Abstract
BackgroundHuman myeloid-derived suppressor cells (MDSC) have been described as a group of immature myeloid cells which exert immunosuppressive action by inhibiting function of T lymphocytes. While there is a huge scientific interest to study these cells in multiple human diseases, the methodological approach varies substantially between published studies. This is problematic as human MDSC seem to be a sensible cell type concerning not only cryopreservation but also time point after blood draw. To date data on delayed blood processing influencing cell numbers and phenotype is missing. We therefore evaluated the kinetics of granulocytic MDSC (gMDSC) and monocytic MDSC (mMDSC) frequencies after blood draw in order to determine the best time point for analysis of this recently defined cell type.MethodsIn this study, we isolated peripheral blood mononuclear cells (PBMC) of patients with HIV infection or solid tumors directly after blood draw. We then analyzed the frequencies of gMDSC and mMDSC 2, 4 and 6 h after blood draw and after an overnight rest by FACS analysis using the standard phenotypic markers. In addition, part of the cells was frozen directly after PBMC preparation and was measured after thawing.ResultsgMDSC levels showed no significant difference using fresh PBMC over time with a limitation for the overnight sample. However they were massively diminished after freezing (p = 0.0001 for all subjects). In contrast, frequencies of fresh mMDSC varied over time with no difference between time point 2 and 4 h but a significantly reduction after 6 h and overnight rest (p = 0.0005 and p = 0.005 respectively). Freezing of PBMC decreased the yield of mMDSC reaching statistical significance (p = 0.04). For both MDSC subgroups, FACS analysis became more difficult over time due to less sharp divisions between populations.ConclusionsAccording to our data human MDSC need to be studied on fresh PBMC. gMDSC can be studied with delay, mMDSC however should be studied no later than 4 h after blood draw. These results are crucial as an increasing number of clinical trials aim at analyzing MDSC nowadays and the logistics of blood processing implies delayed sample processing in some cases.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-015-0755-y) contains supplementary material, which is available to authorized users.
Highlights
Human myeloid-derived suppressor cells (MDSC) have been described as a group of immature myeloid cells which exert immunosuppressive action by inhibiting function of T lymphocytes
In studies in infectious diseases (HIV, HCV), granulocytic MDSC (gMDSC) were defined to lack the expression of CD14 but to express CD15/ CD33/CD11b [8, 10] whereas monocytic MDSC (mMDSC) were characterized as CD14+/CD11b+/HLA-DR−/low cells [8, 10, 11] or CD33+ cells [12, 13]
In order to determine the best time point for MDSC analysis after blood draw, we first studied the kinetics of gMDSC frequencies
Summary
Human myeloid-derived suppressor cells (MDSC) have been described as a group of immature myeloid cells which exert immunosuppressive action by inhibiting function of T lymphocytes. While there is a huge scientific interest to study these cells in multiple human diseases, the methodological approach varies substantially between published studies This is problematic as human MDSC seem to be a sensible cell type concerning cryopreservation and time point after blood draw. Grützner et al J Transl Med (2016) 14:2 approach For this reason, translational studies on human MDSC are difficult to compare especially regarding two criteria: phenotypic markers and time point of analysis after blood draw. In studies in infectious diseases (HIV, HCV), gMDSC were defined to lack the expression of CD14 but to express CD15/ CD33/CD11b [8, 10] whereas mMDSC were characterized as CD14+/CD11b+/HLA-DR−/low cells [8, 10, 11] or CD33+ cells [12, 13]. To date the most established markers for gMDSC are CD14−, CD33+, CD66b+/CD15+ and CD11b+ and for mMDSC CD33+, CD14+ and HLA-DR−/low
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