OMIP-019: Quantification of human γδT-cells, iNKT-cells, and hematopoietic precursors

Authors


  • This article is a US government work and, as such, is in the public domain in the United States of America

Correspondence to: Y. D. Mahnke, Translational and Correlative Studies Laboratory, University of Pennsylvania, Philadelphia, PA 19104, USA. E-mail: Yolanda.Mahnke@uphs.upenn.edu

Purpose and Appropriate Sample Types

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.

Background

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 [1], 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 [3].

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 [6]. γδT-cells, in particular GV9/DV2 cells, are thought to act as a bridge between innate and acquired immunity [7].

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 [10]. 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 [11].

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.

Figure 1.

Example staining and gating. A: Identification of HSC, iNKT-cells, and TCR-GV9+ γδT-cells. After selecting live single lymphocytes (highly auto-fluorescent monocytes appear AqBludim and are excluded from further analyses), eventual dye aggregates are excluded by Boolean gating (gray box) and a lymphocyte gate set. CD34+ cells identify HSC (dark green gate). Within CD3+ cells, CD1d-PBS57 multimer-binding iNKT-cells (red gate) and TCR-GV9+ γδT-cells are then selected for further analysis. Classical T-cells (gray gate) are used to define gates for remaining phenotypic markers, as shown in (B) and (C); the classical T-cells are illustrated in gray-black shades in the overlay graphs to validate gate placement. B: Phenotypic characterization of iNKT-cells. The expression of CD4, CD8, CD27, CD28, CD45RA, CCR5, and CCR7 is investigated on iNKT-cells (red dots) using gates defined according to the corresponding expression on classical T-cells. C: Identification and phenotypic characterization of γδT-cell subsets. Separate GV9+ T-cell subsets were identified due to differential expression of TCR-DV1 and –DV2 (blue, green, and orange dots). The expression of CD4, CD8, CD27, CD28, CD45RA, CCR5, and CCR7 is investigated using gates defined according to the corresponding expression on classical T-cells. D: Exploration of differentiation status of iNKT-cells and γδT-cell subsets. Pie charts illustrate the co-expression pattern of CD27, CD28, CD45RA, CCR5, and CCR7 as defined by Boolean gating. While gray arcs indicate the expression of individual cell surface markers, colored pie slices identify the frequency of subsets expressing varying combinations of these markers; e.g., roughly 50% of iNKT are part of the mustard yellow slice, representing CD27+CD28+CD45RACCR5+CCR7 cells. For the purpose of this OMIP these pies illustrate the relative variety of phenotypes represented within iNKT and different γδT-cell populations.

Table 1. Summary table for application of OMIP-019
PurposeγδT-cells, iNKT-cells, haematopoietic precursors
SpeciesHuman
Cell typesPBMC
Cross-referencesn.a.
Table 2. Reagents used for OMIP-019
SpecificityCloneFluorochromePurpose
  1. 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.

CD3OKT3BV785Lineage
CD1d /PBS-57 multimern.a.PEiNKT
TCR-DV1TS8.2FITCγδT-cells
TCR-DV2B6Ax594 
TCR-GV9B3APC 
CD34HI100BV421Hematopoietic stem cells
CCR52D7/CCR5APC-Cy7Phenotyping
CCR7150503Ax680 
CD4OKT4QD605 
CD8RPA-T8QD585 
CD271A4LDGQD655 
CD28CD28.2PE-Cy5 
CD45RAMEM-56PE-Cy5.5 
Dead cellsAqBluDump

Ancillary