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.

• Authors: Øystein Wendelbo, Øystein Bruserud
• Journal: Journal of hematotherapy & stem cell research (J Hematother Stem Cell Res) Vol. 12 Issue 5 Pg. 525-35 (Oct 2003) ISSN: 1525-8165 [Print] United States
• PMID: 14594509 (Publication Type: Comparative Study, Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Antibodies, Monoclonal
• Antigens, CD
• CD28 Antigens
• CD3 Complex
• Culture Media
• Interleukins
• Proto-Oncogene Proteins
• Granulocyte-Macrophage Colony-Stimulating Factor
• FLT3 protein, human
• Receptor Protein-Tyrosine Kinases
• fms-Like Tyrosine Kinase 3

Topics

• Adolescent
• Adult
• Aged
• Antibodies, Monoclonal (pharmacology)
• Antigens, CD (analysis)
• CD28 Antigens (immunology)
• CD3 Complex (immunology)
• Cell Culture Techniques (methods)
• Cell Division (drug effects)
• Culture Media (pharmacology)
• Drug-Related Side Effects and Adverse Reactions
• Female
• Flow Cytometry
• Granulocyte-Macrophage Colony-Stimulating Factor (pharmacology)
• Humans
• Interleukins (blood, pharmacology)
• Leukemia (blood, therapy)
• Leukocytes, Mononuclear (chemistry, metabolism)
• Lymphocyte Activation (drug effects, immunology)
• Lymphopenia (blood, etiology, immunology)
• Male
• Middle Aged
• Multiple Myeloma (blood, therapy)
• Myelodysplastic Syndromes (blood, therapy)
• Proto-Oncogene Proteins (pharmacology)
• Receptor Protein-Tyrosine Kinases (pharmacology)
• Stem Cell Transplantation (adverse effects)
• T-Lymphocytes (cytology, drug effects, metabolism)
• Time Factors
• fms-Like Tyrosine Kinase 3