PURPOSE AND APPROPRIATE SAMPLE TYPES
The present panel was optimized to investigate the differentiation status of CD4+ and CD8+ T-cells in peripheral blood mononuclear cells (PBMC) fromhealthy individuals. It works well with cryopreserved PBMC and we have observed similar results with fresh specimens. Other tissue types have not been tested.
Knowledge obtained during the optimization of reagent panels developed for previous studies was used as a starting point for the present panel. Some of these precursor panels have been published (1–3). Starting out with a backbone of CD3APC-H7, CD4QD605, CD8QD585, CD28PE-Cy5, CD57QD705, and CCR7Ax680, as well as AqBlu to gate out dead cells, the aim was to include more cell surface markers commonly used to investigate differentiation stages of CD4+ and CD8+ T-cells. In addition to being able to distinguish naïve (TNV), central memory (TCM), transitional memory (TTM), effector memory (TEM), and terminal effector cells (TTE), we also wanted to identify CD4+ recent thymic emigrants (RTE), as well as the recently characterized memory stem cell subset (TSCM), a long-lived memory T-cell population that exhibits enhanced capacity for self-renewal and the ability to differentiate into TCM, TEM, and TTE (4).
CD45RA, CCR7, and CD27 are sufficient for the identification of TCM, TTM, TEM, and TTE, so after AqBlu for the exclusion of dead cells, and CD3, CD4, and CD8 for the gating of CD4+ and CD8+ T-cells, respectively, these markers received highest priority for inclusion in the panel.
Next, CD95 was included to obtain a purer TNV population, and together with CD28, CD57, and CD127 allow the identification of TSCM. A stringent gating tree is employed to identify this latter population. TSCM express high levels of the IL-7Rα chain, CD127, which is important for homeostatic proliferation (5), but not the senescence marker CD57 (6). They are thus phenotypically very similar to TNV, the most prominent difference being the expression of the Fas receptor CD95 (4).
Finally, CD31, also known as PECAM-1, was chosen to distinguish RTE within the CD4+ TNV subset, as CD31+ CD45RA+ CCR7+ cells have been shown to carry more T-cell receptor excision circles (TREC) than CD31− CD45RA+ CCR7+ cells (7).
As an additional marker we included CD244, better known as 2B4, in order to investigate its expression on individual T-cell subsets.
In order to verify the performance of each new reagent in the panel, as well as its potential effects on the detection of unrelated markers, add-in experiments were performed as previously described (8). To this end, a series of PBMC samples were incubated with subsets of the final reagent panel, with each subsequent tube containing the same reagents as the previous one, as well as one additional Ab. This process quickly identifies reagents that are not performing satisfactorily, as well as those that cause interference with unrelated detectors, thus compromising the detection of other reagents. Different reagent combinations were tested in order to identify a panel that provides the best detection (combination of reagent brightness, lack of background, and lack of spillover from other detectors) of all cell surface markers.