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Clonal replacement of tumor-specific T cells following PD-1 blockade [bulk RNA]

ABSTRACT: Immunotherapies that block inhibitory checkpoint receptors on T cells have transformed the clinical care of cancer patients. However, the clonal origin of tumor-specific T cells following checkpoint blockade in patients remains unclear. Here, we performed paired single-cell RNA- and T cell receptor (TCR)- sequencing on site-matched tumors from patients with basal cell carcinoma (BCC) pre- and post-anti-PD-1 therapy. Tracking TCR clonotypes and transcriptome phenotypes revealed a coupling of tumor-recognition, clonal expansion, and T cell dysfunction: the response to treatment was accompanied by a clonal expansion of CD8+CD39+ T cells, which co-expressed markers of chronic T cell activation and exhaustion. However, this response was not accompanied by an expansion of pre-existing tumor-specific T cell clonotypes; rather, expanded T cell clones post-therapy comprised novel clonotypes, which were not previously observed in the same tumor. Clonal replacement of T cells was preferentially observed in tumor-specific exhausted CD8+ T cells, compared to other distinct T cell phenotypes, and was more evident in patients who exhibited a clinical response to treatment. These results, enabled by single-cell multi-omic profiling of clinical samples, demonstrate that the chronic activation of pre-existing tumor-infiltrating T cells may limit their re-invigoration following checkpoint blockade, and that a successful response relies on the expansion of a distinct repertoire of tumor-specific T cell clones.

ORGANISM(S): Homo sapiens

PROVIDER: GSE123812 | GEO | 2019/05/24

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:Infiltrating T-lymphocytes from the peripheral blood into the central nervous system (CNS) play a dynamic role in the development of a neurological immune-mediated diseases. HAM/TSP is a chronic progressive inflammatory neurological disorder associated with human T-cell lymphotropic virus type I (HTLV-I) infection. In this chronic myelopathy, virus-infected circulating T-cells infiltrate the CNS and an immune response is initiated against the components of CNS. As the HTLV-I proviral load (PVL) has been used as the best clinical marker for patient diagnostic with HAM/TSP, we hypothesized there might be a signature on T-cell receptor (TCR) clonal repertoire in these patients, which could distinguish HAM/TSP patients from the healthy population, as well as from patients with a more heterogeneous CNS-reactive inflammatory disease as multiple sclerosis (MS). With this in mind, we applied an innovative unbiased molecular technique – unique molecular identifier (UMI) library-strategy to investigate with high accuracy the TCR clonal repertoire by high throughput sequencing (HTS) technology. cDNA-TCR β-chain libraries were sequenced from 2 million peripheral mononuclear cells (PBMCs) in 14 HAM/TSP patients, 34 MS patients and 20 healthy controls (HC). To address whether the clonal expansion correlates with the patient’s PVL level, analysis of longitudinal TCR repertoire was performed in 2 HAM/TSP patients. Over 5.6 million TCR sequences were generated per sample on HiSeq 2500 Illumina system and analyzed through the molecular identifier groups-based error correction pipeline (MiGEC). Bioinformatic analysis showed that clones with more than 8 reads had a lower coefficient of variation (CV) and then could be used with confidence to evaluate the TCR clonal expansion. While HAM/TSP patients showed the higher clonal T-cell expansion compared to MS and HC, increase of the TCR clonal expansion was inversely correlated with the diversity of TCR repertoire in all subject’s group. In addition, correlation of the PVL with TCR clonal expansion was observed in HAM/TSP patients at longitudinal time-points. Surprisingly, MS patients showed a higher diversity of TCR repertoire along with a very slight clonal T-cell expansion in comparison to either HAM/TSP patients or HC. Despite of the higher TCR clonal expansion in HAM/TSP patients, a non-shared or “private” TCR repertoire was observed in these patients. No clones that shared the same CDR3 amino acid sequences were seen in HC and MS patients. However, a cluster of related CDR3 amino acid sequences were observed for 18 out of 34 MS patients when evaluated by phylogenetic tree analysis. It suggestes that a TCR-repertoire signature might characterize patients with MS. Our findings suggest that even though a unique TCR-b repertoire shapes the immune response in patients with neurological immune-mediated disease, a relatedness on clonal T-cell repertoire exist in MS.

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Clonal replacement of tumor-specific T cells following PD-1 blockade [bulk RNA]

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