Separation of lymphokine-activated killer cell precursors and T-cells: differential growth characteristics and apparent lack of a T-cell suppressor of LAK-cell induction.
Lymphokine activated killer (LAK) cell clinical effectiveness may be limited by the total cell dose and cytotoxic activity. We have, therefore, examined methods to expand the number of LAK-cells by serial passage of unfractionated and fractionated peripheral blood lymphocytes. Human purified lymphocytes were obtained by Ficoll Hypaque gradients followed by exposure of resultant mononuclear cells to phenylalanine methyl ester to remove monocytes. Lymphocytes were then fractionated on a six-step Percoll gradient (50%, 47.5%, 45%, 42.5%, 40%, and 37.5% Percoll). Unfractionated cells and fractions were cultured in standard media (RPMI-1640, 10% human sera, antibiotics and 10 mM HEPES) containing 10 nM of recombinant Interleukin-2 (rIL-2). Lymphocytes were cultured at 1 X 10(6)/ml and recultured every 3 to 4 days in fresh standard media and rIL-2. Utilizing unfractionated and fractionated lymphocytes from seven donors we made the following observations: (1) Continued passage of unfractionated lymphocytes resulted in a loss of LAK-cell activity by greater than or equal to 14 days (e.g., percent lysis of Raji at 10:1 effector:target ratio on days 0, 4, 7, and 21 was 0.38 +/- 11, 41 +/- 17, and 8 +/- 1, n = 4, respectively). (2) LAK-cell functional precursors were predominantly confined to the lymphocytes in the upper (0-4) Percoll fractions (e.g., on day 4, the percent lysis by pooled fractions 1-4 was 63 +/- 5 vs. pooled fraction 5 plus pellet, 18 +/- 7%). (3) As expected, the upper fractions (0-4) were enriched for Leu 19 positive cells (approximately 40%) and large granular lymphocytes (LGL) by morphology (approximately 30%).(ABSTRACT TRUNCATED AT 250 WORDS)
Rinehart
J J
JJ
Ohio State University College of Medicine, Columbus.
Geidenberger
C
C
Aldrich
W
W
D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D003602:Cytotoxicity, Immunologic; D006801:Humans; D007376:Interleukin-2 / Q000502:physiology; D015979:Killer Cells, Lymphokine-Activated / Q000276:immunology*; D008213:Lymphocyte Activation; D013601:T-Lymphocytes / Q000276:immunology*; D050378:T-Lymphocytes, Regulatory / Q000276:immunology
D007376:Interleukin-2