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.
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.
Lantz
O
O
Laboratoire d'Immunologie Cellulaire et de Transplantation, ER 277 CNRS, Villejuif, France.
Alard
P
P
Chavanel
G
G
Fries
D
D
Senik
A
A
Charpentier
B
B
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*
D007376:Interleukin-2; D001973:Bromodeoxyuridine