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The development of T cell receptor-engineered T cells (TCR-T) targeting intracellular antigens is a promising strategy for treating solid tumors; however, the mechanisms underlying their effectiveness remain poorly understood. In this study, we employed advanced techniques to investigate the functional state of T cells engineered with retroviral vectors to express a TCR specific for the NY-ESO-1 157-165 peptide in the HLA-A*02:01 context. Flow cytometry revealed a predominance of naïve T cells. Gene expression profiling using NanoString technology revealed upregulation of genes encoding chemokine receptors CCR2 and CCR5, indicating enhanced migration towards tumor sites. In the SK-Mel-37 xenograft model, these transduced T cells achieved complete tumor eradication. Furthermore, single-cell RNA sequencing (scRNA-seq) conducted 14 days post-TCR T cell infusion provided a comprehensive analysis of the in vivo adaptation of these cells, identifying a distinct subset of CD8+ effector T cells with an NK cell-like gene expression profile. Our findings indicate that NY-ESO-1 TCR-transduced T cells have the potential to mediate dual antitumor effects through both antigen-independent NK-like and antigen-specific CTL-like responses. This study underscores the potential of NY-ESO-1 TCR-T cells as potent tumor-eradicating agents, highlighting the importance of harnessing their versatile functional capabilities to refine and enhance therapeutic strategies.

IntroductionAdoptive cell therapy using TCR-engineered T-cells is one of the most effective strategies against tumor cells. The TCR T-cell approach has been well tested against a variety of blood neoplasms but is yet to be deeply tested against solid tumors. Among solid tumors, cancer-testis antigens are the most prominent targets for tumor-specific therapy, as they are usually found on cells that lie behind blood-tissue barriers.MethodsWe have employed a novel efficient protocol for MAGE-A3-specific T-cell clonal expansion, performed single-cell multi-omic analysis of the expanded T-cells via BD Rhapsody, engineered a selected T-cell receptor into a lentiviral construct, and tested it in an in vitro LDH-cytotoxicity test.Results and discussionWe have observed a 191-fold increase in the MAGE-A3-specific T-cell abundance, obtained a dominant T-cell receptor via single-cell multi-omic BD Rhapsody data analysis in the TCRscape bioinformatics tool, and observed potent cytotoxicity of the dominant-clonotype transduced TCR T-cells against a MAGE-A3-positive tumor. We have demonstrated the efficiency of our T-cell enrichment protocol in obtaining potent anti-tumor T-cells and their T-cell receptors, especially when paired with the modern single-cell analysis methods.

The role of Erythroid cells in immune regulation and immunosuppression is one of the emerging topics in modern immunology that still requires further clarification as Erythroid cells from different tissues and different species express different immunoregulatory molecules. In this study, we performed a thorough investigation of human bone marrow Erythroid cells from adult healthy donors and adult acute lymphoblastic leukemia patients using the state-of-the-art single-cell targeted proteomics and transcriptomics via BD Rhapsody and cancer-related gene copy number variation analysis via NanoString Sprint Profiler. We found that human bone marrow Erythroid cells express the ARG1, LGALS1, LGALS3, LGALS9, and C10orf54 (VISTA) immunosuppressive genes, CXCL5, CXCL8, and VEGFA cytokine genes, as well as the genes involved in antimicrobial immunity and MHC Class II antigen presentation. We also found that ARG1 gene expression was restricted to the single erythroid cell cluster that we termed ARG1-positive Orthochromatic erythroblasts and that late Erythroid cells lose S100A9 and gain MZB1 gene expression in case of acute lymphoblastic leukemia. These findings show that steady-state erythropoiesis bone marrow Erythroid cells express myeloid signature genes even without any transdifferentiating stimulus like cancer.

Erythroid cells, serving as progenitors and precursors to erythrocytes responsible for oxygen transport, were shown to exhibit an immunosuppressive and immunoregulatory phenotype. Previous investigations from our research group have revealed an antimicrobial gene expression profile within murine bone marrow erythroid cells which suggested a role for erythroid cells in innate immunity. In the present study, we focused on elucidating the characteristics of human bone marrow erythroid cells through comprehensive analyses, including NanoString gene expression profiling utilizing the Immune Response V2 panel, a BioPlex examination of chemokine and TGF-beta family proteins secretion, and analysis of publicly available single-cell RNA-seq data. Our findings demonstrate that an erythroid cell subpopulation manifests a myeloid-like gene expression signature comprised of antibacterial immunity and neutrophil chemotaxis genes which suggests an involvement of human erythroid cells in the innate immunity. Furthermore, we found that human erythroid cells secreted CCL22, CCL24, CXCL5, CXCL8, and MIF chemokines. The ability of human erythroid cells to express these chemokines might facilitate the restriction of immune cells in the bone marrow under normal conditions or contribute to the ability of erythroid cells to induce local immunosuppression by recruiting immune cells in their immediate vicinity in case of extramedullary hematopoiesis.

Hemorrhage, a condition that accompanies most physical trauma cases, remains an important field of study, a field that has been extensively studied in the immunological context for myeloid and lymphoid cells, but not as much for erythroid cells. In this study, we studied the immunological response of murine erythroid cells to acute blood loss using flow cytometry, NanoString immune transcriptome profiling, and BioPlex cytokine secretome profiling. We observed that acute blood loss forces the differentiation of murine erythroid cells in both bone marrow and spleen and that there was an up-regulation of several immune response genes, in particular pathogen-associated molecular pattern sensing gene Clec5a in post-acute blood loss murine bone marrow erythroid cells. We believe that the up-regulation of the Clec5a gene in bone marrow erythroid cells could help bone marrow erythroid cells detect and eliminate pathogens with the help of reactive oxygen species and antimicrobial proteins calprotectin and cathelicidin, the genes of which (S100a8, S100a9, and Camp) dominate the expression in bone marrow erythroid cells of mice.

Hypoxia leads to metabolic changes at the cellular, tissue, and organismal levels. The molecular mechanisms for controlling physiological changes during hypoxia have not yet been fully studied. Erythroid cells are essential for adjusting the rate of erythropoiesis and can influence the development and differentiation of immune cells under normal and pathological conditions. We simulated high-altitude hypoxia conditions for mice and assessed the content of erythroid nucleated cells in the spleen and bone marrow under the existing microenvironment. For a pure population of CD71+ erythroid cells, we assessed the production of cytokines and the expression of genes that regulate the immune response. Our findings show changes in the cellular composition of the bone marrow and spleen during hypoxia, as well as changes in the composition of the erythroid cell subpopulations during acute hypoxic exposure in the form of a decrease in orthochromatophilic erythroid cells that are ready for rapid enucleation and the accumulation of their precursors. Cytokine production normally differs only between organs; this effect persists during hypoxia. In the bone marrow, during hypoxia, genes of the C-lectin pathway are activated. Thus, hypoxia triggers the activation of various adaptive and compensatory mechanisms in order to limit inflammatory processes and modify metabolism.

Adoptive T-cell therapies tailored for the treatment of solid tumors encounter intricate challenges, necessitating the meticulous selection of specific target antigens and the engineering of highly specific T-cell receptors (TCRs). This study delves into the cytotoxicity and functional characteristics of in vitro-cultured T-lymphocytes, equipped with a TCR designed to precisely target the cancer-testis antigen NY-ESO-1. Flow cytometry analysis unveiled a notable increase in the population of cells expressing activation markers upon encountering the NY-ESO-1-positive tumor cell line, SK-Mel-37. Employing the NanoString platform, immune transcriptome profiling revealed the upregulation of genes enriched in Gene Ontology Biological Processes associated with the IFN-γ signaling pathway, regulation of T-cell activation, and proliferation. Furthermore, the modified T cells exhibited robust cytotoxicity in an antigen-dependent manner, as confirmed by the LDH assay results. Multiplex immunoassays, including LEGENDplex™, additionally demonstrated the elevated production of cytotoxicity-associated cytokines driven by granzymes and soluble Fas ligand (sFasL). Our findings underscore the specific targeting potential of engineered TCR T cells against NY-ESO-1-positive tumors. Further comprehensive in vivo investigations are essential to thoroughly validate these results and effectively harness the intrinsic potential of genetically engineered T cells for combating cancer.

Nucleated erythroid cells (NECs) are the precursors of erythrocytes. They can be found in various hematopoietic tissues or in the blood. Recently, they have been shown to be active players in immunosuppression through the synthesis of arginase-2 and reactive oxygen species. In this work, we studied NECs in adult bone marrow, umbilical cord blood, and foetal liver parenchyma using single-cell RNA sequencing and found that: (1) all studied NECs expressed the same set of genes, which was enriched in “GO biological process” immunity-related terms; (2) early and late NECs had differential expression of the genes associated with immunosuppression, cell cycle progression, apoptosis, and glycolysis; (3) NECs from different tissues of origin had differential expression of the genes associated with immunosuppression.