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Mononuclear cells (10--20 X 10(6)) obtained from human peripheral blood by a standard Ficoll-Hypaque technique were suspended in RPMI 1640 media at 4 degrees C containing 10% foetal calf serum and 7-5% dimethyl sulphoxide (DMSO). Two-millilitre aliquots were cooled at -1 degree C/min in a Cryoson BV-4 programmed freezing system to -30 degrees C, then -5 degrees C/min to -80 degrees C and stored in liquid nitrogen vapor. On the day of testing, cell suspensions were thawed rapidly in a 37 degree C water bath. DMSO was diluted slowly out of the sample and cells resuspended in fresh RPMI 1640. It was found that frozen stored human lymphocytes (FSHL) demonstrated all the characteristics of fresh unfrozen cells. These included their ability to form spontaneous rosettes with sheep erythrocytes ('E' rosettes) and sheep erythrocyte--antibody--complement rosettes ('EAC' rosettes). The presence of surface immunoglobulins and Fc receptors were shown by membrane immunofluorescence to be comparable. In addition, the results show that FSHL respond to mitogens, specific antigens; act as both stimulators and responders in the mixed lymphocyte culture reaction; and exhibit cell-mediated lymphocytotoxicity following in vitro sensitization, or against antibody-coated target cells.

Monoclonal antibodies (DA-2 and CA-2) and xenoantisera (rabbit anti-human, p23,30) specific for HLA-DR framework determinants were added to primary and secondary mixed lymphocyte cultures. Although such antisera were shown to inhibit primary MLC, primed lymphocytes were much less sensitive to the blocking effects of these antibodies. In the studies shown here, the concentration of antibody required to inhibit primary MLC reactions was 0.1-1.5 micrograms/ml).

With anti-Ly antisera and complement it has been possible to demonstrate that cytotoxic effector cells generated in vitro against allogeneic cells carry the alloantigen Ly-5 and Ly-6. The studies show that antisera directed against the Ly-6 antigens, together with complement, eliminate essentially all of the killer cells, of the appropriate strain, while killing only 50 to 60% of Thy-1+ cells. Anti-Ly-5 antsera and complement lysed 55 to 65% of Thy-1+ cells and killed a significant portion, but not all, of the cytotoxic cells. The later finding was investigated in more detail since it suggested a degree of heterogeneity within the killer cell subpopulation. However, the data did not support that conclusion, at least for allogeneic killer cells.