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

Abstract

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.

Immunologic Response to Antiretroviral Therapy in Hepatitis C Virus–Coinfected Adults in a Population‐Based HIV/AIDS Treatment Program

“…A blunted immune response in HIV/HCV-coinfected patients may be due to the nonspecific immune stimulation driven by chronic HCV infection, or it may be that infection of immune cells by HCV could favor the depletion of CD4 cells [35,36]. Our data support the hypothesis that HCV infection is related to smaller increases in both the absolute CD4 cell count and the CD4 cell fraction than those that occur in HCV antibody-negative patients.…”

The role of arginine and arginine-metabolizing enzymes during Giardia – host cell interactions in vitro

“…Cellular proliferative responses were measured by the incorporation of 3 H-thymidine into newly synthesized DNA by conventional proliferation assay [42]. After 5 days of culture cells were pulsed with 37 kBq/well of 3 H-thymidine (Perkin Elmer, Boston, MA, USA) and harvested 18 h later onto glass-fibre pads.…”

Distinct single cell signal transduction signatures in leukocyte subsets stimulated with khat extract, amphetamine-like cathinone, cathine or norephedrine

“…The assay used to determine in vitro lymphocyte proliferation is based on a whole blood protocol [45]. Experiments were performed in triplicates in 96-well plates, and each well added (i) 5 × 10 4 PBMCs; (ii) CD3 antibody (mouse IgE Moab; CLB-T3/4.E), final concentration 316 ng ml -1 ; (iii) CD28 antibody (mouse IgG1 Moab, CLB-CD28/1), final concentration 842 ng ml -1 ; and (iv) test compound (cathinone, cathine, norephedrine, khat-extract) or control medium.…”