Double labeling immunohistologic and flow cytometric analysis of human B cells with particular reference to Leu-8...

Previous results have shown that human lymphocyte subpopulations are heterogeneous as to Leu-8 expression. In the present study, we performed a heretofore unreported immunohistologic analysis and flow cytometric double labeling investigation focused on Leu-8+ and Leu-8- human B cells, with special reference to their expression of other B cell lineage antigens (Leu-14, B1, OKB7 or B2, OKB2, BA-1, BA-2, IgD, and IgM) or of a functional marker of cell proliferation (Ki-67). Immunohistologic analysis was performed on frozen sections of nine normal or reactive lymph node and tonsil biopsy specimens tested with either single or paired antibodies, the latter procedure (double labeling) being directed at revealing positively subtracted Leu-8+ or Leu-8- cells expressing a given marker depending on the antibody staining sequence used. Dual flow cytometry with paired antibodies was performed on cell suspensions from four normal or reactive lymph nodes and tonsils. As to the follicular district, immunohistologic analysis suggests that germinal center (proliferating) B lymphocytes, identifiable in cell suspension as Leu-8-B1+ and representing 80% of the B1+ cells, are Leu-8- and Leu-14+, BA-2+, OKB7+, and IgM+, being rarely IgD+. Most lymphocytes located in the mantle zone (resting cells), identifiable in cell suspension as Leu-8+B1+ and representing the remaining 20% of the B1+ cells, are Leu-8+, Leu-14+. OKB7+, OKB2+, BA-1+, IgD+, and IgM+. Furthermore, from our results, there is indirect evidence to support the existence of a Leu-8+BA-2+ lymphocyte subset located in the mantle zone. Immunohistologic study of four lymph nodes by Leu-8 and Ki-67 antibody (that recognizes a nuclear, cell proliferation-associated antigen absent only in the Go phase of the cell cycle) showed that these markers are mutually exclusive and added further evidence that Leu-8 antigen is a marker of resting lymphocytes.