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Transcriptomic analysis of ChAdOx/MVA P1A vaccine-induced P1A-35-43-specific CD8+ T-cells by single-cell RNA-sequencing

ABSTRACT: This study aimed to analyze the transcriptional profiles of ChAdOx/MVA vaccine induced P1A-35-43-specific CD8+ T-cells and how this is affected by both tissue location and anti-PD-1 treatment. 15V4T3 tumour-bearing DBA/2 mice were vaccinated with ChAdOx/MVA P1A with or without anti-PD-1 treatment. Following treatment, spleens and tumours were collected, processed to single-cell suspension and P1A-35-43-specific CD8+ T-cells were then isolated by MHC-class I multimer staining and FACS. Single-cell RNA-seq experiments were performed using the 10X Genomics platform to analyze P1A-specific CD8+ T-cell transcriptional profiles. Results of this transcriptomic analysis showed that cells segregated into distinct phenotypic subtypes reflecting either effector or stem-like states, the distribution of which was dependent on tissue of origin and treatment status. These results provide an in depth phenotypic analysis of MAGE-type-antigen-specific CD8+ T-cells induced by ChAdOx/MVA vaccination and how phenotype is influenced by physiological location and immune checkpoint blockade treatment.

ORGANISM(S): Mus musculus

PROVIDER: GSE181183 | GEO | 2021/08/01

REPOSITORIES: GEO

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Project description:This study aimed to analyse the effect of immunotherapeutic treatment with ChAdOx/MVA MAGE vaccine and anti-PD-1 on the transcriptional mRNA profiles of murine 15V4T3 tumours. 15V4T3 tumour-bearing mice were treated with either a PBS control, anti-PD-1 inhibitor (3-doses of 100 µg 3-days apart), ChAdOx/MVA P1A vaccine (10^7 IU / 10^6 PFU doses 1-week apart), or with a combination of vaccine + anti-PD-1. Following treatment, RNA was isolated from tumour tissue samples and then tumour transcriptomic gene expression profiles were analysed by RNA-sequencing. Sequenced reads were mapped to the mouse genome GRCm38. A counts matrix reporting the numbers of reads mapping to each gene was then generated, and abundance measurements performed using TMM-normalized (EdgeR) counts data to quantify gene expression levels. 14937 expressed transcripts were identified as expressed above a minimum threshold. Results from the transcriptomic analysis indicated that ChAdOx/MVA P1A vaccination had a profound effect on the gene expression profiles of 15V4T3 tumours. Approximately 1250 differentially expressed genes were identified in tumours from vaccinated compared to unvaccinated mice using a threshold for differential expression of fold change > 2 and Padj < 0.05. Furthermore, gene expression signatures corresponding to a CD8+ T-cell driven inflammatory response and an IFNg response were found to be highly enriched in tumours from vaccinated mice. Such gene expression signatures have been found in other studies to be predictive of response to immunotherapy and anti-PD-1 immune checkpoint blockade. GSVA further confirmed the enrichment of gene sets corresponding to immune activation, a pro-inflammatory response and T-cell activity in the mRNA expression data from tumours of vaccinated mice compared to unvaccinated. Taken together these findings show that a ChAdOx/MVA vaccine targeting a MAGE type antigen can generate and drive tumour gene expression patterns associated with a T-cell inflammatory response and positive response to cancer immunotherapy.

Project description:Background Clinical success of T cell receptor (TCR) gene therapy has previously been demonstrated for NY-ESO-1 TCR gene therapy. To increase numbers of cancer patients that can be treated with TCR gene therapy we aimed to identify a novel set of high-affinity cancer specific TCRs targeting different cancer testis (CT) antigens in prevalent HLA class I alleles. Methods In this study, we selected based on publicly available gene expression databases the most promising CT genes to target. From these selected genes we identified by HLA peptidomics the naturally processed and presented HLA class I peptides. With these peptide-HLA tetramers were generated, and by single cell sorting CT specific CD8+ T cells were selected, and expanded from the allo-HLA repertoire of healthy donors. By several functional assays high avidity CT-specific T cell clones with safe recognition pattern were selected. To evaluate the potential for clinical application in TCR gene therapy, TCRs were sequenced and transferred into peripheral blood derived CD8+ T cells. Results In total we identified, 7 novel CT-specific TCRs that effectively target MAGE-A1, MAGE-A3, MAGE-A6 and MAGE-A9 in the context of human leukocyte antigen(HLA) -A1, -A2, -A3, -B7, -B35 and -C7. TCR gene transfer into CD8⁺ T cells resulted in efficient cytokine production and cytotoxicity of variety of different tumor types without detectable cross-reactivity. In addition, major in vivo antitumor effects of MAGE-A1 specific TCR engineered CD8⁺ T cells was observed in an orthotopic xenograft model for established multiple myeloma, in which bone marrow located tumor cells were completely eradicated after T cell injection. Conclusion The identification of 7 novel CT-specific TCRs, reactive against CT antigens presented in a variety of different HLA class I alleles, allows selection of therapeutic TCRs for an increased number of cancer patients, and will improve development of personalized TCR gene therapy.

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Transcriptomic analysis of ChAdOx/MVA P1A vaccine-induced P1A-35-43-specific CD8+ T-cells by single-cell RNA-sequencing

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