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

Authors


Purpose and Appropriate Sample Types

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.

Background

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).

Table 1. Summary table for application of OMIP-017
PurposeCD4+ helper T-cell subsets (Th1, Th2, Th9, Th17, Th17Th1, Th22 and TFH)
SpeciesHuman
Cell typesPBMC
Cross-referencesn.a.
Table 2. Reagents used for OMIP-017
SpecificityCloneFluorochromePurpose
  1. Ax, Alexa; QD, quantum dot; PE, R-phycoerythrin; Cy, cyanine; BV, brilliant violet; FITC, fluorescein; AqBlu, LIVE/DEAD Fixable Aqua Dead Cell Stain.

CD3UCHT1Ax594Lineage
CD4OKT4QD800 
CD8RPA-T8QD585 
CXCR5RF8B2Ax647TFH
CCR4TG6/CCR4PE-Cy7Th subsets
CCR6G034E3BV605 
CCR106588-5PE 
CXCR31C6/CXCR3PE-Cy5 
CCR7150503Ax680Differentiation
CD45RA5H9QD655 
CD161DX12FITCExploratory
PD-1EH12.2H7BV421 
Dead cellsAqBluDump

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.

Figure 1.

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).

Similarity to Published OMIPs

None to date.

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