Project description:We developed a modular, high-throughput discovery platform to simultaneously test whether Mut PIK3Ca is immunogenic and to retrieve paired a/b TCR gene sequences that confer specificity to this NeoAg. This method, termed Stimulation Induced Functional TCR sequencing (SIFT-seq), combines single-cell (sc) TCR V(D)J and transcriptome sequencing. Here, microwell cultures of in vitro stimulated T cells with confirmed neoantigen-specific recognition are selected for SIFT-seq. Matched aliquots of selected wells are acutely stimulated with autologous antigen-presenting cells presenting WT or Mut PI3Ka and the transcriptomic profile of individual clonotypes is assessed to identify neoantigen-specific T cells and retrieve their TCR gene sequences.
Project description:We employed a hybrid method based on Oxford Nanopore long-read sequencing and Illumina sequencing to characterize the AS landscapes, and to identify AS-derived neoantigens. Furthermore, we used the AlphaFold2 algorithm to analyze the structure of these putative neoantigens, thus offering structural insights for further drug development. These results lay the foundation for the development of therapies targeting UM-specific, AS-derived neoantigens.
Project description:In this study we investigated the presence of endogenous T-cells recognizing private tumor-associated antigens and predicted neoantigens. To this end, HLA-I and HLA-II restricted tumor associated antigens (TAAs) and neoantigens were respectively identified and predicted. Of interest, circulating T-cells targeting both Mass spectrometry (MS)-identified TAAs and predicted neoantigens were identified in virtually all pediatric BCP-ALL patients, extending up to two years on remission.
Project description:Targeting tumor-specific neoantigens is promising for cancer immunotherapy, yet their ultra-low expression on tumor cells poses significant challenges for T cell therapies. Here, we found that chimeric antigen receptors (CARs) exhibited 10-100 times lower sensitivity compared to T cell receptors (TCRs) when targeting p53R175H common neoantigen. To enhance CAR functionality, we introduce T cell receptor fusion construct (TRuC) and synthetic TCR and antigen receptor (STAR). Our data demonstrate that STAR, which incorporates TCR-mimic antibody fragments and complete TCR signaling machinery, optimally reproduces antigen sensitivity of TCRs. STAR outperforms both CAR and TRuC in redirecting both CD8+ and CD4+ T cells to recognize HLA class I neoantigens. In vitro, human primary T cells engineered with STAR kill multiple cancer cell lines with low neoantigen density better than CAR-T and TRuC-T cells. In tumor mouse models, STAR-T cells outperform CAR-T and TRuC-T cells in controlling neoantigen-low breast cancer and leukemia. Taken together, our findings highlight severe defects in CAR sensitivity and introduce STAR as a more sensitive synthetic receptor, providing a new framework for T cell-based immunotherapy targeting tumors with low neoantigen density.
Project description:For high-throughput sequencing and quantification of immunoglobulin repertoires, most methodologies utilise RNA. However, output varies enormously between recombined genes due to different promoter strengths and differential activation of lymphocyte subsets, precluding quantitation of recombinants on a per cell basis. To date, DNA-based approaches have used V gene primer cocktails, with substantial inherent biases. Here we describe VDJ-seq, which accurately quantitates immunoglobulin diversity at the DNA level in an unbiased manner. This is accomplished with a single primer extension step using biotinylated J gene primers. By addition of unique molecular identifiers (UMI) before primer extension, we reliably remove duplicate sequences and correct for sequencing and PCR errors. Furthermore, VDJ-seq captures productive and non-productive VDJ and DJ recombination events on a per cell basis. Library preparation takes 3 days, with 2 days of sequencing, and 1 day of data processing and analysis.
Project description:Mutations in RNA splicing factors are prevalent across cancers and generate recurrently mis-spliced mRNA isoforms. Here we identified a series of bona fide neoantigens translated from highly stereotyped splicing alterations promoted by neomorphic, leukemia-associated somatic mutations in the splicing machinery. We utilized feature-barcoded peptide-MHC dextramers to isolate neoantigen-specific T cell receptors (TCR) from both healthy donors and patients with leukemia. While circulating neoantigen-specific CD8+ T cells were identified in patients with active disease, they were dysfunctional with reduced inflammatory response gene signatures. In contrast, donor CD8+ T cells with tumor-reactive TCRs were present following curative allogeneic hematopoietic cell transplant. T cells engineered with TCRs recognizing an SRSF2 mutant-induced neoantigen in CLK3 resulted in specific recognition and cytotoxicity of SRSF2 mutant leukemia. These data identify RNA mis-splicing derived neoantigens and neoantigen-specific TCRs across patients and provide proof-of-concept to genetically redirect T cells to public mis-splicing derived neoantigens in myeloid leukemias.
Project description:Lynch syndrome (LS), a common hereditary cancer syndrome, provides a unique model to study immune surveillance at the earliest stages of tumorigenesis. A hallmark of LS carcinogenesis is the generation of highly immunogenic mutated neoantigens derived from insertion deletions, yet the transcriptomic states of the neoantigen-specific T cells that recognize them remain poorly characterized. Here, we characterized neoantigen-specific T cells from LS carriers using functional assays, single-cell RNA/TCR sequencing, and repertoire integration with existing data sets. Recurrent neoantigens elicited strong immune responses, with neoantigen-specific T cells mediating cytotoxicity against patient-derived tumor organoids. Single-cell analysis revealed oligoclonal expansions spanning effector and memory states with LS cancer survivors showing enrichment for metabolically exhausted subsets and previvor LS carriers retaining polyfunctional effectors. Cross-cohort analyses identified public clonotypes overlapping with tumor-infiltrating repertoire. These findings define the architecture of circulating neoantigen-specific immunity in LS and identify public TCRs as biomarkers for immune monitoring and immunoprevention.
Project description:Lynch syndrome (LS), a common hereditary cancer syndrome, provides a unique model to study immune surveillance at the earliest stages of tumorigenesis. A hallmark of LS carcinogenesis is the generation of highly immunogenic mutated neoantigens derived from insertion deletions, yet the transcriptomic states of the neoantigen-specific T cells that recognize them remain poorly characterized. Here, we characterized neoantigen-specific T cells from LS carriers using functional assays, single-cell RNA/TCR sequencing, and repertoire integration with existing data sets. Recurrent neoantigens elicited strong immune responses, with neoantigen-specific T cells mediating cytotoxicity against patient-derived tumor organoids. Single-cell analysis revealed oligoclonal expansions spanning effector and memory states with LS cancer survivors showing enrichment for metabolically exhausted subsets and previvor LS carriers retaining polyfunctional effectors. Cross-cohort analyses identified public clonotypes overlapping with tumor-infiltrating repertoire. These findings define the architecture of circulating neoantigen-specific immunity in LS and identify public TCRs as biomarkers for immune monitoring and immunoprevention.
Project description:Analysis of the antibody repertoire composition is now possible using VDJ-seq. We used this recently developed method for unbiased amplification from genomic DNA (gDNA) to directly compare the Igh repertoire of C57Bl/6 (WT), NE1-/- and Site1.3 -/- pro-B cells. We find that a group of contiguous proximal and intermediate VH genes are under-utilized in V->DJ rearrangement in the absence of NE1 revealing a NE1 zone of influence. We report the VH gene usage profile from WT, NE1-/- and Site1.3 -/- primary pro-B cells from the VDJ-seq data.