PERIPHERAL-BLOOD MONONUCLEAR CELL SONICATES AS AN ALTERNATIVE TO IRRADIATED ALLOGENEIC CELLS TO STIMULATE A MIXED LYMPHOCYTE-REACTION AND TO ENUMERATE CD69-CELLS( ALLOREACTIVE T)

An alloreactive reaction similar to that occurring during GvHD can be generated in a mixed lymphocyte culture. The presence of both stimulat or and responder cells in these cultures makes the identification and enumeration of alloreactive cells difficult and unreliable. We describ e the use of PBMC sonicates as an alternative to the standard MLC meth od to stimulate an allogeneic reaction. Using combinations of autologo us or allogeneic PBMC sonicates, we showed that the lymphocyte prolife rative response to cell sonicates was comparable to the response using irradiated cells. The proliferative response was concentration depend ent and reached maximum levels at day 6. Both irradiated cells and PBM C sonicates induced significantly lower responses when the stimulating cells were partially HLA-DR matched rather than completely mismatched . Alloreactive T cells stimulated with sonicates were enumerated by th e flow cytometric detection of CD69 or CD25. In HLA-mismatched culture s, approximately 7% of CD3+ T cells were CD69+ or CD25+, suggesting al loreactivity. Although there was a significant correlation between the expression of these activation markers and lymphocyte proliferative r esponses, significant individual variations in the results of these tw o assays were observed. The results in this study demonstrate the pote ntial of using PBMC sonicates instead of irradiated lymphocytes for th e study and identification of alloreactive cells at the cellular and m olecular level. (C) American Society for Histocompatibility and Immuno genetics, 1997. Published by Elsevier Science Inc.