A model for the differentiation of human natural killer cells. Studies on the in vitro activation of Leu-11+ granular lymphocytes with a natural killer-sensitive tumor cell, K562.

A subpopulation of low density granular lymphocytes that express the natural killer (NK) cell-associated Leu-11 antigen (IgG Fc receptor) were stimulated directly by coculture with an NK-sensitive tumor cell, K562. T lymphocytes (Leu-11-) responded only weakly when cocultured with K562. The response of Leu-11+ cells apparently did not require exogeneous factors or accessory cells. The K562-activated cells retained expression of Leu-11 antigen, acquired activation antigens, and were highly cytotoxic against NK-sensitive and -insensitive tumor cells. Anti-IL-2 receptor monoclonal antibody minimally inhibited the activation of Leu-11+ cells by K562, but completely inhibited the phytohemagglutinin-induced activation of the Leu-11- cells from the same individual. Leu-11+ cells can be divided into Leu-7-11+ and Leu-7+11+ subpopulations using anti-Leu-7 antibody. These subsets were separated by two-color fluorescence-activated cell sorting and cocultured with K562. Proliferation by Leu-7-11+ cells was significantly greater than by Leu-11+7+ cells. Leu-7+11- granular lymphocytes and T lymphocytes (Leu-7-11-) typically proliferated only weakly when cocultured with K562. A proportion of the Leu-7-11+ cells acquired Leu-7 antigen after stimulation with K562, whereas the phenotype of Leu-7+11+, Leu-7+11-, and Leu-7-11- subsets was unaffected. These results demonstrate a developmental relationship between the Leu-7-11+ and Leu-7+11+ lymphocytes and suggest that Leu-7 antigen may be expressed late in the differentiation pathway of NK cells. The direct activation of highly purified Leu-11+ cells by coculture with K562 provides an in vitro model with which to study the activation and maturation of human NK cells

Phosphotyrosines in the killer cell inhibitory receptor motif of NKB1 are required for negative signaling and for association with protein tyrosine phosphatase 1C.

NKB1 is one member of a growing family of killer cell inhibitory receptors (KIR). It is expressed on natural killer (NK) cells and T cells, and has been shown to inhibit cytolytic functions of these cells upon interacting with its ligand, HLA-B (Bw4). We demonstrate here that the cytoplasmic region of NKB1 is capable of inhibiting T cell activation in Jurkat cells. The tyrosine phosphorylation of the NKB1 KIR consensus motif, YxxL(x)26 YxxL, induces an association with the protein tyrosine phosphatase 1C (PTP1C). Importantly, mutation of both tyrosines in the motif abolished the inhibitory functions of NKB1 and abrogated PTP1C association. Mutational analysis of the individual tyrosines suggest that the membrane proximal tyrosine may play a crucial role in mediating the inhibitory signal. These results demonstrate that KIR can not only inhibit cytolytic activity, but can also negatively regulate T cell receptor activation events that lead to downstream gene activation, and further supports a model that implicates PTP1C as a mediator in the KIR inhibitory signal

Constitutive expression of high affinity interleukin 2 receptors on human CD16-natural killer cells in vivo.

The majority of human NK cells express low affinity IgG Fc receptors (CD16+), whereas a minor subset of NK cells lack Fc receptor expression (CD16-). In contrast to CD16+ NK cells that express only p75 IL-2 receptors, CD16- NK cells constitutively co-express both p75 and p55 IL-2 receptors in vivo and preferentially respond to low concentrations of IL-2 with increased cytolytic activation and proliferation. Scatchard analysis demonstrated the presence of approximately 1,200 high affinity (approximately 25 pM kD) and approximately 9,600 intermediate affinity (approximately 2 nM kD) IL-2 receptors on CD16- NK cells. CD16+ NK cells expressed only a single intermediate affinity IL-2 receptor of approximately 1.9 nM kD (approximately 9,000 sites per cell). The IL-2 binding data thus substantiated the phenotypic and functional studies and definitively show that the differential responsiveness of CD16- and CD16+ NK cells to IL-2 is manifested through different affinity IL-2 receptors

Concomitant induction of the cell surface expression of Ia determinants and accessory cell function by a murine macrophage tumor cell line.

This study demonstrates that an uncharacterized soluble factor produced in concanavalin A-induced rat spleen cell suspensions has the capacity to induce the increased expression of cell surface H-2K and H-2D molecules and the expression of I-region gene products on murine monocyte-macrophage lineage tumors that are not Ia positive in the absence of the factor. In parallel with induction of serologically defined Ia specificities, Ia-induced WEHI-3 macrophage tumor cells are capable of providing accessory cell function in stimulating IL-2 production by T-T hybridomas that are activated in a major histocompatibility complex-restricted, antigen-dependent fashion. The uninduced Ia-negative WEHI-3 tumor cells do not trigger a comparable response in this assay system

Human natural killer cell committed thymocytes and their relation to the T cell lineage.

Recent studies have demonstrated that mature natural killer (NK) cells can be grown from human triple negative (TN; CD3-, CD4-, CD8-) thymocytes, suggesting that a common NK/T cell precursor exists within the thymus that can give rise to both NK cells and T cells under appropriate conditions. In the present study, we have investigated human fetal and postnatal thymus to determine whether NK cells and their precursors exist within this tissue and whether NK cells can be distinguished from T cell progenitors. Based on the surface expression of CD56 (an NK cell-associated antigen) and CD5 (a T cell-associated antigen), three phenotypically distinctive populations of TN thymocytes were identified. CD56+, CD5-; CD56-, CD5-, and CD56-, CD5+. The CD56+, CD5- population of TN thymocytes, although displaying a low cytolytic function against NK sensitive tumor cell targets, were similar in antigenic phenotype to fetal liver NK cells, gave rise to NK cell clones, and were unable to generate T cells in mouse fetal thymic organ cultures (mFTOC). This population of thymocytes represents a relatively mature population of lineage-committed NK cells. The CD56-, CD5- population of TN thymocytes were similar to thymic NK cells in antigenic phenotype and NK cell clonogenic potential. Clones derived from this population of TN thymocytes acquired CD56 surface expression and NK cell cytolytic function. CD56-, CD5- TN thymocytes thus contain a novel population of NK cell-committed precursors. The CD56-, CD5- population of TN thymocytes also contains a small percentage of CD34+ cells, which demonstrate no in vitro clonogenic potential, but possess T cell reconstituting capabilities in mFTOC. The majority of TN thymocytes do not express CD56, but coexpress CD34 and CD5. These CD56-, CD5+, CD34+ cells demonstrate no NK or T cell clonogenic potential, but are extremely efficient in repopulating mFTOC and differentiating into CD3+, CD4+, CD8+ T cells. The results of this investigation have identified NK cells and NK cell precursors in the human thymus and have shown that these cell types are unable to differentiate along the T cell lineage pathway. Thus, while a common NK/T cell progenitor likely exists, once committed to the NK cell lineage these cells no longer have the capacity to develop along the T cell developmental pathway

Regulation of T cell lymphokine production by killer cell inhibitory receptor recognition of self HLA class I alleles.

The killer cell inhibitory receptors (KIRs) are surface glycoproteins expressed by natural killer (NK) and T cells that specifically recognize defined groups of polymorphic human histocompatibility leukocyte antigen (HLA) class I molecules. Interactions between KIRs on NK or T cells and major histocompatibility complex (MHC) class I molecules on potential target cells inhibit cell-mediated cytotoxicity, presumably by delivering a negative signal preventing lymphocyte activation. In this study we examined whether KIRs also regulate cytokine production induced in response to T cell receptor-dependent T cell activation. CD4+ and CD8+ T cell clones were stimulated by bacterial superantigens in the presence or absence of monoclonal antibodies (mAbs) against the KIR NKB1 or MHC class I molecules, and production of tumor necrosis factor alpha and interferon gamma was evaluated. When bacterial superantigen was presented by an autologous antigen-presenting cell (APC) to a KIR+ T cell clone, cytokine production was always enhanced in the presence of anti-MHC class I mAb. Similarly, anti-KIR mAb also augmented cytokine production, provided that the APC expressed a HLA class I allele recognized by the KIR. These results suggest that recognition of autologous MHC class I molecules by KIR+ T cells provides a regulatory mechanism acting to modulate the potency of their responses to antigenic challenge

Identity of Leu-19 (CD56) leukocyte differentiation antigen and neural cell adhesion molecule.

Neural cell adhesion molecule (N-CAM) is a membrane glycoprotein expressed on neural and muscle tissues that is involved in homotypic adhesive interactions. We have demonstrated that N-CAM also is expressed on hematopoietic cells, and is recognized by the anti-Leu-19 mAb. Leu-19 is preferentially expressed on NK cells and T lymphocytes that mediate MHC-unrestricted cytotoxicity, but is also present on some myeloid leukemia cell lines. On NK cells, T cells, the KG1a.5 hematopoietic cell line, and a neuroblastoma cell line, Leu-19 is a approximately 140-kD polypeptide with N-linked carbohydrates and abundant sialic acid residues. Sequential immunoprecipitation and peptide mapping demonstrated that the Leu-19 and N-CAM molecules expressed on leukocyte and neuroblastoma cell lines are similar structures. These findings suggest that the Leu-19 antigen on leukocytes may be involved in cell adhesion, analogous to the function on N-CAM on neural cells

CD28 interaction with B7 costimulates primary allogeneic proliferative responses and cytotoxicity mediated by small, resting T lymphocytes.

Engagement of the CD3/T cell antigen receptor complex on small, resting T cells is insufficient to trigger cell-mediated cytotoxicity or to induce a proliferative response. In the present study, we have used genetic transfection to demonstrate that interaction of the B7-BB1 B cell activation antigen with the CD28 T cell differentiation antigen costimulates cell-mediated cytotoxicity and proliferation initiated by either anti-CD2 or anti-CD3 monoclonal antibody (mAb). Moreover, a B7-negative Burkitt's lymphoma cell line that fails to stimulate an allogeneic mixed lymphocyte response is rendered a potent stimulator after transfection with B7. The mixed leukocyte reaction proliferative response against the B7 transfectant is inhibited by either anti-CD28 or B7 mAb. We also demonstrate that freshly isolated small, resting human T cells can mediate anti-CD3 or anti-CD2 mAb-redirected cytotoxicity against a murine Fc receptor-bearing mastocytoma transfected with human B7. These preexisting cytotoxic T lymphocytes in peripheral blood are present in both the CD4 and CD8 subsets, but are preferentially within the CD45RO+ "memory" population. While small, resting T cells apparently require costimulation by CD28/B7 interactions, this requirement is lost after T cell activation. Anti-CD3 initiates a cytotoxic response mediated by in vitro cultured T cell clones in the absence of B7 ligand. The existence of functional cytolytic T cells in the small, resting T cell population may be advantageous in facilitating rapid responses to immune challenge

Evidence that the T cell antigen receptor may not be involved in cytotoxicity mediated by gamma/delta and alpha/beta thymic cell lines.

After culture in IL-2, thymocytes expressing either TCR-alpha/beta or -gamma/delta acquired the ability to lyse hematopoietic and solid tumor cell targets without deliberate immunization or apparent restriction by the MHC. Moreover, TCR-alpha/beta- and TCR-gamma/delta-bearing thymic cell lines demonstrated an essentially identical spectrum of cytolysis against several tumor cell targets. Cytotoxicity was not inhibited by antibodies against CD3 or CD2 and modulation of the CD3/TCR complex also failed to affect cytotoxicity. Thus, non-MHC-restricted cytotoxicity can be mediated by thymocytes with either TCR-alpha/beta or TCR-gamma/delta, but the TCR may not be responsible for target recognition