Limiting dilution analysis of IL-2 producing cells: I. Studies of normal human peripheral blood.
BACKGROUND
After marrow transplantation, the interaction of helper T lymphocytes from the donor with the patient alloantigens leads to cellular activation and release of IL-2 as initial events of the graft versus host reaction. A method for assessing the size of the pool containing allospecific helper T cells capable of producing IL-2 could be applied in the selection of better donors for marrow transplantation.


METHODS
PBMC are added to replicate sets of wells each containing various amounts of EBV-LCL cells and PHA. After culture for some days the supernatant is removed from each well and added to IL-2 dependent CTLL-2 line. The proliferation of the CTLL-2 line is assessed by pulse labeling with 3H-thimidine. The precursor frequency of cell capable of producing IL-2 per ml/blood is estimated from the minimum X2 regression of the function of non-responding wells plotted as logarithmic function of the number of PBMC added per well.


RESULTS
Approximately 30-40% of PBMC are found to produce IL-2 under the following conditions in culture: the optimal PHA concentration is 1.25 micrograms/ml, the optimal number of stimulator EBV-LCL cells is 1 x 10(3) and 3 days of culture are required.


CONCLUSIONS
Here we report a rapid and quantitative technique of limiting dilution analysis that can estimate the frequency of peripheral blood mononuclear cells capable of secreting interleukin-2 following interaction with specific alloantigen.
Moretti
L
L
Divisione di Ematologia, USL 3, Pesaro, Italy.
Giuliodori
L
L
Stramigioli
S
S
Luchetti
F
F
Baldi
A
A
Sparaventi
G
G
D002461:Cell Line, Transformed; D002478:Cells, Cultured; D015925:Cryopreservation; D006801:Humans; D007376:Interleukin-2 / Q000096:biosynthesis*; D007519:Isoantigens / Q000276:immunology; D007958:Leukocyte Count; D008213:Lymphocyte Activation; D010835:Phytohemagglutinins; D006377:T-Lymphocytes, Helper-Inducer; D014019:Tissue Donors
D007376:Interleukin-2; D007519:Isoantigens; D010835:Phytohemagglutinins