Project description:We introduced single-chain trimer (SCT) technologies into a high throughput platform for pMHC library generation that can be used for screening antigen specific CD8+ T cells. We compared the diversity of T cell receptor repertoire captured by SCT and folded peptide-MHC multimer presenting HLA-A02:01 restricted CMV peptide. We then constructed SCT libraries designed to capture SARS-CoV-2 spike specific CD8+ T cells from COVID-19 participants and healthy donors. TCR sequencing with antigen specificity was analyzed. The immunogenicity of these epitopes was validated by functional assays of T cells with cloned TCRs captured using SCT libraries.
Project description:We identified epitopes derived from FOXM1 which were immunogenic on HLA-A*02:01, HLA-A*24:02, and HLA-A*23:01, endogenously-processed and presented, and resulted in T cell activation and cytotoxic T cell responses. Following the generation of TCR-T cells, sensitivity and specificity were confirmed by peptide dose-response and X-scan, respectively. Most importantly, adoptive transfer of TCR engineered T cells led to a significant reduction in tumor growth, as well as significantly prolonged survival in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) tumor-bearing murine model. Our studies confirm the immunogenicity of FOXM1 and feasibility of targeting this antigen using TCR-engineering.
Project description:Few non-classical HLA-E restricted HIV-specific epitopes have been described, and even less is known about the functional profile of responding CD8 T cells (CD8s). This study evaluates the functional characteristics of CD8s targeting the Gag epitope (KAFSPEVIPMF or KF11) based on their restriction by either HLA-E (E-CD8s) or HLA-B57 (B57-CD8s). CD8s from 8 people with HIV (PWH) were cocultured with KF11 peptide presented by cell lines expressing HLA-B*57:01, HLA-E*01:01 or E*01:03. CD8s were analyzed through single-cell (sc) RNA and TCR sequencing. Additionally, supernatants were analyzed for soluble proteomics using a Luminex assay. B57-CD8s secreted higher levels of cytotoxic cytokines such as IFNγ, while E-CD8s produced more chemotactic cytokines, including RANTES, CXCL10 (IP-10), and IL27confirmed through scRNAseq. Despite distinct cytokine profiles, TCR clonotypes stimulated by KF11 were cross-restricted by HLA-B*57 and HLA-E*01/03. In vitro T cell reporter assays clearly demonstrated this cross-restriction. A TRAV5-containing metaclonotype cluster was seen in PWH with lower viral loads. These findings demonstrate that HIV-specific CD8s in PWH exhibit cross HLA-B*57 and HLA-E*01/03 restriction, resulting in functionally distinct immune responses that may contribute to HIV control.
Project description:Few non-classical HLA-E restricted HIV-specific epitopes have been described, and even less is known about the functional profile of responding CD8 T cells (CD8s). This study evaluates the functional characteristics of CD8s targeting the Gag epitope (KAFSPEVIPMF or KF11) based on their restriction by either HLA-E (E-CD8s) or HLA-B57 (B57-CD8s). CD8s from 8 people with HIV (PWH) were cocultured with KF11 peptide presented by cell lines expressing HLA-B*57:01, HLA-E*01:01 or E*01:03. CD8s were analyzed through single-cell (sc) RNA and TCR sequencing. Additionally, supernatants were analyzed for soluble proteomics using a Luminex assay. B57-CD8s secreted higher levels of cytotoxic cytokines such as IFNγ, while E-CD8s produced more chemotactic cytokines, including RANTES, CXCL10 (IP-10), and IL27confirmed through scRNAseq. Despite distinct cytokine profiles, TCR clonotypes stimulated by KF11 were cross-restricted by HLA-B*57 and HLA-E*01/03. In vitro T cell reporter assays clearly demonstrated this cross-restriction. A TRAV5-containing metaclonotype cluster was seen in PWH with lower viral loads. These findings demonstrate that HIV-specific CD8s in PWH exhibit cross HLA-B*57 and HLA-E*01/03 restriction, resulting in functionally distinct immune responses that may contribute to HIV control.
Project description:HLA-E molecules can present self and pathogen-derived peptides to both NK-cells and T-cells. T-cells that recognize HLA-E peptides via their T-cell receptor (TCR) are termed donor-unrestricted T-cells due to restricted allelic variation of HLA-E. The composition and repertoire of HLA-E TCRs is not known so far. We performed TCR sequencing on CD8+ T-cells from 21 individuals recognizing HLA-E tetramers (TM) folded with 2 Mtb HLA-E restricted peptides. We sorted HLA-E Mtb TM+ and TMCD8+ T-cells directly ex vivo and performed bulk RNA-sequencing and single cell TCR sequencing. The identified TCR repertoire was diverse and showed no conservation between and within individuals. TCRs selected from our single cell TCR sequencing data could be activated upon HLA-E/peptide stimulation, although not robust, reflecting potentially weak interactions between HLA-E peptide complexes and TCRs. Thus, HLA-E Mtb specific T-cells have a highly diverse TCR repertoire.
Project description:Malignant tumors with TP53 mutations exhibit poor therapeutic outcomes and high recurrence rates. T cell receptor (TCR)-based T cell therapy shows great promise for targeting intracellular cancer neoantigens. However, the immunogenic potential of TP53 hotspot mutations remain poorly characterized. Here, we identify a immunogenic neoantigen derived from the recurrent TP53R248Q mutation, presented by the prevalent Human Leukocyte Antigen (HLA)-A*11:01 allele. Additionally, we isolated a TP53R248Q reactive TCR that specifically recognize the TP53R248Q mutation without any discernable cross-activity to cognate wild-type TP53 or other TP53 mutants at the same codon position. Functional characterization revealed that TP53R248Q TCR-T cells exhibited selectively cytotoxicity against tumor cells expressing both TP53R248Q mutation and HLA-A*11:01 in vitro. Importantly, the adoptive transfer of TP53R248Q TCR-T cells exhibited significant anti-tumor activity in a clinically relevant patient-derived xenograft (PDX) model engrafted with TP53R248Q/HLA-A*11:01 positive human tumor tissues. Collectively, our study validates the immunogenicity of the TP53R248Q hotspot mutation and provides a TCR with high therapeutic potential for the development of T cell therapies targeting TP53R248Q/HLA-A*11:01 positive cancers.
Project description:Tapasin is a member of the peptide loading complex which is responsible for stabilizing empty HLA molecules and facilitating the loading of antigenic peptides. Many neuroblastoma cell lines contain low baseline levels of HLA due to defects in antigen processing and presentation machinery. Supplementing the neuroblastoma cell line EBc1 with tapasin-TM, an altered form of tapasin containing the transmembrane domain of HLA-G, enhances surface HLA expression. Here, we have applied our previously used deep mutational scanning library (GSE159247) to the tapasin-TM construct. We then introduced this library into EBc1 cells and isolated cells with the highest surface HLA-A2 expression.
Project description:FOXM1 is highly expressed in various cancer types and considered a key driver of cancer progression. Accordingly, we evaluated the immunogenicity of FOXM1 and investigated the feasibility of targeting this transcription factor using T cell receptor (TCR) engineering. We identified epitopes derived from FOXM1 which were immunogenic on HLA-A*02:01, HLA-A*24:02, and HLA-A*23:01, endogenously-processed and presented, and resulted in T cell activation and cytotoxic T cell responses. Following the generation of TCR-T cells, sensitivity and specificity were confirmed by peptide dose-response and X-scan, respectively. Most importantly, adoptive transfer of TCR engineered T cells led to reduced tumor growth and prolonged survival in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) strain murine models bearing FOXM1 expressing subcutaneous tumors. Our studies confirm the immunogenicity of FOXM1 and feasibility of targeting this tumor-associated antigen using TCR-engineering.