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Background Characterizing the longevity and quality of cellular immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enhances understanding of coronavirus disease 2019 (COVID-19) immunity that influences clinical outcomes. Prior studies suggest SARS-CoV-2–specific T cells are present in peripheral blood 10 months after infection. Analysis of the function, durability, and diversity of cellular response long after natural infection, over a range of ages and disease phenotypes, is needed to identify preventative and therapeutic interventions. Methods We identified participants in our multisite longitudinal, prospective cohort study 12 months after SARS-CoV-2 infection representing a range of disease severity. We investigated function, phenotypes, and frequency of T cells specific for SARS-CoV-2 using intracellular cytokine staining and spectral flow cytometry, and compared magnitude of SARS-CoV-2–specific antibodies. Results SARS-CoV-2–specific antibodies and T cells were detected 12 months postinfection. Severe acute illness was associated with higher frequencies of SARS-CoV-2–specific CD4 T cells and antibodies at 12 months. In contrast, polyfunctional and cytotoxic T cells responsive to SARS-CoV-2 were identified in participants over a wide spectrum of disease severity. Conclusions SARS-CoV-2 infection induces polyfunctional memory T cells detectable at 12 months postinfection, with higher frequency noted in those who experienced severe disease.

BackgroundCharacterizing the longevity and quality of cellular immune responses to SARS-CoV-2 is critical to understanding immunologic approaches to protection against COVID-19. Prior studies suggest SARS-CoV-2-specific T cells are present in peripheral blood 10 months after infection. Further analysis of the function, durability, and diversity of the cellular response long after natural infection, over a wider range of ages and disease phenotypes, is needed to further identify preventative and therapeutic interventions.MethodsWe identified participants in our multi-site longitudinal, prospective cohort study 12-months post SARS-CoV-2 infection representing a range of disease severity. We investigated the function, phenotypes, and frequency of T cells specific for SARS-CoV-2 using intracellular cytokine staining and spectral flow cytometry. In parallel, the magnitude of SARS-CoV-2-specific antibodies was compared.ResultsSARS-CoV-2-specific antibodies and T cells were detected at 12-months post-infection. Severity of acute illness was associated with higher frequencies of SARS-CoV-2-specific CD4 T cells and antibodies at 12-months. In contrast, polyfunctional and cytotoxic T cells responsive to SARS-CoV-2 were identified in participants over a wide spectrum of disease severity.ConclusionsOur data show that SARS-CoV-2 infection induces polyfunctional memory T cells detectable at 12-months post-infection, with higher frequency noted in those who originally experienced severe disease.

Lactate dehydrogenase (LDH) catalyzes the conversion of pyruvate to lactate, with concomitant oxidation of reduced nicotinamide adenine dinucleotide as the final step in the glycolytic pathway. Glycolysis plays an important role in the metabolic plasticity of cancer cells and has long been recognized as a potential therapeutic target. Thus, potent, selective inhibitors of LDH represent an attractive therapeutic approach. However, to date, pharmacological agents have failed to achieve significant target engagement in vivo, possibly because the protein is present in cells at very high concentrations. We report herein a lead optimization campaign focused on a pyrazole-based series of compounds, using structure-based design concepts, coupled with optimization of cellular potency, in vitro drug–target residence times, and in vivo PK properties, to identify first-in-class inhibitors that demonstrate LDH inhibition in vivo. The lead compounds, named NCATS-SM1440 (43) and NCATS-SM1441 (52), possess desirable attributes for further studying the effect of in vivo LDH inhibition.

A novel application of [DTBNpP] Pd(crotyl)Cl (DTBNpP = di-tert-butylneopentylphosphine) (P2), an air-stable, commercially available palladium precatalyst that allows rapid access to a monoligated state, has been identified for room-temperature, copper-free Sonogashira couplings of challenging aryl bromides and alkynes. The mild reaction conditions with TMP in dimethyl sulfoxide afford up to 97% yields, excellent functional group tolerability, and broad reaction compatibility with access to one-pot indole formation.

Immune homeostasis depends upon effective clearance of pathogens while simultaneously preventing autoimmunity and immunopathology in the host. Restimulation-induced cell death (RICD) is one such mechanism where by activated T cells receive subsequent antigenic stimulation, reach a critical signal threshold through the T cell receptor (TCR), and commit to apoptosis. Many details of this process remain unclear, including the role of co-stimulatory and co-inhibitory proteins that influence the TCR signaling cascade. Here we characterize the role of T cell immunoglobulin and mucin domain containing 3 (TIM-3) in RICD regulation. TIM-3 protected newly activated CD8+ effector T cells from premature RICD during clonal expansion. Surprisingly, however, we found that TIM-3 potentiated RICD in late-stage effector T cells. The presence of TIM-3 increased proximal TCR signaling and proapoptotic protein expression in late-stage effector T cells, with no consistent signaling effects noted in newly activated cells with or without TIM-3. To better explain these differences in TIM-3 function as T cells aged, we characterized the temporal pattern of TIM-3 expression in effector T cells. We found that TIM-3 was expressed on the surface of newly activated effector T cells, but remained largely intracellular in late-stage effector cells. Consistent with this, TIM-3 required a ligand to prevent early RICD, whereas ligand manipulation had no effects at later stages. Of the known TIM-3 ligands, carcinoembryonic antigen‐related cell adhesion molecule (CEACAM1) showed the greatest difference in surface expression over time and also protected newly activated cells from premature RICD, with no measurable effects in late-stage effectors. Indeed, CEACAM1 enabled TIM-3 surface expression on T cells, implying a co-dependency for these proteins in protecting expanding T cells from premature RICD. Our findings suggest that co-signaling proteins like TIM-3 and CEACAM1 can alter RICD sensitivity at different stages of the effector T cell response, with important implications for checkpoint blockade therapy.

The active sites of hundreds of human α-ketoglutarate (αKG) and Fe(II)-dependent dioxygenases are exceedingly well preserved, which challenges the design of selective inhibitors. We identified a noncatalytic cysteine (Cys481 in KDM5A) near the active sites of KDM5 histone H3 lysine 4 demethylases, which is absent in other histone demethylase families, that could be explored for interaction with the cysteine-reactive electrophile acrylamide. We synthesized analogs of a thienopyridine-based inhibitor chemotype, namely, 2-((3-aminophenyl)(2-(piperidin-1-yl)ethoxy)methyl)thieno[3,2- b]pyridine-7-carboxylic acid (N70) and a derivative containing a (dimethylamino)but-2-enamido)phenyl moiety (N71) designed to form a covalent interaction with Cys481. We characterized the inhibitory and binding activities against KDM5A and determined the cocrystal structures of the catalytic domain of KDM5A in complex with N70 and N71. Whereas the noncovalent inhibitor N70 displayed αKG-competitive inhibition that could be reversed after dialysis, inhibition by N71 was dependent on enzyme concentration and persisted even after dialysis, consistent with covalent modification.

Cyclic GMP-AMP (cGAMP) synthase (cGAS) stimulator of interferon genes (STING) senses pathogen-derived or abnormal self-DNA in the cytosol and triggers an innate immune defense against microbial infection and cancer. STING agonists induce both innate and adaptive immune responses and are a new class of cancer immunotherapy agents tested in multiple clinical trials. However, STING is commonly silenced in cancer cells via unclear mechanisms, limiting the application of these agonists. Here, we report that the expression of STING is epigenetically suppressed by the histone H3K4 lysine demethylases KDM5B and KDM5C and is activated by the opposing H3K4 methyltransferases. The induction of STING expression by KDM5 blockade triggered a robust interferon response in a cytosolic DNA-dependent manner in breast cancer cells. This response resulted in resistance to infection by DNA and RNA viruses. In human tumors, KDM5B expression is inversely associated with STING expression in multiple cancer types, with the level of intratumoral CD8+ T cells, and with patient survival in cancers with a high level of cytosolic DNA, such as human papilloma virus (HPV)-positive head and neck cancer. These results demonstrate a novel epigenetic regulatory pathway of immune response and suggest that KDM5 demethylases are potential targets for antipathogen treatment and anticancer immunotherapy., Author summary Pathogens often find ways to turn down cell-intrinsic antipathogen immune responses by the host. Similarly, cancer cells use various mechanisms to evade attack by immune cells. One of the common mechanisms is suppression of the stimulator of interferon genes (STING)-dependent innate immune response. Using potent and specific small-molecule inhibitors and genetic-depletion approaches, we found that the silenced STING pathway can be reactivated in breast cancer cells by suppressing KDM5 demethylases. Activation of the STING pathway led to a robust interferon response, which blocked viral infection, and was associated with increased tumor-infiltrated lymphocytes and better patient survival in multiple cancer types. This discovery has major clinical implications for treating both pathogen infection and cancer because KDM5 inhibition provides a fast, robust, and reversible control of innate immune response. Since the discovery of histone demethylase activity of KDM5 proteins a decade ago, significant efforts have been dedicated to developing KDM5 inhibitors for clinical applications. In fact, a KDM5 inhibitor recently entered phase I clinical trial for treatment of hepatitis B infection. Here, we provide mechanistic insights on how KDM5 inhibitors block viral infection. Moreover, our results suggest that KDM5 inhibitors can also be combined with other cancer immunotherapies.

Isomers of chiral drugs can exhibit marked differences in biological activities. We studied the binding and inhibitory activities of 12 compounds against KDM5A. Among them are two pairs of enantiomers representing two distinct inhibitor chemotypes, namely, ( R)- and ( S)-2-((2-chlorophenyl)(2-(piperidin-1-yl)ethoxy)methyl)-1 H-pyrrolo[3,2- b]pyridine-7-carboxylic acid (compounds N51 and N52) and ( R) - and ( S) -N-(1-(3-isopropyl-1 H-pyrazole-5-carbonyl)pyrrolidin-3-yl)cyclopropanecarboxamide (compounds N54 and N55). In vitro, the S enantiomer of the N51/N52 pair (N52) and the R enantiomer of the N54/N55 pair (N54) exhibited about 4- to 5-fold greater binding affinity. The more potent enzyme inhibition of KDM5A by the R-isoform for the cell-permeable N54/N55 pair translated to differences in growth inhibitory activity. We determined structures of the KDM5A catalytic domain in complex with all 12 inhibitors, which revealed the interactions (or lack thereof) responsible for the differences in binding affinity. These results provide insights to guide improvements in binding potency and avenues for development of cell permeable inhibitors of the KDM5 family.

Distinct mutations in the centrosomal-cilia protein CEP290 lead to diverse clinical findings in syndromic ciliopathies. We show that CEP290 localizes to the transition zone in ciliated cells, precisely to the region of Y-linkers between central microtubules and plasma membrane. To create models of CEP290-associated ciliopathy syndromes, we generated Cep290(ko/ko) and Cep290(gt/gt) mice that produce no or a truncated CEP290 protein, respectively. Cep290(ko/ko) mice exhibit early vision loss and die from hydrocephalus. Retinal photoreceptors in Cep290(ko/ko) mice lack connecting cilia, and ciliated ventricular ependyma fails to mature. The minority of Cep290(ko/ko) mice that escape hydrocephalus demonstrate progressive kidney pathology. Cep290(gt/gt) mice die at mid-gestation, and the occasional Cep290(gt/gt) mouse that survives shows hydrocephalus and severely cystic kidneys. Partial loss of CEP290-interacting ciliopathy protein MKKS mitigates lethality and renal pathology in Cep290(gt/gt) mice. Our studies demonstrate domain-specific functions of CEP290 and provide novel therapeutic paradigms for ciliopathies.

Up to 30% of patients with hemophilia A develop inhibitory antibodies to therapeutic FVIII and these antibodies block the efficacy of this critical protein. To target the B cells specific for FVIII, we previously generated cytotoxic cells expressing the FVIII C2 or A2 immunodominant domains as chimeric receptors. We termed these antigen-expressing engineered T cells, "BARs", for B-cell Antibody Receptor. These could directly interact and kill with A2 or C2 FVIII-specific B cells as well as FVIII-specific hybridomas in vitro and in vivo. To increase the number of B-cell targets that could be killed, we recently engineered human and murine BAR CD8 T cells to express the FVIII light chain (LC), which includes A3 and C1, as well as C2 domains. In addition, since C2-expressing BAR T cells showed tonic signaling, we hypothesized that LC expressing BARs might not. Our data suggest that LC-expressing BAR T cells do not display tonic signaling but they can be stimulated to proliferate by anti-LC monoclonal antibody (mAb 5G12), for example. Flow cytometry data showed that LC-BAR T cells are stained by anti-C2 mAb (3G6) but this mAb did not stimulate them to proliferate. These data reflect the accessibility of different FVIII domains in BAR T cells, and are important in terms of the utility of FVIII domain expressing BARs to target specific B cells in vivo in the presence of inhibitors. (Supported by NIH grant R01 HL126727) Disclosures No relevant conflicts of interest to declare.