Single-cell T-cell Receptor Sequencing Research Articles

Shared lung and joint T cell repertoire in early rheumatoid arthritis driven by cigarette smoking

ObjectivesSmoking has been associated with an increased risk of developing rheumatoid arthritis (RA) in individuals carrying shared epitope (SE) HLA-DRB1 alleles. Yet, little is known about the regional and systemic...

TIRTL-seq: Deep, quantitative, and affordable paired TCR repertoire sequencing.

ɑ/β T cells are key players in adaptive immunity. The specificity of T cells is determined by the sequences of the hypervariable T cell receptor (TCR) ɑ and β chains. Although bulk TCR sequencing offers a cost-effective approach for in-depth TCR repertoire profiling, it does not provide chain pairings, which are essential for determining T cell specificity. In contrast, single-cell TCR sequencing technologies produce paired chain data, but are limited in throughput to thousands of cells and are cost-prohibitive for cohort-scale studies. Here, we present TIRTL-seq (Throughput-Intensive Rapid TCR Library sequencing), a novel approach that generates ready-to-sequence TCR libraries from live cells in less than 7 hours. The protocol is optimized for use with non-contact liquid handlers in an automation-friendly 384-well plate format. Reaction volume miniaturization reduces library preparation costs to <$0.50 per well. The core principle of TIRTL-seq is the parallel generation of hundreds of libraries providing multiple biological replicates from a single sample that allows precise inference of both frequencies of individual clones and TCR chain pairings from well-occurrence patterns. We demonstrate scalability of our approach up to 1 million unique paired αβTCR clonotypes corresponding to over 30 million T cells per sample at a cost of less than $2000. For a sample of 10 million cells the cost is ~$200. We benchmarked TIRTL-seq against state-of-the-art 5'RACE bulk TCR-seq and 10x Genomics Chromium technologies on longitudinal samples. We show that TIRTL-seq is able to quantitatively identify expanding and contracting clonotypes between timepoints while providing accurate TCR chain pairings, including distinct temporal dynamics of SARS-CoV-2-specific and EBV-specific CD8+ T cell responses after infection. While clonal expansion was followed by sharp contraction for SARS-CoV-2 specific TCRs, EBV-specific TCRs remained stable once established. The sequences of both ɑ and β TCR chains are essential for determining T cell specificity. As the field moves towards greater applications in diagnostics and immunotherapy that rely on TCR specificity, we anticipate that our scalable paired TCR sequencing methodology will be instrumental for collecting large paired-chain datasets and ultimately extracting therapeutically relevant information from the TCR repertoire.

DEPICT-seq: Single-Cell Transcriptomic Analysis of Rare Cell Subsets Isolated via Nucleic Acid Cytometry.

The ability to dive deep into specific rare cell populations is critical for understanding tissue physiology and pathology across various biological domains. As single-cell RNA-seq flourishes, many newly discovered cell subtypes are defined by their transcriptomic markers. However, our ability to retrieve and analyze cells based on their nucleic acid markers remains underdeveloped. Here, we present Double Emulsion PCR-Initiated Cell sorting and Transcriptomic Sequencing (DEPICT-seq), a high-throughput droplet nucleic acid cytometry method that integrates batch cell fixation for cellular information preservation, double emulsion digital PCR-based cell sorting to target nucleic acid markers of interest, and in-depth full-length transcriptomic analyses at single-cell resolution. We utilize DEPICT-seq to isolate and characterize T cell receptor (TCR)-engineered T cells within a mixed population and also demonstrate a variation of the workflow by incorporating an RNase H-dependent PCR step to enrich full-length TCR sequences for paired single-cell TCR sequencing and transcriptomic profiling.

Dissecting the Distinct Tumor Microenvironments of HRD and HRP Ovarian Cancer: Implications for Targeted Therapies to Overcome PARPi Resistance in HRD Tumors and Refractoriness in HRP Tumors.

High-grade serous tubo-ovarian cancer (HGSTOC) is an aggressive gynecological malignancy including homologous recombination deficient (HRD) and homologous recombination proficient (HRP) groups. Despite the therapeutic potential of poly (ADP-ribose) polymerase inhibitors (PARPis) and anti-PDCD1 antibodies, acquired resistance in HRD and suboptimal response in HRP patients necessitate more precise treatment. Herein, single-cell RNA and single-cell T-cell receptor sequencing on 5 HRD and 3 HRP tumors are performed to decipher the heterogeneous tumor immune microenvironment (TIME), along with multiplex immunohistochemistry staining and animal experiments for validation. HRD tumors are enriched with immunogenic epithelial cells, FGFR1+PDGFRβ+ myCAFs, M1 macrophages, tumor reactive CD8+/CD4+ Tregs, whereas HRP tumors are enriched with HDAC1-expressing epithelial cells, indolent CAFs, M2 macrophages, and bystander CD4+/CD8+ T cells. Significantly, customized therapies are proposed. For HRD patients, targeting FGFR1+PDGFRβ+ myCAFs via tyrosine kinase inhibitors, targeting Tregs via anti-CCR8 antibodies/TNFRSF4 stimulation, and targeting CXCL13+ exhausted T cells by blocking PDCD1/CTLA-4/LAG-3/TIGIT are proposed. For HRP patients, targeting indolent CAFs, targeting M2 macrophages via CSF-1/CSF-1R inhibitors, targeting bystander T cells via tumor vaccines, and targeting epithelial cells via HDAC inhibitors. The study provides comprehensive insights into HRD and HRP TIME and tailored therapeutic approaches, addressing the challenges of PARPi-resistant HRD and refractory HRP tumors.

Streamlined 96-well plate method for simultaneous single-cell TCR immune receptor repertoire analysis and immunophenotyping

Abstract This study describes an efficient immune profiling method by FACS-sorting single cells in a 96-well plate to assess clonotype repertoire diversity, paired-chain information, and cell phenotypes in a single, multiplex RT-PCR-based assay without the use of any specialized equipment. It is a cost-effective alternative to conventional single-cell technologies such as microwell arrays or droplet microfluidics. The 96-well plate contains primers for either T-cell receptor (TCR) / or TCR / chains, together with primers for 30 key T-cell markers. T cells from PBMCs are sorted in a single-cell-per-well format. We conduct RT-PCR library prep using the DriverMapTM Adaptive Immune Receptor (AIR) Repertoire Profiling Assay and sequence the CDR3 region. The results show clonotype frequencies, chain pairing details for / and / chains, and T-cell subtype classifications based on gene expression profiling. This enables in-depth analysis of TCR gene rearrangements at the single-cell level, enhancing understanding of T cell development, proliferation, and clonality, which are important in studying cancer, immunodeficiencies, and autoimmune disorders. Additionally, we can effectively identify and characterize γδ T cells by integrating single-cell TCR sequencing with RNA sequencing data, potentially useful in the development of γδ cancer immunotherapies. Overall, this method provides an efficient way to simultaneously analyze clonotypes and immunophenotypes in a single assay.

Deep learning predictions of TCR-epitope interactions reveal epitope-specific chains in dual alpha T cells

T cells have the ability to eliminate infected and cancer cells and play an essential role in cancer immunotherapy. T cell activation is elicited by the binding of the T cell receptor (TCR) to epitopes displayed on MHC molecules, and the TCR specificity is determined by the sequence of its α and β chains. Here, we collect and curate a dataset of 17,715 αβTCRs interacting with dozens of class I and class II epitopes. We use this curated data to develop MixTCRpred, an epitope-specific TCR-epitope interaction predictor. MixTCRpred accurately predicts TCRs recognizing several viral and cancer epitopes. MixTCRpred further provides a useful quality control tool for multiplexed single-cell TCR sequencing assays of epitope-specific T cells and pinpoints a substantial fraction of putative contaminants in public databases. Analysis of epitope-specific dual α T cells demonstrates that MixTCRpred can identify α chains mediating epitope recognition. Applying MixTCRpred to TCR repertoires from COVID-19 patients reveals enrichment of clonotypes predicted to bind an immunodominant SARS-CoV-2 epitope. Overall, MixTCRpred provides a robust tool to predict TCRs interacting with specific epitopes and interpret TCR-sequencing data from both bulk and epitope-specific T cells.

Abstract 3976: Single-cell TCRɑ/β and TCRγ/δ immune receptor profiling and immunophenotyping using a 96-well plate sorted-cell approach

Abstract Single-cell immune receptor profiling is a revolutionary approach that allows investigators to combine clonotype repertoire identification with paired-chain information and the phenotype of cells (e.g., cell subtype). Single-cell immune receptor profiling can be performed using a medium-throughput approach (1,000-5,000 cells) using microwell arrays or droplet microfluidics technologies. However, these assays are more complicated to run and require expensive reagents and limited sequencing throughput when compared to bulk immune receptor profiling methods. Here, we describe a low-throughput, single-cell immune profiling strategy using sorted cells in 96-well plates. The plate is pre-aliquoted with either T-cell receptor (TCR) ɑ/β or TCR γ/δ primers along with 30 crucial T-cell markers. We perform multiplex RT-PCR amplification and sequencing of the CDR3 regions. The resulting data provides the abundant clonotype counts along with the chain pairing information for these ɑ/β and γ/δ chains, together with the T-cell subtype information using gene-expression profiles. By analyzing the TCR gene rearrangement at the single-cell level, researchers can better understand T cell development, proliferation, and clonality, which are crucial for studying diseases such as cancer, immunodeficiency, and autoimmunity. Furthermore, single-cell TCR sequencing when combined with RNA sequencing datasets, facilitates the identification of γδ T cells. This method provides a standardized tool for identifying potential γδ T cell-based cancer immunotherapies. The technology's cost-effectiveness and ability to analyze clonotypes and immunophenotypes of cells in a single assay make it a valuable tool for unraveling the immune dynamics in various diseases. Citation Format: Alex Chenchik, Tianbing Liu, Mikhail Makhanov, Dongfang Hu, Lester Kobzik, Khadija Ghias, Paul Diehl. Single-cell TCRɑ/β and TCRγ/δ immune receptor profiling and immunophenotyping using a 96-well plate sorted-cell approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3976.

Abstract 25: Multimodal single-cell analysis of lifileucel tumor-infiltrating lymphocyte (TIL) product identifies additional high-resolution potential features

Abstract Introduction: Autologous TIL cell therapies such as lifileucel have demonstrated durable responses in patients (pts) with various solid tumors including melanoma and lung cancer. The TIL drug product (DP) phenotype was previously assessed and extensively characterized based on the bulk population. The current research further characterizes the TIL DP at a single-cell level using high-dimension multimodal sequencing and explores features that have a potential to inform future development of TIL therapies. Methods: Manufactured lifileucel TIL DP from 20 pts (best overall response [BOR]: 6 complete response [CR], 4 partial response, 5 stable disease, 5 progressive disease [PD]) from the registrational melanoma C144-1 trial were analyzed using single-cell RNA and T-cell receptor (TCR) sequencing, resulting in 153K single-cell transcriptomes. To aid with cell-type annotation, 130 surface proteins on TIL DP samples from a subset of 4 pts (BOR: 2 CR, 2 PD) were detected via Cellular Indexing of Transcriptomes and Epitopes by sequencing. Multimodal weighted nearest-neighbor analysis combined with manual cluster annotation (via known marker genes and proteins), cell-type-specific gene signatures, and automated annotation were used to develop a single-cell TIL (scTIL) reference map, resulting in high-resolution subsets of cells. TCR clonotypes from lifileucel TIL DP were compared to those from respective pretreatment tumors previously identified by bulk TCR sequencing. Results: Mapping of the full dataset to the novel scTIL reference revealed a CD8+ TIL-dominant composition of responder TIL DP. TIL from pts with BOR PD exhibited reduced CD8+ effector memory (Tem)-like and resident memory (Trm)-like T cells. Differential expression analysis showed that responder CD8+ TIL had increased expression of TCR signaling genes while responder CD4+ TIL exhibited gene expression consistent with a more cytotoxic-like phenotype. Relative to responders, CD8+ TIL of pts with BOR PD had lower expression of the CD39−CD69− (stem-like) gene signature and higher expression of the CD39+CD69+ (terminal differentiation) gene signature. Pseudotime trajectory analysis found that both CD8+ and CD4+ TIL of pts with BOR PD were more terminally differentiated. Single-cell TCR sequencing revealed that CD8+ T-cell clusters had greater clonal overlap than CD4+ T-cell clusters. Expanded clonotypes in pretreatment tumor tissues were more prevalent in TIL TCR repertoires of responders and were more likely to be expressed by CD8+ (specifically CD8+ Tem- and Trm-like TIL) than CD4+ T cells. Conclusions: Collectively, these results shed additional light on the cellular and molecular characteristics of ex vivo expanded lifileucel TIL DP and signal additional features with a potential to guide future TIL product development. Citation Format: Joe Dean, Joe Yglesias, Hequn Yin, Rongsu Qi. Multimodal single-cell analysis of lifileucel tumor-infiltrating lymphocyte (TIL) product identifies additional high-resolution potential features [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 25.

Virus-Associated CD8+ T-Cells Are Not Activated Through Antigen-Mediated Interaction Inside Atherosclerotic Lesions.

Viral infections have been associated with the progression of atherosclerosis and CD8+ T-cells directed against common viruses, such as influenza, Epstein-Barr virus, and cytomegalovirus, have been detected inside human atherosclerotic lesions. These virus-specific CD8+ T-cells have been hypothesized to contribute to the development of atherosclerosis; however, whether they affect disease progression directly remains unclear. In this study, we aimed to characterize the activation status of virus-specific CD8+ T-cells in the atherosclerotic lesion. The presence, clonality, tissue enrichment, and phenotype of virus-associated CD8+ T-cells in atherosclerotic lesions were assessed by exploiting bulk T-cell receptor-β sequencing and single-cell T-cell receptor (α and β) sequencing datasets on human endarterectomy samples and patient-matched blood samples. To investigate if virus-specific CD8+ T-cells can be activated through T-cell receptor stimulation in the atherosclerotic lesion, the immunopeptidome of human plaques was determined. Virus-associated CD8+ T-cells accumulated more in the atherosclerotic lesion (mean=2.0%), compared with patient-matched blood samples (mean=1.4%; P=0.05), and were more clonally expanded and tissue enriched in the atherosclerotic lesion in comparison with nonassociated CD8+ T-cells from the lesion. Single-cell T-cell receptor sequencing and flow cytometry revealed that these virus-associated CD8+ T-cells were phenotypically highly similar to other CD8+ T-cells in the lesion and that both exhibited a more activated phenotype compared with circulating T-cells. Interestingly, virus-associated CD8+ T-cells are unlikely to be activated through antigen-specific interactions in the atherosclerotic lesion, as no virus-derived peptides were detected on HLA-I in the lesion. This study suggests that virus-specific CD8+ T-cells are tissue enriched in atherosclerotic lesions; however, their potential contribution to inflammation may involve antigen-independent mechanisms.

Terminally differentiated cytotoxic CD4+ T cells were clonally expanded in the brain lesion of radiation-induced brain injury.

Accumulating evidence supports the involvement of adaptive immunity in the development of radiation-induced brain injury (RIBI). Our previous work has emphasized the cytotoxic function of CD8+ T cells in RIBI. In this study, we aimed to investigate the presence and potential roles of cytotoxic CD4+ T cells (CD4+ CTLs) in RIBI to gain a more comprehensive understanding of adaptive immunity in this context. Utilizing single-cell RNA sequencing (scRNA-seq), we analyzed 3934 CD4+ T cells from the brain lesions of four RIBI patients and identified six subclusters within this population. A notable subset, the cytotoxic CD4+ T cells (CD4+ CTLs), was marked with high expression of cytotoxicity-related genes (NKG7, GZMH, GNLY, FGFBP2, and GZMB) and several chemokine and chemokine receptors (CCL5, CX3CR1, and CCL4L2). Through in-depth pseudotime analysis, which simulates the development of CD4+ T cells, we observed that the CD4+ CTLs exhibited signatures of terminal differentiation. Their functions were enriched in protein serine/threonine kinase activity, GTPase regulator activity, phosphoprotein phosphatase activity, and cysteine-type endopeptidase activity involved in the apoptotic signaling pathway. Correspondingly, mice subjected to gamma knife irradiation on the brain showed a time-dependent infiltration of CD4+ T cells, an increase of MHCII+ cells, and the existence of CD4+ CTLs in lesions, along with an elevation of apoptotic-related proteins. Finally, and most crucially, single-cell T-cell receptor sequencing (scTCR-seq) analysis at the patient level determined a large clonal expansion of CD4+ CTLs in lesion tissues of RIBI. Transcriptional factor-encoding genes TBX21, RORB, and EOMES showed positive correlations with the cytotoxic functions of CD4+ T cells, suggesting their potential to distinguish RIBI-related CD4+ CTLs from other subsets. The present study enrichesthe understanding of the transcriptional landscape of adaptive immune cells in RIBI patients. It provides the first description of a clonally expanded CD4+ CTL subset in RIBI lesions, which may illuminate new mechanisms in the development of RIBI and offer potential biomarkers or therapeutic targets for the disease.

Type-I protein arginine methyltransferase inhibition primes anti-programmed cell death protein 1 immunotherapy in triple-negative breast cancer.

Immune-checkpoint blockade (ICB) therapy shows promise for treating aggressive triple-negative breast cancer (TNBC). However, only some patients benefit from ICB, revealing an urgent need for identifying novel strategies for sensitizing patients to ICB. Previously, the authors demonstrated that type-I protein arginine methyltransferases (PRMTs) regulated antiviral innate-immune responses in TNBC by altering RNA splicing. This study aimed to explore the effects of targeting type-I PRMTs on the tumor microenvironment (TME) and the efficacy of ICB therapy against TNBC. Single-cell transcriptomic analysis was performed to investigate the effects of type-I PRMT inhibition on the TME, especially T-cell subsets. Single-cell T-cell receptor sequencing was performed to analyze the diversity and dynamics of the T-cell repertoire. A syngeneic murine model of TNBC was used to evaluate the therapeutic efficacy and immune memory effect of combining a type-I PRMT inhibitor (MS023) with an anti-programmed cell death protein 1 (PD-1) antibody. Type-I PRMT inhibition combined with anti-PD-1 therapy reduced tumor growth. Mechanistically, type-I PRMT inhibition reshaped the TME. Increased CD8 T-cell infiltration was verified using flow cytometry. Increased clonotypes and clonal diversity were also observed after MS023 treatment, which contributed to immune memory following combination treatment. Targeting type-I PRMT can potentially improve immunotherapeutic efficacies in patients with TNBC. By enhancing the tumor immunogenicity and promoting a more favorable immune microenvironment, this combined approach may enable more patients with TNBC to benefit from immunotherapies.

Aging drives chronic exacerbated interactions of disease associated microglia and CD8+ T cells following traumatic brain injury

AbstractBackgroundAged individuals are highly susceptible to adverse outcomes following traumatic brain injury (TBI). Despite worsened correlates related to functional outcomes following injury, as well as increased likelihood to acquire a TBI, our understanding of the cellular mechanisms governing the poorer outcomes associated with aged individuals post‐TBI are not well‐characterized.MethodIn the current study, we examined microglial heterogeneity following TBI, in our mouse model of focal TBI across both acute (3d) and chronic (28d) intervals in both young (4m) and aged (18m) male C57B6J mice. At the appropriate interval, brains were collected and processed for single cell RNAseq (scRNAseq), spatial transcriptomics, and histology. A follow up study examining the interaction of TBI and aging upon T cell receptor (TCR) sequences was utilized to examine clonal expansion and lymphocyte heterogeneity in young and aged mice chronically after TBI.ResultOur findings demonstrate that TBI drives diverse transcriptional heterogeneity of microglia in both the young and aged condition. Acutely at 3 days post injury an inflammatory response is seen both in the young and aged mice. However, chronically at 28 days post injury, the microglia from young animals more closely resemble their pre‐injury (i.e. Sham) phenotypes, in comparison to the aged brain where there is a persistence of Apoe‐linked ‘DAM‐like’ microglial response. Inferential analyses point toward these aging‐related chronic populations of microglia as having distinct transcription factor utilization and metabolic pathway enrichment, including Hif1α upregulation and interferon gamma (INFγ) signaling. Furthermore, linkage with chronically accumulated CD8+ T cells and interferon signaling may underlie these phenotypes. Using single cell TCR sequencing, we demonstrate stark contrasts in the responses due to aging and TBI in the clonal expansion of infiltrated CD8+ T cells chronically following TBI.ConclusionOur studies demonstrate that aging is a centrally associated with the persistence of chronically reactive microglia in the brain and that these responses are linked, in part, with the protracted accumulation of CD8+ T cells following TBI.

Single-Cell RNA Sequencing of Circulating Tumor Cells in Precursor Myeloma Patients Reveals Mechanisms of Disease Dissemination

Introduction Multiple Myeloma (MM) is preceded by the precursors Monoclonal Gammopathy of Undetermined Significance (MGUS) and Smoldering Multiple Myeloma (SMM), where some patients with precursor disease progress to active MM faster than others. BM biopsies are useful to monitor disease progression, however, they cannot be repeated often for continuous monitoring of tumor burden especially in the precursor disease management setting. Numerous studies have identified circulating tumor cell (CTC) levels in peripheral blood (PB) are a powerful biomarker of disease aggressiveness; CTC levels have associated with disease stage, PFS and OS in various trials and even outperformed BM to assess tumor burden in the SMM setting. Genomic profiling also demonstrated CTCs harbor the same copy number abnormalities, translocations and mutations as BM tumor cells, and CTCs can be serially monitored to detect the emergence of high-risk subclones in the blood. Further investigations of the molecular profile of both normal plasma cells (NPCs) and CTCs are needed to improve our understanding of myeloma pathogenesis and mechanisms of CTC dissemination across the MM disease continuum. Methods A cohort of 309 samples including CD138-enriched paired PB and BM aspirates and matched CD138- BM aspirates were collected from 103 individuals, including patients with MGUS (n=11), SMM (n=46), NDMM (n=17) and healthy donors (n=29). Malignant PCs from BM and PB underwent 5' single-cell RNA sequencing (scRNA-seq) and single-cell B-cell receptor sequencing (scBCR-seq) (10x Genomics), and CD138- BM immune cells underwent 5' scRNA-seq and single-cell T-cell receptor sequencing (scTCR-seq) to study immune alterations related the tumors' ability to circulate. To differentiate malignant from normal PCs, we used clonal V(D)J rearrangements. Differential expression (DE) and composition analyses were conducted using Wilcoxon's rank-sum tests. Results We successfully captured and profiled 774,647 BM tumor cells and 93,878 CTCs from the PB. The percent malignant plasma cells within the PB increased with disease stage, where NDMM patients had significantly more CTCs compared to MGUS patients (p=0.017) and low-risk SMM patients (p=0.0052). A median of 13.51%, 15.53%, and 18.34% CTCs were present from low (n=20), intermediate (n=10), and high-risk (n=16) SMM patients as defined by the International Myeloma Working Group's 2/20/20 criteria, suggesting sequencing-based CTC enumeration captures prognostically relevant differences in tumor burden. High expression of driver genes upregulated in patients with translocations, including CCND1, NSD2, and MAF, were detected in both BM tumor cells and CTCs in patients with t(11;14), t(4;14), and t(14;16) as identified by fluorescence in situ hybridization (FISH). In 5 patients with normal or inconclusive FISH results, we observed high levels of CCND2 and MAF in both BM and CTCs, indicating scRNA-seq can detect missed prognostically relevant cytogenetic abnormalities. A cytogenetic classifier model was developed to determine the accuracy of translocation calling using minimal numbers of CTCs, where the classifier's performance was able to consistently identify translocations downsampled to 50 CTCs. DE analysis of CTCs vs. BM tumor cells highlighted transcriptional similarity between BM and CTCs, validating their utility as a surrogate for analyzing BM tumor cells. DE analysis also revealed 8 genes significantly upregulated and 3 genes significantly downregulated in CTCs compared to BM tumor cells, providing novel insights into genes involved in PC circulatory potential. Pathway enrichment analysis revealed genes upregulated in CTCs were associated with epithelial mesenchymal transition, interferon response, and inflammation, consistent with CTC studies on MM patients, suggesting these pathways are dysregulated earlier in the disease continuum. Conclusions In the largest scRNA-seq study on CTCs to date, we demonstrate the utility of CTC-based molecular profiling for prognostication of patients with early-stage disease and provide novel insights into PC circulatory potential. Additional analyses are ongoing to gain further insight into intra-patient CTC heterogeneity and define high-risk disease CTC signatures that emerge throughout the MM disease continuum.

EPCO-19. TUMOR-WIDE NEOANTIGEN-SPECIFIC T-CELLS INFILTRATING MUTANT IDH1 LOW-GRADE GLIOMAS AND PERSISTING IN PERIPHERAL BLOOD ALLOW FOR PERSONALIZED TCR-BASED IMMUNOTHERAPIES

Abstract BACKGROUND The low mutational burden and immunologically “cold” microenvironment of mutant IDH1 low-grade gliomas (LGG) are considerable challenges in immunotherapy for these tumor types. However, we hypothesize that LGG-targeting T-cells may exist at low frequency and with limited regional infiltration within the tumor. Through multi-region tumor sampling coupled with high-throughput T-cell receptor (TCR) profiling, we identified tumor-wide neoantigens and corresponding neoantigen-specific T-cells regionally infiltrating the tumor and persisting in peripheral blood. METHODS Maximally-distanced anatomical sampling of at least 10 distinct tumor regions was performed at the initial resection for three WHO Grade II diffuse astrocytoma patients for exome-based prediction of clonally and subclonally expressed neoantigens, RNAseq analysis of regional immune cell composition, and TCR beta deep sequencing. We used these predictions to generate a barcoded library of patient-specific peptide-HLA multimers loaded with predicted neoepitopes. With this library, neoantigen-specific CD8+ T-cells were captured and isolated from patients’ peripheral blood. Single-cell TCR sequencing allowed us to identify the neoantigen-reactive TCR clonotypes which were transduced subsequently into Jurkat76 cell lines for functional validation. RESULTS We screened patient-derived peripheral blood drawn two years after initial resection in 3 mutant IDH1 LGG patients and detected a total of 20 TCR clonotypes recognizing neoepitopes derived from truncal, tumor-wide mutations in CNTNAP1 (n=8), TP53 (n=3), and MRPL46 (n=2) as well as subclonal mutations in PRMT5 (n=1) and ZDHHC5 (n=6). Jurkat76 cells transduced with the mutant-PRMT5-specific TCR demonstrated dose-dependent neoantigen-specific immune responses when co-cultured with mutant-PRMT5 pulsed-antigen presenting cells expressing HLA-A*0201. CONCLUSION Our study demonstrates the existence and persistence of neoantigen-targeting T-cells within the blood and tumor of mutant IDH1 LGG patients. We identified a TCR clonotype that successfully recognizes and induces an immune response against mutant-PRMT5. These findings suggest a feasible methodology to develop personalized T-cell-based immunotherapies for patients with mutant IDH1 LGGs.

NLGN4X TCR transgenic T cells to treat gliomas.

Neuroligin 4 X-linked (NLGN4X) harbors a human leukocyte antigen (HLA)-A*02-restricted tumor-associated antigen, overexpressed in human gliomas, that was found to induce specific cytotoxic T cell responses following multi-peptide vaccination in patients with newly diagnosed glioblastoma. T cell receptor (TCR) discovery was performed using droplet-based single-cell TCR sequencing of NLGN4X-tetramer-sorted T cells postvaccination. The identified TCR was delivered to Jurkat T cells and primary human T cells (NLGN4X-TCR-T). Functional profiling of NLGN4X-TCR-T was performed by flow cytometry and cytotoxicity assays. Therapeutic efficacy of intracerebroventricular NLGN4X-TCR-T was assessed in NOD scid gamma (NSG) major histocompatibility complex (MHC) I/II knockout (KO) (NSG MHC I/II KO) mice bearing NLGN4X-expressing experimental gliomas. An HLA-A*02-restricted vaccine-induced T cell receptor specifically binding NLGN4X131-139 was applied for preclinical therapeutic use. Reactivity, cytotoxicity, and polyfunctionality of this NLGN4X-specific TCR are demonstrated in various cellular models. Intracerebroventricular administration of NLGN4X-TCR-T prolongs survival and leads to an objective response rate of 44.4% in experimental glioma-bearing NSG MHC I/II KO mice compared to 0.0% in control groups. NLGN4X-TCR-T demonstrate efficacy in a preclinical glioblastoma model. On a global scale, we provide the first evidence for the therapeutic retrieval of vaccine-induced human TCRs for the off-the-shelf treatment of glioblastoma patients.Keywords cell therapy | glioblastoma | T cell receptor | tumor antigen.

Abstract B019: Clinical and molecular characterization of chromophobe renal cell carcinoma: A focus on immunotherapy based regimens and the tumor immune microenvironment

Abstract Background: Chromophobe renal cell carcinoma (ChRCC) represents 5% of all kidney cancers. In contrast to clear cell RCC (ccRCC), the immune landscape of ChRCC and its response to immunotherapy remain poorly characterized. We sought to evaluate the clinical outcomes of patients with ChRCC treated with immuno-oncology (IO)-based regimens, and assess the immune cell composition, phenotypic state, and T cell specificity in the tumor microenvironment of ChRCC. Methods: Using real-world data from the International Metastatic RCC Database Consortium, we analyzed the survival outcomes and objective responses of patients with advanced ChRCC to currently adopted IO-based regimens (i.e. dual IO therapy or IO + vascular endothelial growth factor targeted therapy [VEGF-TT]) in the first-line setting, as compared to patients with ccRCC. Single-cell RNA sequencing (scRNA-seq) and single-cell T-cell receptor sequencing (scTCR-seq) were performed on ChRCC and related oncocytic neoplasms (i.e. renal oncocytoma [RO] and low-grade oncocytic tumor [LOT]) samples with matched normal kidney specimens. The infiltration of CD45+ immune cells in renal oncocytic tumors and ccRCC samples was quantified using immunohistochemistry (IHC). Results: Compared to patients with ccRCC (n=856) treated with first-line IO-based regimens, patients with ChRCC (n=31) had a lower overall survival (median: 24.7 vs. 50.5 months, p&amp;lt;0.001) and lower time to treatment failure (median: 4.5 vs. 11.0 months, p&amp;lt;0.001). Similarly, patients with ChRCC had a significantly lower overall response rate than those with ccRCC (12.0 vs. 47.1%, respectively; p&amp;lt;0.001). When evaluating immune cell infiltration, renal oncocytic tumors (ChRCC, RO, and LOT) exhibited a low density of CD45+ cells (mean: 739 ± 114 cells/mm2; n=5) compared to ccRCC (mean: 3,420 ± 1,979 cells/mm2; n=5) (p&amp;lt;0.05). Single-cell analysis was performed on 46,817 cells from 5 tumors (ChRCC: n=3, RO: n=1 and LOT: n=1) and 4 samples from adjacent normal kidney. Across all tumors, CD8+ T cell clusters displayed a lower expression of immune checkpoints (i.e. PDCD1, CTLA4, LAG3, HAVCR2, and TIGIT) as compared to CD8+ T-cells from ccRCC. This was further validated in the analysis of bulk RNA-seq data from the TCGA, with a significantly lower expression of all immune checkpoints in ChRCC compared to both ccRCC (p&amp;lt;0.01) and papillary RCC (pRCC; p&amp;lt;0.01). Analysis of the T cell receptor repertoire (scTCR-seq) of ChRCC, RO and LOT samples did not show any pattern of clonal expansion, and a higher proportion of T cells in ChRCC were inferred to have a viral specificity, compared to ccRCC (0.79 vs. 0.1%, respectively). Conclusions: Patients with metastatic ChRCC appear to display poor clinical outcomes when treated with IO-based regimens, compared to ccRCC. Renal oncocytic tumors, including ChRCC, exhibit a low infiltration of immune cells, and a non-exhausted immune phenotype. Citation Format: Michel Alchoueiry, Chris Labaki, Long Zhang, Yue Hou, Kevin Bi, Charbel Hobeika, J. Connor Wells, Kosuke Takemura, Ziad Bakouny, Sabrina Camp, Carmen Priolo, Damir Khabibullin, Nicholas Schindler, Renee Maria Saliby, Eddy Saad, Samer Salem, Melissa Daou, Rana McKay, Sumanta Pal, Daniel Heng, Eliezer Van Allen, Sachet Shukla, Toni Choueiri, David Braun, Elizabeth Henske. Clinical and molecular characterization of chromophobe renal cell carcinoma: A focus on immunotherapy based regimens and the tumor immune microenvironment [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr B019.

The pseudokinase Trib1 regulates the transition of exhausted T cells to a KLR+ CD8+ effector state, and its deletion improves checkpoint blockade

CD8+ Tcell exhaustion (TEX) impairs the ability of Tcells to clear chronic infection or cancer. While TEX are hypofunctional, some TEX retain effector gene signatures, a feature associated with killer lectin-like receptor (KLR) expression. Although KLR+ TEX (TKLR) may improve control of chronic antigen, the signaling molecules regulating this population are poorly understood. Using single-cell RNA sequencing (scRNA-seq), flow cytometry, RNA velocity, and single-cell Tcell receptor sequencing (scTCR-seq), we demonstrate that deleting the pseudokinase Trib1 shifts TEX toward CX3CR1+ intermediates with robust enrichment of TKLR via clonal Tcell expansion. Adoptive transfer studies demonstrate this shift toward CD8+ TKLR in Trib1-deficient cells is CD8 intrinsic, while CD4-depletion studies demonstrate CD4+ Tcells are required for improved viral control in Trib1 conditional knockout mice. Further, Trib1 loss augments anti-programmed death-ligand 1 (PD-L1) blockade to improve viral clearance. These data identify Trib1 as an important regulator of CD8+ TEX whose targeting enhances the TKLR effector state and improves checkpoint inhibitor therapy.

Characterization of the immunophenotype and tumor specificity of CD8+ T-cells in chromophobe renal cell carcinoma (ChRCC) and renal oncocytic neoplasms.

4558 Background: ChRCC is an uncommon kidney cancer variant that has a poor prognosis in the metastatic setting, with limited response to current standard-of-care immune checkpoint inhibitors (ICIs) used for other RCC histologies. We evaluated the tumor-immune microenvironment of ChRCC and other related oncocytic neoplasms to better understand the immunophenotype of these tumors. Methods: We performed paired single-cell RNA sequencing (scRNA-seq), single-cell T-cell receptor sequencing (scTCR-seq), and CD45 immunohistochemistry (IHC) of ChRCC, renal oncocytoma (RO) and low-grade oncocytic tumor (LOT) tumor and matched normal samples. Bulk RNA-sequencing (RNA-seq) data of clear cell RCC (ccRCC), papillary RCC (pRCC) and ChRCC were additionally analyzed using The Cancer Genome Atlas (TCGA) kidney cancer cohorts. T cell antigenic specificities from scTCR-seq were inferred using a comprehensive database of annotated T-cell receptor sequences (VDJdb). Single-cell transcriptomic signatures were used to infer the tumor specificity (Oliveira G, Nature, 2021 and Lowery FJ, Science, 2022) and viral specificity (Oliveira G, Nature, 2021) of CD8+ T-cells from ChRCC, as compared to those from ccRCC (Braun DA, Cancer Cell, 2021). Results: ChRCC and other oncocytic tumors had a lower infiltration of CD45+ immune cells as compared to ccRCC (p&lt;0.01). Single-cell analysis was performed on 46,817 cells from 5 tumors (ChRCC: n=3, RO: n=1 and LOT: n=1) and 4 normal samples. Across all tumors, CD8+ T cell clusters displayed a lower expression of immune checkpoints (i.e. PDCD1 [PD-1], CTLA4, LAG3, HAVCR2 [TIM-3], and TIGIT) as compared to ccRCC. This was further validated in a bulk RNA-seq analysis using TCGA data, with a significantly lower expression of all immune checkpoints in ChRCC compared to both ccRCC (p&lt;0.01) and papillary RCC (pRCC; p&lt;0.01). Analysis of the T cell receptor repertoire (scTCR-seq) of ChRCC, RO and LOT samples did not show any pattern of clonal expansion, and a higher proportion of T cells in ChRCC were inferred to have a viral specificity, as compared to ccRCC (0.79 vs. 0.1%, respectively). CD8+ T cells from ChRCC (vs. ccRCC) displayed a significantly lower expression of two signatures of tumor specificity (p&lt;0.01), and a higher expression of the viral-specific signature (p&lt;0.01). Conclusions: In ChRCC, there is low infiltration by CD45+ immune cells. Although infiltrating CD8+ T cells have a predominantly non-exhausted immune phenotype, they likely lack anti-tumor specificity (i.e. are “bystander” T cells). These findings may help to understand the molecular basis for the lack of response to immunotherapy recently identified among patients with advanced ChRCC, and support future therapeutic strategies to increase infiltration of tumor-specific T cells into the tumor microenvironment.

IRIS: Discovery of cancer immunotherapy targets arising from pre-mRNA alternative splicing

Alternative splicing (AS) is prevalent in cancer, generating an extensive but largely unexplored repertoire of novel immunotherapy targets. We describe Isoform peptides from RNA splicing for Immunotherapy target Screening (IRIS), a computational platform capable of discovering AS-derived tumor antigens (TAs) for T cell receptor (TCR) and chimeric antigen receptor T cell (CAR-T) therapies. IRIS leverages large-scale tumor and normal transcriptome data and incorporates multiple screening approaches to discover AS-derived TAs with tumor-associated or tumor-specific expression. In a proof-of-concept analysis integrating transcriptomics and immunopeptidomics data, weshowed that hundreds of IRIS-predicted TCR targets are presented by human leukocyte antigen (HLA) molecules. We applied IRIS to RNA-seq data of neuroendocrine prostate cancer (NEPC). From 2,939 NEPC-associated AS events, IRIS predicted 1,651 epitopes from 808 events as potential TCR targets for two common HLA types (A*02:01 and A*03:01). A more stringent screening test prioritized 48 epitopes from 20 events with "neoantigen-like" NEPC-specific expression. Predicted epitopes are often encoded by microexons of ≤30 nucleotides. To validate the immunogenicity and T cell recognition of IRIS-predicted TCR epitopes, we performed invitro T cell priming in combination with single-cell TCR sequencing. Seven TCRs transduced into human peripheral blood mononuclear cells (PBMCs) showed high activity against individual IRIS-predicted epitopes, providing strong evidence of isolated TCRs reactive to AS-derived peptides. One selected TCR showed efficient cytotoxicity against target cells expressing the target peptide. Our study illustrates the contribution of AS to the TA repertoire of cancer cells and demonstrates the utility of IRIS for discovering AS-derivedTAs and expanding cancer immunotherapies.

Detection of mutant antigen-specific T cell receptors against multiple myeloma for T cell engineering

Multiple myeloma (MM) remains an incurable hematological neoplasm. Neoantigen-specific Tcell receptor (TCR)-engineered T(TCR-T) cell therapy is a potential alternative treatment. Particularly, TCRs derived from a third-party donor may cover broad ranges of neoantigens, whereas TCRs in patients suffering from immune disorders are limited. However, the efficacy and feasibility of treating MM have not been evaluated thoroughly. In this study, we established a system for identifying immunogenic mutant antigens on MM cells and their corresponding TCRs using healthy donor-derived peripheral blood mononuclear cells (PBMCs). Initially, the immune responses to 35 candidate peptides predicted by the immunogenomic analysis were investigated. Peptide-reactive T lymphocytes were enriched, and subsequently, TCR repertoires were determined by single-cell TCR sequencing. Eleven reconstituted TCRs showed mutation-specific responses against 4 peptides. Particularly, we verified the HLA-A∗24:02-binding QYSPVQATF peptide derived from COASY S55Y as the naturally processed epitope across MM cells, making it a promising immune target. Corresponding TCRs specifically recognized COASY S55Y+HLA-A∗24:02+ MM cells and augmented tumoricidal activity. Finally, adoptive cell transfer of TCR-T cells showed objective responses in the xenograft model. We initiatively proposed the utility of tumor mutated antigen-specific TCR genes to suppress MM. Our unique strategy will facilitate further identification of neoantigen-specific TCRs.