John R. Ortaldo

Natural killer cells. Characteristics and regulation of activity.

Recently there has been increasing recognition of natural cell-mediated cytotoxicity as a potentially important antitumor effector mechanism in addition to that of specifically immune T cells and of activated macrophages. Although natural cellular cytotoxicity was first recognized only a few years ago (Herberman et al. 1973,1974, McCoy et al. 1973b, Oldham et al. 1973, Rosenberg et al. 1972,1974), there has already been extensive research in many laboratories on the nature of the effector cells, the possible mechanisms of cytotoxicity, the factors regulating the levels of reactivity, and the relevance of natural immunity to in vivo resistance against tumor growth and immune surveillance. A principal component of natural cell-mediated cytotoxicity in rodents and man has been found to be a particular subpopulation of lymphocytes which have been termed natural killer (NK) cells. We have recently reviewed in detail much of the available information on NK cells (Herberman & Holden 1978). In this paper we will only summarize our views on the characteristics of NK cells and focus on a few issues of current interest in our laboratory.

Beta chemokines costimulate lymphocyte cytolysis, proliferation, and lymphokine production.

We report here the ability of the β chemokines MIP‐1α, MP‐1β, RANTES, and MCP‐1 to enhance some lymphocyte effector functions. Initial studies focused on the effects of chemokines on human and mouse cytotoxic T lymphocyte (CTL)–and natural killer (NK) cell–specific cytolytic responses. The results demonstrate that β chemokines are capable of augmenting mouse and human CTL and human NK–but not lymphokine‐activated killer cell– or antibody‐dependent cell cytotoxicity–specific cytolytic responses. Neutralisation analysis utilizing integrin‐specific antibodies revealed that CTL/NK–tumor cell conjugate formation is required for chemokine‐induced killing. In addition, both CTLs and NK cells incubated with various β chemokines were induced to degranulate and release granule‐derived serine esterases, suggesting that chemokines may be important costimulators of CTL and NK cell degranulation and may thus augment local target cell destruction. Chemokines also modulate antigen‐driven T cell proliferative responses as well as effects on lymphokine production. Many of the β chemokines were found to potentiate human and mouse antigen‐specific Th1 and Th2 clone activation promoting cellular proliferation and the release of various lymphokines. This chemokine‐mediated T cell proliferation was chemokine and antigen dose dependent as well as clone dependent. Chemokine pretreatment analyses with T cells and antigen‐presenting cells (APCs) revealed that chemokines up‐regulate both T cell and APC functions. Costimulation assays using immobilized anti‐CD3 monoclonal antibody–coated plates and purified human and mouse T cells and T cell clones in the presence of various chemokines also exhibited enhanced proliferation and lymphokine secretion. This costimulation was interleukin‐2 dependent and required the presence of free extracellular calcium. Examination of chemokine‐treated APCs revealed that the T cell costimulatory molecule B7‐1 was induced by various β chemokines. Neutralization of endogenously produced chemokines with specific antibodies during an antigen‐specific T cell response blocked cellular proliferation, suggesting that the chemokines have an autocrine and/or paracrine role in antigen‐induced T cell proliferative responses. Together, these results suggest that chemokines play a significant role in the activation of polyclonal as well as antigen‐specific helper and cytotoxic T cells during the genesis of an immune response.

Combined Natural Killer Cell and Dendritic Cell Functional Deficiency in KARAP/DAP12 Loss-of-Function Mutant Mice

KARAP/DAP12 is a transmembrane polypeptide with an intracytoplasmic immunoreceptor tyrosine-based activation motif (ITAM). KARAP/DAP12 is associated with several activating cell surface receptors in hematopoietic cells. Here, we report that knockin mice bearing a nonfunctional KARAP/DAP12 ITAM present altered innate immune responses. Although in these mice NK cells are present and their repertoire of inhibitory MHC class I receptors is intact, the NK cell spectrum of natural cytotoxicity toward tumor cell targets is restricted. KARAP/DAP12 loss-of-function mutant mice also exhibit a dramatic accumulation of dendritic cells in muco-cutaneous epithelia, associated with an impaired hapten-specific contact sensitivity. Thus, despite its homology with CD3zeta and FcRgamma, KARAP/DAP12 plays a specific role in innate immunity, emphasizing the nonredundancy of these ITAM-bearing polypeptides in hematopoietic cells.

Glucocorticoid amplifies IL‐2‐dependent expansion of functional FoxP3+CD4+CD25+ T regulatory cells in vivo and enhances their capacity to suppress EAE

IL‐2 is crucial for the production of CD4+CD25+ T regulatory (Treg) cells while important for the generation of effective T cell‐mediated immunity. How to exploit the capacity of IL‐2 to expand Treg cells, while restraining activation of T effector (Teff) cells, is an important and unanswered therapeutic question. Dexamethasone (Dex), a synthetic glucocorticoid steroid, has been reported to suppress IL‐2‐mediated activation of Teff cells and increase the proportion of Treg cells. Thus, we hypothesized that glucocorticoids may be useful as costimulants to amplify IL‐2‐mediated selective expansion of Treg cells. We show in this study that short‐term simultaneous administration of Dex and IL‐2 markedly expanded functional suppressive Foxp3+CD4+CD25+ T cells in murine peripheral lymphoid tissues. In a myelin oligodendrocyte glycoprotein‐induced experimental autoimmune encephalomyelitis (EAE) mouse model, we observed that splenic CD4+CD25+ T cells failed to suppress the proliferation of CD4+CD25– T cells. Pretreatment with Dex/IL‐2 remarkably increased the proportion of CD4+FoxP3+ cells and partially restored the function of splenic CD4+CD25+ T cells, and inhibited the development of EAE. Therefore, the combination of glucocorticoid and IL‐2, two currently used therapeutics, may provide a novel approach for the treatment of autoimmune diseases, transplant rejection and graft‐vs.‐host disease.

The nuclear factor YY1 suppresses the human gamma interferon promoter through two mechanisms: inhibition of AP1 binding and activation of a silencer element.

Our group has previously reported that the nuclear factor Yin-Yang 1 (YY1), a ubiquitous DNA-binding protein, is able to interact with a silencer element (BE) in the gamma interferon (IFN-gamma) promoter region. In this study, we demonstrated that YY1 can directly inhibit the activity of the IFN-gamma promoter by interacting with multiple sites in the promoter. In cotransfection assays, a YY1 expression vector significantly inhibited IFN-gamma promoter activity. Mutation of the YY1 binding site in the native IFN-gamma promoter was associated with an increase in the IFN-gamma promoter activity. Analysis of the DNA sequences of the IFN-gamma promoter revealed a second functional YY1 binding site (BED) that overlaps with an AP1 binding site. In this element, AP1 enhancer activity was suppressed by YY1. Since the nuclear level of YY1 does not change upon cell activation, our data support a model that the nuclear factor YY1 acts to suppress basal IFN-gamma transcription by interacting with the promoter at multiple DNA binding sites. This repression can occur through two mechanisms: (i) cooperation with an as-yet-unidentified AP2-like repressor protein and (ii) competition for DNA binding with the transactivating factor AP1.

Isolation of human and rat natural killer cells

We describe a method for the purification of human and rat large granular lymphocytes (LGL), which are known to be the mediators of natural killer (NK) activity in these species. Plastic non-adherent and nylon wool passed blood mononuclear cells were separated into 7 fractions by discontinuous density gradient centrifugation on Percoll. Low density cells were highly enriched in LGL (up to 85% purity), whereas high density cells were typical small and medium sized lymphocytes devoid of NK activity. Human LGL could further be enriched by depleting high affinity sheep erythrocyte rosette-forming cells from the LGL-enriched Percoll fractions (resulting in up to greater than 90% purity). One critical variable in the separation technique was osmolarity, since the separation did not work optimally, if 290 mOsmoles/kg H2O in the Percoll solution was exceeded.

In vivo administration of IL-18 can induce IgE production through Th2 cytokine induction and up-regulation of CD40 ligand (CD154) expression on CD4+ T cells.

IL‐18 is considered to be a strong cofactor for CD4+ T helper 1 (Th1) cell induction. We have recently reported that IL‐18 can induce IL‐13 production in both NK cells and T cells in synergy with IL‐2 but not IL‐12, suggesting IL‐18 can induce Th1 and Th2 cytokines when accompanied by the appropriate first signals for T cells. We have now found that IL‐18 can act as a cofactor to induce IL‐4, IL‐10 and IL‐13 as well as IFN‐γ production in T cells in the presence of anti‐CD3 monoclonal antibodies (mAb). IL‐18 can rapidly induce CD40 ligand (CD154) mRNA and surface expression on CD4+ but not CD8+ T cells. The administration of IL‐18 alone in vivo significantly increased serum IgE levels in C57BL/6 (B6) and B6 IL‐4 knockout mice. Furthermore, the administration of IL‐18 plus IL‐2 induced approximately 70‐fold and 10‐fold higher serum levels of IgE and IgG1 than seen in control B6 mice, respectively. IgE and IgG1 induction in B6 mice by administration of IL‐18 plus IL‐2 was eliminated by the pretreatment of mice with anti‐CD4 or anti‐CD154, but not anti‐CD8 or anti‐NK1.1 mAb. These results suggest that IL‐18 can induce Th2 cytokines and CD154 expression, and can contribute to CD4+ T cell‐dependent, IL‐4‐independent IgE production.

Effect of human recombinant interferon on cytotoxic activity of natural killer (NK) cells and monocytes

Abstract Studies have been performed on the in vitro immunologic effects of homogeneous recombinant human leukocyte interferon, IFLrA. Large granular lymphocytes, enriched for natural killer (NK) cell activity, were pretreated wtih IFLrA or natural interferon preparations and then tested for augmentation of NK activity and of antibody-dependent cell-mediated cytoxicity (ADCC). Monocytes were tested for cytolytic and cytostatic activity in 48–72 hr radioisotopic assays performed in the presence or absence of interferon. Treatment with IFLrA caused significant augmentation of NK, ADCC, and monocyte-mediated cytotoxic activities. Even 10 units of IFLrA induced augmentation of NK activity, and 100 units or more boosted monocyte-mediated activity. The effects in each of these assays were species-specific, with no detectable effects on the activity of mouse effector cells. These results indicate that homogeneous recombinant interferon has potent in vitro immunomodulating effects and thus provide a basis for carefully examining the in vivo effects of this protein on host defenses in forthcoming clinical trials with cancer patients.

The ever-expanding Ly49 gene family: repertoire and signaling.

Summary: The mouse lectin‐related Ly49 family and the human killer cell Ig‐like receptor (KIR) family represent structurally distinct, yet functionally analogous, class I MHC receptors that are expressed on natural killer cells and some T cells. The functional similarity of these two families has been borne out by the demonstration of identical signal transduction pathways associated with each receptor family. The Ly49 family therefore provides a useful model system to study the role of this class of receptors in the regulation of the immune system. Recent data relating to the Ly49 repertoire in several mouse strains has revealed an additional evolutionary parallel between KIR and Ly49 receptor families. There is now an appreciation of the variation in the number and type of Ly49s expressed in different mouse strains, similar to the previously demonstrated differences in the number of KIR genes found in humans. This review summarizes the current members of the Ly49 gene family, their MHC class I recognition and associated signal transduction pathways.

Natural cytotoxicity uncoupled from the Syk and ZAP-70 intracellular kinases

The intracellular signals that trigger natural cytotoxicity have not been clearly determined. The Syk and ZAP-70 tyrosine kinases are essential for cellular activation initiated by B and T cell antigen receptors and may drive natural killer (NK) cell cytotoxicity via receptors bearing immunoreceptor tyrosine-based activation motifs (ITAMs). However, we found that, unlike B and T cells, NK cells developed in Syk−/−ZAP-70−/− mice and, despite their nonfunctional ITAMs, lysed various tumor targets in vitro and eliminated tumor cells in vivo, including those without NKG2D ligands. The simultaneous inhibition of phosphotidyl inositol 3 kinase and Src kinases abrogated the cytolytic activity of Syk−/−ZAP-70−/− NK cells and strongly reduced that of wild-type NK cells. This suggests that distinct and redundant signaling pathways act synergistically to trigger natural cytotoxicity.