EXTENSIVE ANALYSIS OF LYMPHOCYTE SUBSETS IN NORMAL SUBJECTS BY 3-COLOR IMMUNOFLUORESCENCE

In the present work, 30 normal controls were studied with 11 combination of 3 monoclonal antibodies in order to define CD20, CD4, CD8, CD16 and CD56 subsets as well as their activation status. Our results indicated that among B cells, CD5- cells predominated (about 92%), the minor CD5+ fractionshowed variations between individuals but sometimes was as high as 20% in B cells; the CD45RA + CD29- subset (naive cells) accounted for 31.56 +/- 10.03% in CD4 cells, as compared to CD4 + CD29 + CD45RA- (memory cells) (46.54 +/- 10.35%), while CD4 + CD29 + CD45RA + (transition cells between naive and memory state) represented 9.6 +/- 5.3% as compared to double-negatives (12.3 +/- 5.24%). Some uncertainties persist in defining CD8 subsets. NK CD8 cells accounted for about 20% of total CD8 according to CD16 and CD56 expression. Cytotoxic T lymphocytes could be delineated by S6F1 (76.22 +/- 11.28% within CD8 cells), though this marker appeared to be expressed by both cytotoxic and NK cells. Phenotypic definition of suppressive CD8 cells remains uncertain despite the fact that suppressor activity was found among CD8 lymphocytes expressing CD11b and probably CD57. NK cells coexpressed CD16 and CD56 markers in about 5% of all lymphocytes, while NK cells expressing CD16 alone accounted about 6%. Exclusive expression of CD56 was found in about 5%. Finally, while activated cells characterized by DR expression represented about 4% of CD4 and 9% of CD8 cells, CD25 appeared to be poorly represented among CD4, CD8 and CD20. These results demonstrate that phenotypic studies by using triple labeling can be performed for routine purpose in total blood. By better defining minor subpopulations, they could aid in understanding pathologic status.