Simultaneous Occurrence of Immunoglobulin-Dependent and Immunoglobulin-Independent Mechanisms in Natural Cytotoxicity of Human Lymphocytes
Authors: Pape Gerd R. , Troye Marita , Axelsson Bernt , Perlmann Peter 
Published: December 2022

Peripheral blood lymphocytes from healthy human donors are cytotoxic to various tumor cells in vitro. The mechanism of this natural cytotoxicity was studied in a 51Cr-release assay. The possible involvement of antibody in the reactions was studied by addition 1) of fragments of rabbit or goat antibodies to human IgG (a-Ig), 2) of aggregated IgG, or 3) of protein A from Staphylococcus aureus (SpA). Six Fab- and one F(ab′)2-preparations were used as inhibitors. The preparations were made specific for either F(ab′)2-determinants or for F(ab′)2-and Fc-determinants by adsorption to Sepharose-bound human F(ab′)2 or Sepharose-bound IgG and subsequent elution. One goat Fab preparation specific for Fc-determinants of human IgG1 was also used. The specificity of the inhibitors was ascertained by several tests. When coupled to Sepharose and reacted with lysates of lymphocytes, surface labeled with 125I, the only components bound were light and heavy chains of B cell-derived immunoglobulin as established by SDS-PAGE and autoradiography. When present in the incubation mixtures, all fragments inhibited natural cytotoxicity to a variety of tumor target cells, including cells from the myeloid cell line K562. No inhibition was obtained when either lymphocytes or target cells were pretreated with the inhibitors and washed before the assay. Depletion of B cells from the lymphocyte preparations by fractionation through anti-immunoglobulin columns did not consistently decrease their cytotoxicity, which was as strongly inhibited by the a-Ig fragments as in the presence of B cells. Inhibition ranged from 15 to ∼80% but was incomplete in almost all instances even when the inhibitor concentration was sufficient to give maximal inhibition. Trypsin treatment of the lymphocytes abolished natural cytotoxicity but this was restored by incubation at 37°C overnight. This restored cytotoxicity was inhibited by a-Ig to a degree similar to that seen before trypsin treatment.

Depletion of lymphocytes with Fc-receptors (FcR+) by fractionation on immune complex columns abolished natural cytotoxicity. In contrast, depletion of FcR+ target cells (K562) had no effect. Addition of aggregated IgG, which completely abolished ADCC in model systems, caused a significant but incomplete inhibition of natural cytotoxicity. SpA added over a wide dose range either increased or did not change natural cytotoxicity. When added to the same target cells but in the presence of anti-target cell antibodies, ADCC was only weakly or not at all inhibited by SpA. This was different from the strong inhibiting effect of SpA on ADCC in a model system in which the target cells (bovine erythrocytes) were not lysed by the lymphocytes in the absence of external antibodies.

The results suggest that a significant part, but not all, of the natural cytotoxicity studied herein reflects K cell reactions, probably induced by natural antibodies, carried over from the lymphocyte donor and/or released into the incubation mixtures during the assay. However, part of the cytotoxicity is immunoglobulin independent. The relative importance of these two mechanisms, which usually occur simultaneously, varies for different lymphocyte donors.