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The histone methyltransferase enhancer of zeste homolog 2 (EZH2)-mediated epigenetic regulation of T cell differentiation in acute infection has been extensively investigated. However, the role of EZH2 in T cell exhaustion remains under-explored. Here, usingin vitroexhaustion models, we demonstrated that transient inhibition of EZH2 in T cells before the phenotypic onset of exhaustion with a clinically approved inhibitor, Tazemetastat, delayed their dysfunctional progression and maintained T cell stemness and polyfunctionality while having no negative impact on cell proliferation. Tazemetestat induced T cell epigenetic reprogramming and increased the expression of the self-renewing T cell transcription factor TCF1 by reducing its promoter H3K27 methylation preferentially in rapidly dividing T cells. In a murine melanoma model, T cells pre-treated with tazemetastat exhibited a superior response to anti-PD-1 blockade therapy after adoptive transfer. Collectively, these data unveil the potential of transient epigenetic reprogramming as a potential intervention to be combined with checkpoint blockade for immune therapy.

SummaryMost human proteins lack chemical probes, and several large-scale and generalizable small-molecule binding assays have been introduced to address this problem. How compounds discovered in such “binding-first” assays affect protein function, nonetheless, often remains unclear. Here, we describe a “function-first” proteomic strategy that uses size exclusion chromatography (SEC) to assess the global impact of electrophilic compounds on protein complexes in human cells. Integrating the SEC data with cysteine-directed activity-based protein profiling identifies changes in protein-protein interactions that are caused by site-specific liganding events, including the stereoselective engagement of cysteines in PSME1 and SF3B1 that disrupt the PA28 proteasome regulatory complex and stabilize a dynamic state of the spliceosome, respectively. Our findings thus show how multidimensional proteomic analysis of focused libraries of electrophilic compounds can expedite the discovery of chemical probes with site-specific functional effects on protein complexes in human cells.

SLC15A4 is an endolysosome-resident transporter that is intimately linked with autoinflammation and autoimmunity. Specifically, SLC15A4 is critical for Toll-like receptor (TLR) 7, 8, and 9 as well as the nucleotide-binding oligomerization domain-containing protein (NOD) 2 signaling in several immune cell subsets. Notably, SLC15A4 is essential for the development of systemic lupus erythematosus in murine models and is associated with autoimmune conditions in humans. Despite its therapeutic potential, to our knowledge no pharmacological tools have been developed that target SLC15A4. Here, we use an integrated chemical proteomics approach to develop a suite of chemical tools, including first-in-class functional inhibitors, for SLC15A4. We demonstrate SLC15A4 inhibitors suppress endosomal TLR and NOD functions in a variety of human and mouse immune cells and provide early evidence of their ability to suppress inflammation in vivo and in clinical settings. Our findings establish SLC15A4 as a druggable target for the treatment of autoimmune/autoinflammatory conditions.One-Sentence SummaryDiscovery and characterization of SLC15A4 inhibitors with anti-inflammatory activity.

Effector T cells comprise the cellular arm of the adaptive immune system and are essential for mounting immune responses against pathogens and cancer. To reach effector status, costimulation through CD28 is required. Here, we report that sialic acid-containing glycans on the surface of both T cells and APCs are alternative ligands of CD28 that compete with binding to its well-documented activatory ligand CD80 on the APC, resulting in attenuated costimulation. Removal of sialic acids enhances antigen-mediated activation of naïve T cells and also increases the revival of effector T cells made hypofunctional or exhausted via chronic viral infection. This occurs through a mechanism that is synergistic with antibody blockade of the inhibitory PD-1 axis. These results reveal a previously unrecognized role of sialic acid ligands in attenuation of CD28-mediated costimulation of T cells., T cells require multiple signals to activate, including costimulation via CD28. We report that sialoglycans are alternative ligands for CD28 and attenuate costimulation by competing with CD80.

SARS-CoV-2 virus has recently given rise to the current COVID-19 pandemic where infected individuals can range from being asymptomatic, yet highly contagious, to dying from acute respiratory distress syndrome. Although the world has mobilized to create antiviral vaccines and therapeutics to combat the scourge, their long-term efficacy remains in question especially with the emergence of new variants. In this work, we exploit a class of compounds that has previously shown success against various viruses. A salicylanilide library was first screened in a SARS-CoV-2 activity assay in Vero cells. The most efficacious derivative was further evaluated in a prophylactic mouse model of SARS-CoV-2 infection unveiling a salicylanilide that can reduce viral loads, modulate key cytokines, and mitigate severe weight loss involved in COVID-19 infections. The combination of anti-SARS-CoV-2 activity, cytokine inhibitory activity, and a previously established favorable pharmacokinetic profile for the lead salicylanilide renders salicylanilides in general as promising therapeutics for COVID-19.

Unleashing anti-tumor T cell activity by checkpoint inhibition is effective in many cancer patients but clinical response rates remain limited. Myeloid derived suppressor cells erode antitumor lymphocyte numbers and function, and correlate with resistance to checkpoint inhibitors. By screening small molecule libraries, we identified JAK inhibitors’ ability to rescue T cell function. Despite its documented immune suppressive properties, the prototypical JAK inhibitor ruxolitinib enhanced the efficacy of immune checkpoint blockade in cancer. This effect correlated with loss of suppressive gene expression, and acquisition of immunostimulatory molecular markers and T cell stimulatory activity in myeloid cells. In preclinical models, ruxolitinib significantly improved the function and increased the total numbers of activated tumor-infiltrating NK and CD4 T cells compared to checkpoint blockade alone and the efficacy was conditional on granulocytic cells. In addition to myeloid reprogramming in the tumor, ruxolitinib blunts G-CSF signaling in the bone marrow to prevent expression of suppressive and chemotaxis genes in neutrophils. In a clinical trial of Hodgkin lymphoma patients resistant to checkpoint inhibitors, treatment with ruxolitinib significantly reduced neutrophil-to-lymphocyte ratios and levels of suppressive markers in myeloid cells but increased numbers of cytokine-producing T cells. These results support the therapeutic potential of JAK inhibition in combination with checkpoint inhibitors in cancer and highlight the potential of reshaped myeloid immunity to improve immunotherapy.One sentence summary: Ruxolitinib reshapes myeloid immunity to synergize with checkpoint inhibitors

BACKGROUND/AIMS: During an immune response, type I interferon (IFN-I) signaling induces a wide range of changes, including those which are required to overcome viral infection and those which suppress cytotoxic T cells to avoid immunopathology. During certain bacterial infections, IFN-I signaling exerts largely detrimental effects. Although the IFN-I family of proteins all share one common receptor, biologic responses to signaling vary depending on IFN-I subtype. Here, we asked if one IFN-I subtype dominates the pro-bacterial effect of IFN-I signaling and found that control of Listeria monocytogenes (L.m.) infection is more strongly suppressed by IFN-β than IFN-α. METHODS: To study this, we measured bacterial titers in IFNAR-/-, IFN-β‑/‑, Stat2-/-, Usp18fl/fl and Usp18fl/fl x CD11c-Cre mice models in addition to IFN-I blocking antibodies. Moreover, we measured interferon stimulated genes in bone marrow derived dendritic cells after treatment with IFN-α4 and IFN-β. RESULTS: Specifically, we show that genetic deletion of IFN-β or antibody-mediated IFN-β neutralization was sufficient to reduce bacterial titers to levels similar to those observed in mice that completely lack IFN-I signaling (IFNAR-/- mice). However, IFN-α blockade failed to significantly reduce L.m. titers, suggesting that IFN-β is the dominant IFN-I subtype responsible for the pro-bacterial effect of IFN-I. Mechanistically, when focusing on IFN-I signals to dendritic cells, we found that IFN-β induces ISGs more robustly than IFN-α, including USP18, the protein we previously identified as driving the pro-bacterial effects of IFN-I. Further, we found that this induction was STAT1/STAT2 heterodimer- or STAT2/STAT2 homodimer-dependent, as STAT2-deficient mice were more resistant to L.m. infection. CONCLUSION: In conclusion, IFN-Β is the principal member of the IFN-I family responsible for driving the pro-bacterial effect of IFN-I.

Background: Checkpoint inhibitors have achieved a wide adoption for treatment of advanced cancer, but the majority of patients fail to respond. Among the underlying causes are redundant mechanisms driving cytotoxic lymphocyte dysfunction. Motivated by the discovery of JAK inhibitors as hit compounds in a screen for the reversal of T cell exhaustion, this study investigates the hypothesis that JAK inhibition could enhance the efficacy of checkpoint inhibitors. Methods: Murine immunocompetent models of cancer were utilized (A20, EL4, LLC1, MC38). The combination therapy of systemic treatment with the JAK inhibitor ruxolitinib with anti-PD1 + anti-CTLA4 was evaluated for efficacy and biomarkers compared to checkpoint inhibitors (ICI) alone. Tumor-infiltrating, blood and lymphoid organ immune cells were phenotyped using single-cell transcriptomics, functional assays and flow cytometry. The combination therapy was clinically tested in an investigator-initiated Phase I/II clinical trial of ruxolitinib with nivolumab in relapsed or refractory Hodgkin lymphoma (NCT03681561). Patients who previously failed to respond to ICI received ruxolitinib for 1 week then nivolumab every 4 weeks concurrent with ruxolitinib twice daily. Peripheral blood was collected at baseline, 1 week after ruxolitinib and 4 weeks after 1st nivolumab dose. Hematologic and transcriptomic analyses were performed on peripheral blood samples. Results: The ruxolitinib + ICI combination was superior to ICI in 3/4 of the tumor models examined in controlling tumor growth. Tumor sizes were reduced by >50% in the MC38 (mean volume 123.1 vs 283.2 mm3, ICI + rux vs ICI, respectively, n=9 per group, ANOVA p=0.0094), LLC1 and A20 models (survival hazard ratio 0.24, ICI + rux vs ICI, n=8 per group, p=0.048). Remarkably, we observed a broad shift of tumor monocytes and granulocytes from a suppressive into an immunostimulatory state characterized by the expression of MHC molecules and the ability to stimulate T cell proliferation. Hodgkin lymphoma patients in the ruxolitinib with nivolumab trial exhibited an interim disease control rate of 76% (13/17). Ruxolitinib treatment in these patients did not impair T cell cytokine production but significantly reduced the neutrophil-to-lymphocyte ratio (NLR, mean difference -0.82, n=14, p=0.0023) and the expression of myeloid derived suppressor cell markers in monocytes (normalized enrichment score -1.74, n=14, q Conclusions: The combination of ruxolitinib with ICI was effective in preclinical models of cancer and in a Phase I/II Hodgkin lymphoma clinical trial. Unexpectedly, JAK inhibitor-mediated reprogramming of myeloid cells and the associated enhanced T cell activity may be important for the observed efficacy. Citation Format: Jaroslav Zak, Isaraphorn Pratumchai, Brett S. Marro, Kristi L. Marquardt, Michael B. Oldstone, Judith A. Varner, Veronika Bachanova, John R. Teijaro. Reprogramming myeloid cells by JAK inhibition to enhance checkpoint blockade immunotherapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5743.

N-Acetylneuraminic acid is the most abundant sialic acid (SA) in humans and is expressed as the terminal sugar on intestinal mucus glycans. Several pathogenic bacteria harvest and display host SA on their own surfaces to evade Siglec-mediated host immunity. While previous studies have identified bacterial enzymes associated with SA catabolism, no reported methods permit the selective labeling, tracking, and quantitation of SA-presenting microbes within complex multi-microbial systems. We combined metabolic labeling, click chemistry, 16S rRNA gene, and whole-genome sequencing to track and identify SA-presenting microbes from a cultured human fecal microbiome. We isolated a new strain of Escherichia coli that incorporates SA onto its own surface and encodes for the nanT, neuA, and neuS genes necessary for harvesting and presenting SA. Our method is applicable to the identification of SA-presenting bacteria from human, animal, and environmental microbiomes, as well as providing an entry point for the investigation of surface-expressed SA-associated structures.

Bispecific T-cell engager (BiTE)-based cancer therapies that activate the cytotoxic T cells of a patient’s own immune system have gained momentum with the recent FDA approval of Blinatumomab for treating B cell malignancies. However, this approach has had limited success in targeting solid tumors. Here, we report the development of BiTE-sialidase fusion proteins that enhance tumor cell susceptibility to BiTE-mediated cytolysis by T cells via targeted desialylation at the BiTE-induced T cell-tumor cell interface. Targeted desialylation results in better immunological synapse formation, T-cell activation and effector function. As a result, BiTE-sialidase fusion proteins show remarkably increased efficacy in inducing T-cell-dependent tumor cell cytolysis in response to target antigens compared to the parent BiTE molecules alone. This enhanced function is seen bothin vitroand inin vivoxenograft and syngeneic solid tumor mouse models. Our findings highlight BiTE-sialidase fusion proteins as promising candidates for the development of next-generation bispecific T-cell engaging molecules for cancer immunotherapy.

A function-impairing mutation (feeble) or genomic deletion of SLC15A4 abolishes responses of nucleic acid–sensing endosomal toll-like receptors (TLRs) and significantly reduces disease in mouse models of lupus. Here, we demonstrate disease reduction in homozygous and even heterozygous Slc15a4 feeble mutant BXSB male mice with a Tlr7 gene duplication. In contrast to SLC15A4, a function-impairing mutation of SLC15A3 did not diminish type I interferon (IFN-I) production by TLR-activated plasmacytoid dendritic cells (pDCs), indicating divergence of function between these homologous SLC15 family members. Trafficking to endolysosomes and function of SLC15A4 were dependent on the Adaptor protein 3 (AP-3) complex. Importantly, SLC15A4 was required for trafficking and colocalization of nucleic acid–sensing TLRs and their ligands to endolysosomes and the formation of the LAMP2+VAMP3+ hybrid compartment in which IFN-I production is initiated. Collectively, these findings define mechanistic processes by which SLC15A4 controls endosomal TLR function and suggest that pharmacologic intervention to curtail the function of this transporter may be a means to treat lupus and other endosomal TLR-dependent diseases.

The JAK family of non-receptor tyrosine kinases includes four subtypes (JAK1, JAK2, JAK3, and TYK2) and is responsible for signal transduction downstream of diverse cytokine receptors. JAK inhibitors have emerged as important therapies for immuno(onc)ological disorders, but their use is limited by undesirable side effects presumed to arise from poor subtype selectivity, a common challenge for inhibitors targeting the ATP-binding pocket of kinases. Here, we describe the chemical proteomic discovery of a druggable allosteric cysteine present in the non-catalytic pseudokinase domain of JAK1 (C817) and TYK2 (C838), but absent from JAK2 or JAK3. Electrophilic compounds selectively engaging this site block JAK1-dependent transphosphorylation and cytokine signaling, while appearing to act largely as “silent” ligands for TYK2. Importantly, the allosteric JAK1 inhibitors do not impair JAK2-dependent cytokine signaling and are inactive in cells expressing a C817A JAK1 mutant. Our findings thus reveal an allosteric approach for inhibiting JAK1 with unprecedented subtype selectivity.

The rapid spread of SARS-CoV-2 variants poses a constant threat of escape from monoclonal antibody and vaccine countermeasures. Mutations in the ACE2 receptor binding site on the surface S protein have been shown to disrupt antibody binding and prevent viral neutralization. Here, we use a directed evolution-based approach to engineer three neutralizing antibodies for enhanced binding to S protein. The engineered antibodies showed increased in vitro functional activity in terms of neutralization potency and/or breadth of neutralization against viral variants. Deep mutational scanning revealed that higher binding affinity reduced the total number of viral escape mutations. Studies in the Syrian hamster model showed two examples where the affinity matured antibody provided superior protection compared to the parental antibody. These data suggest that monoclonal antibodies for anti-viral indications could benefit from in vitro affinity maturation to reduce viral escape pathways and appropriate affinity maturation in vaccine immunization could help resist viral variation.

Summary: Inhibitory receptors (IRs) function as critical regulators of immune responses by tempering T cell activity. In humans, several persisting viruses as well as cancers exploit IR signaling by upregulating IR ligands, resulting in suppression of T cell function (i.e., exhaustion). This allows escape from immune surveillance and continuation of disease. Here, we report the design, implementation, and results of a phenotypic high-throughput screen for molecules that modulate CD8+ T cell activity. We identify 19 compounds from the ReFRAME drug-repurposing collection that restore cytokine production and enhance the proliferation of exhausted T cells. Analysis of our top hit, ingenol mebutate, a protein kinase C (PKC) inducing diterpene ester, reveals a role for this molecule in overriding the suppressive signaling cascade mediated by IR signaling on T cells. Collectively, these results demonstrate a disease-relevant methodology for identifying modulators of T cell function and reveal new targets for immunotherapy. : Discovery of pharmacologic drugs that target exhausted T cells is essential to overcome the limitations of current checkpoint blockade therapies. Marro et al. utilize a high-throughput screening method to identify small-molecule modulators of T cells and describe a role for protein kinase C in resurrecting T cell effector activity. Keywords: T cell exhaustion, chronic infection, high-throughput flow cytometry, checkpoint blockade, CD8 T cell, PKC, ingenol mebutate

Unlike SARS-CoV-1 and MERS-CoV, infection with SARS-CoV-2, the viral pathogen responsible for COVID-19, is often associated with neurologic symptoms that range from mild to severe, yet increasing evidence argues the virus does not exhibit extensive neuroinvasive properties. We demonstrate SARS-CoV-2 can infect and replicate in human iPSC-derived neurons and that infection shows limited anti-viral and inflammatory responses but increased activation of EIF2 signaling following infection as determined by RNA sequencing. Intranasal infection of K18 human ACE2 transgenic mice (K18-hACE2) with SARS-CoV-2 resulted in lung pathology associated with viral replication and immune cell infiltration. In addition, ∼50% of infected mice exhibited CNS infection characterized by wide-spread viral replication in neurons accompanied by increased expression of chemokine (Cxcl9, Cxcl10, Ccl2, Ccl5andCcl19) and cytokine (Ifn-λandTnf-α) transcripts associated with microgliosis and a neuroinflammatory response consisting primarily of monocytes/macrophages. Microglia depletion via administration of colony-stimulating factor 1 receptor inhibitor, PLX5622, in SARS-CoV-2 infected mice did not affect survival or viral replication but did result in dampened expression of proinflammatory cytokine/chemokine transcripts and a reduction in monocyte/macrophage infiltration. These results argue that microglia are dispensable in terms of controlling SARS-CoV-2 replication in in the K18-hACE2 model but do contribute to an inflammatory response through expression of pro-inflammatory genes. Collectively, these findings contribute to previous work demonstrating the ability of SARS-CoV-2 to infect neurons as well as emphasizing the potential use of the K18-hACE2 model to study immunological and neuropathological aspects related to SARS-CoV-2-induced neurologic disease.ImportanceUnderstanding the immunological mechanisms contributing to both host defense and disease following viral infection of the CNS is of critical importance given the increasing number of viruses that are capable of infecting and replicating within the nervous system. With this in mind, the present study was undertaken to evaluate the role of microglia in aiding in host defense following experimental infection of the central nervous system (CNS) of K18-hACE2 with SARS-CoV-2, the causative agent of COVID-19. Neurologic symptoms that range in severity are common in COVID-19 patients and understanding immune responses that contribute to restricting neurologic disease can provide important insight into better understanding consequences associated with SARS-CoV-2 infection of the CNS.

Improved understanding of host antiviral defense and antitumor immunity have elucidated molecular pathways important to both processes. During viral infection, RNA or DNA in the host cell serves as a danger signal that initiates the antiviral response. Recent studies have elucidated similarities in the signaling pathways activated by viruses and the signaling pathways induced by tumor DNA that is released into the cytoplasm of irradiated tumor cells. Both the host defense to viral infection and the sterile inflammation provoked by radiotherapy induce a type I interferon response that is necessary for pathogen control and immune-mediated tumor control, respectively. These findings have led to the hypothesis that radiotherapy employs a form of viral mimicry.

Acute respiratory distress syndrome (ARDS), an inflammatory condition with high mortality rates, is common in severe COVID-19, whose risk is reduced by metformin rather than other anti-diabetic medications. Detecting of inflammasome assembly in post-mortem COVID-19 lungs, we asked whether and how metformin inhibits inflammasome activation while exerting its anti-inflammatory effect. We show that metformin inhibited NLRP3 inflammasome activation and interleukin (IL)-1β production in cultured and alveolar macrophages along with inflammasome-independent IL-6 secretion, thus attenuating lipopolysaccharide (LPS)- and SARS-CoV-2-induced ARDS. By targeting electron transport chain complex 1 and independently of AMP-activated protein kinase (AMPK) or NF-κB, metformin blocked LPS-induced and ATP-dependent mitochondrial (mt) DNA synthesis and generation of oxidized mtDNA, an NLRP3 ligand. Myeloid-specific ablation of LPS-induced cytidine monophosphate kinase 2 (CMPK2), which is rate limiting for mtDNA synthesis, reduced ARDS severity without a direct effect on IL-6. Thus, inhibition of ATP and mtDNA synthesis is sufficient for ARDS amelioration., Graphical abstract, The exact mechanism by which metformin exerts its anti-inflammatory effects is still not known. Xian et al. shows that metformin’s inhibition of ETCCI blocks ATP-dependent mtDNA synthesis, cytoplasmic ox-mtDNA generation and NLRP3 inflammasome activation in macrophages, independent of AMPK and NF-κB. By limiting IL-1β production, metformin blunts pulmonary inflammation.

The ongoing pandemic caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), necessitates strategies to identify prophylactic and therapeutic drug candidates for rapid clinical deployment. Here, we describe a screening pipeline for the discovery of efficacious SARS-CoV-2 inhibitors. We screen a best-in-class drug repurposing library, ReFRAME, against two high-throughput, high-content imaging infection assays: one using HeLa cells expressing SARS-CoV-2 receptor ACE2 and the other using lung epithelial Calu-3 cells. From nearly 12,000 compounds, we identify 49 (in HeLa-ACE2) and 41 (in Calu-3) compounds capable of selectively inhibiting SARS-CoV-2 replication. Notably, most screen hits are cell-line specific, likely due to different virus entry mechanisms or host cell-specific sensitivities to modulators. Among these promising hits, the antivirals nelfinavir and the parent of prodrug MK-4482 possess desirable in vitro activity, pharmacokinetic and human safety profiles, and both reduce SARS-CoV-2 replication in an orthogonal human differentiated primary cell model. Furthermore, MK-4482 effectively blocks SARS-CoV-2 infection in a hamster model. Overall, we identify direct-acting antivirals as the most promising compounds for drug repurposing, additional compounds that may have value in combination therapies, and tool compounds for identification of viral host cell targets., Here, the authors perform repurposing screens of the ReFRAME drug library in two cell lines and identify inhibitors of SARS-CoV-2 infection. Antiviral activity of prodrug MK-4482 is confirmed in hamsters.

Chronic infection and cancer are associated with suppressed T cell responses in the presence of cognate antigen. Recent work identified memory-like CXCR5+ TCF1+ CD8+ T cells that sustain T cell responses during persistent infection and proliferate upon anti-PD1 treatment. Approaches to expand these cells are sought. We show that blockade of interferon type 1 (IFN-I) receptor leads to CXCR5+ CD8+ T cell expansion in an IL-27– and STAT1-dependent manner. IFNAR1 blockade promoted accelerated cell division and retention of TCF1 in virus-specific CD8+ T cells. We found that CD8+ T cell–intrinsic IL-27 signaling safeguards the ability of TCF1hi cells to maintain proliferation and avoid terminal differentiation or programmed cell death. Mechanistically, IL-27 endowed rapidly dividing cells with IRF1, a transcription factor that was required for sustained division in a cell-intrinsic manner. These findings reveal that IL-27 opposes IFN-I to uncouple effector differentiation from cell division and suggest that IL-27 signaling could be exploited to augment self-renewing T cells in chronic infections and cancer.

It is unclear whether individuals with enormous diversity in B cell receptor repertoires are consistently able to mount effective antibody responses against SARS-CoV-2. We analyzed antibody responses in a cohort of 55 convalescent patients and isolated 54 potent neutralizing monoclonal antibodies (mAbs). While most of the mAbs target the angiotensin-converting enzyme 2 (ACE2) binding surface on the receptor binding domain (RBD) of SARS-CoV-2 spike protein, mAb 47D1 binds only to one side of the receptor binding surface on the RBD. Neutralization by 47D1 is achieved independent of interfering RBD-ACE2 binding. A crystal structure of the mAb-RBD complex shows that the IF motif at the tip of 47D1 CDR H2 interacts with a hydrophobic pocket in the RBD. Diverse immunoglobulin gene usage and convergent epitope targeting characterize neutralizing antibody responses to SARS-CoV-2, suggesting that vaccines that effectively present the receptor binding site on the RBD will likely elicit neutralizing antibody responses in a large fraction of the population., Graphical abstract, Zhou et al. isolate 54 potent neutralizing antibodies from convalescent COVID-19 patients, revealing diverse immunoglobulin gene usage and convergent epitope targeting of neutralizing antibody responses to SARS-CoV-2. Vaccines that effectively present the RBD receptor binding site will likely elicit neutralizing antibody responses in a large fraction of the population.

Broadly neutralizing antibodies (bnAbs) to coronaviruses (CoVs) are valuable in their own right as prophylactic and therapeutic reagents to treat diverse CoVs and, importantly, as templates for rational pan-CoV vaccine design. We recently described a bnAb, CC40.8, from a coronavirus disease 2019 (COVID-19)-convalescent donor that exhibits broad reactivity with human beta-coronaviruses (β-CoVs). Here, we showed that CC40.8 targets the conserved S2 stem-helix region of the coronavirus spike fusion machinery. We determined a crystal structure of CC40.8 Fab with a SARS-CoV-2 S2 stem-peptide at 1.6 Å resolution and found that the peptide adopted a mainly helical structure. Conserved residues in β-CoVs interacted with CC40.8 antibody, thereby providing a molecular basis for its broad reactivity. CC40.8 exhibited in vivo protective efficacy against SARS-CoV-2 challenge in two animal models. In both models, CC40.8-treated animals exhibited less weight loss and reduced lung viral titers compared to controls. Furthermore, we noted CC40.8-like bnAbs are relatively rare in human COVID-19 infection and therefore their elicitation may require rational structure-based vaccine design strategies. Overall, our study describes a target on β-CoV spike proteins for protective antibodies that may facilitate the development of pan-β-CoV vaccines.SUMMARYA human mAb isolated from a COVID-19 donor defines a protective cross-neutralizing epitope for pan-β-CoV vaccine design strategies

The new vaccines against SARS-CoV-2 are novel in terms of specificity, their wide dissemination across the global population and the inclusion of newly licensed mRNA platforms. We discuss here how the approved vaccines trigger innate immunity to promote durable immunological memory and consider the future implications of protecting populations with these vaccines., This Comment outlines how the recently licensed vaccines for COVID-19 activate innate immune mechanisms to promote immune memory to SARS-CoV-2. The authors also consider future challenges that could limit vaccine efficacy.

Effector T cells comprise the cellular arm of the adaptive immune system and are essential for mounting immune responses against pathogens and cancer. To reach effector status, co-stimulation through CD28 is required. Here, we report that sialic acid-containing glycans on the surface of both T cells and APCs are alternative ligands of CD28 that compete with binding to its well-documented activatory ligand CD80 on the APC, resulting in attenuated co-stimulation. Removal of sialic acids enhances T cell activation and enhances the activity of effector T cells made hypofunctional via chronic viral infection through a mechanism that is synergistic with antibody blockade of the inhibitory PD-1 axis. These results reveal a previously unrecognized role for sialic acids in attenuation of CD28 mediated co-stimulation of T cells.One Sentence SummarySialic acids attenuate the second signal required for T cell activation.

Type 1 interferon provides potent antiviral defense but unchecked IFN-I signaling can lead to pathology or immune suppression. The mechanisms by which IFN-I contributes to disease pathogenesis remain poorly understood. Here, using Usp18-deficient, USP18 enzymatic-inactive and Isg15-deficient mouse models, we report that lack of USP18 enzymatic function during persistent viral infection leads to severe immune pathology characterized by hematological disruptions, pulmonary cytokine amplification, lung vascular leakage and death. Lack of Usp18 in myeloid cells mimicked the pathological manifestations observed in Usp18-/- or Usp18C61A knock-in mice and required Isg15. Mechanistically, interrupting the enzymes that conjugate/deconjugate ISG15 led to accumulation of ISG15 which was accompanied by inflammatory neutrophil accumulation and lung pathology. Moreover, neutrophil depletion reversed pathological manifestations, morbidity and mortality in Usp18C61A mice. Our results suggest that dysregulated ISG15 signaling during viral infection can produce lethal immune pathology and could be a therapeutic target during severe viral infections with pulmonary pathological manifestations.

Therapeutics against coronavirus disease 2019 (COVID-19) are urgently needed. Granulocyte–macrophage colony-stimulating factor (GM-CSF), a myelopoietic growth factor and pro-inflammatory cytokine, plays a critical role in alveolar macrophage homeostasis, lung inflammation and immunological disease. Both administration and inhibition of GM-CSF are currently being therapeutically tested in COVID-19 clinical trials. This Perspective discusses the pleiotropic biology of GM-CSF and the scientific merits behind these contrasting approaches., Recombinant granulocyte–macrophage colony-stimulating factor (GM-CSF) as well as antibodies targeted at GM-CSF or its receptor are being tested in clinical trials for coronavirus disease 2019 (COVID-19). This Perspective introduces the pleiotropic functions of GM-CSF and explores the rationale behind these different approaches.

The sympathetic nervous system (SNS) contributes to immune balance by promoting anti-inflammatory B cells. However, whether B cells possess a self-regulating mechanism by which they modulate regulatory B cell (Breg) function is not well understood. In this study, we investigated the ability of B cells to synthesize catecholamines upon stimulation with different B cell activators. We found, that expression of the enzymes required to generate catecholamines, is upregulated by TLR9. TLR-9-specific expression of tyrosine hydroxylase (TH) correlated with upregulation of adrenergic receptors, enhanced IL-10 production, and with an overexpression of the co-inhibitory ligands PD-L1 and FasL. Moreover, concomitant stimulation of The sympathetic nervous system (SNS) contributes to immune balance by promoting anti-inflammatory B cells. However, whether B cells possess a self-regulating mechanism by which they modulate regulatory B cell (Breg) function is not well understood. In this study, we investigated the ability of B cells to synthesize catecholamines upon stimulation with different B cell activators. We found, that expression of the enzymes required to generate catecholamines, is upregulated by TLR9. TLR-9-specific expression of tyrosine hydroxylase (TH) correlated with upregulation of adrenergic receptors, enhanced IL-10 production, and with an overexpression of the co-inhibitory ligands PD-L1 and FasL. Moreover, concomitant stimulation of beta 1-3-adrenergic receptors together with a BCR/TLR9 stimulus enhances the anti-inflammatory potential of Bregs to suppress CD4 T cells, a crucial population in the pathogenesis of autoimmune diseases, like rheumatoid arthritis. In conclusion, our data show that B cells possess autonomous mechanisms to modulate their regulatory function. These findings help to better understand the function of Bregs in autoimmune diseases and the interplay of sympathetic nervous system and B cell function. Beta 1-3-adrenergic receptors together with a BCR/TLR9 stimulus enhances the anti-inflammatory potential of Bregs to suppress CD4 T cells, a crucial population in the pathogenesis of autoimmune diseases, like rheumatoid arthritis. In conclusion, our data show that B cells possess autonomous mechanisms to modulate their regulatory function. These findings help to better understand the function of Bregs in autoimmune diseases and the interplay of sympathetic nervous system and B cell function.

Author(s): Rogers, Thomas F; Zhao, Fangzhu; Huang, Deli; Beutler, Nathan; Burns, Alison; He, Wan-Ting; Limbo, Oliver; Smith, Chloe; Song, Ge; Woehl, Jordan; Yang, Linlin; Abbott, Robert K; Callaghan, Sean; Garcia, Elijah; Hurtado, Jonathan; Parren, Mara; Peng, Linghang; Ricketts, James; Ricciardi, Michael J; Rawlings, Stephen A; Smith, Davey M; Nemazee, David; Teijaro, John R; Voss, James E; Andrabi, Raiees; Briney, Bryan; Landais, Elise; Sok, Devin; Jardine, Joseph G; Burton, Dennis R | Abstract: The development of countermeasures to prevent and treat COVID-19 is a global health priority. In under 7 weeks, we enrolled a cohort of SARS-CoV-2-recovered participants, developed neutralization assays to interrogate serum and monoclonal antibody responses, adapted our high throughput antibody isolation, production and characterization pipeline to rapidly screen over 1000 antigen-specific antibodies, and established an animal model to test protection. We report multiple highly potent neutralizing antibodies (nAbs) and show that passive transfer of a nAb provides protection against high-dose SARS-CoV-2 challenge in Syrian hamsters. The study suggests a role for nAbs in prophylaxis, and potentially therapy, of COVID-19. The nAbs define protective epitopes to guide vaccine design.

Cytokine storm during respiratory viral infection is an indicator of disease severity and poor prognosis. Type 1 interferon (IFN-I) production and signaling has been reported to be causal in cytokine storm-associated pathology in several respiratory viral infections, however, the mechanisms by which IFN-I promotes disease pathogenesis remain poorly understood. Here, using Usp18-deficient, USP18 enzymatic-inactive and Isg15-deficient mouse models, we report that lack of deISGylation during persistent viral infection leads to severe immune pathology characterized by hematological disruptions, cytokine amplification, lung vascular leakage and death. This pathology requires T cells but not T cell-intrinsic deletion of Usp18. However, lack of Usp18 in myeloid cells mimicked the pathological manifestations observed in Usp18-/- or Usp18C61A mice which were dependent on Isg15. We further mechanistically demonstrate that interrupting the ISGylation/deISGylation circuit increases extracellular levels of ISG15 which is accompanied by inflammatory neutrophil accumulation to the lung. Importantly, neutrophil depletion reversed morbidity and mortality in Usp18C61A mice. In summary, we reveal that the enzymatic function of Usp18 is crucial for regulating extracellular release of ISG15. This is accompanied by altered neutrophil differentiation, cytokine amplification and mortality following persistent viral infection. Moreover, our results suggest that extracellular ISG15 may drive the inflammatory pathology observed and could be both a prospective predictor of disease outcome and a therapeutic target during severe respiratory viral infections.

SummaryRe-activation and clonal expansion of tumor specific antigen (TSA)-reactive T cells are critical to the success of checkpoint blockade and adoptive transfer of tumor-infiltrating lymphocyte (TIL) based therapies. There are no reliable markers to specifically identify the repertoire of TSA-reactive T cells due to their heterogeneous composition. Here we introduce FucoID as a general platform to detect endogenous antigen-specific T cells and study their biology. Through this interaction dependent labeling approach, TSA-reactive T cells can be detected and separated from bystander T cells in primary tumor digests based on their cell-surface enzymatic fucosyl-biotinylation. Compared to bystander TILs, TSA-reactive TILs possess a distinct TCR repertoire and unique gene features. Though exhibiting a dysfunctional phenotype, this subset of TILs possesses substantial capabilities of proliferation and tumor specific killing. FucoID features genetic manipulation-free procedures and a quick turnover cycle, and therefore should have the potential of accelerating the pace of personalized cancer treatment.HighlightsInteraction dependent fucosylation enables the detection and isolation of bona fide intratumoral tumor specific antigen-reactive T cellsTumor specific antigen-reactive CD8+ T cells possess capabilities to be expanded and adoptively transferred for tumor controlTumor specific antigen-reactive CD8+ T cells feature oligoclonal expansion and upregulate genes for the steroid biosynthesis and metabolic processIntratumoral bystander CD8+ T cells can be separated into two groups based on PD-1 expression that feature distinct gene modules

IFNAR1 signaling in NK cells compromises cellular and humoral immune responses to hamper virus control., Inhibition of type 1 interferon (IFN-I) signaling promotes the control of persistent virus infection, but the underlying mechanisms remain poorly understood. Here, we report that genetic ablation of Ifnar1 specifically in natural killer (NK) cells led to elevated numbers of T follicular helper cells, germinal center B cells, and plasma cells and improved antiviral T cell function, resulting in hastened virus clearance that was comparable to IFNAR1 neutralizing antibody treatment. Antigen-specific B cells and antiviral antibodies were essential for the accelerated control of LCMV Cl13 infection following IFNAR1 blockade. IFNAR1 signaling in NK cells promoted NK cell function and general killing of antigen-specific CD4 and CD8 T cells. Therefore, inhibition of IFN-I signaling in NK cells enhances CD4 and CD8 T cell responses, promotes humoral immune responses, and thereby facilitates the control of persistent virus infection.

Stimulator of interferon genes (STING) links innate immunity to biological processes ranging from antitumor immunity to microbiome homeostasis. Mechanistic understanding of the anticancer potential for STING receptor activation is currently limited by metabolic instability of the natural cyclic dinucleotide (CDN) ligands. From a pathway-targeted cell-based screen, we identified a non-nucleotide, small-molecule STING agonist, termed SR-717, that demonstrates broad interspecies and interallelic specificity. A 1.8-angstrom cocrystal structure revealed that SR-717 functions as a direct cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) mimetic that induces the same "closed" conformation of STING. SR-717 displayed antitumor activity; promoted the activation of CD8+ T, natural killer, and dendritic cells in relevant tissues; and facilitated antigen cross-priming. SR-717 also induced the expression of clinically relevant targets, including programmed cell death 1 ligand 1 (PD-L1), in a STING-dependent manner.

N-acetylneuraminic acid is the most abundant sialic acid in humans and is generally expressed as the terminal sugar on intestinal mucus glycans. Several pathogenic bacterial species harvest sialic acid from the mucus, diet, as well as other intestinal sources and display this sugar on their own surface to evade sialic acid-binding immunoglobulin-type lectin (Siglec)-mediated host immune surveillance. While previous studies have identified bacterial enzymes associated with sialic acid catabolism, no reported methods permit the selective labeling, tracking, and quantitation of sialic acid-presenting microbes within complex multi-microbial systems. Here, we apply an interdisciplinary approach combining metabolic labeling, click chemistry, metagenomic, and whole-genome sequencing to selectively track and identify sialic acid-presenting microbes from a cultured healthy human fecal microbiome. We isolated and identified a new strain of Escherichia coli that incorporates sialic acid onto its own surface. Analysis of the sequence data reveals that this E. coli strain encodes for the NanT, NeuA, and NeuS genes necessary for harvesting environmental sialic acid and generating the capsular polysialic acid. We envision that this method is applicable to the detection and quantitation of sialic acid-presenting bacteria from human, animal, and environmental microbiomes, as well as investigating the importance of other carbohydrates to commensal and pathogenic bacteria.

ABHD12 metabolizes bioactive lysophospholipids, including lysophosphatidylserine (lyso-PS). Deleterious mutations in human ABHD12 cause the neurological disease PHARC, and ABHD12(−/−) mice display PHARC-like phenotypes, including hearing loss, along with elevated brain lyso-PS and features of stimulated innate immune cell function. Here, we develop a selective and in vivo-active inhibitor of ABHD12 termed DO264 and show that this compound elevates lyso-PS in mouse brain and primary human macrophages. Unlike ABHD12(−/−) mice, adult mice treated with DO264 exhibited minimal perturbations in auditory function. On the other hand, both DO264-treated and ABHD12(−/−) mice displayed heightened immunological responses to lymphocytic choriomeningitis virus (LCMV) clone 13 infection that manifested as severe lung pathology with elevated proinflammatory chemokines. These results reveal similarities and differences in the phenotypic impact of pharmacological versus genetic blockade of ABHD12 and point to a key role for this enzyme in regulating immunostimulatory lipid pathways in vivo.

The sensing of microbial genetic material by leukocytes often elicits beneficial pro-inflammatory cytokines, but dysregulated responses can cause severe pathogenesis. Genome-wide association studies have linked the gene encoding phospholipase D3 (PLD3) to Alzheimer's disease and have linked PLD4 to rheumatoid arthritis and systemic sclerosis. PLD3 and PLD4 are endolysosomal proteins whose functions are obscure. Here, PLD4-deficient mice were found to have an inflammatory disease, marked by elevated levels of interferon-γ (IFN-γ) and splenomegaly. These phenotypes were traced to altered responsiveness of PLD4-deficient dendritic cells to ligands of the single-stranded DNA sensor TLR9. Macrophages from PLD3-deficient mice also had exaggerated TLR9 responses. Although PLD4 and PLD3 were presumed to be phospholipases, we found that they are 5' exonucleases, probably identical to spleen phosphodiesterase, that break down TLR9 ligands. Mice deficient in both PLD3 and PLD4 developed lethal liver inflammation in early life, which indicates that both enzymes are needed to regulate inflammatory cytokine responses via the degradation of nucleic acids.

The TAM family of receptor tyrosine kinases (MERTK, AXL, and TYRO3) are expressed on DCs and macrophages, and they serve a critical role in efferocytosis, by which apoptotic bodies are cleared by engulfment. Their activation drives phagocytic activity concomitant to promoting an immuno-suppressive, tolerogenic state in DCs and the M2 polarization of macrophages. This dual signaling mechanism allows for the clearance of cells undergoing non-pathologic apoptotic turnover without inappropriately breaking self-tolerance and driving auto-immunity. Unlike most immuno-therapy targets, the KOs of the TAM kinases, PD-1, and CTLA-4 share common auto-immune disease phenotypes such as splenomegaly, immune infiltration of the liver, arthritis, and lupus. Furthermore, viruses have evolved to promote PD-1/CTLA-4 signaling to suppress the anti-viral immune response, and they similarly target the TAM receptors via apoptotic mimicry to reduce the inflammatory response. This confluence of immune phenotypes provided the biological rationale for the discovery of PF-5807/ARRY-067, a potent and selective inhibitor of MERTK/AXL as a DC-activating immuno-oncology therapeutic. Based on the IC50 for inhibition of phospho-MERTK/AXL in cell-based assays, our mouse PK and PK/PD studies determined that 50 mg/kg PF-5807 provided IC90 plasma coverage for 12 hours. This PK profile supported a twice-daily dosing schedule for maintaining continuous suppression of MERTK and AXL, which was used for mechanistic and efficacy studies in mouse models. To investigate DC and T cell responses to PF-5807, mice were adoptively-transferred with OT-I T cells and challenged with ovalbumin immunization; flow cytometry analysis indicated that PF-5807 treatment increased the fraction of activated, Class II MHC-positive DCs, as well as cross-primed IFNγ+ TNFα+ OT-I T cells. Also, we found that cytotoxic T lymphocytes were necessary for tumor control in the E0771 mouse syngeneic tumor model, as depletion of CD8+ T cells abrogated all activity by PF-5807 in inhibiting tumor growth. This anti-tumor activity was further demonstrated in the MC-38 and EMT6 tumor models, where single agent PF-5807 effected 57-88% TGI and a 13-38% cure rate, while combination with PD-1 blockade drove 99-100% TGI and a 75-100% cure rate. Finally, these complete responders rejected tumor re-challenge without further treatment, resulting in extended survival and durable cures that corroborate the development of immunologic memory. Therefore, we find that PF-5807 activates DCs to cross-prime CD8+ T cells, which are necessary for tumor control and are likely responsible for the establishment of tumor-specific memory T cells. PF-5807 is currently in an ongoing Phase I clinical trial in patients with advanced or metastatic solid tumors, with the intent to explore combination with anti-PD-1/anti-PD-L1 antibodies. Citation Format: Jim Wong, Jacqueline Harrison, Karyn Bouhana, Nhan Nguyen, Namir Shaabani, Vincent F. Vartabedian, LouAnn Cable, Robert Rieger, Lisa Pieti Opie, Shelley Allen, Adam Cook, Joshua Dahlke, Ronald Hinklin, David Chantry, Shannon Winski, John Teijaro. The potent and selective MERTK/AXL inhibitor PF-5807/ARRY-067 activates dendritic cells to cross-prime CD8+ T cells for anti-tumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1735.

Electrophilic compounds originating from nature or chemical synthesis have profound effects on immune cells. These compounds are thought to act by cysteine modification to alter the functions of immune-relevant proteins; however, our understanding of electrophile-sensitive cysteines in the human immune proteome remains limited. Here, we present a global map of cysteines in primary human T cells that are susceptible to covalent modification by electrophilic small molecules. More than 3000 covalently liganded cysteines were found on functionally and structurally diverse proteins, including many that play fundamental roles in immunology. We further show that electrophilic compounds can impair T cell activation by distinct mechanisms involving direct functional perturbation and/or ligand-induced degradation of proteins. Our findings reveal a rich content of ligandable cysteines in human T cells, underscoring the potential of electrophilic small molecules as a fertile source for chemical probes and ultimately therapeutics that modulate immunological processes and their associated disorders.

CXCR5+ TCF1+ CD8+ T cells sustain responses during persistent viral infection and mediate the proliferative burst following anti-PD1 treatment. Huang et al. show that IL-27 supports rapid division of these cells by competing with type 1 interferon for STAT1, driving IRF1 expression and preventing cell death., Chronic infection and cancer are associated with suppressed T cell responses in the presence of cognate antigen. Recent work identified memory-like CXCR5+ TCF1+ CD8+ T cells that sustain T cell responses during persistent infection and proliferate upon anti-PD1 treatment. Approaches to expand these cells are sought. We show that blockade of interferon type 1 (IFN-I) receptor leads to CXCR5+ CD8+ T cell expansion in an IL-27– and STAT1-dependent manner. IFNAR1 blockade promoted accelerated cell division and retention of TCF1 in virus-specific CD8+ T cells. We found that CD8+ T cell–intrinsic IL-27 signaling safeguards the ability of TCF1hi cells to maintain proliferation and avoid terminal differentiation or programmed cell death. Mechanistically, IL-27 endowed rapidly dividing cells with IRF1, a transcription factor that was required for sustained division in a cell-intrinsic manner. These findings reveal that IL-27 opposes IFN-I to uncouple effector differentiation from cell division and suggest that IL-27 signaling could be exploited to augment self-renewing T cells in chronic infections and cancer., Graphical Abstract