Multiple populations of T lymphocytes are distinguished by the level of CD4 and CD8 coexpression and require individual consideration

Expression of CD4 or CD8 on T cells is pivotal in defining them as T helper or T cytotoxic cells, respectively. Aside from an intermediate stage during T cell maturation in the thymus, CD4 and CD8 molecules were thought to be mutually exclusive on mature T cells. However, accumulating evidence has proven that these two molecules can be coexpressed on mature T cells [1–7]. Populations of CD8 T cells re-expressing CD4 have been designated CD4CD8 or CD4CD8 T cells, and likewise, populations of CD4 T cells re-expressing CD8 have been designated CD4CD8 or CD4CD8 T cells. Much of what we know about CD4 CD8 T cells is based on literature that has concentrated on the CD4CD8 T cell population. These studies collectively established the importance of studying CD4 CD8 T cell populations through demonstrating that the CD4 molecule expressed on CD8 T cells is functional, as indicated by several lines of evidence, including the ability to coprecipitate CD4 with the Scr family protein tyrosine kinase p56lck [1] and the ability of CD4 on CD8 T cells to mediate chemotaxis in response to interleukin-16, a CD4-specific ligand [3]. We have shown that CD4CD8 T cells recognize viral [cytomegalovirus (CMV)] antigen more potently than CD8 T cells, which do not re-express CD4 on their surface (CD4CD8 T cells) [6]. Recent data suggest that CD4CD8 T cells function as CD8 effector cells, as assessed in the severe combined immunodeficient (SCID) model of lymphocytic choriomeningitis virus (LCMV) and allogenic immunity [8, 9] and in the chimpanzee model of hepatitis C virus immunity [7]. In analyzing the role of CD4 on CD8 T cells and the biologic significance of this cell population, it is critical that these cells are defined appropriately and that no contaminating populations are included in the analysis. Based on a limited number of markers shown to be shared by both populations, some investigators have combined the analysis of CD4CD8 and CD4CD8 T cells as one population, defining all double-positive (CD4 CD8 ) T cells [7]. Combining these populations to deduce functional potential is problematic. Our own analyses, when collecting a minimum of 100,000 events per sample for immunostaining and using CD4 antibodies conjugated to phycoerythrin (PE) or allophycocyanin (APC), demonstrate that multiple subpopulations of T cells, defined by the level of their expression of CD4 and CD8, exist within the CD4 CD8 double-positive T cell population. As seen in Figure 1A, based on the intensity of CD4 and CD8 expression, six different populations are apparent: the singlepositive populations include CD8 CD4 (denoted as P1) and CD4 CD8 (denoted as P6), and the double-positive populations include CD4CD8 (denoted as P2), CD4CD8 (denoted as P3), CD4 CD8 (denoted as P4), and CD4CD8 (denoted as P5). Given that NKT cells can be CD4 CD8 , CD4CD8, CD4CD8 , or CD4 CD8, it is possible that CD4 CD8 NKT cells may be present within the subpopulations of CD4 CD8 T cells to varying degrees. Therefore, we examined the frequency of NKT cells within the CD4 CD8 T cell subpopulations. We demonstrate that invariant NKT cells, defined by coexpression of CD3 and 6B11 [10], and non-invariant NKT cells, defined by coexpression of CD3 and CD16/56, are present within these CD4 CD8 T cell subpopulations at varying frequencies (Fig. 1, C and D). Expression of invariant NKT and non-invariant NKT markers is most evident in the CD4CD8 (P5) subpopulation, where up to 29% of the cells were found to be invariant CD3 6B11 NKT cells, and up to 26% of the cells were found to be non-invariant CD3 CD16/56 NKT cells. Our studies here show that in characterizing the role of cells coexpressing CD4 and CD8, it is critical that multiple CD4 CD8 T cell subpopulations (CD4CD8, CD4CD8, CD4 CD8 , and CD4CD8) are not evaluated together as one double-positive population. Combining these populations, which may not share similar characteristics and may express differentiation and activation surface markers to varying degrees, and not depleting certain populations of contaminating cells (i.e., NKT cells) will mask the lineage and functional potential analysis of each individual subpopulation of CD4 CD8 T cells.