CD40 Ligation Research Articles

Suppression of antigen-specific CD4+ T cell activation by SRA/CD204 through reducing the immunostimulatory capability of antigen-presenting cell

Pattern recognition scavenger receptor SRA/CD204, primarily expressed on specialized antigen-presenting cells (APCs), including dendritic cells (DCs) and macrophages, has been implicated in multiple physiological and pathological processes, including atherosclerosis, Alzheimer's disease, endotoxic shock, host defense, and cancer development. SRA/CD204 was also recently shown to function as an attenuator of vaccine response and antitumor immunity. Here, we, for the first time, report that SRA/CD204 knockout (SRA(-/-)) mice developed a more robust CD4(+) T cell response than wild-type mice after ovalbumin immunization. Splenic DCs from the immunized SRA(-/-) mice were much more efficient than those from WT mice in stimulating naïve OT-II cells, indicating that the suppressive activity of SRA/CD204 is mediated by DCs. Strikingly, antigen-exposed SRA(-/-) DCs with or without lipopolysaccharide treatment exhibited increased T-cell-stimulating activity in vitro, which was independent of the classical endocytic property of the SRA/CD204. Additionally, absence of SRA/CD204 resulted in significantly elevated IL12p35 expression in DCs upon CD40 ligation plus interferon gamma (IFN-γ) stimulation. Molecular studies reveal that SRA/CD204 inhibited the activation of STAT1, mitogen activated protein kinase p38, and nuclear factor-kappa B signaling activation in DCs treated with anti-CD40 antibodies and IFN-γ. Furthermore, splenocytes from the generated SRA(-/-) OT-II mice showed heightened proliferation upon stimulation with OVA protein or MHC-II-restricted OVA(323-339) peptide compared with cells from the SRA(+/+) OT-II mice. These results not only establish a new role of SRA/CD204 in limiting the intrinsic immunogenicity of APCs and CD4(+) T cell activation but also provide additional insights into the molecular mechanisms involved in the immune suppression by this molecule.

Erratum: The functional in vitro response to CD40 ligation reflects a different clinical outcome in patients with chronic lymphocytic leukemia

Erratum: The functional in vitro response to CD40 ligation reflects a different clinical outcome in patients with chronic lymphocytic leukemia

Protein Kinase R Is a Novel Mediator of CD40 Signaling and Plays a Critical Role in Modulating Immunoglobulin Expression during Respiratory Syncytial Virus Infection

Effective immunoglobulin responses play a vital role in protection against most pathogens. However, the molecular mediators and mechanisms responsible for signaling and selective expression of immunoglobulin types remain to be elucidated. Previous studies in our laboratory have demonstrated that protein kinase R (PKR) plays a crucial role in IgE responses to double-stranded RNA (dsRNA) in vitro. In this study, we show that PKR plays a critical role in IgG expression both in vivo and in vitro. PKR(-/-) mice show significantly altered serum IgG levels during respiratory syncytial virus (RSV) infection. IgG2a expression is particularly sensitive to a lack of PKR and is below the detection level in mock- or RSV-infected PKR(-/-) mice. Interestingly, we show that upon activation by anti-CD40 and gamma interferon (IFN-γ), B cells from PKR(-/-) mice show diminished major histocompatibility complex class II (MHC II), CD80, and CD86 levels on the cell surface compared to wild-type (WT) mice. Our data also show that PKR is necessary for optimal expression of adhesion molecules, such as CD11a and ICAM-1, that are necessary for homotypic aggregation of B cells. Furthermore, in this report we demonstrate for the first time that upon CD40 ligation, PKR is rapidly phosphorylated and activated, indicating that PKR is an early and novel downstream mediator of CD40 signaling pathways.

Dissociation of actin polymerization and lipid raft accumulation by ligation of the Inducible Costimulator (ICOS, CD278)

T lymphocyte antigen activation is facilitated by clustering of membrane glycosphingolipid-enriched microdomains (GEMs, lipid “rafts”) at the T cell/APC contact that is linked to changes in actin cytoskeleton and is one major mechanism of CD28 costimulation. Ligation of CD28 alone, or ligation of the CD28-like molecules CTLA-4 (CD152) and ICOS (CD278) induces actin polymerization with cell elongation and generation of lamellipodia and filopodia in T cells. These changes are dependent on Src, PI3-kinase, Vav, and Rho family GTPases. Whereas CD28 and CTLA-4 have been shown to be functional and physically associated with lipid rafts, the presence of ICOS in lipid rafts or its effect in raft clustering is not known. In this work, we have activated the T cell line D10 with anti-ICOS antibodies, alone or combined with anti-CD3 antibodies, bound or unbound to polystyrene microbeads or glass coverslips. The possible relationship of ICOS-induced changes in actin cytoskeleton to the ICOS localization in membrane rafts was then analyzed by fluorescence microscopy, or by immunoblot of detergent insoluble (“raft”) or soluble (“non-raft”) fractions of cell lysates. Our data show that ICOS promotes TCR/CD3 induction of raft clustering at the site of activation. However, ICOS, which, on its own, can induce accumulations of polymerized actin, is undetectable in membrane rafts, even when using CD3 or ICOS, ligands capable of inducing clear changes in the actin cytoskeleton.

Cholesterol: An Inflammatory Compound

Obesity is one of the main rising causes of health problems in modern society and is correlated to type 2 diabetes mellitus, hypertension, heart disease and atherosclerosis. Bacterial products, endogenous substances such as oxidized LDL (ox-LDL) and heat shock proteins mediate activation of Toll-like receptors and reinforce the view that the innate immune system plays a key role in the genesis of atherosclerosis. In addition, natural killer T (NKT) cells respond to lipids presented via CD1d on APCs, and may also be able to affect atherosclerosis. All the main cell types involved in atherosclerosis such as endothelial cells, macrophages, T cells, smooth muscle cells and platelets express proinflammatory cytokines. In addition, CD4 ligation triggers the expression of adhesion molecules, cytokines and matrix metalloprotinease. IL-6 cytokines travels to the liver where it elicits acute phase response resolving in the release of serum amyloid-A C-reactive protein, fibrogen and plasminogen activator inhibitor-1. Therefore increasing body fat mass is associated with high levels of inflammatory cytokines such as IL-1 and TNF. In this study we revisit the interrelationship between fat and inflammation.

CD34+-derived Langerhans cell-like cells are different from epidermal Langerhans cells in their response to thymic stromal lymphopoietin

Thymic stromal lymphopoietin (TSLP) endows human blood-derived CD11c+ dendritic cells (DCs) and Langerhans cells (LCs) obtained from human epidermis with the capacity to induce pro-allergic T cells. In this study, we investigated the effect of TSLP on umbilical cord blood CD34+-derived LC-like cells. These cells are often used as model cells for LCs obtained from epidermis. Under the influence of TSLP, both cell types differed in several ways. As defined by CD83, CD80 and CD86, TSLP did not increase maturation of LC-like cells when compared with freshly isolated LCs and epidermal émigrés. Differences were also found in the production of chemokine (C-C motif) ligand (CCL)17. LCs made this chemokine only when primed by TSLP and further stimulated by CD40 ligation. In contrast, LC-like cells released CCL17 in response to CD40 ligation, irrespective of a prior treatment with TSLP. Moreover, the CCL17 levels secreted by LC-like cells were at least five times higher than those from migratory LCs. After maturation with a cytokine cocktail consisting of tumour necrosis factor-α, interleukin (IL)-1β, IL-6 and prostaglandin (PG)E2 LC-like cells released IL-12p70 in response to CD40 ligation. Most importantly and in contrast to LC, TSLP-treated LC-like cells did not induce a pro-allergic cytokine pattern in helper T cells. Due to their different cytokine secretion and the different cytokine production they induce in naïve T cells, we conclude that one has to be cautious to take LC-like cells as a paradigm for ‘real’ LCs from the epidermis.

Tumour‐loaded α‐type 1‐polarized Dendritic Cells from Patients with Chronic Lymphocytic Leukaemia Produce a Superior NK‐, NKT‐ and CD8+ T Cell‐attracting Chemokine Profile

Tumour-loaded dendritic cells (DCs) from patients with chronic lymphocytic leukaemia (CLL) matured using an α-type 1-polarized DC cocktail (IL-1β/TNF-α/IFN-α/IFN-γ/poly-I:C;αDC1) were recently shown to induce more functional CD8(+) T cells against autologous tumour cells in vitro than DCs matured with the 'standard' cocktail (IL-1β/TNF-α/IL-6/PGE(2) ;PGE(2) DCs). However, the ability of vaccine DCs to induce a type 1-polarized immune response in vivo probably relies on additional features, including their ability to induce a CXCR3-dependent recruitment of NK cells into vaccine-draining lymph nodes. Moreover, their guiding of rare tumour-specific CD8(+) T cells to sites of DC-CD4(+) T cell interactions by secretion of CCL3 and CCL4 is needed. We therefore analysed the chemokine profile and the lymphocyte-attracting ability in vitro of monocyte-derived PGE(2) DCs and αDC1s from patients with CLL. αDC1s produced much higher levels of CXCR3 ligands (CXCL9/CXCL10/CXCL11) than PGE(2) DCs. Functional studies further demonstrated that αDC1s were superior recruiters of both NK and NKT cells. Moreover, αDC1s produced higher levels of CCL3/CCL4 upon CD40 ligation. These findings suggest that functional αDC1s, derived from patients with CLL, produce a desirable NK-, NKT- and CD8(+) T cell-attracting chemokine profile which may favour a guided and Th1-deviated priming of CD8(+) T cells, supporting the idea that αDC1-based vaccines have a higher immunotherapeutic potential than PGE(2) DCs.

HIV latency is influenced by regions of the viral genome outside of the long terminal repeats and regulatory genes

We have previously described an in vitro primary thymocyte model for HIV latency that recapitulates several important aspects of latently infected cells obtained from patients. Our original model included a truncated HIV genome expressing only Tat, Rev, and Vpu along with a reporter gene. We have now expanded these studies to include reporter viruses encoding more complete viral genomes. We show here that regions of the viral genome outside of the long terminal repeat promoter and Tat/Rev regulatory genes can substantially affect both the basal level of HIV transcription prior to stimulation, and also the level of viral expression following costimulation via CD3 and CD28 ligation. These differences in latency phenotype between truncated and more complete HIV genomes demonstrate the importance of accessory genes in the context of HIV latency and indicate that care should be taken when interpreting data derived from heavily modified HIV genomes in latency models.

Toll-like receptor 9, transmembrane activator and calcium-modulating cyclophilin ligand interactor, and CD40 synergize in causing B-cell activation

B cells receive activating signals from T cells through CD40, from microbial DNA through Toll-like receptor (TLR) 9, and from dendritic cells through transmembrane activator and calcium-modulating cyclophilin ligand interactor (TACI). TLR9 and CD40 ligation augment TACI-driven B-cell activation, but only the mechanism of synergy between CD40 and TACI has been explored. Synergy between CD40 and TLR9 in B-cell activation is controversial. We sought to examine the mechanisms by which TLR9 modulates CD40- and TACI-mediated activation of B cells and to determine whether all 3 receptors synergize to activate B cells. Naive murine B cells and human PBMCs were stimulated with combinations of anti-CD40, CpG, and a proliferation inducing ligand in the presence of IL-4. Proliferation was measured by means of tritiated thymidine incorporation. Immunoglobulin production was measured by means of ELISA. Class-switch recombination (CSR) was examined by measuring mRNA for germline transcripts, activation-induced cytidine deaminase (AICDA), and mature immunoglobulin transcripts. Plasma cell differentiation was examined by using syndecan-1/CD138 staining and mRNA expression of B lymphocyte-induced maturation protein 1 (Blimp-1). TLR9 synergized with CD40 and TACI in driving CSR and inducing IgG(1) and IgE secretion by naive murine B cells and synergized with TACI in driving B-cell proliferation and plasma cell differentiation. All 3 receptors synergized together in driving murine B-cell proliferation, CSR, plasma cell differentiation, and IgG(1) and IgE secretion. TLR9 synergized with CD40 and TACI in driving IgG secretion in IL-4-stimulated human B cells. Signals from TLR9, TACI, and CD40 are integrated to promote B-cell activation and differentiation.

Calcineurin-dependent negative regulation of CD94/NKG2A expression on naive CD8+ T cells

AbstractImmune responses lead to expression of immunoregulatory molecules on T cells, including natural killer (NK) receptors, such as CD94/NKG2A on CD8+ T cells; these receptors restrain CD8+ responses, thereby preventing T-cell exhaustion in chronic infections and limiting immunopathology. Here, we examined the requirements for inducing CD94/NKG2A on T cells responding to antigen. In vitro, moderate induction of CD94/NKG2A expression occurred after exposure of naive CD8+ (but not CD4+) cells to CD3 ligation or specific peptide. Surprisingly, expression was inhibited by CD28/B7 costimulation. Such inhibition applied only to CD94/NKG2A and not other NK receptors (NKG2D) and was mediated by IL-2. Inhibition by IL-2 occurred via a NFAT cell-independent component of the calcineurin pathway, and CD94/NKG2A induction was markedly enhanced in the presence of calcineurin blockers, such as FK506 or using calcineurin-deficient T cells, both in vitro and in vivo. In addition to CD28-dependent inhibition by IL-2, CD94/NKG2A expression was impaired by several other cytokines (IL-4, IL-23, and transforming growth factor-β) but enhanced by others (IL-6, IL-10, and IL-21). The complex interplay between these various stimuli may account for the variable expression of CD94/NKG2A during responses to different pathogens in vivo.

The functional in vitro response to CD40 ligation reflects a different clinical outcome in patients with chronic lymphocytic leukemia

Malignant B lymphocytes from chronic lymphocytic leukemia (CLL) patients maintain the capacity to respond to CD40 ligation, among other microenvironmental stimuli. In this study, we show that (i) leukemic CLL cells stimulated with the soluble form of CD40L in vitro show differential responses in terms of upregulation of surface markers (CD95 and CD80) and induction of chemokines (CCL22 and CCL17) expression/secretion, and that (ii) these changes are mirrored by a distinct activation of intracellular signalling pathways including increase in IKKalpha/beta phosphorylation and upregulation of antiapoptotic proteins (BCL-2 and MCL-1). CLL patients can then be segregated into two distinct functional subsets. We defined the responsive subset of cases CD40L dependent, considering the capacity to respond as a sign of persistent need of this stimulation for the leukemic expansion. Conversely, we named the unresponsive cases CD40L independent, considering them less dependent on this microenvironmental signal, presumably because of a higher autonomous proliferative and survival potential. Importantly, we report that (iii) the two functional subsets show an opposite clinical outcome, with CD40L-independent cases having a shorter time to progression. This indicates that the functional differences observed in vitro may reflect a different leukemic potential in vivo likely responsible for a distinct clinical course.

Ligation of CD4 on human blood monocytes triggers macrophage differentiation (158.8)

Abstract Human monocytes/macrophages (M/M) have an important role in innate immune responses and in controlling adaptive immune responses. One unique aspect of human M/M is that they express the CD4 molecule. The role of the CD4 molecule in human monocyte/macrophage function and development is not known. To investigate the role of CD4 expressed on human M/M, human blood monocytes were treated with soluble HLA Class II (sHLA II), a natural CD4 ligand. We determined that sHLA II:CD4 ligation triggered the differentiation of human monocytes into functional mature macrophages. These newly developed macrophages had an increased ability to induce T cell activation, suggesting enhanced immune function following CD4 ligation of these cells. We observed a calcium flux and the modulation of gene and cytokine protein expression following CD4 ligation on peripheral monocytes. This indicates that ligation of CD4 transmits a functional signal in this cell type. We found that intracellular signaling of CD4 in primary monocytes occurs through src family kinases as well as via the MAPK pathway to trigger macrophage differentiation. We show that HLA II on IFN-γ-induced HUVECs can ligate CD4 on monocytes and trigger differentiation of these cells into functional macrophages. In all, our results have identified a new function for the CD4 molecule on human peripheral monocytes in triggering the differentiation of these cells into functional macrophages following their interaction with HLA II.

Role of co-stimulatory molecules in macrophage regulation of T cell activation (147.3)

Abstract Murine peritoneal cavity (PerC) T cells from C57BL/6J mice respond poorly in vitro to CD3 ligation due to the high concentration of macrophages (Mϕs) found in this cell source. Mϕs suppressed T cell activation primarily by iNOS catabolism of arginine. Rather than costimulating T cell activation, CD28 ligation increased T cell suppression (“cosuppressed”). IFNγ is necessary for suppression as evidenced by the absence of co-suppression with IFNγRKO PerC Mϕs. To further investigate the co-suppression biology of this system, PerC cells from CD28KO, CD80KO, CD86KO, & CD80/86KO (B7KO) mice were studied. PerC T cells from CD28KO & B7KO mice had the lowest number of IFNγ-secreting cells (IFNγSC) & were the least susceptible to Mϕ-mediated suppression. CD86KO PerC cells included more IFNγSC & their T cells were more suppressed than CD80KO T cells. CD28 ligation costimulated B7KO PerC T cells yet cosuppressed both CD80KO & CD86KO PerC T cells, generating more IFNγSC in the latter. These data, consistent with the higher affinity of CD80 for CD28, suggest that CD80 might represent a biomarker for tumor progression, particularly when considered in conjunction with the density of Mϕs/myeloid-derived suppressor cells.

CD28: a novel therapeutic target for the treatment of multiple myeloma (66.10)

Abstract Multiple myeloma (MM) is a plasma cell neoplasm which resides in the bone marrow (BM) and is critically dependent upon the BM microenvironment (BMM) for its survival. It has been observed that CD28 is expressed on MM cells and that expression levels correlate with worsening progression and worse prognosis. We have also observed that ligation of CD28 protects MM from chemotherapy. Taken together, these data imply that CD28 is a critical pro-survival molecule for the MM. The ligands for CD28, CD80 and CD86, are expressed on antigen-presenting cells, particularly dendritic cells (DC). We have observed that DCs infiltrate myelomatous portions of patient BM biopsies. We therefore hypothesize that it is the DC in the BMM which is providing the pro-survival signal to the MM via a CD28:CD80/86 interaction. Co-culture of murine or human DC with MM increased MM survival in melphalan. We then treated MM/DC co-cultures with CTLA4-Ig (which blocks CD80/86) and cultured them with or without melphalan. In the presence of melphalan, DC alone increased the survival of MM cells from 30% to 75%; however, addition of CTLA4-Ig completely abrogated this protection. We observed similar results using a CD28 blocking antibody rather than CTLA4-Ig. Mechanistically, such pro-survival signals often involve regulation of apoptosis. We have preliminary data which suggest that CD28 ligation decreases the levels of the pro-apoptotic molecule Bim, suggesting a mechanism for CD28-mediated survival.

Transcriptional profile of CD28 costimulation and Dec1 relevance in T-cell activation (113.14)

Abstract Antigen recognition by the T cell receptor and costimulatory signal through CD28 receptor are required for a complete T-cell activation. CD28 ligation regulates a wide range of biochemical processes that drive T-cell proliferation, prevent apoptosis and enhance IL-2 production. However, the direct effect of CD28 signaling on the global gene transcription after T-cell activation remains controversial. We described the transcriptional profile of CD28 costimulation employing microarray analysis of human naïve CD4+ T cells. Although we demonstrated that CD28-dependent signaling had quantitative effects on gene expression during the first stages of activation, strong qualitative changes on transcription were observed by 24 hours after activation. Among the genes directly induced by CD28 costimulation we identified the transcription factor Dec1. We described the critical role of Dec1 for the transcriptional response to CD28 costimulation in both Jurkat T cells and primary mouse CD4+ T cells. Importantly, we showed that the effects of Dec1 deficiency impact specifically on CD28 costimulation-dependent gene expression. Further, we proved that Dec1 is necessary to induce experimental autoimmune encephalitis in mice after MOG peptide immunization. This observation validates the in vivo importance of Dec1 for immune system function in a CD28-dependent disease.

The death domain kinase RIP1 links the immunoregulatory CD40 receptor to apoptotic signaling in carcinomas

CD40, a tumor necrosis factor (TNF) receptor family member, is widely recognized for its prominent role in the antitumor immune response. The immunostimulatory effects of CD40 ligation on malignant cells can be switched to apoptosis upon disruption of survival signals transduced by the binding of the adaptor protein TRAF6 to CD40. Apoptosis induction requires a TRAF2-interacting CD40 motif but is initiated within a cytosolic death-inducing signaling complex after mobilization of receptor-bound TRAF2 to the cytoplasm. We demonstrate that receptor-interacting protein 1 (RIP1) is an integral component of this complex and is required for CD40 ligand-induced caspase-8 activation and tumor cell killing. Degradation of the RIP1 K63 ubiquitin ligases cIAP1/2 amplifies the CD40-mediated cytotoxic effect, whereas inhibition of CYLD, a RIP1 K63 deubiquitinating enzyme, reduces it. This two-step mechanism of apoptosis induction expands our appreciation of commonalities in apoptosis regulatory pathways across the TNF receptor superfamily and provides a telling example of how TNF family receptors usurp alternative programs to fulfill distinct cellular functions.

Enforced hematopoietic cell E- and L-selectin ligand (HCELL) expression primes transendothelial migration of human mesenchymal stem cells

According to the multistep model of cell migration, chemokine receptor engagement (step 2) triggers conversion of rolling interactions (step 1) into firm adhesion (step 3), yielding transendothelial migration. We recently reported that glycosyltransferase-programmed stereosubstitution (GPS) of CD44 on human mesenchymal stem cells (hMSCs) creates the E-selectin ligand HCELL (hematopoietic cell E-selectin/L-selectin ligand) and, despite absence of CXCR4, systemically administered HCELL(+)hMSCs display robust osteotropism visualized by intravital microscopy. Here we performed studies to define the molecular effectors of this process. We observed that engagement of hMSC HCELL with E-selectin triggers VLA-4 adhesiveness, resulting in shear-resistant adhesion to ligand VCAM-1. This VLA-4 activation is mediated via a Rac1/Rap1 GTPase signaling pathway, resulting in transendothelial migration on stimulated human umbilical vein endothelial cells without chemokine input. These findings indicate that hMSCs coordinately integrate CD44 ligation and integrin activation, circumventing chemokine-mediated signaling, yielding a step 2-bypass pathway of the canonical multistep paradigm of cell migration.

Ligation of CD40 receptor in human B lymphocytes triggers the 5-lipoxygenase pathway to produce reactive oxygen species and activate p38 MAPK

Previously, we reported that CD40-induced production of reactive oxygen species (ROS) by NADPH oxidase requires the TNF receptor-associated factor (TRAF) 3, as well as the activities of phosphatidylinositol 3-kinase (PI3K) and Rac1. Here we investigated the possible mechanisms of the production of ROS after CD40 ligation in B cells. We describe an alternative ROS production pathway that is triggered by CD40 ligation, involves 5-lipoxygenase (5-LO), and results in activation of p38 MAPK. Our studies in Raji human B lymphomas revealed that CD40-induced ROS production by 5-LO also requires the activities of PI3K and Rac1. In contrast to the NADPH oxidase pathway, however, TRAF molecules are not required for the CD40-induced ROS production by 5-LO. The association of CD40 with 5-LO is dependent on CD40 ligation in Raji B cells, and co-immunoprecipitation experiments using epitope- tagged proteins transiently expressed in human embryonic kidney 293T cells revealed the role of the regulatory subunit of PI3K, p85, in this association. Collectively, these data suggest a separate pathway for the CD40-induced ROS production in B cells and demonstrate that this pathway requires 5-LO via direct association of p85 with both CD40 and 5-LO.

The influence of CD40 ligation and interferon-γ on functional properties of human monocyte-derived dendritic cells activated with polyinosinic-polycytidylic acid

Ligation of a Toll-like receptor (TLR) by specific TLR agonists is a powerful tool for maturation induction of monocyte-derived dendritic cells (MoDCs). Studies so far have shown that the treatment of dendritic cells (DCs) with a TLR3 ligand, polyinosinic-polycytidylicacid [Poly(I:C)], may be an appropriate activation agent for obtaining mature MoDCs, competent to prime effective immune responses. However, little is known about how subsequent interaction of MoDCs with T cell-derived stimuli, such as CD40 or interferon-gamma (IFN-gamma), modulates MoDC functions. Therefore, this problem was the main objective of this study. Immature MoDCs were prepared by cultivation of monocytes from peripheral blood mononuclear cells with granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin (IL)-4 for 5 days. After that, maturation was induced by the treatment of these cells with Poly(I:C) for 2 days. At day 6, immature MoDCs and Poly(I:C)-activated MoDCs were incubated either with CD40 ligand (L)-transfected J558 cells or IFN-gamma for additional 24 hours. Cytokine production was measured by ELISA and FlowCytomix Human T helper Th1/Th2 11plex. Allostimulatory capability of MoDCs was tested using an allogeneic mixed leukocyte reaction (MLR) assay. Immature MoDCs showed a moderate potential for stimulation of proliferation of CD4+ T cells, which was enhanced by the treatment with Poly(I:C). Ligation of CD40 or treatment with IFN-gamma of immature or Poly(I:C)-treated MoDCs significantly up-regulated their allostimulatory activity. MoDCs matured in the presence of Poly(I:C) up-regulated the production of IL-12 and IL-10, which was followed by increased levels of IFN-gamma and decreased levels of IL-5 in co-cultures with allogeneic CD4+ T cells. Ligation of CD40 on immature MoDCs upregulated the production of IL-12 and IL-23 which was accompanied by increased secretion of IFN-gamma in co-culture. Stimulation of CD40 on Poly(I:C)-treated MoDCs significantly enhanced the production of IL-12, IL-23 and IL-10. However, such treated MoDCs decreased the production of IFN-gamma and IL-10 and up-regulated the secretion of IL-17. Immature MoDCs treated with IFN-gamma up-regulated IL-12, but lowered the production of IL-5 and IL-17 by CD4+ T cells. Treatment of Poly(I:C)-activated MoDCs with IFN-gamma down-regulated the production of IL-12 and up-regulated IL-10 by these cells and increased/decreased the levels of IL-10/ IFN-gamma, respectively, in co-culture with CD4+ T cells. Treatment with Poly(I:C) or ligation of CD40 on immature MoDCs induces maturation of these cells into a phenotype that supports Th1 response. Activation of CD40 on Poly(I:C)-treated MoDCs shifts the immune response towards Th17. Treatment of immature MoDCs with IFN-gamma down-regulated Th2 and Th17 responses. However, addition of IFN-gamma to Poly(I:C)-activated MoDCs down-regulated Th1 response and promote T regulatory mechanisms. Each of these results may have functional and therapeutic implications.

CD4 ligation excludes the Carma1–Bcl10–MALT1 complex from GM1-positive membrane rafts in CD3/CD28 activated T cells

The antibody 13B8.2, which is directed against the CDR3-like loop on the D1 domain of CD4, induces CD4/ZAP-70 reorganization and ceramide release in membrane rafts. Here, we investigated whether CD4/ZAP-70 compartmentalization could be mediated by an effect of 13B8.2 on the Carma1–Bcl10–MALT1 complex in membrane rafts. We report that treatment of CD3/CD28-activated Jurkat T cells with 13B8.2, but not rituximab, excluded Carma1–Bcl10–MALT1 proteins from GM1 + membrane rafts and concomitantly decreased NF-κB activation. Fluorescence confocal imaging confirmed that Carma1–Bcl10 and Carma1-MALT1 co-patching, observed in GM1 + membrane rafts following CD3/CD28 activation, were abrogated after a 24 h-treatment with 13B8.2. The CD4/ZAP-70 compartmentalization in membrane rafts induced by 13B8.2 is thus related to Carma1–Bcl10–MALT1 raft exclusion.