Functional evaluation of proliferative T cell responses in patients with severe T lymphopenia: characterization of optimal culture conditions and standardized activation signals for a simple whole blood assay

In this methodological study, we describe an assay for analysis of proliferative T cell responses in patients with severe leukopenia. Severe treatment-induced cytopenia is observed in patients with malignant disorders who receive conventional intensive chemotherapy or autologous stem cell transplantation. The quantitative T cell defect can then be characterized by flow cytometric analysis of membrane molecule expression, whereas the functional status of the remaining T cell population is more difficult to evaluate. In the present study, we describe a standardized whole blood assay that requires small sample volumes and can be used for repeated analysis even in severely ill patients. The assay is based on the following strategy: (i) blood samples are diluted with the serum-free medium X-vivo 10, (ii) T cells are activated either with monoclonal immunoglobulin E (IgE) anti-CD3 or anti-CD3 plus anti-CD28; (iii) T cell proliferation is assayed by (3)H]thymidine incorporation after 4 days of in vitro culture. These proliferative responses are not affected by the plasma levels of interleukin-2 (IL-2), sIL-2-R alpha, IL-7 and IL-15, and the kinetics of the response are not altered by the presence of exogenous cytokines. Detectable proliferation is observed for most patients with treatment-induced cytopenia. We conclude that the assay can be used for functional characterization of remaining T lymphocytes in patients with severe T lymphopenia.