Surface-bound bovine serum albumin carrier protein as present in recombinant cytokine preparations amplifies T helper 17 cell polarization.

Lei Dong,Hermann Haller,Alexandra Helmke,Andreas Pich,Ari Waisman,Sibylle Von Vietinghoff

doi:10.1038/srep36598

Abstract

Understanding of T helper 17 lineage (TH17) polarization has been significantly promoted by cell culture experiments that reduce the complexity of the in vivo environment. We here investigated TH17 amplification by coating of cytokine preparations. Cytokine preparations coated to the surface compared to the same amount given in solution significantly enhanced TH17 polarization assessed by flow cytometry and interleukin (IL)-17A, IL-17F and RORγt mRNA expression. T cell proliferation and TH1 polarization were similarly enhanced while TREG polarization was impeded. TH17 amplification was replicated by coating the plate with low amounts of FCS or albumin as used as carrier protein for cytokines (0.5 μl 0.1%). It was unaltered by filtration, protein digestion and arylhydrocarbon receptor blockade, not replicated by LPS and independent of integrin stimulation. TH17 amplification required anti-CD3 stimulation and was T cell intrinsic. Supernatants of CD4+ cells polarized on coated cytokine preparations with carrier albumin conferred amplification to fresh splenocytes. Coating markedly elevated CD4+ IL-22 mRNA expression and IL-22 blockade significantly reduced TH17 amplification. Our data show TH17 amplification by coated albumin in the low amounts present in recombinant cytokine preparations. This unexpected adjuvant like effect underscores the need for controls also for temporal and spatial factors in cell culture.

Highlights

Adjuvants in vivo, some of which directly stimulate T cells[25]
Given the effect of fractalkine receptor CX3CR1 on T cell polarization demonstrated by others[37] and impeded TH17 polarization in specific gene deficient cells found by us[39], we investigated the effect of recombinant fractalkine on TH17 polarization in vitro
To study the role of T cell receptor stimulation in the TH17 amplification caused by cytokine preparations, we investigated the protocol of anti-CD3 and anti-CD28 stimulation

Summary

Introduction

Beyond enhancing antigen specific response, some adjuvants favor distinct TH lineages, for example, alum induces an innate response that promotes TH2 polarization[26,27]. Fractalkine, the unique ligand of CX3CR1, is a stalked cytokine that exists in soluble and surface bound form in vivo and modulates immune cell migration and function[32]. Fractalkine effects in vitro have largely been studied using surface-bound recombinant cytokine[33,34,35]. We recently demonstrated its expression on both TH17 and TREG cells and induction by TGFβduring lymphocyte culture[39]. This led us to investigate the effect of coated and soluble recombinant fractalkine in TH17 cell polarization. To define appropriate controls for further TH17 polarization experiments, where specific gene deficient controls might not be available, we here explored the underlying mechanism

Methods

Results

Conclusion