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Single-cell transcriptome analysis reveals gene signatures associated with T-cell persistence following adoptive cell therapy

ABSTRACT: A patient (4095) with metastatic colorectal cancer was treated with polyclonal tumor infiltrating lymphocytes (TILs) targeting the KRAS(G12D) hotspot mutation. Three dominant clonotypes specifically recognizing KRAS(G12D) epitopes were identified, but we found that only two clonotypes persisted 40 days after TIL treatment. Because of these findings, in this study, we utilized a 10X system to perform single-cell TCR/transcriptome analysis for the TILs isolated from this patient, together with 4 additional TILs isolated from 4 patients (4007, 4069, 4071, 4081) treated within 5-month window. In addition, we performed single-cell TCR/transcriptome analysis on Patient 4095's peripheral blood lymphocytes (PBL) day 12 or day 40 after TIL treatment. We also utilized a low-throughput Fluidigm C1 system to perform single-cell RNA-Seq on TIL4095. Please use accession number GSE136610 to find the dataset.

ORGANISM(S): Homo sapiens

PROVIDER: GSE136394 | GEO | 2019/09/10

REPOSITORIES: GEO

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Project description:Background: Anti-tumor immunity is highly heterogeneous between individuals; however, underlying mechanisms remain elusive, despite their potential to improve personalized cancer immunotherapy. Head and neck squamous cell carcinomas (HNSCCs) vary significantly in immune infiltration and therapeutic responses between patients, demanding a mouse model with appropriate heterogeneity to investigate mechanistic differences. Methods: We developed a unique HNSCC mouse model to investigate underlying mechanisms of heterogeneous anti-tumor immunity. This model system may provide a better control for tumor-intrinsic and host-genetic variables, thereby uncovering the contribution of the adaptive immunity to tumor eradication. We employed single-cell T-cell receptor (TCR) sequencing coupled with single-cell RNA sequencing to identify the difference in TCR repertoire of CD8 tumor infiltrating lymphocytes (TILs) and the unique activation states linked with different TCR clonotypes. Results: We discovered that genetically identical wild-type recipient mice responded heterogeneously to the same SCC tumors orthotopically transplanted into the buccal mucosa. While tumors initially grew in 100% of recipients and most developed aggressive tumors, ~25% of recipients reproducibly eradicated tumors without intervention. Heterogeneous anti-tumor responses were dependent on CD8 T cells. Consistently, CD8 TILs in regressing tumors were significantly increased and more activated. Single-cell TCR-sequencing revealed that CD8 TILs from both growing and regressing tumors displayed evidence of clonal expansion compared with splenic controls. However, top TCR clonotypes and TCR specificity groups appear to be mutually exclusive between regressing and growing TILs. Furthermore, many TCRa/TCRb sequences only occur in one recipient. By coupling single-cell transcriptomic analysis with unique TCR clonotypes, we found that top TCR clonotypes clustered in distinct activation states in regressing vs. growing TILs. Intriguingly, the few TCR clonotypes shared between regressors and progressors differed greatly in their activation states, suggesting a more dominant influence from tumor microenvironment than TCR itself on T cell activation status. Conclusions: We reveal that intrinsic differences in the TCR repertoire of TILs and their different transcriptional trajectories may underlie the heterogeneous anti-tumor immune responses in different hosts. We suggest that anti-tumor immune responses are highly individualized and different hosts employ different TCR specificities against the same tumors, which may have important implications for developing personalized cancer immunotherapy.

Project description:Recent reports have detailed the efficacy of the adoptive cell transfer (ACT) of autologous anti-tumor lymphocytes for the treatment of patients with melanoma refractory to standard therapy. Here we report the microarray analysis of tumor infiltrating lymphocytes (TIL) generated from twenty-eight melanoma patients enrolled in a treatment protocol that combines non-myeloablative lymphodepletion followed by adoptive cell transfer. The patients were divided into two groups Ÿ twelve who experienced an objective response according to RECIST criteria and sixteen who did not. Using several methods of data analysis, no consistent overt changes in gene expression, pathways or classical markers of lymphocyte development were detected between TIL administered to responding versus non-responding patients. Only one non Y chromosome gene, phosphoserine phophatase-like (PSPHL), was found to be over expressed primarily in lymphocytes that did not result in tumor regression and seemed to negatively correlate with in vivo survival of transferred TIL. As persistence of lymphocyte clonotypes in vivo was correlated with patient response, cyro-preserved treatment TIL cultures were bead-separated into in vivo persisting and non-persisting fractions. Subsequent microarray analysis of these persisting and non-persisting clonotypes also did not reveal any consistent differences in gene expression. Finally, to determine if the lymphocytes used in our adoptive cell transfer studies had similar gene expression profiles, the twenty-eight TIL samples were compared to 10 samples generated through limiting dilution cloning from an early ACT trial. This analysis revealed differences in the gene expression of lymphocytes that have been used in these two different ACT trials. However, the inability to find differential gene expression in tumor infiltrating lymphocytes that correlate to patient response or persistence suggests that differences affecting these clinical events lie in host or tumor factors rather than transferred cells. Keywords: cell type comparison design

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Single-cell transcriptome analysis reveals gene signatures associated with T-cell persistence following adoptive cell therapy

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