A pilot trial targeting the ICOS-ICOS-L pathway in nonhuman primate kidney transplantation.

Costimulation blockade with the B7-CD28 pathway-specific agent belatacept is now used in clinical kidney transplantation, but its efficacy remains imperfect. Numerous alternate costimulatory pathways have been proposed as targets to synergize with belatacept, one of which being the inducible costimulator (ICOS)-ICOS ligand (ICOS-L) pathway. Combined ICOS-ICOS-L and CD28-B7 blockade has been shown to prevent rejection in mice, but has not been studied in primates. We therefore tested a novel ICOS-Ig human Fc-fusion protein in a nonhuman primate (NHP) kidney transplant model alone and in combination with belatacept. ICOS-Ig did not prolong rejection-free survival as a monotherapy or in combination with belatacept. In ICOS-Ig alone treated animals, most graft-infiltrating CD4(+) and CD8(+) T cells expressed ICOS, and ICOS(+) T cells were present in peripheral blood to a lesser degree. Adding belatacept reduced the proportion of graft-infiltrating ICOS(+) T cells and virtually eliminated their presence in peripheral blood. Graft-infiltrating T cells in belatacept-resistant rejection were primarily CD8(+) CD28(-) , but importantly, very few CD8(+) CD28(-) T cells expressed ICOS. We conclude that ICOS-Ig, alone or combined with belatacept, does not prolong renal allograft survival in NHPs. This may relate to selective loss of ICOS with CD28 loss.

Structure-activity relationships for inhibition of inosine monophosphate dehydrogenase by nuclear variants of mycophenolic acid.

Structure-activity relationships in the region of the phthalide ring of the inosine monophosphate dehydrogenase inhibitor mycophenolic acid have been explored. Replacement of the lactone ring with other cyclic moieties resulted in loss of potency, especially for larger groups. Replacement of the ring by acyclic substituents also indicated a strong sensitivity to steric bulk. A phenolic hydroxyl group, with an adjacent hydrogen bond acceptor, was found to be essential for high potency. The aromatic methyl group was essential for activity; the methoxyl group could be replaced by ethyl to give a compound with 2-4 times the potency of mycophenolic acid in vitro and in vivo.

Induction of murine fibrosarcomas by low dose treatment with 3-methylcholanthrene followed by promotion with 12-O-tetradecanoyl-phorbol-13-acetate.

Repeated application of the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA) to the skin of mice previously treated with an initiating dose of the carcinogen 7,12-dimethylbenz[a] anthracene has been shown to lead to an increased incidence of papilloma. The studies presented here describe a modified murine two-stage carcinogenesis model in which a single subcutaneous administration of the carcinogen 3-methylcholanthrene (3-MC) is followed by multiple applications of TPA administered subcutaneously or intraperitoneally. TPA was observed to act as a promoter under these conditions when given either subcutaneously or intraperitoneally. When a carcinogenic dose of 3-MC was administered (0.5 mg/mouse) followed by regular treatment with TPA (10 micrograms/mouse) the percent of tumor-bearing mice increased and the length of time until tumors developed significantly shortened. At a subcarcinogenic dose of 3-MC (0.025 mg/mouse), repeated treatment with TPA led to tumor development whereas no tumors were observed in mice not treated with TPA. All tumors were found to be fibrosarcomas. Thus, TPA is capable of acting as a systemic promoter of mesenchymally derived tumors.

Immune response to weakly immunogenic virally induced tumors. IX. Mice injected with the in vitro variant of YAC tumor (YAC-1) resist lethal doses of the tumorigenic YAC cells.

The ability of the in vitro propagated syngeneic tumor YAC-1 and the allogeneic tumor RBL5 to stimulate cytotoxic cells in A mice, was in correlation with the ability of these tumors to impose in vivo resistance to the viable YAC cells. Similarly, the ability of allogeneic YAC-1 tumor and syngeneic RBL5 tumor to stimulate cytotoxic cells in C57BL/6 mice was in correlation with the ability of these tumors to impose in vivo resistance to the viable RBL5 cells. In contrast, the original in vivo carried YAC tumor cells that induce the appearance of suppressor cells, as measured by in vitro assay, stimulated the in vivo enhancement of tumor growth in A mice. Adult thymectomy delayed the death of A mice injected with viable YAC cells, suggesting that at least some of the cells participating in the tumor enhancement are thymus derived cells.

Immune responses to weakly immunogenic virally induced tumors. III. Genetically unrestricted cytolysis of allogeneic tumor target cells.

Splenocytes from A mice injected with YAC-1 or RBL5 could generate, after in vitro culture with or without stimulation, a genetically nonrestricted cytotoxic response against the allogenic tumor RBL5. YAC-1 tumor is an in vitro carried tumor induced in A mice (H-2a) by Moloney virus. RBL5 tumor is a Rauscher virus-induced tumor of C57BL/6 mice (H-2b). These tumors cross-react serologically. The effector cells that were generated after the in vitro cultivation recognized tumor-associated antigens on the target cells. H-2 alloantigens were not recognized by the effector cells. The effector cells that killed RBL5 tumor in a genetically nonrestricted manner were identified as T cells. The in vivo carried tumor YAC, in contrast to the in vitro carried tumor YAC-1, could not induce anti-RBL5 reactive cells in A mice. Instead, YAC tumor induced suppressor cells in A mice, which could abrogate the anti-RBL5 cytotoxic response of RBL5-primed splenocytes, but not that of YAC-1 primed splenocytes.

Phenotypic identification of specific and nonspecific suppressor T-cell populations involved in the in vivo response to alloantigen.

The monoclonal antibody 984.D4.6 (ma984) has been previously shown to recognize antigen-specific suppressor T-cells in an in vitro alloantigenic mixed-lymphocyte response system. In addition, an antiserum generated in rabbits to the N-terminal sequence of the nonspecific suppressor factor SIRS (soluble immune response suppressor) has been shown to block the suppressive activity of nonspecific, concanavalin A-activated suppressor cells. In the present studies we have used these antibodies to investigate the development of T-suppressor activity in mice immunized with alloantigen. These studies demonstrate the development of two populations of suppressor cells, one of which is antigen nonspecific and inhibitable with anti-SIRS and a second that is antigen-specific and sensitive to removal by lysis with ma984 plus complement. These populations of suppressor cells arise well after the peak of cytolytic T-cell activity in response to alloantigen indicating asynchrony in the development of the immune response to alloantigen.

Immune responses to weakly immunogenic murine-leukemia-virus-induced tumors. VII. Kinetic studies on various parameters of effects induced with suppressor cells.

YAC, a Moloney-virus-induced tumor of A mice, caused an inhibition of specific immunologic responses in A and C57BL/6 mice, which was mediated by suppressor cells. In contrast, YAC-1, the in vitro-carried tumor derived from cultivated YAC cells, stimulated the appearance of antitumor reactive cells in A mice. Splenocytes from YAC-1-injected mice generated anti-YAC cytotoxic cells after six days of culture. The suppressor cells from YAC-injected mice efficiently inhibited the cytotoxic responses of the cultivated reactive cells (YAC-1-primed cells) when added at the start of the culture, but not when added at later times. Suppressor cells appeared in A mice three days after injecion of YAC cells and persisted in the animals for at least 50 days. YAC-1-primed cells, derived from A mice 1, 3, 9 and 20 days after injection of YAC-1 cells, were sensitive to the suppressor cells.

Immune responses to weakly immunogenic murine-leukemia-virus-induced tumors. VIII. Characterization of suppressor cells.

YAC is a Moloney-virus-induced tumor of A mice. YAC-injected A and C57BL/6 mice generated suppressor cells. The suppressive function of the cells was tested by determining the ability of splenocytes from YAC-injected mice to inhibit the in vitro cytotoxic responses of primed splenocytes. It was found that suppressor cells passed through a nylon-wool column did not adhere to the plastic surface and resisted treatment with rabbit antimouse brain serum and guinea-pig complement. Therefore, the suppressor cells were defined operationally as "null" complement. Therefore, the suppressor cells did not demonstrate high efficiency; a relatively high concentration of suppressor cells was required to achieve an effective inhibiting action. These findings are discussed in terms of the heterogeneity of suppressor cells.

Organophosphorus pesticide immunotoxicity: effects of O,O,S-trimethyl phosphorothioate on cellular and humoral immune response systems.

The time course of immunosuppression induced by acute treatment with O,O,S-trimethyl phosphorothioate (OOS-TMP), an impurity in technical formulations of malathion, was examined in female C57B1/6 mice. Both cell-mediated and humoral immune responses were examined and included allospecific cytotoxic T cells, proliferative response to mitogens, interleukin-2 production and antibody production to sheep red blood cells. OOS-TMP pretreatment led to a reversible suppression of the generation of cytotoxic T lymphocytes and antibody-secreting cells to sheep erythrocytes. However, the mitogenic response of splenocytes from animals treated with nontoxic doses of OOS-TMP (as measured by body weight loss, serum cholinesterase levels and splenic lymphocyte number) to concanavalin A was not significantly suppressed, but the response to the B cell mitogen lipopolysaccharide was slightly decreased on day 1 following treatment. In contrast, interleukin-2 production was elevated by 24 h following treatment, but had returned to control levels by day 7. These data suggest that OOS-TMP was able to block the generation of cytotoxic T lymphocytes and antibody responses at doses of OOS-TMP that did not affect body weight or splenic lymphocyte number and this suppression was reversible.

Investigations into the mechanism of immunosuppression caused by acute treatment with O,O,S-trimethyl phosphorothioate. I. Characterization of the immune cell population affected.

Acute administration of O,O,S-trimethyl phosphorothioate (OOS-TMP), an impurity in technical formulations of malathion, to female C57B1/6 mice was previously shown to suppress the generation of both cytotoxic T lymphocyte to alloantigen and antibody-secreting cells to sheep red blood cells. In this report, macrophages were shown to be the immune cell population most affected by acute OOS-TMP pretreatment by cell separation and reconstitution experiments. Macrophages from OOS-TMP-treated animals had increased levels of nonspecific esterases. In addition, the size distribution of macrophages from treated animals was slightly larger and more heterogeneous than macrophages from control animals. However, macrophages from OOS-TMP-treated animals did not exhibit tumoricidal activity. These data suggest that macrophages from OOS-TMP-treated animals were similar to those located in nonimmune inflammatory sites.

Effects of subchronic treatment with O,O,S-trimethyl phosphorothioate on cellular and humoral immune response systems.

The effect of a 14-day treatment with low doses of O,O,S-Trimethyl phosphorothioate (OOS-TMP), an impurity in technical malathion, on the generation of cell-mediated and humoral immune responses was examined in female C57B1/6 mice. At a dose of 0.5 mg/kg/day OOS-TMP, the generation of antibody-secreting cells to sheep red blood cells (SRBC), the production of interleukin 2 (IL-2), and proliferative responses to the mitogens concanavalin A (Con A) and lipopolysaccharide (LPS) were elevated. In contrast, the cytotoxic T-lymphocyte (CTL) response to alloantigen was unchanged. At 5.0 mg/kg/day OOS-TMP, both the CTL and specific antibody response were unchanged, but all other immune parameters examined were elevated. Data from cell separation and reconstitution experiments indicated that both macrophages and B cells were affected by this treatment regime. These data suggest that long-term exposure to low amounts of OOS-TMP may enhance the ability of an animal to generate an immune response.

Investigations into the mechanism of immunosuppression caused by acute treatment with O,O,S-trimethyl phosphorothioate: generation of suppressive macrophages from treated animals.

At concentrations normally found in the spleen, macrophages from animals treated with O,O,S-trimethyl phosphorothioate (OOS-TMP) for 24 hr were previously shown to be immunosuppressive (Rodgers et al., 1985b). In addition, it was shown that macrophages from OOS-TMP-treated animals had a diminished capacity to present antigen (Rodgers et al., 1985c). In this report, it was shown that lowering the number of splenic adherent cells (95% macrophages by morphology) utilized in cell-mixing experiments to reconstitute the nonadherent splenic populations returned the humoral immune response to control levels. One day following acute administration of OOS-TMP, resident peritoneal cells were able to suppress the proliferation of P815 tumor cells. In addition, proliferative responses to concanavalin A and lipopolysaccharide were decreased at suboptimal concentrations of mitogen. Fresh supernatants from splenocytes cultured for 24 hr from OOS-TMP-treated animals blocked the generation of a humoral immune response. However, supernatants from splenocytes of control animals generated in the same manner did not block the generation of a humoral immune response. These data suggest that OOS-TMP induced the generation of suppressive macrophages which may potentially act through the release of labile factors which block proliferation or antigen- or mitogen-induced lymphocyte stimulation.

Antipeptide antibody specific for the N-terminal of soluble immune response suppressor neutralizes concanavalin A and IFN-induced suppressor cell activity in an in vitro cytotoxic T lymphocyte response.

Soluble immune response suppressor (SIRS) is a nonspecific immunosuppressive lymphokine produced by Lyt-2+ lymphocytes after exposure to Con A, IFN-alpha, or IFN-gamma. The N-terminal 21 residues of SIRS have recently been elucidated and antisera specific for this sequence have been raised in rabbits by using a synthetic peptide coupled to an immunogenic carrier protein. In a series of experiments, we have established that this antiserum blocks the suppressive activity of Con A- or IFN-activated suppressor cells. These data establish that Con A- or IFN-activated suppressor cells exert some or all of their suppressive effects via SIRS. Further studies show that SIRS acts primarily during the induction of CTL and not at the effector phase. Last, we show that SIRS is not involved in the radiation-resistant Ag-specific suppressor cell circuit that can be induced during the in vitro MLR.

Production of human-human hybridomas secreting antibody to sheep erythrocytes after in vitro immunization.

This report describes the formation of human hybridomas after in vitro immunization of tonsillar lymphocytes and fusion of the stimulated lymphocytes with a human lymphoblastoid cell line, WI-L279HF2. Lymphocytes were stimulated in vitro with sheep erythrocytes (SRBC) and polyclonal activators (PCA). By varying the duration of in vitro immunization before fusion and varying the PCA concentrations, the optimal conditions for cell fusion, hybrid cell growth, nonspecific immunoglobulin (Ig) secretion, and secretion of antibody to SRBC were determined. Lymphocytes cultured allogeneically at 5 X 10(6) cells per well in 24-well plates with SRBC and PCA (PWM, 1/10,000 dilution or LPS, 5 micrograms/ml) fused with the greatest efficiency. PWM-stimulated cultures fused on day 6 of in vitro stimulation yielded the greatest number of wells containing Ig-secreting hybridomas, whereas LPS-stimulated cultures produced more wells with hybridomas secreting Ig when fused on day 7 of stimulation. In vitro stimulation conducted in tissue culture flasks produced fewer wells with hybrid cell growth and Ig secretion. A fusion ratio of 1:1 (lymphocyte:WI-L2729HF2) yielded the most hybrid formation. Ig levels in wells with hybrid cell growth varied greatly, from less than 10 ng/ml up to 30 micrograms/ml. Ig of IgG, IgA, and IgM isotypes were produced. The addition of lymphocyte conditioned medium to in vitro immunization cultures does not improve the yield of Ig-secreting hybridomas. Hybrid cells secreting antibody to SRBC were cloned and analyzed for HLA specificities and chromosome numbers. Additionally hybridoma cells were formed after in vitro stimulation of peripheral blood lymphocytes (PBL) of normal individuals by fusion of the stimulated lymphocytes with WI-L2-729HF2. Hybridoma formation with PBL was less successful, but hybridoma cells secreting antibody to SRBC after in vitro stimulation were isolated.

Investigations into the mechanism of immunosuppression caused by acute treatment with O,O,S-trimethyl phosphorothioate. II. Effect on the ability of murine macrophages to present antigen.

Acute administration of 10 mg/kg O,O,S-trimethyl phosphorothioate (OOS-TMP) for 24 h has been shown to suppress the in vitro generation of cytotoxic T lymphocyte responses and antibody-secreting cells to sheep red blood cells and to increase interleukin-2 production. Macrophages were shown to be the splenic cell population most affected by OOS-TMP pretreatment. In this report, the ability of macrophages from OOS-TMP-treated animals to function in antigen presentation was shown to be significantly decreased. In addition, macrophages from treated animals had increased phagocytic capability and interleukin-l production. However, the percentage of Ia-positive macrophages present in splenic populations was decreased following OOS-TMP treatment. A decrease in antigen presenting ability and the number of Ia-positive macrophages may explain the reversible suppression in cytotoxic T lymphocytes and antibody responses reported previously.

Transduction of the IFN-gamma signal for HLA-DR expression in the promonocytic line THP-1 involves a late-acting PKC activity.

Interferon gamma (IFN-gamma) is the most potent known lymphokine for activating macrophages and has been shown to induce expression of HLA-DR in THP-1 cells, a monocytic tumor cell line which expresses many of the properties of monocytes, in a dose- and time-dependent manner. Experiments were designed to examine, by FACS analysis and by measurement of messenger RNA levels, the molecular mechanism regulating the expression of HLA-DR molecules. The expression of HLA-DR molecules induced by IFN-gamma was blocked by the protein kinase C (PKC) inhibitors sphingosine, staurosporine, and H7. H7 when added up to 20 hr after the initial stimulation with IFN-gamma prevented the further expression of HLA-DR. The general kinase inhibitors H8, H9, and HA1004, all less potent PKC inhibitors than H7, did not block the IFN-gamma-induced expression of HLA-DR at the concentrations employed. W7, a calmodulin antagonist, but not a PKC inhibitor, was also unable to prevent the IFN-gamma-induced expression of HLA-DR. Treatment of THP-1 with phorbol 12-myristate 13-acetate (PMA), a direct activator of PKC, alone or with Ca2+ ionophore A23187, was unable to induce HLA-DR expression. However, pretreatment with PMA for 24 hr prior to IFN-gamma stimulation decreased the IFN-gamma-induced expression of HLA-DR without decreasing IFN-gamma receptor levels. These results suggest that PKC plays a significant role in the IFN-gamma-induced signal transduction pathway leading to the expression of HLA-DR in cells of the mononuclear phagocytic lineage, and that PKC activity is required throughout the course of events leading to the actual expression of HLA-DR.

Immune responses to weakly immunogenic virally induced tumors. II. Suppressive effects of the in vivo carried tumor YAC.

Unprimed spleen cells from A and C57BL/6 mice could not produce cytotoxic responses to their syngeneic tumors: a Moloney virus-induced in vitro subline YAC-1 and a Rauscher virus-induced in vitro subline RBL5, respectively. Spleen cells from A and C57BL/6 mice immunized with YAC-1 OR RBL5 (which cross-react serologically) generated significant syngeneic cytotoxicities after cultivation in vitro. The in vivo carried tumor of A mice, unlike the in vitro sublines, could not stimulate a priming effect. In contrast, YAC stimulated the formation of suppressor cells in both A and C57BL/6 mice. The suppressor cells abrogated the priming effect of the syngeneic tumors, but not the priming effect of the allogeneic tumors. Furthermore, YAC did not suppress normal allogeneic anti-tumor responses. The theoretical and the practical implications of these studies are discussed.