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
Table 1. Summary table for OMIP-002
Specificity and phenotype of CD8 + T-cells
Fresh or cryopreserved PBMC, bone marrow mononuclear cells
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).
SIMILARITY TO PUBLISHED OMIPs
None to date.
The authors thank Emma Gostick for production ofpMHCI multimers and Junichi Kawada for assistance with early troubleshooting and panel development experiments.