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.

HITECH spurs EHR vendor competition and innovation, resulting in increased adoption.

The Health Information Technology for Economic and Clinical Health (HITECH) Act was enacted to increase electronic health record (EHR) adoption by providers and hospitals. Experts expressed skepticism about whether the program would indeed hasten adoption and could be implemented in time for the initial reporting period. Could EHR vendors meet the certification requirements, and could the industry innovate to meet small-practice needs? This study, in addition to documenting increased provider adoption, provides the first evidence of increased competitiveness and innovation in the EHR industry spurred by HITECH. For example, the number of EHR vendors certified for e-prescribing with Surescripts increased from 96 to 229 over the program's first 3 years. We also find that prescribers in small practices increasingly adopted lower-cost, Web-based e-prescribing and EHR applications at significantly higher rates (15%-35%) than did large practices (3%-4%), which generally have more human and capital resources to make significant investments. These findings suggest that EHR vendors were highly responsive to HITECH requirements and have been adapting their strategies to meet nuanced market needs, providing reason to be optimistic about the Programs' future.

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.

Willingness of health care personnel to work in a disaster: an integrative review of the literature.

Effective hospital surge response in disaster depends largely on an adequate number of personnel to provide care. Studies appearing since 1991 indicate health care personnel may not be willing to work in all disaster situations-and if so, this could degrade surge response. A systematic review of the literature was conducted to determine the state of the evidence concerning the willingness of health care personnel to work in disaster. The aims of this review are to collate and assess the literature concerning willingness of health care personnel to work during a disaster, to identify gaps in the literature as areas for future investigation, and to facilitate evidence-based disaster planning. Twenty-seven studies met inclusion criteria (25 quantitative and 2 qualitative studies). The current evidence indicates there may be certain factors related to willingness to work (or lack of willingness) in disaster including the type of disaster, concern for family, and concerns about personal safety. Barriers to willingness to work have been identified including pet care needs and the lack of personal protective equipment. This review describes the state of an emerging area of science. These findings have significant implications for community and organizational emergency planning and policymaking in an environment defined by limited resources.

Hospital response to acute-onset disasters: the state of the science in 2005.

The level of emergency preparedness in US hospitals is a concern in light of the steady threat of natural disasters, transportation and industrial accidents, and the possibility of terror attack resulting in mass casualties. The science of hospital emergency preparedness is in an early stage of development. For research to logically expand knowledge, an accurate assessment--or examination of the state of the science--is conducted to determine the current state of knowledge, gaps in knowledge, and opportunities for future research. Milsten reviewed the literature on hospital response to acute-onset disasters from 1977 to 1999. His review of 107 articles contains research studies, case studies,and lessons learned pieces largely published in the medical literature.Milsten's analysis provides a substantial starting point. This article examines Milsten's review, identifies articles that have been published that add to this knowledge base, and identifies additional phenomena of interest.

Reaching beyond the familiar.

Sue Thomas Hegyvary built a rich and rewarding life as a nurse-scientist, educator, administrator, author, wife and mother, achieving appointment as dean of the University of Washington School of Nursing at 42. When an accident changed her life, she demonstrated the same remarkable resilience she had shown many times before when faced with challenges.