WNT7a as a new feature of the T mature cells; expression of the WNT7a diminish in a highly activated and proliferative T cells after TCR activation and IL2 stimulus while canonical targets of WNT signaling pathway are overexpress.

Abstract

Event Abstract Back to Event WNT7a as a new feature of the T mature cells; expression of the WNT7a diminish in a highly activated and proliferative T cells after TCR activation and IL2 stimulus while canonical targets of WNT signaling pathway are overexpress. MONSERRAT ALVAREZ-ZAVALA1, ADRIANA AGUILAR-LEMARROY1 and LUIS F. JAVE-SUAREZ1* 1 Centro de Investigación Biomédica de Occidente, Immunology, Mexico WNT ligands are essential in the hematopoietic process, some of them are express in different maturation stages of the hematopoietic cells [1, 2]; WNT7a expression has been detected in single positive T cells in thymus[3]. Previous study of our research group reported that WNT7a expresses in PBMCs, particularly in the T cell fraction also we have shown that WNT7a is a non-canonical ligand [4]. However, the biological role of WNT7a in mature T-cells has not been explored, in mature T-cells it has been reported that canonical targets are express and previously produce, in fact the canonical WNT pathway is trigger after the TCR activation through the inhibition of GSK3β by AKT, however is unknown the status of the non canonical pathways in the T activated cells [5]. The main objective of this research was to investigate whether WNT7a expression is directly modulated by the TCR signaling activation and if the activation by TCR modulates the WNT pathways. We hypothesized that the presence of WNT7a in mature circulating T cells induces cell cycle arrest, and subsequently a non-proliferative status. Moreover, we thought that the control exerted by WNT7a can be avoided by T cell activation process, and that given that WNT7a is a non canonical ligand it needs to be sub expresses to allow the activation of the WNT canonical pathway in T activated cells. To corroborate this hypothesis, we used PBMCs from healthy individuals, first lymphocytes were separated from monocytes. The lymphocyte fraction was harvested and the activation assay was performed using two stimulus, one consist in a set of antibodies directed against CD3, CD28 and CD2 loaded to a bead particle and the other in a superparamagnetic bead loaded with CD3 and CD28, both kind of beads allow the interaction of the antibodies with the receptors in a clustering fashion. RNA isolation was performed on lymphocytes at basal conditions and after 24, 48, 72 hours of TCR activation; additionally at 48 hours of TCR activation plus 24 and 96 hours in the presence of IL2 (20UI ). WNT pathway gene targets were measure after 48 hours of TCR activation by qPCR. First we tested whether T cells were correctly activated. To achieve this objective, we analyzed by qPCR a panel of genes that are overexpress during the activation process such as CD25, CD69, CD71, CD95 and CD40L. Once activation was verified, we pursue to measure the expression of WNT7a. We observed that TCR activation by both combinations of antibodies (CD28/CD3/CD2 and CD28/CD3) induced a profound lowering expression of WNT7a. It was also evident that the stimuli with CD3/CD28/CD2 regulates more strongly the expression of WNT7a, meaning that the regulation depends on the structure and complexity of the TCR clustering in the immunological synapse. Meanwhile down modulation of WNT7a continued in a time dependent manner, we didn’t observed a synergic effect of the IL2 on the modulation of WNT7a. This results indicate that the down-modulation of the WNT7a expression is an obligated mechanism after T cells activation, but also that this down-modulation is necessary in order to achieve the clonal expansion promoted by IL2. We also measure several target genes of the canonical pathway -TCF4, c-MYC, c-JUN, MMP7, CCND1- that were overexpressed after TCR activation, it is well known that the WNT canonical pathway is associated with proliferation, so it might be helping to achieve the T cell clonal proliferation. The target genes of the Calcium pathway MMP9 and CAPN2 present no major changes after activation and PCP target genes CCN2 and CELSR1 were down modulated after the TCR activation. This confirms that WNT canonical pathway is triggered after the TCR activation, since WNT7a is a non canonical ligand the signaling that triggers might antagonized the WNT canonical pathway and this might affect the proliferation rates, in this sense WNT7a modulation might be an early event in the T cell activation, but also an obligated event to allow proliferation. Our results postulated this ligand as a regulator that keeps T cells in a non-proliferative state and that WNT7a might represent a feature of the T mature resting cells.