756 Assessment of the immune checkpoint landscape in head and neck squamous cell carcinoma by single-cell RNA sequencing and multispectral imaging

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A804-A805
Author(s):  
Lazar Vujanovic ◽  
Aditi Kulkarni ◽  
Cornelius Kürten ◽  
Anthony Cillo ◽  
Patricia Santos ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Squamous Cell Carcinoma ◽  
T Cells ◽  
Cd8 T Cells ◽  
Immune Cells ◽  
Immune Checkpoint ◽  
Multispectral Imaging ◽  
Cell Types ◽  
Cell Type ◽  
Single Cell Rna Sequencing

BackgroundResistance to the current generation of immunotherapies is mediated by complex relations between stromal, cancer and immune cells found within the tumor microenvironment (TME). Development of more efficacious drugs is predicated on improved understanding of these multi-spatial interactions. With emergence of new immune checkpoint receptor (ICR)-targeting therapies, a better understanding of topological expression of immune checkpoint ligand (ICL) on suppressive cell types in the TME may allow for improved strategies to treat cancer patients.MethodsSingle cell RNA sequencing (scRNAseq) was performed from head and neck squamous cell carcinoma (HNSCC) specimens (n=18) with matched blood from treatment-naïve patients. Immune and non-immune cells were enriched from tumor cell suspensions. Novel transcriptomic cell-to-cell interactions were predicted between heterogeneous cell populations. Histologic inflammation was corroborated with scRNAseq and multiplex flow cytometry. Cell type-specific PD-L1 contributions within the TME were quantified using multispectral imaging.ResultsMajor cell type clusters (immune, epithelial, fibroblast and endothelial cells) were identified. Expression patterns for PD-1, TIGIT, LAG-3 and TIM-3 ligands were evaluated on these suppressive TME cell types. By modeling receptor-ligand interactions between CD8+ T cells and the rest of the major TME cell types, CD8+ T cells were predicted to form more ICR-ICL interactions with tumor-associated macrophages (TAMs) than with any other cell type. With focus on LGALS9/galectin-9 and CD274/PD-L1, flow cytometric analyses validated the scRNAseq observation that both ligands were expressed on TAMs from both inflamed and non-inflamed tumors. Furthermore, flow cytometry and multispectral imaging analyses implicated macrophages as one of the major contributors of CD274/PD-L1 within the TME.ConclusionsOur data suggest that in the setting of HNSCC, TAMs are one of the major contributors of ICL in the HNSCC TME. Strategies that selective target this immunosuppressive population may be necessary to break tolerance to PD-1-targeting therapies.Ethics ApprovalThe study was approved by the UPMC Hillman Cancer’s Ethics Board, approval number 99-069.

scholarly journals Reference Transcriptomes of Porcine Peripheral Immune Cells Created Through Bulk and Single-Cell RNA Sequencing

2021 ◽  
Vol 12 ◽  
Author(s):  
Juber Herrera-Uribe ◽  
Jayne E. Wiarda ◽  
Sathesh K. Sivasankaran ◽  
Lance Daharsh ◽  
Haibo Liu ◽  
...  
Keyword(s):  
T Cells ◽  
Single Cell ◽  
Immune Cells ◽  
Peripheral Blood ◽  
Cell Types ◽  
Cell Populations ◽  
Rna Seq ◽  
Cell Type ◽  
Transcriptomic Data ◽  
Single Cell Rna Sequencing

Pigs are a valuable human biomedical model and an important protein source supporting global food security. The transcriptomes of peripheral blood immune cells in pigs were defined at the bulk cell-type and single cell levels. First, eight cell types were isolated in bulk from peripheral blood mononuclear cells (PBMCs) by cell sorting, representing Myeloid, NK cells and specific populations of T and B-cells. Transcriptomes for each bulk population of cells were generated by RNA-seq with 10,974 expressed genes detected. Pairwise comparisons between cell types revealed specific expression, while enrichment analysis identified 1,885 to 3,591 significantly enriched genes across all 8 cell types. Gene Ontology analysis for the top 25% of significantly enriched genes (SEG) showed high enrichment of biological processes related to the nature of each cell type. Comparison of gene expression indicated highly significant correlations between pig cells and corresponding human PBMC bulk RNA-seq data available in Haemopedia. Second, higher resolution of distinct cell populations was obtained by single-cell RNA-sequencing (scRNA-seq) of PBMC. Seven PBMC samples were partitioned and sequenced that produced 28,810 single cell transcriptomes distributed across 36 clusters and classified into 13 general cell types including plasmacytoid dendritic cells (DC), conventional DCs, monocytes, B-cell, conventional CD4 and CD8 αβ T-cells, NK cells, and γδ T-cells. Signature gene sets from the human Haemopedia data were assessed for relative enrichment in genes expressed in pig cells and integration of pig scRNA-seq with a public human scRNA-seq dataset provided further validation for similarity between human and pig data. The sorted porcine bulk RNAseq dataset informed classification of scRNA-seq PBMC populations; specifically, an integration of the datasets showed that the pig bulk RNAseq data helped define the CD4CD8 double-positive T-cell populations in the scRNA-seq data. Overall, the data provides deep and well-validated transcriptomic data from sorted PBMC populations and the first single-cell transcriptomic data for porcine PBMCs. This resource will be invaluable for annotation of pig genes controlling immunogenetic traits as part of the porcine Functional Annotation of Animal Genomes (FAANG) project, as well as further study of, and development of new reagents for, porcine immunology.

scholarly journals Reference transcriptomes of porcine peripheral immune cells created through bulk and single-cell RNA sequencing

2021 ◽  
Author(s):  
Juber Herrera-Uribe ◽  
Jayne E Wiarda ◽  
Sathesh K Sivasankaran ◽  
Lance Daharsh ◽  
Haibo Liu ◽  
...  
Keyword(s):  
T Cells ◽  
Single Cell ◽  
Immune Cells ◽  
Peripheral Blood ◽  
Cell Types ◽  
Cell Populations ◽  
Rna Seq ◽  
Cell Type ◽  
Transcriptomic Data ◽  
Single Cell Rna Sequencing

Pigs are a valuable human biomedical model and an important protein source supporting global food security. The transcriptomes of peripheral blood immune cells in pigs were defined at the bulk cell-type and single cell levels. First, eight cell types were isolated in bulk from peripheral blood mononuclear cells (PBMCs) by cell sorting, representing Myeloid, NK cells and specific populations of T and B cells. Transcriptomes for each bulk population of cells were generated by RNA-seq with 10,974 expressed genes detected. Pairwise comparisons between cell types revealed specific expression, while enrichment analysis identified 1,885 to 3,591 significantly enriched genes across all 8 cell types. Gene Ontology analysis for the top 25% of significantly enriched genes (SEG) showed high enrichment of biological processes related to the nature of each cell type. Comparison of gene expression indicated highly significant correlations between pig cells and corresponding human PBMC bulk RNA-seq data available in Haemopedia. Second, higher resolution of distinct cell populations was obtained by single-cell RNA-sequencing (scRNA-seq) of PBMC. Seven PBMC samples were partitioned and sequenced that produced 28,810 single cell transcriptomes distributed across 36 clusters and classified into 13 general cell types including plasmacytoid dendritic cells (DC), conventional DCs, monocytes, B cell, conventional CD4 and CD8 αβ T cells, NK cells, and γδ T cells. Signature gene sets from the human Haemopedia data were assessed for relative enrichment in genes expressed in pig cells and integration of pig scRNA-seq with a public human scRNA-seq dataset provided further validation for similarity between human and pig data. The sorted porcine bulk RNAseq dataset informed classification of scRNA-seq PBMC populations; specifically, an integration of the datasets showed that the pig bulk RNAseq data helped define the CD4CD8 double-positive T cell populations in the scRNA-seq data. Overall, the data provides deep and well-validated transcriptomic data from sorted PBMC populations and the first single-cell transcriptomic data for porcine PBMCs. This resource will be invaluable for annotation of pig genes controlling immunogenetic traits as part of the porcine Functional Annotation of Animal Genomes (FAANG) project, as well as further study of, and development of new reagents for, porcine immunology.

scholarly journals Crosstalk Between Tumor-Associated Microglia/Macrophages and CD8-Positive T Cells Plays a Key Role in Glioblastoma

2021 ◽  
Vol 12 ◽  
Author(s):  
Sheng Tu ◽  
Xu Lin ◽  
Jili Qiu ◽  
Jiaqi Zhou ◽  
Hui Wang ◽  
...  
Keyword(s):  
T Cells ◽  
Cd8 T Cells ◽  
Immune Cells ◽  
Malignant Disease ◽  
Immune Therapy ◽  
Cell Types ◽  
Different Types ◽  
The Central Nervous System ◽  
And Function ◽  
The Relationship

Glioblastoma is considered to be the most malignant disease of the central nervous system, and it is often associated with poor survival. The immune microenvironment plays a key role in the development and treatment of glioblastoma. Among the different types of immune cells, tumor-associated microglia/macrophages (TAM/Ms) and CD8-positive (CD8+) T cells are the predominant immune cells, as well as the most active ones. Current studies have suggested that interaction between TAM/Ms and CD8+ T cells have numerous potential targets that will allow them to overcome malignancy in glioblastoma. In this review, we summarize the mechanism and function of TAM/Ms and CD8+ T cells involved in glioblastoma, as well as update on the relationship and crosstalk between these two cell types, to determine whether this association alters the immune status during glioblastoma development and affects optimal treatment. We focus on the molecular factors that are crucial to this interaction, and the role that this crosstalk plays in the biological processes underlying glioblastoma treatment, particularly with regard to immune therapy. We also discuss novel therapeutic targets that can aid in resolving reticular connections between TAM/Ms and CD8+ T cells, including depletion and reprogramming TAM/Ms and novel TAM/Ms-CD8+ T cell cofactors with potential translational usage. In addition, we highlight the challenges and discuss future perspectives of this crosstalk between TAM/Ms and CD8+ T cells.

scholarly journals Single-Cell Profiling of Kidney Transplant Recipients With Immunosuppressive Treatment Reveals the Dynamic Immune Characteristics

2021 ◽  
Vol 12 ◽  
Author(s):  
Yongguang Liu ◽  
Xiaoyou Liu ◽  
Song Zhou ◽  
Ruiquan Xu ◽  
Jianmin Hu ◽  
...  
Keyword(s):  
T Cells ◽  
Kidney Transplantation ◽  
Cd8 T Cells ◽  
Immune Cells ◽  
Peripheral Blood ◽  
Cd4 T Cells ◽  
Immune Cell ◽  
Immunosuppressive Agents ◽  
Cell Types ◽  
Before And After

Kidney transplantation is currently the first choice of treatment for various types of end-stage renal failure, but there are major limitations in the application of immunosuppressive protocols after kidney transplantation. When the dose of immunosuppressant is too low, graft rejection occurs easily, while a dose that is too high can lead to graft loss. Therefore, it is very important to explore the immune status of patients receiving immunosuppressive agents after kidney transplantation. To compare the immune status of the recipient’s whole peripheral blood before and after receipt of immunosuppressive agents, we used single-cell cytometry by time-of-flight (CyTOF) to detect the peripheral blood immune cells in five kidney transplant recipients (KTRs) from the Department of Organ Transplantation of Zhujiang Hospital of Southern Medical University before and after receiving immunosuppressive agents. Based on CyTOF analysis, we detected 363,342 live single immune cells. We found that the immune cell types of the KTRs before and after receipt of immunosuppressive agents were mainly divided into CD4+ T cells, CD8+ T cells, B cells, NK cells/γδ T cells, monocytes/macrophages, granulocytes, and dendritic cells (DCs). After further reclustering of the above cell types, it was found that the immune cell subclusters in the peripheral blood of patients underwent major changes after receipt of immunosuppressants. After receiving immunosuppressive therapy, the peripheral blood of KTRs had significantly increased levels of CD57+NK cells and significantly decreased levels of central memory CD4+ T cells, follicular helper CD4+ T cells, effector CD8+ T cells, effector memory CD8+ T cells and naive CD8+ T cells. This study used CyTOF to classify immune cells in the peripheral blood of KTRs before and after immunosuppressive treatment, further compared differences in the proportions of the main immune cell types and immune cell subgroups before and after receipt of immunosuppressants, and provided relatively accurate information for assessment and treatment strategies for KTRs.

Evaluating the repertoire of immune checkpoint markers expressed on peripheral and ascites CD8+ T cells in ovarian cancer.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. 5571-5571 ◽  
Author(s):  
Stephanie Gaillard ◽  
Chelsae Dumbauld ◽  
Alyssa Bilewski ◽  
Jessie A Ehrisman ◽  
Angeles Alvarez Secord ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Flow Cytometry ◽  
T Cells ◽  
Cd8 T Cells ◽  
Immune Checkpoint ◽  
Peripheral Blood ◽  
Ascites Fluid ◽  
High Dimensional ◽  
Inhibitory Receptors ◽  
Ifn Γ

5571 Background: Understanding the immune checkpoint marker repertoire can facilitate development of therapeutic strategies to improve efficacy of immune-based therapies. We used a novel high-dimensional flow cytometry panel to determine co-expression patterns of immune checkpoint markers and effector function of CD8+ T cells from peripheral blood and ascites of patients newly diagnosed with ovarian cancer. Methods: Peripheral blood and ascites samples were collected from patients with epithelial ovarian cancer (n=8). Cells isolated from peripheral blood and ascites were used for immune profiling by multiparameter flow cytometry of 5 inhibitory receptors (PD-1, LAG-3, TIM-3, TIGIT, and BTLA) on CD8+ T cells, along with 4 functional parameters (production of each of the following: TNF-α, IFN-γ, IL-2, and upregulation of CD107a). A complementary multiplex analysis on plasma and ascites fluid was performed to quantify 14 soluble checkpoint markers. Results: The concentrations of soluble PD-1, TIM-3, LAG-3, CTLA-4, BTLA, IDO, and CD137 were increased in ascites fluid compared to plasma from patients with ovarian cancer. Ascites CD8+ T cells co-express higher levels of inhibitory receptors than peripheral CD8+ T cells. In total, CD8+ T cells in ascites retained the ability to produce effector functions at levels similar to peripheral blood. However, IFN-γ production was retained in PD-1 only expressing CD8+ T cells and decreased in CD8+ T cells co-expressing multiple receptors. Conclusions: High-dimensional flow cytometry allowed for the phenotypic and functional characterization of CD8+ T cells from ovarian cancer patients. The profile of receptor co-expression was distinct in peripheral blood compared to ascites. Collectively, our study suggests that co-expression of factors beyond PD-1 influences CD8+ T cell activity. Thus blocking PD-1 and PD-L1 alone may not be sufficient for CD8+ T cells expressing multiple inhibitory receptors.

scholarly journals Multiscale fluorescent tracking of immune cells in the liver with a highly biocompatible far-red emitting polymer probe

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Malo Daniel ◽  
Laurence Dubreil ◽  
Romain Fleurisson ◽  
Jean-Paul Judor ◽  
Timothée Bresson ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Cd8 T Cells ◽  
Immune Cells ◽  
Cell Tracking ◽  
Classical Model ◽  
Stokes Shift ◽  
Water Soluble ◽  
Two Photon Microscopy ◽  
Two Photon

Abstract The development of innovative immune cell therapies relies on efficient cell tracking strategies. For this, multiscale fluorescence-based analyses of transferred cells into the host with complementary techniques, including flow cytometry for high-throughput cell analysis and two-photon microscopy for deep tissue imaging would be highly beneficial. Ideally, cells should be labelled with a single fluorescent probe combining all the properties required for these different techniques. Due to the intrinsic autofluorescence of most tissues and especially the liver, far-red emission is also an important asset. However, the development of far-red emitting probes suitable for two-photon microscopy and compatible with clearing methods to track labelled immune cells in thick samples, remains challenging. A newly-designed water-soluble far-red emitting polymer probe, 19K-6H, with a large Stokes shift, was thus evaluated for the tracking of primary immune CD8 T cells. These cells, prepared from mouse spleen, were efficiently labelled with the 19K-6H probe, which was internalized via endocytosis and was highly biocompatible at concentrations up to 20 μM. Labelled primary CD8 T cells were detectable in culture by both confocal and two-photon microscopy as well as flow cytometry, even after 3 days of active proliferation. Finally, 19K-6H-labelled primary CD8 T cells were injected to mice in a classical model of immune mediated hepatitis. The efficient tracking of the transferred cells in the liver by flow cytometry (on purified non-parenchymal cells) and by two-photon microscopy on 800 μm thick cleared sections, demonstrated the versatility of the 19K-6H probe.

scholarly journals Whole Blood Transcriptome Analysis Reveals the Correlation between Specific Immune Cells and Septicemic Melioidosis

2021 ◽  
Vol 2021 ◽  
pp. 1-13
Author(s):  
Ke Xu ◽  
Dahua Xu ◽  
Hua Pei ◽  
Yunfan Quan ◽  
Jun Liu ◽  
...  
Keyword(s):  
Immune Response ◽  
T Cells ◽  
Cd8 T Cells ◽  
Immune Cells ◽  
Immune Cell ◽  
Expression Patterns ◽  
Multivariate Logistic Regression Analysis ◽  
Cell Types ◽  
Host Immune System ◽  
Resting Cells

Melioidosis is a serious infectious disease caused by the environmental Gram-negative bacillus Burkholderia pseudomallei. It has been shown that the host immune system, mainly comprising various types of immune cells, fights against the disease. The present study was to specify correlation between septicemic melioidosis and the levels of multiple immune cells. First, the genes with differential expression patterns between patients with septicemic melioidosis (B. pseudomallei) and health donors (control/healthy) were identified. These genes being related to cytokine binding, cell adhesion molecule binding, and MHC relevant proteins may influence immune response. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed 23 enriched immune response pathways. We further leveraged the microarray data to investigate the relationship between immune response and septicemic melioidosis, using the CIBERSORT analysis. Comparison of the percentages of 22 immune cell types in B. pseudomallei vs. control/healthy revealed that those of CD4 memory resting cells, CD8+ T cells, B memory cells, and CD4 memory activated cells were low, whereas those of M0 macrophages, neutrophils, and gamma delta T cells were high. The multivariate logistic regression analysis further revealed that CD8+ T cells, M0 macrophages, neutrophils, and naive CD4+ cells were strongly associated with the onset of septicemic melioidosis, and M2 macrophages and neutrophils were associated with the survival in septicemic melioidosis. Taken together, these data point to a complex role of immune cells on the development and progression of melioidosis.

scholarly journals Tumor-Infiltrating Immune Cells and PD-L1 as Prognostic Biomarkers in Primary Esophageal Small Cell Carcinoma

2020 ◽  
Vol 2020 ◽  
pp. 1-15
Author(s):  
Xiao Wu ◽  
Xiurong Ke ◽  
Yangpeng Ni ◽  
Liping Kuang ◽  
Fan Zhang ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Carcinoma ◽  
Small Cell Carcinoma ◽  
Cd8 T Cells ◽  
Immune Cells ◽  
Immune Checkpoint ◽  
Small Cell ◽  
Prognostic Indicators ◽  
Survival Prediction ◽  
Poor Survival

Primary esophageal small cell carcinoma (PESCC) is a weakly prevalent but lethal malignancy with early metastasis and a poor prognosis. Currently, neither effective prognostic indicators nor curative therapies are available for PESCC. Immunotherapy has now evolved into one of the most promising therapies for cancer patients. Tumor-infiltrating immune cells which are integral to the tumor immune microenvironment (TIME) are recognized as highly important for prognosis prediction, while the responsiveness to immune checkpoint blockade may be subject to the features of TIME. In this study, we aim to identify the TIME and provide indication for the applicability of immune checkpoint therapy in PESCC. We found that PD-L1 expression was detected in 33.33% (27/81) of all the patients, mostly exhibiting a stroma-only pattern and that it was positively associated with tumor-infiltrating immune cells (CD4+, CD8+, and CD163+). In 74.07% of PD-L1-positive specimens, PD-L1+CD163+ cells were colocalized more with CD4+ than CD8+ T cells. 83.95% (68/81) of all the specimens were infiltrated with more CD4+ than CD8+ T cells. Further analysis showed FoxP3+ Tregs constituted 13-27% of the total CD4+ T cell population. The Kaplan-–Meier analysis indicated several factors that contribute to poor survival, including negative PD-L1 expression, rich CD4 expression, rich FoxP3 expression, a low CD8/CD4 ratio, and a high FoxP3/CD8 ratio. A nomogram model was constructed and showed good performance for survival prediction. These results highlight that a suppressive TIME contributes to poor survival of patients with PESCC. TIME analyses might be a promising approach to evaluate the possibility and effect of immune checkpoint-based immunotherapeutics in PESCC patients.

scholarly journals Metabolic Molecule PLA2G2D Is a Potential Prognostic Biomarker Correlating With Immune Cell Infiltration and the Expression of Immune Checkpoint Genes in Cervical Squamous Cell Carcinoma

2021 ◽  
Vol 11 ◽  
Author(s):  
Hong Liu ◽  
Ruiyi Xu ◽  
Chun Gao ◽  
Tong Zhu ◽  
Liting Liu ◽  
...  
Keyword(s):  
Squamous Cell Carcinoma ◽  
T Cells ◽  
Immune Cells ◽  
Immune Checkpoint ◽  
Immune Cell ◽  
Patient Survival ◽  
Cervical Squamous Cell Carcinoma ◽  
Immune Cell Infiltration ◽  
Immune Microenvironment ◽  
Checkpoint Genes

Cervical squamous cell carcinoma (CSCC) is the major pathological type of cervical cancer (CC), the second most prevalent reproductive system malignant tumor threatening the health of women worldwide. The prognosis of CSCC patients is largely affected by the tumor immune microenvironment (TIME); however, the biomarker landscape related to the immune microenvironment of CSCC and patient prognosis is less characterized. Here, we analyzed RNA-seq data of CSCC patients from The Cancer Genome Atlas (TCGA) database by dividing it into high- and low-immune infiltration groups with the MCP-counter and ESTIMATE R packages. After combining weighted gene co-expression network analysis (WGCNA) and differentially expressed gene (DEG) analysis, we found that PLA2G2D, a metabolism-associated gene, is the top gene positively associated with immune infiltration and patient survival. This finding was validated using data from The Cancer Genome Characterization Initiative (CGCI) database and further confirmed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Finally, multiplex immunohistochemistry (mIHC) was performed to confirm the differential infiltration of immune cells between PLA2G2D-high and PLA2G2D-low tumors at the protein level. Our results demonstrated that PLA2G2D expression was significantly correlated with the infiltration of immune cells, especially T cells and macrophages. More importantly, PLA2G2D-high tumors also exhibited higher infiltration of CD8+ T cells inside the tumor region than PLA2G2D-low tumors. In addition, PLA2G2D expression was found to be positively correlated with the expression of multiple immune checkpoint genes (ICPs). Moreover, based on other immunotherapy cohort data, PLA2G2D high expression is correlated with increased cytotoxicity and favorable response to immune checkpoint blockade (ICB) therapy. Hence, PLA2G2D could be a novel potential biomarker for immune cell infiltration, patient survival, and the response to ICB therapy in CSCC and may represent a promising target for the treatment of CSCC patients.

scholarly journals 208 RTX-224, an engineered allogeneic red cell therapeutic expressing 4–1BBL and IL-12, activates immune cells in blood and spleen to promote tumor growth inhibition in mice

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A219-A219
Author(s):  
Anne-Sophie Dugast ◽  
Shannon McArdel ◽  
Zafira Castano ◽  
Maegan Hoover ◽  
Arjun Reddy Bollampalli ◽  
...  
Keyword(s):  
T Cells ◽  
Tumor Growth ◽  
Growth Inhibition ◽  
Nk Cells ◽  
Mechanism Of Action ◽  
Cd8 T Cells ◽  
Immune Cells ◽  
Cell Types ◽  
Tumor Growth Inhibition ◽  
Tumor Bearing

BackgroundAgonist antibodies and recombinant cytokines have had limited success in the clinic due to three factors: severe toxicity leading to a narrow therapeutic index, the diminished activity of an agonistic antibody compared with natural ligand, and the lack of multiple signals needed to effectively activate most cell types. To address these limitations, Rubius Therapeutics has developed RTX-224, an allogeneic red cell therapeutic genetically engineered to express hundreds of thousands of copies of 4-1BBL and IL-12 in their natural conformation on the cell surface. RTX-224 is designed to activate four key target cell types: CD4+ and CD8+ T cells, antigen presenting cells and NK cells for a broad and effective anti-tumor response while providing improved safety due to the restricted biodistribution of red blood cells to the vasculature and spleen. Here we investigated the potential efficacy and mechanism of action of RTX-224 using the mouse surrogate mRBC-224.MethodsmRBC-224 was administered intravenously (i.v.) to normal or tumor-bearing mice (B16F10 tumor models). Blood, spleen and tumors were harvested and the pharmacodynamic effects of mRBC-224 on immune cells were evaluated.ResultsmRBC-224 administered to mice inoculated i.v. with B16F10 melanoma reduced the number of metastases (p<0.0001 and 76.8% tumor growth inhibition on Day 14). This was accompanied by increased proliferation (Ki67+) and cytotoxicity (GzmB+) of tumor-infiltrating CD8+ T cells and NK cells, and an increased CD8+ effector memory (TEM) phenotype. Similarly, mRBC-224 reduced tumor growth in the B16F10 s.c. model (p<0.0001 and 56.2% tumor growth inhibition on Day 9), and this was associated with increased frequency of activated (MHC-II+) tumor-infiltrating macrophages. Consistent with the known biodistribution of red cells, mRBC-224 did not distribute to the tumor but was predominantly localized in the blood and spleen raising the question about mRBC-224 mechanism of action in mediating antitumor responses. In normal and B16F10 s.c. tumor-bearing mice, mRBC-224 induced the activation of CD8+ T cells, NK cells and monocytes/macrophages in blood and spleen in a dose-dependent manner. PD studies in the tumor suggest that these activated immune cells are capable of trafficking from blood/spleen to the tumor. These results align with published data suggesting that activated T cells in the spleen or blood can replenish exhausted tumor-infiltrating cells.ConclusionsTaken together, these data unveil the mechanism of action of mRBC-224 and suggest that mRBC-224 activate immune cells in the spleen and blood, leading to their trafficking into the tumor microenvironment to promote efficacy.

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