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

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

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

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

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

The Bw4 public epitope of HLA-B molecules confers reactivity with natural killer cell clones that express NKB1, a putative HLA receptor.

Although inhibition of natural killer (NK) cell-mediated lysis by the class I HLA molecules of target cells is an established phenomenon, knowledge of the features of class I molecules which induce this effect remains rudimentary. Using class I alleles HLA-B*1502 and B*1513 which differ only at residues 77-83 which define the Bw4 and Bw6 serological epitopes, we tested the hypothesis that the presence of the Bw4 epitope on class I molecules determines recognition by NKB1+ NK cells. HLA-B*1513 possesses the Bw4 epitope, whereas B*1502 has the Bw6 epitope. Lysis by NKB1+ NK cell clones of transfected target cells expressing B*1513 as the only HLA-A, -B, or -C molecule was inhibited, whereas killing of transfectants expressing B*1502 was not. Addition of an an anti-NKB1 monoclonal antibody reconstituted lysis of the targets expressing B*1513, but did not affect killing of targets bearing B*1502. The inhibitory effect of B*1513 could be similarly prevented by the addition of an anti-class I monoclonal antibody. These results show that the presence of the Bw4 epitope influences recognition of HLA-B molecules by NK cells that express NKB1, and suggest that the NKB1 molecule may act as a receptor for Bw4+ HLA-B alleles. Sequences outside of the Bw4 region must also affect recognition by NKB1+ NK cells, because lysis of transfectants expressing HLA-A*2403 or A*2501, which possess the Bw4 epitope but are in other ways substantially different from HLA-B molecules, was not increased by addition of the anti-NKB1 antibody. Asparagine 86, the single site of N-linked glycosylation on class I molecules, is in close proximity to the Bw4/Bw6 region. The glycosylation site of the Bw4-positive molecule B*5801 was mutated, and the mutant molecules tested for inhibition of NKB1+ NK cells. Inhibition that could be reversed by addition of the anti-NKB1 monoclonal antibody was observed, showing the presence of the carbohydrate moiety is not essential for class I recognition by NKB1+ NK cell clones

The T cell antigen receptor complex expressed on normal peripheral blood CD4-, CD8- T lymphocytes. A CD3-associated disulfide-linked gamma chain heterodimer.

IL-2-dependent cell lines were established from normal peripheral blood T lymphocytes that express neither CD4 nor CD8 differentiation antigens. CD3+,4-,8- cell lines from 15 different donors failed to react with WT31, an mAb directed against the T cell antigen receptor alpha/beta heterodimer. Anti-Leu-4 mAb was used to isolate the CD3/T cell antigen receptor complex from 125I-labeled CD3+,4-,8- (WT31-) T cells. Using detergent conditions that preserved the CD3/T cell antigen receptor complex, an approximately 90 kD disulfide-linked heterodimer, composed of approximately 45- and approximately 40- (or approximately 37-) kD subunits, was coimmunoprecipitated with the invariant 20-29-kD CD3 complex. Analysis of these components by nonequilibrium pH gradient electrophoresis indicated that the approximately 40-kD and approximately 37-kD subunits were similar, and quite distinct from the more basic approximately 45-kD subunit. None of these three subunits reacted with an antibody directed against a beta chain framework epitope. Heteroantiserum against a T cell receptor gamma chain peptide specifically reacted with both the approximately 37- and approximately 40-kD CD3-associated proteins, but not with the approximately 45-kD subunit. CD3+,4-,8- cells failed to transcribe substantial amounts of functional 1.3-kb beta or 1.6-kb alpha mRNA, but produced abundant 1.6-kb gamma mRNA. Southern blot analysis revealed that these CD3+,4-,8- cell lines rearranged both gamma and beta genes, and indicated that the populations were polyclonal. The expression of a CD3-associated disulfide-linked heterodimer on CD3+,4-,8- T cell lines established from normal, adult peripheral blood contrasts with prior reports describing a CD3-associated non-disulfide-linked heterodimer on CD3+/WT31- cell lines established from thymus and peripheral blood obtained from patients with immunodeficiency diseases. We propose that this discrepancy may be explained by preferential usage of the two C gamma genes in T lymphocytes