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:Several current immunotherapy approaches target private neoantigens derived from mutations that are unique to individual patients' tumors. However, immunotherapeutic agents can also be developed against public neoantigens derived from recurrent mutations in cancer driver genes. The latter approaches target proteins that are indispensable for tumor growth, and each therapeutic agent can be applied to numerous patients. Here we review the opportunities and challenges involved in the identification of suitable public neoantigen targets and the development of therapeutic agents targeting them.
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: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) and NE1-/- 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 and NE1-/- primary pro-B cells from the VDJ-seq data.
Project description:Rhesus macaques (RMs) are widely employed as a pre-clinical model in vaccination and infectious disease studies, yet their B cell immunobiology and immunogenetics remain ill-characterized. In this study, single-cell RNA/VDJ-seq (scRNA/VDJ-seq) was conducted on peripheral blood mononuclear cell (PBMC) samples from six RMs to describe the transcriptomic and V(D)JC repertoires of B cells and subtypes. 12 RM B cell clusters of distinct transcriptional states were identified, including IgM+ memory B cells (MBC), class-switched MBC, CD11c+ MBC and activated B cells. Novel gene signatures were also characterized for each B cell subtype, such as FCRL2 and CD24 for circulating marginal-zone-like B cells. In addition, VDJ repertoire properties of the global B cell population and each B cell subtype were elucidated, including IGH/K/L-V(D)JC gene family and subtype usage, class-switch recombination (CSR) status, somatic hypermutation (SHM) rate and levels, complementarity-determining region heavy chain (CDRH3) amino acid (AA) length and CDRH3 AA hydrophobicity scores. Interesting insights included the 1:1 ratio of kappa and lambda light chain usage and a preferential decreased IGHV3 but increased IGHV1 and 5 gene family usage in IGHG1 than IGHM-bearing B cells. Altogether, this study through comprehensive transcriptomic analyses identifies 12 distinct RM B cell subtypes paired with their respective V(D)JC repertoire, unraveling the complexity of B cell heterogeneity and improving future pre-clinical studies that can translate insights from this important non-human primate model to the understanding of human immunobiology.
Project description:Energy metabolism and extracellular matrix function are closely connected to orchestrate and maintain tissue organization, but the crosstalk is poorly understood. Here, we used scRNA-seq analysis to uncover the importance of respiration for extracellular matrix homeostasis in mature cartilage. Genetic inhibition of respiration in cartilage results in the expansion of a central area of 1-month-old mouse femur head cartilage showing disorganized chondrocytes and increased deposition of extracellular matrix material. scRNA-seq analysis identified a cluster-specific decrease in mitochondrial DNA-encoded respiratory chain genes and a unique regulation of extracellular matrix-related genes in nonarticular chondrocyte clusters. These changes were associated with alterations in extracellular matrix composition, a shift in the collagen/non-collagen protein content and an increase of collagen crosslinking and ECM stiffness. The results demonstrate, based on findings of the scRNA-seq analysis, that respiration is a key factor contributing to ECM integrity and mechanostability in cartilage and presumably also in many other tissues.
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: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.