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CITE seq on ALS4 and control CD8 T cells

ABSTRACT: The goal of this study is to compare the transcriptional program and the TCR sequences of different subsets of CD8 T cells purified from the peripheral blood of 3 patienst with Amyotrophic Lateral Sclerosis 4 (ALS4) and 3 age-matched controls (unreleated). Peripheral blood mononuclear cells (PBMCs) were purified from 3 ALS4 patients and 3 age-matched healthy controls.By combining RNA and surface protein expression followed by multidimensional reduction using the Uniform Manifold Approximation and Projection (UMAP), we could identified 9 clusters corresponding to different CD8 T cell subsets: naïve/naïve like (clusters 0, 2, 5, 8), central memory (cluster 7), effector memory (clusters 4 and 6), TEMRA (clusters 1 and 3), and mucosal associated invariant T (MAIT) cells (cluster 9). ALS4 patients exhibited a higher proportion of TEMRA, and a decreased frequency of effector/effector memory cells, as compared to controls. Downregulation of IL7R and CCR7, and upregulation of GZMM and GZMB (encoding for Granzyme M and B, respectively), KLRB1 (Killer Cell Lectin Like Receptor B1), NKG7 (Natural Killer Cell Granule Protein 7), FGFBP2 (Fibroblast Growth Factor Binding Protein 2) and CCL4 (Chemokine (C-C motif) ligands 4), all of which correlate with a TEMRA phenotype are among the top deregulated genes in ALS4 CD8 T cells. The proportion of naïve cells seemed to be increased as well; however only 1 patient has an abnormal high frequency of CCR7+CD45RA+CD28+ cells, while the TEMRA signature was consistent among all 3 ALS4 donors. More than 90% of expanded clones (≥5) are TEMRA cells in ALS4 patients, in comparison to ~ 60% in healthy controls. By matching UMAP coordinates from RNA and TCR analyses, we observed a colocalization between the most expanded clones, i.e. CD8 T cells in which the same TCR sequence was shared by more than 20 cells, and the TEMRA subset that we found enriched in patients. We quantified this clonal expansion and found that over half of all TEMRA cells comprised of highly expanded clones in patients. Hyperexpanded (>100 clones) TEMRA cells were detected in 2 of the 3 patients, with one clone reaching as high as 407 cells. No hyperexpanded clones were found in other CD8 T cell subsets or in control patients. Comparison of surface protein expression from our CITE-seq analysis in “Low” and “High” (>20) expanded clones from ALS4 patients revealed a stronger TEMRA phenotype in the latter, as indicated by CD27 downregulation and CD45RA and CD57 upregulation.

ORGANISM(S): Homo sapiens

PROVIDER: GSE180410 | GEO | 2022/04/22

REPOSITORIES: GEO

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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.

Project description:Our team had previously explored the potential of expanding cord blood (CB) derived γδ T cells (CB-gdT) as well as their corresponding ability to target primary acute myeloid leukemia (AML) cells. Using a feeder cell line-based in vitro expansion protocol, we achieved a clinically relevant scale expansion of γδ T cells over a period of 14 days. These cells exhibit variable degree of potency against a range of human AML cell lines and primary patient samples. In order to dissect the cellular and molecular programs governing the activation, differentiation and functional states of our in vitro expanded CB-gdT, we performed multiplex single cell sequencing analysis using the 10X Genomics Chromium System. After initial quality check and filtering, data from a total of 4,276 cells were retrieved. Among which, we identified 742 unique TCRγδ clonotypes, representing 18.6% of the starting 4,000 FACS purified γδ T cells seeded for expansion. Consistent to our FACS analysis, Vδ1 is the predominant TRD chain in the expanded cultures, accounting for 61.2% of all clones. Based on uniform manifold approximation and projection for dimension reduction (UMAP), all cells were clustered into 11 subsets. Key cytotoxic genes including GZMB, GZMA and NKG7 were all highly expressed across all clusters, indicating that the expanded cells were indeed functionally cytotoxic. Comparing against multiple curated gene sets, we have identified 3 main subsets of γδ T cells: the Proliferative, Cytotoxic γδ T cells (P-CT), Differentiated Cytotoxic γδ T cells (D-CT) and Late Activated Cytotoxic γδ T cells (LA-CT). P-CT (~46% of all cells) shows an expression profile positively associated with cell proliferation as well as increased cell surface expression of memory T cell markers CD27, CCR7 and CD62L. Similar to cytotoxic genes, genes associated with TCR signaling and interferon response were found to be expressed across all cell clusters, yet with elevated levels in D-CT and LA-CT. Furthermore, cell surface expression of different NK receptors including NKG2D, DNAM1 and NKp30 are more enriched in LA-CT compared to the other 2 subsets, suggesting the acquisition of additional NK receptor related functions in this group of cells. Consistent with the concept of progressive γδ T cell differentiation and activation in culture, we found that in 85 (11.5%) of the γδ T cell clones bearing more than 10 cells each, all clones contain cells distributed across the 3 different γδ T cell subsets. This is the first report on single cell immune profiling of in vitro expanded gdT cells derived from human CB.

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CITE seq on ALS4 and control CD8 T cells

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