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

Clin Diagn Lab Immunol. 2000 Jul;7(4):687-92. doi: 10.1128/cdli.7.4.687-692.2000.

Abstract

T-cell proliferation is an important in vitro parameter of in vivo immune function and has been used as a prognostic marker of human immunodeficiency virus type 1 (HIV-1) disease progression. The proliferative capacity of T cells in response to various stimuli is commonly determined by a radioactive assay based on incorporation of [(3)H]thymidine ([(3)H]TdR) into newly generated DNA. In order to assess techniques for application in laboratories where radioactive facilities are not present, two alternative methods were tested and compared to the [(3)H]TdR assay as a "gold standard." As an alternative, T-cell proliferation was measured by flow cytometric assessment of CD38 expression on T cells and by an enzyme-linked immunosorbent assay (ELISA) based on bromo-2'-deoxyuridine (BrdU) incorporation. Peripheral blood mononuclear cells (PBMCs), either in whole blood or Ficoll-Isopaque separated, from a total of 26 HIV-1-positive and 18 HIV-1-negative Dutch individuals were stimulated with CD3 monoclonal antibody (MAb) alone, a combination of CD3 and CD28 MAbs, or phytohemagglutinin. BrdU incorporation after 3 days of stimulation with a combination of CD3 and CD28 MAbs correlated excellently with the [(3)H]TdR incorporation in both study groups (HIV-1 positives, r = 0.96; HIV-1 negatives, r = 0.83). A significant correlation of absolute numbers of T cells expressing CD38 with [(3)H]TdR incorporation, both in HIV-1-positive (r = 0.96) and HIV-1-negative (r = 0.84) individuals, was also observed under these conditions. The results of this study indicate that determination 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 proliferative capacity. The measurement of CD38 expression on T cells provides the additional possibility to further characterize the proliferating T-cell subsets for expression of other surface markers.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • ADP-ribosyl Cyclase
  • ADP-ribosyl Cyclase 1
  • Acquired Immunodeficiency Syndrome / immunology*
  • Acquired Immunodeficiency Syndrome / pathology
  • Acquired Immunodeficiency Syndrome / physiopathology*
  • Antigens, CD*
  • Antigens, Differentiation*
  • Cell Division
  • Flow Cytometry / methods*
  • HIV-1*
  • Humans
  • Lymphocyte Activation
  • Membrane Glycoproteins
  • NAD+ Nucleosidase*
  • Predictive Value of Tests
  • Prognosis
  • T-Lymphocytes / immunology*
  • T-Lymphocytes / pathology

Substances

  • Antigens, CD
  • Antigens, Differentiation
  • Membrane Glycoproteins
  • ADP-ribosyl Cyclase
  • CD38 protein, human
  • NAD+ Nucleosidase
  • ADP-ribosyl Cyclase 1