Biomedical subjects
Publications and source records attributed to B Bonavida.
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A xenogeneic serum directed at alloimmune CTL is shown to block H-2-restricted anti-viral CTL activity. Extensive absorption studies with targets and effector cells indicate that the blocking is at the level of the CTL and not the target cells. Inhibition is demonstrated for influenza-specific cytotoxicity operating in the context of 3 distinct H-2 haplotypes, suggesting that the serum is not directed at antigen-recognition structures. Kinetic studies on the addition of RAT to an ongoing 51Cr release assay further support the hypothesis that inhibition is not operating via the idiotypic region of the antigen receptor. We suggest that RAT is specific for CTL surface structures involved in nonrecognition events--possibly the lytic sites or a conserved region (idiotype-negative) of th antigen-binding receptor.
The frequency of cytotoxic NK cells has been directly measured. The cytotoxic assay is performed by preparing conjugates of effector cells bound to target cells and assessing under the microscope target cell lysis by the trypan blue dye exclusion test. With Ficoll-Hypaque purified cells, it was found that 11--13% of lymphocytes formed conjugates with K562 or Molt-4 target cells, but only 3--4% of the total lymphocytes actually killed. Thus, the NK cell's ability to bind targets cannot be equated to the ability to kill. The nature of the noncytotoxic binding lymphocytes is discussed. The kinetics of lysis by NK cells show a random distribution of killing in relationship to time and plateaus at 3--4 hours. The frequency of killing is the same for a wide variety of populations tested at different times. We conclude that some of the target binding cells are antigen specific NK cells that can be converted to cytotoxic natural killer cells.
Radiolabelled antitumor reactive T lymphocytes (ARC) were prepared in vivo by immunization of SJL/J mice with mitomycin C inactivated syngeneic LA-6 tumor cells followed by injection of 125IUdR to label dividing cells. These ARC were specifically diverted to the liver when injected in LA-6 tumor-bearer serum and injected i.v. into normal SJL/J mice. Likewise, SJL/J anti-Balb/c ARC were diverted to the liver of SJL/J mice bearing spontaneous reticulum cell sarcomas (RCS) carrying Balb/c cross-reactive antigens but not in mice with Balb/c negative neoplasms. Mice with Balb/c positive tumors also had circulating ARC opsonizing factors. These results suggest a mechanism for the survival of antigenic tumors involving macrophages and ARC opsonizing (ARCO) factors. A novel approach to immunotherapy is discussed.
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We have demonstrated that within a given system Cy may alter the immune response in different ways. It may augment the response by inactivating CSS cells or induce immunosuppression by activating CRS. Furthermore, our studies provide a means of analyzing the mechanism of suppression, that is, delineating the target cell for suppression, the properties and life span of the suppressed cells, and the nature of the cell interactions involved. Furthermore, with the available information on the immunosuppressive activity of Cy, clinical studies may be best monitored in transplantation immunology and tumor chemotherapy. It is possible that the induction of CRS cells, which are antigen-nonspecific and non-H-2-restricted, may be useful for inducing specific immunosuppression to alleviate GVH reactions. These studies are currently in progress.
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The kinetics and rate of T cell-mediated cytolysis was assessed by measuring the times required for lysis of isolated target cells by single cytotoxic lymphocytes. Single target cell lysis was determined microscopically by observing trypan blue uptake as a function of time of incubation of effector-target conjugates in agarose. Lysis of EL-4 target cells by alloimmune peritoneal exudate lymphocytes was initiated without a lag and was essentially complete at 2 hr. Both zero-order and first-order kinetics equations were analyzed for fit to the 0 to 2 hr lysis values. Statistically, the zero-order kinetic function could be rejected (p greater than 0.05), but the first-order kinetics function (p less than 0.01) could not. This strong evidence for first-order kinetics of T cell-mediated cytolysis implies that within each CTL-target cell population, cytolysis occurs exponentially as a random decay process and that one event in the entire process of cytolysis is rate limiting. The first-order equation was then applied to measurements of the rate of cytolysis in many different individual effector-target cell combinations. Significant differences in the lytic rate were apparent when either the effector or target cell were varied, with the rate constants spanning a 5-fold range. The heterogeneity of lytic rates is consistent with the hypothesis that lytic efficiency is a function of both the effector and target cells used.
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The generation of cytotoxic T cells by normal and primed lymphocytes fractionated on spleen cell monolayers was examined by two methods. In the first method, sublethally irradiated recipients were inoculated with the fractionated cells, and 5 to 6 days later the spleens were tested for cytotoxicity against 51Cr tumor target cells syngeneic to the host. Significant reduction of cytotoxic activity was observed in one-half of the experiments. In the second method, the fractionated cells were cultured with mitomycin C-treated stimulated cells and tested 6 days later for cytotoxicity. A slight reduction of cytotoxic activity was observed. In both methods, the cytotoxic response generated by the fractionated alloantigen-primed lymphocytes was similar to the response obtained with unprimed cells.
Antibody-forming cells with specificities against syngeneic and allogeneic thymocytes are detected in the spleens of normal mice after activation in vitro or in vivo with lipopolysaccharide (LPS). The activity of such cells was measured in a complement-dependent plaque assay employing trypan blue dye to assess zones of lysis. Plaques were rarely seen in the absence of LPS treatment. Anti-immunoglobulin added to the plaque assay abrogated the appearance of plaques, but the addition of LPS had no effect. Furthermore, plaque formation was 2-mercaptoethanol sensitive indicating that the antibody responsible was of the IgM class. Plaque forming cells (PFC) were also detected against syngeneic and allogeneic lymph node cells and to a much lesser extent against splenocytes. The numbers of PFC found against syngeneic, allogeneic, or a mixture of thymocytes was similar and ranged from 1000 to 3000 PFC/10(8) viable spleen cells tested. All murine strains tested, including congenitally athymic nude mice, exhibited anti-thymocyte PFC after LPS activation. C3H/HeJ mice, genetically unresponsive to LPS, did not respond mitogenically to LPS and no anti-thymocyte plaques were observed. These findings suggest that clones of autoreactive B cells are present in normal mice and can be activated by LPS.
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We have studied both specific and non-specific suppression in allogeneic combinations. On the one hand, achievement of specific suppression may circumvent both HVGR and GVHR, while on the other, Cy-resistant suppressor cells may exacerbate the GVHR in bone marrow graft recipients. A greater understanding of both of these aspects of modulation of immune responsiveness will be important in clinical bone marrow transplantation.
A secondary specific cytotoxic response is obtained when lymphocytes primed in vivo to a tumor allograft are exposed to Con A in culture. The secondary cytotoxic cells generated are specific to target cells bearing antigens of the primary sensitizing cells and are qualitatively indistinguishable from the response obtained upon secondary antigenic stimulation. The cell-mediated cytotoxicity is independent of concanavalin A (Con A) and is not affected by the Con A-specific inhibitor, alpha-methyl-D-mannose pyranoside. Furthermore, cultures containing a mixture of submitogenic concentrations of Con A and stimulating antigens showed synergy and augmentation of cytotoxic activity. It is suggested that activation of prekiller cells by Con A into CTL may be mediated via the same or similar receptors normally triggered by the stimulating antigens. Functional similarities between ConA and the lymphocyte-defined antigens of the major histocompatibility complex region are discussed.
Human peripheral blood leukocytes are cytotoxic to 51Cr-labeled target cells provided phytohemagglutinin is present in the reaction mixture. The lectin-dependent cellular cytotoxicity (LDCC) phenomenon is characterized by (1) the short duration of the assay (2 to 4 hr), (2) revealing potential cytotoxic cells present in the cell preparation and excluding participation of mitogen-induced polyclonal activation of effector cells generated during prolonged assays used in mitogen-induced cytotoxicity, and (3) using nonerythrocyte target cells. The effector cells that mediate LDCC were characterized by several cell fractionation procedures. Fractionation of Ficoll-Hypaque separated peripheral blood leukocytes on polystyrene bead columsn or on nylon fiber columns which remove monocytes, granulocytes, and immunoglobulin-bearing leukocytes did not remove the LDCC activity. Polymorphonuclear leukocytes preparations were devoid of LDCC activity. Enrichment for T cells by E rosette sedimentation demonstrated that LDCC activity is present in both the T cell-enriched fraction and the non-T cell fraction. Nylon column eluted non-Ig-bearing cells which were subsequently depeleted of T cells by E rosette separation were LDCC positive. Depletion of Fc-bearing leukocytes on EA monolayers removed the LDCC activity. These results demonstrated that two populations of Fc-bearing cells of thymus and nonthymus origin mediate LDCC. Normal and malignant cells of immune and human origin were good targets in LDCC. The wide range of sensitive target cells in LDCC makes this test feasible in a completely autologous or syngeneic system. The significance of the LDCC phenomenon in assessing cell-mediated immunity is discussed.
Mice sensitized against a tumor allograft and given cyclophosmamide 6 days later failed to generate an immune response to the allograft. Spleen cells derived from these mice suppressed the generation of cytotoxic T cell response by normal spleen cells in mixed leukocyte cultures. The suppressor cells were not specific, thymus dependent, and resistant to 2000 R.