OMIP-099: 31-color spectral flow cytometry panel to investigate the steady-state phenotype of human T cells.

We have developed a 31-color panel to define the steady-state phenotype of T cells in human peripheral blood (Table 1). The panel presented here was optimized using cryopreserved peripheral blood mononuclear cells (PBMC). The markers included in this panel were chosen in order to characterize the steady-state phenotype of T cells and includes markers (CD45RA, CD45RO, CCR7, CD95) to distinguish the main subsets (e.g., naïve, TEM , TCM , TEMRA , TSCM etc.) of CD4, CD8, and γδ T cells. This panel also includes markers for the identification of differentiation status (CD27, CD28), activation/antigen experience status (CD11a, CD49d, CD38, HLA-DR, CD56, and CD39), co-inhibitory marker expression (PD-1, TIM-3), and CD4 T helper subsets (CXCR3, CXCR5, CCR4, CCR6, Foxp3, CD25, and CD127). This optimized panel provides a broad assessment of the steady-state phenotype of human T cells.

Immunomodulatory Effects of Subacute Inhalation Exposure to Copper Oxide Nanoparticles in House Dust Mite-Induced Asthma.

It has been shown that inhalation exposure to copper oxide nanoparticles (CuO NPs) results in pulmonary inflammation. However, immunomodulatory consequences after CuO NP inhalation exposure have been less explored. We tested the effect of CuO NP aerosols on immune responses in healthy, house dust mite (HDM) asthmatic, or allergen immunotherapy (AIT)-treated asthmatic mice (BALB/c, females). The AIT consisted of a vaccine comprising HDM allergens and CpG-loaded nanoparticles (CpG NPs). AIT treatment involved mice being immunized (via subcutaneous (sc) injection; 2 doses) while concomitantly being exposed to CuO NP aerosols (over a 2 week period), starting on the day of the first vaccination. Mice were then sensitized twice by sc injection and subsequently challenged with HDM extract 10 times by intranasal instillation. The asthmatic model followed the same timeline except that no immunizations were administered. All mice were necropsied 24 h after the end of the HDM challenge. CuO NP-exposed healthy mice showed a significant decrease in TH1 and TH2 cells, and an elevation in T-bet+ Treg cells, even 40 days after the last exposure to CuO NPs. Similarly, the CuO NP-exposed HDM asthma model demonstrated decreased TH2 responses and increased T-bet+ Treg cells. Conversely, CuO NP inhalation exposure to AIT-treated asthmatic mice resulted in an increase in TH2 cells. In conclusion, immunomodulatory effects of inhalation exposure to CuO NPs are dependent on immune conditions prior to exposure.

GRB2 dimerization mediated by SH2 domain-swapping is critical for T cell signaling and cytokine production.

GRB2 is an adaptor protein required for facilitating cytoplasmic signaling complexes from a wide array of binding partners. GRB2 has been reported to exist in either a monomeric or dimeric state in crystal and solution. GRB2 dimers are formed by the exchange of protein segments between domains, otherwise known as "domain-swapping". Swapping has been described between SH2 and C-terminal SH3 domains in the full-length structure of GRB2 (SH2/C-SH3 domain-swapped dimer), as well as between α-helixes in isolated GRB2 SH2 domains (SH2/SH2 domain-swapped dimer). Interestingly, SH2/SH2 domain-swapping has not been observed within the full-length protein, nor have the functional influences of this novel oligomeric conformation been explored. We herein generated a model of full-length GRB2 dimer with an SH2/SH2 domain-swapped conformation supported by in-line SEC-MALS-SAXS analyses. This conformation is consistent with the previously reported truncated GRB2 SH2/SH2 domain-swapped dimer but different from the previously reported, full-length SH2/C-terminal SH3 (C-SH3) domain-swapped dimer. Our model is also validated by several novel full-length GRB2 mutants that favor either a monomeric or a dimeric state through mutations within the SH2 domain that abrogate or promote SH2/SH2 domain-swapping. GRB2 knockdown and re-expression of selected monomeric and dimeric mutants in a T cell lymphoma cell line led to notable defects in clustering of the adaptor protein LAT and IL-2 release in response to TCR stimulation. These results mirrored similarly-impaired IL-2 release in GRB2-deficient cells. These studies show that a novel dimeric GRB2 conformation with domain-swapping between SH2 domains and monomer/dimer transitions are critical for GRB2 to facilitate early signaling complexes in human T cells.

Endogenous SOD2 (Superoxide Dismutase) Regulates Platelet-Dependent Thrombin Generation and Thrombosis During Aging.

BACKGROUND: Reactive oxygen species (ROS) contribute to platelet hyperactivation during aging. Several oxidative pathways and antioxidant enzymes have been implicated; however, their mechanistic contributions during aging remain elusive. We hypothesized that mitochondria are an important source of platelet ROS and that mitochondrial SOD2 (superoxide dismutase) protects against mitochondrial ROS-driven platelet activation and thrombosis during aging. METHODS: We studied littermates of platelet-specific SOD2-knockout (SOD2fl/flPf4Cre, pSOD2-KO) and control (SOD2fl/fl) mice at young (4-5 months) or old (18-20 months) ages. We examined agonist-induced platelet activation, platelet-dependent thrombin generation potential, and susceptibility to in vivo thrombosis. RESULTS: Platelet αIIbß3 activation, aggregation, and adhesion were increased to similar extents in aged mice of both genotypes compared with young mice. In contrast, the age-dependent increases in mitochondrial and total cellular ROS, calcium elevation, and phosphatidylserine exposure were augmented in platelets from pSOD2-KO mice compared with control mice. Aged pSOD2-KO mice showed increased platelet-dependent thrombin generation compared with aged control mice. In vivo, aged pSOD2-KO mice exhibited enhanced susceptibility to carotid artery and pulmonary thrombosis compared to aged control mice. Adoptive transfer of platelets from aged pSOD2-KO but not aged control mice increased thrombotic susceptibility in aged host mice, suggesting a prothrombotic effect of platelet pSOD2 deficiency. Treatment with avasopasem manganese (GC4419), a SOD mimetic, decreased platelet mitochondrial pro-oxidants, cellular ROS levels, and inhibited procoagulant platelet formation and arterial thrombosis in aged mice. CONCLUSIONS: Platelet mitochondrial ROS contributes to age-related thrombosis and endogenous SOD2 protects from platelet-dependent thrombin generation and thrombosis during aging.

Pharmacological ascorbate as a novel therapeutic strategy to enhance cancer immunotherapy.

Pharmacological ascorbate (i.e., intravenous infusions of vitamin C reaching ~ 20 mM in plasma) is under active investigation as an adjuvant to standard of care anti-cancer treatments due to its dual redox roles as an antioxidant in normal tissues and as a prooxidant in malignant tissues. Immune checkpoint inhibitors (ICIs) are highly promising therapies for many cancer patients but face several challenges including low response rates, primary or acquired resistance, and toxicity. Ascorbate modulates both innate and adaptive immune functions and plays a key role in maintaining the balance between pro and anti-inflammatory states. Furthermore, the success of pharmacological ascorbate as a radiosensitizer and a chemosensitizer in pre-clinical studies and early phase clinical trials suggests that it may also enhance the efficacy and expand the benefits of ICIs.

Glycerol monolaurate inhibition of human B cell activation.

Glycerol monolaurate (GML) is a naturally occurring antimicrobial agent used commercially in numerous products and food items. GML is also used as a homeopathic agent and is being clinically tested to treat several human diseases. In addition to its anti-microbial function, GML suppresses immune cell proliferation and inhibits primary human T cell activation. GML suppresses T cell activation by altering membrane dynamics and disrupting the formation of protein clusters necessary for intracellular signaling. The ability of GML to disrupt cellular membranes suggests it may alter other cell types. To explore this possibility, we tested how GML affects human B cells. We found that GML inhibits BCR-induced cytokine production, phosphorylation of signaling proteins, and protein clustering, while also changing cellular membrane dynamics and dysregulating cytoskeleton rearrangement. Although similar, there are also differences between how B cells and T cells respond to GML. These differences suggest that unique intrinsic features of a cell may result in differential responses to GML treatment. Overall, this study expands our understanding of how GML impacts the adaptive immune response and contributes to a broader knowledge of immune modulating monoglycerides.

Comparative Study of Bacterial SPOR Domains Identifies Functionally Important Differences in Glycan Binding Affinity.

Bacterial SPOR domains target proteins to the divisome by binding septal peptidoglycan (PG) at sites where cell wall amidases have removed stem peptides. These PG structures are referred to as denuded glycans. Although all characterized SPOR domains bind denuded glycans, whether there are differences in affinity is not known. Here, we use isothermal titration calorimetry (ITC) to determine the relative PG glycan binding affinity (<i>K</i>_d) of four Escherichia coli SPOR domains and one Cytophaga hutchinsonii SPOR domain. We found that the <i>K</i>_d values ranged from approximately 1 µM for E. coli DamX^SPOR and <i>C. hutchinsonii</i> CHU2221^SPOR to about 10 µM for E. coli FtsN^SPOR. To investigate whether these differences in PG binding affinity are important for SPOR domain protein function, we constructed and characterized a set of DamX and FtsN "swap" proteins. As expected, all SPOR domain swap proteins localized to the division site, and, in the case of FtsN, all of the heterologous SPOR domains supported cell division. However, for DamX, only the high-affinity SPOR domain from CHU2221 supported normal function in cell division. In summary, different SPOR domains bind denuded PG glycans with different affinities, which appears to be important for the functions of some SPOR domain proteins (e.g., DamX) but not for the functions of others (e.g., FtsN). <b>IMPORTANCE</b> SPOR domain proteins are prominent components of the cell division apparatus in a wide variety of bacteria. The primary function of SPOR domains is targeting proteins to the division site, which they accomplish by binding to septal peptidoglycan. However, whether SPOR domains have any functions beyond septal targeting is unknown. Here, we show that SPOR domains vary in their PG binding affinities and that, at least in the case of the E. coli cell division protein DamX, having a high-affinity SPOR domain contributes to proper function.

Pharmacological ascorbate improves the response to platinum-based chemotherapy in advanced stage non-small cell lung cancer.

PURPOSE: Platinum-based chemotherapy with or without immunotherapy is the mainstay of treatment for advanced stage non-small cell lung cancer (NSCLC) lacking a molecular driver alteration. Pre-clinical studies have reported that pharmacological ascorbate (P-AscH-) enhances NSCLC response to platinum-based therapy. We conducted a phase II clinical trial combining P-AscH- with carboplatin-paclitaxel chemotherapy. EXPERIMENTAL DESIGN: Chemotherapy naïve advanced stage NSCLC patients received 75 g ascorbate twice per week intravenously with carboplatin and paclitaxel every three weeks for four cycles. The primary endpoint was to improve tumor response per Response Evaluation Criteria in Solid Tumors (RECIST) v1.1 compared to the historical control of 20%. The trial was conducted as an optimal Simon's two-stage design. Blood samples were collected for exploratory analyses. RESULTS: The study enrolled 38 patients and met its primary endpoint with an objective response rate of 34.2% (p = 0.03). All were confirmed partial responses (cPR). The disease control rate was 84.2% (stable disease + cPR). Median progression-free and overall survival were 5.7 months and 12.8 months, respectively. Treatment-related adverse events (TRAE) included one grade 5 (neutropenic fever) and five grade 4 events (cytopenias). Cytokine and chemokine data suggest that the combination elicits an immune response. Immunophenotyping of peripheral blood mononuclear cells demonstrated an increase in effector CD8 T-cells in patients with a progression-free survival (PFS) ≥ 6 months. CONCLUSIONS: The addition of P-AscH- to platinum-based chemotherapy improved tumor response in advanced stage NSCLC. P-AscH- appears to alter the host immune response and needs further investigation as a potential adjuvant to immunotherapy.

T-Cell Expression of Angiotensin-Converting Enzyme 2 and Binding of Severe Acute Respiratory Coronavirus 2.

The pathogenesis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is not completely understood. SARS-CoV-2 infection frequently causes significant immune function consequences including reduced T cell numbers and enhanced T cell exhaustion that contribute to disease severity. The extent to which T cell effects are directly mediated through infection or indirectly result from infection of respiratory-associated cells is unclear. We show that primary human T cells express sufficient levels of angiotensin converting enzyme 2 (ACE-2), the SARS-CoV-2 receptor, to mediate viral binding and entry into T cells. We further show that T cells exposed to SARS-CoV-2 particles demonstrate reduced proliferation and apoptosis compared to uninfected controls, indicating that direct interaction of SARS-CoV-2 with T cells may alter T cell growth, activation, and survival. Regulation of T cell activation and/or turnover by SARS-CoV-2 may contribute to impaired T cell function observed in patients with severe disease.

TRAF3 in T Cells Restrains Negative Regulators of LAT to Promote TCR/CD28 Signaling.

The adaptor protein TNFR-associated factor 3 (TRAF3) is required for in vivo T cell effector functions and for normal TCR/CD28 signaling. TRAF3-mediated enhancement of TCR function requires engagement of both CD3 and CD28, but the molecular mechanisms underlying how TRAF3 interacts with and impacts TCR/CD28-mediated complexes to enhance their signaling remains an important knowledge gap. We investigated how TRAF3 is recruited to, and regulates, CD28 as a TCR costimulator. Direct association with known signaling motifs in CD28 was dispensable for TRAF3 recruitment; rather, TRAF3 associated with the CD28-interacting protein linker of activated T cells (LAT) in human and mouse T cells. TRAF3-LAT association required the TRAF3 TRAF-C domain and a newly identified TRAF2/3 binding motif in LAT. TRAF3 inhibited function of the LAT-associated negative regulatory protein Dok1, which is phosphorylated at an inhibitory tyrosine residue by the tyrosine kinase breast tumor kinase (Brk/PTK6). TRAF3 regulated Brk activation in T cells, limiting the association of protein tyrosine phosphatase 1B (PTP1B) with the LAT complex. In TRAF3-deficient cells, LAT complex-associated PTP1B was associated with dephosphorylation of Brk at an activating tyrosine residue, potentially reducing its ability to inhibit Dok1. Consistent with these findings, inhibiting PTP1B activity in TRAF3-deficient T cells rescued basal and TCR/CD28-mediated activation of Src family kinases. These results reveal a new mechanism for promotion of TCR/CD28-mediated signaling through restraint of negative regulation of LAT by TRAF3, enhancing the understanding of regulation of the TCR complex.

Suppression of human T cell activation by derivatives of glycerol monolaurate.

Glycerol monolaurate (GML), a naturally occurring monoglyceride, is widely used commercially for its antimicrobial properties. Interestingly, several studies have shown that GML not only has antimicrobial properties but is also an anti-inflammatory agent. GML inhibits peripheral blood mononuclear cell proliferation and inhibits T cell receptor (TCR)-induced signaling events. In this study, we perform an extensive structure activity relationship analysis to investigate the structural components of GML necessary for its suppression of human T cell activation. Human T cells were treated with analogs of GML, differing in acyl chain length, head group, linkage of acyl chain, and number of laurate groups. Treated cells were then tested for changes in membrane dynamics, LAT clustering, calcium signaling, and cytokine production. We found that an acyl chain with 12-14 carbons, a polar head group, an ester linkage, and a single laurate group at any position are all necessary for GML to inhibit protein clustering, calcium signaling, and cytokine production. Removing the glycerol head group or replacing the ester linkage with a nitrogen prevented derivative-mediated inhibition of protein cluster formation and calcium signaling, while still inhibiting TCR-induced cytokine production. These findings expand our current understanding of the mechanisms of action of GML and the of GML needed to function as a novel immunosuppressant.

The membrane proximal proline-rich region and correct order of C-terminal tyrosines on the adaptor protein LAT are required for TCR-mediated signaling and downstream functions.

The primary activating receptor for T cells is the T cell receptor (TCR), which is stimulated upon binding to an antigen/MHC complex. TCR activation results in the induction of regulated signaling pathways vital for T cell differentiation, cellular adhesion and cytokine release. A critical TCR-induced signaling protein is the adaptor protein LAT. Upon TCR stimulation, LAT is phosphorylated on conserved tyrosines, which facilitates the formation of multiprotein complexes needed for propagation of signaling pathways. Although the role of the conserved tyrosines in LAT-mediated signaling has been investigated, few studies have examined the role of larger regions of LAT in TCR-induced pathways. In this study, a sequence alignment of 97 mammalian LAT proteins was used to identify several "functional" domains on LAT. Using LAT mutants expressed in Jurkat E6.1 cells, we observed that the membrane proximal, proline-rich region of LAT and the correct order of domains containing conserved tyrosines are necessary for optimal TCR-mediated early signaling, cytokine production, and cellular adhesion. Together, these data show that LAT contains distinct regions whose presence and correct order are required for the propagation of TCR-mediated signaling pathways.

The lipid membrane of HIV-1 stabilizes the viral envelope glycoproteins and modulates their sensitivity to antibody neutralization.

The envelope glycoproteins (Envs) of HIV-1 are embedded in the cholesterol-rich lipid membrane of the virus. Chemical depletion of cholesterol from HIV-1 particles inactivates their infectivity. We observed that diverse HIV-1 strains exhibit a range of sensitivities to such treatment. Differences in sensitivity to cholesterol depletion could not be explained by variation in Env components known to interact with cholesterol, including the cholesterol-recognition motif and cytoplasmic tail of gp41. Using antibody-binding assays, measurements of virus infectivity, and analyses of lipid membrane order, we found that depletion of cholesterol from HIV-1 particles decreases the conformational stability of Env. It enhances exposure of partially cryptic epitopes on the trimer and increases sensitivity to structure-perturbing treatments such as antibodies and cold denaturation. Substitutions in the cholesterol-interacting motif of gp41 induced similar effects as depletion of cholesterol. Surface-acting agents, which are incorporated into the virus lipid membrane, caused similar effects as disruption of the Env-cholesterol interaction. Furthermore, substitutions in gp120 that increased structural stability of Env (i.e. induced a "closed" conformation of the trimer) increased virus resistance to cholesterol depletion and to the surface-acting agents. Collectively, these results indicate a critical contribution of the viral membrane to the stability of the Env trimer and to neutralization resistance against antibodies. Our findings suggest that the potency of poorly neutralizing antibodies, which are commonly elicited in vaccinated individuals, may be markedly enhanced by altering the lipid composition of the viral membrane.

High-resolution structure of RGS17 suggests a role for Ca2+ in promoting the GTPase-activating protein activity by RZ subfamily members.

Regulator of G protein signaling (RGS) proteins are negative regulators of G protein-coupled receptor (GPCR) signaling through their ability to act as GTPase-activating proteins (GAPs) for activated Gα subunits. Members of the RZ subfamily of RGS proteins bind to activated Gαo, Gαz, and Gαi1-3 proteins in the nervous system and thereby inhibit downstream pathways, including those involved in Ca2+-dependent signaling. In contrast to other RGS proteins, little is known about RZ subfamily structure and regulation. Herein, we present the 1.5-Å crystal structure of RGS17, the most complete and highest-resolution structure of an RZ subfamily member to date. RGS17 cocrystallized with Ca2+ bound to conserved positions on the predicted Gα-binding surface of the protein. Using NMR chemical shift perturbations, we confirmed that Ca2+ binds in solution to the same site. Furthermore, RGS17 had greater than 55-fold higher affinity for Ca2+ than for Mg2+ Finally, we found that Ca2+ promotes interactions between RGS17 and activated Gα and decreases the Km for GTP hydrolysis, potentially by altering the binding mechanism between these proteins. Taken together, these findings suggest that Ca2+ positively regulates RGS17, which may represent a general mechanism by which increased Ca2+ concentration promotes the GAP activity of the RZ subfamily, leading to RZ-mediated inhibition of Ca2+ signaling.

Polymicrobial sepsis influences NK-cell-mediated immunity by diminishing NK-cell-intrinsic receptor-mediated effector responses to viral ligands or infections.

The sepsis-induced cytokine storm leads to severe lymphopenia and reduced effector capacity of remaining/surviving cells. This results in a prolonged state of immunoparalysis, that contributes to enhanced morbidity/mortality of sepsis survivors upon secondary infection. The impact of sepsis on several lymphoid subsets has been characterized, yet its impact on NK-cells remains underappreciated-despite their critical role in controlling infection(s). Here, we observed numerical loss of NK-cells in multiple tissues after cecal-ligation-and-puncture (CLP)-induced sepsis. To elucidate the sepsis-induced lesions in surviving NK-cells, transcriptional profiles were evaluated and indicated changes consistent with impaired effector functionality. A corresponding deficit in NK-cell capacity to produce effector molecules following secondary infection and/or cytokine stimulation (IL-12,IL-18) further suggested a sepsis-induced NK-cell intrinsic impairment. To specifically probe NK-cell receptor-mediated function, the activating Ly49H receptor, that recognizes the murine cytomegalovirus (MCMV) m157 protein, served as a model receptor. Although relative expression of Ly49H receptor did not change, the number of Ly49H+ NK-cells in CLP hosts was reduced leading to impaired in vivo cytotoxicity and the capacity of NK-cells (on per-cell basis) to perform Ly49H-mediated degranulation, killing, and effector molecule production in vitro was also severely reduced. Mechanistically, Ly49H adaptor protein (DAP12) activation and clustering, assessed by TIRF microscopy, was compromised. This was further associated with diminished AKT phosphorylation and capacity to flux calcium following receptor stimulation. Importantly, DAP12 overexpression in NK-cells restored Ly49H/D receptors-mediated effector functions in CLP hosts. Finally, as a consequence of sepsis-dependent numerical and functional lesions in Ly49H+ NK-cells, host capacity to control MCMV infection was significantly impaired. Importantly, IL-2 complex (IL-2c) therapy after CLP improved numbers but not a function of NK-cells leading to enhanced immunity to MCMV challenge. Thus, the sepsis-induced immunoparalysis state includes numerical and NK-cell-intrinsic functional impairments, an instructive notion for future studies aimed in restoring NK-cell immunity in sepsis survivors.

Glycerol monolaurate induces filopodia formation by disrupting the association between LAT and SLP-76 microclusters.

Glycerol monolaurate (GML) is a monoglyceride with potent antimicrobial properties that suppresses T cell receptor (TCR)-induced signaling and T cell effector function. Actin rearrangement is needed for the interaction of T cells with antigen-presenting cells and for migration to sites of infection. Because of the critical role actin rearrangement plays in T cell effector function, we analyzed the effect of GML on the rearrangement of the actin cytoskeleton after TCR activation. We found that GML-treated human T cells were less adherent than untreated T cells and did not form actin ring structures but instead developed numerous inappropriate actin-mediated filopodia. The formation of these filopodia was not due to disruption of TCR-proximal regulators of actin or microtubule polymerization. Instead, total internal reflection fluorescence microscopy demonstrated mislocalization of actin nucleation protein Arp2 microclusters, but not those containing the adaptor proteins SLP-76 and WASp, or the actin nucleation protein ARPC3, which are necessary for TCR-induced actin rearrangement. Additionally, SLP-76 microclusters colocalized with WASp and WAVE microclusters but not with LAT. Together, our data suggest that GML alters actin cytoskeletal rearrangements and identify diverse functions for GML as a T cell-suppressive agent.

SelexGLM differentiates androgen and glucocorticoid receptor DNA-binding preference over an extended binding site.

The DNA-binding interfaces of the androgen (AR) and glucocorticoid (GR) receptors are virtually identical, yet these transcription factors share only about a third of their genomic binding sites and regulate similarly distinct sets of target genes. To address this paradox, we determined the intrinsic specificities of the AR and GR DNA-binding domains using a refined version of SELEX-seq. We developed an algorithm, SelexGLM, that quantifies binding specificity over a large (31-bp) binding site by iteratively fitting a feature-based generalized linear model to SELEX probe counts. This analysis revealed that the DNA-binding preferences of AR and GR homodimers differ significantly, both within and outside the 15-bp core binding site. The relative preference between the two factors can be tuned over a wide range by changing the DNA sequence, with AR more sensitive to sequence changes than GR. The specificity of AR extends to the regions flanking the core 15-bp site, where isothermal calorimetry measurements reveal that affinity is augmented by enthalpy-driven readout of poly(A) sequences associated with narrowed minor groove width. We conclude that the increased specificity of AR is correlated with more enthalpy-driven binding than GR. The binding models help explain differences in AR and GR genomic binding and provide a biophysical rationale for how promiscuous binding by GR allows functional substitution for AR in some castration-resistant prostate cancers.

Yellow Fever Virus, but Not Zika Virus or Dengue Virus, Inhibits T-Cell Receptor-Mediated T-Cell Function by an RNA-Based Mechanism.

The Flavivirus genus within the Flaviviridae family is comprised of many important human pathogens including yellow fever virus (YFV), dengue virus (DENV), and Zika virus (ZKV), all of which are global public health concerns. Although the related flaviviruses hepatitis C virus and human pegivirus (formerly named GBV-C) interfere with T-cell receptor (TCR) signaling by novel RNA and protein-based mechanisms, the effect of other flaviviruses on TCR signaling is unknown. Here, we studied the effect of YFV, DENV, and ZKV on TCR signaling. Both YFV and ZKV replicated in human T cells in vitro; however, only YFV inhibited TCR signaling. This effect was mediated at least in part by the YFV envelope (env) protein coding RNA. Deletion mutagenesis studies demonstrated that expression of a short, YFV env RNA motif (vsRNA) was required and sufficient to inhibit TCR signaling. Expression of this vsRNA and YFV infection of T cells reduced the expression of a Src-kinase regulatory phosphatase (PTPRE), while ZKV infection did not. YFV infection in mice resulted in impaired TCR signaling and PTPRE expression, with associated reduction in murine response to experimental ovalbumin vaccination. Together, these data suggest that viruses within the flavivirus genus inhibit TCR signaling in a species-dependent manner.

Natural Products Discovered in a High-Throughput Screen Identified as Inhibitors of RGS17 and as Cytostatic and Cytotoxic Agents for Lung and Prostate Cancer Cell Lines.

Regulator of G Protein Signaling (RGS) 17 is an overexpressed promoter of cancer survival in lung and prostate tumors, the knockdown of which results in decreased tumor cell proliferation in vitro. Identification of drug-like molecules inhibiting this protein could ameliorate the RGS17's pro-tumorigenic effect. Using high-throughput screening, a chemical library containing natural products was interrogated for inhibition of the RGS17-Gαo interaction. Initial hits were verified in control and counter screens. Leads were characterized via biochemical, mass spectrometric, Western blot, microscopic, and cytotoxicity measures. Four known compounds (1-4) were identified with IC50 values ranging from high nanomolar to low micromolar. Three compounds were extensively characterized biologically, demonstrating cellular activity determined by confocal microscopy, and two compounds were assessed via ITC exhibiting high nanomolar to low micromolar dissociation constants. The compounds were found to have a cysteine-dependent mechanism of binding, verified through site-directed mutagenesis and cysteine reactivity assessment. Two compounds, sanguinarine (1) and celastrol (2), were found to be cytostatic against lung and prostate cancer cell lines and cytotoxic against prostate cancer cell lines in vitro, although the dependence of RGS17 on these phenomena remains elusive, a result that is perhaps not surprising given the multimodal cytostatic and cytotoxic activities of many natural products.

TRAF3 enhances TCR signaling by regulating the inhibitors Csk and PTPN22.

The adaptor protein TNF receptor associated factor (TRAF) 3 is required for effective TCR signaling and normal T cell effector functions, and associates with the CD3/CD28 complex upon activation. To determine how TRAF3 promotes proximal TCR signaling, we studied TRAF3-deficient mouse and human T cells, which showed a marked reduction in activating phosphorylation of the TCR-associated kinase Lck. The impact of TRAF3 on this very early signaling event led to the hypothesis that TRAF3 restrains one or both of two known inhibitors of Lck, C-terminal Src kinase (Csk) and protein tyrosine phosphatase N22 (PTPN22). TRAF3 associated with Csk, promoting the dissociation of Csk from the plasma membrane. TRAF3 also associated with and regulated the TCR/CD28 induced localization of PTPN22. Loss of TRAF3 resulted in increased amounts of both Csk and PTPN22 in T cell membrane fractions and decreased association of PTPN22 with Csk. These findings identify a new role for T cell TRAF3 in promoting T cell activation, by regulating localization and functions of early TCR signaling inhibitors.