OMIP-017: Human CD4⁺Helper T-cell Subsets Including Follicular Helper Cells

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. 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 1) 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 IFN-γ(1,2), while CCR4⁺ CCR6⁻ CXCR3⁻ Th₂cells produce 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 M. tuberculosis infection (4). CCR4⁺ CCR6⁺CCR10⁻CXCR3⁻Th₁₇cells are crucial for the host defense against extracellular pathogens, and mainly produce IL-17A, IL-22 and 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 are 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.

purpose	CD4+ helper T-cell subsets (Th1, Th2, Th9, Th17, Th17Th1, Th22 and TFH)
species	Human
cell types	PBMC
cross-references	n.a.

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 Fig. 1. First, live CD4⁺ T-cells are identified (Fig. 1A), before gating on T_FH (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.

Fig. 1.

Example staining and gating. A Identification of CD4⁺ T-cell. After selecting live CD3⁺ single cells, eventual dye aggregates are excluded (grey box), followed by identification of lymphocytes and CD4⁺CD8⁻ cells for further analysis as shown in B, C and D. B Identification of CXCR5⁺T_FH within CD4⁺ T-cells. The phenotype of T_FH is investigated by overlaying these cells (blue) onto total CD4⁺ T-cells (grey). 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.

Table 2.

Reagents used for OMIP-017.

Specificity	Clone	Fluorochrome	Purpose
CD3	UCHT1	Ax594	lineage
CD4	OKT4	QD800
CD8	RPA-T8	QD585
CXCR5	RF8B2	Ax647	TFH
CCR4	TG6/CCR4	PE-Cy7	Th subsets
CCR6	G034E3	BV605
CCR10	6588-5	PE
CXCR3	1C6/CXCR3	PE-Cy5
CCR7	150503	Ax680	differentiation
CD45RA	5H9	QD655
CD161	DX12	FITC	exploratory
PD-1	EH12.2H7	BV421
Dead cells	--	AqBlu	dump

Ax – Alexa; QD – quantum dot; PE – R-phycoerythrin; Cy – cyanine; BV – brilliant violet; FITC – fluorescein; AqBlu – LIVE/DEAD Fixable Aqua Dead Cell Stain.