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T cell dysfunction is crucial to the pathogenesis of systemic lupus erythematosus (SLE); however,the molecular mechanisms involved in the deficient expression of the T cell receptor-associated CD3zeta chain in SLE are not clear. SLE T cells express abnormally increased levels of an alternatively spliced isoform of CD3zeta that lacks a 562-bp region in its 3'-untranslated region (UTR). We showed previously that two adenosine/uridine-rich elements (ARE) in this splice-deleted region of CD3zeta transcript are critical for the mRNA stability and protein expression of CD3zeta. In this study we show for the first time that the mRNA-stabilizing protein HuR binds to these two ARE bearing regions of CD3zeta 3'-UTR. Knockdown of HuR resulted in decreased expression of the CD3zeta chain,whereas overexpression led to the increase of CD3zeta chain levels. Additionally,overexpression of HuR in human T cells resulted in increased mRNA stability of CD3zeta. Our results identify the 3'-UTR of CD3zeta as a novel target for the mRNA-stabilizing protein HuR. Thus,the absence of two critical AREs in the alternatively spliced CD3zeta 3'-UTR found in SLE T cells may result in decreased HuR binding,representing a possible molecular mechanism contributing to the reduced stability and expression of CD3zeta in SLE. View Publication

CD200 is a transmembrane protein broadly expressed on a variety of cell types,which delivers immunoregulatory signals through binding to receptors (CD200Rs) expressed on monocytes/myeloid cells and T lymphocytes. Signals delivered through the CD200:CD200R axis have been shown to play an important role in the regulation of anti-tumor immunity,and overexpression of CD200 has been reported in a number of malignancies,including CLL,as well as on cancer stem cells. We investigated the effect of CD200 blockade in vitro on a generation of CTL responses against a poorly immunogenic CD200+ lymphoma cell line and fresh cells obtained from CLL patients using anti-CD200 mAb and CD200-specific siRNAs. Suppression of functional expression of CD200 augmented killing of the CD200+ cells,as well as production of the inflammatory cytokines IFN-gamma and TNF-alpha by effector PBMCs. Killing was mediated by CD8+ cytotoxic T cells,and CD4+ T cells play an important role in CD200-mediated suppression of CTL responses. Our data suggest that CD200 blockade may represent a novel approach to clinical treatment of CLL. View Publication

The interaction between the linker for activation of T cells (LAT) with PLC-gamma1 is important for TCR-mediated Ca(2+) signaling and MAPK activation. Knock-in mice harboring a mutation at the PLC-gamma1 binding site (Y136) of LAT develop a severe lymphoproliferative syndrome. These mice have defective thymic development and selection and lack natural regulatory T cells,implicating a breakdown of both central and peripheral tolerance. To bypass this developmental defect,we developed a conditional knock-in line in which only LATY136F is expressed in mature T cells after deletion of the wild type LAT allele. Analysis of LATY136F T cells indicated that the interaction between LAT and PLC-gamma1 plays an important role in TCR-mediated signaling,proliferation,and IL-2 production. Furthermore,the deletion of LAT induced development of the lymphoproliferative syndrome in these mice. Although Foxp3(+) natural Treg cells were present in these mice after deletion,they were unable to suppress the proliferation of conventional T cells. Our data indicate that the binding of LAT to PLC-gamma1 is essential for the suppressive function of CD4(+)CD25(+) regulatory T cells. View Publication

TRAF1 is a signaling adaptor known for its role in tumor necrosis factor receptor-induced cell survival. Here we show that monocytes from healthy human subjects with a rheumatoid arthritis-associated single-nucleotide polymorphism (SNP) in the TRAF1 gene express less TRAF1 protein but greater amounts of inflammatory cytokines in response to lipopolysaccharide (LPS). The TRAF1 MATH domain binds directly to three components of the linear ubiquitination (LUBAC) complex,SHARPIN,HOIP and HOIL-1,to interfere with the recruitment and linear ubiquitination of NEMO. This results in decreased NF-κB activation and cytokine production,independently of tumor necrosis factor. Consistent with this,Traf1(-/-) mice show increased susceptibility to LPS-induced septic shock. These findings reveal an unexpected role for TRAF1 in negatively regulating Toll-like receptor signaling,providing a mechanistic explanation for the increased inflammation seen with a disease-associated TRAF1 SNP. View Publication

T cell activation is predicated on the interaction between the T cell receptor and peptide-major histocompatibility (pMHC) ligands. The factors that determine the stimulatory potency of a pMHC molecule remain unclear. We describe results showing that a peptide exhibiting many hallmarks of a weak agonist stimulates T cells to proliferate more than the wild-type agonist ligand. An in silico approach suggested that the inability to form the central supramolecular activation cluster (cSMAC) could underlie the increased proliferation. This conclusion was supported by experiments that showed that enhancing cSMAC formation reduced stimulatory capacity of the weak peptide. Our studies highlight the fact that a complex interplay of factors determines the quality of a T cell antigen. View Publication

The B cell-activating factor of the TNF family receptor (BAFF-R),encoded by the TNFRSF13C gene,is critically important for transitional B cell survival to maturity. Thus,ligation of BAFF-R by BAFF delivers a potent survival signal. Reports implicating the BAFF/BAFF-R signaling axis in the pathogenesis of autoimmune human diseases and B lineage malignancies have largely prompted studies focusing on BAFF expression; however,there is an equally critical need to better understand BAFF-R expression. Initial BAFF-R expression,although characterized in murine B cells,has not yet been reported in human B lymphopoiesis. In this study,we first demonstrate that BAFF-R expression is absent from early precursors and is acquired by bone marrow B cells newly expressing the BCR. We next focused on identifying the specific genomic region that controls BAFF-R expression in mature B cells (i.e.,the TNFRSF13C promoter). To accomplish this,we used in silico tools examining interspecies genomic conservation in conjunction with reporter constructs transfected into malignant B and plasma cell lines. DNase protection assays using nuclear extracts from BAFF-R-expressing cells suggested potential regulatory sites,which allowed the generation of EMSA probes that bound NFs specific to BAFF-R-expressing cells. With a more stringent analysis of interspecies homology,these assays identified a site at which a single nucleotide substitution could distinctly impact promoter activity. Finally,chromatin immunoprecipitation assays revealed the in vivo binding of the specific transcription factor c-Rel to the most proximal genomic region,and c-Rel small interfering RNA transfections in BAFF-R-expressing lines demonstrated a coincident knockdown of both c-Rel and BAFF-R mRNA. View Publication

Host defense peptides have recently gained much interest as novel anti-infectives owing to their ability to kill bacteria and simultaneously modulate host cell responses. The cationic host defense peptide GKY25 (GKYGFYTHVFRLKKWIQKVIDQFGE),derived from the C terminus of human thrombin,inhibits proinflammatory responses in vitro and in vivo,but the mode of action is unclear. In this study,we show that GKY25,apart from binding bacterial LPS,also interacts directly with monocytes and macrophages in vitro,ex vivo,and in vivo. Moreover,GKY25 inhibits TLR4- and TLR2-induced NF-κB activation in response to several microbe-derived agonists. Furthermore,GKY25 reduces LPS-induced phosphorylation of MAPKs p38α and JNK1/2/3. FACS and electron microscopy analyses showed that GKY25 interferes with TLR4/myeloid differentiation protein-2 dimerization. The results demonstrate a previously undisclosed activity of the host defense peptide GKY25,based on combined LPS and cell interactions leading to inhibition of TLR4 dimerization and subsequent reduction of NF-κB activity and proinflammatory cytokine production in monocytes and macrophages. View Publication

Transcripts expressed in cytotoxic T lymphocytes (CTL) have mechanistic and diagnostic importance in transplantation. We used microarrays to select CTL-associated transcripts (CATs) expressed in human CD4(+) CTL,CD8(+) CTL and NK cells,excluding transcripts expressed in B cells,monocytes and kidney. This generated three transcript sets: CD4(+)-associated,CD8(+)-associated and NK-associated. Surprisingly,many CATs were expressed in effector memory cells e.g. granzyme B/GZMB,interferon-gamma/IFNG. Transcript expression was very similar between CD4(+) and CD8(+) CTL. There were no transcripts highly selective for CD4(+) CTL or CD8(+) CTL: for example,cytotoxic molecule transcripts (perforin,granzymes,granulysin) were shared between CD8(+) CTL and CD4(+) CTL although expression remained higher in CD8(+) CTL. Transcripts that differentiated between CD8(+) CTL and CD4(+) CTL were primarily those shared between CD8(+) CTL and NK cells (e.g. NK receptors KLRC1,KLRC3,KLRD1,KLRK1). No transcripts could differentiate CD4(+) CTL from CD8(+) CTL but NK cell-associated transcripts could differentiate NK cells from CTL. This study serves as a foundation for the interpretation of CATs in rejecting allografts and highlights the extensive sharing of CATs among CD4(+) CTL,CD8(+) CTL and effector memory T cells. View Publication

Tryptophan (Trp) catabolism mediated by indoleamine 2,3-dioxygenase (IDO) plays a central role in the regulation of T-cell-mediated immune responses. In this study,we also demonstrate that natural killer (NK)-cell function can be influenced by IDO. Indeed,l-kynurenine,a Trp-derived catabolite resulting from IDO activity,was found to prevent the cytokine-mediated up-regulation of the expression and function of specific triggering receptors responsible for the induction of NK-cell-mediated killing. The effect of l-kynurenine appears to be restricted to NKp46 and NKG2D,while it does not affect other surface receptors such as NKp30 or CD16. As a consequence,l-kynurenine-treated NK cells display impaired ability to kill target cells recognized via NKp46 and NKG2D. Instead,they maintain the ability to kill targets,such as dendritic cells (DCs),that are mainly recognized via the NKp30 receptor. The effect of l-kynurenine,which is effective at both the transcriptional and the protein level,can be reverted,since NK cells were found to recover their functional competence after washing. View Publication

Lupus nephritis (LN) is a potentially dangerous end organ pathology that affects upwards of 60% of lupus patients. Bruton's tyrosine kinase (BTK) is important for B cell development,Fc receptor signaling,and macrophage polarization. In this study,we investigated the effects of a novel,highly selective and potent BTK inhibitor,BI-BTK-1,in an inducible model of LN in which mice receive nephrotoxic serum (NTS) containing anti-glomerular antibodies. Mice were treated once daily with vehicle alone or BI-BTK-1,either prophylactically or therapeutically. When compared with control treated mice,NTS-challenged mice treated prophylactically with BI-BTK-1 exhibited significantly attenuated kidney disease,which was dose dependent. BI-BTK-1 treatment resulted in decreased infiltrating IBA-1+ cells,as well as C3 deposition within the kidney. RT-PCR on whole kidney RNA and serum profiling indicated that BTK inhibition significantly decreased levels of LN-relevant inflammatory cytokines and chemokines. Renal RNA expression profiling by RNA-seq revealed that BI-BTK-1 dramatically modulated pathways related to inflammation and glomerular injury. Importantly,when administered therapeutically,BI-BTK-1 reversed established proteinuria and improved renal histopathology. Our results highlight the important role for BTK in the pathogenesis of immune complex-mediated nephritis,and BTK inhibition as a promising therapeutic target for LN. View Publication

Persistent viral infections are a major health concern worldwide. During persistent infection,overwhelming viral replication and the rapid loss of antiviral T-cell function can prevent immune-mediated clearance of the infection,and therapies to reanimate the immune response and purge persistent viruses have been largely unsuccessful. Adoptive immunotherapy using memory T cells is a highly successful therapeutic approach to eradicate a persistent viral infection. Understanding precisely how therapeutically administered memory T cells achieve clearance should improve our ability to terminate states of viral persistence in humans. Mice persistently infected from birth with lymphocytic choriomeningitis virus are tolerant to the pathogen at the T-cell level and thus provide an excellent model to evaluate immunotherapeutic regimens. Previously,we demonstrated that adoptively transferred memory T cells require recipient dendritic cells to effectively purge an established persistent viral infection. However,the mechanisms that reactivate and sustain memory T-cell responses during clearance of such an infection remain unclear. Here we establish that therapeutic memory T cells require CD80 and CD86 costimulatory signals to efficiently clear an established persistent viral infection in vivo. Early blockade of costimulatory pathways with CTLA-4-Fc decreased the secondary expansion of virus-specific CD8(+) and CD4(+) memory T cells as well as their ability to produce antiviral cytokines and purge the persistent infection. Late costimulation blockade also reduced virus-specific T-cell numbers,illustrating that sustained interactions with costimulatory molecules is required for efficient T-cell expansion. These findings indicate that antiviral memory T cells require costimulation to efficiently clear a persistent viral infection and that costimulatory pathways can be targeted to modulate the magnitude of an adoptive immunotherapeutic regimen. View Publication

Leukocyte microvilli are flexible projections enriched with adhesion molecules. The role of these cellular projections in the ability of T cells to probe antigen-presenting cells has been elusive. In this study,we probe the spatial relation of microvilli and T-cell receptors (TCRs),the major molecules responsible for antigen recognition on the T-cell membrane. To this end,an effective and robust methodology for mapping membrane protein distribution in relation to the 3D surface structure of cells is introduced,based on two complementary superresolution microscopies. Strikingly,TCRs are found to be highly localized on microvilli,in both peripheral blood human T cells and differentiated effector T cells,and are barely found on the cell body. This is a decisive demonstration that different types of T cells universally localize their TCRs to microvilli,immediately pointing to these surface projections as effective sensors for antigenic moieties. This finding also suggests how previously reported membrane clusters might form,with microvilli serving as anchors for specific T-cell surface molecules. View Publication