Analysis of lymphocyte subpopulations stimulated in vitro with human dialyzable leukocyte extracts (DLE) (49.33)

Abstract

Abstract Dialyzable leukocyte extracts (DLE) are obtained from freezed-thawed buffy coats and subsequent dialysis ( <12 KDa). They can transfer cellular immune response from an immune donor to a naïve recipient. DLE have been used as immunomodulators for the last 50 years with good results. Until now little research has been done regarding their mechanism of action. The aim of this work was to analyze, using flow cytometry, if in vitro stimulation of PBMC with a range of DLE concentrations (50 microg, 50 ng, 50 pg, 50fg and 50ag) induced proliferation or activation of different lymphocyte subpopulations. Peripheral blood mononuclear cells (PBMC) were purified using a density gradient and viability was assessed using trypan blue exclusion stain. CFSE stain was performed to measure proliferation of CD3+/CD4+ (Th), CD3+/CD8+ (Tc), CD19+ (B) and CD56+ (NK) cells after 24, 48 and 72h. CD69 expression was measured on the same cell subpopulations after 3, 6, 24 and 48 h. Controls included PMA+ionomicin and non stimulated cells. Results showed that DLE increased CD69 expression only in the Th (CD3+/CD4+) population (50 microg, 50 ng, 50 pg). This subpopulation also proliferate more in comparison with Tc, B or NK cells. Next experiments will analyze if these cells that activated and proliferate upon DLE stimulation express other markes, such as CD25 and Foxp3. Our results may explain in part the immunomodulatory effects that these extracts have. This work was supported in part by SIP/IPN. 1Are fellows of COFAA, EDI and SNI.