Large libraries of single-chain trimer peptide-MHCs enable antigen-specific CD8+ T cell discovery and analysis.

The discovery and characterization of antigen-specific CD8+ T cell clonotypes typically involves the labor-intensive synthesis and construction of peptide-MHC tetramers. We adapt single-chain trimer (SCT) technologies into a high throughput platform for pMHC library generation, showing that hundreds can be rapidly prepared across multiple Class I HLA alleles. We use this platform to explore the impact of peptide and SCT template mutations on protein expression yield, thermal stability, and functionality. SCT libraries were an efficient tool for identifying T cells recognizing commonly reported viral epitopes. We then construct SCT libraries to capture SARS-CoV-2 specific CD8+ T cells from COVID-19 participants and healthy donors. The immunogenicity of these epitopes is validated by functional assays of T cells with cloned TCRs captured using SCT libraries. These technologies should enable the rapid analyses of peptide-based T cell responses across several contexts, including autoimmunity, cancer, or infectious disease.

Large libraries of single-chain trimer peptide-MHCs enable rapid antigen-specific CD8+ T cell discovery and analysis.

CD8 + cytotoxic T cell responses against viral infection represent a major element of the adaptive immune response. We describe the development of a peptide antigen - major histompatibility complex (pMHC) library representing the full SARS-CoV-2 viral proteome, and comprised of 634 pMHC multimers representing the A*02.01, A*24.02, and B*07.02 HLA alleles, as well as specific antigens associated with the cytomegalovirus (CMV). These libraries were used to capture non-expanded CD8 + T cells from blood samples collected from 64 infected individuals, and then analyzed using single cell RNA-seq. The discovery and characterization of antigen-specific CD8 + T cell clonotypes typically involves the labor-intensive synthesis and construction of peptide-MHC tetramers. We adapted single-chain trimer (SCT) technologies into a high throughput platform for pMHC library generation, showing that hundreds can be rapidly prepared across multiple Class I HLA alleles. We used this platform to explore the impact of peptide and SCT template mutations on protein expression yield, thermal stability, and functionality. SCT libraries were an efficient tool for identifying T cells recognizing commonly reported viral epitopes. We then constructed SCT libraries designed to capture SARS-CoV-2 specific CD8 + T cells from COVID-19 participants and healthy donors. The immunogenicity of these epitopes was validated by functional assays of T cells with cloned TCRs captured using SCT libraries. These technologies should enable the rapid analyses of peptide-based T cell responses across several contexts, including autoimmunity, cancer, or infectious disease.

Targeting an alternate Wilms' tumor antigen 1 peptide bypasses immunoproteasome dependency.

Designing effective antileukemic immunotherapy will require understanding mechanisms underlying tumor control or resistance. Here, we report a mechanism of escape from immunologic targeting in an acute myeloid leukemia (AML) patient, who relapsed 1 year after immunotherapy with engineered T cells expressing a human leukocyte antigen A*02 (HLA-A2)-restricted T cell receptor (TCR) specific for a Wilms' tumor antigen 1 epitope, WT1126-134 (TTCR-C4). Resistance occurred despite persistence of functional therapeutic T cells and continuous expression of WT1 and HLA-A2 by the patient's AML cells. Analysis of the recurrent AML revealed expression of the standard proteasome, but limited expression of the immunoproteasome, specifically the beta subunit 1i (ß1i), which is required for presentation of WT1126-134. An analysis of a second patient treated with TTCR-C4 demonstrated specific loss of AML cells coexpressing ß1i and WT1. To determine whether the WT1 protein continued to be processed and presented in the absence of immunoproteasome processing, we identified and tested a TCR targeting an alternative, HLA-A2-restricted WT137-45 epitope that was generated by immunoproteasome-deficient cells, including WT1-expressing solid tumor lines. T cells expressing this TCR (TTCR37-45) killed the first patients' relapsed AML resistant to WT1126-134 targeting, as well as other primary AML, in vitro. TTCR37-45 controlled solid tumor lines lacking immunoproteasome subunits both in vitro and in an NSG mouse model. As proteasome composition can vary in AML, defining and preferentially targeting these proteasome-independent epitopes may maximize therapeutic efficacy and potentially circumvent AML immune evasion by proteasome-related immunoediting.

The effects of low-dose irradiation on inflammatory response proteins in a 3D reconstituted human skin tissue model.

Skin responses to moderate and high doses of ionizing radiation include the induction of DNA repair, apoptosis and stress response pathways. Additionally, numerous studies indicate that radiation exposure leads to inflammatory responses in skin cells and tissue. However, the inflammatory response of skin tissue to low-dose radiation (≤10 cGy) is poorly understood. To address this, we have utilized a reconstituted human skin tissue model (MatTek EpiDermFT™) and assessed changes in 23 cytokines, 24 and 48 h after treatment of skin with either 3 or 10 cGy low dose of radiation. Three cytokines, IFN-γ, IL-2, MIP-1α, were significantly altered in response to low-dose radiation. In contrast, seven cytokines were significantly altered in response to a high radiation dose of 200 cGy (IL-2, IL-10, IL-13, IFN-γ, MIP-1α, TNFα and VEGF) or the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (G-CSF, GM-CSF, IL-1α, IL-8, MIP-1α, MIP-1ß and RANTES). Additionally, radiation induced inflammation appears to have a distinct cytokine response relative to the nonradiation induced stressor, TPA. Overall, these results indicate that there are subtle changes in the inflammatory protein levels after exposure to low-dose radiation and this response is a subset of what is seen after a high dose in a human skin tissue model.