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The proliferative capacity of T cells infiltrating human tumors is known to be impaired, possibly through their interaction with tumor. Here we demonstrate that soluble products derived from renal cell carcinoma (RCC-S) explants but not normal kidney can inhibit an IL-2-dependent signaling pathway that is critical to T cell proliferation. A major target of the immunosuppression was the IL-2R-associated protein tyrosine kinase, Janus kinase 3 (Jak3). RCC-S suppressed basal expression of Jak3 and its increase following stimulation with anti-CD3/IL-2. Jak3 was most sensitive to suppression by RCC-S; however, reduction in expression of p56(lck), p59(fyn), and ZAP-70 was observed in some experiments. Expression of other signaling elements linked to the IL-2R (Jak1) and the TCR (TCR-zeta, CD3-epsilon, and phospholipase C-gamma) were minimally affected. In naive T cells, RCC-S also partially blocked induction of IL-2R alpha-, beta- and gamma-chain expression when stimulating via the TCR/CD3 complex with anti-CD3 Ab. To determine whether RCC-S suppressed IL-2-dependent signaling, primed T cells were employed since RCC-S had no effect on IL-2R expression but did down-regulate Jak3 expression and, to a lesser degree, p56(lck) and p59(fyn). Reduction in Jak3 correlated with impaired IL-2-dependent proliferation and signal transduction. This included loss of Jak1 kinase tyrosine phosphorylation and no induction of the proto-oncogene, c-Myc. These findings suggest that soluble products from tumors may suppress T cell proliferation through a mechanism that involves down-regulation of Jak3 expression and inhibition of IL-2-dependent signaling pathways.

CD94 (Kp43) is a member of the human C-type lectin superfamily encoding type II membrane glycoproteins expressed on NK cells and a subset of T cells. Ligation of CD94 has been shown to either potentiate or inhibit NK cell proliferation and cytolytic effector function. Here we show that CD94 ligation triggers apoptosis in IL-2-primed NK cells. Evidence for CD94-induced apoptosis includes: 1) chromatin condensation as measured by increased fluorescence of Hoechst dye, 2) induction of DNA fragmentation, and 3) characteristic morphology by transmission electron microscopy. IL-2 priming (at least 12 h) is required for activation-induced NK cell death triggered by CD94. Activation-induced NK cell death triggered by CD94 ligation is extremely rapid (DNA fragmentation is first observed at 120 min). Unlike activation-induced T cell death, it is not inhibited by neutralizing Abs reactive with TNF-alpha or Fas ligand. Our results suggest that CD94 may play a role in the elimination of activated NK cells during the transition from the innate to the Ag-specific immune response.

IL-12 stimulates both T and NK cells and is pivotal in the development of the Th1 immune response. In this work, we show that an interaction between CD2 and CD58 on activated T cells and monocytes, respectively, regulates the T cell response to IL-12. B cells provide little IL-12-specific costimulation, and this correlates with the low level of CD58 on B cells relative to monocytes and the lack of significant up-regulation in response to IFN-gamma or PHA activation. CHO cell transfectants expressing CD58 at a level comparable with that found on monocytes restore IL-12 responsiveness to APC-depleted T cells. This effect is not observed with CHO cells expressing CD48, a second CD2 ligand with a low avidity for CD2 relative to CD58. Thus, in addition to augmenting adhesion between T cells and their cognate APCs and facilitating TCR-triggered activation, the CD2-CD58 interaction uniquely optimizes the T cell response to IL-12.

CD56dim NK cells, which comprise approximately 90% of human peripheral blood NK cells, respond to IL-2 with cytokine production, up-regulation of functionally relevant surface molecules, and augmented cytolytic activity. Nevertheless, CD56dim NK cells proliferate poorly in response to IL-2 alone. We found that other NK cell mitogens, including IL-4, IL-7, and IL-12, also induced little proliferation of CD56dim NK cells. Indeed, IL-2 stimulated at least 10-fold more NK cell proliferation than did IL-4, IL-7, or IL-12. In contrast, leukocyte-conditioned medium (LCM) induced two- to threefold greater proliferation of CD56dim NK cells than did optimal concentrations of IL-2. Although the calcium ionophore ionomycin did not stimulate proliferation by itself, it markedly augmented LCM-induced proliferation of CD56dim NK cells. Proliferation in response to either LCM alone or LCM together with ionomycin was almost completely abrogated by anti-IL-2R antibodies. Thus, IL-2 appears to be necessary but not sufficient for optimal proliferation of CD56dim NK cells. LCM-induced proliferation of ionomycin-activated CD56dim NK cells was inhibited 24% by anti-IL-1 heteroantisera and 57% by anti-TNF antisera; a combination of both antisera inhibited proliferation by 73%. Furthermore, although rIL-1 and TNF did not induce proliferation by themselves, both cytokines could augment IL-2-induced proliferation of resting or ionomycin-activated NK cells. Hence IL-1 and TNF do not appear to be primary NK cell mitogens, but rather accessory factors that can enhance IL-2-dependent NK cell proliferation. Stimulation through CD2 or CD16 Ag did not enhance LCM-induced NK cell proliferation. However, stimulation with NK-sensitive K562 cells strongly augmented CD56dim NK cell proliferation to LCM or to IL-2, IL-1, and TNF in combination. NK-resistant Daudi cells did not promote the proliferation of highly purified NK cells. Thus, NK cell proliferation may be enhanced by triggering through putative receptors for natural killing, and ionomycin may mimic such triggering. Although IL-2 by itself can induce NK cell proliferation, most NK cells resemble T and B lymphocytes in that they require multiple signals for optimal proliferation.

The CD27 Ag is expressed by the majority of resting T lymphocytes and appears to play a crucial role in T cell activation. We found that some resting peripheral blood NK cells also express CD27. Furthermore, CD27 expression was up-regulated on NK cells stimulated by IL-2. The cytolytic activity of IL-2-activated, but not resting, NK cells was inhibited by an anti-CD27 mAb (anti-1A4). However, anti-1A4 did not affect conjugate formation between IL-2-activated NK cells and tumor cell targets. In contrast, anti-1A4 inhibited CD2-mediated calcium mobilization and the serine esterase activity of NK cell granules. These inhibitory effects could be mediated in part by increase in intracellular cAMP levels induced by anti-1A4. Our results suggest that the CD27 Ag plays an important role in the regulation of activated NK cells.

Cell adhesion molecules (CAM) participate in interactions between lymphocytes, accessory cells, and target cells that are critical in the generation of effective immune responses. To characterize the involvement of CAM in NK and lymphokine activated killer (LAK) activities, we examined the expression of several CAM by freshly isolated human NK cells and by NK cells activated in vitro with IL-2, and compared this to CAM expression by T lymphocytes under similar conditions. Freshly isolated human NK cells were uniformly LFA-3 (CD58)+ and expressed two to three-fold higher surface levels of LFA-1 (CD11a/CD18) than resting T lymphocytes. More NK cells than T cells also expressed phenotypically detectable levels of intercellular adhesion molecule-1 (CD54). After in vitro incubation with IL-2, human NK cells demonstrated four- to sixfold increases in surface levels of CD11a/CD18, CD2, CD54, CD58, and the NK cell-associated Ag NKH-1 (CD56). Furthermore, essentially all NK cells became CD54+ within 3 days of exposure to IL-2. T cells did not demonstrate comparable up-regulation of CAM after incubation with IL-2. Increases in NK cell CAM expression were associated with enhanced formation of E:T cell conjugates, enhanced killing of NK-sensitive targets, and the induction of cytotoxicity for previously NK-resistant targets (LAK activity). The LAK activity induced by exogenous IL-2 could be partially inhibited by anti-CD2, anti-CD11a, or anti-CD54 antibodies and almost completely abrogated by anti-CD2 and anti-CD11a in combination. These studies suggest that CAM play a central role in the regulation of NK cytolysis, and that changes in CAM expression may alter the target cell specificity of activated NK effectors.

We studied the mechanisms whereby human T cells and NK cells are activated and directed to lyse tumor targets through the CD2 (T11/E-rosette) Ag. Using two cloned NK lines, we showed that these cells, as had previously been shown for T cells, could be directed to lyse an "NK-resistant" tumor target in the presence of antibody heterodimers. These heterodimers consisted of a (mAb) to CD2 (anti-T11(2) or anti-T11(3] linked to a mAb recognizing the tumor cell (J5, anti-CALLA). However, distinct differences between NK cells and T cells were observed with regard to the requirements for such directed lysis: first, only one epitope of CD2 on NK cells (either T11(2) or T11(3] needed to be recognized by the antibody heterodimer in order for directed lysis to occur, whereas for T cells both T11(2) and T11(3) epitopes had to be recognized. Second, in confirmation of previous data with monomeric anti-T11(2) or anti-T11(3) antibody, heterodimers constructed with these reagents enhanced conjugate formation between NK cells and tumor targets, whereas no such enhancement was seen with T cells. All types of heterodimer directed lysis were dependent on the adhesion molecule LFA-1, as an anti-LFA-1 antibody-blocked lysis. Third, whereas in T cells lysis mediated through CD2 appeared to be regulated by CD3 but not vice versa, all types of lysis by NK cells appeared to be regulated through CD2. Finally we showed that F(ab')2 fragments of the anti-T11(2) and anti-T11(3) antibodies could activate NK cells, but were unable to activate T cells either as cloned cytolytic lines, or in populations of PBL. The implications of our findings with regard to the role of CD2 in the activation of cytolytic cells is discussed.

Modulation of a human common acute lymphoblastic leukemia antigen (CALLA) by specific monoclonal antibody (J5) has been studied with the immune precipitation method to identify radiolabeled antigen. Surfaces of leukemic cells have been labeled using 125I both before and after modulation by J5 antibody for different time intervals. Leukemic cells have also been metabolically labeled with 35S-methionine before modulation. These studies indicate that the 100,000-dalton glycoprotein expressing CALLA (gp 100-CALLA) cannot be detected in cells that were modulated with J5 antibody before surface labeling but that it is easily detectable in cells that were surface labeled before modulation for 10 hr. At later time points, gp 100-CALLA is selectively lost from cells that were surface labeled before modulation. Gp 100-CALLA is not detected in the supernatants from cultures of these modulated cells. We conclude that gp 100-CALLA is rapidly internalized during modulation and that CALLA is degraded. Gp 100-CALLA is not shed into the culture media, nor does it remain on the cell surface in an altered form. Incubation of leukemic cells with antisera to beta2-microglobulin or IgM does not affect the expression of gp 100-CALLA.

A heteroantiserum raised to leukemic cells of a patient with non-T cell acute lymphoblastic leukemia (ALL) has been extensively absorbed with cells from a leukemic T cell line and an autologous B lymphoblastoid cell line to produce a common ALL antiserum (CALLA). CALLA is specific for leukemic cells of most patients with non-T cell ALL and chronic myelogenous leukemia (CML) in lymphoid blast crisis. It has been extensively tested on a wide variety of normal cells and is unreactive with them. CALLA identifies a surface glycoprotein having a m.w. of approximately 100,000 on reactive cell populations. In contrast, partially absorbed anti-ALL sera detect a similar glycoprotein band on CALLA-negative B and T cell lines. The glycoprotein identified by CALLA has been isolated and used as an immunogen. This new antiserum (C129) detects a 100,000-dalton glycoprotein not only on CALLA-positive cell populations but also on most CALLA-negative normal and malignant hematopoietic cells and on B and T cell lines. We conclude that there exists a family of 100,000-dalton glycoproteins that are present on a variety of normal, transformed, and malignant cells and that possess shared as well as unique antigenic regions. The expression of at least one of these antigens, detected by CALLA, may be tumor specific.

The NKH1 Ag is expressed on all cells in human peripheral blood capable of mediating spontaneous non-MHC restricted cytolytic function (i.e., natural killing). The majority of NK cells do not express CD3 Ag and do not express TCR gene products. However, approximately 20 to 25% of NKH1+ cells coexpress CD3 and TCR proteins. Both NKH1+CD3+ and NKH1+CD3- effectors can proliferate in response to IL-2 which also results in enhancement of cytolytic function. In the present studies, we examined thymocytes after incubation with rIL-2 for the presence of NKH1+ cells and for the development of non-MHC restricted cytolytic function. NKH1+ cells and NK activity could not be detected in fresh thymus. After culture with rIL-2 only, NK activity appeared in 3 days, reached a maximum after 7 days, and was effective against a panel of NK-sensitive targets. NK activity was correlated with the expression of NKH1 on the surface of in vitro proliferating thymocytes and immunofluorescent cell sorting demonstrated that almost all cytolytic activity was mediated by NKH1+ cells. As expected given the thymic origin of these cells, the majority of NKH1+ cells in culture expressed CD3. However, all cultures contained NKH1+CD3- effector cells which represent 15 to 40% of the NKH1+ population. As in peripheral blood, both NKH1+CD3- and NKH1+CD3+ exhibited non-MHC-restricted cytotoxicity, but only CD3+ effectors could be inhibited by anti-T3 mAb. These findings demonstrate that rIL-2 alone can induce subpopulations of thymocytes to proliferate, to express the NKH1 marker and become NK active in vitro. Furthermore, they suggest that the thymus which plays a role in the differentiation of NKH1+CD3+ NK effectors may also play a role in the differentiation or maturation of NKH1+CD3- NK effectors.

Natural killer (NK)3 cells are large granular lymphocytes that appear to play a significant role in the host's defense against viral infection. We performed an extensive phenotypic and functional characterization of NK cells on 41 patients with the chronic fatigue syndrome (CFS), or "chronic active Epstein-Barr virus infection" syndrome, and on 23 age- and sex-matched asymptomatic control subjects in an attempt to further characterize this illness. These studies demonstrated that a majority of patients with CFS have low numbers of NKH1+T3- lymphocytes, a population that represents the great majority of NK cells in normal individuals. CFS patients had normal numbers of NKH1+T3+ lymphocytes, a population that represents a relatively small fraction of NK cells in normal individuals. When tested for cytotoxicity against a variety of different target cells, patients with CFS consistently demonstrated low levels of killing. After activation of cytolytic activity with recombinant interleukin 2, patients were able to display increased killing against K562 but most patients remained unable to lyse Epstein-Barr virus-infected B cell targets. Additional cytotoxicity experiments were carried out utilizing anti-T3 monoclonal antibody to block killing by NKH1+T3+ cells. These experiments indicated that the NK cell that appears to be responsible for much of the functional activity remaining in patients with CFS belongs to the NKH1+T3+ subset, which under normal circumstances represents only approximately 20% of the NK cell population.

Although considerable data have recently been accumulated regarding the functional role of natural killer (NK) cells, relatively little is known about the factors that regulate NK cell activity. In these studies, we evaluated the role of interleukin 2 (IL 2) and the expression of the IL 2 receptor in the activation and proliferation of human NK cloned cell lines. By using a series of cloned cell lines, we were able to analyze homogeneous populations of NK cells that ordinarily comprise only a small fraction of peripheral blood lymphocytes and are extremely heterogeneous with respect to phenotypes and cytotoxic specificities. In comparison with several T cell clones, we found a much lower density of IL 2 receptors on NK clones, regardless of whether or not these cloned cells had a mature T cell phenotype. Correspondingly, NK clones needed a 10-fold higher concentration of recombinant IL 2 for maximal proliferation. Moreover, blocking studies with specific monoclonal IL 2 receptor antibodies indicated that IL 2 is both necessary and sufficient to induce the proliferation of NK clones. Because the majority of peripheral blood NK cells and NK clones express the T11 E rosette receptor antigen, which has been shown to be an antigen-independent activation pathway for T cells, we were able to study the role of monoclonal anti-T11 antibodies in the activation of various NK clones for which a specific target antigen is not known. In contrast to T cell clones, the induction of IL 2 receptor expression after T11 activation was possible only for some NK clones such as JT10 and JT3, but not for CNK5. Before activation, the IL 2 receptor expression of NK clones was confined to cells in the G2 - M phase, but after T11 activation the more pronounced IL 2 receptor expression became independent of the cell cycle. With respect to the direct proliferative effect of anti-T11 activation that has been noted with T cell clones, only the T3+ (JT10) and not the T3- NK clones could be directly stimulated. Nevertheless, IL 2 receptor expression could be triggered on some T3- clones such as JT3. Because T11-induced proliferation of T cells has been shown to be dependent on both the expression of the IL 2 receptor and on the interaction of this receptor with IL 2, it is proposed that the different responses of NK cells to T11 activation may reflect the ability of the individual clone to produce endogenous IL 2, as well as its ability to express the IL 2 receptor.

In the present studies we analyzed the role of LFA-1 antigens in the interaction between NK clones and target cells. The use of various cloned NK cell lines allowed us to analyze homogeneous populations of NK cells which ordinarily comprise only a small fraction of peripheral blood lymphocytes and are extremely heterogeneous with respect to phenotype and specificity. Indirect immunofluorescence with monoclonal antibodies against the alpha (MHM24) and beta (MHM23) chains of the LFA-1 antigen revealed similar patterns of positive reactivity with all NK clones. Both monoclonal antibodies exerted a significant blocking effect on NK cytotoxicity against target cells such as Molt-4 and CEM, whereas the inhibition was very weak against other targets such as K562 and HSB cells. Additive blocking effects were seen when both monoclonal antibodies MHM23 and MHM24 were added to the cytotoxicity assays. When we compared the inhibitory effect of MHM23 and MHM24 on uncultured peripheral blood NK cells and IL 2-activated NK cells, inhibition of cytotoxicity also was found to be primarily dependent on the individual target cells. Thus, the inhibitory activity of anti-LFA-1 antibody was shown to be independent of the phenotypic and functional heterogeneity of the NK clones, activated NK cells, and unstimulated NK cells utilized in these studies. These blocking effects were found to be independent of the LFA-1 antigen expression on the target cell membrane and inhibition occurred only when antibody was bound to the effector cells. Comparison of the effects of anti-LFA-1, anti-T3, and anti-clonotypic antibodies against a Ti-like structure of different NK clones with a mature T cell phenotype demonstrated that each of these antibodies acts on the effector cells in an independent and additive fashion. However, unlike T3 and NKTa antigen, LFA-1 antigen expression is not modulated by cell surface interaction with antibodies specific for this molecule.

Early human pre-B cells were isolated from fetal bone marrow and induced to differentiate in vitro under the stimulus of phorbol myristic acid or leukocyte-conditioned medium during a 48-hr culture period. Tritiated thymidine culture experiments substantiated that changes in surface marker phenotypes were not the results of outgrowth of subsets responsive to these stimuli. Interestingly, the addition of monoclonal antibodies directed against CALLA resulted in neither proliferation nor differentiation of the fetal lymphoid progenitor cells. Distinct changes in cell surface phenotypes were observed without evidence of cellular enrichment or depletion. The number of CALLA- and TdT-positive cells decreased, whereas the number of B1- and sIgM-positive cells increased. Moreover, a small number of pre-B cells could be driven to a more mature phenotype with the appearance of B2 and sIgG. In contrast, the pan-B B4 antigen did not alter significantly. These changes were even more pronounced when both induction stimuli were present. These studies, and previous studies on the subsets and differentiation of non-T cell acute lymphoblastic leukemias, suggest an orderly acquisition of B cell antigens during the stages of pre-B cell differentiation in man.

The immunologic responses to anti-T4 and anti-T11 mAb infusions were assessed in subjects with chronic progressive multiple sclerosis as part of phase I clinical studies. Eight subjects received five daily infusions (0.2 mg/kg/day) of either anti-T11 (CD2) or anti-T4 (CD4) murine mAb. It was found that in vivo anti-T cell mAb infusions specifically suppress in vitro measurements of the human immune response; anti-T11 mAb blocked T cell activation via the CD2 SRBC-binding protein, and anti-T4 mAb infusions abolished PWM-induced Ig synthesis without lysis of the CD4+ T cell subpopulations. With repeated infusions of the anti-T11 mAb in three subjects, anti-mouse antibodies were found in the circulation. Although the majority of human anti-mouse antibody was not isotype specific, significant anti-idiotypic-like activity was observed after repeated infusions in two of three subjects. The human anti-mouse antibodies were almost exclusively of the IgG isotype. The magnitude of the human anti-mouse response was significantly less after administration of either anti-T11 or anti-T4 as compared with anti-T12 mAb. Murine anti-T cell mAb can provide a specific, benign form of acute immunosuppression in humans. However, repeated administration of these reagents in more chronic diseases can result in anti-Id-like antibodies that block binding of the anti-T cell mAb to the T cell surface.

Human cloned 35S-labeled NK cells were disrupted by nitrogen cavitation, and their secretory granules were obtained by filtration through 5-micron and 3-micron membrane filters followed by Percoll density-gradient centrifugation. These granule preparations, which contained 35S-labeled chondroitin sulfate A proteoglycans, were sonicated and were analyzed for carboxypeptidase activity and tryptic serine esterase activity. A carboxypeptidase activity that digested angiotensin I to des-Leu-angiotensin I, Ile-His-Pro-Phe to Ile-His-Pro and Phe, and hippuryl-L-phenylalanine to hippuric acid and Phe was detected in the granules of these NK cells. As determined by cleavage of the tetrapeptide, the pH optimum of the carboxypeptidase was 7.0. As assessed by the cleavage of N-benzyloxycarbonyl-L-lysine thiobenzyl ester (BLTe), the granule preparations also contained a serine esterase with trypsin-like specificity that had a pH optimum of 8.5. When the isolated secretory granules were disrupted and chromatographed on columns of Sepharose CL-2B in PBS, greater than 60% of the BLTe serine esterase activity and essentially all of the carboxypeptidase activity filtered as a macromolecular complex with approximately 8% of the 35S-labeled proteoglycans. Whereas treatment with 4 M urea or nonionic detergent failed to disrupt the macromolecular complex, the serine esterase activity was dissociated from the macromolecular complex in the presence of 3 M NaCl, demonstrating an ionic interaction with the proteoglycans. No difference was observed in the disaccharide composition of the chondroitin sulfate glycosaminoglycans of the 35S-labeled proteoglycans that were complexed with the enzymes as compared to those that were not complexed. These studies indicate that the secretory granules of human NK cells contain serine esterase activity and carboxypeptidase activity, both of which have neutral pH optima, and both of which are bound to protease-resistant chondroitin sulfate proteoglycans.

Expression of the common acute lymphoblastic leukemia antigen (CALLA) on the surface of individual cells of the human lymphoblastoid lines CW678, Namalwa, and Nalm-6, and the distribution of the antigen epitopes within the cell populations have been determined quantitatively with the murine monoclonal anti-CALLA antibody J5. The distribution of CALLA epitopes in the cell populations was analyzed by indirect immunofluorescence measured by using flow cytometry. The average number of CALLA epitopes per cell were measured by two assays: in a direct assay by binding 125I-labeled antibody J5 to cells, and indirectly by binding 125I-labeled protein A from Staphylococcus aureus to J5-coated cells. On average, CW678, Namalwa, and Nalm-6 cells bore about 1 X 10(4), 6 X 10(4), and 8 X 10(4) CALLA epitopes per cell respectively. Histograms of the absolute number of CALLA epitopes expressed by individual cells in the populations of CW678, Namalwa, and Nalm-6 cultures were generated by a combined analysis of all the binding data. This is the first example of histograms showing quantitative distribution of antigen epitopes. Previously, the expression of antigens by individual cells as obtained by flow cytometry was only presented in terms of relative fluorescence intensity of individual cells in cell populations.

The T11 (CD2) antigen has been found to be an alternate pathway for antigen-independent activation of resting T cells. T11 triggering also results in activation of NK cells and enhancement of their cytolytic function. The present studies were carried out to further define the mechanisms whereby cytotoxicity is enhanced after T11 activation. A series of clonal human NK cell lines were analyzed after incubation with monoclonal anti-T112 and anti-T113 antibodies specific for different epitopes of the CD2 protein. Anti-T112/3 triggering resulted in increased cytotoxicity against a variety of target cells. Similar results were obtained with F(ab')2 fragments of anti-T112/3, indicating that this effect was not mediated through binding of FcR. The induction of cytotoxicity was found to be associated with increased formation of effector cell-target cell conjugates and with release of secretory granule-localized 35S-labeled proteoglycans. Both enhanced conjugate formation and cytotoxicity could be blocked by anti-lymphocyte function-associated antigen (LFA-1) mAb. Ultrastructural analysis of NK cells after T11 activation demonstrated increased adherence of effector cells to targets and other NK cells as well as a directional reorientation of cytoplasm and intracellular granules toward the area of contact between cells. Discharge of granules occurred into pockets bounded by closely apposed plasma membranes. In the presence of anti-LFA-1 and anti-T112/3, the close apposition and formation of pockets between effector cells and target cells did not occur but the cells exocytosed their intracellular granules. T11 activation of NK cloned cells also resulted in the formation of the homotypic conjugates and autocytotoxicity. As seen with resistant allogeneic targets, autocytotoxicity was mediated by F(ab')2 fragments of T112/3 antibodies and could be blocked by anti-LFA-1 antibody. Ultrastructural analysis of NK cloned cells after T11 activation confirmed the presence of homotypic conjugates with reorientation of effector cells toward one another and discharge of cytolytic granules into pockets formed between NK cloned cells. Taken together, these results indicate that T11-induced cytolytic function of NK cells is, in part, mediated through increased binding of effector cells and targets and that enhanced conjugate formation is at least in part mediated by the LFA-1 antigen. In addition, T11 activation results in the triggering of the cytolytic mechanism of NK cells and the exocytosis of cytolytic granules and their constituents.