Natural killer cells are a recently discovered subpopulation of lymphoid cells that are present in most normal individuals of a range of mammalian and avian species. Natural killer cells have spontaneous cytolytic activity against a variety of tumor cells and some normal cells, and their reactivity can be rapidly augmented by interferon. They have characteristics distinct from other types of lymphoid cells and are closely associated with large granular lymphocytes, which comprise about 5 percent of blood or splenic leukocytes. There is increasing evidence that natural killer cells, with the ability to mediate natural resistance against tumors in vivo, certain virus and other microbial diseases, and bone marrow transplants, may play an important role in immune surveillance.

Recent evidence, has demonstrated an association between a subpopulation of peripheral blood mononuclear cells, morphologically identified as large granular lymphocytes (LGL), and natural killer (NK) activity. We have now evaluated more directly the role of LGL in both NK activity and antibody- dependent cellular cytotoxicity (ADCC), by using highly enriched populations of LGL, obtained by centrifugation of peripheral blood mononuclear cells on Percoll discontinuous density gradients. Both spontaneous and interferon- augmented NK and ADCC activities were exclusively associated with the LGL- enriched, low density fractions. The majority of LGL formed conjugates with NK-susceptible and antibody-coated target cells. Approximately 20 percent of small conventional lymphocytes also formed conjugates with the target cells for NK, but this was not associated with cytotoxic activity. Virtually all LGL were found to have receptors for the Fc portion of IgG (FcγR). The frequency of LGL among blood leukocytes was 2-6 percent. LGL could be enriched to an average purity of 95 percent by combining discontinuous density gradient centrifugation with subsequent adsorptions of the low density fractions on monolayers of immobilized immune complexes. About 50 percent of LGL were found to be FcγR-bearing T cells (T(G)), forming low affinity rosettes with sheep erythrocytes at 4 degrees C. Only 10-20 percent of LGL formed high affinity rosettes with sheep erythrocytes at 29 degrees C. LGL could be enriched to a purity of more than 90 percent by depleting high affinity rosette-forming cells from low density Percoll fractions. LGL were only a subpopulation of T(G) cells, because some lymphocytes with conventional morphology also adhered to the immobilized immune complex monolayers and formed high affinity rosettes with sheep erythrocytes. Separation of these cells from LGL by discontinuous density gradient centrifugation indicated that they are not cytotoxic, suggesting a morphological and functional subdivision of T(G) cells. The verification in this study that virtually all human NK and K cells have a characteristic morphology adds a useful parameter to the monitoring of human lymphocytes, and the ability to purify these cells by simple physical procedures should be invaluable in their further characterization.

A rapid in vivo clearance of tumor cells was found in normal mice following intravenous inoculation of [125l]dUrd‐labelled YAC‐1 and RBL‐5 cultured cell lines derived from lymphomas. The ability of mice to eliminate tumor cells from spleen, liver and lungs during the first 4 h, as evaluated by the recovery of radioactivity in these organs, was found to correlate with the level of natural killer (NK) cell reactivity in their spleen and lungs, as measured simultaneously in vitro in a shortterm 51Cr release assay (CRA). Lower recovery of radioactivity was found in mouse strains with high spontaneous levels of NK activity. The degree of clearance was also found to be age‐dependent and older mice of several strains, whose NK activity had declined to low levels, were less effective in eliminating tumor cells. In vivo treatment with interferon and interferon inducers (poly I:C, pyran copolymer, Corynebacterium parvum, murine sarcoma virus) increased the levels of NK activity in the spleen and lungs and also augmented the in vivo clearance of tumor cells from the lungs and liver. Immunopharmacological treatments with antimacrophage agents (silica, iota‐carrageenan, Seakem‐carrageenan), antineoplastic drugs (dexamethasone, cyclophosphamide, 5‐(3,‐3′dimethyl‐I‐triazeno)‐imidazole‐4‐carboxamide, 4‐amino‐L‐D‐arabinofuranosyl‐2‐(IH)‐pyrimidone, adriamycin) or irradiation (850 R γ‐ray) had comparable effects on both in vitro cytolytic activity and in vivo clearance of tumor cells. When the susceptibility to in vitro and in vivocytotoxicity by several other tumors was examined, the lines with detectable sensitivity to lysis by NK cells were found to be cleared in vivo to a greater degree in a high NK strain (CBA) than in a low NK strain (SJL). In contrast, NK‐resistant lines were cleared at approximately the same rate in both strains. However, the actual levels of in vivo clearance and the degree of difference between the strains for the various NK‐sensitive lines did not correlate well with their relative sensitivities to lysis in vitro. In the various situations in which the in vivo recovery of a particular NK‐sensitive line was studied relative to the levels of NK reactivity in the recipients, the best correlations were seen with clearance from the lungs. In several instances, clearance from the spleen did not correlate well with splenic NK activity. These data indicate that rapid in vivo clearance of radiolabelled NK‐sensitive tumor cell lines is appreciably influenced by levels of NK reactivity, but that other factors are probably also involved.

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