PKC-\u0473 is dispensable for OX40L-induced TCR-independent Treg proliferation but contributes by enabling IL-2 production from effector T-cells

Khaled Alharshawi,Palash Bhattacharya,Zuoming Sun,Alan L Epstein,Ajay V Maker,Bellur S Prabhakar,Osama El-Sayed,Prabhakaran Kumar,Alejandra Marinelarena

doi:10.1038/s41598-017-05254-8

Abstract

We have previously shown that OX40L/OX40 interaction is critical for TCR-independent selective proliferation of Foxp3+ Tregs, but not Foxp3− effector T-cells (Teff), when CD4+ T-cells are co-cultured with GM-CSF derived bone marrow dendritic cells (G-BMDCs). Events downstream of OX40L/OX40 interaction in Tregs responsible for this novel mechanism are not understood. Earlier, OX40L/OX40 interaction has been shown to stimulate CD4+ T-cells through the formation of a signalosome involving TRAF2/PKC-Ѳ leading to NF-kB activation. In this study, using CD4+ T-cells from WT and OX40−/− mice we first established that OX40 mediated activation of NF-kB was critical for this Treg proliferation. Although CD4+ T-cells from PKC-Ѳ−/− mice were also defective in G-BMDC induced Treg proliferation ex vivo, this defect could be readily corrected by adding exogenous IL-2 to the co-cultures. Furthermore, by treating WT, OX40−/−, and PKC-Ѳ−/− mice with soluble OX40L we established that OX40L/OX40 interaction was required and sufficient to induce Treg proliferation in vivo independent of PKC-Ѳ status. Although PKC-Ѳ is dispensable for TCR-independent Treg proliferation per se, it is essential for optimum IL-2 production by Teff cells. Finally, our findings suggest that OX40L binding to OX40 likely results in recruitment of TRAF1 for downstream signalling.

Highlights

CD4+ T-cell proliferation occurs predominantly through T-cell receptor (TCR) dependent activation and associated co-stimulation[1]
We investigated whether the underlying mechanism of TCR-independent proliferation of Tregs observed in ex vivo GM-CSF derived bone marrow dendritic cells (G-bone marrow derived dendritic cells (BMDCs))/T-cell co-cultures involved OX40-dependent activation of NF-kB through formation of TRAF2/Protein Kinases C (PKC)-Ѳ signalosome
OX40 ligand (OX40L)/OX40 interaction is indispensable for G-BMDC-induced ex vivo expansion of Tregs

Summary

Introduction

CD4+ T-cell proliferation occurs predominantly through T-cell receptor (TCR) dependent activation and associated co-stimulation[1]. We have earlier shown that bone marrow derived dendritic cells (BMDCs) generated ex vivo using GM-CSF (G-BMDCs) can selectively cause proliferation of Foxp3+ Tregs when co-cultured with total CD4+ T-cells[13] This proliferation was found to be TCR-independent, but critically dependent on OX40 ligand (OX40L) expression on G-BMDCs; and required IL-2 production in those co-cultures by Teff[13]. Apart from effector T-cell activation, OX40L/OX40 interaction has been shown to influence adaptive Treg generation and proliferation[21], and thymic Treg differentiation[22] These findings have been attributed to OX40L/OX40 mediated co-stimulation in the presence of primary activation signal delivered upon TCR ligation to MHC presented antigens. Proliferation of PKC-Ѳ−/− Tregs upon supplementation with exogenous IL-2 suggested that expression of PKC-Ѳ per se was dispensable for TCR-independent Treg proliferation

Methods

Results

Conclusion