Arginine-based cationic liposomes accelerate T cell activation and differentiation in vitro.

Cationic liposomes are versatile lipid nanocarriers to improve the pharmacological properties of drug payloads. Recent advantages include the application of their intrinsic immunostimulatory effects to enhance immune activation. Herein, we report for the first time the structural effect of cationic lipids in promoting T cell activation and differentiation in vitro. Two types of cationic liposomes R3C14 and R5C14 were prepared from single type of lipids Arg-C3-Clu2C14 or Arg-C5-Clu2C14, which bear arginine head group and ditetradecyl tails but vary in the carbon number of the spacer in between. Murine CD8 or CD4 T cells were pretreated with 50 µM of each type of liposomes for 2 h, followed by stimulation with anti-CD3/CD28 antibodies for 24 h. In comparison to liposome-untreated T cells, R5C14-pretreatment induced a robust T cell activation (IL-2, CD25+) and differentiation into effector cells (CD44high, CD62Llow), whereas R3C14 did not show comparable effect. Furthermore, a weak activation of nuclear factor of activated T cells (NFAT) was detected in Jurkat-Lucia NFAT cells (InvivoGen), suggesting a potential signaling pathway for the liposomal effect. Although R5C14 liposomes did not activate T cells without subsequent CD3/CD28 stimulation, this study implied a recessive effect of some cationic adjuvant in priming T cells to enhance their responsiveness to antigens.

New Murine Model of Early Onset Autoimmune Thyroid Disease/Hypothyroidism and Autoimmune Exocrinopathy of the Salivary Gland.

Sixty to seventy percent of IFN-γ(-/-) NOD.H-2h4 mice given sodium iodide (NaI)-supplemented water develop a slow onset autoimmune thyroid disease, characterized by thyrocyte epithelial cell (TEC) hyperplasia and proliferation (H/P). TEC H/P develops much earlier in CD28(-/-) mice and nearly 100% (both sexes) have severe TEC H/P at 4 mo of age. Without NaI supplementation, 50% of 5- to 6-mo-old CD28(-/-)IFN-γ(-/-) mice develop severe TEC H/P, and 2-3 wk of NaI is sufficient for optimal development of severe TEC H/P. Mice with severe TEC H/P are hypothyroid, and normalization of serum thyroxine levels does not reduce TEC H/P. Activated CD4(+) T cells are sufficient to transfer TEC H/P to SCID recipients. Thyroids of mice with TEC H/P have infiltrating T cells and expanded numbers of proliferating thyrocytes that highly express CD40. CD40 facilitates, but is not required for, development of severe TEC H/P, as CD40(-/-)IFN-γ(-/-)CD28(-/-) mice develop severe TEC H/P. Accelerated development of TEC H/P in IFN-γ(-/-)CD28(-/-) mice is a result of reduced regulatory T cell (Treg) numbers, as CD28(-/-) mice have significantly fewer Tregs, and transfer of CD28(+) Tregs inhibits TEC H/P. Essentially all female IFN-γ(-/-)CD28(-/-) NOD.H-2h4 mice have substantial lymphocytic infiltration of salivary glands and reduced salivary flow by 6 mo of age, thereby providing an excellent new model of autoimmune exocrinopathy of the salivary gland. This is one of very few models where autoimmune thyroid disease and hypothyroidism develop in most mice by 4 mo of age. This model will be useful for studying the effects of hypothyroidism on multiple organ systems.

Glycerol Monolaurate (GML) inhibits human T cell signaling and function by disrupting lipid dynamics.

Glycerol Monolaurate (GML) is a naturally occurring fatty acid widely utilized in food, cosmetics, and homeopathic supplements. GML is a potent antimicrobial agent that targets a range of bacteria, fungi, and enveloped viruses but select findings suggest that GML also has immunomodulatory functions. In this study, we have mechanistically examined if GML affects the signaling and functional output of human primary T cells. We found that GML potently altered order and disorder dynamics in the plasma membrane that resulted in reduced formation of LAT, PLC-γ, and AKT microclusters. Altered membrane events induced selective inhibition of TCR-induced phosphorylation of regulatory P85 subunit of PI3K and AKT as well as abrogated calcium influx. Ultimately, GML treatment potently reduced TCR-induced production of IL-2, IFN-γ, TNF-α, and IL-10. Our data reveal that the widely used anti-microbial agent GML also alters the lipid dynamics of human T cells, leading to their defective signaling and function.

Human ß-defensin 3 induces STAT1 phosphorylation, tyrosine phosphatase activity, and cytokine synthesis in T cells.

The AMP hBD-3 stimulates numerous immune effector functions in myeloid cells and keratinocytes, predominantly through the MAPK signaling cascade. In contrast, hBD-3 was reported to neutralize the activation of T cells by antagonizing MAPK signaling initiated by SDF-1α through CXCR4. With the use of complementary proteomic and immunochemical approaches, we investigated possible stimulatory effects of hBD-3 on T cells and demonstrate that hBD-3 induces STAT1 tyrosine phosphorylation within 5 min yet is unable to induce MAPK activation. Inclusion of a PTPase inhibitor increased hBD-3-induced phosphorylation dramatically, suggesting that hBD-3 also stimulates PTPase activity concurrently. The increase in PTPase activity was confirmed by demonstrating that hBD-3 suppresses IFN-γ-induced STAT1 tyrosine phosphorylation but not STAT1 serine and ERK1/2 threonine phosphorylation and stimulates the translocation of SHP-2 into the nucleus within 15 min. The signaling pathways initiated by hBD-3 may lead to the observed enhancement of distinct T cell effector functions during TCR activation, such as the increase in IL-2 and IL-10, but not IFN-γ secretion. Thus, hBD-3 initiates distinct lineage-specific signaling cascades in various cells involved in host defense and induces a concurrent tyrosine kinase and tyrosine phosphatase signaling cascade that may activate simultaneously the targeted T cells and inhibit their response to other immune mediators. Furthermore, these results suggest that this evolutionarily conserved peptide, which exhibits a broad spectrum of antimicrobial and immunomodulatory activities, serves to integrate innate and adaptive immunity.

Nuevas dianas terapéuticas en el lupus sistémico (parte 2/2) / New targets in systemic lupus (part 2/2)

Glucocorticoides, aspirina, antipalúdicos e inmunosupresores convencionales constituyen la base del tratamiento del lupus eritematoso sistémico (LES). Hasta recientemente, los 3 primeros eran los únicos agentes aprobados para su tratamiento. El mejor conocimiento de la fisiopatología del sistema inmunitario ha permitido identificar nuevas dianas terapéuticas. De hecho, belimumab, un anticuerpo monoclonal humano inhibidor de BLyS, se ha convertido hace pocos meses en el primer fármaco aprobado para el tratamiento del LES desde 1957, lo que subraya las dificultades de todo tipo, incluyendo las económicas y organizativas, inherentes a los ensayos clínicos sobre esta enfermedad. Otras muchas moléculas se encuentran en distintas fases de desarrollo y en poco tiempo dispondremos de resultados concretos. En esta revisión repasamos el mecanismo de acción y los datos clínicos más relevantes de estas moléculas (AU)

Glucocorticoids, aspirin, conventional antimalarials and immunosuppressants are the mainstay of treatment of Systemic Lupus Erythematosus (SLE). Until recently, the first three were the only agents approved for treatment. A better understanding of the pathophysiology of the immune system has identified new therapeutic targets. In fact, belimumab, a human monoclonal antibody to BLyS inhibitor has become, in recent months, the first drug approved for the treatment of SLE since 1957, underscoring difficulties of all kinds, including economic and organizational ones inherent to clinical trials on this disease. Many other molecules are in various stages of development and soon will have concrete results. In this review, we examined the mechanism of action and most relevant clinical data for these molecules (AU)

ATP release and autocrine signaling through P2X4 receptors regulate γδ T cell activation.

Purinergic signaling plays a key role in a variety of physiological functions, including regulation of immune responses. Conventional αß T cells release ATP upon TCR cross-linking; ATP binds to purinergic receptors expressed by these cells and triggers T cell activation in an autocrine and paracrine manner. Here, we studied whether similar purinergic signaling pathways also operate in the "unconventional" γδ T lymphocytes. We observed that γδ T cells purified from peripheral human blood rapidly release ATP upon in vitro stimulation with anti-CD3/CD28-coated beads or IPP. Pretreatment of γδ T cells with (10)panx-1, CBX, or Bf A reversed the stimulation-induced increase in extracellular ATP concentration, indicating that panx-1, connexin hemichannels, and vesicular exocytosis contribute to the controlled release of cellular ATP. Blockade of ATP release with (10)panx-1 inhibited Ca(2+) signaling in response to TCR stimulation. qPCR revealed that γδ T cells predominantly express purinergic receptor subtypes A2a, P2X1, P2X4, P2X7, and P2Y11. We found that pharmacological inhibition of P2X4 receptors with TNP-ATP inhibited transcriptional up-regulation of TNF-α and IFN-γ in γδ T cells stimulated with anti-CD3/CD28-coated beads or IPP. Our data thus indicate that purinergic signaling via P2X4 receptors plays an important role in orchestrating the functional response of circulating human γδ T cells.

Belatacept: from rational design to clinical application.

Gradually improved immunosuppression has contributed significantly to the progress achieved in transplantation medicine so far. Nevertheless, current drug regimens are associated with late graft loss--in particular as a result of immunologic damage or drug toxicity--and substantial morbidity. Recently, the costimulation blocker belatacept (marketed under the name Nulojix®) has been approved for immunosuppression in renal transplantation. Belatacept (a mutated version of CTLA4Ig) is a fusion protein rationally designed to block CD28, a critical activating receptor on T cells, by binding and saturating its ligands B7-1 and B7-2. In phase II and III trials, belatacept was compared with cyclosporine (in combination with basiliximab, MMF, and steroids). Advantages observed with belatacept include superior graft function, preservation of renal structure and improved cardiovascular risk profile. Concerns associated with belatacept are a higher frequency of cellular rejection episodes and more post-transplant lymphoproliferative disorder (PTLD) cases especially in EBV seronegative patients, who should be excluded from belatacept-based regimens. Thus, after almost three decades of calcineurin inhibitors as mainstay of immunosuppression, belatacept offers a potential alternative. In this article, we will provide an overview of belatacept's preclinical development and will discuss the available evidence from clinical trials.

A novel monoclonal antibody against human CD80 and its immune protection in a mouse lupus-like disease.

Blockade of the interactions between CD28/CTLA-4 and their ligands, CD80 (B7, B7.1)/CD86 (B70, B7.2), is an attractive means to induce antigen-specific peripheral tolerance in autoimmune disease and organ transplantation. In this study, we generated and characterized a monoclonal antibody (Clone 4E5) against human CD80. 4E5 could recognize both human and mouse CD80 and suppress mixed lymphocyte reaction in vitro. To investigate their potency for clinical use, we further administrated 4E5 to a mouse lupus-like disease model (C57BL/J6) induced by Pristane. 4E5 could inhibit the immune response and attenuate the severity of lupus-like disease. The data showed 4E5 function and suggested that blockade of CD80/CD28 co-stimulatory signal pathway with 4E5 is a promising strategy to decelerate the progression of lupus-like disease and other autoimmune diseases.

The CD28/CTLA-4-B7 signaling pathway is involved in both allergic sensitization and tolerance induction to orally administered peanut proteins.

Dendritic cells are believed to play an essential role in regulating the balance between immunogenic and tolerogenic responses to mucosal Ags by controlling T cell differentiation and activation via costimulatory and coinhibitory signals. The CD28/CTLA-4-CD80/CD86 signaling pathway appears to be one of the most important regulators of T cell responses but its exact role in responses to orally administered proteins remains to be elucidated. In the present study, the involvement of the CD28/CTLA-4-CD80/CD86 costimulatory pathway in the induction of allergic sensitization and oral tolerance to peanut proteins was investigated. In both an established C3H/HeOuJ mouse model of peanut hypersensitivity and an oral tolerance model to peanut, CD28/CTLA-4-CD80/CD86 interactions were blocked using the fusion protein CTLA-4Ig. To examine the relative contribution of CD80- and CD86-mediated costimulation in these models, anti-CD80 and anti-CD86 blocking Abs were used. In the hypersensitivity model, CTLA-4Ig treatment prevented the development of peanut extract-induced cytokine responses, peanut extract-specific IgG1, IgG2a, and IgE production and peanut extract-induced challenge responses. Blocking of CD80 reduced, whereas anti-CD86 treatment completely inhibited, the induction of peanut extract-specific IgE. Normal tolerance induction to peanut extract was found following CTLA-4Ig, anti-CD86, or anti-CD80 plus anti-CD86 treatment, whereas blockade of CD80 impaired the induction of oral tolerance. We show that CD28/CTLA-4-CD80/CD86 signaling is essential for the development of allergic responses to peanut and that CD86 interaction is most important in inducing peanut extract-specific IgE responses. Additionally, our data suggest that CD80 but not CD86 interaction with CTLA-4 is crucial for the induction of low dose tolerance to peanut.

Immuno-suppressive effect of blocking the CD28 signaling pathway in T-cells by an active component of Echinacea found by a novel pharmaceutical screening method.

AFTIR (after flowing through immobilized receptor) is a novel method for screening herbal extracts for pharmaceutical properties. Using AFTIR, we identified Cynarin in Echinacea purpurea by its selective binding to chip immobilized CD28, a receptor of T-cells, which is instrumental to immune functioning. The results of surface plasma resonance show that binding between immobilized CD28 and Cynarin is stronger than the binding between CD28 and CD80, a co-stimulated receptor of antigen presenting cells. Cynarin's function was verified by its ability to downregulate CD28-dependent interleukin-2 (IL-2) expression in a T-cell culture line. AFTIR offers promise as an efficient screening method for herbal medicines.

CD28 signals in the immature immunological synapse.

T cell recognition of peptide-MHC complexes on APCs results in the aggregation of TCRs at a central supramolecular activation complex (c-SMAC) within a mature immunological synapse. T cells require a second "costimulatory" signal for activation, the most important of which, for naive T cells, is from CD28. However the time at which CD28-derived signals are induced relative to c-SMAC formation is not well understood. In this study, we have assessed the kinetics of CD28 localization and function relative to well-established aspects of c-SMAC formation. CD28 accumulates at the immature synapse alongside the TCR and is likewise enriched at the synapse at the onset of the calcium signal. In addition, using CD28 deficient or reconstituted murine cells in a single-cell recording approach shows that CD28 regulates this signal within seconds of a TCR-mediated rise in intracellular calcium levels. Finally, CD28 exerts effects on both the initiation and stabilization of the synapse in parallel with its effects on the downstream proliferation of T cells. Together, the data show that CD28 functions in the immunological synapse before the formation of the c-SMAC.

Non-CD28 costimulatory molecules present in T cell rafts induce T cell costimulation by enhancing the association of TCR with rafts.

While CD28 functions as the major T cell costimulatory receptor, a number of other T cell molecules have also been described to induce T cell costimulation. Here, we investigated the mechanisms by which costimulatory molecules other than CD28 contribute to T cell activation. Non-CD28 costimulatory molecules such as CD5, CD9, CD2, and CD44 were present in the detergent-insoluble glycolipid-enriched (DIG) fraction/raft of the T cell surface, which is rich in TCR signaling molecules and generates a TCR signal upon recruitment of the TCR complex. Compared with CD3 ligation, coligation of CD3 and CD5 as an example of DIG-resident costimulatory molecules led to an enhanced association of CD3 and DIG. Such a DIG redistribution markedly up-regulated TCR signaling as observed by ZAP-70/LAT activation and Ca2+ influx. Disruption of DIG structure using an agent capable of altering cholesterol organization potently diminished Ca2+ mobilization induced by the coligation of CD3 and CD5. This was associated with the inhibition of the redistribution of DIG although the association of CD3 and CD5 was not affected. Thus, the DIG-resident costimulatory molecules exert their costimulatory effects by contributing to an enhanced association of TCR/CD3 and DIG.

TCR and CD28 are coupled via ZAP-70 to the activation of the Vav/Rac-1-/PAK-1/p38 MAPK signaling pathway.

CD28 costimulation amplifies TCR-dependent signaling in activated T cells, however, the biochemical mechanism(s) by which this occurs is not precisely understood. The small GTPase Rac-1 controls the catalytic activity of the mitogen-activated protein kinases (MAPKs) and cell cycle progression through G1. Rac-1 activation requires the phospho-tyrosine (p-Tyr)-dependent recruitment of the Vav GDP releasing factor (GRF) to the plasma membrane and assembly of GTPase/GRF complexes, an event critical for Ag receptor-triggered T cell activation. Here, we show that TCR/CD28 costimulation synergistically induces Rac-1 GDP/GTP exchange. Our findings, obtained by using ZAP-70-negative Jurkat T cells, indicate that CD28 costimulation augments TCR-mediated T cell activation by increasing the ZAP-70-mediated Tyr phosphorylation of Vav. This event regulates the Rac-1-associated GTP/GDP exchange activity of Vav and downstream pathway(s) leading to PAK-1 and p38 MAPK activation. CD28 amplifies TCR-induced ZAP-70 activity and association of Vav with ZAP-70 and linker for activation of T cells (LAT). These results favor a model in which ZAP-70 regulates the intersection of the TCR and CD28 signaling pathways, which elicits the coupling of TCR and CD28 to the Rac-1, PAK-1, and p38 MAPK effector molecules.