Multicolor cytoenzymatic evaluation of dipeptidyl peptidase IV (CD26) function in normal and neoplastic human T-lymphocyte populations

Abstract

Dipeptidyl peptidase IV (DPP IV), also identified as the glycoprotein CD26, is a transmembrane 110- to 120-kDa serine aminopeptidase involved in immune responses by influencing T-cell costimulation and by cleaving cytokines. Additionally, CD26 is a nonintegrin receptor that contains a binding site for extracellular matrix and other molecules. In order to further define the expression and functional activity of this membrane exopeptidase in human T cells, we developed a nondisruptive, four-color cytofluorogenic assay that utilizes three separate antibodies to cell-surface molecules (e.g., CD4/CD8/CD26 and CD19/CD56/CD26) along with a rhodamine 110-conjugated dipeptide substrate that allows the measurement of DPP IV activity in phenotypically defined cells. We found normal human thymi to have notable differences in time-dependent DPP IV activity among the thymocyte subsets defined by their CD4/CD8 phenotype, with CD4-/CD8- thymocytes containing less DPP IV activity than cells expressing CD4 and/or CD8 (i.e., maturing). CD26 positivity was moderately intense in thymocytes and tended to identify cells with higher DPP IV activity. The four-color technique was also used to examine mature peripheral blood lymphocytes, along with an assortment of leukemias and transformed T-cell lines. These experiments revealed that while DPP IV was consistently evident in normal T cells, neoplastic T cells could vary in their expression patterns. Furthermore, the presence (or intensity) of surface CD26 in some abnormal T cells and certain normal peripheral blood mononuclear cells was separable from the level of DPP IV measured intracellularly. Our results established that multicolor cytofluorographic analysis can be a practical means to measure DPP IV activity in various human cell populations. Furthermore, we found that DPP IV activity could vary in T cells according to their differentiation status and that under certain circumstances surface CD26 expression can be disassociated from the level of measured enzyme (i.e., DPP IV) activity.

FIG. 1

Kinetic study of total DPP IV activity in normal peripheral blood, as determined by single-color flow cytometry analysis of lymphocytes, gated with typical low-forward and side-scatter characteristics. Values are means of separate determinations of lymphocytes from five subjects. Error bars indicate standard deviations.

FIG. 2

Four-color flow cytometry analysis of human thymus cells for the simultaneous determination of DPP IV activity and expression of other surface molecules, as described in Materials and Methods. Shown are two-parameter histograms with DPP IV activity in CD4⁺, CD8⁺, and CD26⁺ cells at 1 (top) and at 10 (middle) min of incubation. The predominant pattern of CD4 and CD8 coexpression is demonstrated in the bottom histogram.

FIG. 3

DPP IV activity in human thymocyte subsets distinguished by their CD4 and CD8 expression patterns. Values are means of eight separate determinations of DPP IV activity (top) and CD26 expression (bottom) in unstimulated, freshly isolated human thymus lymphocytic cells. ∗, P ≤ 0.05 compared to other subsets within either the 1- or the 10-min group. Error bars indicate standard deviations.

FIG. 4

DPP IV activity in human thymocyte subsets distinguished by their CD4 and CD8 expression patterns. Values are means of eight separate determinations of DPP IV activity (top) and CD26 expression (bottom) in unstimulated, freshly isolated human thymus cells and thymus cells cultured with IL-2 as indicated (see Materials and Methods). ∗, P ≤ 0.05 compared with the control group (medium [med] alone). Error bars indicate standard deviations.

FIG. 5

Four-color flow cytometry analysis of human peripheral blood mononuclear cells for the simultaneous determination of DPP IV activity and expression of other surface molecules, as described in Materials and Methods. Shown are two-parameter histograms with DPP IV activity (10-min incubation) and CD26 expression in CD4⁺ and CD8⁺ cells.

FIG. 6

Four-color flow cytometry analysis of human leukemias for the simultaneous determination of DPP IV activity and expression of other surface molecules, as described in Materials and Methods. Gating was performed on populations almost exclusively comprised of tumor cells. Shown are two-parameter histograms with DPP IV activity (after 10 min of incubation) (right) or CD26 expression (left) in either CD4⁺, CD8⁺, or CD19⁺ cells. (A to D) Four separate T-cell acute lymphoblastic leukemias; (E) B-cell acute lymphoblastic leukemia.