Prieto A., Reyes E., García-Suarez., Carrión F., Hernández MP., Moragues B., Alvarez-Mon M.

Department of Medicine. University of Alcala. Clinical Inmunology and Hematology Services. Principe de Asturias University Hospital. Alcala de Henares. Madrid (Spain).

Impaired T cell proliferation response to polyclonal mitogens such as vegetal lectins has been described in B-CLL patients. Several studies failed to find an intrinsic defect in T lymphocytes from B-CLL patients that would explain the defective proliferative response to mitogens.

Materials and methods : CD2+ cells were obtained from peripheral blood by double rosetting method. Subpopulational Viability assays : Cells were cultured for 1 to 3 days stained with antiCD4 FITC/anti-CD45RO PE; and incubated for 30 min at 4ºC and resuspended in PBS. After two washes, PBS containing 7aminoactinomicin D (7aaD) at a final concentration of 1ūg/ml was added and incubated for 3 minutes before FACScan analysis. Cells that were permeable to 7aa (nonviable) and cells that were not permeable to 7aa (viable) were distinguished by fluorescence analysis on a flow cytometer. Determination of apoptotic cells : PHA stimulated lymphocytes after 24h. of culture were treated to extract low molecular weight DNA from nuclei of apoptotic cells. DNA was stained with Propidium iodide.

Results : Increased percentages of CD4+CD45RO+ lymphocytes were found in CD2+ cells from B-CLL patients. The percentages of CD4+CD45RO+ viable cells in phytohemagglutinin stimulated CD2+ cultures of lymphocytes from four B-CLL at 24h. of culture were significantly lower than those of viable CD4+CD45RO- lymphocytes found in cultures. The percentages of nuclei with reduced DNA content in lymphocyte cultures from B-CLL patients were higher than those found in cultures of lymphocytes from healthy controls; thus suggesting an apoptotic cell death mechanism.

Conclusions : 1) T cells from B-CLL patients are in vivo activated. 2) In vivo activation of CD4 subset with subsequent decrease in CD4+CD45RA+ cells appears to be involved in the biological basis of their defective proliferative response to mitogens. 3) Viability data from CD4 subpopulations demonstrate that CD4+CD45RO+ cells die after activation and they do it by a mechanism that implies nuclear DNA degradation.