Variants in CXCR4 associate with juvenile idiopathic arthritis susceptibility.

BACKGROUND: Juvenile idiopathic arthritis (JIA) is the most common chronic rheumatic disease among children, the etiology of which involves a strong genetic component, but much of the underlying genetic determinants still remain unknown. Our aim was to identify novel genetic variants that predispose to JIA. METHODS: We performed a genome-wide association study (GWAS) and replication in a total of 1166 JIA cases and 9500 unrelated controls of European ancestry. Correlation of SNP genotype and gene expression was investigated. Then we conducted targeted resequencing of a candidate locus, among a subset of 480 cases and 480 controls. SUM test was performed to evaluate the association of the identified rare functional variants. RESULTS: The CXCR4 locus on 2q22.1 was found to be significantly associated with JIA, peaking at SNP rs953387. However, this result is subjected to subpopulation stratification within the subjects of European ancestry. After adjusting for principal components, nominal significant association remained (p < 10(-4)). Because of its interesting known function in immune regulation, we carried out further analyses to assess its relationship with JIA. Expression of CXCR4 was correlated with CXCR4 rs953387 genotypes in lymphoblastoid cell lines (p = 0.014) and T-cells (p = 0.0054). In addition, rare non-synonymous and stop-gain sequence variants in CXCR4, putatively damaging for CXCR4 function, were significantly enriched in JIA cases (p = 0.015). CONCLUSION: Our results suggest the association of CXCR4 variants with JIA, implicating that this gene may be involved in the pathogenesis of autoimmune disease. However, because this locus is subjected to population stratification within the subjects of European ancestry, additional replication is still necessary for this locus to be considered a true risk locus for JIA. This cell-surface chemokine receptor has already been targeted in other diseases and may serve as a tractable therapeutic target for a specific subset of pediatric arthritis patients with additional replication and functional validation of the locus.

Tumor necrosis factor receptor-associated factor 1 influences KRN/I-Ag7 mouse arthritis autoantibody production.

PURPOSE: Recently, genomewide association analysis has revealed that the Tumor Necrosis Factor Receptor-associated factor 1-Complement 5 (TRAF1-C5) containing locus on chromosome 9 was associated with an increased risk for RA. Studies in model systems suggested that either gain- or loss-of-function TRAF1 mutations have immune effects that could plausibly lead to or exacerbate the arthritis phenotype. KRN/I-A(g7) (KxB/N) is a genetic mouse model of inflammatory arthritis. We aimed to assess the impact of TRAF1 deficiency on KRN/I-A(g7) mice. METHODS: We have bred KRN/I-A(g7) mice onto a TRAF1-deficient background and followed cohorts for the spontaneous appearance of arthritis. We have also transferred KxB/N serum to B6.I-A(g7) TRAF1KO recipients. In addition, systemic autoimmunity was induced through cGVH by injecting bm12 splenocytes into TRAF1KO recipient mice. RESULTS: TRAF1-deficient KRN/I-A(g7) mice spontaneously developed severe, progressive arthritis, comparable to that seen in TRAF1-intact KRN/I-A(g7) mice. However, the anti-GPI antibody titer was significantly lower in the former group. Interestingly, the TRAF1KO mice that had background levels of anti-GPI antibodies still showed severe arthritis, although with a brief delay compared to TRAF1 sufficient mice. In addition, TRAF1KO mice were fully susceptible to passive, serum transfer experiments. In another model of autoimmunity, TRAF1KO had no effect on cGVH autoantibodies production; nor was the response to an exogenous antigen impaired. CONCLUSION: The pathogenesis of spontaneous KRN/I-A(g7) arthritis can largely proceed by TRAF1-independent pathways. The production of anti-GPI autoantibody, but not other autoantibody or antibody responses, was markedly impaired by TRAF1 deficiency. The spontaneous arthritis model in KRN mice appears to be much less antibody dependent than previously believed.

HIV-1 Vif promotes the G1- to S-phase cell-cycle transition.

HIV-1 depends on host-cell resources for replication, access to which may be limited to a particular phase of the cell cycle. The HIV-encoded proteins Vpr (viral protein R) and Vif (viral infectivity factor) arrest cells in the G2 phase; however, alteration of other cell-cycle phases has not been reported. We show that Vif drives cells out of G1 and into the S phase. The effect of Vif on the G1- to-S transition is distinct from its effect on G2, because G2 arrest is Cullin5-dependent, whereas the G1- to-S progression is Cullin5-independent. Using mass spectrometry, we identified 2 novel cellular partners of Vif, Brd4 and Cdk9, both of which are known to regulate cell-cycle progression. We confirmed the interaction of Vif and Cdk9 by immunoprecipitation and Western blot, and showed that small interfering RNAs (siRNAs) specific for Cdk9 inhibit the Vif-mediated G1- to-S transition. These data suggest that Vif regulates early cell-cycle progression, with implications for infection and latency.

T cell receptor triggering by force.

Antigen recognition through the interaction between the T cell receptor (TCR) and peptide presented by major histocompatibility complex (pMHC) is the first step in T cell-mediated immune responses. How this interaction triggers TCR signalling that leads to T cell activation is still unclear. Taking into account the mechanical stress exerted on the pMHC-TCR interaction at the dynamic interface between T cells and antigen presenting cells (APCs), we propose the so-called receptor deformation model of TCR triggering. In this model, TCR conformational change induced by mechanical forces initiates TCR signalling. The receptor deformation model, for the first time, explains all three aspects of the TCR triggering puzzle: mechanism, specificity, and sensitivity.

Glucocorticoid elevation of dexamethasone-induced gene 2 (Dig2/RTP801/REDD1) protein mediates autophagy in lymphocytes.

Glucocorticoid hormones, including dexamethasone, induce apoptosis in lymphocytes and consequently are used clinically as chemotherapeutic agents in many hematologic malignancies. Dexamethasone also induces autophagy in lymphocytes, although the mechanism is not fully elucidated. Through gene expression analysis, we found that dexamethasone induces the expression of a gene encoding a stress response protein variously referred to as Dig2, RTP801, or REDD1. This protein is reported to inhibit mammalian target of rapamycin (mTOR) signaling. Because autophagy is one outcome of mTOR inhibition, we investigated the hypothesis that Dig2/RTP801/REDD1 elevation contributes to autophagy induction in dexamethasone-treated lymphocytes. In support of this hypothesis, RNAi-mediated suppression of Dig2/RTP801/REDD1 reduces mTOR inhibition and autophagy in glucocorticoid-treated lymphocytes. We observed similar results in Dig2/Rtp801/Redd1 knock-out murine thymocytes treated with dexamethasone. Dig2/RTP801/REDD1 knockdown also leads to increased levels of dexamethasone-induced cell death, suggesting that Dig2/RTP801/REDD1-mediated autophagy promotes cell survival. Collectively, these findings demonstrate for the first time that elevation of Dig2/RTP801/REDD1 contributes to the induction of autophagy.

Two Distinct Illnesses Consistent With MIS-C in a Pediatric Patient.

Multisystem inflammatory syndrome in children (MIS-C) is a severe inflammatory response described in children after infection with severe acute respiratory syndrome coronavirus 2. We present a case of a 9-year-old African American boy with 2 distinct illnesses that were both consistent with MIS-C. He first presented in the early stages of our understanding of MIS-C with predominantly neurologic and gastrointestinal symptoms and demonstrated elevated inflammatory markers consistent with MIS-C. He was treated with intravenous immunoglobulin with complete resolution of signs and symptoms. After 7 months of good health, he returned with a second, distinct illness characterized by fever, rash, gastrointestinal symptoms, and elevated inflammatory markers that met the criteria for MIS-C. In addition, we identified new dilatation of the left anterior descending coronary artery. He improved rapidly after treatment with intravenous immunoglobulin, aspirin, and steroids. Our report highlights the need to achieve a better understanding of this entity's pathogenesis and clinical course and to improve anticipatory guidance for children with MIS-C.

Surface-anchored monomeric agonist pMHCs alone trigger TCR with high sensitivity.

At the interface between T cell and antigen-presenting cell (APC), peptide antigen presented by MHC (pMHC) binds to the T cell receptor (TCR) and initiates signaling. The mechanism of TCR signal initiation, or triggering, remains unclear. An interesting aspect of this puzzle is that although soluble agonist pMHCs cannot trigger TCR even at high concentrations, the same ligands trigger TCR very efficiently on the surface of APCs. Here, using lipid bilayers or plastic-based artificial APCs with defined components, we identify the critical APC-associated factors that confer agonist pMHCs with such potency. We found that CD4+ T cells are triggered by very low numbers of monomeric agonist pMHCs anchored on fluid lipid bilayers or fixed plastic surfaces, in the absence of any other APC surface molecules. Importantly, on bilayers, plastic surfaces, or real APCs, endogenous pMHCs did not enhance TCR triggering. TCR triggering, however, critically depended upon the adhesiveness of the surface and an intact T cell actin cytoskeleton. Based on these observations, we propose the receptor deformation model of TCR triggering to explain the remarkable sensitivity and specificity of TCR triggering.

The receptor deformation model of TCR triggering.

Through T cell receptors (TCRs), T cells can detect and respond to very small numbers of foreign peptides among a huge number of self-peptides presented by major histocompatibility complexes (pMHCs) on the surface of antigen-presenting cells (APCs). How T cells achieve such remarkable sensitivity and specificity through pMHC-TCR binding is an intensively pursued issue in immunology today; the key question is how pMHC-TCR binding initiates, or triggers, a signal from TCRs. Multiple competing models have been proposed, none of which fully explains the sensitivity and specificity of TCR triggering. What has been omitted from existing theories is that the pMHC-TCR interaction at the T cell/APC interface must be under constant mechanical stress, due to the dynamic nature of cell-cell interaction. Taking this condition into consideration, we propose the receptor deformation model of TCR triggering. In this model, TCR signaling is initiated by conformational changes of the TCR/CD3 complex, induced by a pulling force originating from the cytoskeleton and transmitted through pMHC-TCR binding interactions with enough strength to resist rupture. By introducing mechanical force into a model of T cell signal initiation, the receptor deformation model provides potential mechanistic solutions to the sensitivity and specificity of TCR triggering.

The HIV-1 Vif protein mediates degradation of Vpr and reduces Vpr-induced cell cycle arrest.

Prior work has implicated viral protein R (Vpr) in the arrest of human immunodeficiency virus type 1 (HIV-1)-infected cells in the G2 phase of the cell cycle, associated with increased viral replication and host cell apoptosis. We and others have recently shown that virion infectivity factor (Vif ) also plays a role in the G2 arrest of HIV-1-infected cells. Here, we demonstrate that, paradoxically, at early time points postinfection, Vif expression blocks Vpr-mediated G2 arrest, while deletion of Vif from the HIV-1 genome leads to a marked increase in G2 arrest of infected CD4 T-cells. Consistent with this increased G2 arrest, T-cells infected with Vif-deleted HIV-1 express higher levels of Vpr protein than cells infected with wild-type virus. Further, expression of exogenous Vif inhibits the expression of Vpr, associated with a decrease in G2 arrest of both infected and transfected cells. Treatment with the proteasome inhibitor MG132 increases Vpr protein expression and G2 arrest in wild-type, but not Vif-deleted, NL4-3-infected cells, and in cells cotransfected with Vif and Vpr. In addition, Vpr coimmunoprecipitates with Vif in cotransfected cells in the presence of MG132. This suggests that inhibition of Vpr by Vif is mediated at least in part by proteasomal degradation, similar to Vif-induced degradation of APOBEC3G. Together, these data show that Vif mediates the degradation of Vpr and modulates Vpr-induced G2 arrest in HIV-1-infected T-cells.

Being Overweight or Obese and the Development of Asthma.

OBJECTIVES: Adult obesity is linked to asthma cases and is estimated to lead to 250 000 new cases yearly. Similar incidence and attributable risk (AR) estimates have not been developed for children. We sought to describe the relationship between overweight and obesity and incident asthma in childhood and quantify AR statistics in the United States for overweight and obesity on pediatric asthma. METHODS: The PEDSnet clinical data research network was used to conduct a retrospective cohort study (January 2009-December 2015) to compare asthma incidence among overweight and/or obese versus healthy weight 2- to 17-year-old children. Asthma incidence was defined as ≥2 encounters with a diagnosis of asthma and ≥1 asthma controller prescription. Stricter diagnostic criteria involved confirmation by spirometry. We used multivariable Poisson regression analyses to estimate incident asthma rates and risk ratios and accepted formulas for ARs. RESULTS: Data from 507 496 children and 19 581 972 encounters were included. The mean participant observation period was 4 years. The adjusted risk for incident asthma was increased among children who were overweight (relative risk [RR]: 1.17; 95% confidence interval [CI]: 1.10-1.25) and obese (RR: 1.26; 95% CI: 1.18-1.34). The adjusted risk for spirometry-confirmed asthma was increased among children with obesity (RR: 1.29; 95% CI: 1.16-1.42). An estimated 23% to 27% of new asthma cases in children with obesity is directly attributable to obesity. In the absence of overweight and obesity, 10% of all cases of asthma would be avoided. CONCLUSIONS: Obesity is a major preventable risk factor for pediatric asthma.

Effective gene suppression using small interfering RNA in hard-to-transfect human T cells.

RNA interference (RNAi) is an evolutionarily conserved cellular defense mechanism that protects cells from hostile genes and regulates the function of normal genes during growth and development. In this study, we established proof of principle of small interfering RNA (siRNA) silencing in hard-to-transfect human T cell lines and primary human CD4 T cells. We used public and in-house programs to design four siRNAs each for GFP, for our novel cellular gene HALP, and for their corresponding scrambled siRNA controls. We generated siRNA expression cassettes (SECs) by PCR and directly transfected the PCR products into T cells using amaxa Nucleofector technology. The most effective SECs were selected and cloned into a TA cloning vector and titered with their respective controls to increase transfection efficiency. Flow cytometry and fluorescence microscopy analyses were performed for GFP siRNAs, and Northern blot analysis was done to assess the HALP silencing effect. These experiments demonstrate that SECs are an excellent screening tool to identify siRNA sequences effective in silencing expression of genes of interest. The vector expressing the most effective siRNA robustly inhibited GFP expression (up to 92%) in the context of co-transfection in human T cell lines and primary CD4 T cells. The optimized siRNA for our endogenous cellular gene HALP also silenced its target RNA expression by more than 90%. These studies demonstrate that the combination of SEC, siRNA expression vectors and Nucleofector technology can be successfully applied to hard-to-transfect human T cell lines and primary T cells to effectively silence genes.

REDD1 Is Essential for Optimal T Cell Proliferation and Survival.

REDD1 is a highly conserved stress response protein that is upregulated following many types of cellular stress, including hypoxia, DNA damage, energy stress, ER stress, and nutrient deprivation. Recently, REDD1 was shown to be involved in dexamethasone induced autophagy in murine thymocytes. However, we know little of REDD1's function in mature T cells. Here we show for the first time that REDD1 is upregulated following T cell stimulation with PHA or CD3/CD28 beads. REDD1 knockout T cells exhibit a defect in proliferation and cell survival, although markers of activation appear normal. These findings demonstrate a previously unappreciated role for REDD1 in T cell function.

Epistasis amongst PTPN2 and genes of the vitamin D pathway contributes to risk of juvenile idiopathic arthritis.

Juvenile idiopathic arthritis (JIA) is a leading cause of childhood-onset disability. Although epistasis (gene-gene interaction) is frequently cited as an important component of heritability in complex diseases such as JIA, there is little compelling evidence that demonstrates such interaction. PTPN2, a vitamin D responsive gene, is a confirmed susceptibility gene in JIA, and PTPN2 has been suggested to interact with vitamin D pathway genes in type 1 diabetes. We therefore, tested for evidence of epistasis amongst PTPN2 and the vitamin D pathway genes GC, VDR, CYP24A1, CYP2R1, and DHCR7 in two independent JIA case-control samples (discovery and replication). In the discovery sample (318 cases, 556 controls), we identified evidence in support of epistasis across six gene-gene combinations (e.g., GC rs1155563 and PTPN2 rs2542151, ORint=0.45, p=0.00085). Replication was obtained for three of these combinations. That is, for GC and PTPN2, CYP2R1 and VDR, and VDR and PTPN2, similar epistasis was observed using the same SNPs or correlated proxies in an independent JIA case-control sample (1008 cases, 9287 controls). Using SNP data imputed across a 4 MB region spanning each gene, we obtained highly significant evidence for epistasis amongst all 6 gene-gene combinations identified in the discovery sample (p-values ranging from 5.6×10(-9) to 7.5×10(-7)). This is the first report of epistasis in JIA risk. Epistasis amongst PTPN2 and vitamin D pathway genes was both demonstrated and replicated.

Differential gene expression during HIV-1 infection analyzed by suppression subtractive hybridization.

OBJECTIVE: Characterization of the effects of HIV-1 infection and apoptosis on cellular and viral gene expression. METHODS: Flow cytometry was used to analyze infection and apoptosis concurrently in HIV-1IIIB-infected CEM-SS T cells. Suppression subtractive hybridization (SSH) was applied to cells from different time points of infection to construct subtracted complementary DNA (cDNA) libraries. Differential screening and Northern blots confirmed differential gene expression and these genes were sequenced and compared with database. RESULTS: T cells undergo apoptosis at early stages of HIV-1IIIB infection (days 5-7 post-infection). Surprisingly, cells begin to recover after day 9 and by day 18 almost all infected cells are viable, even though they maintain the same level of infection. By SSH, differential gene expression profiles between day 7 and day 18 after HIV-1IIIB infection were characterized. SSH yielded two subtracted cDNA libraries; differential screening of the subtracted cDNA libraries suggested that 200 out of 864 colonies were highly expressed at their respective time points. DNA sequence analysis identified specific apoptosis-related genes, HIV-1 viral genes, and other candidate genes of interest. Northern blot analysis confirmed that some of these genes were expressed predominantly at the 'apoptotic' or 'non-apoptotic' time points. CONCLUSIONS: Known and novel cellular gene products have been identified that are directly (or inversely) correlated with apoptosis and may regulate cell death in HIV-1 infection. These results provide a framework for functional studies on the differentially expressed genes and may suggest novel therapeutic approaches for treatment of HIV-1-infected individuals.

T cell signaling and apoptosis in HIV disease.

Groundbreaking research has led to an understanding of some of the pathogenic mechanisms of HIV-1 infection. Surprisingly, an unanswered question remains the mechanism(s) by which HIV-1 inactivates or kills T cells. Our goals are to define candidate T cell signaling cascades altered by HIV infection and to identify mechanisms whereby HIV-infected cells escape the apoptosis triggered by this aberrant signaling. In earlier work, we found that HIV reprograms healthy T cells to self-destruct by a process called apoptosis. We asked whether apoptosis occurs in organs of infected people and made a surprising discovery-this cell death occurs predominantly in healthy bystander cells and only rarely in infected cells. We hypothesize that HIV may be doubly diabolical-healthy T cells are killed in HIV infection, while infected cells resist killing. Thus, the virus protects its viral factory and allows HIV to turn the cell into a "Trojan Horse," with the virus in hiding or "latent." In this review, we discuss the role of viral and cellular proteins in HIV induced T cell anergy and death. We also discuss mechanisms by which HIV may protect infected T cells from apoptosis. These studies will yield new insights into the pathogenesis of AIDS, identify cellular targets that regulate HIV-1 infection, and suggest novel therapeutic approaches to cure HIV infection.

Ligation of human CD4 interferes with antigen-induced activation of primary T cells.

The CD4 molecule functions to enhance T cell activation when it is co-aggregated with the T cell receptor for antigen (TCR) by MHC class II antigenic peptide complexes. However, independent ligation of CD4 has been shown to negatively effect signaling through the TCR in vitro. The interaction between the HIV-1 envelope glycoprotein gp120 and CD4 is a central event in the pathogenesis of AIDS and may contribute to immune deficiency via both direct and indirect mechanisms, including lytic infection of T cells and induction of CD4 signaling events resulting in apoptosis and anergy. Analysis of the consequences of interactions between CD4 and gp120 have yielded contradictory results presumably because most of these studies have focused on T cell clones of questionable relevance to the in vivo target of the virus. Here, we analyzed the effects of CD4 ligation on freshly isolated cells of human CD4 transgenic mice, and show that huCD4 preligation, in the absence of human CXCR4, has an inhibitory effect on both early and late T cell activation events. CD4 signaling negatively regulates the response to antigen, as well as to anti-TCR mAb. In addition, we show here that this negative signal requires the cytoplasmic tail of CD4. These results suggest that in HIV infected patients the interaction of gp120 with CD4 induces unresponsiveness of CD4+ T cells to subsequent activation by antigen.

The impact of Nucleofection® on the activation state of primary human CD4 T cells.

Gene transfer into primary human CD4 T lymphocytes is a critical tool in studying the mechanism of T cell-dependent immune responses and human immunodeficiency virus-1 (HIV-1) infection. Nucleofection® is an electroporation technique that allows efficient gene transfer into primary human CD4 T cells that are notoriously resistant to traditional electroporation. Despite its popularity in immunological research, careful characterization of its impact on the physiology of CD4 T cells has not been documented. Herein, using freshly-isolated primary human CD4 T cells, we examine the effects of Nucleofection® on CD4 T cell morphology, intracellular calcium levels, cell surface activation markers, and transcriptional activity. We find that immediately after Nucleofection®, CD4 T cells undergo dramatic morphological changes characterized by wrinkled and dilated plasma membranes before recovering 1h later. The intracellular calcium level also increases after Nucleofection®, peaking after 1h before recovering 8h post transfection. Moreover, Nucleofection® leads to increased expression of T cell activation markers, CD154 and CD69, for more than 24h, and enhances the activation effects of phytohemagglutinin (PHA) stimulation. In addition, transcriptional activity is increased in the first 24h after Nucleofection®, even in the absence of exogenous stimuli. Therefore, Nucleofection® significantly alters the activation state of primary human CD4 T cells. The effect of transferred gene products on CD4 T cell function by Nucleofection® should be assessed after sufficient resting time post transfection or analyzed in light of the activation caveats mentioned above.

TCR triggering by pMHC ligands tethered on surfaces via poly(ethylene glycol) depends on polymer length.

Antigen recognition by T cells relies on the interaction between T cell receptor (TCR) and peptide-major histocompatibility complex (pMHC) at the interface between the T cell and the antigen presenting cell (APC). The pMHC-TCR interaction is two-dimensional (2D), in that both the ligand and receptor are membrane-anchored and their movement is limited to 2D diffusion. The 2D nature of the interaction is critical for the ability of pMHC ligands to trigger TCR. The exact properties of the 2D pMHC-TCR interaction that enable TCR triggering, however, are not fully understood. Here, we altered the 2D pMHC-TCR interaction by tethering pMHC ligands to a rigid plastic surface with flexible poly(ethylene glycol) (PEG) polymers of different lengths, thereby gradually increasing the ligands' range of motion in the third dimension. We found that pMHC ligands tethered by PEG linkers with long contour length were capable of activating T cells. Shorter PEG linkers, however, triggered TCR more efficiently. Molecular dynamics simulation suggested that shorter PEGs exhibit faster TCR binding on-rates and off-rates. Our findings indicate that TCR signaling can be triggered by surface-tethered pMHC ligands within a defined 3D range of motion, and that fast binding rates lead to higher TCR triggering efficiency. These observations are consistent with a model of TCR triggering that incorporates the dynamic interaction between T cell and antigen-presenting cell.

Improved method of preparation of supported planar lipid bilayers as artificial membranes for antigen presentation.

T cell activation is the result of direct cell-cell contact between T cells and antigen presenting cells (APCs), and of interactions between membrane-bound ligands and receptors at the contact interface, the "immunological synapse." Model APCs based upon supported fluid lipid bilayers have been used to dissect these complex molecular interactions and to facilitate real-time microscopic observations. Nearly all studies have used liposome fusion-based methods to make supported bilayers, and the biophysical properties of these membranes were not characterized in detail. Here, using both Langmuir-Blodgett and liposome fusion techniques, we explored five different methods of lipid bilayer preparation on glass, mica, or dextran cushion substrates and characterized the stability, homogeneity, and fluidity of the bilayers with fluorescence microscopy and fluorescence recovery after photobleaching (FRAP). Most combinations of techniques and substrates led to unsatisfactory results, notably, a lack of homogeneity for liposome fusion on glass, low stability of bilayers on mica, and loss of fluidity of dextran-cushioned bilayers in solutions containing protein. To overcome these deficits, we developed a technique that combines liposome fusion on glass and thermally enhanced bilayer expansion. The newly expanded pristine bilayer showed high degrees of stability, homogeneity, and fluidity. MHC and ICAM-1 molecules anchored on the bilayer diffused freely and stimulated T cell calcium flux and adhesion, respectively.