Human neutrophils can mimic myeloid-derived suppressor cells (PMN-MDSC) and suppress microbead or lectin-induced T cell proliferation through artefactual mechanisms

Dmitri Negorev,Tianyi Zhang,Evgeniy Eruslanov,Wayne W Hancock,Joshua M Diamond,Falk W Lohoff,Matthew H Levine,Jon G Quatromoni,Sunil Singhal,Ulf H Beier,Pratik Bhojnagarwala,Steven M Albelda,Jason D Christie,Tatiana Akimova

doi:10.1038/s41598-018-21450-6

Abstract

We report that human conventional CD15+ neutrophils can be isolated in the peripheral blood mononuclear cell (PBMC) layer during Ficoll gradient separation, and that they can impair T cell proliferation in vitro without concomitant neutrophil activation and killing. This effect was observed in a total of 92 patients with organ transplants, lung cancer or anxiety/depression, and in 18 healthy donors. Although such features are typically associated in the literature with the presence of certain myeloid-derived suppressor cell (PMN-MDSC) populations, we found that commercial centrifuge tubes that contained membranes or gels for PBMC isolation led to up to 70% PBMC contamination by CD15+ neutrophils, with subsequent suppressive effects in certain cellular assays. In particular, the suppressive activity of human MDSC should not be evaluated using lectin or microbead stimulation, whereas assays involving soluble or plate-bound antibodies or MLR are unaffected. We conclude that CD15+ neutrophil contamination, and associated effects on suppressor assays, can lead to significant artefacts in studies of human PMN-MDSC.

Highlights

Characterization of immune cells with suppressive properties is of major interest in transplantation[1,2], autoimmunity[3,4] and tumor immunology[5,6,7,8]
As polymorphonuclear neutrophil (PMN)-myeloid-derived suppressor cells (MDSC) were a promising candidate cell type for the observed effects, and MDSC are commonly associated with malignancies[6,7,8,17,18,19,20], we evaluated the number of CD15+ cells in blood and tumor samples from lung cancer patients
We demonstrate some important and surprising features of normal human neutrophils, including that they may be isolated within the peripheral blood mononuclear cell (PBMC) layer, and that they can suppress T cell proliferation induced using monoclonal antibody (mAb)-coated bead or lectin stimulation

Summary

Introduction

Characterization of immune cells with suppressive properties is of major interest in transplantation[1,2], autoimmunity[3,4] and tumor immunology[5,6,7,8]. The use of anti-CD3 or anti-CD3/28 monoclonal antibody (mAb)-coated microbeads were developed and has gained in popularity, given its greater physiologic relevance All such forms of T cell stimulation are perceived by researchers as tools to study corresponding biological processes in T cells, with little or no attention paid to the effects of such stimulation on non-T cells. We describe unexpected and previously unnoticed effects of lectins on conventional neutrophils, and novel interactions of neutrophils with microbeads Both features lead to remarkable but artefactual suppression of T cell division, a result that could be erroneously interpreted as reflecting suppression by PMN-MDSC. The goal of this paper is to inform researchers studying human MDSC that CD15+ neutrophils may co-localize with cellular fractions expected to be enriched for MDSC and mediate artefactual suppression

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