Characterization of the effector mechanism of help for antigen-presenting and bystander resting B cell growth mediated by Ia-restricted Th2 helper T cell lines

The mechanism of help for resting B cell growth in MHC-restricted T-B collaboration was investigated using an in vitro polyclonal model for these T cell-B cell interactions. In the presence of rabbit anti-mouse Ig, small, size-selected B cells elicit help from syngeneic Ia-restricted Th2 cell lines specific for F(ab')2 rabbit globulin. Both Ag-presenting and bystander B cells receive signals from stimulated Th cells that lead to B cell proliferation. The results suggest that the direct activation of resting Ag-presenting and bystander B cells by Th2 cells is mediated by a similar effector mechanism. Although proliferative responses by Ag-presenting B cells are of greater magnitude, help for both Ag-presenting and bystander B cell populations is characterized by the lack of a requirement for membrane Ig cross-linking, by identical kinetics, and by the necessity for direct cell contact or close proximity with Th cells. B cell proliferation is not induced by exposure to the sequence of diffusable mediators released from a synchronized Ag-specific T-B interaction. The T cell-dependent proliferation by both B cell populations can be inhibited by excess mitomycin C-treated syngeneic "cold target" B cells, demonstrating a requirement for a short-range T cell-B cell interaction. mAb inhibition experiments fail to identify a role for class II, LFA-1, or CD4 membrane molecules in the delivery of help to bystander B cells. Antibody against H2d bystander class II molecules has no effect on bystander B cell proliferation at concentrations that completely block Ag presentation by H2d B cells to an H2d-restricted Th cell line. Antibodies against the cell adhesion molecule LFA-1 or the Th cell molecule CD4 do inhibit bystander B cell proliferation, but only to the extent that they block T cell activation and the induction of help. The inductive stimulus leading to resting B cell growth results from an early, short-range interaction with Th cells. B cell proliferation is supported by T cell soluble mediators as a consequence of this interaction, which is required for at least 8 hr after T cell recognition of Ag/Ia on the surface of Ag-presenting B cells.     

Dilution of irradiated cell conditioned medium and the bystander effect

While nontargeted and low-dose effects such as the bystander effect are now accepted, the mechanisms underlying the response have yet to be elucidated. It has been shown that the transfer of irradiated cell conditioned medium (ICCM) can kill cells that are not directly irradiated; however, to date the effect of ICCM concentration on cell killing has not been reported. The occurrence of a bystander effect was determined by measuring cell survival after exposure to various ICCM dilutions, using the colony-forming assay, in cells of six human cell lines with varied bystander responses and tumor/ p53 status. Autologous ICCM transfer for these cell lines induced a bystander effect as reported previously. ICCM from these cell lines was transferred to cells of a common reporter cell line (HPV-G) to investigate whether the lack of an induced bystander effect was due to their inability to generate or to respond to a bystander signal(s). ICCM from cells of four cell lines induced a bystander effect in HPV-G reporter cells, confirming that signal production is a critical factor. A saturation response was observed when ICCM was diluted. Survival was found to increase linearly until a plateau was reached and the bystander effect was abolished at 2x dilution. The effect of ICCM from the different cell lines reached a plateau at different dilutions, which were found to correlate with the cell line's radiosensitivity.     

Human peripheral blood lymphocytes targeted with bispecific antibodies release cytokines that are essential for inhibiting tumor growth

We have compared the mechanisms by which human PBL targeted with bispecific antibodies either lyse tumor cells or block their growth in culture or in mice. We found that resting PBL were unable to mediate lysis, but were able to block tumor growth. Moreover, targeted PBL were unable to lyse bystander cells, whereas targeted PBL did block the growth of bystander tumor cells in culture and in nude mice. Supernatants from cultures of targeted PBL, or from PBL grown on anti-CD3-coated flasks, blocked the growth of tumor cells in the absence of added effector cells, and antibodies against TNF-alpha and IFN-gamma reversed the inhibition of tumor growth, but had no effect upon cytolysis mediated by targeted by PBL. Our results show that targeted human PBL mediate two different antitumor activities: lysis, which occurs rapidly and requires the direct attachment of the target cell to the cytotoxic cell, and tumor growth inhibition, which is mediated by cytokines released into the medium as a result of receptor cross-linking. The inhibition of bystander tumor growth in mice by targeted PBL suggests that factor release is sufficient to block tumor growth in vivo. Targeted factor release therefore provides a mechanism by which targeted PBL could block the growth of tumor cells in vivo that were not bound by the effector cells, but which were located in the vicinity of tumor cells that were bound.     

THE KINETICS OF 51,Cr RELEASE FROM TARGET CELLS IN CELL MEDIATED CYTOTOXICITY AND THE RELATIONSHIP TO THE KINETICS OF KILLING

The kinetics of T-cell mediated cytotoxicity (CMC) have been re-examined. It is clear that per cent ⁵¹Cr release is linearly related to effector cell number and not to its log as often suggested. Data are presented showing that killing can occur within 1 min of lymphocyte-target cell contact and that ⁵¹Cr is released rapidly from killed cells. Inactivation experiments aimed to stop ongoing cytolysis indicate that all target cells are brought into contact with effector lymphocytes within 45 min. At a ratio of 50:1 the kinetics of contact resemble saturation kinetics, indicating a number of effector cells similar to or in excess of the number of target cells (that is, at least 2 % of total lymphocytes). Although CMC is causally related to cell contact, it is chronometrically unrelated and occurs as a random reaction (that is it can occur immediately or up to hours later than contact). The mean time depends on the number of effector cells. The killing reaction is temperature dependent and proportional to the number of effector cells but does not depend on the intact effector cell. An intact effector cell is only required to bring about contact. It is suggested that this final cytolytic event is brought about by the target cell itself, perhaps as a result of attempts to free itself from the attached lymphocyte.     

Exposure of normal monocyte-derived dendritic cells to human immunodeficiency virus type-1 particles leads to the induction of apoptosis in co-cultured CD4+ as well as CD8+ T cells

The depletion of immune T cells by human immunodeficiency virus type-1 (HIV-1) infection is a major mechanism involved in the pathogenesis of AIDS. Here, we examined a possible effector function of blood monocyte-derived dendritic cells (DCs) to induce apoptosis in bystander CD4+ and CD8+ T cells. The DCs were generated by culturing monocytes in the presence of granulocyte-macrophage colony-stimulating factor and interleukin-4. The DCs exposed to HIV-1 particles were co-cultured with healthy donor-derived blood T cells at a ratio of 1:20. Analyses by percent cell mortality, staining with propidium iodide and reactivity with Annexin V revealed the induction of apoptosis in both CD4+ and CD8+ target T cells. Further, this apoptosis occurred without stimulation with mitogens when the cell cycle of target T cells shifted from G0 to G1, probably due to the mitogenic effect of the DCs. Thus, induction of apoptosis in both CD4+ and CD8+ T cells occurred via interaction with DCs adsorbed with HIV-1 particles.     

Migratory neural stem cells for improved thymidine kinase-based gene therapy of malignant gliomas

Gene therapy of glioma based on viral delivery of herpes simplex virus type I thymidine kinase (HSV-TK) has failed in the clinic because of low transduction efficacy. To circumvent this problem, this study evaluated highly migratory HSV-TK-transduced neural stem cells (NSC) for their ability to kill untransduced glioma cells by a gap junction-mediated bystander effect. The admixture of HSV-TK-transduced NSC to U87MG and LN-18 human malignant glioma cell lines at ratios of 1:10 or 1:1 eliminated more than 50% or 90% of glioma cells in the presence of ganciclovir (25 microM). Glioma cell cytotoxicity required cell-cell contact. Similarly, tumor cell cytotoxicity was observed in two of three primary glioblastoma cell cultures, and the presence of this bystander effect correlated with the expression of connexin 43 in the untransduced glioma target cells. In conclusion, we delineate a role for migratory HSV-transfected NSC to eliminate glioma cells purely by means of the bystander effect.     

A stochastic model of cancer initiation including a bystander effect

A stochastic model of cancer initiation is considered. The model is used to evaluate whether a bystander effect may be important in the pre-malignant and malignant stages of carcinogenesis, and furthermore, on the basis of epidemiological data, to estimate the mutation rates of genes involved in the development of oral leukoplakias. The bystander effect is defined here as the capability of oncogenic mutations to increase the mutation probability of neighbouring (bystander) cells, thus leading potentially to a cascade of neighbouring mutated and neoplastic cells as a pre-stage in the development to leukoplakias and cancer. We find that incidence data for oral cancer are indeed in accordance with a significant bystander effect, operating either alone or in combination with genomic instability in the early stages of carcinogenesis, i.e. the development of neoplasia. Simulations performed gave a picture of how mutations and neoplasia may spread in a tissue, to form characteristic leukoplakias with a core of neoplastic cells. The model also showed that the probability of finding at least one neoplastic cell in the tissue after a given number of years is more sensitive to changes in genomic instability within the cell itself than to changes in a bystander effect. Based on epidemiological data we also calculate the maximum number of oncogenic genes that may be involved in the bystander effect and development of genomic instability. Even if capable of explaining the initial development of oncogenic mutations towards neoplastic cells, the bystander model could not reproduce the observed incidence rates of leukoplakia without assuming a carcinogen mutation probability per cell per year of neoplastic cells practically equal to one. This means that the bystander effect, to be of substantial importance in the final development of neoplastic cells towards leukoplakias, requires a very significant increase in mutation probabilities for bystanders to neoplastic cells. Alternatively, additional mechanisms such as abnormal cell differentiation and uncontrolled proliferation and apoptotis in the neoplastic stage may be of major importance during the development to cancerization.     

Bystander killing of breast cancer MCF-7 cells by MDA-MB-231 cells exposed to 5-fluorouracil is mediated via Fas

The major drawback with cancer therapy is the development of resistant cells within tumors due to their heterogeneous nature and due to inadequate drug delivery during chemotherapy. Therefore, the propagation of injury ("bystander effect" (BE)) from directly damaged cells to other cells may have great implications in cancer chemotherapy. The general advantage of the bystander cell killing phenomenon is the large therapeutic index that can be achieved. Experiments suggest that this phenomenon is detected in radiation therapy as well as in gene therapy in conjunction with chemotherapy. In the present study, we developed an original in vitro model dedicated to the exploration of bystander cytotoxicity induced during breast carcinoma chemotherapy. In brief, we investigated this perpetuation of injury on untreated bystander MCF-7 breast cancer cells which were coplated with 5-fluorouracil (5-FU)-treated MDA-MB-231 breast cancer cells. To achieve this goal, a specific in vitro coculture model which involved mixing of aggressive MDA-MB-231 breast cancer cells with enhanced green fluorescent protein (EGFP) expressing stable clone of non-metastatic MCF-7 breast cancer cells (MCF-EGFP), was used. A bystander killing effect was observed in MCF-EGFP cells cocultured with MDA-MB-231 cells pretreated with 5-FU. The striking decrease in MCF-EGFP cells, as detected by assaying for total GFP intensity, is mediated by activation of Fas/FasL system. The implication of Fas in MCF-EGFP cell death was confirmed by using antagonistic anti-FasL antibody that reverses bystander cell death by blocking FasL on MDA-MB-231 cells. In addition, inhibition of CD95/Fas receptor on the cell surface of MCF-EGFP cells by treatment with Pifithrin-alpha, a p53 specific transactivation inhibitor, partially abrogated the sensitivity of bystander MCF-EGFP cells. Our data, therefore, demonstrates that the Fas/FasL system could be considered as a new determinant for chemotherapy-induced bystander cell death in breast cancers.     

Tissue exit: a novel control point in the accumulation of antigen-specific CD8 T cells in the influenza a virus-infected lung

Memory/effector T cells efficiently migrate into extralymphoid tissues and sites of infection, providing immunosurveillance and a first line of defense against invading pathogens. Even though it is a potential means to regulate the size, quality, and duration of a tissue infiltrate, T cell egress from infected tissues is poorly understood. Using a mouse model of influenza A virus infection, we found that CD8 effector T cells egressed from the infected lung in a CCR7-dependent manner. In contrast, following antigen recognition, effector CD8 T cell egress decreased and CCR7 function was reduced in vivo and in vitro, indicating that the exit of CD8 T cells from infected tissues is tightly regulated. Our data suggest that the regulation of T cell egress is a mechanism to retain antigen-specific effectors at the site of infection to promote viral clearance, while decreasing the numbers of bystander T cells and preventing overt inflammation.     

Tumor-specific T-bodies: towards clinical application

 Unlike antibodies, T cells are well suited to penetrate and destroy solid tumors. The T-body approach combines antibody recognition and T cells effector function. It is based on T cells expressing chimeric receptors composed of antibody-derived Fv or scFv as their extracellular recognition elements joined to lymphocyte triggering molecules. This receptors can redirect the specificity of T cells in an MHC independent manner. Upon encountering their target cells, T-bodies are able to undergo specific stimulation for interleukin/cytokine production, and kill hapten-modified or tumor cells in model systems both in vitro and in vivo. T cells expressing chimeric receptors made of antitumor antibodies are able to discriminate between a tumor and normal cell with negligible bystander cytotoxicity. Further studies should be carried out to evaluate and optimize the persistence, homing patterns and reactivation potential of T-bodies in the body. Accepted: 14 October 1997     

System of cell irradiation with a defined number of heavy ions (III).

A single cell irradiation system has been developed at JAERI-Takasaki to study radiobiological processes in single-ion-hit mammalian cells and bystander cells, in ways that cannot be achieved using conventional broad field exposures. Individual mammalian cultured cells are irradiated in the atmosphere on the cell dish, the bottom of which is made of ion-track-detector CR-39, with a single or defined numbers of 13.0 MeV/amu 20Ne and 11.5 MeV/amu 40Ar ions. Targeting and irradiation of the cells are performed automatically at the on-line microscope of the microbeam apparatus according to the positional data of the target cells obtained at the off-line microscope before irradiation. Using this system, Chinese hamster ovary (CHO-K1) cells were irradiated with counted number of 20Ne and 40Ar ions. Thereafter, the growth of the cells was observed individually and repeatedly during post-irradiation incubation. The cells hit by a single 40Ar ion on their nucleus showed strong growth inhibition. Meanwhile, the cells in the irradiated dish but not hit by the ion (bystander cells) showed limited cell growth. This might be a bystander effect caused by heavy ion hit cell co-existing in the same dish.     

Antigen-dependent release of IFN-gamma by cytotoxic T cells up-regulates Fas on target cells and facilitates exocytosis-independent specific target cell lysis

Effector cytolytic T (Tc) lymphocytes, deficient in the exocytosis-mediated pathway of target cell lysis, induce Fas on target cells and, in turn, delayed cell death and apoptosis via the Fas ligand-Fas interaction. The induction of Fas can be blocked by anti- IFN-gamma Abs. This Fas up-regulation on initially Fas-negative target cells is not mediated by TCR-MHC/peptide signaling per se, but by secreted IFN-gamma from Tc cells after Ag engagement. The Fas up-regulation by Tc cells can be mimicked by treatment of target cells with rIFN-gamma. Tc cells from IFN-gamma knockout mice do not induce Fas expression on target cells. Tc cell-mediated Fas expression on third party, bystander, target cells does not enhance their susceptibility to lysis by these nominal effector cells. The results are discussed as to the possible relevance of the phenomenon in efficiency and regulation of the Tc cell response to infections by viruses.     

Cloned protein antigen-specific, Ia-restricted T cells with both helper and cytolytic activities: mechanisms of activation and killing

Myoglobin-specific, Iad-restricted cloned helper T cells and T hybridomas were found to directly kill Iad-bearing, myoglobin-pulsed B lymphoma targets and could also kill bystander targets, but only in the presence of antigen-pulsed antigen presenting cells (APC). The induction of the killing requires recognition of processed antigen in the context of class II major histocompatibility complex (MHC) molecules. Despite the specificity of induction, the bystander killing suggests a nonspecific lytic mechanism. The direct killing can be inhibited only by cold specific targets, whereas the bystander killing can be blocked by both specific and nonspecific targets. The cold target inhibition seems to be due to interference with effector-to-target contact or proximity rather than due to high-dose suppression of T-cell activation. Experiments using T-cell supernatants or cyclosporin A suggested that the helper T cells kill targets by synthesizing short-range soluble factor(s) with nonspecific killing activity de novo during the effector phase, but only while antigen-specific signal transduction is occurring. The mechanism of cold target inhibition appears to be absorption or consumption of a short-acting cytotoxic lymphokine by cells which must be able to interact closely with the effector cell. Normal spleen B cells, despite their capability for activating the helper T cells, cannot inhibit specific killing or be killed by helper T cells, even after lipopolysaccharide stimulation. Thus, although killing by helper T cells may play a negative feedback role in the normal immune response, our data raise the possibility that the helper T-cell-mediated killing may contribute to the immune surveillance against malignancy by virtue of the preferential killing of tumor cells either directly or indirectly.     

Antibodies to adhesion molecules inhibit the lytic function of MHC-unrestricted cytotoxic cells by preventing their activation.

We evaluated the effect of the antibodies to adhesion molecules CD2, CD11a/CD18 (LFA-1), and CD56 (N-CAM) on MHC-unrestricted cytotoxicity mediated by polyclonal NK cells and LAK cells or by CD3+ or CD3- cytolytic cell clones against a panel of tumor cell targets selected according to expression or absence of the corresponding ligands. We show that (i) antibodies to CD11a/CD18 and, to a lesser extent, antibodies to CD2 inhibit target cell lysis, whereas anti-CD56 antibodies exert little if any effect; (ii) in a model system using polyclonal NK/LAK cells as effectors and K562 or HL60-R (NK-resistant) cells as targets, inhibition of cytotoxicity occurs without a significant impairment of effector to target cell binding; (iii) the cytotoxic function of CD3+ or CD3- cytotoxic cell clones is inhibited differentially by antibodies to adhesion molecules; (iv) conjugates formed in the presence of antibodies which inhibit target cell lysis display a significant reduction of target to effector cell contact surface; and (v) this may lead to defective activation of effector cells, as indicated by lack of redistribution of the microtubular apparatus. We conclude that (i) MHC-unrestricted cytotoxicity is regulated by a number of molecular interactions that span far beyond our present knowledge and that it is strictly dependent on the surface phenotype of the effector cell and of the target cell; (ii) in certain types of effector/target cell interactions, antibodies to adhesion molecules do not prevent conjugate formation but reduce the extent of cell-to-cell surface contact which, in turn, leads to defective activation of the effector cell and, therefore, to inhibition of target cell lysis.     

Macrophage-Based Anti-Cancer Therapy: Modelling Different Modes of Tumour Targeting

Tumour hypoxia is associated with poor drug delivery and low rates of cell proliferation, factors that limit the efficacy of therapies that target proliferating cells. Since macrophages localise within hypoxic regions, a promising way to target hypoxic tumour cells involves engineering macrophages to express therapeutic genes under hypoxia. In this paper we develop mathematical models to compare the responses of avascular tumour spheroids to two modes of action: either the macrophages deliver an enzyme that activates an externally applied prodrug (bystander model), or they deliver cytotoxic factors directly (local model). The models we develop comprise partial differential equations for a multiphase mixture of tumour cells, macrophages and extracellular fluid, coupled to a moving boundary representing the spheroid surface. Chemical constituents, such as oxygen and drugs, diffuse within the multiphase mixture. Simulations of both models show the spheroid evolving to an equilibrium or to a travelling wave (multiple stable solutions are also possible). We uncover the parameter dependence of the wave speed and steady-state tumour size, and bifurcations between these solution forms. For some parameter sets, adding extra macrophages has a counterintuitive deleterious effect, triggering a bifurcation from bounded to unbounded tumour growth. While these features are common to the bystander and local models, the crucial difference is where cell death occurs. The bystander model is comparable to traditional chemotherapy, with poor targeting of hypoxic tumour cells; however, the local mode of action is more selective for hypoxic regions. We conclude that effective targeting of hypoxic tumour cells may require the use of drugs with limited mobility or whose action does not depend on cell proliferation.     

B cell response to fresh and effector T helper cells. Role of cognate T-B interaction and the cytokines IL-2, IL-4, and IL-6

The helper activity of resting T cells and in vitro generated effector T cells and the relative roles of cognate interaction, diffusible cytokines, and non-cognate T-B contact in B cell antibody responses were evaluated in a model in which normal murine CD4+ T cells (Th), activated with alloantigen-bearing APC, were used to support the growth and differentiation of unstimulated allogeneic B cells. Both "fresh" T cells, consisting of memory and naive cells, stimulated for 24 h, and "effector" T cells, derived from naive cells after 4 days of in vitro stimulation, induced the secretion of IgM, IgG3, IgG1, IgG2a, and IgA. Effector T cells were significantly better helpers of the response of small dense B cells, inducing Ig at lower numbers and inducing at optimal numbers 2- to 3-fold more Ig production than fresh T cells. The predominant isotype secreted was IgM. Supernatants derived from fresh T cell cultures contained moderate levels of IL-2, whereas those from effector cultures contained significant levels of IL-6 and IFN-gamma in addition to IL-2. The involvement of soluble factors in the B cell response was demonstrated by the ability of antibodies to the cytokines IL-2, IL-4, and IL-6 to each block Ig secretion. Antibodies to IL-5 and IFN-gamma had no effect on the T cell-induced response. Kinetic studies suggested that IL-4 acted during the initial stages of the response, whereas the inability of anti-IL-6 to block B cell proliferation suggested that IL-6 was involved in part in promoting differentiation of the B cells. The relative contributions of cognate (MHC-restricted) and bystander (MHC-unrestricted) T-B cell contact vs cytokine (non-contact)-mediated responses were assessed in a transwell culture system. The majority of the IgM, IgG3, IgG1, and IgG2a response induced by both fresh and effector T cells was dependent on cognate interaction with small, high density B cells. In contrast, a small proportion of these isotypes and most of the IgA secreted resulted from the action of IL-6 on large, presumably preactivated, B cells. The IgA response did not require cell contact or vary when fresh and effector cells were the helpers. The contribution of bystander contact in the overall antibody response to both T cell populations was minimal.(ABSTRACT TRUNCATED AT 400 WORDS)     

Bystander effect: biological endpoints and microarray analysis

In cell populations exposed to ionizing radiation, the biological effects occur in a much larger proportion of cells than are estimated to be traversed by radiation. It has been suggested that irradiated cells are capable of providing signals to the neighboring unirradiated cells resulting in damage to these cells. This phenomenon is termed the bystander effect. The bystander effect induces persistent, long-term, transmissible changes that result in delayed death and neoplastic transformation. Because the bystander effect is relevant to carcinogenesis, it could have significant implications for risk estimation for radiation exposure. The nature of the bystander effect signal and how it impacts the unirradiated cells remains to be elucidated. Examination of the changes in gene expression could provide clues to understanding the bystander effect and could define the signaling pathways involved in sustaining damage to these cells. The microarray technology serves as a tool to gain insight into the molecular pathways leading to bystander effect. Using medium from irradiated normal human diploid lung fibroblasts as a model system we examined gene expression alterations in bystander cells. The microarray data revealed that the radiation-induced gene expression profile in irradiated cells is different from unirradiated bystander cells suggesting that the pathways leading to biological effects in the bystander cells are different from the directly irradiated cells. The genes known to be responsive to ionizing radiation were observed in irradiated cells. Several genes were upregulated in cells receiving media from irradiated cells. Surprisingly no genes were found to be downregulated in these cells. A number of genes belonging to extracellular signaling, growth factors and several receptors were identified in bystander cells. Interestingly 15 genes involved in the cell communication processes were found to be upregulated. The induction of receptors and the cell communication processes in bystander cells receiving media from irradiated cells supports the active involvement of these processes in inducing bystander effect.     

The bystander effect in radiation oncogenesis: II. A quantitative model

There is strong evidence that biological response to ionizing radiation has a contribution from unirradiated "bystander" cells that respond to signals emitted by irradiated cells. We discuss here an approach incorporating a radiobiological bystander response, superimposed on a direct response due to direct energy deposition in cell nuclei. A quantitative model based on this approach is described for alpha-particle-induced in vitro oncogenic transformation. The model postulates that the oncogenic bystander response is a binary "all or nothing" phenomenon in a small sensitive subpopulation of cells, and that cells from this sensitive subpopulation are also very sensitive to direct hits from alpha particles, generally resulting in a directly hit sensitive cell being inactivated. The model is applied to recent data on in vitro oncogenic transformation produced by broad-beam or microbeam alpha-particle irradiation. Two parameters are used in analyzing the data for transformation frequency. The analysis suggests that, at least for alpha-particle-induced oncogenic transformation, bystander effects are important only at small doses-here below about 0.2 Gy. At still lower doses, bystander effects may dominate the overall response, possibly leading to an underestimation of low-dose risks extrapolated from intermediate doses, where direct effects dominate.     

Target cell lysis by cytotoxic T lymphocytes redirected by antibody-coated polystyrene beads

Cytotoxic T lymphocytes were found to mediate rapid lysis of target cells not normally recognized in the presence of small polystyrene beads coated with a combination of anti-T3 and antitarget cell antibodies. Lysis was not seen with beads bearing one of these antibodies alone, nor with a mixture of two types of beads each coated with a single antibody. The effector cells mediating this lysis include long term allospecific human CTL, and both human and mouse CTL clones recognizing mouse class I MHC Kb Ag. TNP-modified mouse tumor cells, a human lymphoblastoid line, and human red cells were found to be good targets for this cytotoxicity. Polystyrene beads with diameters of 3 to 15 mu caused target lysis, with a dose-response curve which typically went through a maximum and declined at high bead numbers. Maximal bead-redirected lysis by CTL was less efficient than that mediated by soluble antibody heteroconjugates of the same two antibodies. Bead-redirected target lysis was calcium dependent. These results are interpreted as a form of bystander lysis induced by the beads, since the target cell membrane is not directly crosslinked to the region of CTL activation. These observations thus favor a mechanism of lysis involving the polarized secretion of a locally acting lytic agent by CTL.     

Evidence for an involvement of T4+ cytotoxic T cells in tumor immunity

Cell-mediated immunity against cancer cells primarily involves class I major histocompatibility complex (MHC)-restricted cytotoxic T cells and natural killer (NK) cells. To investigate whether T4+ cytotoxic T cells also have a role in tumor-specific immunity, mice were immunized with a B cell lymphoma. T cell hybridomas were constructed from the immune spleen cells and analyzed for their cytotoxic ability against the immunizing lymphoma. A T4+, Lyt-1+ hybridoma cell line was developed (103L2) which specifically killed the immunizing tumor cells but not normal B cells or a range of other tumor cells of B or non-B origin. This cytotoxic hybridoma cell line differed from Lyt-2+ cytotoxic T lymphocyte cells and NK cells, commonly identified with cytotoxicity, in a number of important ways. First, the cells were class II MHC restricted; second, interleukin-2 was released from activated effector cells; and finally but most importantly, innocent nonparticipating bystander cells were also killed. The significance of this observation was that normal cells were protected, although a broad range of tumor cell types, including tumor antigen-negative mutants, were killed. It is therefore conceivable that T4+ cytotoxic T cells might play an important role in tumor immunity through the direct recognition and lysis of tumor cells while any tumor variants, arising due to antigen loss, would remain susceptible through the bystander killing effect and normal cells would remain unaffected. These results strongly suggest that tumor-reactive T4+ cytotoxic T cells belong to a new category of effector cells with an important role in tumor-specific immunity.