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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 for the evaluation of CD4+ and CD8+ T-cell responses to various HIV-1–derived peptide pools in peripheral blood mononuclear cells (PBMC) from HIV-1+ individuals with differences in clinical progression. It works well with cryopreserved PBMC, and we have observed similar results with fresh specimens. Other tissue types have not been tested.1, 21

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Figure 1. Example staining and gating. A: Identification of T-cell subsets. After selecting live CD3+CD14CD19 single cells, eventual dye aggregates are excluded (gray box) and a lymphocyte gate set. CD4+ and CD8+ T-cells are then selected for further analysis. B: Selection of cytokine-producing cells after gating as shown in (A). CD4+ and CD8+ T-cells positive for either IFN-γ, TNF-α, or IL-2 are separately gated. Besides analyzing the cytokine pattern (combination of cytokines produced on a per cell basis) produced in response to antigenic stimulation, a Boolean gate encompassing all cytokine positive cells (cyt+) is created to evaluate the total Ag-specific response. C: Phenotypic analysis of Ag-specific cells gated as described in (B). Total CD4+ and CD8+ T-cells (in gray) are used as a reference when analyzing the cell surface phenotype of cyt+ cells (in red). Shown are cryopreserved PBMC from an HIV-1+ subject stimulated with an HIV Gag peptide pool.

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Table 1. Summary table for application of OMIP-001
PurposeT-cell cytokine production after in vitro stimulation and phenotyping of cytokine-producing T-cells
SpeciesHuman
Cell typesPBMC
Cross referencesn.a.
Table 2. Reagents used for OMIP-001
SPECIFICITYCLONEFLUOROCHROMEPURPOSE
  1. APC, allophycocyanin; Ax, Alexa; Cy, cyanine; QD, quantum dot; PE, R-phycoerythrin; Bi, biotin; PacBlu, pacific blue; ViViD, LIVE/DEAD fixable violet dead cell stain.

IFN-γB27APCFunction
IL-2MQ1-17H12Ax488
TNF-αMAb11Ax594
CD3SK7APC-Cy7Lineage
CD4M-T477QD605
CD8RPA-T8QD585
CCR7150503Ax680Memory/ differentiation
CD27M-T271PE-Cy7
CD28CD28.2PE-Cy5
CD45ROUCHL1QD545
CD57NK-1QD705
CD127R34.34PE
PD-1MIH4Bi
BiotinStreptavidinQD655 
CD14M5E2PacBluDump
CD19HIB19PacBlu
Dead cellsViViD

BACKGROUND

  1. Top of page
  2. PURPOSE AND APPROPRIATE SAMPLE TYPES
  3. BACKGROUND
  4. SIMILARITY TO PUBLISHED OMIPs
  5. LITERATURE CITED
  6. Supporting Information

The approach used for the development of this panel has been described in detail (1). Briefly, a large number of Ab-conjugates were screened for each antigen of interest, as available, to select those Ab-conjugates providing best detection. As the focus of the panel was the detection of cytokine-producing T-cells, the brightest fluorochromes were used for interleukin-2 (IL-2), interferon (IFN)-γ, and tumor necrosis factor (TNF)-α. Next, priority was given to PD-1 and CCR7, as these antigens are expressed at low molecular densities. After selecting a dump channel to exclude dead cells, B-cells and monocytes/macrophages from the analysis, a range of Ab-conjugates for other markers used for T-cell subset definition and determination of activation status were tested in the free detectors until optimal detection of all antigens was achieved. To this end, CD4-QD655, which was included in early panels, was replaced with CD4-QD605 to improve the detection threshold of CD28-PE-Cy5 on CD4+ T-cells. PD-1, which is labeled with QD655 in the final panel, does not influence the detection threshold of PD-1+ CD28+ cells in the same way. This is because PD-1 has a lower expression level (and thus a lower measured mean fluorescence intensity) than CD4, thereby causing less spillover into other detectors.

CCR7 was labeled at 37°C (2), while CD3 was labeled after fixing and permeabilizing the cells, so as not to inadvertently exclude any relevant cells that might have internalized their T-cell receptor/CD3 complexes after activation (3). Fluorescently conjugated CD28 Ab was added to the stimulation cultures, thus serving as a costimulator during the cultures while at the same time, labeling CD28 molecules.

The total number of cells acquired determines the detection sensitivity of cytokine-producing cells. Thus, to reliably quantify cytokine responses, higher number of cells should be acquired as the frequency of responding cells decreases.

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

Technical details may be found in Supporting Information in the online version of this article.

FilenameFormatSizeDescription
CYTO_20944_sm_suppinfo.pdf1097KSupporting Information

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