Generation of killer lymphocytes in vitro against human autologous leukemia cells with leukemic blasts and BCG extract

Summary Acute lymphoblastic leukemia patients under 18 years of age were studied to determine the ability of their remission lymphocytes to kill autologous leukemic blasts (ALB) following in vitro sensitization with their leukemic cells and/or soluble extract of BCG (BCG-SE). Remission lymphocytes, when cultured together with the mitomycin-treated ALB, became significantly lytic for ALB but not for autologous remission lymphocytes. The ALB were usually immunogenic at low concentrations and no cytotoxic lymphocytes were generated at a ratio of 1:1 of responding lymphocytes to stimulating leukemic cells. T-leukemic cells appeared to immunize more effectively than null-cell leukemic cells. In some cases, when ALB alone could not generate killer lymphocytes (KL) the combination of ALB and BCG-SE induced more intense cytotoxicity than was induced by BCG-SE alone. In a few other cases, the addition of BCG-SE to mixed lymphocyte leukemic cell cultures potentiated the immunization of lymphocytes by leukemic cells. Inhibition of cytotoxicity induction was noted in one case when remission lymphocytes were cultured together with ALB and BCG-SE. Leukemic cellssensitized lymphocytes from some cancer patients and normal persons were cytotoxic to several but not all patients' leukemic cells tested. Nylon wool-nonadherent, non-E-rosette-forming, and E-rosette-forming cells became cytotoxic following in vitro stimulation with autologous leukemic cells.     

Generation of functional dendritic cells for potential use in the treatment of acute lymphoblastic leukemia

Immunotherapy of malignant diseases mediated by dendritic cells (DC) pulsed with tumor antigens ex vivo is a promising new tool in the individual treatment of malignant diseases. The present study focuses on the problem of how to optimize in vitro culture conditions and induce the maturation of DC with the capacity to induce antitumor immunity toward leukemic cells. DC were generated from peripheral mononuclear cells by co-cultivation with granulocyte/macrophage-colony stimulating factor (GM-CSF) and interleukin-4 (IL-4). Tumor antigens were added for 2 h after 7 days in culture. Irradiated leukemic blasts, blast lysate, apoptotic cells from the Jurkat cell line (T ALL) and their lysate were used in various concentrations for antigen pulsing. Harvested DC were phenotyped by flow cytometry, and viability was assessed using trypan blue exclusion (Annexin test). After the cells had been pulsed with tumor antigens and co-cultured with autologous lymphocytes, the production of interferon-gamma (IFN-gamma) and IL-12 was analyzed, and lymphocyte proliferative response and cytotoxicity against the target tumor cell line were assessed. The cultivation of monocytes under the described conditions led to the expression of surface markers typical of DC (i.e. CD83, CD86, HLA-DR, CD11c and CD40). Pulsation by antigens from leukemic cells further increased the cell populations expressing these markers. Antigen pulsation decreased the viability of generated DC depending on the increase in concentration of tumor antigens. Pulsed DC-lymphocyte interaction increased the proliferative response of lymphocytes and IFN-gamma production depending on the type of tumor antigens used for pulsation. The highest proliferative response was detected with DC pulsed with Jurkat cell-line lysate. Similarly to the proliferation assay, cytotoxic testing showed the highest efficiency of DC pulsed with Jurkat cell-line lysate in killing the target malignant cells. Our results show that an appropriate antigen concentration used for DC pulsing is one of the crucial factors in an effective treatment strategy, as high concentrations of tumor antigens induce apoptosis of DC, thereby rendering them non-functional. Under optimal conditions, pulsation by lysate from leukemic blasts induced the maturation of DC and led to an increase in the proliferation of autologous lymphocytes, to the production of Th1-cytokines and to the induction of cytotoxicity toward the leukemic cell line. These results are encouraging for the possible application of pulsed DC in the therapy of acute lymphoblastic leukemia.     

The progenitor cell inhibition assay to measure the anti-leukemic reactivity of T cell clones against acute and chronic myeloid leukemia

Adoptive immunotherapy with donor T lymphocytes may be used as a treatment for relapsed leukemia after allogeneic hematopoietic stem cell transplantation (SCT). In vitro selected and expanded anti-leukemic T cells may be more effective in inducing a response in vivo. To identify the anti-leukemic reactivity of in vitro generated T cells, standard target cell read-out assays like the 51Cr-release assay are not always appropriate. We developed an assay in which the ability of T cells to antigen specifically inhibit the in vitro growth of leukemic progenitor cells in the presence of cytokines can be measured. This assay allows the evaluation of the cytolytic or suppressive potential of leukemia reactive T cells for prolonged periods of time. The assay is based on inhibition of [3H]thymidine incorporation by the leukemic progenitor cells induced by multiple hematopoietic growth factors. T cell clones with a known specificity were used to compare the analytic potential of the new assay with those of other cytotoxicity assays. Based on the results of the T cell clones, a modification of a limiting dilution assay was developed to identify anti-leukemic allogeneic T cells in HLA identical donor-recipient combinations selected on their ability to inhibit the in vitro growth of CML or AML progenitor cells, to be used for the generation of leukemia-reactive CTL lines for clinical use.     

Generation of killer lymphocytes in vitro against human autologous leukemia cells with soluble bacterial extraxts

Summary Ten patients with acute lymphoblastic leukemia (ALL) were studied to determine the ability of their remission lymphocytes to kill autologous leukemic blasts (ALB) following in vitro exposure to soluble extracts (SE) of BCG, Staphylococcus aureus (SA) or Listeria monocytogenes (LM). Remission lymphocytes from some patients became markedly cytotoxic to ALB after stimulation with BCG-SE, LM-SE, or SA-SE. These bacterially stimulated lymphocytes, although specifically lytic for ALB, were usually not cytotoxic to autologous remission lymphocytes. Bacterial extracts were able to generate killer lymphocytes at low concentrations. Generally, large amounts either had no stimulatant effect or were less stimulating. Bacteria-stimulated lymphocytes of ALL patients were cytotoxic not only to their leukemia cells, but also to leukemia cells from ALL and AML patients who were allogeneic to stimulated lymphocytes.     

Cell-mediated lympholysis of trinitrophenyl-derivatized autologous human cells: in vitro triggering by nonspecific signals.

This report describes the primary in vitro generation of human CTL that lyse TNP-derivatized autologous cells. Although in the majority of these studies, a direct cytotoxic response to the TNP-modified autologous stimulators was not achieved, in all experiments the addition of either allogeneic cells or soluble antigen triggered the generation of killer cells which destroy TNP-modified, but not unaltered, autologous targets. Fractionation of responder lymphocyte populations demonstrated that the cytotoxic activity was mediated by T cells. Killer cell specificity was tested by assaying for cytotoxicity to a variety of targets, and by blocking the cytolysis of TNP-altered autologous targets with various populations of nonradiolabeled cells. Results indicated that these CTL were cytotoxic for TNP-modified autologous cells but not unaltered autologous or TNP-modified allogeneic targets. The capacity of soluble antigen and alloantigens to facilitate the in vitro generation of altered-self reactive human CTL is not an isolated phenomenon. This "helper" effect has now been observed for the cytotoxic response to chemically modified autologous cells and MHC identical human leukemic blasts. It is possible that in vivo, similar responses to nonspecific antigenic stimuli may play a role in the maintenance of immune surveillance.     

Interactions between lymphocytes and hematopoietic progenitor cells in normal and leukemic human bone marrow (phase contrast observations)

Single cell observations of normal and of leukemic human bone marrow cells demonstrated cell-cell interactions of lymphocytes with hematopoietic progenitor cells. In all cases lymphocytes and target cells were from the same individual. Lymphocyte-target cell interactions occurred more frequently with normal committed progenitor cells and leukemic blast cells from acute myeloid leukemia than with precursor cells of the proliferative cell pool, including myeloblasts, promonocytes, erythroblasts and megakaryocytes. Both induction of mitosis and degeneration of the progenitor cells occurred after cell-cell interaction with almost the same frequency. Acute myeloid leukemic blast cells degenerated after contact with lymphocytes with the same frequency as normal progenitor cells (i. e. in 16% of cell contacts), but especially during mitosis. In contrast, normal and regenerating bone marrow progenitor cells from myeloproliferative diseases demonstrated no degeneration after cell-cell interaction with lymphocytes during mitosis. Otherwise the induction of mitoses by lymphocyte-target cell interactions was more frequently observed in normal progenitor cells than in leukemic blasts.     

Methods for amplifying the induction and expression of cytotoxic response in vitro to syngeneic and autologous freshly-isolated solid tumors of mice

Summary Spontaneously arising tumors are frequently poorly immunogenic and exhibit a limited capacity to induce cytotoxic effector lymphocytes. In the present study, various approaches have been used to amplify the induction and expression of cytotoxic responses in vitro toward freshly isolated, autologous, and syngeneic solid neoplasms of spontaneous origin in mice. Cytotoxic lymphocytes were generated in one-way mixed lymphocyte-tumor cell cultures (MLTC) consisting of splenocytes or lymph node cells from normal and from tumor-bearing mice co-cultured with inactivated tumor cells. Optimal culture conditions have been established for the number of responder (R) cells, the method of inactivation of the stimulating (S) tumor cells, the responder/stimulator (R/S) cell ratio, and the duration of sensitization. Under optimal sensitization conditions only weak cytotoxic responses, as measured by the ⁵¹Cr-release assay, were generated. The antitumor cytotoxic activity could be augmented 2- to 12-fold by using each of the following procedures: (a) addition of crude or of partially purified interleukin-2 (IL-2) to the sensitization cultures; (b) depletion of nylon-adherent cells from the responding cell population; (c) enrichment of large lymphoblasts from the sensitized effector cell population by Percoll density gradient; and (d) treatment of mice donating the responder lymphocytes with low doses of either cyclophosphamide, adriamycin, or indomethacin. Although the highly reactive effector cells generated under the improved conditions also reacted appreciably with unrelated tumor target cells, only low levels of cytotoxicity could be demonstrated against normal target cells. The antitumor cytotoxic cells in sensitized splenocyte cultures were exclusively Thy1⁺, Lyt1^–2⁺, whereas in lymph node cell cultures some cytotoxicity was also exerted by Thy1⁺, Lyt1⁺2⁺ cells.     

Human immune responses to hapten-conjugated cells. II. The roles of autologous and allogeneic histocompatibility determinants in proliferative responses in vitro.

Proliferative responses of human lymphocytes primed in vitro to autologous TNP-cells were found to be associated with autologous D-region determinants irrespective of HLA-B locus antigens. Family studies of secondary TNP-conjugate proliferative responses demonstrated a gene dosage effect in this phenomenon. Moreover, co-culture with allogeneic cells did not affect the net TNP-conjugate proliferative responses of primed responder cells, suggesting that HLA-D region preference was due to a requirement for representation of TNP-molecules in association or combination with autologous MHC structures. Alloantigens were found to influence the sensitization of lymphocytes to autologous hapten-conjugated cells. Co-culture of allogeneic and TNP-modified autologous stimulator cells in primary cultures enhanced the secondary TNP proliferative response. Sensitization of human lymphocytes to allogeneic cells alone did not prime responses to autologous modified cells. However, priming lymphocytes to modified autologous cells potentiated responses to allogeneic cells. The data suggest a complex relationship between responses to alloantigens and modified autologous cells.     

Lysis of autologous human melanoma cells by in vitro allosensitized peripheral blood lymphocytes

Summary Peripheral blood lymphocytes (PBL) of melanoma patients were sensitized in vitro with lymphocytes of a single donor or with a pool of lymphocytes of 5–20 different donors. After 6–7 days, the cytotoxic activity of the sensitized PBL was tested against cultured autologous tumor cells and lymphocytes in a ⁵¹Cr-release assay. Tumor lysis was observed in 13 of 16 cases in which patients' PBL (Pt-PBL) were stimulated by a pool of allogeneic lymphocytes and in five out of seven cases when single sensitization was performed. In no case was lysis of autologous normal lymphocytes or blasts seen. Cultivation of Pt-PBL with irradiated autologous tumor cells never led to the induction of lymphocytes cytotoxic to melanoma cells. Lysability by pool-activated autologous Pt-PBL of fresh cryopreserved tumor cells was compared to that of short-term cultured tumor cells, and no significant differences were observed. Cold-target inhibition experiments indicated that the cytotoxicity of Pt-PBL was tumor-restricted since only autologous melanoma cells but not lymphocytes were able to inhibit the reaction. These results indicate that activation of Pt-PBL is necessary in order to elicit or amplify their antitumor activity.     

Human B cell differentiation. II. Suppression by T cells of T-dependent and T-independent plasma cell maturation.

In vitro human plasma cell generation induced by both T-dependent (PWM) and T-independent (NWSM) mitogens was found to be suppressed by peripheral blood lymphocytes preincubated with human aggregated IgG. T cells, but not B lymphocytes, were able to mediate the suppressive activity; since aggregated (Fab)'2 fragments were found unable to generate suppressor cells, it was concluded that the suppressor cell was a T lymphocyte bearing Fcgamma receptors. These cells appeared to be largely radiosensitive. In most cases the proliferative responses remained unchanged. Since NWSM-induced activation is not dependent on the presence of T cells, these results show that, at least in this case, T cells exert their suppressor function directly on B lymphocytes. Whether PWM-induced B cell differentiation is suppressed by the same mechanism or/and by inactivation of T helper lymphocytes remains under investigation.     

LY-2+ effectors cytotoxic for syngeneic tumor cells: generation by allogeneic stimulation and by supernatants from mixed leukocyte cultures

The present study was aimed at gaining insight into means by which stimulation of mouse spleen cells with allogeneic normal cells in mixed leukocyte cultures (MLC) can result in the generation of effector cells cytotoxic for syngeneic tumor or transformed cells. Stimulation of lymphocytes from BALB/c or C3H mice for 5 days with cells from mice of every allogeneic strain tested, in medium containing mouse serum and lacking xenogeneic serum, resulted in the activation of effectors cytotoxic for syngeneic cells transformed spontaneously or by SV40, polyoma or adenovirus. In each experiment, all of the syngeneic transformed cell lines, as well as clones derived from these lines, were lysed to the highest degree by effectors obtained from the same culture, and therefore stimulated with cells from the same allogeneic strain. Although the particular allogeneic sensitizing strain that induced the highest cytolytic activity varied between experiments, effectors obtained from the culture with the highest cell recovery always exhibited the greatest cytotoxicity against all the syngeneic transformed cells and clones. Lysis was mediated predominantly by Ly-2+ effectors; total lytic units of cytotoxicity recovered after treatment with monoclonal anti-Ly-2 antibody and complement (C) were reduced by 85 to 90% compared to cells treated with C alone. Lysis of syngeneic tumor cells by the allosensitized effectors in cytotoxicity assays was not inhibited by the addition of unlabeled "blocking" lymphocytes from the allogeneic strain used for sensitization. In addition, it was found that lymphocytes cultured without stimulating cells for 5 days in medium supplemented with supernatants from secondary MLC that are known to contain high levels of lymphokines, mediated high levels of cytotoxicity on all the transformed cells tested, but lacked detectable cytotoxic activity for syngeneic or allogeneic Con A blasts. The MLC supernatant-activated effectors that lyse the transformed cells are phenotypically CTL, because treatment with anti-Ly-2 and C reduced lytic activity by approximately 75%. Taken together, these findings suggest that the generation in MLC of Ly-2+ effector cells cytotoxic for syngeneic transformed cell lines might not be due, in some cases, to lymphocyte responses to particular alloantigens on the stimulating cells that are cross-reactive with "alien" histocompatibility antigens on transformed cells, but rather is due to effector cell activation by lymphokines produced during allogeneic stimulation.     

Functional characteristics of T cell receptors during sensitization against histocompatibility antigens in vitro

Using the mixed leukocyte culture (MLC) reaction as a model system for the generation of cytotoxic effector lymphocytes, we have examined changes which occur in recognition-binding function during T cell sensitization. Properties of recognition-binding units on normal and immune T lymphocytes were assessed through the ability of T cells to bind to specific cellular immunoadsorbants at various stages of in vitro sensitization. While antigen-specific recognition-binding function was readily detected on fully cytotoxic effector cells, we were unable to detect functionally specific binding of unsensitized lymphocytes to cellular immunoadsorbants. The ability of cells undergoing sensitization in MLC to bind specifically to target cell monolayers appeared congruent in time with cytotoxic function. These results suggest that a fundamental membrane-associated change occurring during T cell sensitization may be the development of a strong and specific target-cell binding function.     

Normal T-cell receptors for alloantigens

To study the diversity of normal mouse T lymphocytes capable of mediating allograft immunity, we modified a cell culture system so that both induction of sensitization and target cell damage could be studied in vitro. Mouse lymph node lymphocytes were sensitized in vitro against allogeneic fibroblasts. The sensitized lymphocytes produced immunospecific cytotoxic effects against target fibroblasts in vitro. We found that T lymphocytes were directly involved in both sensitization and cytotoxicity.We used this allograft system to separate nonsensitized mouse lymphocytes on the basis of their ability to bind to allogeneic fibroblasts. Adhering lymphocytes were found to be enriched in effector cells following sensitization. The nonadhering lymphocytes showed a decreased ability to undergo sensitization against fibroblasts that were syngeneic to the ones used for adsorption. However, they were able to become sensitized against unrelated fibroblasts of another H-2 phenotype.These findings indicate that specific receptors for histocompatibility antigens pre-exist on diverse populations of normal mouse T lymphocytes.     

Stimulation of peripheral human lymphocytes by autologous EBV genome-carrying lymphoblastoid cell lines.

EBV-carrying lymphoblastoid cell lines (LCL) can stimulate lymphocytes of the autologous donor [autologous stimulation (AS) assay] to blast transformation and generation of killer cells of broad-range cytotoxicity. We have tested the possibility of developing an EBV-specific AS assay for use in the demonstration of EBV-specific memory cells in the peripheral blood of normal donors. For this purpose, the stimulating lines were treated by heat and protein synthesis inhibitors to prevent the release of possible nonspecifically mitogeneic factors. Moreover, an extensive purification of the effector cells was achieved in the hope of removing a possible cellular contributor to the observed nonspecific cytotoxicity. None of these approaches was able to narrow down this nonspecific cytotoxicity to an EBV-specific response. We have shown that AS reaction (1) is not related to the release of lymphocyte mitogeneic factors by stimulating LCL, (2) is mediated by Fc receptor-negative T cells, and (3) does not require macrophage nor any other non-T helper cells.     

Antigenic requirements for the generation of secondary cytotoxicity.

The in vitro role of products of the H-2 complex in restimulation of secondary proliferative and cytotoxic responses has been studied. In this paper we have studied in particular the role of CD antigens, the target antigens for cytotoxic T lymphocytes. We have used UV-treated cells that express their CD antigens but no LD. Like others, we demonstrate that CD antigens alone are able to generate a secondary cytotoxic response and furthermore that these CD antigens must be the same as those used for primary sensitization. However, in contrast to the results of others, in some cases UV-treated cells were unable to restimulate such a response even when appropriate controls showed that the CD antigens on UV-treated cells were functional. Furthermore, in these cases the addition of an LD stimulus in the presence of UV-treated cells results in a secondary cytotoxic response that is significantly greater than that elicited by LD restimulation alone. Such potentiation is not observed if the CD antigens used for restimulation are different from those used for the primary sensitization. In addition, a non-H 2 differences(s), presumably the M1s locus, appears to be able to generate a secondary response. The implication of these results are discussed in terms of "memory" T lymphocytes in cell-mediated immunity.     

EBV-gp350 confers B-cell tropism to tailored exosomes and is a neo-antigen in normal and malignant B cells--a new option for the treatment of B-CLL

gp350, the major envelope protein of Epstein-Barr-Virus, confers B-cell tropism to the virus by interacting with the B lineage marker CD21. Here we utilize gp350 to generate tailored exosomes with an identical tropism. These exosomes can be used for the targeted co-transfer of functional proteins to normal and malignant human B cells. We demonstrate here the co-transfer of functional CD154 protein on tailored gp350+ exosomes to malignant B blasts from patients with B chronic lymphocytic leukemia (B-CLL), rendering B blasts immunogenic to tumor-reactive autologous T cells. Intriguingly, engulfment of gp350+ exosomes by B-CLL cells and presentation of gp350-derived peptides also re-stimulated EBV-specific T cells and redirected the strong antiviral cellular immune response in patients to leukemic B cells. In essence, we show that gp350 alone confers B-cell tropism to exosomes and that these exosomes can be further engineered to simultaneously trigger virus- and tumor-specific immune responses. The simultaneous exploitation of gp350 as a tropism molecule for tailored exosomes and as a neo-antigen in malignant B cells provides a novel attractive strategy for immunotherapy of B-CLL and other B-cell malignancies.     

CD8+ suppressor T cell clone capable of inhibiting the antigen- and anti-T cell receptor-induced proliferation of Th clones without cytolytic activity

A CD8+ Ts clone 13G2 was established from lymph node cells of bovine alpha s1-casein-primed C57BL/6 mice by in vitro antigenic stimulation followed by maintenance with IL-2-containing medium. The clone suppressed the Ag-induced proliferative responses of CD4+ Th cell clones without detectable cytotoxicity for both APC and responding T cells. The clone was able to suppress the in vitro proliferative response and antibody formation of Ag-primed lymph node cells. The suppression was Ag-nonspecific and not restricted to the MHC. The clone was able to suppress the proliferation of Th clones induced by an immobilized anti-TCR antibody in which APC was absent. The clone was, however, unable to suppress the proliferation of Th clones induced by anti-CD3 or IL-2. Thus, the mechanism of suppression by 13G2 was found to be due to a direct action on Th by inhibiting a consequence of signal transduction initiated through the TCR.     

MLC-activation by acute lymphatic leukemia blasts.

The MLC-activating potential of 25 ALL blasts (16 "common" ALL, 6 T-ALL, 3 not identified) was investigated. Mitomycin-treated leukemic blasts or X-irradiated lymphocytes were cultured with heparinized whole blood from different healthy donors. MLC activation by blast cells was expressed as percentage of MLC activation by X-irradiated lymphocytes. Leukemic blasts showed a heterogeneous pattern of MLC activation, ranging from 2% to 245%. Eleven out of 25 cases of ALL poorly stimulated the MLC (2% to 33% response). Twelve ALL stimulated a normal response (50% to 120%); and 2/25 ALL stimulated a supranormal response (more than 200%). Four of six cases of T-ALL stimulated the MLC as efficiently as irradiated lymphocytes, 2/6 were among the poor stimulators. Most poor stimulator blasts had, however, normal MLC-activating properties if, instead of whole blood, isolated lymphocytes were used as the responding cells. The poor activation of lymphocytes by some leukemic blasts in whole blood appeared to be associated with impaired release of blastogenic factor(s) during the MLBC. No evidence for active suppressor mechanisms was found. The significance of the MLC-activating properties of leukemic blasts for the classification and immunotherapeutic use of ALL is discussed.     

T-rosettes in hemodialysis patients and renal allograft recipients

Subcellular fractions, isolated from the lymphoid cell line IM-1, are capable of stimulating a weak proliferative response in allogeneic lymphocytes. They also stimulate the generation of cytotoxic effector lymphocytes. The proliferative response to subcellular fractions, as measured by ³H-thymidine incorporation, is only one-fourth to one-sixth as great as that to intact IM-1 cells, suggesting that a component(s) synthesized during the mixed lymphocyte reaction (MLR), or a short-lived cellular constituent, may be responsible for the ability of intact cells to stimulate a lymphocyte proliferative response. This component appears to be lacking or in limiting quantity in subcellular fractions, including the soluble fractions. In contrast to the decreased proliferative response to subcellular fractions, the cytotoxic capacity of the stimulated lymphocytes is comparable to that after stimulation by intact IM-1 cells. The data demonstrate that, in this system, cytotoxic effector lymphocytes can be generated in the absence of the extensive proliferative response normally observed in the MLR. The antigenic stimulus responsible for the generation of cytotoxic effector cells appears to reside on intracellular components as well as on plasma membrane. In these reactions, specificity is shown by the failure of the cytotoxic cells to release ⁵¹Cr from autologous target cells. In fact, reactivity of lymphocytes stimulated by subcellular fractions is more specific than the reactivity of cells stimulated by intact IM-1 as judged by their lytic capacity for another target cell, RPMI 4265.     

Cyclosporine-induced tolerance in experimental organ transplantation. Evidence of diminished donor-specific cytotoxicity relative to donor-specific proliferative response

Alloreactivity of intragraft and peripheral blood lymphocytes from tolerant canine lung allograft recipients was examined. Tolerance was induced by variable periods of treatment with cyclosporine. Analysis of effector cells from lung allografts (obtained by bronchoalveolar lavage) revealed the absence of specific cytolytic T lymphocyte (CTL) activity and the presence of a low level of cytolytic activity detected in a lectin-dependent cell-mediated cytotoxicity assay. In contrast, high levels of specific CTL activity and lectin-dependent activity were detected in cell preparations from lung allografts undergoing rejection. Tolerant recipients retained normal ability to generate specific CTL activity to third party alloantigens in mixed lymphocyte cultures (MLC) but had diminished ability to generate CTL to donor alloantigens in recipient X donor MLC. Addition of exogenous interleukin 2 to these MLC was unable to restore donor-specific CTL activity. Lymphocytes from tolerant recipients were, however, capable of generating proliferative responses and lectin-dependent cytotoxicity on exposure to donor alloantigens in MLC. Evidence presented in this report suggests that the lectin-dependent cytolytic activity generated in these MLC is mediated by lymphokine-activated killer cells. Such cells are likely to be activated by interleukin 2 released in the proliferative response. The results support the proposal that the cyclosporine-induced tolerant state is characterized by the relative inability to respond against major histocompatibility complex class I antigens in contrast to class II antigens and/or minor histocompatibility antigens since MLC-induced CTL are directed, for the most part, against class I molecules.