Lymphokine-activated killer cells. Analysis of progenitors and effectors.

IL-2 has been examined for its ability to regulate lymphokine-activated killer (LAK) activity. IL-2 is a potent activator of cytolytic activity against a wide array of tumor cells, including those from fresh autologous and allogeneic tumors. Using subpopulations of lymphoid cells that were separated on Percoll density gradients, and subsequently purified by immunoadsorbance, studies were performed to examine the phenotypes of progenitor and effector cells of human LAK cells and to compare them with the phenotype of activated NK cells. From these studies, it was evident that several lymphoid subsets, including CD3+, CDw16- and CD3-, CDw16+ cells could mediate LAK lysis of fresh tumor cells. Our examination of the kinetics of activation revealed that CDw16+, NKH1+ (NK-active) cells were maximally activated by 1-2 d. In contrast, CD3+ cells appeared not to achieve maximal cytolytic activity against fresh and cultured tumor cells until days 2-3. Using limiting-dilution frequency analysis, we showed that a large percentage of cytolytically active progenitors was present among the CDw16+, NKH1+ cells. The progenitor and effector cell frequencies appear to be 10-50 times higher in these populations compared to CD3+ cells. In addition, the selective blockage by mAb to the CD3 determinant of the T cell receptor complex indicated that these two effector cell phenotypes relied on different receptors to mediate their cytotoxic activity against tumor cells. Therefore, the accumulated data suggest that there is not a single unique progenitor of LAK activity, but rather that multiple subsets of lymphocytes become cytotoxic in response to IL-2. However, the NK cell population forms the largest single component of LAK cell activity in human peripheral blood.

Characteristics of human large granular lymphocytes and relationship to natural killer and K cells.

Recent evidence, has demonstrated an association between a subpopulation of peripheral blood mononuclear cells, morphologically identified as large granular lymphocytes (LGL), and natural killer (NK) activity. We have now evaluated more directly the role of LGL in both NK activity and antibody- dependent cellular cytotoxicity (ADCC), by using highly enriched populations of LGL, obtained by centrifugation of peripheral blood mononuclear cells on Percoll discontinuous density gradients. Both spontaneous and interferon- augmented NK and ADCC activities were exclusively associated with the LGL- enriched, low density fractions. The majority of LGL formed conjugates with NK-susceptible and antibody-coated target cells. Approximately 20 percent of small conventional lymphocytes also formed conjugates with the target cells for NK, but this was not associated with cytotoxic activity. Virtually all LGL were found to have receptors for the Fc portion of IgG (FcgammaR). The frequency of LGL among blood leukocytes was 2-6 percent. LGL could be enriched to an average purity of 95 percent by combining discontinuous density gradient centrifugation with subsequent adsorptions of the low density fractions on monolayers of immobilized immune complexes. About 50 percent of LGL were found to be FcgammaR-bearing T cells (T(G)), forming low affinity rosettes with sheep erythrocytes at 4 degrees C. Only 10-20 percent of LGL formed high affinity rosettes with sheep erythrocytes at 29 degrees C. LGL could be enriched to a purity of more than 90 percent by depleting high affinity rosette-forming cells from low density Percoll fractions. LGL were only a subpopulation of T(G) cells, because some lymphocytes with conventional morphology also adhered to the immobilized immune complex monolayers and formed high affinity rosettes with sheep erythrocytes. Separation of these cells from LGL by discontinuous density gradient centrifugation indicated that they are not cytotoxic, suggesting a morphological and functional subdivision of T(G) cells. The verification in this study that virtually all human NK and K cells have a characteristic morphology adds a useful parameter to the monitoring of human lymphocytes, and the ability to purify these cells by simple physical procedures should be invaluable in their further characterization.

Receptor-induced death in human natural killer cells: involvement of CD16.

Propriocidal regulation of T cells refers to apoptosis induced by interleukin 2 (IL-2) activation with subsequent antigen receptor stimulation. We examined whether natural killer (NK) cells exhibited cytokine- and ligand-induced death similar to activated T cells. Peripheral NK cells were examined for ligand-induced death using antibodies to surface moieties (CD2, CD3, CD8, CD16, CD56), with and without prior activation of IL-2. Only those NK cells stimulated first with IL-2 and then with CD16 exhibited ligand-induced death; none of the other antibody stimuli induced this phenomenon. Next we examined various cytokines (IL-2, IL-4, IL-6, IL-7, IL-12, IL-13, interferon alpha and gamma) that can activate NK cells and determined if CD16-induced killing occurred. Only IL-2 and IL-12 induced NK cell death after occupancy of this receptor by aggregated immunoglobulin or by cross-linking with antireceptor antibody. The CD16-induced death was inhibited by herbimycin A, indicating that cell death was dependent upon protein tyrosine kinases. Identical to T cells, the form of cell death for NK cells was demonstrated to be receptor-induced apoptosis. Overall these data indicate that highly activated NK cells mediate ligand-induced apoptosis via signaling molecules like CD16. Whereas the propriocidal regulation of T cells is antigen specific, this is not the case for NK cells due to the nature of the receptor. The clinical implications of this finding are considered.

Chédiak-Higashi gene in humans I. Impairment of natural-killer function.

Natural-killer (NK)-cell function was profoundly depressed in donors homozygous for the Chediak-Steinbrinck-Higashi syndrome (C-HS) gene when compared with age- and sex-matched normals. This apparent defect was not simply a result of a delayed response because little cytolysis was evident in kinetics experiments even after 24 h of incubation. NK cells from C-HS donors failed to lyse adherent (MDA, CEM, and Alab) or nonadherent (K562 and Molt-4) tumor cell lines or nontransformed human fetal fibroblasts. Therefore, the apparent C-HS defect was not a result of a shift in target selectivities. In addition, the depressed reactivity did not appear to be a result of suppressor cells or factors because: (a) enriched NK populations (nonadherent lymphocytes bearing receptors for the Fc portion of IgG) from C-HS donors were low in NK-cell function, (b) C-HS mononuclear cells did not inhibit the cytotoxicity of normal cells in coculture experiments, and (c) cells from the C-HS donors remained poorly reactive even after culture for up to 7 d. The nature of the defective NK activity in C-HS patients is not clear but may lie within the lytic mechanism rather than at the level of the recognition structure or population size because the frequency of target-binding cells was normal. In vitro NK activity could be partially restored by interferon treatment. Combined with the results presented in the following paper (4), these observations suggest that the C-HS gene causes a selective immunodeficiency disorder, mainly involving NK cells. This finding, in conjunction with the high incidence of spontaneous possibly malignant, lymphoproliferative disorders in these patients, may have important implications regarding the theory of immune surveillance mediated by NK cells.

Mechanism of target cell recognition by natural killer cells: characterization of a novel triggering molecule restricted to CD3- large granular lymphocytes.

In an attempt to identify a molecule in target recognition by CD3- large granular lymphocytes (LGL), we have generated a rabbit antiidiotypic (anti-ID) serum against a monoclonal antibody (mAb 36) that reacted with the cell membrane of K562. Flow cytometry analysis demonstrated that the anti-ID serum bound selectively to CD3- LGL and that F(ab')2 fragments of the anti-ID serum blocked both target cell binding and lysis by NK cells. Stimulation of CD3- LGL with F(ab')2 fragments resulted in the release of serine esterases and the secretion of interferon gamma. Furthermore, anti-ID F(ab')2 antibodies crosslinked to anti-DNP F(ab')2 mediated directed cytotoxicity of a non-natural killer (NK)-susceptible mouse target (YAC-1) via this surface ligand. These functional reactivities were only removed by adsorption with the specific idiotype. Protein analysis showed that the anti-ID serum immunoprecipitated 80-, 110-, and 150-kD proteins. Using this anti-ID, a partial cDNA was cloned and an antipeptide antiserum was made against the portion of the predicted amino acid sequence that corresponded to a portion of the ID binding region. This antipeptide serum exhibited similar functional and biochemical reactivities to those observed with the anti-ID serum. These data suggest that the cell surface moiety recognized by the anti-ID and anti-p104 is novel and is selectively involved in both recognition and triggering of NK-mediated lytic function.

Cloning and functional characteristics of murine large granular lymphocyte-1: a member of the Ly-49 gene family (Ly-49G2)

Large granular lymphocyte (LGL) 1 is a cell surface glycoprotein expressed on a subset (50%) of C57BL/6 natural killer (NK) cells. Immunoprecipitation experiments reveal that the LGL-1 protein exists as a disulfide-linked 40-kD homodimer. Functional studies of LGL-1+ cells indicate that selected H-2d target cells are not lysed efficiently by these interleukin (IL)-2-cultured NK cells. These findings suggested that LGL-1 may be a member of the Ly-49 gene family. Here we report the molecular cloning of the LGL-1 cDNA from a severe combined immunodeficient-adherent lymphokine-activated killer cell library transfected into Cos-7 cells and find LGL-1 to be homologous to the Ly-49 gene at both the nucleotide (85%) and amino acid levels (73%). Sequencing of our LGL-1 cDNA has revealed it to be nearly identical to the Ly-49G2 cDNA recently isolated by cross-hybridization with an Ly-49 probe. LGL-1 represents a type II transmembrane protein of 267 amino acids with its carboxyl end exposed extracellularly. The LGL-1 protein contains 11 highly conserved cysteine residues and a 25-amino acid transmembrane region. Southern blot analysis demonstrates that there are a number of homologous genes in mouse DNA that hybridize strongly to LGL-1. Northern analyses using poly A+ RNA from LGL-1+ NK cells indicate that LGL-1 is expressed as a 1.4 kb mRNA. Two-color flow cytometry analysis (FCA) of C57BL/6 splenic NK cells demonstrates that LGL-1 and Ly-49 label overlapping subsets of cells. FCA identifies four subsets of NK cells as defined by LGL-1 versus Ly-49 staining. We have sorted these individual subsets, expanded them in IL-2, and performed cytotoxicity experiments to determine their target cell profiles in relation to class I expression. Results of these studies are complex, but indicate that Ly-49 may not be the only molecule that recognizes class I as an inhibitory signal for cytotoxicity. LGL-1+ cells also fail to lyse several H-2d-expressing tumor targets and concanavalin A lymphoblasts from BALB/c but not C57BL/6 mice. This inhibition of lysis by LGL-1+ NK cells is negated by addition of monoclonal antibody (mAb) 4D11 that recognizes the LGL-1 protein. When mAbs to the class I molecules H-2Dd and H-2Ld (alpha 1 alpha 2 domains only) are added to cytotoxicity assays, LGL-1+ cells lyse H-2d targets very effectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Interleukin 12 (IL-12) induces tyrosine phosphorylation of JAK2 and TYK2: differential use of Janus family tyrosine kinases by IL-2 and IL-12.

Interleukin (IL-12) has many effects on the function of natural killer and T cells, and is important in the control of cell-mediated immunity. IL-2 and IL-12 display many similar activities, yet each also induces a distinct set of responses. A human IL-12 receptor subunit has recently been cloned and, like the IL-2R beta and IL-2R gamma, is a member of the hematopoietic receptor superfamily; however, the molecular mechanisms of IL-12 action are unknown. In this report we show that IL-12 and IL-2 induce tyrosine phosphorylation of distinct members of the Janus (JAK) family of protein tyrosine kinases in human T lymphocytes. IL-12, but not IL-2, stimulates the tyrosine phosphorylation of TYK2 and JAK2, whereas JAK1 and JAK3, which are phosphorylated in response to IL-2, are not phosphorylated after IL-12 treatment. The use of distinct but related JAK family tyrosine kinases by IL-12 and IL-2 may provide a biochemical basis for their different biological activities.

The Ly-49D receptor activates murine natural killer cells.

Proteins encoded by members of the Ly-49 gene family are predominantly expressed on murine natural killer (NK) cells. Several members of this gene family have been demonstrated to inhibit NK cell lysis upon recognizing their class I ligands on target cells. In this report, we present data supporting that not all Ly-49 proteins inhibit NK cell function. Our laboratory has generated and characterized a monoclonal antibody (mAb) (12A8) that can be used to recognize the Ly-49D subset of murine NK cells. Transfection of Cos-7 cells with known members of the Ly-49 gene family revealed that 12A8 recognizes Ly-49D, but also cross-reacts with the Ly-49A protein on B6 NK cells. In addition, 12A8 demonstrates reactivity by both immuno-precipitation and two-color flow cytometry analysis with an NK cell subset that is distinct from those expressing Ly-49A, C, or G2. An Ly-49D+ subset of NK cells that did not express Ly-49A, C, and G2 was isolated and examined for their functional capabilities. Tumor targets and concanovalin A (ConA) lymphoblasts from a variety of H2 haplotypes were examined for their susceptibility to lysis by Ly-49D+ NK cells. None of the major histocompatibility complex class I-bearing targets inhibited lysis of Ly-49D+ NK cells. More importantly, we demonstrate that the addition of mAb 12A8 to Ly-49D+ NK cells can augment lysis of Fc gamma R+ target cells in a reverse antibody-dependent cellular cytotoxicity-type assay and induces apoptosis in Ly-49D+ NK cells. Furthermore, the cytoplasmic domain of Ly-49D does not contain the V/Ix-YxxL immunoreceptor tyrosine-based inhibitory motif found in Ly-49A, C, or G2 that has been characterized in the human p58 killer inhibitory receptors. Therefore, Ly-49D is the first member of the Ly-49 family characterized as transmitting positive signals to NK cells, rather than inhibiting NK cell function.

IL-4 and IL-13 induce Lsk, a Csk-like tyrosine kinase, in human monocytes.

Lsk is a protein tyrosine kinase with homology to the COOH-terminal Src kinase (Csk). Unlike Csk that is ubiquitously expressed, Lsk has limited tissue distribution. Here we have examined the expression and regulation of Lsk and Csk in peripheral human monocytes. We have found that Lsk mRNA and protein were not expressed in resting monocytes but were induced by treatment with interleukin 4 (IL-4) or IL-13 but not by interferon gamma (IFN-gamma) or IL-2. In fact, IFN-gamma, but not IL-2, efficiently blocked Lsk induction by IL-4 or IL-13. In contrast, Csk was constitutively present in human monocytes and was upregulated by IFN-gamma but not by IL-4 or IL-13. These results suggest that despite their structural similarities, Lsk and Csk may play distinct regulatory roles in monocyte functions elicited by cytokines, with Lsk functioning specifically within the context of a Th2-type immune response.

Augmentation of organ-associated natural killer activity by biological response modifiers. Isolation and characterization of large granular lymphocytes from the liver.

Natural killer (NK) activity in the rat and human has been attributed to cells having the morphology of large granular lymphocytes (LGL). However, this association has been less clear in the mouse, largely because of difficulties in obtaining highly enriched populations of LGL from normal spleen and blood. We have previously observed that the administration of the biological response modifier (BRM) maleic anhydride divinyl ether (MVE-2) strongly augmented NK activity in lung and liver, and the augmented NK activity coincided with increased resistance to the formation of experimental metastases in these organs. The degree of NK augmentation was most striking in the liver, an unexpected and previously unreported observation. In the present study, both MVE-2 or Corynebacterium parvum induced a dramatic augmentation of liver NK activity, which reached maximum levels 3-5 d after treatment. This augmentation of NK activity in the liver coincided with a large increase in the number of lymphoid cells with the morphological characteristics of LGL that could be isolated from enzymatically digested suspensions of perfused liver. The yield of LGL per liver following BRM treatment corresponded to a 10-50-fold increase as compared to normal mice. LGL were purified from these enzymatically digested suspensions of perfused liver by depletion of adherent cells on nylon wool columns and subsequent enrichment for low-density lymphoid cells by fractionation on Percoll density gradients. The enrichment of LGL correlated with greatly increased NK activity against YAC-1. Conversely, the higher-density fractions were depleted of both LGL and NK activity. This increase in NK activity in the liver was suppressed by in vivo treatment with anti-asialo GM1 (asGM1) serum. This treatment also resulted in a corresponding reduction in both the total number and percentage of LGL. By flow cytometry analysis, the phenotype of the majority of these highly cytolytic LGL isolated from the livers of BRM-treated mice were asGM1+, Thy-1+, Ly-5+, Qa-5+, Mac-1+, and Gma-1+, whereas these LGL were Ly-1-, Lyt-2-, L3T4-, and surface Ig-. We conclude that the livers of BRM-treated mice can provide a rich source of highly active mouse LGL that could be used for further characterization of this lymphocyte subset. Further, these studies imply a potential for BRM therapy of neoplastic or viral diseases through augmentation of organ-associated immune responses.

Mouse Ly-49D recognizes H-2Dd and activates natural killer cell cytotoxicity.

Although activation of natural killer (NK) cytotoxicity is generally inhibited by target major histocompatibility complex (MHC) class I expression, subtle features of NK allorecognition suggest that NK cells possess receptors that are activated by target MHC I. The mouse Ly-49D receptor has been shown to activate NK cytotoxicity, although recognition of MHC class I has not been demonstrated previously. To define Ly-49D-ligand interactions, we transfected the mouse Ly-49D receptor into the rat NK line, RNK-16 (RNK.mLy-49D). As expected, anti- Ly-49D monoclonal antibody 12A8 specifically stimulated redirected lysis of the Fc receptor- bearing rat target YB2/0 by RNK.mLy-49D transfectants. RNK.mLy-49D effectors were tested against YB2/0 targets transfected with the mouse MHC I alleles H-2Dd, Db, Kk, or Kb. RNK.mLy-49D cells lysed YB2/0.Dd targets more efficiently than untransfected YB2/0 or YB2/0 transfected with Db, Kk, or Kb. This augmented lysis of H-2Dd targets was specifically inhibited by F(ab')2 anti-Ly-49D (12A8) and F(ab')2 anti-H-2Dd (34-5-8S). RNK.mLy-49D effectors were also able to specifically lyse Concanavalin A blasts isolated from H-2(d) mice (BALB/c, B10.D2, and DBA/2) but not from H-2(b) or H-2(k) mice. These experiments show that the activating receptor Ly-49D specifically interacts with the MHC I antigen, H-2Dd, demonstrating the existence of alloactivating receptors on murine NK cells.

Interleukin 2 induction of pore-forming protein gene expression in human peripheral blood CD8+ T cells.

Our studies have analyzed pore-forming protein (PFP) mRNA expression in resting and stimulated human peripheral blood CD3- large granular lymphocytes (LGL), CD3+ T cells, and their CD4+ or CD8+ subsets. Signals that stimulate T cells to develop cytotoxic activity (i.e., IL-2 or OKT-3 mAb) led to the induction of PFP mRNA in T cells. The data indicated that IL-2 directly increased PFP mRNA in the CD8+ subset of T cells, in the absence of new DNA or protein synthesis. Abrogation of IL-2-induced PFP mRNA expression and cytotoxic potential of T cells by the anti-p75 IL-2 receptor mAb suggested that low numbers of p75 IL-2 receptors on CD8+ T cells were capable of transducing signals responsible for these IL-2-induced effects. The induction of T cell PFP mRNA via CD3, using OKT-3 mAb, was less rapid but greater than that caused by IL-2; however, a combination of PMA and ionomycin, which bypasses crosslinking of the TCR/CD3 complex, could not mimic this increase in PFP mRNA levels in T cells. The role of second messenger systems in regulating PFP mRNA expression remains to be determined. In contrast, high constitutive PFP mRNA expression was observed in CD3- LGL and these mRNA levels could not be enhanced by stimulation with IL-2. The cytotoxic potential of peripheral blood T cells and LGL induced in response to IL-2 correlated with IL-2-induced PFP mRNA levels in these cells and was consistent with PFP being one of several important molecules involved in the effector function of cytotoxic lymphocytes.

Expression and role of p75 interleukin 2 receptor on human monocytes.

We investigated the expression of IL-2R subunits in human monocytes using the TU27 mAb, which recognizes the p75 chain, and anti-Tac mAb, which recognizes the p55 moiety of the IL-2R. We found that p75 but not p55 is constitutively expressed in more than 90% of fresh human monocytes. Antibody to p75, but not to p55, inhibited the activation of monocytes to a cytotoxic stage induced by IL-2 but did not block IFN-gamma-induced cytotoxicity. Our data demonstrate that the p75 chain is expressed on human monocytes and is involved in the activation of monocytes by IL-2.

Recombinant human interferon-inducible protein 10 is a chemoattractant for human monocytes and T lymphocytes and promotes T cell adhesion to endothelial cells.

The human cytokine interferon-inducible protein 10 (IP-10) is a small glycoprotein secreted by activated T cells, monocytes, endothelial cells, and keratinocytes, and is structurally related to a family of chemotactic cytokines called chemokines. Although this protein is present in sites of delayed-type hypersensitivity reactions and lepromatous leprosy lesions, the biological activity of IP-10 remains unknown. We report here that recombinant human IP-10 stimulated significant in vitro chemotaxis of human peripheral blood monocytes but not neutrophils. Recombinant human IP-10 also stimulated chemotaxis of stimulated, but not unstimulated, human peripheral blood T lymphocytes. Phenotypic analysis of the stimulated T cell population responsive to IP-10 demonstrated that stimulated CD4+ and CD29+ T cells migrated in response to IP-10. This resembles the biological activity of the previously described T cell chemoattractant RANTES. Using an endothelial cell adhesion assay, we demonstrated that stimulated T cells pretreated with optimal doses of IP-10 exhibited a greatly enhanced ability to bind to an interleukin 1-treated endothelial cell monolayer. These results demonstrate that the IP-10 gene encodes for an inflammatory mediator that specifically stimulates the directional migration of T cells and monocytes as well as potentiates T cell adhesion to endothelium.

Natural killer cells: their roles in defenses against disease.

Natural killer cells are a recently discovered subpopulation of lymphoid cells that are present in most normal individuals of a range of mammalian and avian species. Natural killer cells have spontaneous cytolytic activity against a variety of tumor cells and some normal cells, and their reactivity can be rapidly augmented by interferon. They have characteristics distinct from other types of lymphoid cells and are closely associated with large granular lymphocytes, which comprise about 5 percent of blood or splenic leukocytes. There is increasing evidence that natural killer cells, with the ability to mediate natural resistance against tumors in vivo, certain virus and other microbial diseases, and bone marrow transplants, may play an important role in immune surveillance.

Ly49 C/I-dependent NKT cell-derived IL-10 is required for corneal graft survival and peripheral tolerance.

Similar to their activity on NK cells, Ly49 molecules play a pivotal role in influencing how NKT cells respond. It is known that Ly49 C/I is an inhibitory receptor capable of down-modulating proliferation, IFN-gamma response, and cytotoxic activity in cells that express it. In a model of peripheral tolerance induced via the eye, we observed that Ly49 C/I-positive, invariant NKT cells were required. To test if the NK inhibitory receptor functionally contributed to tolerance development, we used blocking antibody, in vivo and in vitro, to interfere with the development of antigen-specific suppression. A result of blocking ligation of Ly49 C/I inhibitory receptor prevented NKT cell production of IL-10 and the subsequent development of tolerance. Ly49 C/I-blocking antibodies also prevented corneal graft survival, a phenomenon dependent on eye-induced tolerance. Furthermore, in the presence of TCR stimulation, cross-linking of Ly49 C/I on CD4(+) NKT cells stimulated an increase in IL-10 mRNA and a decrease in IFN-gamma. The concept of Ly49 inhibitory receptors regulating immune reactivity to self by regulating immune activity of individual cells is thus expanded to include a role for the inhibitory receptors in the more global process of peripheral tolerance to foreign antigens.

Antitumor effects of interleukin-7 and adoptive immunotherapy on human colon carcinoma xenografts.

The antitumor properties of recombinant human IL-7 (rhIL-7) on a human tumor was evaluated by engrafting a human colon carcinoma into immunodeficient mice and then treating the mice with rhIL-7 and adoptively transferred human peripheral blood T cells. It was found that rhIL-7 alone had no effect on the survival of the tumor-bearing recipients. However, the combination of rhIL-7 and human T cells significantly promoted the survival of the recipients compared with mice receiving either treatment by itself. When the surviving mice were analyzed 6 mo later for the degree of human cell engraftment, the recipients receiving both rhIL-7 and human T cells had greater numbers of human CD8+ T cells in the spleens. However, the human T cells recovered from the surviving mice showed low lytic activity against the tumor in vitro. Supernatants from human T cells cultured with the tumor and rhIL-7 in vitro were found to inhibit tumor growth and were demonstrated to contain high levels of IFN-gamma. Antibodies to IFN-gamma neutralized the growth inhibition of the tumor both in vitro and in vivo demonstrating that the in vivo mechanism underlying the antitumor effects of this regimen was partly dependent on the production of IFN-gamma by the T cells and not their cytolytic capability. Interestingly, systemic administration of rhIFN-gamma to tumor-bearing mice yielded little antitumor effect suggesting that adoptive immunotherapy with rhIL-7 was superior possibly because of the continuous local release of the cytokines. Therefore, rhIL-7 may be of clinical use as an antineoplastic agent and the human/mouse model is a potentially important preclinical model for in vivo evaluation of the efficacy of this and other immunotherapies.

Mechanisms of target cell killing by natural killer cells.

Understanding of the lytic process of NK cells has been greatly advanced in the past decade. However, the definition of the primary event required for lysis has been elusive. The recent enthusiasm about the granule exocytosis/PFP model of membrane damage has been tempered by the multitude of factors that cytotoxic effector cells can secrete and the observation of rapid nuclear degradation before membrane damage which is not well explained by the PFP model. Logically, one would expect that this array of cytotoxic, cytostatic and immunoregulatory factors are secreted for a purpose, and should, therefore, be involved in the lysis of target cells. Further studies are necessary to define the role of each of these factors in plasma membrane and nuclear damage. Additional experiments to determine whether these two events are related will be necessary. On the basis of information currently available, one could hypothesize that after recognition of foreign cells, the NK cell is not single-minded in its delivery of lethal factors. The end result is that target cell lysis may occur because of many factors acting not only on the target cell itself but also on other components of the host immune response.

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.

Human natural lymphocyte effector cells: definition, analysis of activity, and clinical effectiveness.

Lymphokine-activated killing has enormous potential in the treatment of cancer. Lymphoid effectors have the potential to recognize and lyse a wide array of tumor cells, a phenomenon which has been seen in vitro and to some extent in vivo. However, studies have indicated that complexity exists not only in the nature of the lymphocyte that can be activated ex vivo for therapeutic use, but also in the delivery of such therapy in a clinical setting. This review attempts to deal with both issues. The nature of the cells mediating lymphokine-activated killer activity, their heterogeneous phenotype, their activation requirements, and a hypothetical mechanism of action are discussed. In addition, previous clinical studies are reviewed and key issues are raised that need to be addressed in upcoming clinical trials.