Faculty Bibliography

Therapeutically targeting pathogenic T cells in autoimmune diseases has been challenging. Although LAG-3, an inhibitory checkpoint receptor specifically expressed on activated T cells, is known to bind to major histocompatibility complex class II (MHC class II), we demonstrate that MHC class II interaction alone is insufficient for optimal LAG-3 function. Instead, LAG-3's spatial proximity to T cell receptor (TCR) but not CD4 co-receptor, facilitated by cognate peptide-MHC class II, is crucial in mediating CD4⁺ T cell suppression. Mechanistically, LAG-3 forms condensate with TCR signaling component CD3ε through its intracellular FSAL motif, disrupting CD3ε/lymphocyte-specific protein kinase (Lck) association. To exploit LAG-3's proximity to TCR and maximize LAG-3-dependent T cell suppression, we develop an Fc-attenuated LAG-3/TCR inhibitory bispecific antibody to bypass the requirement of cognate peptide-MHC class II. This approach allows for potent suppression of both CD4⁺ and CD8⁺ T cells and effectively alleviates autoimmune symptoms in mouse models. Our findings reveal an intricate and conditional checkpoint modulatory mechanism and highlight targeting of LAG-3/TCR cis-proximity for T cell-driven autoimmune diseases lacking effective and well-tolerated immunotherapies.

The factors that install and recognize N⁶-methyladenosine (m⁶A) on RNA to regulate gene expression are well characterized, but how their spatial organization responds to physiological stress, including infection, is unclear. Here, we show that human cytomegalovirus (HCMV) infection induces accumulation of m⁶A methyltransferase subunits, including WTAP, together with nuclear m⁶A reader YTHDC1, into distinctive, membraneless nuclear bodies (NBs) overlapping with incoming virus genomes and immediate-early (IE) RNA transcripts. De novo assembly and integrity of these DNA-associated, IE, virus-activated NBs requires RNAPII transcription, METTL3 m⁶A methyltransferase activity, and m⁶A recognition by YTHDC1, but not new protein synthesis. Depleting YTHDC1 or WTAP limits the accumulation of critical HCMV RNAs required for virus DNA replication, interfering with virus reproduction. This reveals a surprising strategy whereby a discrete sub-nuclear RNA biogenesis compartment replete with RNAPII and m⁶A modification components is swiftly consolidated in proximity to infecting HCMV genomes to initialize and sustain virus gene expression.

Allergic contact dermatitis (ACD) is a delayed-type IV hypersensitivity response driven by innate and adaptive immune cells. While specific immune regulations of these cell types are amply elucidated, their regulations by extracellular matrix (ECM) components and T cell mediated adaptive immunity in ACD remains unclear. Lumican and biglycan are ECM proteoglycans abundant in the dermis and lymph node, known to regulate innate immune myeloid cells, but have not been investigated in lymphoid cell regulations in ACD. By immunohistology we localized lumican and biglycan in skin biopsies of psoriatic patients. Using wild type (WT), lumican and biglycan knockout mice, we investigated CD4⁺T cell infiltration, activation and proliferation in the skin and draining lymph node (dLN) of CHS-challenged mice by immunohistochemistry and flow cytometry. We used the OT-II adoptive transfer model to test antigen specific CD4⁺T cell activation. We assessed interactions of the proteoglycans with LFA-1 on T cells by confocal microscopy. Compared to WTs, the knockouts showed severe ear inflammation, with increased CD4⁺T cells infiltration in the dermis. CHS-challenged knockout mice dLN showed increased T-bet, STAT1 and -STAT4 signaling, indicating enhanced Th1 commitment and proliferation. We found that WT lymph node fibroblastic reticular cells (FRCs) secrete lumican, biglycan and decorin, a related proteoglycan, while none are expressed by naive or activated T cells. Lumican and biglycan interact with LFA-1 on T cell surfaces, and in vitro all three proteoglycans suppress CD4⁺T cell activation. Secreted by dLN FRCs, lumican, biglycan, and possibly decorin interact with LFA-1 on CD4⁺T cells to restrict its activation and reduce dermatitis severity.

BACKGROUND:Plakophilin-2 (PKP2) is a component of the desmosome. Pathogenic variants can lead to arrhythmogenic cardiomyopathy (PKP2-ACM). In PKP2-ACM patients, exercise and catecholamine surges negatively impact arrhythmia incidence and severity. OBJECTIVE:To characterize remodeling of the sympathetic input and adrenergic response in hearts of PKP2-deficient mice (PKP2cKO) subjected to endurance exercise. METHODS:transient dynamics. Separately, we evaluated distribution of sympathetic terminals in PKP2cKO trained hearts vs controls. RESULTS:Exercise led to increased abundance of sarcolemma β1-ARs in control, and decreased abundance in PKP2cKO-myocytes. OCT3 knockdown drastically reduced the response of trained PKP2cKO-myocytes to norepinephrine but not isoproterenol, indicating preserved response to native catecholamines by intracellular (dyad-associated) receptors in the setting of a reduced sarcolemma pool. In tissue, we observed reduced abundance of sympathetic terminals, and heterogeneous distribution across the myocardium. CONCLUSION/CONCLUSIONS:Endurance exercise in PKP2-deficient myocytes leads to reduced pool of functional β1-ARs in the sarcolemma and yet availability of intracellular receptors, which can activate selected (and heterogeneous) routes of intracellular signaling cascades. We speculate that remodeling of nerve terminals affects sympathetic input distribution and hence, regional modulation of excitability and conduction. These changes can facilitate cell-generated triggered activity and heterogeneity of the underlying substrate, setting the stage for life-threatening arrhythmias.

Embryonic development in many species, including case reports in humans, can be temporarily halted before implantation during a process called diapause. Facultative diapause occurs under conditions of maternal metabolic stress such as nursing. While molecular mechanisms of diapause have been studied, a natural inducing factor has yet to be identified. Here, we show that oxytocin induces embryonic diapause in mice. We show that gestational delays were triggered during nursing or optogenetic stimulation of oxytocin neurons simulating nursing patterns. Mouse blastocysts express oxytocin receptors, and oxytocin induced delayed implantation-like dispersion in cultured embryos. Last, oxytocin receptor-knockout embryos transferred into wild-type surrogates had low survival rates during diapause. Our results indicate that oxytocin coordinates timing of embryonic development with uterine progression through pregnancy, providing an evolutionarily conserved mechanism for ensuring successful reproduction.

UNLABELLED: IMPORTANCE/OBJECTIVE:research should consider capsule size, not just its presence and type. The results imply that standardized vaccine efficacy tests may yield variable results depending on the capsule production of target strains.

INTRODUCTION/BACKGROUND:Chinese Americans are one of the fastest growing racial and ethnic groups and represent the largest subgroup of the Asian American population in the US and in New York City (NYC) where they number 573,528 in 2021. Despite their numbers, current pain perceptions, expectations, and attitudes of Chinese Americans remains poorly understood, especially as related to postoperative pain. OBJECTIVE:A better understanding of pain experience among Chinese American patients is needed to inform strategies on improving pain management satisfaction. METHODS:A total of 27 Chinese American postoperative patients from a NYC health system were recruited for face-to-face surveys and interviews with a trained bilingual and bicultural Community Health Worker. Questions from the Survey on Disparities in Quality of Healthcare and Kleinman's Explanatory Model of Illness were integrated into the survey and topic guide. Topics of discussion included satisfaction with healthcare and pain management during hospital stay and health beliefs and practices. RESULTS:More than half of participants experienced language challenges that made it difficult to communicate with healthcare staff. In general, high levels of satisfaction with pain management were reported; however, participants reported feeling less comfortable asking healthcare teams questions. Common themes across interviews included: (1) pain was an expected outcome of the procedure and was thus perceived as tolerable; (2) the wish to not be a burden to others; (3) concerns about side effects of pain medications; and (4) a cultural and language mismatch between healthcare teams and patients on words being used to elicit pain and discomfort. CONCLUSION/CONCLUSIONS:Our project findings can inform pain management strategies and tools to serve the Chinese American patient population.

In response to the antimicrobial resistance crisis, we have developed a powerful and versatile therapeutic platform, the Antibacterial Drone (ABD) system. The ABD consists of a highly mobile staphylococcal pathogenicity island re-purposed to deliver genes encoding antibacterial proteins. The chromosomally located island is induced by a co-resident helper phage, packaged in phage-like particles, and released in very high numbers upon phage-induced lysis. ABD particles specifically adsorb to bacteria causing an infection and deliver their DNA to these bacteria, where the bactericidal cargo genes are expressed, kill the bacteria, and cure the infection. Here, we report a major advance of the system, incorporation of the gene encoding a secreted, bactericidal, species-specific lytic enzyme, lysostsphin. This ABD not only kills the bacterium that has been attacked by the ABD, but also any surrounding bacteria that are sensitive to the lytic enzyme which is released by secretion and by lysis of the doomed cell. So while the killing field is thus expanded, there are no civilian casualties (bacteria that are insensitive to the ABD and its cargo protein(s) are not inadvertently killed). Without amplifying the number of ABD particles (which are not re-packaged), the expression and release of the cargo gene's product dramatically extend the effective reach of the ABD. A cargo gene that encodes a secreted bactericidal protein also enables the treatment of a mixed bacterial infection in which one of the infecting organisms is insensitive to the ABD delivery system but is sensitive to the ABD's secreted cargo protein.

In a previous study, heart xenografts from 10-gene-edited pigs transplanted into two human decedents did not show evidence of acute-onset cellular- or antibody-mediated rejection. Here, to better understand the detailed molecular landscape following xenotransplantation, we carried out bulk and single-cell transcriptomics, lipidomics, proteomics and metabolomics on blood samples obtained from the transplanted decedents every 6 h, as well as histological and transcriptomic tissue profiling. We observed substantial early immune responses in peripheral blood mononuclear cells and xenograft tissue obtained from decedent 1 (male), associated with downstream T cell and natural killer cell activity. Longitudinal analyses indicated the presence of ischemia reperfusion injury, exacerbated by inadequate immunosuppression of T cells, consistent with previous findings of perioperative cardiac xenograft dysfunction in pig-to-nonhuman primate studies. Moreover, at 42 h after transplantation, substantial alterations in cellular metabolism and liver-damage pathways occurred, correlating with profound organ-wide physiological dysfunction. By contrast, relatively minor changes in RNA, protein, lipid and metabolism profiles were observed in decedent 2 (female) as compared to decedent 1. Overall, these multi-omics analyses delineate distinct responses to cardiac xenotransplantation in the two human decedents and reveal new insights into early molecular and immune responses after xenotransplantation. These findings may aid in the development of targeted therapeutic approaches to limit ischemia reperfusion injury-related phenotypes and improve outcomes.

Glycolysis is a fundamental cellular process, yet its regulatory mechanisms remain incompletely understood. Here, we show that a subset of glucose transporter 1 (GLUT1/SLC2A1) co-endocytoses with platelet-derived growth factor (PDGF) receptor (PDGFR) upon PDGF-stimulation. Furthermore, multiple glycolytic enzymes localize to these endocytosed PDGFR/GLUT1-containing vesicles adjacent to mitochondria. Contrary to current models, which emphasize the importance of glucose transporters on the cell surface, we find that PDGF-stimulated glucose uptake depends on receptor/transporter endocytosis. Our results suggest that growth factors generate glucose-loaded endocytic vesicles that deliver glucose to the glycolytic machinery in proximity to mitochondria, and argue for a new layer of regulation for glycolytic control governed by cellular membrane dynamics.