Publisher Summary Studies of cytotoxicity by human lymphocytes revealed not only that both allogeneic and syngeneic tumor cells were lysed in a non-MHC-restricted fashion, but also that lymphocytes from normal donors were often cytotoxic. Lymphocytes from any healthy donor, as well as peripheral blood and spleen lymphocytes from several experimental animals, in the absence of known or deliberate sensitization, were found to be spontaneously cytotoxic in vitro for some normal fresh cells, most cultured cell lines, immature hematopoietic cells, and tumor cells. This type of nonadaptive, non-MHC-restricted cellmediated cytotoxicity was defined as “natural” cytotoxicity, and the effector cells mediating natural cytotoxicity were functionally defined as natural killer (NK) cells. The existence of NK cells has prompted a reinterpretation of both the studies of specific cytotoxicity against spontaneous human tumors and the theory of immune surveillance, at least in its most restrictive interpretation. Unlike cytotoxic T cells, NK cells cannot be demonstrated to have clonally distributed specificity, restriction for MHC products at the target cell surface, or immunological memory. NK cells cannot yet be formally assigned to a single lineage based on the definitive identification of a stem cell, a distinct anatomical location of maturation, or unique genotypic rearrangements.

Biology of natural killer cells.

Many viruses, including retroviruses, are characterized by their specific cell tropism. Lymphadenopathy-associated virus (LAV) is a human lymphotropic retrovirus isolated from patients with acquired immune deficiency syndrome (AIDS) or related syndromes, that displays selective tropism for a subset of T lymphocytes defined by the expression of a surface glycoprotein of relative molecular mass 62,000 (62K) termed T4 (refs 6-8). This glycoprotein delineates a subset of T lymphocytes with mainly helper/inducer functions, while T lymphocytes of the reciprocal subset express a glycoprotein termed T8, have mainly cytotoxic/suppressor activities, and are unable to replicate LAV. Such a tropism may be controlled at the genomic level by regulatory sequences, as described for the human T-cell leukaemia viruses HTLV-I and -II (refs 2, 3). Alternatively or concomitantly, productive cell infection may be controlled at the membrane level, requiring the interaction of a specific cellular receptor with the virus envelope, as demonstrated recently for Epstein-Barr virus (EBV). Therefore, we have investigated whether the T4 molecule itself is related to the receptor for LAV. We report here that preincubation of T4+ lymphocytes with three individual monoclonal antibodies directed at the T4 glycoprotein blocked cell infection by LAV. This blocking effect was specific, as other monoclonal antibodies--such as antibody to histocompatibility locus antigen (HLA) class II or anti-T-cell natural killer (TNK) target--directed at other surface structures strongly expressed on activated cultured T4+ cells, did not prevent LAV infection. Direct virus neutralization by monoclonal antibodies was also ruled out. These results strongly support the view that a surface molecule directly involved in cellular functions acts as, or is related to, the receptor for a human retrovirus.

T-lymphocyte T4 molecule behaves as the receptor for human retrovirus LAV

NATURAL killer cells are a population of T-cell–receptornegative (CD3–) lymphocytes that spontaneously mediate the lysis of sensitive target cells. Natural killer cells are similar morphologically ...

Severe herpesvirus infections in an adolescent without natural killer cells.

Biology and clinical relevance of human natural killer cells.

Activation of a novel human transforming gene, ret, by DNA rearrangement

The initial characterization of two monoclonal antibodies directed at antigens selectively expressed on large granular lymphocytes (LGL) is reported in the present paper. These two reagents, anti-natural killer (NK) H1A and anti-NKH2, were obtained following immunization of mouse spleen cells with a cloned human NK cell line termed JT3. In fresh human peripheral blood, both anti-NKH1A and anti-NKH2 selectively reacted with cells that appeared morphologically as large granular lymphocytes. However, complement lysis studies and two color fluorescence analysis demonstrated that some LGL express both antigens and other cells express only NKH1A or NKH2. Functional analysis of these subsets indicated that the population of NKH1A+ cells contains the entire pool of NK active lymphocytes, whereas expression of NKH2 antigen appeared to delineate a unique subpopulation of LGL which, in a resting state, display a low degree of spontaneous cytotoxicity. Expression of NKH1A and NKH2 was also investigated using a series of nine well characterized human NK clones. All NK clones were found to be NKH1A+ and four out of nine also expressed NKH2. These results strongly supported the view that NKH1A is a "pan-NK" associated antigen, and indicated that at least a fraction of cloned NKH2 + LGL are strongly cytotoxic. Anti-NKH1A was shown to have the same specificity as the previously described N901 antibody and was found here to precipitate a 200,000-220,000-mol wt molecule in SDS-polyacrylamide gel electrophoresis (PAGE) analysis. Anti-NKH2 was specific for a structure that migrates at 60,000 mol wt in SDS-PAGE analysis under reducing conditions. Two color immunofluorescence analysis of NKH1A, NKH2, and other NK-associated antigens (Leu7 and B73.1) demonstrated variable degrees of coexpression of these antigens, which confirmed that NKH1A and NKH2 define distinct cell surface structures. Anti-NKH1A and anti-NKH2 appear to be useful reagents for characterizing LGL present in human peripheral blood and for identifying functionally relevant subsets within this heterogeneous population of cytotoxic lymphocytes.

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Generation of monoclonal antibodies to a human natural killer clone. Characterization of two natural killer-associated antigens, NKH1A and NKH2, expressed on subsets of large granular lymphocytes.

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.

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

In the present study, we have developed a monoclonal antibody termed anti-TNKTAR, able to block cytotoxicity mediated by a human natural killer (NK) clone termed JT9. Analysis of the functional effects of anti-TNKTAR indicated that alteration of the cytotoxic reactions resulted from the binding of the antibody to the membrane of target cells. In addition, it was shown that inhibition of cytotoxicity induced by anti-TNKTAR could be abolished by lectin approximation.



Immunoprecipitation experiments indicated that TNKTAR antigen is a heterodimeric structure which resolves as a single band at 140 kDa under nonreducing conditions and as two bands at approximately 97 kDa and 40 kDa under reducing conditions in sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis. This heterodimer is present on lymphocytes and monocytes in human peripheral blood and perhaps more importantly, the membrane density of TNKTAR antigen increases very early and strongly following lymphocyte activation. In addition, it was shown that TNKTAR is expressed on each single cultured cell line which has been tested, although the density of the antigen varies strongly from one cell line to another.



Even though the 140-kDa molecule was found to be widely distributed on activated cells, anti-TNKTAR had no blocking effects on cytotoxic reactions mediated by a series of either NK or cytotoxic T lymphocyte clones unrelated to JT9. In contrast, anti-TNKTAR blocked, in an identical fashion, cytotoxicity of JT9 and two additional clones, JT10 and JT11, against a series of 8 sensitive targets. JT9, JT10 and JT11 human cloned NK cell lines have been derived from peripheral blood of one individual donor drawn on month 0 (JT9), 12 (JT10) and 18 (JT11). Most importantly, these three clones initially selected for their capacity to kill K562 cells have been found to express the same 90-kDa clonotypic antigen receptor structure (termed NKTa) and display identical specificity when tested against a panel of randomly selected target cell lines.



We have previously demonstrated that a unique subset of NK active mature T lymphocytes interact with target cells via 90-kDa clonotypic determinants in a major histocompatibility complex-independent fashion. Taken together, the present data strongly supports the view that a surface antigen of 140 kDa, linked to cell activation, serves as a specific recognition structure at the target cell level for these NK-active T lymphocytes.

Identification of a 140-kDa activation antigen as a target structure for a series of human cloned natural killer cell lines.

Natural killer-like function of activated T lymphocytes: differential blocking effects of monoclonal antibodies specific for a 90-kDa clonotypic structure.

Agnes Brennan

Papers

Generation of monoclonal antibodies to a human natural killer clone. Characterization of two natural killer-associated antigens, NKH1A and NKH2, expressed on subsets of large granular lymphocytes.

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

Identification of a 140-kDa activation antigen as a target structure for a series of human cloned natural killer cell lines.

Natural killer-like function of activated T lymphocytes: differential blocking effects of monoclonal antibodies specific for a 90-kDa clonotypic structure.