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.
Division for Hematology, Department of Medicine, Haukeland University Hospital, Bergen, Norway. wend@haukeland.no
Øystein
Ø
Wendelbo
Øystein
Ø
Bruserud
D000293:Adolescent; D000328:Adult; D000368:Aged; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D015703:Antigens, CD / Q000032:analysis; D018106:CD28 Antigens / Q000276:immunology; D017252:CD3 Complex / Q000276:immunology; D018929:Cell Culture Techniques / Q000379:methods; D002455:Cell Division / Q000187:drug effects; D003470:Culture Media / Q000494:pharmacology; D064420:Drug-Related Side Effects and Adverse Reactions; D005260:Female; D005434:Flow Cytometry; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D006801:Humans; D007378:Interleukins / Q000097:blood / Q000494:pharmacology; D007938:Leukemia / Q000097:blood / Q000628:therapy; D007963:Leukocytes, Mononuclear / Q000737:chemistry / Q000378:metabolism; D008213:Lymphocyte Activation / Q000187:drug effects / Q000276:immunology*; D008231:Lymphopenia / Q000097:blood* / Q000209:etiology / Q000276:immunology; D008297:Male; D008875:Middle Aged; D009101:Multiple Myeloma / Q000097:blood / Q000628:therapy; D009190:Myelodysplastic Syndromes / Q000097:blood / Q000628:therapy; D011518:Proto-Oncogene Proteins / Q000494:pharmacology; D020794:Receptor Protein-Tyrosine Kinases / Q000494:pharmacology; D033581:Stem Cell Transplantation / Q000009:adverse effects; D013601:T-Lymphocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D013997:Time Factors; D051941:fms-Like Tyrosine Kinase 3
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D018106:CD28 Antigens; D017252:CD3 Complex; D003470:Culture Media; D007378:Interleukins; D011518:Proto-Oncogene Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; C497970:FLT3 protein, human; D020794:Receptor Protein-Tyrosine Kinases; D051941:fms-Like Tyrosine Kinase 3