Multiple Immune Cell Subsets Research Articles

Comprehensive characterization of B7 family members in breast cancer: B7-H5 switch reverses breast cancer from “immuno-cold” into “immuno-hot” status

The members of the classic B7 family regulate the immune microenvironment of several malignant tumors. However, the potential relationship between the B7 family and the breast cancer (BrCa) tumor immune microenvironment has remained elusive. In the present study, we provide a comprehensive explanation of the expression, clinical significance, mutation, and immune cell infiltration of B7 family molecules in BrCa. First, we recruited 10 patients with BrCa surgery from the Wuxi Maternal and Child Health Hospital and performed single-cell RNA sequencing (scRNA-seq) analysis to investigate the distribution of B7 family members in multiple immune cell subsets. We focused on B7-2, B7-H3, and B7-H5 molecules of the B7 family and constructed tumor microarrays by self-recruiting patients to perform multiple immunohistochemical (mIHC) analyses and study tumor expression of B7-2, B7-H3, B7-H5 and CD8+ immune cell infiltration. B7-H5 displayed a strong correlation with CD8+ immune cell infiltration. In summary, B7-H5 provides a new perspective for the identification of immunothermal subtypes of BrCa and could function as a switch to reverse BrCa from an “immunologically cold” state to an “immunologically hot” state.Graphical abstract

Mgp High-Expressing MSCs Orchestrate the Osteoimmune Microenvironment of Collagen/Nanohydroxyapatite-Mediated Bone Regeneration.

Activating autologous stem cells after the implantation of biomaterials is an important process to initiate bone regeneration. Although several studies have demonstrated the mechanism of biomaterial-mediated bone regeneration, a comprehensive single-cell level transcriptomic map revealing the influence of biomaterials on regulating the temporal and spatial expression patterns of mesenchymal stem cells (MSCs) is still lacking. Herein, the osteoimmune microenvironment is depicted around the classical collagen/nanohydroxyapatite-based bone repair materials via combining analysis of single-cell RNA sequencing and spatial transcriptomics. A group of functional MSCs with high expression of matrix Gla protein (Mgp) is identified, which may serve as a pioneer subpopulation involved in bone repair. Remarkably, these Mgp high-expressing MSCs (MgphiMSCs) exhibit efficient osteogenic differentiation potential and orchestrate the osteoimmune microenvironment around implanted biomaterials, rewiring the polarization and osteoclastic differentiation of macrophages through the Mdk/Lrp1 ligand-receptor pair. The inhibition of Mdk/Lrp1 activates the pro-inflammatory programs of macrophages and osteoclastogenesis. Meanwhile, multiple immune-cell subsets also exhibit close crosstalk between MgphiMSCs via the secreted phosphoprotein 1 (SPP1) signaling pathway. These cellular profiles and interactions characterized in this study can broaden the understanding of the functional MSC subpopulations at the early stage of biomaterial-mediated bone regeneration and provide the basis for materials-designed strategies that target osteoimmune modulation.

Abstract 1237: IGM-2644, a CD38xCD3 bispecific IgM T cell engager, shows enhanced anti-tumor activity compared to daratumumab in preclinical models of multiple myeloma

Abstract Multiple myeloma (MM) is a hematologic malignancy characterized by the accumulation of monoclonal plasma cells in the bone marrow. CD38 is an attractive therapeutic target for MM due to its high expression on plasma cells, but it is also expressed on multiple subsets of normal immune cells. Approved therapies including anti-CD38 monoclonal antibodies such as daratumumab are available, however resistance inevitably develops, and toxicities such as reduced immune cell counts put patients at increased risk for infections. To address this and other issues associated with current therapies, we are developing IGM-2644, an engineered high affinity, high avidity bispecific anti-CD38xCD3 IgM T cell engager (TCE) antibody for the treatment of plasma cell disorders. IGM-2644 kills CD38-expressing tumor cells through complement-dependent cytotoxicity (CDC) and T cell-dependent cellular cytotoxicity (TDCC). Live cell imaging of tumor cells in the presence of normal human serum as a source of complement has shown that IGM-2644, compared to daratumumab, has exhibited superior CDC with faster kinetics and higher maximal killing, as well as increased activity on tumor cell lines that express low levels of CD38. IGM-2644 also has displayed enhanced cellular dependent cytotoxicity over daratumumab across tumor cell lines with a range of CD38 expression levels in vitro. In ex vivo assays with MM patient bone marrow mononuclear cells (BMMC), IGM-2644 has shown superior tumor cell depletion compared to daratumumab even with low T cell to tumor cell ratios. Furthermore, IGM-2644 can deplete tumor cells in BMMC from daratumumab-refractory MM patients. Similarly, using in vivo humanized xenograft tumor mouse models with various levels of CD38 expression, greater anti-tumor activity with IGM-2644 over daratumumab was observed. When compared to daratumumab, IGM-2644 demonstrated lower immune cell fratricide in an in vivo xenograft tumor mouse model, suggesting a potentially differentiated safety profile. In summary, IGM-2644 is a bispecific anti-CD38xCD3 IgM TCE antibody that demonstrates superior tumor cell killing when compared to daratumumab in vitro and especially in xenograft tumor models in vivo with low CD38 expression while having low immune cell depletion, potentially reducing the risk of infections. The safety and efficacy of IGM-2644 has been initially studied in a phase 1 clinical trial for relapsed/refractory (r/r) MM. Applicability of IGM-2644 for the treatment of other plasma cell disorders is currently being explored, including autoimmune diseases. Citation Format: Kevin C. Hart, Daniel Santos, Keyu Li, Rui Yun, Min Chai, Andras Kabai, Gene Li, Poonam Yakkundi, Roel P. Funke, Yinghui Guan, Thomas Manley, Liqin Liu, Angus M. Sinclair, Albert F. Candia, Bruce Keyt, Maya F. Kotturi. IGM-2644, a CD38xCD3 bispecific IgM T cell engager, shows enhanced anti-tumor activity compared to daratumumab in preclinical models of multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1237.

Synergistic combination therapy using cowpea mosaic virus intratumoral immunotherapy and Lag-3 checkpoint blockade

Immune checkpoint therapy (ICT) for cancer can yield dramatic clinical responses; however, these may only be observed in a minority of patients. These responses can be further limited by subsequent disease recurrence and resistance. Combination immunotherapy strategies are being developed to overcome these limitations. We have previously reported enhanced efficacy of combined intratumoral cowpea mosaic virus immunotherapy (CPMV IIT) and ICT approaches. Lymphocyte-activation gene-3 (LAG-3) is a next-generation inhibitory immune checkpoint with broad expression across multiple immune cell subsets. Its expression increases on activated T cells and contributes to T cell exhaustion. We observed heightened efficacy of a combined CPMV IIT and anti-LAG-3 treatment in a mouse model of melanoma. Further, LAG-3 expression was found to be increased within the TME following intratumoral CPMV administration. The integration of CPMV IIT with LAG-3 inhibition holds significant potential to improve treatment outcomes by concurrently inducing a comprehensive anti-tumor immune response, enhancing local immune activation, and mitigating T cell exhaustion.

Single cell transcriptomics shows that malaria promotes unique regulatory responses across multiple immune cell subsets

Plasmodium falciparum malaria drives immunoregulatory responses across multiple cell subsets, which protects from immunopathogenesis, but also hampers the development of effective anti-parasitic immunity. Understanding malaria induced tolerogenic responses in specific cell subsets may inform development of strategies to boost protective immunity during drug treatment and vaccination. Here, we analyse the immune landscape with single cell RNA sequencing during P. falciparum malaria. We identify cell type specific responses in sub-clustered major immune cell types. Malaria is associated with an increase in immunosuppressive monocytes, alongside NK and γδ T cells which up-regulate tolerogenic markers. IL-10-producing Tr1 CD4 T cells and IL-10-producing regulatory B cells are also induced. Type I interferon responses are identified across all cell types, suggesting Type I interferon signalling may be linked to induction of immunoregulatory networks during malaria. These findings provide insights into cell-specific and shared immunoregulatory changes during malaria and provide a data resource for further analysis.

Cxxc finger protein 1 maintains homeostasis and function of intestinal group 3 innate lymphoid cells with aging.

Aging is accompanied by homeostatic and functional dysregulation of multiple immune cell subsets. Group 3 innate lymphoid cells (ILC3s) constitute a heterogeneous cell population that plays pivotal roles in intestinal immunity. In this study, we found that ILC3s in aged mice exhibited dysregulated homeostasis and function, leading to bacterial and fungal infection susceptibility. Moreover, our data revealed that the enrichment of the H3K4me3 modification in effector genes of aged gut CCR6+ ILC3s was specifically decreased compared to young mice counterparts. Disruption of Cxxc finger protein 1 (Cxxc1) activity, a key subunit of H3K4 methyltransferase, in ILC3s led to similar aging-related phenotypes. An integrated analysis revealed Kruppel-like factor 4 (Klf4) as a potential Cxxc1 target. Klf4 overexpression partially restored the differentiation and functional defects seen in both aged and Cxxc1-deficient intestinal CCR6+ ILC3s. Therefore, these data suggest that targeting intestinal ILC3s may provide strategies to protect against age-related infections.

Single Cell Analysis of High-Parameter Histology Images Using Histoflow Cytometry.

Immunofluorescence histology is commonly used to study immune cells in tissues where the number of fluorescence parameters is normally limited to four or less. This makes it impossible to interrogate multiple subsets of immune cells in tissue with the same precision as flow cytometry. The latter, however, dissociates tissues and loses spatial information. To bridge the gap between these technologies, we developed a workflow to expand the number of fluorescence parameters that can be imaged on widely available microscopes. We instituted a method for identifying single cells in tissue and exporting the data for flow cytometry-based analysis. This histoflow cytometry technique successfully separates spectrally overlapping dyes and identifies similar numbers of cells in tissue sections as manual cell counts. Populations identified through flow cytometry-like gating strategies are mapped to the original tissue to spatially localize gated subsets. We applied histoflow cytometry to immune cells in the spinal cords of mice with experimental autoimmune encephalomyelitis. We ascertained that B cells, T cells, neutrophils, and phagocytes differed in their frequencies in CNS immune cell infiltrates and were increased relative to healthy controls. Spatial analysis determined that B cells and T cells/phagocytes preferentially localized to CNS barriers and parenchyma, respectively. By spatially mapping these immune cells, we inferred their preferred interacting partners within immune cell clusters. Overall, we demonstrate the ease and utility of histoflow cytometry, which expands the number of fluorescent channels used in conventional immunofluorescence and enables quantitative cytometry and spatial localization of histological analyses.

Mass Spectrometry-Based Tissue Imaging of the Tumor Microenvironment.

Multiplex ion beam imaging (MIBI) and imaging mass cytometry (IMC) enable highly multiplexed antibody (40+) staining of frozen or formalin fixed, paraffin-embedded (FFPE) human or murine tissues through detection of metal ions liberated from primary antibodies by time-of-flight mass spectrometry (TOF). These methods make detection of more than 50 targets theoretically possible while maintaining spatial orientation. As such, they are ideal tools to identify the multiple immune, epithelial, and stromal cell subsets in the tumor microenvironment and to characterize spatial relationships and tumor-immune status in either murine models or human samples. This chapter summarizes methods for antibody conjugation and validation, staining, and preliminary data collection using IMC or MIBI in both human and mouse pancreatic adenocarcinoma samples. These protocols are intended to facilitate use of these complex platforms in not only tissue-based tumor immunology studies but also tissue-based oncology or immunology studies more broadly.

Comprehensive analyses of one-carbon metabolism related genes and their association with prognosis, tumor microenvironment, chemotherapy resistance and immunotherapy in lung adenocarcinoma.

Background: Lung adenocarcinoma (LUAD) is the most common type of lung cancer and is a global public health concern. One-carbon (1C) metabolism plays a crucial role in the occurrence and development of multiple cancer types. However, there are limited studies investigating 1C metabolism in LUAD. This study aims to evaluate the prognostic value of 1C metabolism-related genes in LUAD and to explore the potential correlation of these genes with gene methylation, the tumor microenvironment, and immunotherapy. Methods: We identified 26 1C metabolism-related genes and performed a Kaplan-Meier and Cox regression analysis to evaluate the prognostic value of these genes. Consensus clustering was further performed to determine the 1C metabolism-related gene patterns in LUAD. The clinical and molecular characteristics of subgroups were investigated based on consensus clustering. CIBERSORT and ssGSEA algorithms were used to calculate the relative infiltration levels of multiple immune cell subsets. The relationship between 1C metabolism-related genes and drug sensitivity to immunotherapy was evaluated using the CellMiner database and IMvigor210 cohort, respectively. Results: The expression levels of 23 1C metabolism-related genes were significantly different between LUAD tumor tissues and normal tissues. Seventeen of these genes were related to prognosis. Two clusters (cluster 1 and cluster 2) were identified among 497 LUAD samples based on the expression of 7 prognosis-related genes. Distinct expression patterns were observed between the two clusters. Compared to cluster 2, cluster 1 was characterized by inferior overall survival (OS) (median OS = 41 vs. 60months, p = 0.00031), increased tumor mutation burden (15.8 vs. 7.5mut/Mb, p < 0.001), high expression of PD-1 (p < 0.001) and PD-L1 (p < 0.001), as well as enhanced immune infiltration. 1C metabolism-related genes were positively correlated with the expression of methylation enzymes, and a lower methylation level was observed in cluster 1 (p = 0.0062). Patients in cluster 1 were resistant to chemotherapy drugs including pemetrexed, gemcitabine, paclitaxel, etoposide, oxaliplatin, and carboplatin. The specific expression pattern of 1C metabolism-related genes was correlated with a better OS in patients treated with immunotherapy (median OS: 11.2 vs. 7.8months, p = 0.0034). Conclusion: This study highlights that 1C metabolism is correlated with the prognosis of LUAD patients and immunotherapy efficacy. Our findings provide novel insights into the role of 1C metabolism in the occurrence, development, and treatment of LUAD, and can assist in guiding immunotherapy for LUAD patients.

Manipulation of the crosstalk between tumor angiogenesis and immunosuppression in the tumor microenvironment: Insight into the combination therapy of anti-angiogenesis and immune checkpoint blockade.

Immunotherapy has been recognized as an effective and important therapeutic modality for multiple types of cancer. Nevertheless, it has been increasing recognized that clinical benefits of immunotherapy are less than expected as evidenced by the fact that only a small population of cancer patients respond favorably to immunotherapy. The structurally and functionally abnormal tumor vasculature is a hallmark of most solid tumors and contributes to an immunosuppressive microenvironment, which poses a major challenge to immunotherapy. In turn, multiple immune cell subsets have profound consequences on promoting neovascularization. Vascular normalization, a promising anti-angiogenic strategy, can enhance vascular perfusion and promote the infiltration of immune effector cells into tumors via correcting aberrant tumor blood vessels, resulting in the potentiation of immunotherapy. More interestingly, immunotherapies are prone to boost the efficacy of various anti-angiogenic therapies and/or promote the morphological and functional alterations in tumor vasculature. Therefore, immune reprograming and vascular normalization appear to be reciprocally regulated. In this review, we mainly summarize how tumor vasculature propels an immunosuppressive phenotype and how innate and adaptive immune cells modulate angiogenesis during tumor progression. We further highlight recent advances of anti-angiogenic immunotherapies in preclinical and clinical settings to solidify the concept that targeting both tumor blood vessels and immune suppressive cells provides an efficacious approach for the treatment of cancer.

Genetic variants associated with psychiatric disorders are enriched at epigenetically active sites in lymphoid cells

Multiple psychiatric disorders have been associated with abnormalities in both the innate and adaptive immune systems. The role of these abnormalities in pathogenesis, and whether they are driven by psychiatric risk variants, remains unclear. We test for enrichment of GWAS variants associated with multiple psychiatric disorders (cross-disorder or trans-diagnostic risk), or 5 specific disorders (cis-diagnostic risk), in regulatory elements in immune cells. We use three independent epigenetic datasets representing multiple organ systems and immune cell subsets. Trans-diagnostic and cis-diagnostic risk variants (for schizophrenia and depression) are enriched at epigenetically active sites in brain tissues and in lymphoid cells, especially stimulated CD4+ T cells. There is no evidence for enrichment of either trans-risk or cis-risk variants for schizophrenia or depression in myeloid cells. This suggests a possible model where environmental stimuli activate T cells to unmask the effects of psychiatric risk variants, contributing to the pathogenesis of mental health disorders.

Influenza vaccination features revealed by a single-cell transcriptome atlas.

Emerging and re-emerging viruses like influenza virus pose a continuous global public health threat. Vaccines are one of the most effective public health strategies for controlling infectious diseases. However, little is known about the immunological features of vaccination at the single-cell resolution, including for influenza vaccination. Here, we report the single-cell transcriptome atlas of longitudinally collected peripheral blood mononuclear cells(PBMCs) in individuals immunized with an inactivated influenza vaccine. Overall, vaccination with the influenza vaccine only had a small impact on the composition of peripheral immune cells, but elicited global transcriptional changes in multiple immune cell subsets. In plasma and B cell subsets, transcriptomic changes, which were mostly involved in antibody production as well as B cell activation and differentiation, were observed after influenza vaccinations. In influenza-vaccinated individuals, we found a reduction in multiple biological processes (e.g., interferon response, inflammatory response, HLA-I/II molecules, cellular apoptosis, migration, and cytotoxicity, etc.,) 7 days postvaccination in multiple immune cell subsets. However, 14 days postvaccination, these levels returned to similar levels observed in prevaccination samples. Additionally, we did not observe significant upregulation of pro-inflammatory response genes and key thrombosis-related genes in influenza-vaccinated individuals. Taken together, we report a cell atlas of the peripheral immune response to influenza vaccination and provide a resource for understanding the immunological response mechanisms of influenza vaccination.

Role of chemokines in breast cancer.

Chemokines belong to a family of chemoattractant cytokines and are well known to have an essential role in various cancer aetiologies. Multiplesubsets of immune cells are recruited and enrolled into the tumor microenvironment through interactions between chemokines and their specific receptors. These populations and their interactions have a distinct impact on tumor growth, progression, and treatment outcomes. While it is clear that many chemokines and their cognate receptors can be detected in breast and other cancers, the role of each chemokine and receptor has yet to be determined. This review focuses on the main chemokines that play a crucial role in the tumor microenvironment, emphasizing breast cancer. We have also discussed the techniques used to identify the chemokines and their future implication in the early diagnosis of cancer. In-depth knowledge of chemokines and their role in breast cancer progression can provide specific targets for breast cancer biotherapy.

Abstract 5518: Single cell RNA sequencing revealed that the mechanism of the synergistic efficacy of STA551 and immune checkpoint inhibitor results from the induction of highly activated T cells in tumor

Abstract Background: CD137 (4-1BB) is a co-stimulatory molecule which exerts activation signals in multiple immune cell subsets. However, agonist antibodies targeting CD137 have not been clinically successful due to severe systemic toxicity. Therefore, we generated STA551, a tumor selective CD137 agonist antibody. STA551 binds to CD137 only in the presence of ATP, which is known to be present at high levels in tumor tissue, and activates immune cells such as T cells. Previous studies have shown that STA551 exhibits antitumor efficacy without inducing a systemic immune response in a murine model. Combination studies with anti-PD-L1 antibody have also shown synergistic efficacy without inducing a systemic immune response. However, it was unclear why they exhibit strong combination effects. In this study, we elucidated the mechanism of STA551 and anti-PD-L1 antibody efficacy through comprehensive analysis of gene expression using single cell RNA sequencing. Method &amp; Result: In a colon 38-bearing human CD137 knock-in mouse model, combination therapy with STA551 and anti-PD-L1 antibody showed potent anti-tumor efficacy. From immunohistochemistry of tumor tissue, increased numbers of CD8+ T cells and elevated expression of PD-L1 molecules were observed. To perform a detailed mechanistic analysis, tumor tissue was collected after antibody administration and CD3 positive and CD3 negative fractions were separated and gene expression was comprehensively analyzed using single cell RNA-seq. Next, the fraction expressing Cd8a gene was extracted from CD3 positive fraction and clustered. One cluster was found to be markedly increased in STA551 and anti-PD-L1 antibody combination compared to vehicle or monotherapy treatment. Differential gene expression analysis revealed that the cluster was enriched for genes related to cytokines and chemokines. This result revealed that the combination of STA551 and anti-PD-L1 antibody induced highly activated CD8+T cells. Conclusion: The combination of STA551 and PD-L1 showed synergistic anti-tumor efficacy in a colon38-bearing human CD137 knock-in mouse model. Detailed mechanistic analysis showed that the combination of STA551 and PD-L1 induced highly activated CD8+T cells expressing chemokines and cytokines compared to monotherapy. This cluster also express chemokines, suggesting that other immune cells have been recruited. These results suggest that even the activation of only tumor tissue, which is a characteristic of STA551, can induce a strong immune response. Citation Format: Ryo Uchikawa, Kazuya Takakuwa, Hirofumi Mikami, Shoichi Metsugi, Kenji Taniguchi, Yoshinori Narita, Mika Kamata-Sakurai. Single cell RNA sequencing revealed that the mechanism of the synergistic efficacy of STA551 and immune checkpoint inhibitor results from the induction of highly activated T cells in tumor [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 5518.

Abstract 2882: Selective activation of CD8+ T cells by a CD8-targeted IL-21 results in enhanced anti-tumor efficacy and safety

Abstract Interleukin-21 (IL-21) is a pleiotropic cytokine that activates multiple lymphoid and myeloid immune cell subsets. IL-21 can have beneficial effects on anti-tumor immune responses due to its ability to activate STAT3 in CD8+ T cells and promote cytotoxicity, memory cell differentiation and survival. However, IL-21 can also induce immunosuppressive effects, such as suppression of antigen presentation, which may directly oppose its anti-tumor effects on CD8+ T cells. Although recombinant IL-21 did show monotherapy activity in the clinic, overall response rates were modest. In addition to the pharmacological sink created by the broad expression of IL-21R and potential immunosuppressive effects, clinical utility of IL-21 was further limited by its low bioavailability and dose-limiting toxicities. We hypothesized that maximizing IL-21’s activity on CD8+ T cells while avoiding non-CD8 cells would enhance efficacy and improve tolerability. We developed cis-targeted IL-21 (CD8-IL21) fusion proteins that exhibit improved bioavailability and selectively activate CD8+ T cells while exhibiting minimal activity on non-CD8 cells. In vitro phospho-STAT3 assays with CD8-IL21 in mouse splenocytes and human peripheral blood cells showed &amp;gt;1000-fold selective activation of CD8+ T cells over non-CD8 cells. A mouse CD8-IL21 surrogate demonstrated profound single agent efficacy in multiple syngeneic tumor models at considerably lower doses than an untargeted IL-21 control, despite similar in vitro potency. CD8-IL21 mediated anti-tumor efficacy without inducing body weight loss. Whereas prior data showed that repeated high doses of tumor-targeted IL-21 fusion protein (&amp;gt;3 mg/kg) had single agent efficacy in the MC38 tumor model overexpressing a tumor antigen [1], here we show that a single dose of CD8-targeted IL-21 as low as 0.1 mg/kg induced curative responses in mice bearing established MC38 tumors. We demonstrated using the lymphocyte migration inhibitor FTY720 that this enhanced potency of low dose CD8-IL21 is predominately driven by the activation of peripheral CD8 T cells. Furthermore, curative effects in the MC38 model were elicited in the absence of substantial CD8+ T cell expansion (&amp;lt;1.5-fold) suggesting that CD8-IL21 mediated anti-tumor efficacy through modulation of T cell phenotype. A CD8-IL21 selective for human and cynomolgus CD8+ T cells with improved bioavailability also exhibited improved tolerability over untargeted IL-21 in cynomolgus monkeys, showing that there is a tolerability benefit to CD8-selective activation. In summary, CD8-IL21 showed improved efficacy and safety over untargeted IL-21, demonstrating clear promise as a novel therapeutic agent which by activating STAT3 signaling in CD8+ T cells may offer complementary benefits to activation of STAT5 signaling by cytokines such as IL-2 and IL-15.Ref: 1. Deng et al., 2020 Citation Format: Renee Greer, David Liu, Henry Nguyen, Ruth Lan, Wei Wei Prior, Meghana Sukthankar, Paul Mesko, irene Ni, Mike Chin, Kelly Moynihan, Ton Schumacher, Ivana Djuretic, Andy Yeung. Selective activation of CD8+ T cells by a CD8-targeted IL-21 results in enhanced anti-tumor efficacy and safety [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 2882.

The role of VISTA in the tumor microenvironment

V-domain Ig Suppressor of T cell Activation (VISTA) is a negative immune checkpoint that is expressed on multiple immune cell subsets and has been characterized in T cells, macrophages, and myeloid-derived suppressor cells. As the only immune checkpoint expressed on naïve T cells, VISTA contributes to the maintenance of T cell quiescence and tolerance. VISTA also regulates multiple myeloid cell activities such as chemotaxis, differentiation, and migration. In the context of cancer, antagonistic monoclonal antibody targeting of VISTA has been shown to aid anti-tumor immunity. Furthermore, combination therapies that include other immune checkpoints such as PD-1 or CTLA-4 with VISTA blockade may enhance therapeutic efficacy in a variety of cancers. Combination therapy may help overcome adaptive resistance to individual checkpoint therapies, thereby improving patient outcomes and survival. Here, we summarize the role of VISTA in myeloid cells and T cells within the tumor microenvironment. We discuss the proposed counter-receptors for VISTA, VISTA antibodies currently in development, and the potential for combination therapies with checkpoint inhibitors such as PD-1 and CTLA-4.

Liuwei Buqi capsule modulates immune function by targeting multiple immune cell subsets in lung tissue of patients with COPD

To investigate the characteristics of immune cell subsets in the lung tissues of patients with chronic obstructive pulmonary disease (COPD) and the mechanism of Liuwei Buqi capsule in modulating immune and inflammatory imbalance in COPD. We downloaded COPD-related single-cell RNA sequencing data from Gene Expression Omnibus (GEO) and identified COPD immune cell subsets using the Seurat package in the R software to construct an immune cell subsets-differential genes network. The target genes and active ingredients of Liuwei Buqi capsule were obtained from the Chinese Medicine System Pharmacology Database and Analysis Platform (TCMSP), and the Liuwei Buqi capsule-immune cell subsets-target genes network was constructed by mapping the target genes to the differentially expressed genes in each immune cell subset. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was performed to analyze significantly enriched pathways of the target genes, and the key genes involved in the top 20 pathways were identified. In a rat model of COPD, we investigated the effects of Liuwei Buqi capsule on pulmonary function, lung tissue pathology, serum levels of IL-1β, NF-κB, and TNF-α, and expressions of IKBα, JNK, c-JUN, and c-FOS proteins in the lung tissue. A total of 18 immune-related cell subsets, including macrophages and alveolar macrophages, were identified in both COPD patients and healthy control subjects, and the patients with COPD showed significant changes the percentages of macrophages, cDC1, pDC, mast cells, T cells, and mature dendritic cells (P < 0.05). Liuwei Buqi capsules targeted multiple immune cell subsets, and the identified target genes were enriched mostly in such immune and inflammation-related signaling pathways as lipids and atherosclerosis, IL-17 signaling pathway, Toll-like receptor signaling pathway, and TNF signaling pathway; the genes CXCL8, IL1B, JUN, NFKBIA, MAPK8, and FOS were the key genes involved in the significantly enriched pathways. In the rat models of COPD, treatment with Liuwei Buqi capsule significantly improved pulmonary function, alleviated lung pathologies, reduced serum levels of IL-1β, TNF-α, and NF-κB (P < 0.05) and pulmonary expressions of JNK, c-JUN, and c-FOS (P < 0.01) protein, and increased pulmonary expression of IκBα (P < 0.01). Liuwei Buqi capsule may play an immunomodulatory role by targeting multiple immune cell subsets in the lung tissue of COPD patients.

A Peripheral Immune Signature of Labor Induction.

Approximately 1 in 4 pregnant women in the United States undergo labor induction. The onset and establishment of labor, particularly induced labor, is a complex and dynamic process influenced by multiple endocrine, inflammatory, and mechanical factors as well as obstetric and pharmacological interventions. The duration from labor induction to the onset of active labor remains unpredictable. Moreover, prolonged labor is associated with severe complications for the mother and her offspring, most importantly chorioamnionitis, uterine atony, and postpartum hemorrhage. While maternal immune system adaptations that are critical for the maintenance of a healthy pregnancy have been previously characterized, the role of the immune system during the establishment of labor is poorly understood. Understanding maternal immune adaptations during labor initiation can have important ramifications for predicting successful labor induction and labor complications in both induced and spontaneous types of labor. The aim of this study was to characterize labor-associated maternal immune system dynamics from labor induction to the start of active labor. Serial blood samples from fifteen participants were collected immediately prior to labor induction (baseline) and during the latent phase until the start of active labor. Using high-dimensional mass cytometry, a total of 1,059 single-cell immune features were extracted from each sample. A multivariate machine-learning method was employed to characterize the dynamic changes of the maternal immune system after labor induction until the establishment of active labor. A cross-validated linear sparse regression model (least absolute shrinkage and selection operator, LASSO) predicted the minutes since induction of labor with high accuracy (R = 0.86, p = 6.7e-15, RMSE = 277 min). Immune features most informative for the model included STAT5 signaling in central memory CD8+ T cells and pro-inflammatory STAT3 signaling responses across multiple adaptive and innate immune cell subsets. Our study reports a peripheral immune signature of labor induction, and provides important insights into biological mechanisms that may ultimately predict labor induction success as well as complications, thereby facilitating clinical decision-making to improve maternal and fetal well-being.

Associations of immune cell homing gene signatures and infiltrates of lymphocyte subsets in human melanomas: discordance with CD163+ myeloid cell infiltrates

BackgroundImmune cells in the tumor microenvironment have prognostic value. In preclinical models, recruitment and infiltration of these cells depends on immune cell homing (ICH) genes such as chemokines, cell adhesion molecules, and integrins. We hypothesized ICH ligands CXCL9-11 and CCL2-5 would be associated with intratumoral T-cells, while CXCL13 would be more associated with B-cell infiltrates.MethodsSamples of human melanoma were submitted for gene expression analysis and immune cells identified by immunohistochemistry. Associations between the two were evaluated with unsupervised hierarchical clustering using correlation matrices from Spearman rank tests. Univariate analysis performed Mann–Whitney tests.ResultsFor 119 melanoma specimens, analysis of 78 ICH genes revealed association among genes with nonspecific increase of multiple immune cell subsets: CD45+, CD8+ and CD4+ T-cells, CD20+ B-cells, CD138+ plasma cells, and CD56+ NK-cells. ICH genes most associated with these infiltrates included ITGB2, ITGAL, CCL19, CXCL13, plus receptor/ligand pairs CXCL9 and CXCL10 with CXCR3; CCL4 and CCL5 with CCR5. This top ICH gene expression signature was also associated with genes representing immune-activation and effector function. In contrast, CD163+ M2-macrophages was weakly associated with a different ICH gene signature.ConclusionThese data do not support our hypothesis that each immune cell subset is uniquely associated with specific ICH genes. Instead, a larger set of ICH genes identifies melanomas with concordant infiltration of B-cell and T-cell lineages, while CD163+ M2-macrophage infiltration suggesting alternate mechanisms for their recruitment. Future studies should explore the extent ICH gene signature contributes to tertiary lymphoid structures or cross-talk between homing pathways.

Abstract 1851: Responses of a novel tumor selective anti-CD137 agonist antibody activated by elevated extracellular ATP in tumor microenvironment

Abstract Background: CD137 (4-1BB) is a co-stimulatory molecule which exerts activation signals in multiple immune cell subsets. However, agonist antibodies targeting CD137 have not been clinically successful due to severe systemic toxicity. Since conferring tumor selectivity through tumor-associated antigen limits its clinical use to cancers that highly express such antigen, we instead exploited extracellular adenosine triphosphate (exATP), which is known to be elevated in tumor microenvironment (TME) while remaining tightly regulated in normal tissues, as a broadly tumor selective switch. Here we report a novel anti-CD137 switch antibody, STA551, which exerts agonistic activity only in the presence of exATP. Methods &amp; Results: STA551 bound to human CD137 in an ATP dependent manner. Consistent with the binding profile, STA551 induced IFN-γ only in the presence of ATP, whereas urelumab induced IFN-γ regardless of ATP concentration in a human T cell assay. We examined the anti-tumor efficacy of Sta-MB, which has the same variable region as STA551 using a surrogate mouse Fc, in human CD137 knock-in (hCD137-KI) mice. STA551 demonstrated potent and broad anti-tumor efficacy against all the tumors we tested both as a monotherapy and in combination with anti-PD-L1 antibody. To confirm the immune responses in tumors, we evaluated gene expression and analyzed tumor infiltrating lymphocytes by flow cytometry. Immune-related gene expression changed after treatment with Sta-MB. We also confirmed that Sta-MB treatment increased the number of activated CD8+ T cells in tumors. For all tumor responses, Sta-MB tended to be more potent than Ure-MB. Whereas other anti-CD137 conventional antibodies showed significant CD137 mediated clearance and systemic T cell activation in draining lymph node (DLN), spleen, and liver, STA551 demonstrated significant intratumor T cell activation, but not in normal tissues and without CD137 mediated systemic clearance in mice. Furthermore, STA551 was well-tolerated in cynomolgus monkeys in a repeated-dose toxicity study*. Conclusion: These results suggest that STA551, due to its tumor selective agonistic activity, has potent anti-tumor efficacy and a wide therapeutic window, providing a strong rationale for its clinical testing against a broad variety of cancers regardless of antigen expression, and for the further application of this novel platform to other targets in cancer therapy. STA551 is currently being tested in a phase 1 clinical study. We will here disclose the clinical study design for the first time. Citation Format: Mika Kamata-Sakurai, Yoshinori Narita, Ryo Uchikawa, Kenji Taniguchi, Sayuri Horikawa, Koki Hamada, Yuji Hori, Takayuki Nemoto, Shoichi Metsugi, Michiyasu Inatani, Tomoyuki Igawa. Responses of a novel tumor selective anti-CD137 agonist antibody activated by elevated extracellular ATP in tumor microenvironment [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 1851.