Properties of syngeneic and allogeneic antisera raised to tumor-specific suppressor factor from DBA/2J mice

Summary Tumor-specific suppressor factor was prepared by injecting soluble membrane extracts of the syngeneic mastocytoma, P815, into DBA/2J mice 4 days prior to sacrifice. The suppressor factor was purified by passage of spleen extracts over an immunoadsorbent containing P815 membrane components. Antisera raised in syngeneic and allogeneic (C57Bl/6) mice by repeated injections of suppressor factor were tested. It was found that these antisera, but not their controls, were capable of absorbing out the suppressor factor. The antisera were also capable, in the presence of complement, of eliminating suppressor cells from suppressive spleen cell populations. However, the antisera were not capable of eliminating syngeneic tumor-specific in vitro-generated killer cells, indicating that the receptor molecules on suppressor and effector cells in this system are distinct from each other.     

Further characterization of thymic suppressor cells and a factor that suppress the generation of cells cytotoxic for a syngeneic tumor in DBA/2 mice.

Antigen-specific suppressor cells and suppressive extracts obtained from the thymuses of DBA/2 mice bearing small syngeneic P815 mastocytomas were compared for their immunogenetic properties and requirements. The assay for specific suppression involved the ability of either cells or extracts to inhibit the primary in vitro cytotoxic response of normal DBA/2 splenocytes to mitomycin-treated P815 cells. It was shown that pretreatment of suooressor cell populations with anti-Iad antiserum plus rabbit complement removed the suppressive activity. Similarly, absorption of the suppressor factor with anti-Iad antiserum removed the suppressive properties of the material. It was found that the suppressor cells, generated in DBA/2 tumor bearers, were capable of specifically suppressing the anti-P815 response of B6D2 F1 radiation chimeras possessing lymphoid cells of the H-2b or H-2t2 haplotype equally as well as they could suppress the response of H-2d-bearing effector cells. This indicates that the suppressor cells are not H-2 restricted with respect to K or D markers on the responder cells in this system.     

Antigen-specific suppression of the in vitro cytotoxic response by a soluble factor produced by Corynebacterium parvum-induced allospecific suppressor cells

The adjuvant Corynebacterium parvum, when administered intravenously during an ongoing alloimmunization, induces alloantigen-specific splenic suppressor cells which inhibit primary and secondary in vitro sensitizations. We have previously shown that these cells produce a soluble suppressor factor in culture. We now further characterize this factor and its mechanism of action. Release of this suppressive factor is dependent upon specific restimulation of the splenic suppressor cell with the sensitizing alloantigen for 24-48 hr in culture. The suppressor factor inhibits primary, but not secondary, in vitro sensitizations in an antigen-specific, genetically unrestricted manner. The suppressive activity is not absorbed by passage through immunoadsorbent columns containing anti-mouse immunoglobulin. The factor does not lyse tumor cells bearing the sensitizing alloantigen. Delay in addition to primary cultures of as little as 4 hr after culture initiation leads to loss of suppressive activity, suggesting that this antigen-specific allosuppressor factor inhibits an early step in the sensitization of precursor cytotoxic T lymphocytes.     

Immunization of DBA/2 mice with a T cell hybridoma-derived TsF increases immune resistance to the syngeneic tumors P815 and L1210

The ability of a tumor-specific T suppressor factor (TsF) isolated from a T cell hybridoma, A10, to act as an immunogen in DBA/2 mice was investigated. The TsF was affinity purified from ascites over an immunoadsorbent column containing a monoclonal antibody (B16G) that has specificity for the TsF molecule, or over columns containing membrane extracts of the P815 mastocytoma (the tumor for which A10 is specific). The specificity control was BW5147 (the fusion partner for A10) membrane extracts treated in the same way as A10. DBA/2 mice were immunized with the affinity-purified material or PBS and were subsequently challenged with either the P815 tumor or the L1210 DBA/2 thymoma. When mice were immunized with material affinity purified over B16G, eluted material from both A10 ascites and BW5147 membrane extracts enhanced resistance to both P815 and L1210 challenge, indicating that B16G was binding immunogenic material derived from both preparations, which exerted a tumor-protective effect. However, when a P815 affinity column was used, protective material was eluted only from A10 ascites, and this bestowed resistance to both P815 and L1210. When irradiated whole cells were used as immunogens, only A10 cells stimulated anti-tumor immunity, and this appeared to be directed specifically to the P815 tumor. The implications of these findings in terms of the potential for immune modulation with anti-suppressor therapy, and the specificity of the B16G monoclonal, are discussed. The demonstration of B16G binding material (TsF) in the membranes (but not the ascites) of the BW5147 line is also of significance to investigators using BW5147 fused suppressor hybridomas.     

Isolation and characterization of a tumor-specific T suppressor factor from a T cell hybridoma

In our laboratory we have described a monoclonal antibody, B16G, which has been shown to bind to suppressive T cell factors (TsF) in DBA/2 mice. Therefore, B16G was used as a probe to identify T cell hybridomas secreting putative TsF. Hybridomas were obtained by the fusion of DBA/2 thymocytes stimulated in vivo by P815 tumor membrane extracts with the thymoma BW5147. One such hybridoma, A10, was selected and used for additional studies. From both the supernatants and ascites fluid of this hybrid a factor could be obtained that could specifically bind to both B16G and P815 antigen immunoadsorbent columns, and that scored positively with B16G in an ELISA after elution. Such reactivity could not be obtained from A10 supernatants or ascites absorbed over irrelevant columns, nor was it obtained from supernatants or ascites from other T cell hybrids that had scored B16G nonreactive in the original screening. In vivo studies indicated that affinity-purified A10 material injected into DBA/2J mice enhanced significantly the growth of P815 tumor cells, but not the growth of other DBA/2 syngeneic tumor lines such as L1210 or M-I. Additionally, this material did not inhibit the in vitro mixed leukocyte reaction (MLR) between DBA/2 splenocytes and allogeneic B10.BR target cells (unlike B16G purified material from whole DBA/2 spleens, which has been demonstrated to be suppressive in this type of MLR). Biochemical analysis of this tumor-specific TsF from A10 was undertaken; the native m.w. was found to be in the region of 80,000 and 90,000. Under reducing conditions, affinity-purified A10 TsF was found to resolve in SDS-PAGE as what appeared to be a heterodimer of 45,000 and 43,000. In most preparations, an associated molecule resolving at about 25,000 was observed. The implications of these observations are discussed.     

Regulation of the immune response to tumor antigen. IV. Tumor antigen-specific suppressor factor(s) bear I-J determinants and induce suppressor T cells in vivo.

The nature and function of suppressor factor(s) elaborated by suppressor T cells in response to certain chemically induced tumors have been further defined. Thus, suppressor factor(s) specific for the S1509a methylchol-anthrene-induced fibrosarcoma have been shown to bear determinants encoded by the I-J subregion of the murine MHC since suppressive activity is removed by passage of the factor through an immunoadsorbent composed of anti-I-Jk coupled to Sepharose. No loss of activity was observed after passage of factor through control columns composed of normal mouse globulin. Furthermore, activity could be recovered from the relevant immunoadsorbent by elution with high salt. The administration of crude suppressor factor(s) to normal animals for 4 days resulted in the development of a population of suppressor cells that act in a manner analogous to the suppressor cell population used for production of factor. These factor-induced suppressor cells are T cells and exhibit an antigen specificity similar to that displayed by the tumor-induced suppressor cells. Thus, tumor-specific suppressor factor(s) bear I-J determinants and are capable of inducing the appearance of suppressor T cells in the nontumor-bearing host, which may then act in a specific manner to limit host responsiveness to tumor antigen.     

Affinity chromatography of terminal deoxynucleotidyl tranferase from calf thymus and human leukemic cells on a solid-phase immunoadsorbent

A solid-phase immunoadsorbent specific for terminal deoxynucleotidyl transferase has been prepared. The enzyme from calf thymus and acute lymphoblastic leukemia cells binds to columns of this material. Bound enzyme can be eluted in an active form. Selective and rapid purification of terminal deoxynucleotidyl transferase from crude extracts of cells containing this enzyme can be achieved by this method since the immunoadsorbent has no affinity for other cellular DNA polymerases.     

Immunologic tolerance to HGG in mice. I. Suppression of the HGG response in normal mice with spleen cells or a spleen cell lysate from tolerant mice.

Adoptive transfer of spleen cells or spleen cell lysates from mice tolerant to human-gamma-globulin (HGG) specifically suppressed the response of normal syngeneic recipients to HGG. The suppressive activity could be transferred for over 100 days after tolerance induction. The suppression induced by both spleen cells and spleen cell lysate was found to be specific as evidenced by a normal response to a challenge with turkey-gamma-globulin or goat erythrocytes. The activity of the suppressive lysate could be removed by passing the material through an HGG immunoadsorbent column but not by passing it through an anti-HGG column or a BSA column. These results indicated that the factor had antigen specificity and was probably not antigen-antibody complexes. That this suppression was not due to a shifting of the kinetics of the antibody response has also been demonstrated. The antigen-specific suppressor factor in the tolerant spleen cell lysates was a protein with a m.w. of approximately 45,000 daltons. The kinetics of the appearance of both suppressor cells and suppressor factor were consistent with a mechanism of active suppression functioning in the maintenance of tolerance to HGG.     

Suppression of growth of a mouse plasmacytoma by the IgG2 fraction of syngeneic antitumor globulin

Summary The effects of syngeneic antitumor antibody on transplanted plasmacytoma cells have been examined. Globulin was prepared from ascites fluids produced in Balb/c mice injected with MOPC 315 tumor cells and bearing the solid tumor. Normal Balb/c mice were given inoculations of tumor cells that had been incubated with the antitumor globulin obtained at various intervals after immunization, or with portions of such globulins. These materials were prepared to express the IgG2 class antibody by three procedures: precipitation with heterologous anti-mouse IgG1, passage through columns of Sepharose anti-IgG1, or adsorption to and elution from heat- and formalin-killed protein A-bearing staphylococci. The original antitumor globulins showed differences with time relative to the second injection of the immunizing tumor, in that a number of the earlier pools led to some suppression of tumor growth, and a number of the later pools led to some enhancement. Of the globulins obtained later, preparations expressing the IgG2 class of antibody by precipitation with anti-IgG1 serum caused some suppression of tumor growth. Pronounced suppression of growth was consistently obtained with anti-MOPC 315 globulin freed of IgG1 by passing it through an anti-IgG1 immunoadsorbent, and with IgG2a preparations obtained by elution from the protein A-bearing staphylococci. Of the anti-IgG1 column-treated preparations, the suppressive effect was maximal in globulin obtained 15–26 days after the second immunization. The suppressive effect of these preparations could be removed by absorption with MOPC 315 cells, but not by cells of another Balb/c tumor nor by two other plasmacytomas.     

CTP: CMP phosphotransferase activity in L1210 cells

Substrate converting enzymes interfering with the measurement of ribonucleotide reductase were assessed in cell-free extracts prepared from L1210 cells. Data show the presence of a myokinase-type enzyme activity (CTP:CMP phosphotransferase) which catalyzes the reaction: 2CDP in equilibrium CMP + CTP. This enzyme is not removed by passage of cell extracts over ATP-agarose columns. Monitoring of nucleoside diphosphate substrate level is, therefore, mandatory for obtaining accurate measurements of CDP reductase activity in crude cell extracts.     

Characterization of suppressor cells in mice bearing syngeneic mastocytoma.

Suppressor cells from syngeneic P815 mastocytoma-bearing DBA/2 mice that inhibit in vitro generation of specific anti-tumor cytotoxicity were characterized. Suppressive activity was almost completely eliminated by treating suppressive spleen cells with anti-theta serum and complement. Treatment with anti-mouse lg serum and complement or with carbonyl iron did not affect their suppressive activity. When suppressive thymocytes from P815 tumor-bearing DBA/2 mice were tested for their capacity to inhibit the generation of cytotoxicity against L1210 cells, a leukemia line in DBa/2 mice, they did not affect the activity, indicating that the supressor cells in the thymocytes of P815 tumor-bearing mice are specific to the tumor. When Ficoll-Hypaque density cell separation was carried out with cytotoxic spleen cells and suppressive spleen cells from 815 tumor-bearing mice, the dense fraction was enriched for kiler cells whereas the suppressive activitty was mainly recovered in the light fraction. Therefore, killer cells and suppressor cells in P815 tumor-bearing mice are thought to be distinct populations.     

Macrophages suppress CTL generation in rat mixed leukocyte cultures.

The generation of CTL in rat MLC was actively suppressed by a cell population present in spleen cell preparations from normal rats. These suppressor cells were characterized by using a variety of cell fractionation techniques. Suppressor cells were removed by passage of spleen cells through nylon wool columns or by treatment with carbonyl iron. Suppressive activity was present in the mononuclear cell fraction of spleen cells obtained by Ficoll-Hypaque density gradient centrifugation. After velocity sedimentation at unit gravity, enrichment of suppressive activity was demonstrated in the fractions containing large cells as compared to the fractions containing small cells. Populations rich in macrophages were shown to have similar suppressive activity upon CTL induction in MLC. These studies suggest that macrophages present in normal rat spleen cell preparations account for the difficulty in generating CTL in MLC prepared with rat cells.     

Multiple biotin-containing proteins in 3T3-L1 cells.

Extracts of 3T3-L1 cells prepared after labelling the monolayer cultures with [3H]biotin contained numerous protein bands that were detected by fluorography of dried SDS/polyacrylamide electrophoresis gels. All labelled proteins in the extracts could be removed by avidin affinity chromatography. The biotin-containing subunits of acetyl-CoA carboxylase, pyruvate carboxylase, methylcrotonyl-CoA carboxylase and propionyl-CoA carboxylase, with molecular masses of approx. 220, 120, 75 and 72 kDa respectively, were detected together with minor bands at 100, 85 and 37 kDa that did not appear to be partial degradation products. Additional labelled bands increased in amount during incubation of cell extracts or did not occur in extracts prepared with trichloroacetic acid, 9.5 M-urea or proteolytic inhibitors, and were tentatively classified as partial degradation products. The unknown bands were not removed by incubation of cell monolayers for 24 h, a treatment that gave degradation rate constants of 0.47 day-1 for acetyl-CoA carboxylase and 0.28 day-1 for pyruvate carboxylase. Upon two-dimensional electrophoresis, pyruvate carboxylase, methylcrotonyl-CoA carboxylase and propionyl-CoA carboxylase had isoelectric points of 6.4, 7.2 and 6.4 respectively. Several additional discrete spots with isoelectric points below 6.2 were also present. All the unknown biotin-containing proteins banded with intact mitochondria during density-gradient centrifugation. We conclude that several unknown biotin-containing proteins are present in the mitochondria of 3T3-L1 cells, whereas others are partial breakdown products of mitochondrial proteolysis.     

Specific suppression of the immune response by a factor obtained from spleen cells of mice tolerant to human gamma-globulin.

An antigen-specific suppressive factor was extracted from spleen cells of mice made tolerant by injection of deaggregated human gamma-globulin (HGG). The active material could be prepared from T cells, obtained by passaging spleen cells through an anti-immunoglobulin column, although not from cells adherent to the column nor from spleen cells pretreated with anti-Thy-1 serum and C. This factor was antigen-specific since it was retained on immunoadsorbents containing HGG, but not on columns coated with antibody to HGG or with irrelevant antigens. Despite its specificity for antigen it did not bear any classical immunoglobulin determinants. Its m.w. ranged between 30,000 and 55,000 daltons. It was a product of the I region of the major histocompatibility complex since it carried Ia determinants. The properties of this factor are very similar to those reported elsewhere for suppressive factors obtained from primed T cells, cells from nonresponder mice, and allotype-specific cells. This suggest the existence of a major class of immunoregulatory molecules, nonimmunoglobulin in nature, and responsible for the mediation of antigen-specific T cell-dependent suppression.     

Tumor-associated blocking factors: isolation from sera of tumor-bearing mice.

An isolation and partial purifications of tumor-associated blocking factors from the sera of tumor-bearing mice is described. Columns for affinity chromatography were prepared by coupling syngeneic tumor-immune antibodies to Sepharose 4B. Passage of serum through such immunoadsorbent columns removed all blocking activity from tumor-bearers' sera; subsequent elution of the absorbent with 3 M NaSCN recovered the activity. The blocking material was further purified on Sephadex G-200. The data provide evidence for the presence of antigen in tumor-associated blocking factors and are compatible with the hypothesis that blocking factors often consist of antigen and antibodies in the form of immune complexes.     

Studies on vacuole regeneration in evacuolated tobacco mesophyll protoplasts. Protein analysis by one- and two-dimensional microgel-electrophoresis

Evacuolated protoplasts, P(–), have been prepared from mesophyll protoplasts. P(+), of Nicotiana tabacum L. (cv. Samsun) by centrifugation in an iso-osmotic Percoil gradient. In comparative analyses performed with these two types of cells, vacuole proteins should appear in P(+) extracts, but be absent from freshly isolated P(–). Such differences were detectable in total protein extracts after two-dimensional electrophoresis. Four spots were located that are likely to represent soluble vacuolar proteins. They have low molecular mass (around 20 kDa) and slightly acidic isoelectric points. In culture, evacuolated tobacco protoplasts regenerated a vacuole de novo. The vacuolation process as observed microscopically correlated welt with the reappearance of vacuolar marker enzymes. Likewise, the protein spots that were missing in the P(–)-pattern immediately after evacuolation reappeared within the first days of protoplast culture. By immunoblotting the same behaviour was demonstrated for the well-known vacuolar protein, tonoplast ATPase.     

Ultraviolet-induced suppressor T cells and factor(s) in murine contact photosensitivity. I. Biological and immunochemical characterization of factor(s) extracted from suppressor T cells

Murine contact photosensitivity (CPS) to 3,3',4',5-tetrachlorosalicylanilide (TCSA) is a highly specific, T-cell-mediated delayed-type hypersensitivity (DTH). Preexposure of the photosensitizing site to low doses of ultraviolet B(UVB) rendered mice unresponsive to challenge reaction. This unresponsiveness was associated with the generation of antigen-specific, afferent limb-acting, Lyt-1+2-,L3T4+ suppressor T cells (Ts-cps) in the spleen, thymus, and lymph node. Cell-free extract(s) obtained by freezing and thawing of these cells contained T-cell-suppressor factor (TsF) that inhibited the development of the induction phase of the CPS response to TCSA in vivo in an antigen-specific fashion. The treatments of TsF both with immunoadsorbent columns and with reduction and alkylation showed that the factor bore photoantigen-binding site(s), was reactive with monoclonal anti-I-Jd, anti-I-E alpha but not anti-I-Ad, and behaved as a single-chain factor containing both photoantigen binding and I-J molecules. By gel chromatography the majority of the suppressive activity was eluted in the fractions corresponding to molecular weights of 60-80 and 100-200 kDa. Our present study demonstrated clearly that UVB-induced unresponsiveness in the DTH reaction was mediated by a soluble suppressive factor derived from T cells.     

Stimulatory and inhibitory effects of extracts from mammalian cell-cycle mutants on DNA replication in partially lysed cells

Heat-sensitive (arrested at 39.5 degrees C, multiplying at 33 degrees C) and cold-sensitive (arrested at 33 degrees C, multiplying at 39.5 degrees C) cell-cycle mutants of the P-815-X2 murine mastocytoma line were used for the preparation of cell extracts. These were tested for their effects on DNA synthesis in 'gently lysed cells' (obtained by treatment with 0.01% Brij-58) or 'highly lysed cells' (obtained by treatment with 0.1% Brij-58). Gently lysed cells prepared from proliferating P-815-X2 or mutant cells incorporated [3H]dTTP efficiently, while highly lysed cells exhibited a low level of [3H]dTTP incorporation which was markedly increased by the addition of extracts from proliferating cells. Extracts prepared from arrested mutant cells, however, were found to inhibit DNA synthesis by gently and highly lysed cells prepared from proliferating cells. After return of arrested mutant cells to the permissive temperature, stimulating activity in cell extract reappeared at the time of reentry of cells into S phase. Both stimulatory and inhibitory activities were associated with material(s) of molecular weight above 25 000, but differed in heat sensitivity and in sensitivity to immobilized proteinase and ribonuclease. Extracts from arrested cells counteracted the stimulating effects of extracts from proliferating cells with kinetics suggesting competitive interaction between stimulating and inhibitory factors.     

Regulation of naturally occurring autoantibody-producing cells: detection of a suppressive substance which inhibits the secretion of antibodies directed against enzyme-treated isologous erythrocytes

Normal mice possess spleen cells capable of forming hemolytic plaques against bromelain-treated autologous red cells (Br MRBC). There is present in the serum of these same mice a substance which can inhibit the formation of these plaques. This substance is inhibitory to the secretion of these antibodies following incubation of spleen cells in 20% serum at 4 degrees C for 5 min. This substance is not inhibitory to the formation of anti-sheep erythrocyte plaques from mice either immunized or nonimmunized with sheep erythrocytes. Characterization of the substance indicates that it is neither soluble antigen nor specific antibody. However, inclusion of nanogram amounts of soluble antigen from bromelain-treated red cells in the assay mixture effectively neutralized the suppression. In addition, passage of serum through a mouse anti-Br MRBC antibody immunoadsorbent effectively removed the suppressive activity of the serum while suppression could be recovered in the acid eluate from such a column. This suggests that the mechanism of suppression brought about by incubation in serum is due to the action of a molecule possessing anti-idiotypic activity directed against the cell surface receptors of anti-Br MRBC B cells. Attempts to isolate the molecule based on the postulate that it is immunoglobulin in nature have been unsuccessful.     

Stimulatory and inhibitory effects of extracts from mammalian cell-cycle mutants on DNA replication in partially lysed cells

Heat-sensitive (arrested at 39.5°C, multiplying at 33°C) and cold-sensitive (arrested at 33°C, multiplying at 39.5°C) cell-cycle mutants of the P-815-X2 murine mastocytoma line were used for the preparation of cell extracts. These were tested for their effects on DNA synthesis in ‘gently lysed cells’ (obtained by treatment with 0.01% Brij-58) or ‘highly lysed cells’ (obtained by treatment with 0.1% Brij-58). Gently lysed cells prepared from proliferating P-815-X2 or mutant cells incorporated [³H]dTTP efficiently, while highly lysed cells exhibited a low level of [³H]dTTP incorporation which was markedly increased by the addition of extracts from proliferating cells. Extracts prepared from arrested mutant cells, however, were found to inhibit DNA synthesis by gently and highly lysed cells prepared from proliferating cells. After return of arrested mutant cells to the permissive temperature, stimulating activity in cell extracts reappeared at the time of reentry of cells into S phase. Both stimulatory and inhibitory activities were associated with material(s) of molecular weight above 25 000, but differed in heat sensitivity and in sensitivity to immobilized proteinase and ribonuclease. Extracts from arrested cells counteracted the stimulating effects of extracts from proliferating cells with kinetics suggesting competitive interaction between stimulating and inhibitory factors.