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Introduction: Human Herpesvirus 6B (HHV-6B) impedes host immune responses by downregulating class I MHC molecules (MHC-I), hindering antigen presentation to CD8+ T cells. Downregulation of MHC-I disengages inhibitory receptors on natural killer (NK) cells, resulting in activation and killing of the target cell if NK cell activating receptors such as NKG2D have engaged stress ligands upregulated on the target cells. Previous work has shown that HHV-6B downregulates three MHC-like stress ligands MICB, ULBP1, and ULBP3, which are recognized by NKG2D. The U20 glycoprotein of the related virus HHV-6A has been implicated in the downregulation of ULBP1, but the precise mechanism remains undetermined., Methods: We set out to investigate the role of HHV-6B U20 in modulating NK cell activity. We used HHV-6B U20 expressed as a recombinant protein or transduced into target cells, as well as HHV-6B infection, to investigate binding interactions with NK cell ligands and receptors and to assess effects on NK cell activation. Small-angle X-ray scattering was used to align molecular models derived from machine-learning approaches., Results: We demonstrate that U20 binds directly to ULBP1 with sub-micromolar affinity. Transduction of U20 decreases NKG2D binding to ULBP1 at the cell surface but does not decrease ULBP1 protein levels, either at the cell surface or in toto. HHV-6B infection and soluble U20 have the same effect. Transduction of U20 blocks NK cell activation in response to cell-surface ULBP1. Structural modeling of the U20 - ULBP1 complex indicates some similarities to the m152-RAE1γ complex., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Weaver, Schneider, Becerra-Artiles, Clayton, Hudson and Stern.)

Seasonal "common-cold" human coronaviruses are widely spread throughout the world and are mainly associated with mild upper respiratory tract infections. The emergence of highly pathogenic coronaviruses MERS-CoV, SARS-CoV, and most recently SARS-CoV-2 has prompted increased attention to coronavirus biology and immunopathology, but the T-cell response to seasonal coronaviruses remains largely uncharacterized. Here we report the repertoire of viral peptides that are naturally processed and presented upon infection of a model cell line with seasonal coronavirus OC43. We identified MHC-bound peptides derived from each of the viral structural proteins (spike, nucleoprotein, hemagglutinin-esterase, membrane, and envelope) as well as non-structural proteins nsp3, nsp5, nsp6, and nsp12. Eighty MHC-II bound peptides corresponding to 14 distinct OC43-derived epitopes were identified, including many at very high abundance within the overall MHC-II peptidome. Fewer and less abundant MHC-I bound OC43-derived peptides were observed, possibly due to MHC-I downregulation induced by OC43 infection. The MHC-II peptides elicited low-abundance recall T-cell responses in most donors tested. In vitro assays confirmed that the peptides were recognized by CD4+ T cells and identified the presenting HLA alleles. T-cell responses cross-reactive between OC43, SARS-CoV-2, and the other seasonal coronaviruses were confirmed in samples of peripheral blood and peptide-expanded T-cell lines. Among the validated epitopes, spike protein S903-917 presented by DPA1*01:03/DPB1*04:01 and S1085-1099 presented by DRB1*15:01 shared substantial homology to other human coronaviruses, including SARS-CoV-2, and were targeted by cross-reactive CD4 T cells. Nucleoprotein N54-68 and hemagglutinin-esterase HE128-142 presented by DRB1*15:01 and HE259-273 presented by DPA1*01:03/DPB1*04:01 are immunodominant epitopes with low coronavirus homology that are not cross-reactive with SARS-CoV-2. Overall, the set of naturally processed and presented OC43 epitopes comprise both OC43-specific and human coronavirus cross-reactive epitopes, which can be used to follow CD4 T-cell cross-reactivity after infection or vaccination, and to guide selection of epitopes for inclusion in pan-coronavirus vaccines., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Becerra-Artiles et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Initial TCR affinity for peptide Ag is known to impact the generation of memory; however, its contributions later, when effectors must again recognize Ag at 5-8 d postinfection to become memory, is unclear. We examined whether the effector TCR affinity for peptide at this "effector checkpoint" dictates the extent of memory and degree of protection against rechallenge. We made an influenza A virus nucleoprotein (NP)-specific TCR transgenic mouse strain, FluNP, and generated NP-peptide variants that are presented by MHC class II to bind to the FluNP TCR over a broad range of avidity. To evaluate the impact of avidity in vivo, we primed naive donor FluNP in influenza A virus-infected host mice, purified donor effectors at the checkpoint, and cotransferred them with the range of peptides pulsed on activated APCs into second uninfected hosts. Higher-avidity peptides yielded higher numbers of FluNP memory cells in spleen and most dramatically in lung and draining lymph nodes and induced better protection against lethal influenza infection. Avidity determined memory cell number, not cytokine profile, and already impacted donor cell number within several days of transfer. We previously found that autocrine IL-2 production at the checkpoint prevents default effector apoptosis and supports memory formation. Here, we find that peptide avidity determines the level of IL-2 produced by these effectors and that IL-2Rα expression by the APCs enhances memory formation, suggesting that transpresentation of IL-2 by APCs further amplifies IL-2 availability. Secondary memory generation was also avidity dependent. We propose that this regulatory pathway selects CD4 effectors of highest affinity to progress to memory., (Copyright © 2023 by The American Association of Immunologists, Inc.)

Seasonal "common-cold" human coronaviruses are widely spread throughout the world and are mainly associated with mild upper respiratory tract infections. The emergence of highly pathogenic coronaviruses MERS-CoV, SARS-CoV, and most recently SARS-CoV-2 has prompted increased attention to coronavirus biology and immunopathology, but identification and characterization of the T cell response to seasonal human coronaviruses remain largely uncharacterized. Here we report the repertoire of viral peptides that are naturally processed and presented upon infection of a model cell line with seasonal human coronavirus OC43. We identified MHC-I and MHC-II bound peptides derived from the viral spike, nucleocapsid, hemagglutinin-esterase, 3C-like proteinase, and envelope proteins. Only three MHC-I bound OC43-derived peptides were observed, possibly due to the potent MHC-I downregulation induced by OC43 infection. By contrast, 80 MHC-II bound peptides corresponding to 14 distinct OC43-derived epitopes were identified, including many at very high abundance within the overall MHC-II peptidome. These peptides elicited low-abundance recall T cell responses in most donors tested. In vitro assays confirmed that the peptides were recognized by CD4+ T cells and identified the presenting HLA alleles. T cell responses cross-reactive between OC43, SARS-CoV-2, and the other seasonal coronaviruses were confirmed in samples of peripheral blood and peptide-expanded T cell lines. Among the validated epitopes, S 903-917 presented by DPA1*01:03/DPB1*04:01 and S 1085-1099 presented by DRB1*15:01 shared substantial homology to other human coronaviruses, including SARS-CoV-2, and were targeted by cross-reactive CD4 T cells. N 54-68 and HE 128-142 presented by DRB1*15:01 and HE 259-273 presented by DPA1*01:03/DPB1*04:01 are immunodominant epitopes with low coronavirus homology that are not cross-reactive with SARS-CoV-2. Overall, the set of naturally processed and presented OC43 epitopes comprise both OC43-specific and human coronavirus cross-reactive epitopes, which can be used to follow T cell cross-reactivity after infection or vaccination and could aid in the selection of epitopes for inclusion in pan-coronavirus vaccines., Author Summary: There is much current interest in cellular immune responses to seasonal common-cold coronaviruses because of their possible role in mediating protection against SARS-CoV-2 infection or pathology. However, identification of relevant T cell epitopes and systematic studies of the T cell responses responding to these viruses are scarce. We conducted a study to identify naturally processed and presented MHC-I and MHC-II epitopes from human cells infected with the seasonal coronavirus HCoV-OC43, and to characterize the T cell responses associated with these epitopes. We found epitopes specific to the seasonal coronaviruses, as well as epitopes cross-reactive between HCoV-OC43 and SARS-CoV-2. These epitopes should be useful in following immune responses to seasonal coronaviruses and identifying their roles in COVID-19 vaccination, infection, and pathogenesis.

Sequence homology between SARS-CoV-2 and common-cold human coronaviruses (HCoVs) raises the possibility that memory responses to prior HCoV infection can affect T cell response in COVID-19. We studied T cell responses to SARS-CoV-2 and HCoVs in convalescent COVID-19 donors and identified a highly conserved SARS-CoV-2 sequence, S 811-831 , with overlapping epitopes presented by common MHC class II proteins HLA-DQ5 and HLA-DP4. These epitopes are recognized by low-abundance CD4 T cells from convalescent COVID-19 donors, mRNA vaccine recipients, and uninfected donors. TCR sequencing revealed a diverse repertoire with public TCRs. T cell cross-reactivity is driven by the high conservation across human and animal coronaviruses of T cell contact residues in both HLA-DQ5 and HLA-DP4 binding frames, with distinct patterns of HCoV cross-reactivity explained by MHC class II binding preferences and substitutions at secondary TCR contact sites. These data highlight S 811-831 as a highly conserved CD4 T cell epitope broadly recognized across human populations., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Human roseolovirus U20 and U21 are type I membrane glycoproteins that have been implicated in immune evasion by interfering with recognition of classical and non-classical MHC proteins. U20 and U21 are predicted to be type I glycoproteins with extracytosolic immunoglobulin-like domains, but detailed structural information is lacking. AlphaFold and RoseTTAfold are next generation machine-learning-based prediction engines that recently have revolutionized the field of computational three-dimensional protein structure prediction. Here, we review the structural biology of viral immunoevasins and the current status of computational structure prediction algorithms. We use these computational tools to generate structural models for U20 and U21 proteins, which are predicted to adopt MHC-Ia-like folds with closed MHC platforms and immunoglobulin-like domains. We evaluate these structural models and place them within current understanding of the structural basis for viral immune evasion of T cell and natural killer cell recognition., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Weaver, Arya, Schneider, Hudson and Stern.)

Aim: E-learning technologies are becoming vital components of medical and health professions education, as highlighted during the current coronavirus disease (COVID-19) pandemic. The National Academy of Medicine (NAM) considers education technologies essential to forming connections between education and healthcare delivery systems, which promote evidence-based practice and continuous learning and quality improvement in healthcare. There is a lack of evidence-based models to guide the integration of technology in medical and health profession education, in particular models that form synergistic linkages between healthcare education and delivery systems. This paper presents the evaluation of an innovative blended learning model, which leverages virtual technology to connect students in the classroom with clinicians in community clinics (C4Tech) for authentic learning related to quality improvement (QI) and social determinants of health (SDH)., Method: This study applied a case study approach to evaluate the efficacy of the C4Tech model in supporting learning outcomes and assessed how virtual collaboration influenced the process of learning., Results: This study contributes to a more comprehensive understanding of how to design effective blended courses that connect the healthcare education and delivery systems through virtual technology. It also demonstrates how to connect students and practicing clinicians virtually to design evidence-based quality improvement projects.

Learning Health Systems (LHSs) seek continuous improvement through the translation and integration of internally and externally generated knowledge across stakeholders within and external to the organization, yet current approaches are primarily described from the healthcare delivery perspective, leaving teaching and research responsibilities underexposed. Academic medical centers offer a unique perspective on LHSs because their mission includes teaching, research, and healthcare. This introduces an opportunity to enact, educate, and study processes and outcomes of LHSs within a single system. Little information is available to describe these processes and outcomes, resulting in a knowledge gap regarding the role of education and research in the quality improvement cycles and learning of LHSs. To close this knowledge gap, The George Washington University School of Medicine and Health Sciences initiated the Health Research and Education Collaboratory (GW Collaboratory) in 2017. The GW Collaboratory was established to study mechanisms supporting continuous quality improvement and learning in health systems within an academic medical center. We envision the GW Collaboratory as interconnected knowledge nodes facilitating collaboration among clinicians, patients, researchers, and educators to study the knowledge generation, dissemination, application, and evaluation required for continuous quality improvement and learning. We employ a project-based approach to foster communities of learning focused on exploring specific health problems of interest. We propose the GW Collaboratory as one model by which academic medical centers can contribute to the science of LHS.

Purpose: This pilot study investigated the level of cognition that physician assistant (PA) students achieved through adoption of an innovative blended learning model that connects the classroom, clinicians, and community clinics through electronic-learning (e-learning) technologies (C4Tech) used in a PA course. This education intervention aimed to facilitate authentic learning collaborations between PA students and practicing clinicians that would result in higher-order cognition related to the manifestations of social determinants of health and health disparities., Methods: A case study approach was adopted to assess levels of cognition and changes in those levels resulting from application of an innovative blended learning model. Content analysis using Bloom's taxonomy of cognitive domains facilitated determination levels of cognition and changes in those levels. The sample of 8 groups comprised 70 PA students and 8 clinical instructors from community clinics with underrepresented patient populations., Results: Analysis of 2 course assignments revealed that application of the C4Tech model yields high levels of cognition. By the course's end, all 8 groups achieved at least the "evaluate" level of cognition and half of the groups achieved the highest level of cognition, the "create" level. A wide variation in the level of cognition was demonstrated between the first and second assignments in each group and among groups., Conclusion: Our findings suggest that e-learning technologies can be effective in blending classrooms and work environments for authentic and collaborative learning. Adoption of the C4Tech model yielded higher-order cognition related to course content.

OPCML, a tumor suppressor gene, is frequently silenced epigenetically in ovarian and other cancers. Here we report, by analysis of databases of tumor sequences, the observation of OPCML somatic missense mutations from various tumor types and the impact of these mutations on OPCML function, by solving the X-ray crystal structure of this glycoprotein to 2.65 Å resolution. OPCML consists of an extended arrangement of three immunoglobulin-like domains and homodimerizes via a network of contacts between membrane-distal domains. We report the generation of a panel of OPCML variants with representative clinical mutations and demonstrate clear phenotypic effects in vitro and in vivo including changes to anchorage-independent growth, interaction with activated cognate receptor tyrosine kinases, cellular migration, invasion in vitro and tumor growth in vivo. Our results suggest that clinically occurring somatic missense mutations in OPCML have the potential to contribute to tumorigenesis in a variety of cancers.

Background: With the remarkable attention being paid to STEM education nationally, with the growing engagement of universities and colleges in STEM education reform, and with the rise of STEM education centers, SECs, assisting universities as they strive to achieve these reforms, this research provides insight into the roles of six SECs. Through a multi-dimensional cross-site comparison, we provide a lens into the ways in which SECs function on their campuses, illuminating possibilities for those seeking to strengthen undergraduate STEM education., Results: SECs play an important networking role on their campuses, where they inform and unify institutional efforts, serving to elevate their visibility and importance both internally and externally. Through their scholarship, SECs contribute to the knowledge base and provide funding, which add resources and incentives for the implementation of evidence-based instructional practices (EBIPs) and STEM education research. SECs augment these efforts with the assessment and evaluation of learning outcomes and curricular innovations. Additionally, SECs act as an internal resource for faculty and instructors providing programs and training to foster the application of EBIPs in STEM courses. Several SECs provide the infrastructure for broader impact activities, and act as an external funding resource., Conclusions: STEM education centers make key contributions to their institutional environments. While the individual roles of these SECs on their campuses are distinctly unique, an in-depth look across six SECs reveals common areas of focus that allow these centers to enhance the undergraduate teaching and learning experience. Our results suggest that the ability of SECs to link STEM education research with teaching and learning initiatives provides a breadth of impact and attention across organizational levels. The analysis describes the ways in which these centers support institutional goals for undergraduate STEM education and relates these to areas of national priority. This research was carried out as part of a broader study, which informs the organizers of NSEC, the network of STEM education centers., Competing Interests: This study was approved by the University of Massachusetts Amherst’s Human Research and Protection Office # 00000017.Not applicable.The authors declare that they have no competing interests.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Activation of immune cells (but not B cells) with lectins is widely known. We used the structurally defined interaction between influenza hemagglutinin (HA) and its cell surface receptor sialic acid (SA) to identify a B cell receptor (BCR) activation modality that proceeded through non-cognate interactions with antigen. Using a new approach to reconstitute antigen-receptor interactions in a human reporter B cell line, we found that sequence-defined BCRs from the human germline repertoire could be triggered by both complementarity to influenza HA and a separate mode of signaling that relied on multivalent ligation of BCR sialyl-oligosaccharide. The latter suggested a new mechanism for priming naïve B cell responses and manifested as the induction of SA-dependent pan-activation by peripheral blood B cells. BCR crosslinking in the absence of complementarity is a superantigen effect induced by some microbial products to subvert production of antigen-specific immune responses. B cell superantigen activity through affinity for BCR carbohydrate is discussed.

Predicting immune responses before vaccination is challenging because of the complexity of the governing parameters. Nevertheless, recent work has shown that B cell receptor (BCR)-antigen engagement in vitro can prove a powerful means of informing the design of antibody-based vaccines. We have developed this principle into a two-phased immunogen evaluation pipeline to rank-order vaccine candidates. In phase 1, recombinant antigens are screened for reactivity to the germline precursors that produce the antibody responses of interest. To both mimic the architecture of initial antigen engagement and facilitate rapid immunogen screening, these antibodies are expressed as membrane-anchored IgM (mIgM) in 293F indicator cells. In phase 2, the binding hits are multimerized by nanoparticle or proteoliposome display, and they are evaluated for BCR triggering in an engineered B cell line displaying the IgM sequences of interest. Key developments that complement existing methodology in this area include the following: (i) introduction of a high-throughput screening step before evaluation of more time-intensive BCR-triggering analyses; (ii) generalizable multivalent antigen-display platforms needed for BCR activation; and (iii) engineered use of a human B cell line that does not display endogenous antibody, but only ectopically expressed BCR sequences of interest. Through this pipeline, the capacity to initiate favorable antibody responses is evaluated. The entire protocol can be completed within 2.5 months.

Acute kidney injury, often caused by an ischemic insult, is associated with significant short-term morbidity and mortality, and increased risk of chronic kidney disease. The factors affecting the renal response to injury following ischemia and reperfusion remain to be clarified. We found that the Stem cell antigen-1 (Sca-1), commonly used as a stem cell marker, is heavily expressed in renal tubules of the adult mouse kidney. We evaluated its potential role in the kidney using Sca-1 knockout mice submitted to acute ischemia reperfusion injury (IRI), as well as cultured renal proximal tubular cells in which Sca-1 was stably silenced with shRNA. IRI induced more severe injury in Sca-1 null kidneys, as assessed by increased expression of Kim-1 and Ngal, rise in serum creatinine, abnormal pathology, and increased apoptosis of tubular epithelium, and persistent significant renal injury at day 7 post IRI, when recovery of renal function in control animals was nearly complete. Serum creatinine, Kim-1 and Ngal were slightly but significantly elevated even in uninjured Sca-1-/- kidneys. Sca-1 constitutively bound both TGFβ receptors I and II in cultured normal proximal tubular epithelial cells. Its genetic loss or silencing lead to constitutive TGFβ receptor-mediated activation of canonical Smad signaling even in the absence of ligand and to KIM-1 expression in the silenced cells. These studies demonstrate that by normally repressing TGFβ-mediated canonical Smad signaling, Sca-1 plays an important in renal epithelial cell homeostasis and in recovery of renal function following ischemic acute kidney injury.

Inquiry-based laboratory instruction has been shown to actively engage students in the content and skills being taught. These courses are further intended to teach students not only what is known, but also the process by which investigators come to know it. We sought to take this approach one step further and incorporate novel research questions into an inquiry-based laboratory model early in the undergraduate course of study. In this research-based introductory laboratory course, first-year students acquired basic lab skills not just for their own sake, but rather within the context of a research question of a member of the faculty. Student projects investigated potential neuroanatomical changes in animal models of dyslexia and aging and included measurements of neuron numbers and levels and distribution of neuronal proteins. Students played an active role in designing and implementing an experimental plan, explored data analysis techniques, and reflected on the results that they obtained in scholarly forms such as research papers and a departmental poster session. Student feedback on this approach has been extremely positive, and the data collected were research quality preliminary data that are being actively pursued for further study. Based on our encouraging experiences, we conclude that designing an introductory course around novel research, including some assessments modeled after scholarly practices, provides motivation and excitement for the students, instills good scientific habits, and can potentially benefit departmental research.