title: EBV cell-lines (LCL) and E- cells as stimulator cells for limiting dilution analysis (LDA) of alloreactive IL-2-secreting cells (IL-2-SC) and cytotoxic precursors (CTLp). Comparison of their stimulator capacity and ability to generate specific alloreactivity.
abstract: The choice of stimulator cells is crucial in determining the frequency of alloreactive cells by limiting dilution analysis (LDA). In humans, E- cells or EBV-lymphoblastoid cell lines (LCL) are available for this purpose. The E- cells have been mostly used until now, but they appear to stimulate much less than LCL in other models. Consequently, we undertook a systematic comparison of the efficiency of both types of cell for the LDA of alloreactive IL-2-secreting cells (IL-2-SC) and cytotoxic precursors (CTLp). We show that LCL are stronger stimulator cells both for IL-2 secretion and for cytotoxicity induction. However, high levels of non-allogeneic reactivity were also induced. Therefore, to measure the frequency of specific alloreactive IL-2-SC and CTLp, T cells were depleted of such irrelevant reactive cells by a suicide technique: culture with autologous LCL in the presence of BUDR and use of the remaining live cells as responding effectors in LDA. Using this methodology, no non-allogeneic reactive T cells remain in the responding cells: after restimulation by autologous LCL, no IL-2-SC could be seen and no cytotoxic activity could be observed against autologous, irrelevant or LAK sensitive targets. In contrast, the number of IL-2-SC and CTLp against allogeneic targets was preserved. Therefore with this methodology, the strong stimulator capacity of LCL could be used whereas non-specific activation could be avoided in order to assess the frequency of allo-specific IL-2-SC and CTLp.
authors.affiliation: Laboratoire d'Immunologie Cellulaire et de Transplantation, ER 277 CNRS, Villejuif, France.
authors.forename: O
authors.initials: O
authors.lastname: Lantz
authors.forename: P
authors.initials: P
authors.lastname: Alard
authors.forename: G
authors.initials: G
authors.lastname: Chavanel
authors.forename: D
authors.initials: D
authors.lastname: Fries
authors.forename: A
authors.initials: A
authors.lastname: Senik
authors.forename: B
authors.initials: B
authors.lastname: Charpentier
mesh_terms: D001973:Bromodeoxyuridine / Q000494:pharmacology; D002458:Cell Fractionation; D002460:Cell Line; D003602:Cytotoxicity, Immunologic; D006412:Hematopoietic Stem Cells / Q000276:immunology*; D004854:Herpesvirus 4, Human; D006801:Humans; D007376:Interleukin-2 / Q000378:metabolism*; D015979:Killer Cells, Lymphokine-Activated / Q000276:immunology; D007959:Lymphocyte Culture Test, Mixed; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*
chemical_list: D007376:Interleukin-2; D001973:Bromodeoxyuridine