Nonradioactive techniques for measurement of in vitro T-cell proliferation: Alternatives to the [H-3]thymidine incorporation assay

T-cell proliferation is an important in vitro parameter of in vivo immune f unction and has been used as a prognostic marker of human immunodeficiency virus type 1 (HIV-1) disease progression. The proliferative capacity of T c ells in response to various stimuli is commonly determined by a radioactive assay based on incorporation of [H-3]thymidine ([H-3]TdR) into newly gener ated DNA. In order to assess techniques for application in laboratories whe re radioactive facilities are not present, two alternative methods were tes ted and compared to the [H-3]TdR assay as a "gold standard." As an alternat ive, T-cell proliferation was measured by flow cytometric assessment of CD3 8 expression on T cells and by an enzyme-linked immunosorbent assay (ELISA) based on bromo-2'-deoxyuridine (BrdU) incorporation. Peripheral blood mono nuclear cells (PBMCs), either in whole blood or Ficoll-Isopaque separated, from a total of 26 HIV-1-positive and 18 HN-l-negative Dutch individuals we re stimulated with CD3 monoclonal antibody (MAb) alone, a combination of CD 3 and CD28 MAbs, or phytohemagglutinin. BrdU incorporation after 3 days of stimulation with a combination of CD3 and CD28 MAbs correlated excellently with the [3H]TdR incorporation in both study groups (HIV-1 positives, r = 0 .96; HIV-1 negatives, r = 0,83). A significant correlation of absolute numb ers of T cells expressing CD38 with [H-3]TdR incorporation, both in HIV-l-p ositive (r = 0.96) and HIV-l-negative (1. = 0.84) individuals, was also obs erved under these conditions. The results of this study indicate that deter mination of both the number of CD38-positive T cells and BrdU incorporation can be used as alternative techniques to measure the in vitro T cell proli ferative capacity, The measurement of CD38 expression on T cells provides t he additional possibility to further characterize the proliferating T-cell subsets for expression of other surface markers.