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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. Acknowledgements
  6. LITERATURE CITED
  7. Supporting Information

This panel was developed to determine the phenotype of human antigen (Ag)-specific CD8+ T-cells. Ag-specificities are identified by four peptide-major histocompatibility complex (MHC) class I (pMHCI) multimers (e.g., against Epstein-Barr virus (EBV) and cytomegalovirus (CMV) epitopes). Six markers of T-cell phenotype are used. This panel has been tested on fresh and cryopreserved peripheral blood mononuclear cells (PBMC), as well as bone marrow samples; staining may be performed in 96-well plates to increase throughput.1, 21

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Figure 1. Staining patterns in PBMC. A: Overall gating strategy. Successive gates are applied to identify single cells, live CD3+ T-cells, and lymphocytes. Gates are also applied to all fluorochromes to remove fluorochrome aggregates (e.g., as shown for PD1 and CD45RO). Subsequent panels are gated on these cells. B: Identification of Ag-specific CD8+ T-cells. Multimer staining is depicted within CD3+ T-cells for various HLA-A2 epitopes of CMV and EBV (see Supporting Information). C: Phenotyping markers in bulk CD8+ T-cells. D: Phenotype of multimer+ CD8+ T-cells (NLV, red; YLL, blue; CLG, green; and FLY, orange). Although CCR7 expression is dim, multimer+ CCR7+ cells can be roughly distinguished from CCR7− cells. By measuring CD27, identification of central memory multimer+ cells is refined.

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Table 1. Summary table for OMIP-002
PurposeSpecificity and phenotype of CD8 + T-cells
SpeciesHuman
Cell typesFresh or cryopreserved PBMC, bone marrow mononuclear cells
Cross referencesNone
Table 2. Reagents used in OMIP-002
SPECIFICITYCLONEFLUOROCHROMEPURPOSE
  1. APC, allophycocyanin; Cy, cyanin; QD, quantum dot; PE, R-phycoerythrin; Ax, alexa; Bi, biotin; SAV, streptavidin; FITC, fluorescein isothiocyanate; PacBlu, pacific blue; ViViD, LIVE/DEAD fixable violet dead cell stain.

CD3SK7APC-Cy7T-cell subset
CD4MT-477QD705 
CD8RPA-T8PE-Ax594 
Multimer 1PEAg-specificity
Multimer 2QD565 
Multimer 3QD605 
Multimer 4QD800 
CD45ROUCHL1APC-Ax700Maturity
CCR7150503PE-Ax750 
CD271A4PE-Cy5 
CD127R34.34PE-Ax700 
PD1MIH4Bi 
BiSAVAPC 
CD57NK-1FITC 
CD14M5E2PacBluExclusion
CD19HIB19PacBlu 
Dead cellsViViD 

BACKGROUND

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

Analysis of Ag-specific T-cells can be difficult because: (A) frequencies are low, (B) phenotypes are diverse, and (C) sample is typically limited. To overcome these issues, this panel assesses multiple Ag-specificities and phenotypes (1). To maximize sensitivity, some multimers are produced using quantum dots. These are bright and detected in channels receiving minimal contaminating light from other dyes. R-phycoerythrin and allophycocyanin are used for an additional multimer and a low-density Ag, respectively. Various CD27, CD127, CCR7, and CD45RO conjugates were tested; the brightest were selected. CCR7 and multimer staining were performed at 37°C (2, 3).

Various strategies are used to ensure accurate identification of Ag-specific cells. First, sources of nonspecific binding (e.g., dead cells, B-cells, and monocytes) are identified in one channel for exclusion from analysis. Second, when the same cells bind multimers against different Ag-specific cell populations, the multimers are discarded and remade. Third, preliminary experiments are repeated until nonspecific binding of the multimer to CD4+ T-cells is no longer observed, thus ensuring the quality of the investigator's staining technique (3).

Acknowledgements

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

The authors thank Emma Gostick for production ofpMHCI multimers and Junichi Kawada for assistance with early troubleshooting and panel development experiments.

LITERATURE CITED

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

Supporting Information

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

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

FilenameFormatSizeDescription
CYTO_20945_sm_suppinfo.doc5203KSupporting Information

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