To follow on to this discussion, I decided to provide some data to prove that using only two colors (CD3, CD8) to identify T cell subsets is prone to significant artefact, especially in measuring cytokine profiles. I looked over our database of recent immunophenotyping (over 800 visits, most of which are HIV+), and tabulated the relative proportions of CD3+ T cell subsets, being CD4 (CD4+CD8-), CD8 (CD8+CD4-), DN (CD4-CD8-), or DP (CD4+CD8+). The following are expressed as a percentage of CD3+ T cells [given as "mean (SD)"]: HIV-: CD4 = 63 (10) CD8 = 29 (9) DP = 0.8 (0.5) DN = 5.1 (3.6) HIV+: CD4 = 19 (12) CD8 = 72 (11) DP = 1.3 (1.4) DN = 6.3 (4.2) Therefore, the use of CD3+CD8- to identify CD4 T cells would include 7.4% DN T cells (for HIV-), and 25% (!!!!) DN T cells for HIV+. In other words, among CD3+CD8- T cells in HIV-infected adults, one-fourth are NOT CD4 T cells. Therefore, you CANNOT consider the DN population to be inconsequential. Because of the unique cytokine profile of this subset, it can significantly impact on cytokine measurements. There is another important consideration when using stimulated cells. Especially with PMA + Ionomycin, a lot of cell death can occur (even in 6 hours). Because dead cells tend to be sticky, the dead cells will appear to be DP--and they have very high binding of (certain) anti-cytokine antibodies. Therefore, even gating on CD3+CD8+ to identify T cells can include a substantial contamination of dead cells after stimulation. If you are uninterested in dead cells, then you can start by simply gating out DP cells in your analysis. Or, you can use a live/dead discriminator that works with fixed cells (Ethidium monoazide, EMA is the only one I know of--again, see Mitra et al., Int Immunol 1999 11: 1801-1810 for details). So, DN, DP, and dead cells can all introduce serious artefacts into FACS cytokine measurements. And, no, I'm sorry, but we have not published this data or interpretation. (I'd be happy to put together a brief letter if some kindly editor requests it, however.) mr (PS: Please note: we are not a clinical lab and I do not wish to characterize these values as "reference ranges". These values should be used only as an instructive example).
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