At least two suggestions for overcoming downmodulation of CD4 included gating for CD3+CD8-: >3. Try staining for CD3+, CD8- cells. Yeah, I don't like it much either, but >it works and has been used in a number of papers. >... stains with CD3 and CD8, then gates the CD3+, CD8- events and assumes >these >to be the cells which would express CD4 (has they not been overstimulated). Having the advantage of 10-color FACS to explore all of the different T cell subsets simultaneously, I can assert definitively that this method can lead to significant artefact. CD3+CD8- T cells are comprised of at least two distinct subsets: CD4+ and CD4- T cells. In healthy adults, the ratio of these two subsets is around 6:1. In HIV-infected adults, especially advanced stages, this ratio can be much lower, for example 1:1. Therefore, the CD3+CD8- gate can be substantially contaminated with "double-negative" (DN) T cells. This is a significant problem, because DN T cells have a very different functional profile from CD4 T cells (and somewhat different from CD8 T cells). A large fraction of these cells make g-IFN, few make IL2, and none make IL4. Therefore, a CD3+CD8- gate, taken to be CD4 T cells, will significant OVER-estimate the gIFN production by CD4 T cells, and somewhat underestimate the IL2 or IL4 production. (Sorry, we haven't done other cytokines as yet). Again, in healthy adults, this is not such a major problem, although it can change the apparent CD4 gIFN production by 25-50% too high. In adults with low CD4 counts, however, the gIFN production by CD3+CD8- T cells is due principally to DN T cells, NOT to CD4 T cells. (Note that DN T cells are not the only contaminants to the CD3+CD8- subset. Depending on the CD8 antibody being used, you may also end up including the CD8-dull T cells. These cells are not that frequent in healthy adults, but, in many situations such as BMT, chemotherapy, and HIV (among others) this population can become quite prevalent. These cells also have a very high proportion of gIFN producers, and very low IL2 and nonexistent IL4 production.) mr
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