OMIP-025: evaluation of human T- and NK-cell responses including memory and follicular helper phenotype by intracellular cytokine staining

Abstract

This panel was developed to assess antigen-specific T cells using peptide pools to various antigens of interest, although other types of antigens such as recombinant proteins or whole pathogens could be considered using different stimulation times. In addition to multiple functional markers, the panel includes differentiation markers and markers to assess follicular helper T cells and NK cells (Table 1). It was optimized using cryopreserved peripheral blood mononuclear cells (PBMC) from human immunodeficiency virus (HIV) uninfected and HIV infected adults with known cytomegalovirus (CMV) responses and it underwent assay qualification. The panel is being used to evaluate the responses to HIV and malaria vaccine candidates in adults and children from different geographic areas.

Figure 1. Example of the staining and gating strategy for PBMC stimulated with staphylococcal enterotoxin B (SEB)

All gates for non-functional markers were defined using fluorescence minus one (FMO) controls whereas gates for functional markers were defined using the unstimulated samples. (A) Gating hierarchy to identify NK cells, NKT-like cells, CD4+ and CD8+ T cells and T_FH-like cells. Initial gating is done on FSC-H and FSC-A to discriminate singlets, followed by the exclusion of events collected during a period of time early in collection when fluctuations may occur. In this example there were no problems of fluctuations and the time gate was minimized to avoid exclusion of any events. Dead cells and monocytes are excluded by an amine reactive dye and the CD14 marker in the same dump channel. Lymphocytes are gated using FSC-A and SSC-A. Subsequent gating discriminates two subsets of NK cells by CD56 and CD3 expression (CD56dimCD3− and CD56hiCD3−) and NKT-like cells (CD56+CD3+). Within the gate of lymphocytes, CD3+ cells are identified, followed by identification of CD4+ and CD8+ T cells. Of note, NKT-like cells are not excluded from classical T cells and therefore are overlapping populations. Finally, T_FH-like cells are identified as CXCR5+ CD45RA- CD4+ T cells that have a low expression of CCR7 and are PD-1+. (B) Functional markers for CD4+ and CD8+ T cells. A gate is applied for each cytokine, not taking into account the coexpression of other markers. Boolean gates are then created based on these gates to identify cells expressing different combinations of markers. (C) The expression level of CCR7 and CD45RA is examined within CD4+ and CD8+ T-cell subsets to later provide insight into the memory phenotype of the antigen specific cells.