Mechanism Of Target Cell Lysis Research Articles

Abstract 5671: AFM26 - A novel CD16A-directed bispecific TandAb targeting BCMA for multiple myeloma

Abstract Multiple myeloma (MM) is the second most common haematological cancer and is characterized by the accumulation of neoplastic plasma cells in the bone marrow and production of high levels of monoclonal immunoglobulin (M-protein). While historically considered incurable, recent approvals and ongoing clinical trials with monoclonal antibodies (mAbs) targeting surface antigens promise greatly improved outcomes and have heralded a new era of MM treatment in which immunotherapies are expected to take center stage. However, an unmet need remains as patients eventually relapse and/or become refractory to currently available treatments. Consequently, novel immunotherapeutic approaches are needed to provide improved treatment options to MM patients. Among the currently explored targets, B-cell maturation antigen (BCMA, CD269) is considered to be particularly attractive due to its limited expression on healthy tissues and almost universal expression on myeloma cells in the majority of patients. Natural killer (NK) cells are cytotoxic effectors of the innate immune system capable of rapidly eradicating infected and transformed cells. The cytolytic activity of NK-cells can be used therapeutically to induce tumor cell lysis by direct engagement of the activating receptor CD16A (FcγRIIIa) using mAbs (ADCC). Despite similar mechanisms of target cell lysis, activation of NK-cells is not associated with the systemic symptoms of high level cytokine release as seen with direct T-cell engagement. Hence, it is considered a potent immunotherapeutic approach with reduced toxicity and a well-manageable safety profile. NK-cells readily infiltrate bone marrow and are thought to contribute to the efficacy of current myeloma treatments. Therefore, redirecting NK-cell cytotoxicity to malignant plasma cells appears to be a suitable therapeutic approach for MM. Here we describe the characterization of AFM26, a novel tetravalent bispecific tandem diabody (TandAb) that specifically targets BCMA and CD16A with high affinity and induces potent and efficacious myeloma cell lysis. AFM26 incorporates an affinity-improved anti-CD16A domain and interacts bivalently with NK-cells, resulting in high avidity and prolonged cell surface retention that is largely unaffected by the presence of polyclonal IgG. Notably, AFM26 potently induces NK-cell-mediated in vitro lysis of target cells expressing low levels of BCMA at low effector:target ratios, even in presence of polyclonal IgG. This may suggest that AFM26, in contrast to classical mAbs, retains ADCC activity at low antibody concentrations in presence of serum IgG and despite high levels of IgG M-protein occurring in about half of MM patients. AFM26 exhibits high protein stability, full cross-reactivity with cynomolgus antigens (BCMA and CD16A) and does not bind APRIL and TACI, two functionally related receptors. These data suggest that TandAb AFM26 is a promising and highly potent drug candidate for MM treatment. Citation Format: Thorsten Gantke, Uwe Reusch, Kristina Ellwanger, Ivica Fucek, Michael Weichel, Martin Treder. AFM26 - A novel CD16A-directed bispecific TandAb targeting BCMA for multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5671. doi:10.1158/1538-7445.AM2017-5671

In Vitro Comparison of ADCC and CAR Sensitivity of Adult HER-2+ B-ALL Using the NK-92 Human Cell Line Transduced with a Human CD16 (ADCC) or an Anti-HER2 Chimeric Antigen Receptor (CAR)

Introduction: Prognosis of acute lymphoblastic leukemia (ALL) in adults remains dismal. Recent advances have consisted of targeted therapies with monoclonal antibodies and chimeric antigen receptor (CAR) T lymphocytes (anti-CD19 CAR T cells), showing very encouraging results. However, relapse still occur after this new therapeutics and increasing the armamentarium for ALL is still required. Her2 is expressed in above one third of adult B-ALL (Chevallier,Haematologica 2009). The anti-HER2 antibodytrastuzumab has been tested prospectively in such patients, showing a low efficacy (Chevallier, Blood 2012).The present work was designed to compare, in vitro, thelysis sensibility of B-ALL HER2+ mediated by two mechanisms of HER2 recognition based on antibody (Ab) specificity: firstly, when the anti-HER2 monoclonalAbtrastuzumab bridges target cells and cytotoxic lymphocytes armed with a Fc receptor (antibodydependant cellular cytotoxicity, ADCC) andsecondly, when HER2-positive target cells are directly recognized by cytotoxic lymphocytes armed with a chimeric antigen receptor (anti-HER2 CAR).Methods: Frozen samples from 7 HER2+ B-ALL adults were available, including onesample expressing also CD20. Characteristics of Patients and controls are presented in Table 1.Three human breast-cancer cell lines (BCCL) expressing different validated (HercepTest, Dako) levels of HER2 (BT474 (HER2 3+), MCF-7 (HER2 0-/1+) and MDA-MB-231 (HER2 0-/1+) were used as positive control. Leukemic HER2-negative B-ALL cells from two patients were used as negative control, including one patient with CD20+ expression. The level of HER2 expression by HER2+ B-ALL was determined by fluorescence-activated cell sorter (FACS) and compared to the HER2 expressing BCCL.Cytotoxic activity was assessed at a 30-to-1 effector-to-target ratio in a 4 hours 51Cr-release assay.We used the same cytotoxic effector lymphocytes (the human NK cell line NK-92), armed with eitheraFcγRIIIa/FcεRIγ receptor (referred to as NK-92CD16) or with an anti-HER2/FcεRIγ CAR receptor (referred to as NK-92CAR) as previously described (Clémenceau, JImmunol Research 2015). For ADCC/CAR assays, target cells were pre-incubated withtrastuzumab and /or rituximab at 10µg/ml each.The study was approved by our local review board and twoalivepatients gave informed consent.Results:The level of HER2 expression by HER2+ B-ALL (n=7) was highly variable with a mean relative fluorescence intensity ratio (RFI) of 13 (range 5 to 30). According to the HercepTestranking used for BCCL lines (see above) all B-ALL would be classified as HER2 0-/1+. In vitro HER2+ BCCL were efficiently lysed by anti-HER2 NK-92CAR in this 4 hours 51Cr-release assay and this direct pathway of killing by CAR was always more efficient than the indirect pathway mediated by ADCC. In addition, thelysis level was correlated to HER2 level expression.Trastuzumabalone has no effect on the spontaneous 51Cr-release of B-ALL cells (not shown). Anti-HER2 CAR mediatedlysis was observed for 5 out of 6 HER2+ B-ALL cells (83%, 1 case (#5) non interpretable). Although lysis levels were low, they werespecificsince the CAR mediatedlysis was inhibited when the B-ALL were pre-incubated with 10 µg/mltrastuzumab (not shown). In contrast, for these 5 HER2+ B-ALL, no anti-HER2 ADCClysis was observed. These results confirmed that in these experimental conditions, HER2 targeting by CAR is more efficient than by ADCC. For the two HER2- B-ALL, no ADCC or CARlysis were observed.Finally, for the two B-ALL expressing CD20, anti-CD20 ADCClysiscan be observed demonstrating that B-ALL cells are not intrinsically resistant to ADCCcytoxicity. Regarding the unique case of B-ALL expressing both CD20/HER2 antigens, only anti-CD20 ADCClysiswas observed. Results are presented in Table 2.Conclusion:Together, these preliminary results suggest that for the CD20-negative and HER2-positive B-ALLs, one cannot rely on ADCC as a mechanism of target celllysis, while the interest of an anti-HER2-CAR approach deserve further studies to better correlate the level of HER2 expression and the sensitivity tolysisby HER2-specific CAR. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Selective Chemical Functional Probes of Granzymes A and B Reveal Granzyme B Is a Major Effector of Natural Killer Cell-Mediated Lysis of Target Cells

The mechanism of target cell lysis in cytotoxic lymphocyte-mediated death is not well understood, and the role of granzymes in this process is unclear. Chemical functional probes were thus prepared for the major granzymes A and B to deconvolute their role in natural killer cell-mediated lysis of target cells. These biotinylated and substrate specificity-based diphenyl phosphonates allowed facile evaluation of selectivity through activity-based profiling in cell lysates and intact cells. Both inhibitors were found to be extremely selective in vitro and in cells. Use of these inhibitors in cell-based assays revealed granzyme A to be a minor effector and granzyme B to be a major effector of target cell lysis by natural killer cells. These studies indicate that the proapoptotic granzyme B functions also as a pronecrotic effector of target cell death.

Study of diverse mechanisms of cell-mediated cytotoxicity in gene-targeted mice using flow cytometric cytotoxicity assay

Cytotoxic lymphocytes kill tumor or virus-infected target cells utilizing two mechanisms—(1) release of lytic granules (containing perforin and granzymes), and (2) Fas ligand (FasL)/Fas or TNF-initiated apoptosis. We have examined mechanisms of target cell lysis by effector T cells from gene-targeted and mutant mice using a new Flow Cytometric Cytotoxicity Assay (FC Assay). Target cells were labeled with PKH67 dye. Cell death was estimated by 7-AAD inclusion and annexin V-PE binding. A direct correlation has been found between the percentage of dead target cells in FC Assay and the results of 111In release cytotoxicity assay when effector T cells from either Pfp −/− (perforin knockout) or gld (non-functional Fas Ligand) mice were used. As shown by the 4 h FC assay, the granule-mediated mechanism was utilized by T cells from gld mice. In contrast, T cells from Pfp −/− mice used death receptor-mediated lysis. Therefore, cytotoxic cells from gene-targeted and mutant mice can serve as valuable tools for studying different mechanisms of cell-mediated cytotoxicity, and the FC assay could be applied irrespective of which cytotoxic effector pathway is involved.

Three-color flow cytometric assay for the study of the mechanisms of cell-mediated cytotoxicity

Cytotoxic lymphocytes kill tumor or virus-infected target cells utilizing two mechanisms (1) release of lytic granules (containing perforin and granzymes) and (2) Fas ligand (FasL)/Fas or TNF initiated apoptosis. We have examined mechanisms of target cell lysis using a new Flow Cytometric Cytotoxicity Assay (FC Assay). Target cells were labeled with PKH 67 dye. Cell death was estimated by 7-amino-actinomycin (7-AAD) inclusion and annexin V-PE binding. A strong direct correlation has been found between the percentage of dead target cells in the FC Assay and the results of 51Cr release assay when human LAK and CTL were used as a model system. We have shown that both NK and CTL kill tumor cells mostly by granule-mediated mechanisms, as lysis was blocked by a perforin inhibitor Concanamycin A (Folimycin) but was significantly less sensitive to zVAD-FMK caspase inhibition. The FC assay allows accurate measurement of cell-mediated cytotoxicity as individual target cell death is detected directly.

Engagement of Natural Cytotoxicity Programs Regulates AP-1 Expression in the NKL Human NK Cell Line

NK cell cytotoxicity is a fast and efficient mechanism of target cell lysis. Using transcription analysis, such as multiplex messenger assays, we show here that natural cytotoxicity exerted by the human NKL cell line correlates with mRNA accumulation of very early activator protein (AP)-1 transcription factor genes such as JunB, FosB and c-Fos. In addition, DNA-binding activities of Jun-Fos heterodimers were observed by electrophoretic mobility shift assays during the course of natural cytotoxicity. Interaction between immunoglobulin-like transcript-2/leukocyte Ig-like receptor 1 on NKL cells and HLA-B27 on target cells leads to an impairment of NKL natural cytotoxicity, which correlates with an absence of JunB, FosB, and c-Fos transcription, as well as an absence of their DNA-binding activity. Our studies thus indicate that, despite the rapidity of NK cell-mediated lysis, AP-1 transcription factor is activated during the early stage of NK cell cytolytic programs and that engagement of NK cell inhibitory receptors for MHC class I molecules impairs the very early activation of AP-1.

Superoxide‐mediated lysis of erythrocytes: The role of colloid‐osmotic forces

Although superoxide anions are a well-known mediator of cytotoxicity, their mechanism of target cell lysis is not clearly understood. In the present study we have used an exogenous source of superoxide to study erythrocyte cytolysis. RBC lysis was studied in buffers containing the cations Li+, Na+, K+, Rb+, and Cs+; superoxide anions were produced and available in these buffers. During this model superoxide-dependent cytolytic process, erythrocytes underwent a shape change from biconcave disk to sphere as shown by scanning electron microscopy. Soret band transmitted light microscopy has confirmed this shape change and shown that it precedes cytosolic oxidation. This evidence is consistent with a colloid-osmotic type lytic mechanism. Erythrocyte lysis was studied by 51Cr-release and light scattering methods. Superoxide-mediated target cytolysis was characterized by: 1) a sigmoidal dose-response curve and 2) a lag time in cytolysis after superoxide addition in kinetic light scattering experiments. The efficacy of cytolysis followed the rank order Cs+ > Rb+ > Na+, Li+ > sucrose = raffinose, which provides additional support for a colloid-osmotic lytic mechanism. Furthermore, the rank order potency correlates with the cations' hydration numbers. We suggest that oxidative events trigger the formation of colloid-osmotic pores approximately 1 nm in diameter.

Clonal analysis in the ultrastructure of cell-to-cell interaction between a human glioma cell line and autologous tumor-specific cytotoxic T lymphocytes

The clonal analysis in the ultrastructure of tumor-lymphocyte interaction was carried out in order to investigate the precise mechanism responsible for CTL-mediated cytolysis of tumor cells. A glioma-derived cell line (GI-1) and autologous tumor-specific cytotoxic T lymphocyte (CTL) clones were established. The CTL lines were composed of the morphologically homogeneous lymphocytes with intracytoplasmic electron-dense secretory granules. After the stimulation by GI-1, the size of the CTLs increased, and the intracytoplasmic organellas were developed. It was noted that the intracytoplasmic secretory granules markedly increased in number and size, and many of them exhibited an “immature” appearance. On the other hand, the tumor cells underwent a progressive degeneration. In contrast, the stimulation by other antigens caused only small morphological changes in the CTLs. It is suggested, therefore, that the secretory function of tumor-specific CTLs is activated by the stimulation of the specific antigen, and that soluble factors in the secretory granules in the CTLs may be closely associated with the mechanism of target Cell lysis.

Mechanistic, functional and immunopharmacological implications of biochemical studies of antigen receptor-triggered cytolytic T-lymphocyte activation.

Biochemical events that follow the engagement of cytotoxic T lymphocytes (CTL) with an Ag-bearing target cell (TC) or triggering by the crosslinking of the Ag-receptor (TcR) by immobilized anti-TcR mAb were studied using cloned CTL and a novel CTL activation assay. The approach described here was undertaken to shed light on the molecular mechanisms of "ON", "STOP" and "OFF" signalling that allow CTL to be activated, kill TC and disengage from the target cell after delivery of the "lethal hit" and then to proceed with the destruction of the next Ag-bearing target encountered. Biochemical studies of TcR-regulated and TcR-triggered constitutive exocytosis in CTL provided a detailed description of the molecular requirements for this important phenomenon in T lymphocytes and provided an alternative CTL activation assay; this assay measures the TcR-dependent response in the absence of a TC. These studies also helped to envision CTLs screening activities as a cycle of engagements-disengagements with the TC, where every surrounding cell is treated by the CTL as a potential Ag-bearing TC. Both constitutive and regulated exocytosis in CTL are triggered through a transmembrane signalling pathway which involves protein kinase C and extracellular Ca2+ that, most likely, is translocated through Ca2+ channels. This is followed by the involvement of calmodulin (CaM)-binding proteins, e.g., calcineurin, a CaM-dependent phosphatase, which was shown to be a major CaM-binding protein in murine lymphocytes. Unexpectedly, these biochemical studies demonstrated that the granule exocytosis model of CTL-mediated cytotoxicity cannot account for the mechanism of target cell lysis by CTL, at least in in vitro conditions in the absence of extracellular Ca2+. These results indicate the existence of an extracellular Ca2+-independent, TcR-regulated CTL response and raise the possibility that second messenger(s) other than Ca2+ and/or products of phosphoinositide turnover are involved in T-cell lysis. Predominance of "non-lethal" engagements between some CTL and TC, revealed during time-lapse cinematographic studies, together with comparative studies of TcR-regulated exocytosis of granules and of constitutive exocytosis of gamma-interferon, suggested that TC destruction by CTL may not be their only or even their most important function in vivo. It is possible that CTL, triggered by Ag recognition to exocytose storage granules and to synthesize and constitutively exocytose macrophage-activating factors, in turn promote tumor destruction by macrophages.(ABSTRACT TRUNCATED AT 400 WORDS)

Mechanism of target cell lysis by cytotoxic T lymphocytes (CTL) employing RSV-induced H-2 congenic and recombinant mouse tumor cells: Demonstration of soluble mediators released from H-2-restricted CTL clones

The secretion and the specificity of cytotoxic mediators from H-2-restricted cytotoxic T lymphocytes (CTL) were examined using non-virus-producing target tumor cells induced by the Schmidt-Ruppin strain of Rous sarcoma virus (SR-RSV) in B10 congenic and recombinant mice. By using rat concanavalin A supernatant, two H-2-restricted CTL clones were established from cytotoxic effector cells of B10.A(5R) mice primed with SR-RSV-induced syngeneic tumor cells. Cell-free supernatants from the H-2-restricted CTL clones cocultured with syngeneic tumor cells had selectively high cytotoxic activity for syngeneic and H-2-compatible tumor cells, but not for H-2-incompatible tumor cells, YAC-1 cells, and B10.A(5R) blasts as defined in the 5-hr 51Cr-release assay. The cytotoxic activity was detected in the cell-free supernatants from the CTL clones cocultured with the CTL-sensitive syngeneic and H-2-compatible tumor cells, but not with the CTL-insensitive tumor cells and YAC-1 cells. The cytotoxic activity of the cell-free supernatant could be adsorbed by the syngeneic tumor cells, but not by YAC-1 and L(s) cells. Thus, the H-2-restricted CTL clones against SR-RSV-induced tumor cells were capable of releasing cytotoxic mediators by coculturing with syngeneic or H-2-compatible tumor cells, and the cytotoxic mediators showed a certain H-2-restricted manner in killing the target cells. These results suggest that the lysis of RSV-induced tumor cells by H-2-restricted CTL can at least in part be mediated by cytotoxic factors.

Exocytosis of cytolytic granules may not be required for target cell lysis by cytotoxic T-lymphocytes.

Secretory processes have been implicated in the mechanism of target-cell lysis by cytotoxic T-lymphocytes (CTL) (refs 1, 2). Both CTL and cytotoxic large granular lymphocytes have cytolytic granules, containing the cytolytic molecules 'perforin' and 'cytolysin'; perforin and cytotoxic lymphocytes can damage target cells through the membrane assembly of pores. The description of proteases which are cytotoxic cell-associated and granule-located has supported the granule exocytosis model of cytotoxicity mediated by cytotoxic lymphocytes, and has emphasized the similarities between cell-mediated and complement-mediated cytotoxicity. But recent experiments have challenged the importance of lytic granules and perforin in CTL activity. Here we report that CTL can be triggered to deliver a lethal hit to target cells even when exocytosis of lytic granules has been abolished by removal of extracellular calcium. This dissociation of exocytosis of granules and delivery of the lethal hit suggests that cytolytic granules may not be involved in target-cell lysis by CTL.

A fast-acting cytotoxin derived from Con A-activated porcine leukocytes: II. Mechanism of target cell lysis

Serum-free culture supernatants of Con A-stimulated pig leukocytes contain cytotoxins (PCT) which have a fast-acting lytic effect on a variety of murine lymphomas. Mathematical analysis revealed that the cytodestructive reaction which leads to the immediate release of 51Cr from labeled target cells follows “single hit”/“first order” kinetics. The release of labeled compounds was found to be size dependent, such that low molecular isotope ( 86Rb) was released at faster rates than high molecular 51Cr-labeled complexes. The 51Cr release was strongly reduced by lowering the temperature to 4 °C. PCT-mediated target cell lysis could be competed by cold target cells and the factor could be absorbed by intact cells, plasma membranes and artificial liposomes. Neither the presence of EDTA or of various monosaccharides, nor exposure of target cells to trypsin, metabolic inhibitors, and cytoskeletal antagonists altered the target cells susceptibility to PCT-mediated lysis. Labeling of target cell DNA and subsequent exposure of these target cells to PCT revealed that target cell DNA is degraded into low-molecular-weight split products which is similar to that seen during T-cell mediated target cell lysis. The analysis of the lytic event mediated by PCT thus revealed similarities to T-cell mediated target cell lysis. PCT was clearly distinct from the well-known cytolytic agents lymphotoxin (LT), tumor necrosis factor (TNF), or complement (C′). Taken together, the described characteristics make PCT a unique-acting cytolytic cytokine with anticancer activity and suggest further research concerning its mode of action and its possible therapeutic application.

Studies on natural, antibody-dependent, and interleukin-2-activated killer-cell activity of a patient with mucolipidosis III as a test of the mannose-6-phosphate lytic acceptor hypothesis.

The natural (NK), antibody-dependent (K), and interleukin-2-generated (LAK) killer-cell activity of a patient with mucolipidosis III (ML III; an autosomal recessive defect in UDP-N-acetylglucosamine: lysosomal enzyme N-acetylglucosamine-1-phosphotransferase) was studied to determine whether or not the defect in the phosphorylation of lysosomal enzyme mannose residues resulted in a failure of target-cell lysis, as would be predicted from recent studies showing NK inhibition by phosphorylated sugars, especially mannose-6-phosphate. The patient was studied in parallel with normal donors known to be at the high and low extremes of NK activity. The following results were obtained: NK activity was markedly elevated against K562, Molt-4, human fibroblasts, HL-60, and MeWo to levels approximately one to two times that of our previous highest donor and five times the mean of normal donors previously tested. Interleukin-2-generated killer-cell activity and antibody-dependent cell-mediated cytotoxicity against antibody-coated P815 cells were normal and increased, respectively. HNK-1-positive cells were normal in frequency (7.3 +/- 1.7%), while lytic conjugates were proportional to activity (3.9 +/- 0.6 vs 2.7 +/- 0.4% for the "high" donor), and this was attributable to an increased proportion of lytic cells. The addition of fresh serum from the ML III patient had no effect on the NK activity of normal donors and the effects of preincubation with interferon (enhancement), monensin (inhibition), fructose-6-phosphate (inhibition), and mannose-6-phosphate (inhibition) were identical to those seen using lymphocytes from normal donors. Studies on the NK activity of the parents and two normal female siblings showed that the father's NK activity was high, the mother's was low, and both siblings' was intermediate but low. The data obtained suggest that the inability of lymphocytes to phosphorylate lysosomal enzyme mannose residues had no effect on NK-, K-, or LAK-cell function and that the mechanism of target-cell lysis is independent of either a mannose-6-phosphate-bearing lytic moiety or a mannose-6-phosphate-dependent ligand mechanism.

Involvement of T11 molecules in antigen receptor-mediated T lymphocyte functions: effect of anti-T11 monoclonal antibody on functional capabilities of alloreactive T cell clones.

As shown by previous studies, the sheep erythrocyte-binding T11 molecule is involved in T cell activation, as well as in mechanisms of specific allogeneic target cell lysis. In this study, we utilized two anti-T11 monoclonal antibodies (mAb) that inhibited the specific cytolytic activity of mixed lymphocyte culture (MLC)-activated T cells to analyze, at the clonal level, the involvement of T11 molecules in (a) antigen-specific vs. nonspecific mechanisms of target cell lysis, and (b) antigen-driven T cell proliferation and interleukin 2 (IL 2) production vs. IL 2-induced cell proliferation. In contrast to anti-T3 or anti-T8 mAb, antibodies to T11 molecules inhibited the cytolytic activity of MLC-derived allospecific clones in a uniform manner. In addition, anti-T11 antibodies inhibited the specific activity of cytotoxic T lymphocyte clones resistant to anti-T3 antibodies, even after antibody-induced modulation of T3 molecules (while anti-T3 mAb had no effect). Similarly, anti-T11 antibodies inhibited the alloantigen-induced proliferation and IL 2 release of alloreactive clones independent of their T4+ or T8+ phenotype. The inhibitory activity of anti-T11 antibodies appears to be confined to antigen-specific T cell functions since neither natural killer-like activity of cytotoxic T lymphocyte clones nor the IL 2-induced clonal proliferation was affected. Thus, our results indicate that T11 molecules are functionally involved in antigen recognition by T cell regardless of their function and T4/T8 phenotype. The possible mechanisms of anti-T11 antibody-mediated inhibition are discussed.

Studies on the mechanism of macrophage cytotoxicity

The interactions with tumor target cells of resident and BCG-activated murine peritoneal macrophages (M phi) as well as of BCG-activated M phi additionally stimulated by a lymphokine-like factor were investigated in order to get some insight into the cytolytic process mediated by activated M phi. The lymphokine-like factor enhancing the cytotoxicity of BCG-activated M phi (MCF) was isolated and partially purified from cell-free fluid of rat Zajdela ascites hepatoma. M phi cytotoxicity was determined by a modified 51Cr release assay. Scanning and transmission electron microscopic findings suggested a two-step mechanism of target cell lysis: a first step of specific attachment of processes of M phi on the target cell surface and a second step with transport of lysosome-like vesicles to the target cells obviously with liberation of these vesicles in the immediate vicinity of target cells resulting in a local accumulation of cytolytic substances. This interpretation was supported by findings after treatment of interacting effector and target cells with amphotericin B and bestatin which substances were modifying M phi cytotoxicity. MCF caused only an augmentation of M phi cytotoxicity without qualitative differences to the cytolytic action of merely BCG-activated M phi.

Mechanism of target cell lysis of cytolytic T lymphocytes. I. Characterization of specific lymphocyte-target cell conjugates separated by velocity sedimentation.

Differential velocity sedimentation was applied for separating alloimmune T lymphocytes bound to target cells (TC) from free lymphocytes. Maximal size differences between lymphocytes and TC were achieved a) by isolating the fraction of small peritoneal lymphocytes (SPL) from an alloimmune peritoneal cell population, and b) by selecting large tumor cells as TC. Under the conditions used, most of the conjugates formed at room temperature consisted of one SPL bound to one TC, and adequate separation of bound from free SPL could be achieved within less than 5 min. Functional studies of the conjugate-enriched fractions showed that a minimum of 60% of TC-bound SPL were indeed cytolytic. Conjugate-depleted fractions, however, were still lytic, suggesting that not all effector cells formed stable conjugates at room temperature. Transmission and scanning electron microscopy studies revealed that binding between SPL and TC was achieved through interpenetrating membrane projections and was characterized by point and broad zone contacts. When lysis was allowed to proceed, prominent changes of the TC membrane morphology, including loss of microvillous projections, appearance of localized blebs, pseudopod-like projections, and membrane defects were documented.