The association of PTPN22 rs2476601 with juvenile idiopathic arthritis is specific to females.

A preponderance of females develop autoimmune disease, including juvenile idiopathic arthritis (JIA), yet the reason for this bias remains elusive. Evidence suggests that genetic risk of disease may be influenced by sex. PTPN22 rs2476601 is associated with JIA and numerous other autoimmune diseases, and has been reported to show female-specific association with type 1 diabetes. We performed main effect and sex-stratified association analyses to determine whether a sex-specific association exists in JIA. As expected, rs2476601 was associated with JIA in our discovery (413 cases and 690 controls) and replication (1008 cases and 9284 controls) samples. Discovery sample sex-stratified analyses demonstrated an association specifically in females (odds ratio (OR)=2.35, 95% confidence interval (CI)=1.52-3.63, P=0.00011) but not males (OR=0.91, 95% CI=0.52-1.60, P=0.75). This was similarly observed in the replication sample. There was evidence for genotype-by-sex interaction (Pinteraction=0.009). The association between rs2476601 and JIA appears restricted to females, partly accounting for the predominance of females with this disease.

Apoptosis occurs predominantly in bystander cells and not in productively infected cells of HIV- and SIV-infected lymph nodes.

Although 13 years have passed since identification of human immunodeficiency virus-1 (HIV-1) as the cause of AIDS, we do not yet know how HIV kills its primary target, the T cell that carries the CD4 antigen. We and others have shown an increase in the percentage of apoptotic cells among circulating CD4+ (and CD8+) T cells of HIV-seropositive individuals and an increase in frequency of apoptosis with disease progression. However, it is not known if this apoptosis occurs in infected or uninfected T cells. We show here, using in situ labelling of lymph nodes from HIV-infected children and SIV-infected macaques, that apoptosis occurs predominantly in bystander cells and not in the productively infected cells themselves. These data have implications for pathogenesis and therapy, namely, arguing that rational drug therapy may involve combination agents targeting viral replication in infected cells and apoptosis of uninfected cells.

Surface expression of a T cell receptor beta (TCR-beta) chain in the absence of TCR-alpha, -delta, and -gamma proteins.

The antigen receptor expressed by mature T cells has been described as a disulfide-linked alpha/beta or gamma/delta heterodimer noncovalently associated with CD3, a complex of transmembrane proteins that communicates signals from the T cell receptor (TCR) to the cell interior. Studies suggest that all component chains must assemble intracellularly before surface expression can be achieved. We described, however, a CD4+/CD8+ transformed murine thymocyte, KKF, that expresses surface TCR-beta chains in the absence of gamma, delta, and alpha proteins; these beta chains are only weakly associated with CD3-epsilon and CD3-zeta. Furthermore, KKF responds differently to stimulation through TCR-beta and CD3-epsilon, a functional dissociation that has been ascribed to a CD4+/CD8+ subpopulation of normal thymocytes. KKF's unique TCR structure may offer an explanation for the functional anomalies observed.

Crosslinking CD4 by human immunodeficiency virus gp120 primes T cells for activation-induced apoptosis.

During human immunodeficiency virus (HIV) infection there is a profound and selective decrease in the CD4+ population of T lymphocytes. The mechanism of this depletion is not understood, as only a small fraction of all CD4+ cells appear to be productively infected with HIV-1 in seropositive individuals. In the present study, crosslinking of bound gp120 on human CD4+ T cells followed by signaling through the T cell receptor for antigen was found to result in activation-dependent cell death by a form of cell suicide termed apoptosis, or programmed cell death. The data indicate that even picomolar concentrations of gp120 prime T cells for activation-induced cell death, suggesting a mechanism for CD4+ T cell depletion in acquired immune deficiency syndrome (AIDS), particularly in the face of concurrent infection and antigenic challenge with other organisms. These results also provide an explanation for the enhancement of infection by certain antibodies against HIV, and for the paradox that HIV appears to cause AIDS after the onset of antiviral immunity.

Indirect mechanisms of HIV pathogenesis: how does HIV kill T cells?

Although twelve years have passed since the identification of HIV as the cause of AIDS, we do not yet know how HIV kills its target, the CD4+ T cell, nor how this killing cripples the immune system. Prominent theories include direct killing of infected CD4+ T cells by the action or accumulation of cytopathic viral DNA, transcripts or proteins, or by virus-specific cytotoxic T lymphocytes, and indirect killing of uninfected CD4+ T cells (and other immune cells) by autoimmune mechanisms, cytokines, superantigens, or apoptosis. In the past year, studies have provided tantalizing clues as to why infected cells may not die and how these infected cells kill innocent bystander cells.

Central nervous system Sjögren's syndrome in a child: case report and review of the literature.

We describe a case of pediatric Sjögren's syndrome with progressive neurologic involvement. At age 4 years, she had been diagnosed with Melkersson-Rosenthal syndrome. After being stable with facial diplegia and swelling for 5 years, she acutely presented with diplopia, vertigo, and ataxia. Cranial magnetic resonance imaging (MRI) showed a left dorsal midbrain lesion. Serologic and histopathologic findings confirmed primary Sjögren's syndrome. She responded well to intravenous methylprednisolone, with subsequent clinical improvement and MRI resolution. This report reviews the pediatric literature and underscores the importance of considering Sjögren's syndrome in a child with unexplained facial weakness and in the differential diagnosis of pediatric stroke.

Novel and enhanced anti-melanoma DNA vaccine targeting the tyrosinase protein inhibits myeloid-derived suppressor cells and tumor growth in a syngeneic prophylactic and therapeutic murine model.

Melanoma is the most deadly type of skin cancer, constituting annually ∼ 75% of all cutaneous cancer-related deaths due to metastatic spread. Currently, because of metastatic spread, there are no effective treatment options for late-stage metastatic melanoma patients. Studies over the past two decades have provided insight into several complex molecular mechanisms as to how these malignancies evade immunological control, indicating the importance of immune escape or suppression for tumor survival. Thus, it is essential to develop innovative cancer strategies and address immune obstacles with the goal of generating more effective immunotherapies. One important area of study is to further elucidate the role and significance of myeloid-derived suppressor cells (MDSCs) in the maintenance of the tumor microenvironment. These cells possess a remarkable ability to suppress immune responses and, as such, facilitate tumor growth. Thus, MDSCs represent an important new target for preventing tumor progression and escape from immune control. In this study, we investigated the role of MDSCs in immune suppression of T cells in an antigen-specific B16 melanoma murine system utilizing a novel synthetic tyrosinase (Tyr) DNA vaccine therapy in both prophylactic and therapeutic models. This Tyr vaccine induced a robust and broad immune response, including directing CD8 T-cell infiltration into tumor sites. The vaccine also reduced the number of MDSCs in the tumor microenvironment through the downregulation of monocyte chemoattractant protein 1, interleukin-10, CXCL5 and arginase II, factors important for MDSC expansion. This novel synthetic DNA vaccine significantly reduced the melanoma tumor burden and increased survival in vivo, due likely, in part, to the facilitation of a change in the tumor microenvironment through MDSC suppression.

Mechanisms of lymphocyte killing by HIV.

One of the remaining mysteries of HIV infection is what causes the destruction of the CD4+ T cells. Several reports in the past year have linked viral load to disease progression, strengthening the conclusion that the virus is responsible for CD4+ T-cell depletion. We discuss several possible mechanisms of T-cell death, including the killing of uninfected CD4+ T cells. We also discuss the possibility that the virus protects the cell it infects at least until viral replication can be completed.

HIV-1 NL4-3, but not IIIB, inhibits JAK3/STAT5 activation in CD4(+) T cells.

HIV-1 infection leads to T cell dysfunction and apoptosis in vivo and in vitro. The shared common gamma chain of IL-2R and its associated Janus kinase, JAK3, are indispensable for normal T cell function and survival. We have reported that CD4 ligation with HIV gp120 inhibits T cell receptor-induced activation and expression of JAK3. We have also shown that while some strains of HIV-1, such as NL4-3, induce apoptosis of infected CD4(+) T cells, other strains, such as HIV-1 IIIB, do not. Interestingly, we show here that infection of CD4(+) T cells with HIV-1 NL4-3, but not IIIB, inhibited activation and expression of JAK3. NL4-3-infected T cells were unable to upregulate JAK3 expression following stimulation through TCR/CD3. In addition, NL4-3, but not IIIB, inhibited tyrosine phosphorylation and expression of STAT5, a downstream target of JAK3. These data suggest a correlation between apoptosis of HIV-1-infected T cells and inhibition of the JAK3/STAT5 activation pathway.

ADP-ribosylation of rho by C3 ribosyltransferase inhibits IL-2 production and sustained calcium influx in activated T cells.

Activation of the T lymphocyte induces dramatic cytoskeletal changes, and there is increasing evidence that disruption of the cytoskeleton inhibits early and late events of T cell signal transduction. However, relatively little is known about the signaling molecules involved in activation-induced cytoskeletal rearrangement. The rho family of small GTP-binding proteins, which include rho, rac, and cdc42, regulates the cytoskeleton and coordinates various cellular functions via their many effector targets. In prior studies, the Clostridium botulinum toxin C3 exoenzyme has been used to ADP-ribosylate and inactivate rho. In this study, we demonstrate that treatment of T cells with C3 exoenzyme inhibits IL-2 transcription following ligation of the TCR. Inhibition of IL-2 expression correlated with loss of sustained increase in [Ca+2]i and mitogen activated protein kinase (MAPK/Erk) activity, but not with activation of the tyrosine kinase, lck. These findings are the first to show that ADP-ribosylation of rho by C3 ribosyltransferase (exoenzyme) inhibits IL-2 production due, in part, to the requirement for sustained calcium influx and MAPK activation after Ag receptor ligation.

Cutting edge: JAK3 activation and rescue of T cells from HIV gp120-induced unresponsiveness.

In early HIV disease, immunodeficiency is characterized by the inability of CD4+ T cells to produce a critical cytokine, IL-2, and to express the receptor for IL-2 (IL-2R) in response to antigenic or mitogenic stimulation. The shared common gamma-chain (gamma(c)) of IL-2R and its associated Janus kinase, JAK3, are indispensable for normal T cell function. Here, we show that the inhibition of IL-2R expression and proliferation induced by ligation of CD4 by HIV envelope glycoprotein, gp120, is correlated with inhibition of expression and activation of JAK3. Stimulation through the gamma(c)-related cytokine receptors restores JAK3 expression and activation and rescues CD4-mediated T cell unresponsiveness. Collectively, these data argue that inhibition of JAK3 expression and activation may, in part, explain the T cell dysfunction seen in early HIV disease. In addition, rescue from gp120-mediated T cell unresponsiveness by activation of JAK3 suggests a novel therapeutic approach for enhancing immune function in HIV disease.

Vpu increases susceptibility of human immunodeficiency virus type 1-infected cells to fas killing.

The importance of the Fas death pathway in human immunodeficiency virus (HIV) infection has been the subject of many studies. Missing from these studies is direct measurement of infected cell susceptibility to Fas-induced death. To address this question, we investigated whether T cells infected with HIV are more susceptible to Fas-induced death. We found that Fas cross-linking caused a decrease in the number of HIV-infected Jurkat T cells and CD4(+) peripheral blood leukocytes (PBLs). We confirmed this finding by demonstrating that there were more apoptotic infected than uninfected cells after Fas ligation. The increase in sensitivity of HIV-infected cells to Fas killing mapped to vpu, while nef, vif, vpr, and second exon of tat did not appear to contribute. Furthermore, expression of Vpu in Jurkat T cells rendered them more susceptible to Fas-induced death. These results show that HIV-infected cells are more sensitive to Fas-induced death and that the Vpu protein of HIV contributes to this sensitivity. The increased sensitivity of HIV-infected cells to Fas-induced death might help explain why these cells have such a short in vivo half-life.

T-cell development and transmembrane signaling: changing biological responses through an unchanging receptor.

The antigen receptor repertoire is conditioned by discriminating forces in the thymus. Cells that see antigen only in the context of self major histocompatibility gene products are positively selected and those that recognize self antigens are deleted. The molecular mechanisms by which this complex conditioning is achieved via a single antigen receptor is one of the most fascinating problems in immunology. Here Terri Helman Finkel and colleagues review the literature and present a unifying mechanistic model of this process.

Immature thymocytes are protected from deletion early in ontogeny.

Self/non-self discrimination occurs within the thymus, where T cells undergo positive selection to produce a repertoire that recognizes foreign antigen in the context of self major histocompatibility complex proteins and negative selection to eliminate from the repertoire those T cells with self-reactive specificities. We have previously shown that two subpopulations of immature antigen receptor-bearing thymocytes exist, one that is susceptible to negative selection induced by ligation of the T-cell receptor for antigen and a second that is resistant to T-cell receptor-mediated negative selection. In the current work, we show that all antigen receptor-bearing thymocytes in early fetal thymuses are resistant to negative selection and that thymocytes susceptible to deletion do not develop until later gestational ages. Thus, deletion is a relatively late event in T-cell ontogeny. In addition, these data suggest that a thymocyte population can be isolated early in ontogeny that is capable of transducing selective signals through the antigen receptor, which do not lead to deletion but may result in positive selection.

Tyrosine-phosphorylated T cell receptor zeta chain associates with the actin cytoskeleton upon activation of mature T lymphocytes.

The multichain T cell antigen receptor (TCR) is composed of an antigen binding (alpha/beta) domain and associated signal-transducing complexes, the CD3 (gamma, delta, and epsilon) and the zeta chains. The zeta chain (TCR zeta) plays a key role in signal transduction. We show here that TCR ligation induces association of tyrosine-phosphorylated TCR zeta with the detergent-insoluble cell fraction. The microfilament poison, cytochalasin D, disrupts this association and enhances the coprecipitation of actin with TCR zeta after receptor ligation. This microfilament association is specific to TCR-associated polypeptides containing at least one intact immunoreceptor tyrosine-based activation motif (ITAM). Mapping studies using transfectants and chimeric TCR zeta chain constructs suggest that the third ITAM is necessary and sufficient for association, if the distal tyrosine is intact. This cytoskeletal association is directly correlated with IL-2 production, and ligation of TCR on immature thymocytes does not induce TCR zeta-cytoskeleton association. These data thus provide direct evidence of a developmentally regulated activation-dependent interaction between a lymphocyte antigen receptor and the actin cytoskeleton.

pp56Lck mediates TCR zeta-chain binding to the microfilament cytoskeleton.

The TCR zeta-chain (zeta) on mature murine T lymphocytes binds to the microfilament cytoskeleton in response to Ag receptor ligation. Here, we report the role of Src family kinases in zeta-cytoskeletal binding, using mutant mice and a cell-free model system. Binding of zeta to actin in the cell-free system has a specific requirement for ATP and divalent cations, with an apparent Michaelis-Menton constant for ATP in the millimolar range, and can be disrupted by either EDTA or the microfilament poison, cytochalasin D, suggesting that microfilaments provide the structural framework for an active process involving cellular kinases. Indeed, tyrosine-phosphorylated zeta is a predominant form of the zeta-chain bound to polymerized actin, while challenge with alkaline phosphatase prevents zeta-chain association in solution and releases zeta-chain from the bound state. Phosphorylated Src-family kinase pp56Lck also associates with membrane skeleton upon TCR engagement and is a component of the reconstituted cytoskeletal pellet. Zeta-chain phosphorylation and zeta-cytoskeletal binding are abrogated in cell lysates with reduced levels of pp56Lck and in activated mutant murine T cells lacking pp56Lck, implicating pp56Lck as the kinase involved in zeta-chain tyrosine phosphorylation and zeta-cytoskeletal binding. Finally, recombinant Lck Src homology 2 domain preferentially inhibits reconstituted zeta-cytoskeleton association, suggesting that zeta-microfilament binding is dependent on interactions between phosphorylated tyrosine residues in zeta-chain activation motifs and the Src homology 2 domain of the Lck protein tyrosine kinase.

Both immature and mature T cells mobilize Ca2+ in response to antigen receptor crosslinking.

T cells develop from prothymocytes which express no detectable antigen receptors to immature thymocytes with few receptors, eventually becoming mature thymocytes and peripheral T cells with 20,000-40,000 receptors per cell. Recent studies suggest that immature thymocytes are immunologically unresponsive. We have suggested that an early step in signal transduction following engagement of the T cell receptor might differ in immature and mature T cells. Here we examine anti-receptor antibody mediated induction of calcium mobilization in immature and mature T cells. Results indicate that antigen receptors on both immature and mature receptor-positive T cells transduce signals via calcium mobilization. Significant differences were observed, however, between these populations in the magnitude of influx of extracellular Ca2+ following binding of antireceptor antibody. Specifically immature cells show a much reduced Ca2+ influx response compared to mature cells which could result from a low Ca2+ channel frequency in the plasma membranes of immature T cells, or from less efficient activation of existing channels.

Alpha beta T cell receptor and CD3 transduce different signals in immature T cells. Implications for selection and tolerance.

Ligand binding to alpha beta TCR has different consequences in thymocytes at different developmental stages, causing alternatively positive selection, clonal deletion, or activation. These various functional consequences may be due to changes in the signaling properties of the receptor complex during development. In this report we show that alpha beta TCR engagement on immature thymocytes has different effects on intracellular free calcium concentrations than alpha beta TCR engagement on mature T cells. In contrast, CD3 engagement on immature thymocytes and mature T cells has the same effect on intracellular free calcium, suggesting that altered signal transduction in immature thymocytes may be due to inefficient alpha beta TCR-CD3 coupling. These studies also suggest that in certain T cell populations, activation events resulting from ligation of CD3 may not accurately reflect the activation events resulting from ligation of the physiologic receptor, alpha beta TCR.