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Keywords:

  • DV1;
  • DV2;
  • CD1d-PBS57 multimers;
  • CD34

Purpose and Appropriate Sample Types

  1. Top of page
  2. Purpose and Appropriate Sample Types
  3. Background
  4. Similarity to Published OMIPs
  5. Literature Cited
  6. Supporting Information

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

  1. Top of page
  2. Purpose and Appropriate Sample Types
  3. Background
  4. Similarity to Published OMIPs
  5. Literature Cited
  6. Supporting Information

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

  1. Top of page
  2. Purpose and Appropriate Sample Types
  3. Background
  4. Similarity to Published OMIPs
  5. Literature Cited
  6. Supporting Information

None to date.

image

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.

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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

Literature Cited

  1. Top of page
  2. Purpose and Appropriate Sample Types
  3. Background
  4. Similarity to Published OMIPs
  5. Literature Cited
  6. Supporting Information

Supporting Information

  1. Top of page
  2. Purpose and Appropriate Sample Types
  3. Background
  4. Similarity to Published OMIPs
  5. Literature Cited
  6. Supporting Information

Additional and updated supporting information including technical details may be found in the online version of this article.

FilenameFormatSizeDescription
cytoa22326-sup-0001-suppfig1A.tiff641KOnline Fig.1 Reagent titrations. Reagents were titrated on healthy donor PBMC by performing 1:2 serial dilutions. (A) Individual. fcs files were concatenated in order to visualize all titrations in a single pseudo-colour plot, where increasing Ab-concentrations are arranged along the x-axis. Titers used in OMIP-019 are highlighted in red. (B) To titrate TCR-DV1FITC and (C) TCR-DV2Ax594 for the detection of γδT-cell subsets, PBMC were co-labeled with TCR-GV9APC. Titers were selected to separate the positive population from negative cells, and maintain background staining at a minimum. (D) Specificity of CD1d-PBS57PE multimers was confirmed by co-staining with TCR-AV24FITC, TCR-BV11APC, and CD45RABV421. Unloaded CD1d multimers did not stain TCR-BV11+ cells. The TCR-AV24FITC vs. CD45RABV421 dot plot in the bottom right shows CD1d multimer+ TCR-BV11+ (green) overlaid onto classical T-cells (grey-black). Samples were gated on live T-cells (B-D).
cytoa22326-sup-0002-suppfig1BCD.tiff477KOnline Fig.1 Reagent titrations. Reagents were titrated on healthy donor PBMC by performing 1:2 serial dilutions. (A) Individual. fcs files were concatenated in order to visualize all titrations in a single pseudo-colour plot, where increasing Ab-concentrations are arranged along the x-axis. Titers used in OMIP-019 are highlighted in red. (B) To titrate TCR-DV1FITC and (C) TCR-DV2Ax594 for the detection of γδT-cell subsets, PBMC were co-labeled with TCR-GV9APC. Titers were selected to separate the positive population from negative cells, and maintain background staining at a minimum. (D) Specificity of CD1d-PBS57PE multimers was confirmed by co-staining with TCR-AV24FITC, TCR-BV11APC, and CD45RABV421. Unloaded CD1d multimers did not stain TCR-BV11+ cells. The TCR-AV24FITC vs. CD45RABV421 dot plot in the bottom right shows CD1d multimer+ TCR-BV11+ (green) overlaid onto classical T-cells (grey-black). Samples were gated on live T-cells (B-D).
cytoa22326-sup-0003-suppfig2.tiff26370KOnline Fig.2 Sample illustrations for choice of reagents. (A) Comparison of CD4QD605 and CD4BV605. (B) Performance of TCR-GV9PE and TCR-GV9APC were compared. Both reagents readily identify TCR-GV9hi cells, but the latter is better at also separating out the TCR-VG9dim cells. (C) Comparison of PE-Cy5.5-, PacBlu- and BV421-conjugates of anti-CD34 Ab. While it is not clear whether the PE-Cy5.5-conjugate stains haematopoietic stem cells (red broken-lined gate), the PacBlu-conjugate clearly identifies this population (red gate) and hints at a CD34dim population (green broken-lined gate), while the BV421-conjugate clearly identifies both these populations (red and green gates). (D) Staining intensity of CCR5PE-Cy7 and CCR5APC-Cy7. Dot plots show total live cells (A, C) or live T-cells (B, D) from healthy donor PBMC.
cytoa22326-sup-0004-suppfig3.tiff316KOnline Fig.3 Effect of incubation temperature on a subset of reagents used in OMIP-019. Staining performance comparison of CD1d-PBS57PE multimers, CCR5APC-Cy7, TCR-GV9APC, TCR-DV1FITC (A), TCR-DV2Ax594 (B), and CD34BV421 (C) after incubation at either room temperature or 37°C. Graphics show total live cells (A, C) or live TCR-DV9+ T-cells (B) from healthy donor PBMC.
cytoa22326-sup-0005-suppinfo.doc23KSupporting Information
cytoa22326-sup-0006-suppinfo.doc144K

Online Table 1 Instrument configuration.

Online Table 2 Commercial reagents used in OMIP-019.

Online Table 3 In-house conjugated reagents used in OMIP-019.

Online Table 4 Priority rating for reagents.

Online Table 5 Reagents tested but not included in final panel.

Please note: Wiley Blackwell 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.