OMIP‐017: Human CD4+ helper T‐cell subsets including follicular helper cells

Abstract

This panel was optimized to measure the relative frequencies of CD4+ T-helper cell subsets and follicular helper T-cells in peripheral blood mononuclear cells (PBMC) from healthy individuals (Table 1). It works well with cryopreserved PBMC and we have observed similar result with fresh specimens. Other tissue types have not been tested. Activated CD4+ T-cells differentiate into lineages, commonly referred to as T-helper (Th) subtypes such as Th2, with unique phenotypes, cytokine signatures, and functions. The present panel (Table 2) was designed to identify major Th subsets described to date according to disparate chemokine receptor expression patterns. Immunity to intracellular infections and viruses requires CCR4− CCR6− CCR10− CXCR3+ Th1 cells that produce interferon-γ (IFN-γ) (1, 2), while CCR4+ CCR6− CXCR3− Th2 cells produce interleukin-4 (IL-4), IL-5, and IL-13 and mediate the host defense against helminthes (3). Th9 cells are CCR4− CCR6+ IL-9-producing cells that could be involved in wound healing of pleural mesothelial cells during Mycobacterium tuberculosis infection (4). CCR4+ CCR6+ CCR10− CXCR3− Th17 cells are crucial for the host defense against extracellular pathogens, and mainly produce IL-17A, IL-22, and granulocyte-macrophage colony-stimulating factor (GM-CSF) (2, 5). Finally, CCR4+ CCR6+ CCR10+ Th22 cells produce IL-22, IL-26, and IL-13 and are thought to be involved in skin immunity (2, 6). However, there is plasticity in the system and some of these phenotypes and functional characteristics, previously thought mutually exclusive, can be expressed by the same cell. One such example is Th17Th1 cells that are CCR4− CCR6+ CXCR3+ and capable of producing both IFN-γ and IL-17 (5). The affinity maturation of plasma cells in the germinal centers of B-cell follicles requires the help from another specialized CD4+ T-cell lineage called follicular helper cells, or TFH (7). These cells express CXCR5, which confers homing to follicular dendritic cell networks within the germinal centers. The gating scheme for evaluating all these subsets with the present panel is illustrated in Figure 1. First, live CD4+ T-cells are identified (Fig. 1A), before gating on TFH (Fig. 1B) or other Th lineage cells (Fig. 1C). Additional non-lineage defining markers, namely CCR7, CD45RA, CD161, and PD-1, were included in order to further characterize the phenotype of individual subsets. Example staining and gating. A: Identification of CD4+ T-cell. After selecting live CD3+ single cells, eventual dye aggregates are excluded (gray box), followed by identification of lymphocytes and CD4+CD8− cells for further analysis as shown in (B, C, D). B: Identification of CXCR5+ TFH within CD4+ T-cells. The phenotype of TFH is investigated by overlaying these cells (blue) onto total CD4+ T-cells (gray). C,D: Enumeration of CD4+ helper T-cell subsets according to differential expression of the chemokine receptors CCR4, CCR6, CCR10, and CXCR3; gates indicate putative subtypes as defined in the literature (see discussion). None to date. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.