Identification of a clonally restricted 90 kD heterodimer on two human cloned natural killer cell lines. Its role in cytotoxic effector function.

Abstract

The present studies were carried out to identify surface molecules involved in the cytotoxic effector function of a human natural killer (NK) clone termed JT9. This clone represents a mature T lymphocyte (T3+T8+T11+) mediating NK-like activity. Using JT9 for immunization, one monoclonal antibody termed anti-NKTa was selected that blocked the cytotoxicity of the clone towards K562 cells. Reactivity of anti-NKTa antibody was assessed using a large panel of lymphoid and nonlymphoid cells including a variety of cloned cell lines with either cytotoxic T lymphocyte (CTL) or NK-like activity. Among all cells tested, only two individual clones, JT9 and JT10, were found to express NKTa antigen. JT10 was derived independently from the same individual as JT9 and also represents a mature T cell (T3+T8+T11+) mediating NK-like activity. Like the Ti structure on CTL clones, the molecule defined by anti-NKTa was shown to be membrane associated with T3 in co-modulation experiments. Moreover, anti-NKTa precipitated a 90 kD heterodimeric structure in sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of 125I surface-labeled JT9 cells. The blocking capacity of anti-NKTa was evaluated in cytotoxicity assays using a panel of target cells. The influence of anti-T3 was tested in parallel and it was found that both anti-NKTa and anti-T3 blocked the cytotoxicity of the cloned cells against all targets. Given the potential role of 90 kD molecules as antigen-receptor structures, the specificity of the two NKTa+ NK clones was compared and found superimposable when assessed using 15 in vitro established cell lines. However, in contrast to conventional CTL clones, the expression of cytotoxicity by JT9 and JT10 was not dependent upon recognition of class I or class II major histocompatibility complex gene products on the target cells. In addition, the cytotoxicity of these T8+ NK active clones could not be blocked by anti-T8 antibodies. Taken together, the present data suggest that the specificity of one population of human NK active lymphocytes is determined by clonotypic structures. The NKTa determinant identified here appears to belong to the same family of molecules as Ti structures, previously identified on antigen-specific T lymphocytes.

Lack of gene rearrangement and mRNA expression of the beta chain of the T cell receptor in spontaneous rat large granular lymphocyte leukemia lines.

“…The results with these cells, however, are in marked contrast to those obtained in previous studies in the mouse (16) and human (17), which have suggested that a T cell receptor-like molecule is present in IL-2-propagated cloned cells with NK-like activity. Several possible reasons for this difference are: (a) differences between in vitro grown clones derived from normal ceils and in vivo derived tumors, (b) differences between species, or, most likely, (c) differences in the origin of the cytotoxic cells.…”

“…Several possible reasons for this difference are: (a) differences between in vitro grown clones derived from normal ceils and in vivo derived tumors, (b) differences between species, or, most likely, (c) differences in the origin of the cytotoxic cells. Since these previous studies (16,17) examined cytotoxic lines with T celllike as well as NK-like characteristics, it is quite possible that these lines were derived from T cells rather than LGL. Several groups (16,17) have reported that clones of cytotoxic T lymphocytes (CTL) can, under appropriate conditions, develop NK-like activity, either together with CTL activity or after the loss of specific reactivity.…”

“…Since these previous studies (16,17) examined cytotoxic lines with T celllike as well as NK-like characteristics, it is quite possible that these lines were derived from T cells rather than LGL. Several groups (16,17) have reported that clones of cytotoxic T lymphocytes (CTL) can, under appropriate conditions, develop NK-like activity, either together with CTL activity or after the loss of specific reactivity. The finding of T cell receptors on such CTL-derived clones would not be surprising; however, this might not be reflective of the receptor expression on in vivo derived LGL.…”

Proteoglycans in cell-mediated cytotoxicity. Identification, localization, and exocytosis of a chondroitin sulfate proteoglycan from human cloned natural killer cells during target cell lysis.

“…The activated PBMC were then washed four times to remove the mitogens, and were resuspended in RPMI 1640 supplemented with 2.5% human AB serum. After 40 h of incubation at 37°C, the cells were pelleted at 500 g, and the LCM was sterilized by passage through 0.45 #m filters and stored at -70°C until it was used for cloning or subcloning the JTB18 cells (21)(22)(23)(24). All NK cells used in these studies were subcloned at least four times at 100 cells/well on a feeder layer of allogeneic irradiated PBMC plus irradiated EBV-transformed B cells.…”

“…Cytotoxicity assays with human tumor lines used as targets were performed according to a standard chromium-release method (21)(22)(23)(24). The K562 cell line was established from a patient with chronic myeiogenous leukemia and the KG1 cell line from a patient with acute myeloblastic leukemia.…”