CD4 and CD8 T-cell subset frequencies determined by three-color flow cytometry. To determine the CD4 and CD8 T-cell frequencies, the staining combination of CD3, CD4, and CD8 was used. The 5% probability plots shown on the left have first been gated on lymphocytes as determined by forward and side scatter, and then they have been gated on CD3 to identify T cells. In addition to CD4 and CD8 single positive T cells, CD4⁻CD8⁻, and CD4⁺CD8⁺ T cells can also be distinguished. In a few individuals treated for HD, we can identify two unusual T-cell subsets that express dull levels of CD8 and are either CD4⁺ or CD4⁻. The naive and memory CD4 and CD8 T-cell frequencies were determined by the staining combinations of CD62L, CD45RA, and either CD4 or CD8. Shown on the right are contour plots first scatter gated on lymphocytes and then gated on either CD4⁺ or CD8^bright cells. Naive cells and three memory subsets (M1, M2, and M3) can be distinguished by this method. Shown are examples for a healthy control and two patients who were treated for HD more than 15 years before analysis. The naive T-cell depletion noted in these two individuals is typical of that seen in the posttreatment HD subjects.