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It has been reported that the assembly of MHC class I molecules in mutagenized cell lines could be induced by specific binding peptides. We have now demonstrated that the defect in assembly between heavy and light chains of class I molecules naturally occurred in tumor cells of one spontaneous ovarian carcinoma detected by one-dimensional isoelectric focusing of immunoprecipitates with anti-monomorphic class I MAb (W6/32) and by immunostaining with free heavy chain and beta 2m-specific MAbs. In vitro treatment of the tumor cells with IFN-gamma induced the assembly and surface expression of majority class I molecules (A2.1, B7, B15, Cw6, Cw7 out of A2.1, A2*, B7, B15, Cw6, Cw7). Moreover, assembly of A2 and Cw6 was induced by exposure of the tumor cells to a HLA A2-binding peptide K62 derived from influenza A matrix protein. Autologous blood T lymphocytes were activated in mixed lymphocyte-tumor cell culture (MLTC) by the IFN-gamma-treated but not by the unmanipulated tumor cells. Although activated lymphocytes damaged both IFN-gamma-treated and untreated tumor cells, the alpha class I MAb (W6/32) efficiently inhibited the lysis of IFN-gamma-treated targets, but not the untreated targets. These results indicate that the defect of MHC class I assembly may result in the escape of tumor cells from immune response.

Epstein-Barr virus (EBV) transformed lymphoblastoid cell lines (LCL) are potent inducers of cytotoxic T-lymphocytes (CTL) in allogeneic mixed lymphocyte cultures (MLC). The contribution of EBV antigens to the induction of cytotoxic responses was investigated by comparing CTL clones derived from allogeneic MLCs of lymphocytes from one EBV seropositive and one seronegative donor for their capacity to lyse paired EBV positive and negative targets. The majority of the clones showed a conventional "HLA-specific" cytotoxicity and lysed equally well HLA-matched LCLs and mitogen-induced T- or B-blasts. A minority of the clones from both donors exhibited an "LCL-selective" killing potential as they lysed poorly T- and B-blasts. The LCL-selective clones did not recognize EBV antigens because they could not discriminate between EBV negative Burkitt lymphoma (BL) lines and their in vitro EBV-converted sublines. MAbs to CD3, CD8, and MHC class I antigens blocked the lysis of LCLs by HLA-specific and LCL-selective CTLs with comparable efficiency suggesting that the two effector types express T-cell receptors of similar affinity. T-blasts were unable to inhibit the lysis of LCLs in cross competition assays. This correlated with a significantly lower expression of the cell adhesion molecules ICAM-1 and LFA-3. The results suggest that stimulation with allogeneic LCLs activates HLA class I-specific CTLs with variable target cell avidity. Only CTLs that act independently of the enhancing effect of cell adhesion molecules are able to lyse mitogen-induced T- and B-blasts.

We exposed human blood lymphocytes to autologous and to allogeneic lymphoblastoid lines (LCLs), each alone or in combination, and analyzed the MHC Class I restriction pattern of the generated auto-LCL reactive cytotoxicity. In the cultures of two EBV-seropositive, HLA All-positive individuals the majority of cytotoxic lymphocytes generated after repeated stimulation with autologous LCL were restricted by this molecule. One of the cultures was subjected to various stimulation strategies. A relatively low proportion of HLA A2- and HLA B7-restricted cytotoxic T cells could be detected in the autostimulated cultures. Such cells were enriched at the expense of All-restricted ones by stimulating with allogeneic LCLs which lacked HLA All but expressed A2 or B7. Interestingly, stimulation of the lymphocytes with only allogeneic LCL also generated autoreactive CTLs. Thus, by including or using exclusively allogeneic LCL stimulators, the CTL fractions represented by few cells could be enriched.

We have shown that intercellular adhesion molecule-1 (ICAM-1) (CD54) positive cells are mainly responsible for the natural cytotoxic function of human blood lymphocytes. The evidences were the inhibition of cytotoxicity by anti-ICAM-1 (LB-2) monoclonal antibodies (mAb) and the loss of lytic activity after removal of the ICAM-1+ cells. In addition, the cytotoxic potential of the separated ICAM-1- lymphocyte population after activation appeared in parallel with the expression of this molecule. The ICAM-1+ lymphocytes lysed both LFA-1 (CD11a/CD18 or Leu-CAMa) positive and negative cell lines, and pretreatment of the effectors with the LB-2 mAb also inhibited the lysis of LFA-1- targets. The results point to a yet unrecognized role of ICAM-1 on the lymphocytes. Kinetics experiments suggested that pretreatment of lymphocytes with alpha-ICAM-1 (LB-2) mAb did not inhibit the promptly established lytic interactions but influenced later events, recycling and/or recruitment of effectors. It is possible that the cytotoxic potential is regulated by contacts between the members of the lymphocyte population and that these events occur via their ICAM-1 and LFA-1. Exposure of lymphocytes to NK-sensitive targets for 16 hr elevated their cytotoxic potential. The function of activated lymphocytes was not inhibited by the LB-2 mAb.

C3 fragments fixed on zymosan particles were presented to resting human B lymphocytes. The opsonized zymosan (Ops-Z) particles induced release of leukocyte migration inhibitory factor, a slight decrease in mIgD, and a slight increase in the activation marker Blast-2. The B cells did not proceed further along the pathway of activation: they did not respond to B cell growth factor (BCGF) and Ops-Z did not synergize with other activators for BCGF response either. Thus, we found that interaction between C3 fragments and CR2 initiates the activation of human B lymphocytes, but this is limited to the early phase.

Treatment of lymphocytes with the tumor promoter P(Bu) 2 enhanced their target-binding capacity. In the conventional 51 Cr-release assay, the cytotoxic potential of lymphocytes pretreated with P(Bu) 2 for a short time was reduced while after prolonged treatment their function was increased. The peak of lytic potential was attained after 15 hr of exposure. Comparison of the cytotoxic results obtained in suspension and immobilized conjugate assays suggested that P(Bu) 2 treatment causes an impairment of the recycling capacity of lymphocytes. After prolonged exposure, the lytic machinery became activated as reflected by the reduced time elapsing after contact between effectors and targets and the delivery of lethal hit. The activation was also observed in the immobilized agarose assay. It was reflected by the elevated proportion of damaged targets that were bound to the treated lymphocytes. The P(Bu) 2 - and interferon-induced augmentation of lytic potential is achieved through different mechanims. Combination treatment applied to the effectors in sequence (first P(Bu) 2 followed by interferon), resulted in an additive effect. Similarly, simultaneous treatment with IL-2 and P(Bu) 2 also gave an additive increase in cytotoxicity. Addition of antibodies directed against IL-2 did not abrogate the P(Bu) 2 effect. Consequently, neither interferon nor IL-2 are involved in the phorbol ester-induced cytotoxic function.

T lymphocytes activated in mixed lymphocyte cultures were found to be sensitive to the lytic effect of glucocorticoids (methylprednisolone and hydrocortisone). Human thymocytes, freshly separated blood lymphocytes, and phytohemagglutinin blasts were resistant. In addition, the activated T lymphocytes isolated from the synovial fluid of arthritic patients were also glucocorticoid sensitive whereas the blood lymphocytes of the same patients were resistant. The glucocorticoid sensitivity was not accompanied by elevation of the cytoplasmatic receptors to steroids. The direct sensitivity of activated T cells to glucocorticoids may explain partly the immunosuppressive activity of these drugs.

Lymphocyte subsets separated on the basis of nylon-wool adherence and E and EA rosetting, and characterized for the presence of esterase-positive phagocytic cells were investigated for production of leukocyte migration inhibitory factor (LIF) in response to polyclonal T- and B-cell activators, PHA, ConA, PWM, and Epstein-Barr virus (EBV). In the nylon-passed population only the high avidity E+EA+ cells responded to ConA, PHA-induced LIF production in all E-rosetting subsets. The nylon-adherent E+ subset, which contains activated T cells, produced LIF spontaneously. B cells produced LIF when exposed to PWM or uv-inactivated EBV. In accordance with the known T-cell dependence of PWM activation, LIF was detected only in supernatants of reconstituted populations containing both B and T cells. In contrast, uv-inactivated EBV, devoid of transforming potential, elicited LIF production in the pure B-cell population. LIF production in response to polyclonal activators seemed to be independent of accessory cells since reconstitution with autologous macrophages or semipurified monokine, high-molecular-weight Interleukin 1 (IL-1), did not alter the results.

The effect of lymphocyte subsets, separated on the basis of cell density, on Epstein-Barr virus (EBV)-induced B-cell proliferation was studied. The experiments were performed with lymphocytes of seropositive individuals. After 2 weeks of culture, the growth of B cells was inhibited by the T subset, which is also active in natural killer assays, i.e., the low-buoyant density lymphocyte fractions. However, if the cultures were observed for a longer time, the initial growth regressed even in cultures containing the subsets which did not have natural killing (NK) function, i.e., those with high cell density. The initial cell concentration at which the cultures were seeded determined the outcome of the experiments and the demonstration of inhibitory effects. An important difference was seen between the subsets with regard to radiosensitivity. The prompt inhibitory effect of the NK-positive subset remained after irradiation, while the function of the NK-negative one was abrogated. In the presence of the irradiated T-enriched total population, infected B cells (BEBV) grew. Consequently, the radiation-resistant effector compartment, represented by the low-density cells, was not sufficient to counteract the establishment of BEBV lines. They contributed, nevertheless, to the regression because the kinetics of B-cell growth were different in cultures containing separated high-density cells or the total population. In the former, growth continued for a longer time and complete regression occurred only in the cultures initiated with high cell concentrations. The experiments showed that two types of cells contribute to the regression of BEBV growth in cultures initiated with lymphocytes of seropositive donors. One acts promptly and is independent of cell proliferation; another is activated for proliferation by encounter with B blasts.

OVA-specific T cells were immortalized by infection with radiation leukemia virus (RadLV). Some clones derived from such population were shown to exhibit helper activity. We then tested clones without such function and found among them some that secreted macrophage migration inhibition factor (MIF) and leukocyte migration inhibition factor (LIF) upon exposure to the antigen in vitro . The lymphokine-producing clones, which were Thy-1 + , Ly-1 + and Ly-2 − , did not secrete MIF and LIF constitutively. Like other antigen-specific T cells, the immortalized clones could not be stimulated by free soluble antigen but required macrophages for presentation and for triggering the lymphokine production. The antigen-activated clones exclusively produced MIF and LIF, but not interleukin 2 or colony-stimulating factor. They neither provided helper activity nor induced delayed-type hypersensitivity. The data suggest that the T-cell clones carry the antigen receptors and that their antigen-inducible biological function is restricted to the migration inhibitory factor production.

Treatment of human blood mononuclear cells with nanomolar concentrations of phorbol 12,13-dibutyrate (P(Bu) 2 ) induced their aggregation. The phenomenon was seen within a few minutes and reached its maximum manifestation after 20 min. At this time, 20–30% of unfractionated and nylon wool-passed mononuclear leukocytes were in the aggregates. The influence of pretreatment with 2-deoxyglucose, NaN 3 , EDTA, cyclohexamide, or incubation at 4 °C on the phenomenon indicated that it is energy and temperature dependent, requires the presence of extracellular divalent cations, and is independent of protein synthesis. Nonaggregating 2-deoxyglucose- and NaN 3 -pretreated cells could still bind [ 3 H]P(Bu) 2 which rules out the possibility that the phorbol ester molecule acts as a bridge between the aggregated cells. Seventeen percent of the P(Bu) 2 -treated T-cell population bound untreated autologous and allogeneic cells. The binding property has a certain species specificity because only 4% of the cells interacted with mouse lymphocytes. At the ultrastructural level, the intercellular binding showed broad areas of surface contact (both between lymphocytes and lymphocyte-monocyte) and “trapping” by surface processes was not seen. Aggregated cells did not show cytopathogenic changes.

Lymphocyte subsets were separated by nylon-wool adherence, E rosetting, and presence of Fc-gamma receptor (as defined by EA-rosetting capacity), and characterized for reactivity with the following monoclonal antibodies: OKT3, OKT4, OKT8 (reactive with T lymphocytes and T-lymphocyte subsets), OKM1 (reactive with cells of myelomonocytic origin), and OKIa-1 (reactive with HLA-Dr antigens). The majority of the OKM1-positive cells were collected in the EA + compartment of the E − , and low-avidity E + subpopulations. In spite of the separation as E rosettes, the EA + low-avidity E + cells did not react with OKT3 and OKT4. However, a proportion of these cells reacted with OKT8. These cells had high anti-K562 cytotoxicity. The EA-receptor-negative part of the E − subset, which had less OKM1 -positive cells, was also active against K562. Activated T cells may have contributed to the activity of this fraction, because OKT3- and OKIa-1-positive cells were present. When Daudi was used as target, only the EA-rosette-negative parts of the E − and low-avidity E + subsets were lytic. This activity was more evident after IFN activation of the effector lymphocytes. After introduction of the markers defined by monoclonal antibodies the heterogeneity of the NK-active population was still a fact. At least three cell types defined by these reagents were enriched in the different cytotoxic fractions: OKM1-, OKT8-, and OKIa-1-positive cells. The fact that part of NK activity was exerted by cells separated on the basis of SRBC resetting and in the SRBC rosette-negative subsets, a high proportion of the cells reacted with monoclonals, which define T markers, confirms previous suggestions that the majority of NK cells belong to the T lineage.

Cell lines derived from Epstein-Barr virus (EBV)-positive and EBV-negative Burkitt lymphoma (BL) have a low or defective expression of polymorphic HLA class I determinants compared to EBV-transformed lymphoblastoid cell lines (LCL) of normal B cell origin and are resistant to lysis by cytotoxic T lymphocytes (CTL) specific for the corresponding determinants (M. G. Masucci, S. Torsteinsdottir, J. Colombani, C. Brautbar, E. Klein, and G. Klein, Proc. Natl. Acad. Sci. USA 84 , 4567, 1987; S. Torsteinsdottir, C. Brautbar, E. Klein, G. Klein, and M. G. Masucci, Int. J. Cancer , 41 , 913, 1988). In order to investigate whether this phenotypic trait of the tumor cells can be modulated by agents known to enhance HLA class I antigen expression, pairs of LCL and BL lines were cultured in the presence of recombinant human interferon (IFN)-γ and tumor necrosis factor (TNF)-α. Three low HLA A11 expressor EBV-negative BL lines, DG 75, BL 28, and BL 41, reacted significantly stronger with the anti-HLA A11 monoclonal antibody (Mab) AUF 5.13 after combined treatment with 500 U/ml IFN-γ and 500 U/ml TNF-α. Reactivity with the AUF 5.13 and with other anti-polymorphic class I Mab's was up-regulated also in in vitro EBV-converted sublines of BL 28 and BL 41. The increment of antigen expression depended on the baseline expression in untreated cells. It was largest for the low expressor lines and decreased proportionally to the level of up-regulation induced by EBV conversion. Up-regulation of HLA A11 was accompanied by induction of sensitivity to HLA A11-specific CTLs in BL 28 and its converted subline E95A BL 28 while BL 41 and E95A BL 41 remained resistant. The treatment did not affect significantly HLA A11 expression of two EBV-carrying, low HLA A11 expressor BL lines, WW-1-BL and WW-2-BL, and of the EBV-carrying BL 72 line that had a high spontaneous expression. The results suggest that the down-regulation of class I antigen expression is reversible in some but not all BL lines.

The properties of lymphocytes cultivated with K562 (MKC)—a cell line which lacks HLA antigens and is sensitive to natural cytotoxicity—was compared to conventional mixed lymphocyte cultures (MLC). The characteristics of the two cultures differed. In the MKC there were fewer E positive blasts and more FcR-positive cells, and a higher proportion of the T cells was nylon adherent. The cells of both cultures were cytotoxic against K562. Against the sensitizer alloblasts, MLC populations were regularly more active than lymphocytes derived from the same donor but cultivated with K562. As indicated by the relative cytotoxic efficiencies of MLC subsets, part of the the killer cells affecting K562 and alloblasts differed in the expression of E receptors. Acting similarly to natural killers in the fresh blood, anti-K562 effectors were more abundant in the subsets which did not sediment as SRBC rosettes. In contrast, the specific alloreactivity was more pronounced with the SRBC-rosetting cells.

The mechanisms, cell surface structures, and cell types involved in the phorbol 12,13-dibutyrate (P(Bu)2)-induced binding between human lymphocytes were studied. Induction of cell aggregation by 20 min treatment with P(Bu)2 required Ca2+, an intact membrane, functional microfilaments, and the possible participation of an esterase or, less likely, a protease. Trypsin-sensitive cell surface structures were needed and neuraminidase (NANase) treatment slightly increased the intercellular binding. Retinoic acid, an anti-tumor promoting agent, was inhibitory. Calmodulin-dependent processes, microtubules, phospholipid methylation, intracellular levels of cyclic adenosine monophosphate, and cellular secretion did not seem to be involved. Cell conjugation between 24 hr P(Bu)2-treated and untreated cells required participation of trypsin-sensitive cell surface structures in each of the interacting cells and NANase treatment of one partner slightly increased the intercellular binding. Thymocytes, T cells, mature B and Epstein-Barr virus-transformed B cells aggregated while pre-B, early B, and intermediate B lymphocytes derived from representative malignancies did not. The lack of aggregation was not due to the absence of phorbol ester receptors. It is concluded that the P(Bu2)-induced intercellular binding is mediated by cell surface proteins, depends on certain enzymatic activities and metabolic events and involves certain cell types.

The T-cell subset mediating tumor graft neutralization was characterized in a methylcholanthrene (MC) tumor system. Lyt 1+ cells were critical for the successful prevention of outgrowth of the tumor cells in graft neutralization assays with irradiated recipients. Elimination of Lyt 1+ cells abolished the outgrowth inhibitory effect exerted by T-cell-enriched populations derived from syngeneic or semisyngeneic mice immunized with the H-2-carrying MC-induced M-A tumor. In accordance, lymphocyte populations containing 98% Lyt 1+ cells derived from M-A-immune mice, mediated a complete transplantation immunity against this tumor. When the immune T cells admixed to the tumor inoculum were syngeneic to the recipient (i.e., A-derived cells were transferred to A recipients, or F1 to F1), elimination of the Lyt2+ cells did not influence the potential to inhibit tumor outgrowth. The presence of Lyt 1+2- cells were thus necessary, and sufficient, in the syngenic combination. A reduction of the graft-inhibiting potential occurred after elimination of Lyt 2+ cells from the A-derived M-A immune T-cell population when the recipients were semisyngeneic (i.e., (A/Sn X A.SW)F1, (A/Sn X CBA)F1, or (A/Sn X C57B1/6)F1). Consequently, only in the semisyngeneic, but not in the syngeneic combinations, was the transfer of Lyt 2+ cells necessary for optimal graft inhibition. It can be concluded that the genotypic relation between the donor and the recipient influences the prerequisites of the tumor cell neutralization.

Activation of human blood T lymphocytes in mixed lymphocyte culture (MLC) causes a reduction in the net negative surface charge, as indicated by the reduction in the electrophoretic mobility. Concomitantly, the activated cells acquire new properties, including the ability to form “stable” E rosettes, and attach to normal and malignant cells of the same species (natural attachment (NA)). These properties were found to be expressed by lymphocytes within the low electrophoretic fractions (cells with low negative charge) of the MLC populations. The formation of stable E rosettes and natural attachment capacities of human thymocytes were also found to correlate with the amount of surface negative charge. The slowly migrating (less negative charged) cortical thymocytes, reported earlier as being able to form stable E rosettes, were found also to exhibit NA activity. Medullary thymocytes carrying a high net negative surface charge lack these characteristics. We consider it likely that the reduction of negative charge during activation of peripheral T cells, facilitates cell-to-cell contacts, and thus prepares the (activated) cells to perform cooperative interactions with other cell types, and express the lytic activity of T cells.

At various times after yellow fever (17 D) vaccination mononuclear blood cells were tested for cytotoxic potential in conjugate with K 562. They were characterized with monoclonal reagents Leu2a, Leu4, and OKM1, using indirect immunofluorescent technique. The cytotoxicity against K 562 increased after vaccination with a peak on Days 8–12 when the [ 3 H]thymidine uptake was also maximal. The majority of cells in conjugates with K 562 were then T cells. Later the conjugates contained more OKM1-positive cells. OKM1 reagent was found to react with K 562 and thereby cause a coagglutination between this target cell and the OKM1-positive monocytic cells. The surface moiety reacting with OKM1 reagent might be of lipid nature.

Natural cytotoxicity against tumor-derived monolayer and lymphoblastoid cell lines was found to be most marked in the non-T non-B subpopulation of human lymphocytes. A high proportion of cells in this population were Fc receptor positive. This receptor seems to be important in exerting the cytotoxic effect. The T-cell fraction had a weak cytotoxic activity for which Fc-positive T cells were responsible. Steps in the cell separation procedure such as EA resetting and ammonium chloride treatment impaired cytotoxic activity. However, the cells recovered to a large degree during the 48-hr assay. Cells incubated at 37 °C prior to the functional test regained part of the activity and could thus be used in a short-term, 8-hr assay. Within a certain effector cell range, the dose-response curves were linear. Thus, it was possible to express the efficiency of a certain fraction in lytic units (the cells which cause 50% lysis of the target cell). Considering the representativeness of the cell type in the total population, the non-T non-B cells were calculated to be responsible for 24% of the total activity though their proportion was only 7%.

The majority of cell lines derived from Burkitt lymphomas carry CR2 on their plasma membrane. Cell lines of haematopoetic origin can activate C3 present in human serum through the alternative pathway. However, only the lines that carry CR2 were shown to bind C3 fragments. This bond can be either fixation to acceptor sites or attachment to the CR. Our studies with Raji cells showed that when the possibility for the covalent acceptor bond was eliminated by using methylamine (MA)- or zymosan-treated serum, considerably lower amounts of C3 were bound. In the zymosan-treated serum C3 fragments are present that can bind to receptors but their capacity for acceptor bond is absent. These results indicate that when Raji cells are incubated in human serum some of the generated C3 fragments are bound to acceptors and a lower proportion through the specific interaction with complement receptors. Pretreatment of the CR2 carrying cell lines with human serum elevated their sensitivity to the lytic effect of human blood lymphocytes. We showed in this work that MA-treated serum did not induce this elevation. Zymosan-treated serum under conditions that excluded activation of the residual native C3 molecules, i.e., in the presence of EDTA, did not have the enhancing effect either. These results suggest that the increased lytic efficiency imposed by human serum was due to cleavage of C3 molecules by Raji and fixation of the C3 fragments by acceptor sites. Natural killer cells carry CR3; therefore it is likely that the attached C3 fragments bind also to the effector cells. The C3 molecules could elevate thereby the avidity between the target and the lytic lymphocytes. The observation that C3 fragments are not bound to the surface of CR2 negative lines in spite of their capacity to activate C3 suggests that the receptor molecule is either involved in the activation and/or serves also as an acceptor.

Two interleukin-2 (IL-2)-dependent cytotoxic T-cell clones were obtained by limiting dilution from a lymphocyte culture stimulated in vitro with the autologous Epstein-Barr virus-transformed lymphoblastoid cell line (LCL) in the presence of fetal calf serum (FCS). Both clones uniformly had a T3 + , T4 + , Dr + phenotype and lysed autologous B blasts, the autologous LCL and allogeneic B cell lines sharing major histocompatibility complex (MHC) class II antigens. The cytotoxic function was triggered by FCS-derived components. There was no killing if the sensitive targets were cultured in serum-free medium or in medium supplemented with human serum. Sensitivity to lysis could be restored by exposing the targets to FCS for at least 6 hr at 37 °C. Monoclonal antibodies directed to T-cell-specific surface antigens and MHC class II antigens inhibited lysis with different efficiencies depending on the target cell origin. Killing of Burkitt's lymphoma (BL)-derived cell lines was blocked more easily than killing of LCLs. LCLs but not BL lines induced proliferation of the T-cell clones in the absence of exogenous IL-2. The differences were not related to quantitative variations in the expression of MHC class II antigens, indicating that BL lines differ from LCLs in other cell membrane properties that may influence antigen presentation. The results suggest that the affinity of effector/target binding, which is probably influenced by the concentration of antigenic determinants expressed on the target cell membrane, determines whether proliferative responses or cytotoxicity are induced in the antigen-recognizing T cells.

Guinea pigs were immunized with cells of a human lymphoblastoid line. While nonsensitized lymph node cells were not influenced, those derived from immunized animals were markedly stimulated in vitro by the human cells. Maximal activity was found with lymphocytes harvested on day 6. The response was paralleled with the strength of skin induration elicited by the human cells and with the efficiency of the cytotoxic effect of lymphocytes on human target cells. The stimulation and cytotoxicity was specific while the skin test demonstrated cross reactivity with mouse cells.

The P3HR-1 Burkitt lymphoma line carries the Epstein-Barr virus (EBV) genome and a small proportion of the cells (1–3%) enter the lytic cycle spontaneously. Treatment with TPA and n-butyrate elevates considerably the number of virus-producing cells (25–35%). Cells which enter the lytic cycle express the EBV early antigen EA, the viral capsid antigen VCA, and the membrane antigen MA. Antibodies against these antigens are present in EBV-immune human sera. The expression of virus envelope protein on the plasma membrane renders the cells sensitive to immune effector mechanisms. These were shown to be initiated by the alternative complement pathway (ACP)-activating capacity of the cells and by their reactivity with antibodies directed to the MA. When incubated with EBV-immune or nonimmune human serum, the induced (P3HR-1-V) cells activated C3 through ACP and fixed the generated C3 fragments. The efficiency of opsonization was higher in immune serum. By varying the experimental conditions we showed the damage of the induced cells by the complement system and by blood lymphocytes, and analysed the involvement of antibodies and the activated C3 fragments in the lymphocyte-mediated lysis. P3HR-1-V cells were lysed by immune serum and also by nonimmune serum though with lower efficiency. The induced cells had elevated sensitivity to the NK effect which was potentiated if the conditions allowed their opsonization. In the presence of antibodies the lymphocyte-mediated lysis was considerably higher and the ADCC mechanism was also potentiated by opsonization. These experiments suggest that B cells which enter the virus-producing cycle may be eliminated in EBV nonimmune host by NK cells. After the antibody response against the virus develops, the attack on these cells is more efficient through complement and lymphocyte-mediated antibody-dependent mechanisms. These effector mechanisms are enhanced by opsonization which is the consequence of the C3-activating capacity of the cells. The multiple ways of the immune attack on the B cells prepared to produce EBV may explain the absence of EA and VCA positive B cells in tumor cell populations and during the acute phase of infectious mononucleosis.

The natural killer (NK) sensitivity of two Epstein-Barr virus (EBV)-carrying Burkitt lymphoma lines (Daudi and Raji) and one EBV-negative acute lymphocytic leukemia-derived T-cell line (Molt-4) increased when they were cultured for 24 hr in the presence of the phorbol ester TPA. In the EBV-carrying lines, this increase occurred independently of the entry of the cells into the viral cycle. 12-O-Tetradecanoylphorbol-13-acetate (TPA)-treated Daudi and Molt-4 cells cross-competed for the effectors in the NK assay as indicated by the cold-target inhibition tests. TPA-treated cells showed an increased binding of human but not of mouse lymphocytes.

We have analyzed the effect of cyclosporin-A (CsA) on the proliferative and cytotoxic responses induced by mixed-lymphocyte cultures (MLC-s) on low and high density T lymphocytes. Allogeneic stimulation had a different impact on the two subsets. Proliferative and cytotoxic responses were inversely correlated; i.e., high density cells proliferated but exerted low levels of cytotoxicity while the lytic activity of the low density subset was stronger and the proliferation was weak. CsA impaired the proliferative and cytotoxic responses of the high density T lymphocytes but influenced less markedly the response of the low density cells. In both subsets CsA inhibited the MLC-induced interleukin 2 (IL-2) production. The generation of specific cytotoxicity was markedly suppressed by CsA, whereas the generation of anomalous activity was less affected. Addition of exogenous IL-2 to the CsA-containing cultures fully restored the proliferation and the generation of nonspecific cytotoxicity. In contrast, addition of interferon gamma (IFN-gamma) restored neither of these responses. However, for complete restoration of the stimulation-specific cytotoxicity, addition of both lymphokines was required. Taken together these results suggest that the CsA-induced suppression of the lymphokine production has different consequences in the low and high density subsets; the expression of anomalous and specific cytotoxicities require different signals; CsA interferes with several steps in the T-cell activation.

Recombinant human interferon alpha (IFN-alpha) and interferon gamma (IFN-gamma) were compared for their ability to influence the proliferative capacity of tumor-derived cell lines and of normal B lymphocytes infected in vitro by Epstein-Barr virus (EBV). EBV-induced B-cell proliferation was suppressed almost completely when 10(2) U/ml IFN-alpha were added to the culture medium while the same dose of IFN-gamma had significantly lower inhibitory activity. The pure IFNs differed in their ability to influence the growth of three Burkitt lymphoma-derived cell lines, Raji, Daudi, and Namalwa, depending on whether the cells were propagated in suspension or in semisolid cultures. IFN-alpha inhibited cell proliferation under both culture conditions with thresholds of sensitivity characteristics for each cell line. In contrast, IFN-gamma had no effect on the growth in suspension but it abolished the clonogenic potential of tumor cell lines in semisolid agarose. The results suggest that the two IFN types may exert their growth inhibitory activity through different mechanisms of action.

Using the indirect leukocyte migration inhibition technique T cells have been identified as being responsible for Epstein-Barr virus nuclear antigen-induced specific leukocyte migration inhibitory factor production. The response was dependent on the presence of macrophages or their product, T-lymphocyte activating factor.

Human B-cell lines derived from normal donors (LCL) or from Burkitt lymphomas (BL) were compared for their sensitivity to natural (NK) and interferon (IFN)-activated (IAK) cytotoxicity, mediated by effector cells from normal human blood. In four cases, a BL and an LCL line were derived from the same donor and had been kept in culture for the same period of time. The BL series included both Epstein-Barr virus (EBV)-carrying and EBV-negative lymphoma lines. The latter were compared with their own EBV-converted, Epstein-Barr nuclear antigen (EBNA)- and EBV-DNA-positive sublines, established by in vitro infection with two different viral substrains. LCL and BL lines from the same donor were lysed with equal efficiency by both NK and IAK effectors. There was no relationship between the NK sensitivity and the nude mouse tumorigenicity of different EBV-converted Ramos sublines, or the expression of differentiation markers such as insulin receptor, surface IgD, and the B2 surface antigen. Moreover, EBV-converted sublines of BJAB differed in their NK sensitivity, in spite of closely similar expression of these markers. NK-sensitive Ramos and BJAB sublines induced a stronger proliferative response upon confrontation with allogeneic lymphocytes than their NK-resistant counterparts. This suggests that the target cell may play an active role in triggering the lytic interaction. There was no correlation between this property and any of the other parameters studied.

Raji and Daudi cells were opsonized with C3b, iC3b, and C3d fragments by using purified complement components. The sensitivity of C3-opsonized cells to lysis mediated by low density blood lymphocytes was studied. Raji and Daudi cells carrying C3b or C3d fragments were lysed with similar efficiencies as the nonopsonized cells. The presence of iC3b on the target surface imposed elevated NK sensitivity. The iC3b-mediated enhancement of NK lysis was inhibited when iC3b fragments or rabbit anti-human C3 antibodies were included into the lytic assays. These results indicate that the iC3b fragments fixed on the targets bind to the CR3 on the lymphocytes. Results obtained in immobilized conjugate-lytic assays showed that iC3b-opsonized targets interact more readily with the lymphocytes. This was reflected by the elevated proportion of lymphocytes that were bound to the iC3b-carrying targets. The proportions of conjugates in which target damage occurred were similar with the control and with the iC3b-carrying cells. It seems therefore that opsonization of targets with iC3b leads to recruitment of effector lymphocytes due to contact with their CR3. However, once the effector-target contact is established, the triggering of lytic function does not seem to be influenced by the iC3b/CR3 bridge.

T cells were exposed in vitro to autologous B cells or monocytes. Tested on the seventh day, the cultured lymphocytes lysed K562, Daudi, autologous, and allogeneic phytohemagglutinin (PHA) blasts. Autologous B blasts were not affected. The supernatants contained gamma interferon (IFN-γ). The quantity of IFN did not correlate with the strength of the proliferative response nor with the strength of the cytotoxic potential.

K562 is sensitive to the natural killer (NK) effect of blood lymphocytes. When cultured alone, the lymphocytes lose this activity. When cultured with K562 (MKC) or with allogeneic lymphocytes (MLC) anti-K562 cytotoxicity is maintained. In order to study the relationship of the cytotoxicity of fresh and cultured lymphocytes, subsets characterized for NK activity were allowed to react in the mixed cultures. Populations rich in T cells and devoid of NK effect responded well in MLC with concomitant generation of cytotoxicity to K562. The same fractions failed to respond or responded weakly in MKC, unless supernatant from MLC was added to the culture. The results showed that at least part of the anti-K562 activity in the mixed cultures is not due to maintenance of NK but is newly generated from precursors which belong to the T subset. The strength of cytotoxicity in various cultures is related to the efficiency of the activation as judged by blastogenesis, which indicates that killing of K562 is a characteristic of T lymphocytes in a certain stage of activation.

The cytotoxic activity of human blood lymphocytes toward Raji cells was strongly elevated when human serum (HS) was included in the cytotoxicity assay. This phenomenon also occurred when the effector cells were activated by interferon (IFN). Hypogammaglobulinemic serum (HyS) and heat-inactivated serum could also augment cytotoxicity, but C3-depleted serum was inefficient. IFN treatment of Raji cells decreased their sensitivity to lysis and this effect was counteracted by addition of HS to the system. It is likely that C3 activation by, and deposition on, Raji cells when used as targets for cytotoxicity facilitate their recognition and lysis by lymphocytes. These events may represent one mechanism operating in the natural killing phenomenon.

The NK sensitivity of Raji cells was elevated after superinfection with the P3HR-1 strain of Epstein-Barr virus (EBV). The superinfected cells reacted with EBV antibody-positive sera and this was detectable by ADCC. F (ab′) 2 fragments from the antibody-positive sera blocked the ADCC reaction but did not affect attachment of lymphocytes and natural killing. This indicates that the EBV-determined membrane antigens that serve as targets for the reactivity with the sera are not responsible for the increased NK sensitivity.

Blood lymphocytes of individuals differ in the spontaneous cytotoxic potential exerted in vitro against certain cell lines (natural killing, NK). In the low NK donors, the activity can be enhanced by short-term IFN pretreatment of the effectors (interferon activated killing, IAK) and by addition of PHA to the short-term assay (lectin-dependent cellular cytotoxicity, LDCC). Lymphocyte subpopulations fractionated on the basis of nylon adherence, SRBC, and EA rosette formation differ in their response to these measures. The results obtained with IFN-treated lymphocytes of low NK donors were similar in strength to the spontaneous activity of the high NK donors. Therefore, the distinction between NK and IAK is only operational. The nylon passed E receptor-negative and low-avidity E receptor-positive cells had the strongest NK activity. These subsets can be triggered for enhanced activity by IFN. In the majority of the cases the high-activity E-receptor-positive subset which did not sediment with EA indicators had low NK effect and was not triggered by IFN. Addition of PHA to the lytic assay, however, induced activity in the subset. Realization of DNA synthesis was not necessary for the lytic performance. The PHA-imposed triggering event was not dependent on IFN production nor on induction of the competence for IFN response. The results showed that all non-B lymphocyte subsets separated on the basis of nylon wool adherence, SRBC, and EA rosetting contain cells with lytic potential if the appropriate stimulus is used. The relative activities of the subsets against K562 and Daudi differed. Cells which rosetted readily with EA indicators had weak effect against Daudi.

Lymphocytes from healthy donors were cultivated with K562, a cell line sensitive to natural cytotoxicity and lacking HLA antigens. Blastogenesis and cytotoxicity were generated in the presence of the serum of the lymphocyte donor. The anti K562 killing effect of the cocultivated populations exceeded considerably that of the fresh lymphocytes. In the majority of cases lymphocytes cultured alone had no cytotoxic activity. We have attempted to characterize the active lymphocyte subset, using the fractionation procedure designed for fresh lymphocytes. Separation of T cells on the basis of SRBC rosetting was not efficient because a high proportion of E rosettes remained in the interface when centrifuged on the Ficoll. Furthermore a high number of E rosetting cells adhered to nylon wool. The cytotoxic potential of various fractions correlated to the presence of blasts and EA positive cells. The least active population was the non-adherent, E rosette sedimented one. The cytotoxicity was not restricted to the sensitizing K562 cell line.

The specificity of in vivo rejection across an H-2 barrier was reinvestigated by mixing a small number of compatible tumor cells with a large number of incompatible cells and comparing the outgrowth of the compatible minority with appropriate control groups, inoculated with compatible cells alone, or compatible cells mixed with irradiated syngeneic cells. Eight mouse lymphomas and nine methylcholanthrene-induced sarcomas were tested in various combinations. There was no inhibition of the compatible minority by a 101–105-fold excess of incompatible cells in 29 evaluatable combinations; in 14 there was, on the contrary, a clear stimulatory effect. In part of the experiments, the hosts were preimmunized against the incompatible cells, but the results were essentially identical. Some nonspecific inhibitory effect was seen in six combinations. Four of them where the EL4 lymphoma entered as the incompatible cell compartment, were relatively consistent. The nonspecific inhibitory effect of the allogeneic EL4 cell depended upon its temporary growth, before rejection, in untreated allogeneic mice; it was prevented by preirradiation of the cells or by preimmunization of the recipient hosts.