Idoia Gimferrer

Relevance of CD6-mediated interactions in T cell activation and proliferation.

CD6 is a cell surface receptor expressed on immature thymocytes and mature T and B1a lymphocytes. The ultimate function of CD6 has not been deciphered yet, but much evidence supports a role for CD6 in T cell activation and differentiation. In this study, we show that a fraction of CD6 molecules physically associates with the TCR/CD3 complex by coimmunoprecipitation, cocapping, and fluorescence resonance energy transfer experiments. Image analysis of Ag-specific T-APC conjugates demonstrated that CD6 and its ligand, activated leukocyte cell adhesion molecule (CD166), colocalize with TCR/CD3 at the center of the immunological synapse, the so-called central supramolecular activation cluster. The addition of a soluble rCD6 form significantly reduced the number of mature Ag-specific T-APC conjugates, indicating that CD6 mediates early cell-cell interactions needed for immunological synapse maturation to proceed. This was in agreement with the dose-dependent inhibition of CD3-mediated T cell proliferation induced by soluble rCD6. Taken together, our data illustrate the important role played by the intra- and intercellular molecular interactions mediated by CD6 during T cell activation and proliferation processes.

CD6 binds to pathogen-associated molecular patterns and protects from LPS-induced septic shock

CD6 is a lymphocyte receptor that belongs to the scavenger receptor cysteine-rich superfamily. Because some members of the scavenger receptor cysteine-rich superfamily act as pattern recognition receptors for microbial components, we studied whether CD6 shares this function. We produced a recombinant form of the ectodomain of CD6 (rsCD6), which was indistinguishable (in apparent molecular mass, antibody reactivity, and cell binding properties) from a circulating form of CD6 affinity-purified from human serum. rsCD6 bound to and aggregated several Gram-positive and -negative bacterial strains through the recognition of lipoteichoic acid and LPS, respectively. The Kd of the LPS–rsCD6 interaction was 2.69 ± 0.32 × 10−8 M, which is similar to that reported for the LPS–CD14 interaction. Further experiments showed that membrane CD6 also retains the LPS-binding ability, and it results in activation of the MAPK signaling cascade. In vivo experiments demonstrated that i.p. administration of rsCD6 before lethal LPS challenge significantly improved mice survival, and this was concomitant with reduced serum levels of the proinflammatory cytokines TNF-α, IL6, and IL-1β. In conclusion, our results illustrate the unprecedented bacterial binding properties of rsCD6 and support its therapeutic potential for the intervention of septic shock syndrome or other inflammatory diseases of infectious origin.

The influence of innate immunity gene receptors polymorphisms in renal transplant infections.

Background. Genetically defined deficiencies in key components of the innate immune system have been associated with a greater risk of infection. The aim of this study was to assess the influence of genetic variability of innate immune receptors (mannose-binding lectin [MBL], mannose-associated serine-protease-2 [MASP-2], and Toll-like receptors [TLR4]) in the risk of infections after a kidney transplantation. Methods. All patients undergoing a kidney or kidney–pancreas transplantation during a 3-year period were included. Functionally relevant mutations in MBL2, MASP2, and TLR4 genes were determined by DNA sequencing. The incidence of major bacterial infections, asymptomatic cytomegalovirus (CMV) infection, and CMV disease were compared among groups. Results. There were no differences regarding major transplant characteristics among groups. Older age, requirements for posttransplant hemodialysis, and pretransplant diabetes, but not gene polymorphisms, were associated with a greater number of bacterial infections. In univariate analysis, low-MBL genotypes were associated with CMV disease in pretransplant CMV seropositive patients (P=0.015), whereas the TLR4 mutation was associated with higher risk of CMV primary infection (P=0.024). TLR4 mutation was an independent factor associated with CMV disease (odds ratio 5.84, 95% confidence interval 1.35−25.20, P=0.018). Conclusion. Polymorphisms of innate immunity receptors, especially TLR4 mutation, were associated with higher risk of CMV disease, while susceptibility to other infectious disorders was not observed.

The Lymphocyte Receptor CD6 Interacts with Syntenin-1, a Scaffolding Protein Containing PDZ Domains

CD6 is a type I membrane glycoprotein expressed on thymocytes, mature T and B1a lymphocytes, and CNS cells. CD6 binds to activated leukocyte cell adhesion molecule (CD166), and is considered as a costimulatory molecule involved in lymphocyte activation and thymocyte development. Accordingly, CD6 partially associates with the TCR/CD3 complex and colocalizes with it at the center of the mature immunological synapse (IS) on T lymphocytes. However, the signaling pathway used by CD6 is still mostly unknown. The yeast two-hybrid system has allowed us the identification of syntenin-1 as an interacting protein with the cytoplasmic tail of CD6. Syntenin-1 is a PDZ (postsynaptic density protein-95, postsynaptic discs large, and zona occludens-1) domain-containing protein, which functions as an adaptor protein able to bind cytoskeletal proteins and signal transduction effectors. Mutational analyses showed that certain amino acids of the most C-terminal sequence of CD6 (-YDDISAA) and the two postsynaptic density protein-95, postsynaptic discs large, and zona occludens-1 domains of syntenin-1 are relevant to the interaction. Further confirmation of the CD6-syntenin-1 interaction was obtained from pull-down and coimmunoprecipitation assays in mammalian cells. Image analyses also showed that syntenin-1 accumulates at CD6 caps and at the IS. Therefore, we propose that syntenin-1 may function as a scaffolding protein coupling CD6 and most likely other lymphocyte receptors to cytoskeleton and/or signaling effectors during IS maturation.

Mitogen-activated protein kinase pathway activation by the CD6 lymphocyte surface receptor.

CD6 is a cell surface receptor primarily expressed on immature thymocytes and mature T and B1a lymphocytes. Through its binding to activated leukocyte cell adhesion molecule (ALCAM/CD166), CD6 is considered to play an important role in lymphocyte development and activation. Accordingly, CD6 associates with the TCR/CD3 complex and colocalizes with it at the center of the mature immunological synapse on T lymphocytes. Moreover, the CD6-ALCAM interaction has been shown to be critical for proper immunological synapse maturation and T cell proliferative responses. However, the precise biological effects of CD6 ligation and its signaling pathway are still not well understood. The present study shows that CD6 ligation with three different specific mAbs (161.8, SPV-L14.2, and MAE1-C10) induces time- and dose-dependent activation of ERK1/2 on normal and leukemic human T cells. This effect was also observed upon CD6 ligation with a chimerical ALCAM protein (ALCAM-Fc). The C-terminal cytoplasmic region of CD6, as well as Src tyrosine kinases, was critical for CD6-induced ERK1/2 activation. Synergistic effects were observed upon coligation of the TCR/CD3 complex with CD6. The ligation of CD6 induced the transcriptional activation of reporter genes under the control of the c-Fos serum responsive element and AP-1. Accordingly, CD6-mediated activation of p38 and JNK was also observed. These findings indicate that the CD6-ALCAM interaction results in activation of the three MAPK cascades, likely influencing the dynamic balance that determines whether resting or activated lymphocytes survive or undergo apoptosis.

Residues Y429 and Y463 of the human CD5 are targeted by protein tyrosine kinases

The human CD5 lymphocyte cell surface co‐receptor modulates activation and differentiation responses mediated by the antigen‐specific receptor of T and B cells. CD5 is phosphorylated followinglymphocyte activation; however, the exact sites and kinases involved are yet to be determined. Jurkat T cell transfectants expressing tyrosine‐mutated CD5 molecules have been used to show that residues Y429 and Y463 are targeted in vivo by protein tyrosine kinases following cell stimulation with anti‐CD3 mAb or pervanadate. This is in agreement with data from direct in vitrokinase assays using purified recombinant Lck and Fyn protein tyrosine kinases. The analysis of Lck‐ and CD3‐deficient Jurkat cells shows that tyrosine phosphorylation of CD5 requires Lck activity. We propose that T cell activation mediates CD5 tyrosine phosphorylation at residues Y429 and Y463 mainly through the activation of Lck.

Role of Two Conserved Cytoplasmic Threonine Residues (T410 and T412) in CD5 Signaling

CD5 is a transmembrane coreceptor that modulates activation and differentiation signals mediated by the Ag-specific receptor present on both T and B1a lymphocytes. CD5 lacks intrinsic catalytic activity, and its immunomodulatory properties result from intracellular interactions mediated by the CD5 cytoplasmic tail. The nature of these interactions is currently a matter of investigation. Here, we present a selective mutagenesis analysis of two conserved threonine residues (T410 and T412) located at the membrane-proximal cytoplasmic region of CD5. These residues are contained within consensus phosphorylation motifs for protein kinase C and are shown here to be critical for in vivo protein kinase C-mediated phosphorylation of CD5. Functional studies revealed that the integrity of T410 and T412 is also critical for CD5-mediated phosphatidylcholine-specific phospholipase C (PC-PLC) activation and phorbol ester-mediated inhibition of Ab-induced internalization of CD5. These results strongly argue in favor of a role for T410 and T412 in the signaling mediated by CD5.

Regulation of GATA-3 Expression during CD4 Lineage Differentiation

GATA-3 is necessary for the development of MHC class II-restricted CD4 T cells, and its expression is increased during positive selection of these cells. TCR signals drive this upregulation, but the signaling pathways that control this process are not well understood. Using genetic and pharmacological approaches, we show that GATA-3 upregulation during thymocyte-positive selection is the result of additive inputs from the Ras/MAPK and calcineurin pathways. This upregulation requires the presence of the transcription factor c-Myb. Furthermore, we show that TH-POK can also upregulate GATA-3 in double-positive thymocytes, suggesting the existence of a positive feedback loop that contributes to lock in the initial commitment to the CD4 lineage during differentiation.