title: Immunophenotypic Dissection of Normal Peripheral Blood NK Associated (NKa) Subpopulations by Flow Cytometry: Morphological Features and Relationships Between Membrane NKa (CD11b, CD 16, CD56 and CD57) arid T-cell (CD2, CD3, TCR, CD5, CD7, CD8 and CD38) Associated Determinant Expression.
abstract: Using single and multiple colour flow cytometry (FACSCAN), this study has examined the expression of NK-associated (NKa) CD11b, CD16, CD56 and CD57 membrane antigens by normal lymphocyte subpopulations. In addition to determining the normal proportions and absolute numbers of NKa(+) cells, this investigation also related the expression of these four NKa markers to the presence of; (a) morphologically-defined large granular lymphocytes, (LGL); and (b) membrane CD2, CD3, CD8 and TCR chain expression. This was achieved by preparing highly enriched normal blood CD4-CD8(+)/CD4 CD8(+) and CD4(-)CD8(-) lymphocyte fractions (n = 6) by immunomagnetic depletion of CD4(+)/CD14(+)/CD19(+) or CD4(+)/CD8(+)/CD14(+)/CD19(+) components respectively. Morphological assessment of these fractions showed that the sequential depletion procedures resulted in the enrichment of LGL, from a mean of 9% for the "whole" lymphocyte (pre-depletion) fraction, to 35% following removal of CD4(+), CD14(+) and CD19(+) cells and to 80%) following the additional removal of CD8(+) cells. FACS analysis revealed the presence of three CD8 subgroups (CD8(-), CD8(dim+) and CD8(+)), defined by differences in membrane staining intensity, and subsequent three-colour (CD2/CD3/CD8) studies indicated that the main composite CD2/CD3 phenotypes for these CD8 groups were (relative frequencies in parenthesis); (a) CD8(+), CD2(+)CD3(+) (94%); (b) CD8(dim+), CD2(+) CD3(+) (31%) and CD2(+)CD3(-) (52%); and (c) CDS(-), CD2(+)CD3(+) (20%), CD2(+)CD3(-) (58%) and CD2(-)CD3(-) (22%). Analysis of paired NKa marker expression (CD11b and CD56; CD11b and CD57; CD16 and CD57), when correlated with CD3 and CD8, showed that (a) the predominant NKa phenotype of CD3(+)CD4(-)CD8(+) cells was CD11b(-)CD16(-)CD56(-)CD57(-); (b) CD3(+)CD4(-)CD8(dim+) and CD3(+)CD4(-)CD8(-) subpopulations showed CD11b(-)CD16(-)CD56(-)CD57(±) and CD11b(±)CD16(-)CD56(-)CD57(-) composite phenotypes respectively (where - and ± denote <20% and 21-60%0 NKa(+) positive cells); and (c) the highest proportions of NKa(+) cells were associated with CD3- subpopulations, with composite phenotypes for CD3-CD4-CD8(dim+) and CD3(-)CD4(-)CD8(-) components being CD11b(-)CD16(±)CD56(+)CD57(±) and CD11b(+)CDl6(+)CD56(+)CD57(±) (where + denotes >60% NKa(+) cells). Expression of NKa determinants by normal CD4(-) lymphoid subpopulations was thus shown to increase in the order CD3(+)CD8(+) > CD3(+) CD8(dim+) or CD3(+)CD8(-) > CD3(-)CD8(dim+) > CD3(-)CD8(-). Studies of TCR chain expression by NKa(+) (CD16/CD56) and NKa(-) subgroups of the CD4(-)CD8(-) fraction additionally showed that the presence of membrane TCRαδ (mean 18%) or TCRγδ (mean 35%) chains were primarily associated with the NKa(-) component and that virtually all (93%) NKa(+) cells were TCRαβ-TCRγδ. Examination of CD4(-)CD8(-)NKa(+) cells for the expression of other T-cell associated markers (CD5, CD7 and CD38) also indicated that this NKa(+) LGL-rich fraction could be further subdivided into CD2(+)CD3-CD5(-)CD7(+)CD38(+) and CD2-CD3(-)CD5(-)-CD7(+)CD38(+) subpopulations.
authors.affiliation: a Department of Haematology, Cookridge Hospital, Leeds, LS16, England.
authors.forename: S J
authors.initials: SJ
authors.lastname: Richards
authors.affiliation: a Department of Haematology, Cookridge Hospital, Leeds, LS16, England.
authors.forename: C S
authors.initials: CS
authors.lastname: Scott
keywords: NK cells; T-cells; flow cytometry; immunophenotyping