothesize that (PVPON/TA)-encapsulation of islets will prolong graft function and delay allotransplantation rejection in the absence of systemic immunosuppression. We recently published that allogeneic transplantation of encapsulated C57BL/6 islets into NOD mice significantly delayed graft failure, decreased free radical-adduct formation, and reduced infiltrating immune cell effector responses. These data suggest local immunosuppression with (PVPON/TA)-encapsulation may be a promising technique to prolong islet graft function in islet allotransplants. To enhance immunosuppression, we conjugated CTLA-4-Ig to the outer surface of (PVPON/TA) layers. (PVPON/TA/CTLA-4)-encapsulated islets display significantly delayed allograft rejection compared to (PVPON/TA) controls. Flow cytometry analysis demonstrated reduced CD80/CD86 costimulatory receptor and MHC-II expression on innate immune populations, enhanced FOXP3+ regulatory T cells, and enhanced PD1+ CD8 T cells within (PVPON/TA/CTLA-4)-encapsulated grafts. To monitor graft integrity in vivo, we conjugated (PVPON/TA) layers with the fluorophore Cy5.5. Transplantation of (PVPON/TA/Cy5.5)-encapsulated islets restored glucose tolerance and was detected in vivo for up to eight weeks post-transplant. The ability to modify our (PVPON/TA) coatings opens the door for endless possibilities to improve clinical islet transplantation. Disclosure J. Barra: None. V. Kozlovskaya: None. E. Kharlampieva: None. H. M. Tse: None. Funding National Science Foundation (1608728); National Institute of Diabetes and Digestive and Kidney Diseases (DK-099550); JDRF (SRA-2016-270-S-B, 2-SRA-2019-692-S-B)

Harnessing the IL-21-BATF Pathway in the CD8+ T Cell Anti-Tumor Response.

Simple SummaryIn cancer, CD8+ T cells enter a state of dysfunction within the tumor that prevents them from targeting and killing tumor cells. Our study aims to uncover how CD8+ T cells can be helped by CD4+ T cells or modified in order to improve their effector function against cancer. Thus, allowing them to better fight and control tumors. Our work shows that the protein Basic Leucine Zipper ATF-Like Transcription Factor (BATF) may be a key regulator of CD8+ T cells and their anti-tumor function. These findings can provide further insight for the development of novel therapeutic treatments for cancer patients.In cancer, CD8+ T cells enter a dysfunctional state which prevents them from effectively targeting and killing tumor cells. Tumor-infiltrating CD8+ T cells consist of a heterogeneous population of memory-like progenitor, effector, and terminally exhausted cells that exhibit differing functional and self-renewal capacities. Our recently published work has shown that interleukin (IL)-21-producing CD4+ T cells help to generate effector CD8+ T cells within the tumor, which results in enhanced tumor control. However, the molecular mechanisms by which CD4+ helper T cells regulate the differentiation of effector CD8+ T cells are not well understood. In this study, we found that Basic Leucine Zipper ATF-Like Transcription Factor (BATF), a transcription factor downstream of IL-21 signaling, is critical to maintain CD8+ T cell effector function within the tumor. Using mixed bone marrow chimeras, we demonstrated that CD8+ T cell-specific deletion of BATF resulted in impaired tumor control. In contrast, overexpressing BATF in CD8+ T cells enhanced effector function and resulted in improved tumor control, bypassing the need for CD4+ helper T cells. Transcriptomic analyses revealed that BATF-overexpressing CD8+ T cells had increased expression of costimulatory receptors, effector molecules, and transcriptional regulators, which may contribute to their enhanced activation and effector function. Taken together, our study unravels a previously unappreciated CD4+ T cell-derived IL-21–BATF axis that could provide therapeutic insights to enhance effector CD8+ T cell function to fight cancer.

Study of immune regulatory receptor expression upon combination of chemotherapy and immune checkpoint blockade in a murine lung cancer model.

e21083 Background: Blockade of inhibitory immune checkpoints namely the programmed cell death protein 1 (PD-1) and the PD-1 ligand (PD-L1) achieved remarkable clinical outcomes in multiple malignant diseases including lung cancer. However, as the dynamic interactions between the cancer cells and the tumor-associated microenvironment evolves, acquired resistance arises. Thus exploration on alternative strategies would be necessary in order to reach long-term tumor suppression. Methods: Murine lung cancer model was established by inoculation of Lewis lung carcinoma cells subcutaneously on the flank of C57BL/6J mice. Chemotherapy (cisplatin or pemetrexed) or anti-PD-1 alone, or in combination started at 7 days post-inoculation as tumor growth was established. Digital caliper was used to measure tumor sizes and survival rates were recorded. Mice were sacrificed upon humane endpoint (tumor size > 17 mm in diameter). Splenocytes and tumor cells were harvested and expression of immune regulatory markers (including PD-L1, PD-1, Tim-3, OX-40, GITR, LAG-3 and CTLA-4) was analyzed by flow cytometry. Results: While PD-1 blockade delayed tumor growth ( p< 0.001), there was no significant difference in overall survival. Systemically, anti-PD-1 immunotherapy induced upregulation of Tim-3, OX-40 and GITR; cisplatin alone and in combination with anti-PD-1 induced upregulation of GITR; OX-40 is upregulated in combinations of chemotherapy and anti-PD-1. Population of tumor-infiltrating T cells were increased in anti-PD-1-treated mice, while combination of pemetrexed and anti-PD-1 prompted augmented expression of co-stimulatory receptors including GITR and OX-40 within tumor. Conclusions: Cisplatin and pemetrexed triggered different systematic and intratumoral immune responses when used alone or in combination with anti-PD-1 treatment. Increased expression of co-stimulatory receptors in the tumor in response to combination therapy provide insights for investigation of synergism on anti-PD-1 and alternative therapeutic targets.

2B4 but not PD-1 blockade improves mortality in septic animals with preexisting malignancy.

In addition to its well-known beneficial effects for the treatment of several types of cancer, PD-1 blockade has shown encouraging results in preclinical models of sepsis and in a recent clinical trial in sepsis. Because cancer is the most common comorbidity in septic patients, here we aimed to determine the efficacy of PD-1 checkpoint blockade in the setting of sepsis complicated with preexisting malignancy. In a model of established lung cancer followed by cecal ligation and puncture-induced (CLP-induced) sepsis, PD-1 blockade exhibited no therapeutic effect on sepsis survival. This diminished efficacy of PD-1 blockade in cancer septic animals (septic animals with cancer) was characterized by a reduction in both the quality and quantity of PD-1+ responder cells. Specifically, CD8+ T cells isolated from cancer septic animals exhibited decreased CD28 expression and a reduction in the CXCR5+PD-1+ subset. In addition, flow cytometric analysis of T cells isolated from cancer septic animals revealed 2B4 as another possible checkpoint under these conditions. Administration of anti-2B4 to cancer septic animals significantly improved sepsis survival and was associated with increased T cell costimulatory receptor expression and decreased coinhibitory receptor expression. These results illustrate functions of coinhibitory receptors in the setting of sepsis complicated with cancer.

4-1BB Delineates Distinct Activation Status of Exhausted Tumor-Infiltrating CD8+ T Cells in Hepatocellular Carcinoma.

Background and AimsTargeting costimulatory receptors with agonistic antibodies is a promising cancer immunotherapy option. We aimed to investigate costimulatory receptor expression, particularly 4‐1BB (CD137 or tumor necrosis factor receptor superfamily member 9), on tumor‐infiltrating CD8+ T cells (CD8+ tumor‐infiltrating lymphocytes [TILs]) and its association with distinct T‐cell activation features among exhausted CD8+ TILs in hepatocellular carcinoma (HCC).Approach and ResultsTumor tissues, adjacent nontumor tissues, and peripheral blood were collected from HCC patients undergoing surgical resection (n = 79). Lymphocytes were isolated and used for multicolor flow cytometry, RNA‐sequencing, and in vitro functional restoration assays. Among the examined costimulatory receptors, 4‐1BB was most prominently expressed on CD8+ TILs. 4‐1BB expression was almost exclusively detected on CD8+ T cells in the tumor—especially on programmed death 1 (PD‐1)high cells and not PD‐1int and PD‐1neg cells. Compared to PD‐1int and 4‐1BBnegPD‐1high CD8+ TILs, 4‐1BBposPD‐1high CD8+ TILs exhibited higher levels of tumor reactivity and T‐cell activation markers and significant enrichment for T‐cell activation gene signatures. Per‐patient analysis revealed positive correlations between percentages of 4‐1BBpos cells among CD8+ TILs and levels of parameters of tumor reactivity and T‐cell activation. Among highly exhausted PD‐1high CD8+ TILs, 4‐1BBpos cells harbored higher proportions of cells with proliferative and reinvigoration potential. Our 4‐1BB–related gene signature predicted survival outcomes of HCC patients in the The Cancer Genome Atlas cohort. 4‐1BB agonistic antibodies enhanced the function of CD8+ TILs and further enhanced the anti‐PD‐1–mediated reinvigoration of CD8+ TILs, especially in cases showing high levels of T‐cell activation.Conclusion4‐1BB expression on CD8+ TILs represents a distinct activation state among highly exhausted CD8+ T cells in HCC. 4‐1BB costimulation with agonistic antibodies may be a promising strategy for treating HCCs exhibiting prominent T‐cell activation.

Extracellular vesicles in human semen modulate antigen-presenting cell function and decrease downstream antiviral T cell responses.

Human semen contains trillions of extracellular vesicles (SEV) similar in size to sexually transmitted viruses and loaded with potentially bioactive miRNAs, proteins and lipids. SEV were shown to inhibit HIV and Zika virus infectivity, but whether SEV are able also to affect subsequent immune responses is unknown. We found that SEV efficiently bound to and entered antigen-presenting cells (APC) and thus we set out to further dissect the impact of SEV on APC function and the impact on downstream T cell responses. In an APC-T cell co-culture system, SEV exposure to APC alone markedly reduced antigen-specific cytokine production, degranulation and cytotoxicity by antigen-specific memory CD8+ T cells. In contrast, inhibition of CD4+ T cell responses required both APC and T cell exposure to SEV. Surprisingly, SEV did not alter MHC or co-stimulatory receptor expression on APCs, but caused APCs to upregulate indoleamine 2,3 deoxygenase, an enzyme known to indirectly inhibit T cells. Thus, SEV reduce the ability of APCs to activate T cells. We propose here that these immune-inhibitory properties of SEV may be intended to prevent immune responses against semen-derived antigens, but can be hi-jacked by genitally acquired viral infections to compromise adaptive cellular immunity.