Antigen-specific memory and virgin B cells differ in their requirements for conjugation to T cells

The physical interaction between carrier-specific T hybridoma cells and long-term primed, TNP-specific memory B cells (TNP-MABC) exposed to TNP-OVA was compared to that of unprimed, TNP-specific virgin B cells (TNP-ABC). The direct conjugation of the T and B cells was visualized at the light microscopic level and the number of T/B conjugates was quantified directly. The results demonstrate that the TNP-MABC, as compared to TNP-ABC, formed T/B conjugates after a shorter exposure time to antigen and at a 10-fold lower concentration of antigen. Conjugate formation was inhibited almost completely by treating the TNP-MABC with concentrations of chloroquine that only partially inhibited the ability of the TNP-ABC to form conjugates. Exposure of the T hybridoma cells or the TNP-ABC to monoclonal antibodies directed against cell surface antigens prior to conjugation indicated that L3T4, Thy-1.2, and LFA-1 antigens on the T cells and LFA-1 and I-A antigens on the TNP-ABC are involved in conjugate formation. However, in contrast to the TNP-ABC where treatment of the B cells with anti-LFA-1 blocked T/B conjugate formation, pretreatment of the TNP-MABC with anti-LFA-1 had no effect.     

The effects of cytokines and adherent cells on the interleukin 4-mediated induction of Ia antigens on resting B cells

In this report we have extended our previous studies on interleukin 4 (IL-4) [previously termed B-cell stimulatory factor-1 (BSF-1)]. Our results demonstrate that 8 hr of exposure to IL-4 is sufficient to induce maximal expression of Ia antigens. This increase in expression of Ia antigens on resting B cells is due to the direct action of IL-4 on the B cells since adding or removing adherent cells or utilizing low density cultures of B cells at 50-100/culture had no effect on the IL-4-mediated increase in Ia. Monoclonal anti-IL-4 antibody completely abrogated the Ia-inducing activity of IL-4. A variety of other purified lymphokines including interleukin 2 (IL-2), interleukin 1 (IL-1), and a source of either B-cell differentiation factor for IgM (BCDF mu), or B-cell growth factor II (BCGF II), did not alter the expression of Ia antigens on resting B cells. However, interferon-gamma can partially inhibit the IL-4-mediated induction of Ia.     

Enhanced antigen-presenting capacity of cultured Langerhans' cells is associated with markedly increased expression of Ia antigen

Recent studies indicate that when epidermal Langerhans' cells (LC) are cultured for 2 to 3 days they, in comparison to freshly prepared LC, exhibit markedly enhanced ability to stimulate T cell proliferative responses in oxidative mitogenesis and in the mixed epidermal-leukocyte reaction. In this study, we determined whether cultured LC enhance antigen-specific T cell responses, and whether such enhanced stimulatory capacity correlates with the level of Ia antigen expressed on LC. We used C3H/He (Iak) epidermal cells as stimulators and, as responder cells, both the trinitrophenyl-specific clones D8 and SE4, which were assayed for [3H]dThd incorporation, and the pigeon cytochrome c specific hybridoma 2C2, which was assayed for interleukin 2 production. Cultured LC induced 10 to 100 times greater proliferation or interleukin 2 production by responder cells than did freshly prepared LC. The intensity of I-Ak and I-Ek, expressed on cultured LC as assessed by immunofluorescence and flow cytometry, was found to be 10 to 36 times greater on a per cell basis than that on freshly prepared LC. Depletion of LC from fresh epidermal cell suspensions by anti-Iak and complement or treatment with 50 mJ/cm2 medium range ultraviolet light or cycloheximide before culture abrogated both the increase in Ia expression and antigen-specific clonal proliferation. The results suggest that when LC are removed from their usual epidermal milieu, they express increased amounts of Ia and become more potent stimulators of T cell responses.     

Inhibition of the helper function of murine T cells with Fab'-anti-L3T4 ricin A chain immunotoxin

The ability of Fab'-anti-L3T4 A chain-containing immunotoxins to inhibit the helper function of keyhole limpet hemocyanin-specific T helper lymphocytes was evaluated. Keyhole limpet hemocyanin-specific T helper cells were prepared from the lymph nodes of primed mice and were enriched for T cells. Enriched populations of trinitrophenyl-specific B cells were prepared from spleens of normal mice. In the presence of antigen, the keyhole limpet hemocyanin-specific T helper cells were able to induce proliferation and differentiation of the trinitrophenyl-specific B cells. However, when the T helper cells were first treated with an immunotoxin composed of Fab' fragments of anti-L3T4 antibody coupled to ricin A chain (Fab'-anti-L3T4-A), they failed to induce proliferation and differentiation of the antigen-specific B cells. The concentrations of Fab'-anti-L3T4-A required to inhibit T cell help for the proliferation and differentiation of trinitrophenyl-specific B cells by 50% were 1 X 10(-9)M and 4 X 10(-10) M, respectively. Fab'-anti-L3T4 antibody alone did not inhibit T cell-induced B cell proliferation and differentiation by 50% at greater than 100-fold higher concentrations.     

Specific lymphocyte-target cell conjugate formation between tumor-specific helper T-cell hybridomas and IA-bearing RCS tumors and IE-bearing allogeneic cells. I. Role of Ia and both L3T4 and LFA-1 antigens in recognition/binding

The studies reported here describe the feasibility of using single cell techniques with nonadherent target cells for the formation of T helper lymphocyte-target cell conjugates in an Ia recognition system. We have taken advantage of four tumor-specific T cell hybridomas lines, two of which respond only to IA-bearing RCS tumor cells of SJL/J (H-2s) origin, and the other two that respond to both RCS and IA- or IE-bearing allogeneic cells of H-2k,d haplotypes. The conjugate frequency between the T cell hybridomas and target cells was scored microscopically and was facilitated by labeling the lymphocyte with fluorescein. The frequency of conjugate formation ranged from 20 to 40% above background. Conjugate formation was antigen specific and correlated well with the hybridoma specificity determined by IL 2 responses after antigenic stimulation. The cross-reactive hybridomas formed conjugates with RCS and LPS blasts derived from CBA or DBA/2 origin, but not with cells of syngeneic or other allogeneic strains. Conjugate formation with RCS was inhibited greater than 50% with mAb directed against IAs determinants on the RCS tumor cells, and conjugate formation with allogeneic cells was blocked only with mAb directed to either IA/IEk or IA/IEd specificities directed against the alpha or beta polypeptide chain. Blocking of conjugate formation was also achieved by various mAb directed against surface membrane molecules associated with the T cell hybridomas. LFA-1 mAb inhibited significantly the formation of conjugates. However, L3T4 mAb blocked only partially the conjugates. Other antibodies directed against Lyt-1 or Thy-1.2 antigens were without blocking effect. The poor blocking observed with L3T4 mAb did not correlate with the almost complete blocking observed in the IL 2 response by the same hybridomas. These studies of the syngeneic anti-RCS tumor response directed against IA-bearing RCS showed that the conjugate assay permits mapping of tumor-associated Ia epitopes. In addition, the results of these studies demonstrate the feasibility of conjugate formation in determining the antigenic specificity of the T helper system. This assay system can be used to establish the minimal frequency of antigen-reactive cells and can divide the T helper response into multiple steps (i.e., recognition/binding, activation, proliferation, and lymphokine release) and determine the surface membrane molecules involved in recognition.     

Antibody conjugates mimic specific B cell presentation of antigen: relationship between T and B cell specificity

We developed antibody conjugates by covalently coupling antibodies against mouse mu-chain and monoclonal antibodies against nominal antigen, myoglobin, as a tool for antigen presentation and as a model of specific presentation of antigen by antigen-specific B cells and T-B interaction. In the presence of the antibody conjugates, myoglobin-specific Iad-restricted cloned T cells proliferated at 1000-fold lower concentration of myoglobin than the stimulatory concentration without the conjugates. This enhanced presentation was observed only when Iad spleen cells were 1000 R-irradiated but not 3300 R-irradiated, consistent with B cell presentation. The simple mixture of each component of the conjugates had no enhancement effects. The conjugates per se had no mitogenic effects on either splenic B cells or the cloned T cells at concentrations employed for antigen presentation. The conjugates reduced the number of antigen-presenting cells required for the maximal response but did not change the kinetics of response. The enhanced presentation by the conjugates required a genetically restricted interaction with B cells. Antigen specificity of the enhanced presentation was confirmed by using various T cell clones or lines with different antigen specificities and different conjugates constructed with monoclonal antibodies of known epitope specificity. The enhanced presentation was significantly inhibited by competition with exogenous mouse IgM or anti-mouse mu-chain but was not significantly inhibited by monoclonal antibodies against Fc receptor. Thus, conjugate-coated B cells serve as models for myoglobin-specific B cells in that they can take up specific antigens at extremely low concentration and can present the antigen to specific T cells. This model system can be applied to any antigen and any species without the need to develop antigen-specific B cell clones, which is not yet possible for most antigens and species of experimental animals. This system allowed us to investigate the relationship between T cell epitope and B cell epitope when these cells interact with each other in an antigen-specific and Ia-restricted manner. Experiments using antibody conjugates of different monoclonal antibodies against myoglobin and various myoglobin-specific cloned T cells of known antigen specificity revealed that there are some particular combinations in which much more limited enhancement of antigen presentation is observed.(ABSTRACT TRUNCATED AT 400 WORDS)     

Preparation and analysis of antigen-specific memory B cells

A procedure has been developed for the enrichment of TNP-binding memory B cells (TNP-MABC) from spleens of immunized mice. More than 75% of the cells expressed surface IgM (sIgM) and IgD (sIgD) and about 9% expressed surface IgG (sIgG). The TNP-MABC consisted of small resting lymphocytes with high affinity antigen-binding receptors. These cells expressed increased densities of Ia antigens and decreased densities of sIgD. Adoptive transfer of the cells into irradiated, carrier-primed syngeneic recipients resulted in their differentiation into IgG anti-TNP antibody-secreting cells. TNP-MABC secreted high affinity IgG anti-TNP antibodies when cultured in vitro with carrier-primed T cells and antigen. Limiting dilution analysis revealed that TNP-MABC contained a relatively low frequency of precursors for IgG-secreting cells that had an exceptionally large clone size. These results show that a highly enriched population of antigen-specific memory B cells can now be prepared and used to analyze their activation requirements.     

Cyclosporine inhibits macrophage-mediated antigen presentation

The influence of cyclosporine on antigen-specific, macrophage-dependent T cell activation was analyzed in vitro. Murine T cell activation by antigens derived from Listeria monocytogenes was monitored by the production of interleukin 2. Pretreatment (2 hr, 37 degrees C) of macrophages with cyclosporine resulted in a cell population with a markedly diminished capacity to support the activation of T lymphocytes. When cyclosporine-pretreated macrophages were added to cultures of untreated T cells and antigen, the dose of cyclosporine that produced 50% inhibition (ID50) was 1.5 micrograms/ml, and if antigen was present during the drug pretreatment, the ID50 was 0.6 micrograms/ml. Pretreatment of T cells also inhibited their subsequent activation by antigen and untreated macrophages, but a higher dose of cyclosporine was required to produce similar inhibition (ID50 = 4.4 micrograms/ml). Additional experiments focused on the mechanism of inhibition of antigen presentation when macrophages were pretreated with the drug. The addition of interleukin 1 or indomethacin to the cultures did not alter the inhibitory effect of cyclosporine. Under conditions that produced greater than 90% inhibition of antigen presentation, macrophage surface Ia expression was not altered, and the uptake and catabolism of radiolabeled antigen remained normal. Thus, cyclosporine had profound effects on antigen presentation that appear to be unrelated to decreases in interleukin 1 production, increases in prostaglandin production, decreases in Ia expression, or changes in antigen uptake and catabolism.     

Activated human T cells can present alloantigens but cannot present soluble antigens

Human T cells, when activated by antigen or mitogen, express Ia antigens. We have examined the capacity of activated T cells to stimulate autologous and allogeneic T cells and their ability to present soluble antigen. Interleukin 2-dependent T-cell lines (TCL), free of accessory cells, were used for antigen-presenting cells. These activated T cells were potent stimulators in an autologous mixed lymphocyte reaction (AMLR), more so than autologous irradiated non-T mononuclear cells. Activated T cells were also able to stimulate proliferation of allogeneic T cells in the absence of any other accessory cells, and this stimulation was blocked by anti-Ia antibodies. Resting unstimulated T cells were unable to stimulate autologous or allogeneic responses. Thus, activated T cells were able to present self antigens and alloantigens. However, activated T cells could not present soluble antigens to autologous T cells or to antigen-specific TCL even if exogenous interleukin 1 was added to cultures. The ability of activated T cells to stimulate an AMLR in vitro may reflect an important immunologic amplification mechanism in vivo. The ability of activated T cells to present alloantigens but not soluble antigens suggests an inability to process antigen, and this may provide further insights into the complexities of antigen presentation.     

Modulation of Ia-like antigen expression and antigen-presenting activity of human monocytes by endotoxin and zymosan A

The environmental agents E. coli endotoxin and zymosan A modulated antigen-specific T cell proliferation in vitro, assessed by 3H-TdR uptake. In the continual presence of these agents, human mononuclear leukocyte responses to the antigens tuberculin PPD, Candida albicans, and mumps were significantly reduced. Treatment of adherent cell-depleted T cells with the agents did not affect their subsequent reactivity to soluble antigens in the presence of normal M phi. However, cultures consisting of pretreated M phi, normal T cells, and soluble antigen gave responses that were only 7 to 38% of control values, indicating that the function of the antigen-presenting cell, not the T cell, was inhibited. This effect was observed only when treatment with endotoxin or zymosan A preceded antigen stimulation by at least 24 hr, suggesting that a gradual inhibition of antigen presentation had occurred. When various ratios of normal antigen-pulsed and agent-treated M phi were cultured with normal T cells, antigen-specific responses were not significantly different from control cultures; this indicated that M phi-mediated suppression was not involved. It did not appear that the inhibition was due to enhanced antigen degradation by the treated M phi because responses were not reconstituted in the presence of excess antigen. After endotoxin or zymosan A treatment of the M phi population the proportion of Ia+ cells was reduced significantly, and surface expression of Ia antigen correlated with the ability of the cell population to present antigens to immune T cells. This suggested that endotoxin and zymosan A induce a loss of surface Ia antigen on antigen-presenting cells that inhibits immune T cell activation.     

LPS and specific T cell responses: interleukin 1 (IL 1)-independent amplification of antigen-specific T helper (TH) cell proliferation

Lipopolysaccharide (LPS) fraction of endotoxin induces a significant potentiation of the antigen-specific proliferative response of T helper (TH) cell lines. This effect was obtained with LPS from different bacterial sources and reproduced with the lipid A moiety of endotoxin. Purified adherent spleen cells used as antigen-presenting cells (APC) support this LPS-enhanced TH cell proliferation. In addition, the effect of endotoxin on specific TH cell responses was found to be absolutely dependent on the interaction between TH lymphocytes and APC through antigen-specific recognition. Thus, it was not observed in the absence of specific antigen or when monoclonal antibodies against class II MHC products or against L3T4 antigens were used to inhibit the T cell-APC interaction. Similarly, it was found that APC from the B6.CH-2bm12 mutant do not support the LPS-mediated enhancing effect. Furthermore, interleukin 1 (IL 1) appears not to be involved in LPS-mediated enhancement, and this effect is not reproduced by muramyl dipeptide (MDP)-mediated activation of APC.     

Antigen-induced T suppressor cells regulate the autoreactive T helper-B cell interaction

The T suppressor (Ts) cell population that functions to regulate antigen-specific MHC-restricted T helper (Th)-B cell interactions also regulates the activation of B cells by cloned autoreactive Th cells. Activated Ts cells were generated by in vivo priming and restimulation in vitro with high concentrations of the specific priming antigen. Once generated, this Ts population inhibits the Th-dependent activation of primed B cells by both antigen-specific and autoreactive T cells in an antigen-nonspecific manner. This suppression requires the participation of both Lyt-1+2- and Lyt-1-2+ T cells. It was also demonstrated that accessory cells were required for the induction of Ts cells. Moreover, the generation of suppression was MHC-restricted and required the recognition by T cells of Ia antigens on accessory cells. These studies demonstrate that the same or a very similar Ts cell population can function to inhibit the activation of B cells by antigen-specific as well as autoreactive T cells.     

Studies of in vitro activation and differentiation of human B lymphocytes. I. Phenotypic and functional characterization of the B cell population responding to anti-Ig antibody

Investigation of the activation of splenic B cells by anti-immunoglobulin (Ig) antibody has enabled us to characterize the anti-Ig-responsive B cell and to analyze the phenotypic changes which accompany proliferation and differentiation. The anti-Ig antibody-responsive B cell population was characterized by the expression of high levels of the B2 antigen and represented approximately 40% of splenic B cells. Brisk mitogenesis which peaked at 3 to 4 days was induced by anti-Ig antibody. The proliferative phase was characterized phenotypically by a dramatic decline in B2 antigen expression, with most cells showing no detectable B2 by 4 days post-activation. The other hallmark of this phase was de novo expression of a group of "activation antigens." These included the B cell-restricted antigens B-LAST 1, BB1, and B5, and the T cell-associated interleukin 2 receptor and T12 antigens. Concomitantly, B1, B4, and Ia expression increased, the increase being roughly proportional to the increase in cell size. After day 4, the mitogenic response progressively diminished, while Ig synthesis increased. During this differentiation phase, cell surface antigens again displayed a distinct sequence of changes. The five activation antigens and the B1, B4, and Ia antigens began to decrease. However, two markers, T10 and PCA-1, which are found on plasmacytomas, appeared and their level of expression steadily increased. These changes and the appearance of morphologically identifiable plasma cells required the presence of T cells in this system. T cell supernatants alone induced Ig secretion but did not induce expression of PCA-1 or the appearance of cells with plasma cell morphology. The culture system developed in this study has allowed us to analyze the antigenic changes following activation by anti-Ig antibody. This sequence of changes has not only permitted the identification of antigens which, by their appearance at distinct stages may have an important role in proliferation and differentiation of B cells, but also provides us with the means of studying the function of each antigen.     

Evidence for effects of interleukin 4 (B cell stimulatory factor 1) on macrophages: enhancement of antigen presenting ability of bone marrow-derived macrophages

We have studied the effects of recombinant mouse interleukin 4 (IL 4) (previously known as B cell stimulatory factor 1) on the antigen-presenting ability of murine splenic B cells and bone marrow macrophages. Our assay is based on the induction of antigen-presenting ability in these cells after incubation with IL 4 for 24 hr. The presenting cells were then used to stimulate IL 2 production by antigen-specific, I-Ad-restricted T cell hybridomas, a response mainly dependent on the induction of Ia antigens. Consistent with our previously published data using partially purified natural IL 4, we show here that recombinant IL 4 (but not interferon-gamma (IFN-gamma) or IL 1) induces antigen-presenting ability in B cells. Recombinant IL 4 was also found to induce antigen-presenting ability in a cloned, bone marrow derived-macrophage cell line (14M1.4), and in normal bone marrow-derived macrophages. These macrophage populations also respond to IFN-gamma showing enhanced antigen-presenting ability (mediated by increased Ia antigen expression). A small but significant increase in Ia antigen expression was also detected in 14M1.4 macrophages induced with IL 4. However, additional analysis suggested that the effect of IL 4 on 14M1.4 is different from that of IFN-gamma, because IL 4 (but not IFN-gamma) is able to maintain the viability and increase the size of and metabolic activity of bone marrow macrophages. However, IL 4 may not affect all macrophages because the macrophage cell line P388D1, which responds to IFN-gamma, failed to show enhanced antigen-presenting function after stimulation with IL 4. These observations indicate that IL 4, a lymphokine previously considered to be B cell lineage specific, has effects on macrophages and may be involved in their activation.     

Antigen presentation by human antigen-presenting cells to antigen-specific xenogeneic murine T cells

Successful antigen presentation by xenogeneic human antigen-presenting cells (APC) to stimulate the proliferation of antigen-specific, keyhole limpet hemocyanin (KLH)-specific, ovalbumin (OVA)-specific, and purified protein derivative of Mycobacterium tuberculosis (PPD)-specific murine T cells was observed. Evidence indicating a direct cell interaction between antigen-specific murine T cells and xenogeneic human APC was given by experiments using antigen-specific murine T cell clones. The OVA-specific B10.S(9R) T cell line (9-0-A1) and PPD-specific B10.A(4R) T cell line (4-P-1) were stimulated by both xenogeneic human APC and murine APC from syngeneic or I-A compatible strains, while the PPD-specific human T cell line (Y-P-5) was stimulated by autologous human APC but not by murine APC. Anti-HLA-DR monoclonal antibodies (MoAb) blocked the xenogeneic human APC-antigen-specific murine T cell clone interaction. Thus, human xenogeneic APC can stimulate antigen-specific murine T cells through HLA-DR molecules in the same manner as syngeneic murine APC do through Ia molecules coded for by the I region of the H-2 complex, while murine APC failed to present antigen to stimulate human antigen-specific T cells.     

Idiotope antigens (Ab2 alpha and Ab2 beta) can induce in vitro B cell proliferation and antibody production

We previously showed that immunization of various strains of mice with three types of antigen--PC-Hy (nominal antigen), F6-Hy (Ab2 alpha-Hy, and 4C11-Hy (Ab2 beta-Hy)--induces a differential PC-specific, T15-Id+ antibody response. In this report, the in vitro phosphorylcholine (PC)-specific B cell responses induced by these three antigens were studied. A hemocyanin-specific long-term T helper cell line was used to provide help for primary and secondary in vitro T cell-dependent B cell responses. At low doses (0.005 to 0.5 micrograms/ml) of antigen, a significant increase in the proliferation of PC-OVA-primed BALB/c B cells was observed with Ab2-Hy or PC-Hy conjugate, but not unconjugate, antigens. Similar low doses of antigen could stimulate naive B cells to secrete IgM and stimulate PC-OVA- or 4C11-Hy-primed B cells to secret IgM and IgG1 anti-PC antibodies. The percentage of T15-Id of the PC-specific antibodies produced in the in vitro T-B culture was found to be less dominant than that produced by in vivo immunization, suggesting that certain regulatory mechanisms occur in the in vivo environment that may help to maintain the T15-Id dominance. Taken together, our in vivo and in vitro results indicate that idiotope antigens can function like nominal antigens to induce antigen-specific B cell responses. The mechanisms of thymic-dependent B cell activation induced by idiotope and nominal antigen are similar in that the T-B interaction is MHC-restricted and requires cognate recognition.     

Specific suppression of the immune response by a factor obtained from spleen cells of mice tolerant to human gamma-globulin.

An antigen-specific suppressive factor was extracted from spleen cells of mice made tolerant by injection of deaggregated human gamma-globulin (HGG). The active material could be prepared from T cells, obtained by passaging spleen cells through an anti-immunoglobulin column, although not from cells adherent to the column nor from spleen cells pretreated with anti-Thy-1 serum and C. This factor was antigen-specific since it was retained on immunoadsorbents containing HGG, but not on columns coated with antibody to HGG or with irrelevant antigens. Despite its specificity for antigen it did not bear any classical immunoglobulin determinants. Its m.w. ranged between 30,000 and 55,000 daltons. It was a product of the I region of the major histocompatibility complex since it carried Ia determinants. The properties of this factor are very similar to those reported elsewhere for suppressive factors obtained from primed T cells, cells from nonresponder mice, and allotype-specific cells. This suggest the existence of a major class of immunoregulatory molecules, nonimmunoglobulin in nature, and responsible for the mediation of antigen-specific T cell-dependent suppression.     

T11/CD2 activation of cloned human natural killer cells results in increased conjugate formation and exocytosis of cytolytic granules

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.