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.

The Immunological Genome Consortium has generated a public resource (www.immgen.org) that provides a compendium of gene expression profiles of ∼270 leukocyte subsets in the mouse. This effort established carefully standardized operating procedures that resulted in a transcriptional dataset of unprecedented comprehensiveness and quality. The findings have been detailed recently in a series of publications providing molecular insights into the development, heterogeneity, and/or function of these cellular lineages and distinct subpopulations. Here, we review the key findings of these studies, highlighting what has been gained and how the knowledge can be used to accelerate progress toward a comprehensive understanding of the immune system. © 2013 Elsevier Ltd. All rights reserved.

In vitro culture of human peripheral blood lymphocytes in IL-2 results in the generation of cytotoxic cells that can lyse fresh and cultured solid tumor cells, as well as hematopoietic tumor cell lines, without deliberate immunization or MHC restriction. This has been referred to as the lymphokine activated killer (LAK) phenomenon. Here, we show that the majority of this activity is mediated by NK cells that express the Leu-19 (NKH-1) antigen, but do not express CD3. The precursor of this effector population also expressed the phenotype CD3-, Leu-19+. Peripheral blood CD3+ T lymphocytes contributed little to the LAK phenomenon, although low levels of non-MHC restricted cytotoxicity against hematopoietic tumor cell targets were mediated by a subset of CD3+ T lymphocytes that coexpressed the Leu-19 antigen. These studies clearly indicated that the LAK phenomenon is not mediated by a unique LAK cell, but is mediated mainly by IL-2-activated peripheral blood NK cells.

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.

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.

A subset of peripheral blood T lymphocytes coexpressing CD3 and IgG Fc receptors (FcR) (CD16/Leu-11 antigen) have been identified, isolated, and functionally characterized. The CD3+, CD16+ cells were established in short-term culture using growth medium containing interleukin 2 (IL-2). Both the freshly isolated cells and the cultured cell line stably expressed the CD3+, CD16+ phenotype. Furthermore, a majority of these T cells lacked either CD4 or CD8 expression. Like in vitro-activated cytotoxic T lymphocytes and natural killer (NK) cells, the CD3+, CD16+ cells showed numerous azurophilic granules. Although these cells failed to mediate significant levels of NK cell-mediated cytotoxicity even after stimulation with IL-2, they efficiently functioned as effectors of antibody-dependent cellular cytotoxicity (ADCC). The Ig isotype specificity of the ADCC was analyzed using an isotype switch-variant family of a murine anti-HLA monoclonal antibody (mAb). Similar to the CD3-, CD16+ NK cell population, the CD3+, CD16+ T cells preferentially used the IgG2a antibody to mediate ADCC. The CD3+, CD16+ cells demonstrated a proliferative response when cocultured with either a NK-sensitive tumor cell line, K562, or a NK-insensitive B lymphoblastoid cell line, CCRF-SB. The response against CCRF-SB was significantly inhibited by anti-IL-2 receptor antibody, whereas the response against K562 was only partially diminished. Cytotoxicity was also induced in the CD3+, CD16+ population by the presence of anti-CD3 mAb, indicating that cytotoxicity can be triggered by stimulation via the CD3-T cell antigen receptor complex. By isolating these CD3+, CD16+ cells from the peripheral blood of a normal, healthy individual, it has been possible to extensively study the morphology, antigenic phenotype, and functional behavior of this unique subset of T lymphocytes expressing IgG FcR.

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.

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.

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.

CD3+ T lymphocytes expressing neither CD4 nor CD8 antigens exist in normal human peripheral blood in low frequency (approximately 3% of lymphocytes). The CD3+,4-,8- phenotype was stably maintained after in vitro culture in IL-2. Culture of CD3+,4-,8- cells in only rIL-2 generated cytotoxic T cells that lysed NK-sensitive and NK-insensitive tumor cell targets without MHC restriction. These experiments clearly show that phenotypically and functionally competent T cells expressing neither CD4 nor CD8 are present in normal peripheral blood.