No lack of regulatory B cells in patients with Multiple Sclerosis

International audienc

The Involvement of SMILE/TMTC3 in Endoplasmic Reticulum Stress Response

The state of operational tolerance has been detected sporadically in some renal transplanted patients that stopped immunosuppressive drugs, demonstrating that allograft tolerance might exist in humans. Several years ago, a study by Brouard et al. identified a molecular signature of several genes that were significantly differentially expressed in the blood of such patients compared with patients with other clinical situations. The aim of the present study is to analyze the role of one of these molecules over-expressed in the blood of operationally tolerant patients, SMILE or TMTC3, a protein whose function is still unknown.We first confirmed that SMILE mRNA is differentially expressed in the blood of operationally tolerant patients with drug-free long term graft function compared to stable and rejecting patients. Using a yeast two-hybrid approach and a colocalization study by confocal microscopy we furthermore report an interaction of SMILE with PDIA3, a molecule resident in the endoplasmic reticulum (ER). In accordance with this observation, SMILE silencing in HeLa cells correlated with the modulation of several transcripts involved in proteolysis and a decrease in proteasome activity. Finally, SMILE silencing increased HeLa cell sensitivity to the proteasome inhibitor Bortezomib, a drug that induces ER stress via protein overload, and increased transcript expression of a stress response protein, XBP-1, in HeLa cells and keratinocytes.In this study we showed that SMILE is involved in the endoplasmic reticulum stress response, by modulating proteasome activity and XBP-1 transcript expression. This function of SMILE may influence immune cell behavior in the context of transplantation, and the analysis of endoplasmic reticulum stress in transplantation may reveal new pathways of regulation in long-term graft acceptance thereby increasing our understanding of tolerance

Differential CD4-dependent inhibition of JNK but not Erk-2 activities in human naive and memory CD4+ T cell populations.

CD4 ligand binding to the CD4 molecules has been shown to inhibit T cell proliferation and IL-2 transcription and synthesis. We have recently shown that this inhibition correlated with a CD4-mediated inhibition of the kinase Erk-2 and c-Jun-N-terminal kinases (JNK) which play a key role in IL-2 transcription. Moreover, we have previously reported that antigen-independent adhesion of CD45RObright/CD4+ T cells to B cells is negatively regulated by CD4 ligands, whereas that of CD45RAbright/CD4+ naive T cells is not. Other groups have described, in murine models, a differential sensitivity of memory and naive T cells to CD4-mediated inhibitory effects on T cell activation. The aim of the present report was to study the sensitivity of the naive and memory CD4+ T cell populations to the CD4-mediated inhibition of Erk-2 and JNK activation. Our data show that preincubation with anti-CD4 mAb, of the CD45RAbright/CD4+ naive and the CD45RObright/CD4+ memory human T cell populations, induces inhibition of both Erk-2 phosphorylation and Erk-2 activation by phorbol ester or anti-CD3 mAb. In contrast, CD3 mediated JNK activation was inhibited in the memory but not in the naive CD4+ T cell population, whereas JNK activation by phorbol ester or phorbol esters plus Ca2+ ionophore was inhibited by anti-CD4 mAb in both T cell populations. These data further demonstrate a differential sensitivity of naive and memory CD4+ T cell populations to the CD4-mediated negative signaling

gp160 of HIV or anti-CD4 monoclonal antibody ligation of CD4 induces inhibition of JNK and ERK-2 activities in human peripheral CD4+ T lymphocytes.

Under physiological conditions, activation of CD4+ T cells by major histocompatibility complex (MHC)antigen complexes requires engagement of both the T cell receptor and the CD4 molecule. However, CD4 ligands binding to the CD4 molecule has also been shown to inhibit T cell proliferation and interleukin (IL)-2 production in human CD4+ T cells, in an MHC-independent way. We have previously shown that this inhibition was associated with a diminished binding activity of the IL-2 transcription factors NF-AT, NF-kappaB, and AP-1. AP-1 plays a key role in the regulation of IL-2 transcription, and ERK and JNK activities are necessary for regulating AP-1 at both the transcriptional and the post-transcriptional levels. We therefore studied, in human peripheral CD4+ T cells, the regulation of the activities of extracellular signal-regulated protein kinases (ERK) and c-Jun N-terminal kinases (JNK) by two CD4 ligands, gp160 the envelope glycoprotein of human immunodeficiency virus (HIV) and an anti-CD4 monoclonal antibody (mAb). Pre-incubation of CD4+ T lymphocytes in the presence of anti-CD4 mAb or gp160 inhibits the activation of JNK in response to phorbol 12-myristate 13-acetate and ionomycin. In the same conditions, phosphorylation and activation of ERK-2 were also inhibited. Inhibition of both JNK and ERK-2 activities are specific for binding of CD4 ligands to the CD4 molecule. They were not observed in CD8+ T lymphocytes. These results suggest that a specific inhibition of JNK and ERK-2 activities contributes to defective IL-2 production in T lymphocytes pre-incubated with CD4 ligands

Sam68 association with p120GAP in CD4+ T cells is dependent on CD4 molecule expression.

p120 GTPase-activating protein (p120GAP) is a major negative regulator of p21ras activity in several cell types including T cells. Catalytic activity of this enzyme is regulated in part by its interaction with several associated tyrosine-phosphorylated proteins. Sam68 was initially described as associated with p120GAP. It has been further established that Sam68 is a substrate of src kinases in mitosis and that it is not associated with p120GAP in transformed fibroblasts. We describe herein that Sam68 associates with p120GAP and PLC gamma 1 in human mature T cells and in a T cell line expressing the CD4 molecule HUT78 CD4+. This association is present in nonactivated cells and increases after anti-CD3 activation. It is dependent on CD4 expression and, in part, on the association of CD4 with p56lck, as shown by the strongly decreased association of Sam68 with p120GAP in the CD4- mutants, HUT78 CD4-, and by the reduced association of Sam68 with both p120GAP and p56lck in the HUT78 T cell line expressing a CD4 mutant unable to interact with p56lck, HUT78 C420/22. We propose that recruitment of Sam68, via CD4/p56lck, to the inner face of the plasma membrane may permit, via its docking properties, the correct association of key signaling molecules including PLC gamma 1 and p120GAP. This formation of transduction modules will enable the activation of different signaling cascades including the p21ras pathway and an array of downstream events, ultimately leading to T cell activation

The DESCARTES-Nantes survey of kidney transplant recipients displaying clinical operational tolerance identifies 35 new tolerant patients and 34 almost tolerant patients

Kidney recipients maintaining a prolonged allograft survival in the absence of immunosuppressive drugs and without evidence of rejection are supposed to be exceptional. The ERA-EDTA-DESCARTES working group together with Nantes University launched a European-wide survey to identify new patients, describe them and estimate their frequency for the first time

Ligands of CD4 inhibit the association of phospholipase Cgamma1 with phosphoinositide 3 kinase in T cells: regulation of this association by the phosphoinositide 3 kinase activity.

We have previously shown that CD4 ligands inhibit interleukin-2 (IL-2) production and T cell proliferation in human peripheral CD4+ T lymphocytes, in an MHC-independent way. Two major pathways implicated in T cell activation are inhibited by binding of CD4 ligands to the CD4 molecule, i.e. Ca2+ signaling by phospholipase Cgamma1 (PLCgamma1), and ERK-2 activation, suggesting a p21ras inhibition. We have correlated these inhibitions with the disruption of multifunctional complexes containing PLCgamma1, p120GAP and Sam68, induced by T cell activation. We report here that T cell activation through the TCR/CD3 induces an association of the phosphoinositide 3 kinase (PI3 kinase) with PLCgamma1, both in peripheral CD4+ T lymphocytes and the HUT-78 CD4+ T cell line. PI3 kinase is present in the multifunctional complexes that we have described previously. Preincubation of human peripheral CD4+ T cells and HUT-78 CD4+ T cells with gp160 or a peptide analogue of the HLA class II DR molecule precludes the association of PLCgamma1 with PI3 kinase. We also demonstrate, using two specific inhibitors of PI3 kinase activity (LY294002 and wortmannin), that this activity plays a key role in the association of PLCgamma1 with PI3 kinase. Moreover, we demonstrate the implication of the PI3 kinase activity in the negative signal mediated by HIV gp160 binding to CD4 molecules. We propose that the products of the PI3 kinase are important mediators of the negative signaling induced by the binding of CD4 ligands to the CD4 molecule implicated in the regulation of the formation of multifunctional complexes

Sialylation of antibodies in kidney recipients with de novo donor specific antibody, with or without antibody mediated rejection

International audienceBACKGROUND: DSA are associated with reduced long-term transplant function and increased prevalence of chronic rejection in some patients, whereas others do not: our goal was to determine whether the sialylation of IgG and DSA could help to explain in these last cases their "non-aggressive" and/or "protective" biological activity.METHODS: The sialylation level of total IgG in blood from two groups of kidney-transplant patients with de novo DSA, one with an AMR (DSA+AMR+), and the other without were studied.RESULTS: In the DSA+AMR- patients total IgG were more sialylated at time of transplant, and at the first detection of DSA, class I DSA were 2.6-fold more sialylated (mean 9.943±1.801 versus 3.898±2.475, p=0.058); DSA+AMR+ patients exhibited higher levels of class II DSA.CONCLUSIONS: In our study, higher levels of sialylated IgG are detectable on day of transplant in patients who do not develop AMR, they have higher sialylated class I DSA at the initial detection of DSA, whereas class II DSA are significantly higher in patients who develop AMR. This is the first report suggesting that transplant outcome, and particularly AMR, is associated with levels of sialylated IgG antibodies. Our data suggest that DSA are functionally heterogeneous and that further studies with an enlarged cohort may improve our understanding of their clinical impact

Upregulation of miR-142-3p in Peripheral Blood Mononuclear Cells of Operationally Tolerant Patients with a Renal Transplant

Achieving drug-free tolerance or successfully using only small doses of immunosuppression is a major goal in organ transplantation. To investigate the potential mechanisms by which some kidney transplant recipients can achieve operational tolerance, we compared the expression profiles of microRNA in peripheral blood mononuclear cells of operationally tolerant patients with those of stable patients treated with conventional immunosuppression. B cells from operationally tolerant patients overexpressed miR-142-3p. The expression of miR-142-3p was stable over time and was not modulated by immunosuppression. In Raji B cells, overexpression of miR-142-3p modulated nearly 1000 genes related to the immune response of B cells, including potential miR-142-3p targets and molecules previously identified in the blood of operationally tolerant patients. Furthermore, our results suggested that a negative feedback loop involving TGF-β signaling and miR-142-3p expression in B cells may contribute to the maintenance of tolerance. In summary, miR-142-3p expression in peripheral blood mononuclear cells correlates with operational tolerance. Whether upregulation of miR-142-3p modulates inflammatory responses to promote tolerance or is a result of this tolerance state requires further study