206 14th International Leucocyte Culture Conference, Heidelberg

Addendum

This abstract has been accepted for the workshop NK Cetts

Laboratory of Immunodiagnosis , National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20205, USA.

Cultures of human natural killer cells in the presence of T cell growth factor (TCGF) containing conditioned medium (CM)

TUOMO TIMONEN, JOHN R. ORTALDO, GUY D. BONNARD, and RONALD B. HERBERMAN

Recent studies have demonstrated natural killer (NK)-like activity among lymphocytes cultured in the presence of TCGF-containing eM. It has been unclear whether this reactivity is exerted by proliferating NK cells. We have directly examined this question by culturing human NK cells (large granular lymphocytes, LGL) in the presence of CM. LGL were purified by discontinuous density gradient centrifugation and subsequently by high affinity sheep erythrocyte rosette formation to remove mature T cells. T cells were isolated by recovering high density cell fractions directly from the gradients. Both LGL and T cells were cultured in the presence of a) 10 % 4X concentrated CM from human peripheral blood lymphocytes stimulated with PHA, b) eM depleted of PHA by absorption on chicken erythrocytes, c) 1 Jlg/ml PHA, and d) 2.5 Jlg/ml Can A. Both LGL and T cells proliferated in response to crude CM and PHA. Only LGL responded to Can A, whereas neither LGL nor T cells responded to the PHA-depleted CM. However, 5-day-old cultures of PHA and Con A­induced LGL and PHA-induced T cells could be further maintained in the presence of absorbed eM. LGL cultures exerted NK, antibody-dependent cellular cytotoxicity (ADCC) and lectin-induced cellular cytotoxicity (LICC), whereas the T cell cultures exhibited only LIce and a low reactivity against allogeneic blasts. The typical morphology of fre.~h LGL could be detected in 70--90 % of cells in the LGL cultures. In a 51 Chromium release cytotoxicity assay, the lytic efficiency of cultured LGL was less than that of the fresh ones. In a single cell agarose cytotoxicity assay, the cultured LGL bound more frequently to NK sensitive target cells, but the number of killer cells among binders was only one third of that of fresh LGL. The results demonstrate that a) human natural killer cells can be cultured in the presence of CM, b) Con A is more selective than PHA in the induction of proliferation of LGL, and c) the cytotoxicity characteristics of NK cells are retained in the cultures.

This abstract has been accepted for the workshop T Lymphocytes: Effector Functions

Laboratoire de Biochimie des Membranes, ER CNRS 228. 8 rue de l'Ecole Normale, F-3407S Montpellier. France

The importance of enzyme unmasking for the characterization of lymphocyte plasma membranes

J. C. BONNAFOUS, J. DORNAND, J . FA VERO, and J. C. MANI

Specific enzyme activities are currently used as markers for subcellular organelle characteri­zation. Unmasking of latent activities must be taken into account, specially for plasma

14th International Leucocyte Culture Conference, Heidelberg 207

membrane enzymatic markers. A large-scale purification of plasma membranes from pig lymph node lymphocytes was carried out by centrifugation on discontinuous sucrose density gradient in a zonal rotor. Adenylate cyclase activity of untreated fractions was shown (1) to display a profile different from that of membrane enzymatic markers (5 'nucleotidase, Na-K­ATPase, Ca-ATPase. phosphodiesterase T, alkaline phosphatase, y-glutamyltranspeptidase); it was maximal at higher density (1. 15-1.16). However, when latent adenylate cyclase was unmasked by Lubrol PX or alamethicin treatment (2), its maximum was shifted to lower density (1. 12) and was no longer significantly different from that of plasma membrane markers. Similarly Na-K-ATPase activity, which is very low in untreated lymphocyte plasma membranes, was strongly increased by unmasking latent active sites; this stimulation was observed after either detergent (DOC, SDS) (3) or alamethicin (2) treatment, or cold storage (1), or magnetic stirring (specially in the presence of Sepharose) (1). These resu lts emphasize the importance of unmasking latent membrane enzymes in order to demonstrate their subcellular localization or to characterize different mem brane subfractions. For this reason we believe that it remains to be demonstr.ued that, if intact lymphocytes display a "functional mosaicism" (4) on their plasma membrane, the resulting biochemical heterogeneity can be evidenced after cell disruption and classical procedures for separation of plasma membrane vesicles. 1. BONNAfOUS, J. c., J. DORNAND, J. FA VERO, and j. C. MAN!. 1981. Unmasking of

membrane enzyme activ ities and the problem of subcellular distribution of adenylate cyclase in pig lymph node lymphocytes. Biochim. Biophys. Acta (in press).

2. BONNAFOUS, J. C., J. DORNAND. and J. C. MANJ. 1979. Detergent-like effects of alamethi­cin on lymphocyte plasma membranes. Biochem. Biophys. Res. Commun. 86: 536-544.

3. DORNAND, J., C. R EMINIAC, and j . C. MANJ. 1978. Studies of (Na + -K +)-sensitive ATPase activity in pig lymphocytes. Biochim. Biophys. Acta 509: 194-200.

4. RODE, H . N. , B. MAHLER, A. LORACHER, and K. RESCH. 1979. Functional mosaicism of the lymphocyte plasma membrane. II . Characterization of membrane subfractions of activated thymocytes. Eur. J. Immunol. 9: 402-408.

This abstract has been accepted for the workshop T Lymphocytes: Eff«tor Functions:

Institut f. Immunologie u. Genecik, Deutsches Krebsforschungszentrum. 6900 H eidelberg, FRG

Functional role of two clusters of antigenic determinants on the H-2Kk molecule(s) for generation of CTL

C. WEYAND, J. GORONZY, and G. j. HAMMULING

Six diffe rent determinants on H -2Kk-encoded antigens can be defined by monoclonal anti H -2-antibodies. In competitive antibody binding assays these determinants were found to be arranged in two separate clusters (A and B). The re:levanee of these twO domains for generation of alloreactive T-killer cells was investigated by target inhibition studies. In a bulk culture system all antibodies specifically inhibited killer cell-target interaction, those monodonaJs however, which describe cluster B reduced target ceHlysis to a much higher degree than the antibodies belonging to cluster A. Consequently a limiting dilution system was established permitting determination of CTL precursor frequencies reactive against different antigenic determinants on the target molecules. Responder spleen cells in limiting numbers were grown

208 14th International Leucocyte Culture Conference, Heidelberg

in the presence of stimulator cells and T-cell growth factor. Target blocking experiments with the different monoclonal antibodics confirmed that different CTL clones recognize different target antigenic determinants, the efficiency of blocking however differed markedly for the six diffcrent antibodies. Moreover, the frequencies determined for CTL precursors interacting with antibodies covered target structures correlated well with the extent of inhibition obtained in bulk culture. The findings provide evidence that alloreactive CTL preferentially recognize one domain of the H-2Kk-molecule(s) which is defined by a group of monoclonal antibodies.

This abstract has been accepted for the workshop Lymphokines

Tnstitute of Clinical Immunology, Insclspital, 3010 Bern, Switzerland

Effects of interleukin 2 on the cell cycle of murine thymocytes

F. KRISTENSFN, C. WALKER, and A. L DE WEeK

In order to proliferate, T lymphocytes require, in addition to various nutritional factors, a lymphokine denominated interleukin 2 (ILl)' Whcn murine thymocytes are stimulated, by PHA in the absence of ILl' they initiate RNA synthesis (ulltil a Cia stage) as a.~sessed by cytofluorometry but very little DNA synthesis as detected by 3H-thymidine uptake. Addition of IL2-containing culture supcrnatans further increases RNA synthesis and enables the cel1 to

enter the S phase. From a cytofluorometric cell distribution analysis in the presence or absence of lL2, it appears that IL2 permits to distinguish between a GI, and GIl. phase and is required for pushing the cells from the Gl~ into the Clh and ultimately the S phase. A kinetic analysis of thymocyte cultures pulsed with 3H-thymidine at 6 hours intervals and adding PHA andlor IL2 at various times show that IL2 required 6-12 hours on GI .• cells in order to initiate detectable prolifertttion. While ILl may appear to be mitogenic when added at the onset of cell cultures containing already a certain number of stimulated cells. Whcn added to resting cells cultured without stimulant for at least 30 hours, ILl is no longer mitogenic. However, TL2 is able without further stimulation to enable proliferating lymphocytes to recycle and seems therefore to act also on cells leaving the M phase. With the same kind of combined cytofluorometric and 3H-thymidine uptake analysis. the effect of steroids on Con A-induced proliferation has been analyzed and found to be correlated with cdl viability rather than with ILl production, as recently postulated. GU.I.ES S. et a!. 1979. J. Immuno!. 123: 1624.