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.

The integrated processing of signals transduced by activating and inhibitory cell surface receptors regulates NK cell effector functions. Here, I review the structure, function, and ligand specificity of the receptors responsible for NK cell recognition.

Nk cell recognition

The biology of human natural killer-cell subsets.

NK cells are regulated by opposing signals from receptors that activate and inhibit effector function. While positive stimulation may be initiated by an array of costimulatory receptors, specificity is provided by inhibitory signals transduced by receptors for MHC class I. Three distinct receptor families, Ly49, CD94/NKG2, and KIR, are involved in NK cell recognition of polymorphic MHC class I molecules. A common pathway of inhibitory signaling is provided by ITIM sequences in the cytoplasmic domains of these otherwise structurally diverse receptors. Upon ligand binding and activation, the inhibitory NK cell receptors become tyrosine phosphorylated and recruit tyrosine phosphatases, SHP-1 and possibly SHP-2, resulting in inhibition of NK cell-mediated cytotoxicity and cytokine expression. Recent studies suggest these inhibitory NK cell receptors are members of a larger superfamily containing ITIM sequences, the inhibitory receptor superfamily (IRS).

Nk cell receptors

Human natural killer cells

We examined the antigenic and functional characteristics of human peripheral blood lymphocytes that differentially express the CD16 (Leu-11) and Leu-19 (NKH-1) antigens. Leu-19 is a approximately 220,000 daltons protein expressed on approximately 15% of freshly isolated peripheral blood lymphocytes. Within the Leu-19+ subset, three distinct populations were identified: CD3-,CD16+,Leu-19+ cells; CD3+,CD16-,Leu-19+ cells; and CD3-,CD16-,Leu-19bright+ cells. Both the CD3+,CD16-,Leu-19+ and CD3-,CD16+,Leu-19+ populations mediated non-major histocompatibility complex (MHC)-restricted cytotoxicity against the NK-sensitive tumor cell K562 and were large granular lymphocytes. CD3-,CD16+,Leu-19+ NK cells were the most abundant (comprising approximately 10% of peripheral blood lymphocytes) and the most efficient cytotoxic effectors. The finding that CD3+,Leu 19+ lymphocytes mediated cytotoxicity against K562 unequivocally demonstrates that a unique subset of non-MHC-restricted cytotoxic CD3+ T lymphocytes are present in the peripheral blood of unprimed, normal individuals. However, CD3+,CD16-,Leu-19+ cells comprised less than 5% of peripheral blood lymphocytes, and the cytotoxic activity of this subset was significantly less than CD3-,CD16+,Leu-19+ NK cells. Most CD3+,Leu-19+ T cells co-expressed the CD2, CD8, and CD5 differentiation antigens. The antigenic and functional phenotype of peripheral blood CD3+,Leu-19+ cytotoxic T lymphocytes corresponds to the interleukin 2-dependent CD3+ cell lines that mediate non-MHC-restricted cytotoxicity against NK-sensitive tumor cell targets. A small population of Leu-19bright+ lymphocytes lacking both CD3 and CD16 was also observed. This population (comprising less than 2% of peripheral blood lymphocytes) contained both large agranular lymphocytes and large granular lymphocytes. CD3-,CD16-,Leu-19bright+ lymphocytes also mediate non-MHC-restricted cytotoxicity. The relationship of these CD3-CD16-,Leu-19bright+ lymphocytes to CD3+ T cells or CD16+ NK cells is unknown.

The relationship of CD16 (Leu-11) and Leu-19 (NKH-1) antigen expression on human peripheral blood NK cells and cytotoxic T lymphocytes

The functional and phenotypic characteristics of cells in human peripheral blood that mediate "natural killer" (NK) cytolysis have been examined with the use of multiparameter flow cytometry analysis and cell sorting. Essentially, all lymphocytes expressing NK and ADCC activity reacted with the anti-Leu-11a monoclonal antibody. The Leu-11a antigen was expressed on cytotoxic large granular lymphocytes (LGL), neutrophils, and basophils, but was not present on B cells, mitogen-activated T lymphoblasts, or Leu-1+ and Leu 4+ resting T cells. Anti-Leu-11a antibody selectively inhibited the binding of FITC heat-aggregated IgG complexes to granulocytes and LGL, and it may recognize a type of Fc receptor on these cells. Two-color FACS cell sorting indicated the existence of four lymphocyte subsets defined by the expression of Leu-11a and Leu-7 antigens. The Leu-11a+, -7- cells were highly active in 4-hr NK assays with the use of 51Cr-labeled K562 as the target. In contrast, the Leu-11a-, -7+ cells demonstrated weak activity and the Leu-11a-, -7- cells demonstrated no activity. The function of the Leu 11a+, -7+ cells varied considerably among several individuals examined. Multiparameter analysis with the use of two-color flow cytometry was used to determine the relationship between the expression of these NK-associated antigens and T and B cell-associated markers. These data indicate that considerable heterogeneity exists within human peripheral lymphocytes with regard to cell phenotype and function, but that several defined cellular subsets can be clearly revealed by using multiparameter FACS analysis and sorting.

Subpopulations of human natural killer cells defined by expression of the Leu-7 (HNK-1) and Leu-11 (NK-15) antigens.

Lymphoid cells with natural killer (NK)-like function, morphology, and antigenic phenotype have been identified in a mixed lymphocyte culture generated by co-culture of human peripheral blood mononuclear cells with an allogeneic human B lymphoblastoid cell line CCRF-SB. The majority of these mixed lymphocyte (MLR)-response activated NK cells express the Leu-11 surface antigen, but do not express certain T cell-associated antigens (Leu-1, Leu-3, and Leu-4) or the mature monocyte specific antigen, Leu-M3. Unlike most freshly isolated Leu-11+ human NK cells, the MLR-activated Leu-11+ cells expressed class II major histocompatibility antigens, DR and DC. Concomitant with expression of class II gene products, the Leu-11+,DR+ NK cells demonstrate enhanced cytotoxicity against the NK-sensitive tumor cell line K562. The presence of mitotic cells in the Leu-11+,DR+ population and the acquisition of increased levels of transferrin receptor on the cell surface were further indicators of activation of these cells. The direct precursors of the MLR-activated Leu-11+,DR+ cell were Leu-11+ cells that lacked expression of another NK-associated antigen Leu-7, i.e., Leu-7-,11+. These studies provided a definitive identification of the "NK-like" cell in MLR cultures and thus allow quantitation and isolation of these cells for further study.

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Natural killer cells activated in a human mixed lymphocyte response culture identified by expression of Leu-11 and class II histocompatibility antigens.

The Leu-19 (NKH-1) antigen is expressed on human peripheral blood NK cells and a subset of peripheral blood cytotoxic T lymphocytes that kill "NK-sensitive" tumor cell targets without major histocompatibility complex restriction. In the present study, we demonstrate that the Leu-19 (NKH-1) antigen is also expressed on most interleukin 2 (IL 2) dependent T cell lines and clones that have been maintained in long term culture. The Leu-19 (NKH-1) antigen expressed on an antigen-specific, class I directed cytotoxic T lymphocyte cell line was an approximately 200,000 to 220,000 dalton protein, similar to Leu-19 (NKH-1) protein expressed on natural killer cells and KG1a, an immature stem cell leukemia cell line. Furthermore, Leu-19 (NKH-1) was expressed on both CD4+ and CD8+ IL 2 dependent T cell clones, and was present on both cytotoxic and non-cytotoxic T cell clones. Thus expression of Leu-19 (NKH-1) antigen on cultured cell lines does not directly correlate with cytotoxic function, antigenic specificity, or cell lineage.

Expression of Leu-19 (NKH-1) antigen on IL 2-dependent cytotoxic and non-cytotoxic T cell lines.

Cytotoxicity not restricted by the major histocompatibility complex (MHC) is mediated by two distinct types of lymphocyte: natural killer (NK) cells and non-MHC-restricted cytotoxic T lymphocytes (CTL). These two types of cytotoxic lymphocytes can be distinguished by antigenic phenotype, function, and molecular genetic studies. In human peripheral blood, NK cells are identified by expression of the Leu-19 and/or CD16 cell surface antigens, and lack of CD3/T cell antigen receptor (Ti) complex expression (i.e., CD3-,Leu-19+). Peripheral blood non-MHC-restricted CTL express both CD3 and Leu-19 (i.e., CD3+, Leu-19+, referred to as Leu-19+ T cells). Both Leu-19+ T cells and NK cells lyse "NK-sensitive" hematopoietic tumor cell targets, such as K562, without deliberate immunization of the host. However, most "NK activity" in peripheral blood is mediated by NK cells, because they are usually more abundant and more efficient cytotoxic effectors than Leu-19+ T cells. The cytolytic activity of both NK cells and Leu-19+ T cells against hematopoietic targets was enhanced by recombinant interleukin 2 (rIL 2). NK cells, but not peripheral blood Leu-19+ T cells, were also capable of lysing solid tumor cell targets after short-term culture in rIL 2. Southern blot analysis of NK cells revealed that both the T cell antigen receptor beta-chain genes and the T cell-associated gamma genes were not rearranged, but were in germ-line configuration. These findings indicate that NK cells are distinct in lineage from T lymphocytes and do not use the T cell antigen receptor genes for target recognition.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigenic, functional, and molecular genetic studies of human natural killer cells and cytotoxic T lymphocytes not restricted by the major histocompatibility complex.

An My Le

Papers

The relationship of CD16 (Leu-11) and Leu-19 (NKH-1) antigen expression on human peripheral blood NK cells and cytotoxic T lymphocytes

Subpopulations of human natural killer cells defined by expression of the Leu-7 (HNK-1) and Leu-11 (NK-15) antigens.

Natural killer cells activated in a human mixed lymphocyte response culture identified by expression of Leu-11 and class II histocompatibility antigens.

Expression of Leu-19 (NKH-1) antigen on IL 2-dependent cytotoxic and non-cytotoxic T cell lines.

Antigenic, functional, and molecular genetic studies of human natural killer cells and cytotoxic T lymphocytes not restricted by the major histocompatibility complex.