Biomedical subjects
Publications and source records attributed to R N Lausch.
HSV-1 sensitized hosts were not resistant to challenge with small numbers of HSV-1 transformed cells. Virus immunization did not inhibit or enhance the incidence of tumour metastases.
Spleen cells obtained from hamsters bearing PARA-7 tumors greater than 1.0 cm were not reactive in microcytotoxicity assays unless preincubated overnight. The events occurring during in vitro incubation which lead to reversal of tumor-mediated suppression of cellular immunity were investigated. After 24 hr of incubation, supernatants overlying spleen cells from tumor-bearing hosts contained a factor which blocked cytotoxicity of simian virus 40 (SV40)3-sensitized spleen cells at the PARA-7 target cell level but not at the effector cell level. The preparations did not mediate antibody-dependent cellular cytotoxicity (ADCC). Opposite results were obtained in assays of culture medium overlying spleen cells from hosts with a tumor burden less than 0.1 cm. Although ADCC activity was present, no significant blocking was detectable. Treatment of inactive spleen cells with anti-hamster gamma-globlin in the presence of complement (anti-HGG + C) prevented activation and formation of blocking factor but did not impair the cytotoxic activity of already activated cells. Addition of SV40 antiserum to anti-HGG + C-treated cells led to effector cell activation, whereas heterologous virus-immune sera did not. Control studies established that the antibody-mediated recovery of cytotoxicity was not due to arming. Further studies showed that PARA-7 tumor antigen extract blocked at the effector cell level, not at the target cell level. Addition of PARA-7 extract to spleen cell supernatants mediating ADCC resulted in formation of a factor which blocked at the target cell level but not at the effector cell level. These data are compatible with the following interpretation. Spleen cell unresponsiveness is due to antigen blockade. Recovery of cytotoxicity occurs because antibody synthesized during the incubation period promotes elution of antigen from the effector cell surface. Thus, activation is accompanied by the generation of tumor antigen-antibody complexes.
PARA-7 membrane-associated antigen was prepared by treatment of the tumor cells with pH 9.4 glycine buffer, or by concentration of spent cell culture medium. When admixed with sensitized effector cells, both preparations could specifically block cellular cytotoxicity for PARA-7 target cells. Pretreatment of target cells with antigen did not result in blocking. Incubation of antigen extract with simian virus 40 (SV40) anti-serum caused neutralization of antibody-dependent cellular cytotoxicity (ADCC) with concomitant formation of a factor which blocked at the target cell level but not at the effector cell level. Serum from tumor-bearing hamsters exhibited blocking characteristics comparable to those of the antigen-SV40 teristics comparable to those of the antigen-SV40 antiserum preparation. Washing experiments indicated that PARA-7 antigen was more efficient than PARA-7 antigen-antibody complexes in blocking cell-mediated immunity in vitro. Material extracted from untransformed hamster embryo fibroblasts either by itself or when admixed with SV40 antiserum exhibited no significant blocking activity. These observations support the concept that loss of serum ADCC during progressive tumor growth is due to immune complex formation.
Sera collected at intervals after PARA-7 tumor-cell inoculation were monitored for blocking, unblocking, and antibody-dependent cellular cytotoxicity (ADCC) using the microcytotoxicity test. Within 7 days after isografting, significant levels of blocking were demonstrated. This activity increased in parallel with tumor growth and reached a maximum at day 21. In contrast, the capacity of the sera to mediate ADCC was maximal at day 7, and no longer detectable at 3 weeks. Unblocking was not demonstrable in tumor-bearer sera. Further studies showed that, when the tumor was exicised, blocking activity fell rapidly while ADCC was elevated. Sera collected 7 days after surgery could neutralize the blocking activity of tumor-bearer sera. Significant levels of unblocking were still evident 10 weeks after surgery while ADCC had generally disappeared after 5 weeks. These findings illustrate the complexity of the hamster's humoral immune response to tumor cells, and suggest that (1) the sequestering of antibody by circulating tumor antigen accounts for the demise of ADCC in tumor-bearer sera and the concomitant appearance of blocking activity; and (2) the serum factors mediating ADCC and unblocking differ.
Spleen cells cytotoxic at high effector cell: target cell ratios specifically enhanced target cell survival at low ratios. Both cytotoxicity and immunostimulation could be specifically blocked by sera from tumor-bearing or virus-sensitized hosts. The results suggested that serum factors participate in the regulation of the cell-mediated immune response.
Serum from hamsters immunized repeatedly with cytomegalovirus (CMV) was able to block spleen-cell cytotoxicity for CMV-infected and CMV-transformed cells. Inhibition was observed regardless of whether spleen cells were obtained from hamsters sensitized to the virus or to isografts of the transformed cells (designated Cx-90-3B, T-2). Such serum did not significantly block effector cell response against transformed cells expressing herpes simplex virus or SV40-associated membrane antigens. The mechanism of blocking was investigated. It was found that the blocking activity could be adsorbed out with Cx-90-3B, T-2 cells but not by untransformed hamster embryo fibroblasts. Cx-90-3B, T-2 target cells treated with serum, and then washed, remained resistant to effector cell attack. On the other hand, if serum-treated attacker cells were washed their cytotoxic activity was not significantly impaired. These results suggest that the blocking factor in the serum is antibody-directed against cytomegalovirus-related membrane antigen. This conclusion is supported by the finding that the serum contained antibody specifically reactive with the transformed cell surface in isotopic antiglobulin tests.
The nature of the host's immune response to isografts of hamster embryo fibroblasts (HEF) transformed by herpes simplex virus type 1 (HSV-1) was investigated by the microcytotoxicity assay. It was found that spleen cells from tumor-bearing hamsters killed homologous tumor cells but not HEF transformed by cytomegalovirus or PARA-(defective SV40)-adenovirus 7 (PARA-7). Cytotoxicity was lost as the tumor increased in size. Spleen cells from animals bearing isografts of HSV type 2 (HSV-2) transformed cells also killed HSV-1 target cells whereas spleen cells from PARA-7 tumor bearers did not. Further studies showed that animals immunized with HSV-1 or HSV-1-infected rabbit kidney cells produced spleen cells specifically cytotoxic for HSV-transformed cells. On the other hand, sera from virus-immunized hosts or tumor bearers had no effect on the cells in the presence of guinea pig complement. However, in blocking experiments such sera could significantly reduce spleen cell cytotoxicity. These experiments established that cells transformed by HSV could elicit a cellular immune response in the syngeneic host, and provided evidence that the immunity was directed against virus-specific antigens on the cell surface.
The role of serum factors in tumor immunity to cells transformed by PARA-(defective SV40)-adenovirus 7 was investigated. It was found that sera from SV40-sensitized hosts did not block the specific cytotoxicity of SV40-sensitized spleen cells for PARA-7 cells. However, such sera could collaborate with nonsensitized spleen cells to produce specific killing. This activity could be absorbed out by PARA-7 cells but not by cells transformed by cytomegalovirus. The activity of sera from hamsters bearing tumor isografts depended upon when, after transplantation, the specimens were obtained. Sera collected greater than or equal to 10 days after grafting completely blocked immune spleen cell cytotoxicity and did not mediate target cell killing in the presence of normal spleen cells. Sera obtained at an earlier time, i.e., 3 to 6 days after transplantation, consistently were active in the antibody-dependent cellular cytotoxicity test and exhibited reduced or no blocking of antibody-independent cellular cytotoxicity. Thus, there appears to be an inverse correlation in the capacity of serum from tumor bearing hosts to block effector cell cytotoxicity and mediate antibody-dependent cellular cytotoxicity.
Evidence is presented that cells transformed by CMV and HSV-1 express virus-associated antigens on the cell surface. The finding that hyper-immune antivirus sera block cellular immunity in vitro may explain why virus-immunized hosts have thus far not been found to reject isografts of these cells.
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A rapid tissue culture assay for detecting the activated metabolites of cyclophosphamide in serum is described. Serum obtained shortly after drug administration was added to microtest wells containing a known number of previously planted target cells. The presence and amount of drug was directly correlated with the degree of inhibition of target cell replication. Either transformed or untransformed cells could be used as the target cells. No inhibition of cell growth was observed if cyclophosphamide was added directly to target cells without prior in vivo activation. The appearance and persistence of activated drug in hamster serum was followed by using in vitro microassay. Cyclophosphamide administered intraperitoneally was metabolized to its active form very rapidly, reaching peak activity in 15 min. After 2 h, activity was no longer detectable. Drug given subcutaneously was activated less rapidly and persisted for a longer period of time. The kinetics of drug activation, as determined by tissue culture assay, appeared to be similar to that obtained by chemical assay for other species.
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