The present panel was optimized to quantify the relative frequencies of γδT-cells, invariant natural killer T-cells (iNKT-cells), and hematopoietic precursors in peripheral blood mononuclear cells (PBMC) from healthy individuals (Table 1). It works well with cryopreserved PBMC and we have observed similar results with fresh specimens. Other tissue types have not been tested.
We developed this panel (Table 2) as part of a large study where we aim to survey the relative proportion of different immune cell subsets, including hematopoietic stem cells (HSC), in human peripheral blood specimens from healthy adults. It addresses HSC, γδT-cells, and iNKT-cells.
HSC are multipotent precursor cells that give rise to all blood cell types, including the myeloid and lymphoid lineages. Though predominantly found in bone marrow and umbilical cord blood, they also occur at reduced frequencies in the blood , and can be identified by their expression of CD34 [1, 2]. In spite of being generally used as a molecular marker of HSCs, the function of CD34 is poorly understood .
While most T-cells express a T-cell receptor (TCR) comprised of an α- and a β-chain, a minority of blood T-cells express the γδTCR. In healthy individuals, the vast majority of these have one of two phenotypes, representing ontologically separate lineages: DV1+ (previously Vδ1) cells are prevalent during fetal and early life, while DV2+ (previously Vδ2) cells usually dominate in adult blood [4, 5]. The latter are usually GV9+ (previously Vγ9), but DV1 associates with a number of different Vγ chains . γδT-cells, in particular GV9/DV2 cells, are thought to act as a bridge between innate and acquired immunity .
iNKT-cells express the AV24/BV11 TCR (previously Vα24/Vβ11) and recognize CD1d-restricted lipid antigens. The classical antigen used to detect these cells is the marine sponge-derived α-galactosylceramide (α-GalCer), though more common environmental Ags have recently been shown to also stimulate iNKT-cells [8, 9]. CD1d molecules loaded with the α-GalCer analogue PBS-57 form more stable multimeric complexes than those loaded with α-GalCer, thus making a good tool to identify iNKT-cells . Three iNKT subsets have been characterized that differ in function, but also in CD4/CD8 expression: cytokine-producing CD4+ CD8− (predominant in fetal and neonatal blood), cytotoxic CD4− CD8−, and the rare IFN-γ-producing CD4− CD8+ iNKT-cells .
Finally, we included Abs to CCR5, CCR7, CD27, CD28, and CD45RA in order to further explore the differentiation phenotypes of both γδT-cells and iNKT-cells (Figure 1).
Similarity to Published OMIPs
None to date.
Table 1. Summary table for application of OMIP-019
γδT-cells, iNKT-cells, haematopoietic precursors
Table 2. Reagents used for OMIP-019
BV, brilliant violet; PBS-57, analogue of α-galactosylceramide; n.a., not applicable; PE, R-phycoerythrin; FITC, fluorescein; Ax, Alexa; APC, allophycocyanin; Cy, cyanine; QD, quantum dot; AqBlu, LIVE/DEAD Fixable Aqua Dead Cell Stain.