Inability of EDTA to prevent damage mediated by cytolytic T-lymphocytes.

To analyze the nature of the target cell determinants recognized and bound by killer lymphocytes during lymphocyte-mediated cytolysis (LMC), the specific binding of serologically active tumor cell membrane fractions to cytotoxic T lymphocytes has been investigated. Particulate membrane fractions and soluble antigen preparations (extracted by papain or 3 M KCl) from tumor target cells were tested for their ability to inhibit the destruction of intact ⁵¹Cr-labeled target cells by killer lymphocytes in vitro. The effect of papain-solubilized tumor cell antigen on the binding of killer lymphocytes to tumor cell monolayers was also evaluated. Direct assays to determine the extent of binding of unlabeled or radioiodinated soluble antigen (extracted by papain or deoxycholate) to cytotoxic lymphocytes were carried out. In marked contrast to their serological activity, all of these particulate and soluble preparations failed to inhibit LMC or bind to killer lymphocytes in an immunologically specific way. It is suggested that killer lymphocytes recognize and bind to an antigenic complex whose organization is dependent upon the integrity of the target cell membrane.     

Reversible Inhibition of Lymphocyte-mediated Cytotoxicity by Cytochalasin B

ALLOGRAFT immunity is characterized by the appearance of sensitized lymphocytes which are specifically able in vitro to destroy target cells carrying the sensitizing alloantigens. These cytotoxic lymphocytes represent thymus-derived effector cells^1,2 and are quite distinct from alloantibody-producing cells, which are also formed during induction of allograft immunity³. Although contact between viable cytotoxic lymphocytes and target cells is necessary for destruction, the events which lead to target cell lysis are still unknown⁵.     

Graft-versus-host reactivity of mouse thymocytes: effect of in vitro treatment with anti-TL serum.

The lymph ducts efferent from prefemoral nodes of sheep were cannulated and the lymph flow monitored during immune responses to injected allogeneic lymphocytes or xenogeneic murine P815 mastocytoma cells. Changes in the lymph began 5–6 days after injection of allogeneic cells but at 3–4 days after injection of xenogeneic cells, in both systems the number of large cells in the lymph increased to reach peak values of up to 40% of the total. The in vitro cytotoxic activity of lymph cells, cell supernatants, or cell free lymph was determined by measuring the release of ⁵¹Cr from prelabeled target lymphocytes or P815 cells. The cytotoxic mechanisms that were detected in the allogeneic and xenogeneic systems were similar; in both cases the lymph cells were cytotoxic only during the large cell response, and when the immunoblast numbers had returned to normal levels in the lymph no further cell-mediated cytotoxic effects were detected. During the blast response lymphocytes alone caused some target cell damage but their cytotoxic effector function was greatly increased in cultures containing complement or normal blood white cells. It was concluded that the lymph immunoblasts caused some target cell damage by direct action, but the majority of their cytotoxic activity was associated with synthesis and secretion of complement-dependent antibody (C.D.A.) and leukocyte-dependent antibody (L.D.A.).     

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.     

The Tumor Targeted Superantigen ABR-217620 Selectively Engages TRBV7-9 and Exploits TCR-pMHC Affinity Mimicry in Mediating T Cell Cytotoxicity

The T lymphocytes are the most important effector cells in immunotherapy of cancer. The conceptual objective for developing the tumor targeted superantigen (TTS) ABR-217620 (naptumomab estafenatox, 5T4Fab-SEA/E-120), now in phase 3 studies for advanced renal cell cancer, was to selectively coat tumor cells with cytotoxic T lymphocytes (CTL) target structures functionally similar to natural CTL pMHC target molecules. Here we present data showing that the molecular basis for the anti-tumor activity by ABR-217620 resides in the distinct interaction between the T cell receptor β variable (TRBV) 7-9 and the engineered superantigen (Sag) SEA/E-120 in the fusion protein bound to the 5T4 antigen on tumor cells. Multimeric but not monomeric ABR-217620 selectively stains TRBV7-9 expressing T lymphocytes from human peripheral blood similar to antigen specific staining of T cells with pMHC tetramers. SEA/E-120 selectively activates TRBV7-9 expressing T lymphocytes resulting in expansion of the subset. ABR-217620 selectively triggers TRBV7-9 expressing cytotoxic T lymphocytes to kill 5T4 positive tumor cells. Furthermore, ABR-217620 activates TRBV7-9 expressing T cell line cells in the presence of cell- and bead-bound 5T4 tumor antigen. Surface plasmon resonance analysis revealed that ABR-217620 binds to 5T4 with high affinity, to TRBV7-9 with low affinity and to MHC class II with very low affinity. The T lymphocyte engagement by ABR-217620 is constituted by displaying high affinity binding to the tumor cells (K_D approximately 1 nM) and with the mimicry of natural productive immune TCR-pMHC contact using affinities of around 1 µM. This difference in kinetics between the two components of the ABR-217620 fusion protein will bias the binding towards the 5T4 target antigen, efficiently activating T-cells via SEA/E-120 only when presented by the tumor cells.     

Studies on the specificity of in vitro-induced lymphocytotoxicity to methylcholanthrene-induced fibrosarcoma cell lines

Cytotoxic effector lymphocytes were induced by in vitro immunization of lymph node and spleen cells from CS7B16(H2^b) and Balb/c(H2^d) mice to syngeneic or allogeneic methylcholanthrene-induced fibrosarcoma (MCAF) cell lines. The T cell-dependent cytotoxicity was specific to target cell lines to which the lymphocytes were immunized in vitro. Normal fibroblasts as stimulator cells did not induce lymphocytotoxicity to syngeneic MCAF cells or to normal syngeneic fibroblasts. The results indicate that the in vitro-immunized lymphocytes recognize individual specific tumor-associated antigens of the MCAF cells. In experiments in which the lymphocytes were immunized in vitro to allogeneic MCAF cells, cytotoxic reactions to alloantigens, but not to tumor-associated antigens, were detected. Incubation with phytohemagglutinin (PHA) during the sensitization period modified the specificity of the cell-mediated lysis of MCAF cells: Allogeneic as well as syngeneic target cells were destroyed by these effector cells. PHA induced a nonspecific cytotoxic effect which increased the specific lysis of target cells. The cytotoxicity of the in vitro-immunized lymphocytes was inhibited by incubation with membrane protein preparations from the syngeneic MCAF cell lines. In contrast to the specificity of the cytotoxic effect to the different syngeneic cell lines, the membrane extract of one individual syngeneic MCAF cell line was able to inhibit the lymphocytotoxicity to all other syngeneic cell lines. Membrane protein preparations from allogeneic MCAF cells or from normal syngeneic fibroblasts were not inhibitory. The in vitro-immunized cytotoxic lymphocytes did not impair the tumor growth in vivo as could be demonstrated by passive transfer of the lymphocytes in a Winn assay.     

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.     

THE MECHANISM OF LYMPHOCYTE-MEDIATED CYTOTOXICITY

1. Three classes of cytotoxic lymphocyte are discussed: thymus-derived T cells, antibody-dependent K cells and NK (natural killer) cells. Each of these cytotoxic lymphocytes has receptors allowing the formation of adhesions (contact) with a target cell (the cell to be killed). The type of receptor and the corresponding ligand on the target cell is different in each class. Cytotoxic T cells (and probably NK cells) react with a target cell antigen, in a manner rather like antibody-antigen reactions (although not involving classical serum antibody). K cells have a receptor for the Fc part of immunoglobulin (IgG) and hence can make contact with antibody-coated target cells. 2. It seems likely that all three classes of cytotoxic lymphocyte have a similar basic mechanism of killing, which is different from the membrane leakage occurring in complement-mediated lysis. Much more information is available on cytotoxic T cells than on the other types of cell. 3. Cytotoxic T cell killing can be divided into two phases. A reversible phase in which the T cell is in contact with the target cell, but causes no apparent damage. This phase can vary from a few minutes up to several hours, when a single T cell interacts with a single target cell. If the T cell detaches or is inactivated the target cell survives. The second phase is irreversible, once the lethal event has occurred, and the target cell will progress to eventual lysis in the absence of the Tc cells. 4. The lethal event initiates a period of zeiosis (membrane blebbing) in the target cell, which is accompanied by increased effiux of ⁸⁶rubidium. Cell lysis occurs at a variable time after the initiation of zeiosis, when the soluble contents of the cytoplasm burst out of the target cell. The fact that both these phases are of variable length leads to the accumulation of cytoplasmic markers (such as ⁵¹chromium) in the medium in an approximately linear fashion. 5. The nature of the lethal event is unknown, but it is suggested that it involves changes inside the target cell rather than at the target cell membrane. Remarkable long projections from the T cell (and also seen from K cells and NK cells), apparently arising as a result of the receptor-ligand interaction, may be involved in the delivery of the lethal event.     

Antibody-dependent cytotoxic activity of the lymphocytes and the cytological characteristics of immune cell complexes in vitro

Bovine blood lymphocytes showed a high antibody-dependent (AD) cytotoxicity against target cultured L cells. The cytological analysis of immune cellular complexes, formed by affector lymphocytes with target cells during the cytotoxic reaction in vitro, revealed that adsorption of AD lymphocytes to target cells was completed within the first hours of incubation. Temporal parameters of contact between effector and target cells and dissociation of adsorbed lymphocytes varied widely, but after 48 hours of incubation the number of immune cellular complexes decreased by 5.6%. The morphological modification of AD effector lymphocytes during interaction with target cells is described.     

In vitro induction of tumor-specific immunity. III. Lack of requirement for H-2 compatibility in lysis of tumor targets by T cells activated in vitro to oncofetal and plasmacytoma antigens.

Many recent studies have demonstrated that cytotoxic T lymphocytes (CL) activated to various antigens other than those of the H-2 complex, will lyse target cells only when H-2 compatibility exists between the CL and target cell. From these observations, it has been inferred that T lymphocytes might only be capable of responding to H-2 antigen or antigens that become associated with H-2 region gene products. Our results suggest that this is not the case, and that in some situations, cytotoxic T lymphocytes can specifically lyse target cells of different H-2 types. Two in vitro systems are described where primary induction of cytotoxic T lymphocytes to oncofetal and plasmacytoma antigens results in CL capable of lysing suitable targets bearing these antigens, of either syngeneic or allogeneic derivation. Thus it is proposed that although interaction antigens involving H-2 components may preferentially activate T lymphocytes, this does not imply a restriction on the recognition potential of T lymphocytes.     

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.     

New insights to the functional role of the T cell-antigen presenting cell immunological synapse

Three innovative and complementary morphological approaches were employed to study the T cell/antigen presenting cell (APC) interaction: (i) high resolution three-dimensional confocal microscopy of the T cell-APC contact site; (ii) time lapse video recording in living T cells of [Ca2+]I and changes in distribution of various GFP fusion proteins with TCR/CD3-zeta complex associated- and other signaling components; (iii) measurement of lateral TCR mobility and that of recruited signaling components using techniques based on fluorescence recovery after photo-bleaching. These approaches were combined with biochemical and functional experiments to investigate two principal issues: (A) Recruitment and the three-dimensional arrangement of receptors and signaling components at the contact site between human CD4+ T lymphocytes and APCs, (B) Structure of the immunological synapse formed at the contact site between cytotoxic T lymphocytes (CTLs) and target cells. We discuss evidence indicating that TCR engagement and triggering can occur in the absence of large-scale molecular segregation into the T cell-APC contact site. Taken together our results indicate that although not required for TCR engagement and triggering, formation of the IS is important to reinforce TCR-mediated signal transduction and achieve full T cell activation.     

T-lymphocyte mediated lysis of tumor cells in the presence of alloantiserum.

The blocking effects of tumor alloantiserum (AS) on the process of T-lymphocyte mediated cytolysis of target cells was investigated with the scanning electron microscope by analyzing the frequency of lymphocyte-target cell interactions and the respective changes in target cell morphology at various time intervals of the cytolytic process. Our results demonstrate that in the presence of AS the frequencies of lymphocyte-target cell conjugates in which the target cells were undergoing active blebbing correlated with the delayed kinetics of ⁵¹Cr release. Our data also show that the AS did not interfere significantly with the binding of lymphocytes to target cells, but delayed the appearance of surface blebbing of the target cells. Thus, effector lymphocytes required a prolonged time of continuous interaction with the target cells in order to exert their cytolytic effects in the presence of AS. This conclusion was further confirmed by experiments in which lymphocyte-target cell interactions were interrupted by the addition of EDTA to the culture medium.     

Polyfunctionality of Allospecific Cytotoxic T-Lymphocytes in in vitro Systems

The data presented demonstrate the capacity of allospecific cytotoxic lymphocytes to fulfill noncytolytic regulatory functions. Cytotoxic lymphocytes suppress cell proliferation induced during development of a response in reactions of a mixed culture of lymphocytes and blast transformation irrespective of haplotypes of the cells involved in these reactions. At the same time, cytotoxic lymphocytes do not practically affect spontaneous proliferation of T-cells. The inhibitory effect of cytotoxic lymphocytes is expressed only upon direct contact with activated T-lymphocytes and is not relate to their apoptosis and death. The results obtained suggest polyfunctionality of allospecific cytotoxic lymphocytes expressed in the capacity of these lymphocytes to fulfill both effector (target lysis) and regulatory functions.     

Multi-functionality of allospecific cytotoxic T-lymphocytes in vitro

The data presented demonstrate the capacity of allospecific cytotoxic lymphocytes to fulfill noncytolytic regulatory functions. Cytotoxic lymphocytes suppress cell proliferation induced during development of a response in reactions of a mixed culture of lymphocytes and blast transformation irrespective of haplotypes of the cells involved in these reactions. At the same time, cytotoxic lymphocytes do not practically affect spontaneous proliferation of T-cells. The inhibitory effect of cytotoxic lymphocytes is expressed only upon direct contact with activated T-lymphocytes and is not relate to their apoptosis and death. The results obtained suggest polyfunctionality of allospecific cytotoxic lymphocytes expressed in the capacity of these lymphocytes to fulfill both effector (target lysis) and regulatory functions.     

Human lymphocyte dependent antibody mediated cytotoxicity: Adherence of lymphocytes to antibody treated target cells

An in vitro human antibody dependent cell mediated cytotoxicity system was used to study the adherence of nonsensitized attacking lymphocytes from peripheral blood to antibody coated melanoma target cells. Specific adherence of attacking cells was documented by labeling the lymphocytes with ⁵¹Cr. The degree of specific adherence was proportional to antibody concentration and incubation time and could be detected before the lysis of target cells. Adsorption of attacking lymphocytes on immune serum treated target cells depleted B cells, enriched T cells, and removed most cytotoxic activity of nonadherent lymphocytes in this system. These results were not found when attacking lymphocytes were adsorbed on normal serum treated target cells.     

Ultrastructure of an influenza virus-specific cytotoxic T-cell clone and its interaction with P815 and macrophage targets

The morphology of a cloned, H-2^d-restricted, type A influenza virus-cross-reactive cytotoxic T-cell line (L4) and its interaction with histocompatible P815 and macrophage targets was studied by transmission electron microscopy. Most characteristic features of L4 cells were organelle-free, filament-rich pseudopods, by which contact was made with target cells, tightly stacked Golgi saccules near the nuclear cleft, and large (1–3 μm) multivesiculate cytoplasmic bodies of unknown function. L4 cells contacted both type A influenza virus-infected and control (uninfected or type B virus-infected) targets, but tight contact involving interdigitating pseudopodal projections was prominent only with type A virus-infected target cells. Target cell lysis was characterised by zeiosis; however, no degranulation, membrane damage, or transfer of ultrastructurally identifiable material from killers to targets was ever observed.     

Imaging effector functions of human cytotoxic CD4+ T cells specific for Plasmodium falciparum circumsporozoite protein

Malaria vaccines, comprised of irradiated Plasmodium falciparum sporozoites or a synthetic peptide containing T and B cell epitopes of the circumsporozoite protein (CSP), elicit multifunctional cytotoxic and non-cytotoxic CD4⁺ T cells in immunised volunteers. Both lytic and non-lytic CD4⁺T cell clones recognised a series of overlapping epitopes within a ‘universal’ T cell epitope EYLNKIQNSLSTEWSPCSVT of CSP (NF54 isolate) that was presented in the context of multiple DR molecules. Lytic activity directly correlated with T cell receptor (TCR) functional avidity as measured by stimulation indices and recognition of naturally occurring variant peptides. CD4⁺ T cell-mediated cytotoxicity was contact-dependent and did not require de novo synthesis of cytotoxic mediators, suggesting a granule-mediated mechanism. Live cell imaging of the interaction of effector and target cells demonstrated that CD4⁺ cytotoxic T cells recognise target cells with their leading edge, reorient their cytotoxic granules towards the zone of contact, and form a stable immunological synapse. CTL attacks induced chromatin condensation, nuclear fragmentation and formation of apoptotic bodies in target cells. Together, these findings suggest that CD4⁺ CTLs trigger target cell apoptosis via classical perforin/granzyme-mediated cytotoxicity, similar to CD8⁺ CTLs, and these multifunctional sporozoite- and peptide-induced CD4⁺ T cells have the potential to play a direct role as effector cells in targeting the exoerythrocytic forms within the liver.     

Quantitative analysis of the 51Cr release cytotoxicity assay for cytotoxic lymphocytes

Using ⁵¹Cr-labelled P-815 mastocytoma cells as target cells and CS7BL/6 spleen cells sensitized against DBA/2 antigens as effector cells, it is shown that the variation in the observed specific ⁵¹Cr release over a broad range of experimental conditions can be explained on the basis of a simple physical model of the interaction process. The model assumes that a target cell can be destroyed only after contact with an effector cell, contact takes place on a random basis, one contact is sufficient, and that one effector cell can kill several targets with unchanged efficiency. The fraction of target cells destroyed (f) depends only on the incubation time (t), the number of effector cells (n) and a constant interaction probability (δ). Thus f = 1 ? e^?nδt. However, the experimental measurement, the fraction of ⁵¹Cr specifically released into the supernatant during the assay, may not be the same as the fraction of target cells destroyed because it takes considerable time for the releasable ⁵¹Cr to be released from a damaged target cell. This can be overcome experimentally by following the standard 37 °C incubation with a further incubation at 45 °C during which there are no new lytic events but all previously damaged target cells release the remainder of their releasable ⁵¹Cr. The model enables one to obtain accurate measurements of relative effector cell frequency over a broad range of experimental conditions.     

Effects of sodium periodate modification of lymphocytes on the sensitization and lytic phases of T cell-mediated lympholysis.

Sensitization of mouse splenic lymphocytes in vitro with sodium borohydride, suggesting that the biologic effects of sodium periodate are-treated autologous spleen cells stimulated a one-way mixed lymphocyte reaction and led to the generation of thymus-derived cytotoxic effector cells. These effectors were capable of lysing in 4 hr periodate-treated syngeneic and, to a lesser extent, periodate-treated allogeneic target cells. These results suggest that sensitization by periodate-treated autologous cells could result either from a specific reaction to modified self components or from a nonspecific mitogenic stimulation. Effector cells generated by allogeneic sensitization were detected on periodate-modified targets, irrespective of the H-2 antigens expressed by the targets. The effects of periodate modification on both stimulator and target cells were reversible by sodium periodate are dependent on the formation of a free aldehyde group on cell surface glycoproteins. Pretreatment of stimulator cells with neuroaminidase prevented the effect of periodate treatment, suggesting that the sensitization involves oxidized sialic acid residues. During the 4-hour 51Cr-release assay periodate-treated targets could be used to detect cytotoxic effector cells of any specificity. Fresh spleen cells and lymphocytes cultured for 5 days without antigen or in the presence of lipopolysaccharide did not lyse periodate-treated targets. An increasing level of cytotoxicity was detected on periodate-treated targets when the effector cells were generated, respectively, by stimulation with concanavalin A, by sensitization with periodate-modified autologous cells. Although the lysis of periodate-treated targets is itself nonspecific, effector cell specificity could be determined by selective blocking of the lytic phase with cells syngeneic to the stimulators. These results indicate that a nonspecific interaction can occur between lymphocytes and periodate-treated target cells, but that this interaction leads to lysis only when the lymphocytes were activated to become cytotoxic effectors.