Antibodies to bacterial and tumor-derived antigens in sera from normal guinea pigs.

Antibodies that react with radiolabeled antigens derived from guinea pig line-10 tumor cells and Mycobacterium bovis (BCG) were detected in sera from normal tumor-free strain-2 guinea pigs (NGPS). Binding by NGPS to the two antigens was inhibited by extracts of either line-10 cells or BCG. Binding by NGPS to the line-10 antigen was inhibited by a number of other bacterial extracts. NGPS was tested after absorption with a variety of cells including line-10, line-1, normal guinea pig spleen, normal adult and fetal liver cells. Results indicated that some of the antibodies in NGPS were directed to line-10-specific determinants. The specific stimulating antigen for these antibodies was not identified but because of the antigenic relationship between BCG, line-10 cells and other bacteria, antibodies to line-10-associated antigens might have been induced by exposure to environmental microorganisms.     

Passive transfer of tumor immunity with spleen cells from guinea pigs cured of hepatocarcinoma by nonspecific immunotherapy

Summary The purpose of this study was to evaluate cell-mediated tumor immunity in strain-2 guinea pigs cured of line-10 hepatocarcinoma by oil-in-water emulsions containing phenol-water extracts from either BCG or the Re mutant of Salmonella typhimurium (Re ET) admixed with mycobacteria glycolipid (P3). Treatment with these emulsions produced complete regression of established tumor nodules and prevented the growth of lymph node metastases in 25 of the 28 animals inoculated intradermally (ID) with 10⁶ line-10 cells and given intralesional immunotherapy 6 days later. No tumor regression was observed in animals given phenol-water extracts alone. Spleen cells, taken from guinea pigs cured of line-10 by BCG extract + P3 or Re ET + P3, were tested for their influence on tumor growth by means of an in vivo adoptive neutralization test (Winn test). Cell transfer was accomplished by the subcutanous injection of various concentrations of spleen cells admixed with 10⁵ viable line-10 cells. The results showed that as few as 10⁷ immune spleen cells completely inhibited the growth of 10⁵ tumor cells in 46–54% of the animals. The best tumor growth inhibition (77–85%) was observed in animals given 5 × 10⁷ immune cells admixed with 10⁵ tumor cells. The onset of transferrable tumor immunity was earlier in animals treated with the BCG extract + P3 than in those given the Re ET + P3. However, the duration of detectable tumor immunity was longer in the latter group. In contrast, no inhibition of tumor growth was observed in animals given spleen cells from normal or tumor-bearing guinea pigs. Moreover, spleen cells obtained from guinea pigs immunized with BCG extract + P3 or Re ET + P3 emulsions only and admixed with line-10 cells failed to transfer tumor immunity to normal animals. Thus, results from this study clearly demonstrated that cell-mediated tumor immunity was elicited in animals cured of line-10 tumor with combinations of P3 and phenol-water extracts of either BCG or Re mutant of S. typhimurium and that sensitized spleen cells effectively transferred systemic tumor immunity to normal recipients.     

Naturally soluble tumor antigens from guinea pig hepatomas: isolation and partial characterization.

Naturally soluble tumor antigens were detected in the ascites fluid of guinea pigs bearing an ascites tumor and from exhausted tissue culture media of cultured tumor cells. Two antigenically distinct cell lines of diethylnitrosamine-induced strain-2 guinea pig hepatomas (line-10 and line-1) served as the source of tumor antigens. Tumor antigen activity was detected by four different techniques: immunodiffusion, inhibition of complement-mediated cytotoxicity, inhibition of membrane immunofluorescence, and delayed cutaneous hypersensitivity. With syngeneic tumor-specific antiserum, line-10 guinea pig tumor antigens were detected by immunofluorescence in the concentrated ascites and tissue culture fluids. With a xenogenic antiserum, demonstrated to be tumor specific, line-10 tumor antigens were detected not only in the concentrated ascites and tissue culture fluids but also in two of the partially purified fractions of these fluids. When the line-10 concentrated ascites and its fraction I were subjected to ultracentrifugation at 300,000 x G for 1 hr, the antigen activity was retained in the supernatant and thus by this criterion the tumor antigens detected in these samples are soluble. Immunodiffusion data indicate that more than one antigen is present in the line-10 system since three lines of precipitation were detected when line-10 concentrated ascites was reacted with the line-10 tumor-specific antiserum. In contrast to this, the line-10-concentrated tissue culture fluid displayed only one line of precipitation. Although tumor antigens could not be demonstrated in the other antigenically distinct tumor cell line, line-1, by immunodiffusion or inhibition of membrane immunofluorescence, inhibition of complement-mediated cytotoxicity was able to detect tumor antigens in the line-1 concentrated ascites and tissue culture fluids.     

Characterization of in vitro reactivity by BCG-treated guinea pigs to syngeneic line-10 hepatocarcinoma

Summary The purpose of this study was to characterize in vitro the systemic tumor immunity induced by a BCG-intratumoral injection in line-10 hepatocarcinoma established in the skin of inbred guinea pigs (strain 2). Macrophages from BCG-tumor-cured guinea pigs at effector to target cell ratios of 10:1 and 100:1 were cytotoxic in vitro to line-10 tumor cells, and this cytotoxicity was potentiated by autologous serum. Significant cytotoxicity of lymphocytes from BCG-tumor-cured guinea pigs could only be achieved at ratios of 10,000:1, and no effect of autologous serum could be demonstrated. Lymphocytes from both normal and BCG-tumor-cured (line-10 immune) guinea pigs had a significant cytotoxic effect on the highly antigenic line-1 cells at ratios of 1:10,000. Macrophages from both normal and line-10 immune guinea pigs were cytotoxic to line-1 target cells at ratios of 1:100. With respect to specific cytotoxicity (cytotoxicity above and beyond levels achieved with effector cells from normal animals), the only significant difference was demonstrated when line-10 served as target cells and the effector cells were isolated from BCG-tumor-cured (line-10 immune) guinea pigs. Abbreviations used in this paper: BCG, Bacillus Calmette-Guérin; CMEM, complete minimum essential medium; cpm, counts per minute; HBSS, Hanks' balanced salt solution; i.d. intradermally; i.p., intraperitoneally; PEC, peritoneal exudate cells; SDA, superficial distal axillary; ¹²⁵IdUrd, [¹²⁵I]iododeoxyuridine.     

Suppression of the guinea-pig line-10 hepatocarcinoma by a factor(s) released from line-10 cells

Summary A factor or factors released from line-10 hepatocarcinoma cells during mixing in a fluid medium suppressed tumor growth when injected intradermally (ID) with viable line-10 cells to strain-2 guinea-pigs. No suppressive factor was released from other syngeneic normal adult, fetal, or tumor cells. The mechanism behind the tumor suppression was not determined, although antigens derived from line-10 cells were demonstrated in media containing the suppressive factor(s).     

Major histocompatibility complex class II antigen expression during potentiation of line-10 tumor immunity after intralesional administration of bacillus Calmette-Guérin

Summary Intralesional injection of BCG into an established line-10 hepatocellular carcinoma in the strain-2 guinea pig causes regression of the tumor and induction of line-10 immunity. We found that the animals were already protected for a second challenge with line-10 tumor cells 7 days after BCG treatment. We studied whether this early induction of immunity was correlated with the expression of MHC class II antigens on line-10 tumor cells and was correlated with an increased expression of MHC class II antigens on leukocytes in the primary tumor and in the regional lymph node (Ln. axillaris accessorius). The MHC class II antigens and the leukocyte subpopulations were measured with monoclonal antibodies and flow cytofluorometry. In the draining lymph node the number of nucleated cells increased about 10-fold during the first 5 days after intralesional injection of BCG. At this time the MHC class II antigen expression of these cells was increased from 21%–32% in the naive controls to 39%–53% in animals with BCG-treated tumors. This implies that the number of MHC-class-II-positive cells increased about 20-fold in the draining lymph node. Surprisingly, the increase in percentage of MHC-class-II-antigen-positive cells was mainly due to an increase of IgM-positive B cells from 8%–11% to 22%–41% and an increase of IgG-positive B cells from 7%–27% to 25%–44%. In the tumor, BCG treatment induced a small increase of MHC-class-II-antigen-positive cells from 11%–12% to 15%–20%. Probably this increase came not from tumor cells but mainly from a BCG-induced infiltration of mononuclear cells, as an increase of T cells from 14% to 20%, an increase of macrophages from 8% to 18%, and an increase of B cells from 0 to 6% was observed. We conclude that the potentiation of anti-(line-10 tumor cell) immunity correlated with a 20-fold increase of MHC-class-II-antigen-positive cells in the lymph nodes and a small increase in the number of MHC-class-II-antigen-positive tumor-infiltrating cells.     

Major-histocompatibility-complex-class-II-positive cells and interleukin-2-dependent proliferation of immune T cells are required to reject carcinoma cells in the guinea pig

Summary Tumor immunity induced by bacillus Calmette-Guérin was studied in the line 10 hepatocellular carcinoma (line 10) in the strain-2 guinea pig. Line 10 immunity was investigatedin vitro with a lymphocyte proliferation assay using line 10 tumor protein extracted with 3 M KCl andin vivo by adoptive transfer of line-10-immune spleen cells. Monoclonal antibodies against guinea pig leucocyte markers were used to block functional properties of the immune cells in order to determine which cell types or cell markers are involved in the immune response to the line 10 tumor.In vitro cells from the spleen, peripheral blood and regional lymph node of immune animals reacted with a proliferative response to line 10 protein. This antigen-specific response was caused by T cells and was regulated by major histocompatibility complex (MHC) class II molecules. In blocking experiments it was found that CT5 (anti-PanT), or MSgp4 [anti-(MHC class I antigen)] monoclonal antibodies did not block but some-times stimulated the proliferative response. The effect of H159 (anti-PanT) was irregular, while H155 [anti-(T helper)], and 5C3 [anti-(IL-2 receptor)] monoclonal antibodies blocked the response almost completely. We studied the relevance of the resultsin vitro obtained and found that mAb 5C3 [anti-(IL-2 receptor)] inhibited the adoptive transfer of line 10 immunity, suggesting that the rejection of line 10 cells is caused by a mechanism that is interleukin-2 (IL-2)-dependent. Moreover, complement lysis of MHC-class-II-antigen-positive immune spleen cells inhibited completely the rejection of the line 10 tumor cell challenge in the adoptive-transfer experiments. In conclusion, our data show that MHC class II molecules or cells possessing these molecules are involved in immunity against line 10 tumor cells, as (a) monoclonal antibodies against MHC class II antigens inhibited thein vitro proliferative response of T cells to tumor antigens and (b) removal of MHC-class-II-positive immune spleen cells abrogated the antitumor effect in the adoptive-transfer experiments. Interleukin-2-dependent proliferation of immune T cells is required for the rejection of line 10 tumor cells.     

Tumour rejection after adoptive transfer of line-10-immune spleen cells is mediated by two T cell subpopulations

Summary The growth of line-10 tumours in naive guinea pigs is prevented by adoptive transfer of spleen cells that are hyperimmune to this hepatocellular carcinoma. To study the T cell subpopulations responsible for the adoptive transfer of immunity, various cell populations were removed from immune spleen cells using monoclonal antibodies (mAbs) and magnetic microspheres. Spleen cell subpopulations were identified by mAb after flow cytometry and rosette formation with the magnetic microspheres. mAb CT5 was confirmed to be a pan T cell marker, while the CT6 (anti-T-suppressor/cytotoxic) and CT7 (anti-T-cell) markers were present on two different T cell subpopulations. So our results show that CT7 mAb cannot be used as a pan T cell marker as was published previously. Moreover, the mAb H155 (anti-T-helper/inducer) reacted with the same T cell subpopulation recognized by CT7. So we designated this H155/CT7-positive subpopulation as T helper/inducer cells. Removal of the CT6-, CT7-, or the H155-positive T cells from the immune spleen cells resulted in loss of the in vitro poliferative response to line-10 tumour protein and tuberculin purified protein derivative (PPD). The H155/CT7(anti-T-helper/inducer)-positive spleen cells did not express MHC class II antigens as determined by mAb 25E3. In most experiments, elimination of MHC-class-II-positive cells did not change the in vitro proliferative response to line-10 protein, whereas the response to tuberculin PPD was completely abrogated. Immune spleen cells after depletion of CT6-, CT7- or H155-positive cells, failed to transfer immunity. However, after depletion of MHC-class-II-antigen-positive cells the line-10 immunity was still present, whereas the immune response to tuberculin PPD was lost. In conclusion, our data indicate that immunity to the line-10 tumour is the result of a cooperation between at least two different T cell subpopulations, the T helper/inducer (CT7/H155) cells and the T suppressor/cytotoxic cells (CT6). If this is a common feature, then the therapeutic approach of in vitro expanded TIL cells should take into consideration the requirement of two T cell subsets.     

The efficacy of BCG-induced tumor immunity in guinea pigs with regional and systemic malignancy

Summary It has been previously demonstrated that transplanted syngeneic line-10 hepatocarcinoma established in the skin of inbred guinea pigs (strain 2) regressed and regional lymph node metastases were eliminated after intratumoral injection of viable Mycobacterium bovis strain BCG. During the course of this reaction there is the development of systemic tumor immunity. The purpose of this study was to determine the relative efficacy of the induced tumor immunity to eliminate regional as well as systemic tumor burden. The approach to evaluate the efficacy of BCG-induced systemic tumor immunity in vivo, for regional as well as systemic tumor, was to develop a competition assay using increasing doses of intravascular disseminated line-10 tumor cells in animals with established regional tumors. The results clearly show that the efficacy of intratumoral BCG injection in producing regression of regional tumor is abrogated by initial intravascular doses of 10³–10⁶ line-10 cells. That the vascular systemic tumor burden diminished the effective systemic tumor immunity was demonstrated by the inability of animals with systemic tumor burdens to reject contralateral challenge of line-10 tumor cells. The capability of BCG-treated animals to reject contralateral line-10 challenge was inversely proportional to the initial intravascular tumor dose. Survival studies clearly demonstrate that a significant therapeutic effect could be achieved in guinea pigs with regional skin tumors and limited vascular metastases when the modality of therapy included BCG-intratumoral injection, followed 6 weeks later by surgery of the established skin tumor and regional lymph node. These results suggest that the development of tumor immunity after BCG-intratumoral injection is not impaired by the systemic tumor burden, but rather that it is preempted at distant sites. Abbreviations used in this paper: BCG, Bacillus Calmette-Guérin; i.a., intraarterially; i.d., intradermally; i.v., intravenously; SDA, superficial distal axillary.     

Complete local tumor regression with antibody to fibrin Fragment E.

Rabbit antibody to fibrin fragment E (FFE) was used in an immunotherapy model for the treatment of the line-10 ascites variant of a diethylnitrosamine-induced hepatoma in strain 2 guinea pigs. When 0.75 or 1.0 mg of an IgG preparation containing anti-FFE antibody was injected s.c. 6 and 16 days after the injection of a uniformly lethal dose of line-10 tumor cells, complete regression of the i.d. growing tumor was observed in all 18 strain 2 guinea pigs treated. Thus, this therapy appears to be more effective than any BCG or other immunotherapeutic regimen thus far reported for this tumor. No significant anti-tumor effect was noted when normal rabbit IgG or smaller doses (0.25 or 0.50 mg) of the anti-FFE IgG preparation were used. The injection sites exhibited an inflammatory response for 7 to 10 days characterized by erythema and hemorrhage. Since all animals were treated after the metastatic progression of the tumor is known to frequently occur, the long-term tumor-free survival of these animals as well as their resistance to subsequent tumor challenge indicate that the anti-FFE antibody therapy led to systemic tumor immunity.     

Passive immunization protects guinea pigs from lethal toxoplasma infection

Abstract The cellular and humoral interactions that contribute to protective immunity in toxoplasmosis were studied by adoptive transfer of selective cell populations or immune serum and its fractions into normal syngeneic strain 2 guinea pigs. The results of this study with the RH strain of Toxoplasma gondii confirm and extend the findings of previous studies by showing that the passive transfer of parasite-sensitized T cells or of immune serum from previously infected donors protected recipient guinea pigs against lethal toxoplasmosis. An additional key finding was that similar levels of complete protection against lethal infection occurred in guinea pigs receiving partially purified anti- Toxoplasma immunoglobulins or immune cells that had been enriched for B cells prior to transfer. Cells residing in the spleen, lymph nodes and peritoneal cavity, but not the thymus, were equally effective in conferring immunity to challenged recipients. In addition, cell titration experiments revealed that guinea pigs could survive T. gondii infection by infusing them with as little as 2 × 10⁷ sensitized T cells or B cells. Unlike protection mediated by T cells, protection against lethal disease occurring in the B cell recipients was associated with the formation of Toxoplasma antibodies. These findings illustrate the major role of both humoral and cell-mediated immunity in affording protection against toxoplasmosis based on a guinea pig model of the human disease.     

Tumor-specific immunity induced by somatic hybrids. II. Elicitation of enhanced immunity against the parent plasmacytoma.

Hybrid cells derived from fusion of a BALB/c plasmacytoma (TEPC-15) and L cells (C3H origin) were used to stimulate tumor-specific immunity against the parental plasmacytoma cells. Live hybrid cells induced tumor-specific immunity against TEPC-15 more effectively than mitomycin-treated hybrid or TEPC-15 tumor cells. Adoptive transfer of immunity with spleen cells of mice immunized with hybrid cells was also more effective than that with mitomycin-treated tumor cells. The immunity induced by the hybrid cells was specific to the TEPC-15 tumor because the mice that received immune spleen cells were not protected against challenge with either HOPC-8 or McPC-603 plasmacytomas. T cell populations were primarily responsible for the transfer of specific immunity based on the sensitivity of immune cells to anti-Thy 1.2 and complement. Mice that had established solid tumors were treated with 5 x 10(7) spleen cells to evaluate the therapeutic value of the hybrid-induced immune cells. Tumors in the mice that received immune cells gradually regressed over a 40-day period, whereas tumors on the control mice continued to grow. These results suggest that a rearrangement of tumor-specific antigens on allogeneic hybrid cells can enhance their immunogenicity.     

Pulmonary metastases in guinea pigs as a consequence of dermal implantation of line-10 tumor cells

Summary Metastases to the lungs of guinea pigs occurred at high frequency as a consequence of intradermal implantation of tumor cells derived from the syngeneic hepatocellular carcinoma line-10. Surgery had a major influence on the proportion of guinea pigs found to have pulmonary metastases at necropsy. Without surgery all guinea pigs died with extensive lymph node metastases; macroscopic pulmonary metastases were present in a minority of the animals. Animals treated by excision of dermal tumors survived longer than untreated animals, and macroscopic pulmonary metastases were present in the majority of the animals. Animals treated by excision of dermal tumor and regional lymph nodes were rendered tumor-free. The data suggest that lymph node metastases were the most likely source of the tumor cells that spread to the lungs in animals from whom the dermal tumor transplant had been removed.     

Heterogeneity of primary and metastatic human malignant melanoma as detected with monoclonal mntibodies in cryostat sections of biopsies

Summary Monoclonal antibodies were generated against established melanoma cell lines and characterized by their reactivity with various sublines. The antibodies selected for their reaction with melanoma-associated antigens were tested on cryostat sections of melanoma tissue from various stages and on other tumors. The reactivity with normal tissues was also determined. Of 30 antibodies reacting with melanoma cell lines 11 did not react with melanoma biopsies. Of the remaining 19 antibodies nine displayed broad cross-reactivity with normal cells and structures and other benign or malignant tumor cells. Among the remaining antibodies five types were defined that detected antigens (nevocellular I, nevocellular II, neural, endothelial, basal cell) found on certain normal tissues and structures and on certain tumor phenotypes. Even though there seems to be a tendency for some antigens to be preferentially associated with certain stages of melanoma, it has not yet been possible to establish any clear-cut correlation between the expression of one of the differentiation antigens and a particular stage or malignancy potential of melanoma.     

Tumor-associated antigens of rat moloney sarcoma cells. I. Cell-surface antigens.

The dissociated cell surface membranes of a rat Moloney sarcoma (MST), derived from a BN rat, were extracted with 2 M KI, with 6 M guanidine thiocyanate, or by papain digestion. Extracts obtained with these three reagents were fractionated on columns of controlled-pore glass, 170 A pore size. A fraction was eluted from each preparation that contained tumor-associated antigens (TAA), viral, fetal, and histocompatibility antigens. With an antibody specific for TAA, the TAA, devoid of detectable viral, fetal, and histocompatibility antigens, were co-precipitated by addition of goat antibody to rat immunoglobulin. After electrophoresis, on slab gels, three bands were detected with estimated m.w. of 185,000, 150,000, and 70,000. No such bands were detected on slab gel electrophoresis with extracts of BC5, a chemically induced tumor, of normal BN lymphoid cells, of M-MuLV, or of fetuses, after incubation with anti-TAA antibody and goat antibody to rat immunoglobulin. TAA extracts prepared with 2 M KI, 6 M guanidine thiocyanate, or papain digestion showed immunologic reactivity. Cold TAA inhibited the co-precipitation of labeled TAA by rat antibody specific for TAA; they elicited antibody in guinea pigs but not in rats; and antibodies specific for TAA were cytotoxic to MST in the presence of C.     

In vitro studies on transplantation immunity. II. The migration inhibition in homograft reactions in guinea pigs

The migration of peritoneal exudate cells from guinea pigs exhibiting transplantation immunity is inhibited in the presence of donor antigens. This inhibition of migration is demonstrable whether the donor transplantation antigens are presented in the form of viable cells (peritoneal exudate cells) or as particulate subcellular antigens (spleen microsomes). A greater degree of inhibition was observed when transplantation immunity was induced with lymphoid cells in Freud's adjuvant compared to sensitization with orthotopic skin grafts. There was no inhibition of migration in mixtures of normal allogeneic cells or when peritoneal cells from guinea pigs exhibiting tuberculin hypersensitivity were mixed with similar cells from normal animals. Finally, supernatants from cultures of sensitive lymphocytes plus donor antigens inhibited the migration of normal peritoneal cells indicating the presence of migration inhibitory factor (MIF) activity.     

Lysis of tumor cells by antibody and complement. VI. Enhanced killing of enzyme-pretreated tumor cells.

The ascites form of a chemically induced guinea pig hepatoma, line-10, was resistant to killing in vitro by xenogeneic antibody and guinea pig complement. Pretreatment of line-10 cells with certain proteolytic enzymes rendered tham susceptible to the killing action of antibody and guinea pig complement. The effects of enzyme pretreatment were dependent on enzyme concentration, temperature, and could be blocked by addition of competitive or non-competitive inhibitors. The effect of the enzyme treatment could reversed by incubating the treated cells at 37 degrees C (but not at 0 degrees C), in the absence of the enzyme. Effective enzymes included ficin, bromelain, pronase, elastase, papain, trypsin, collagenase, lipases type I and type VI, and the neuraminidase preparation isolated from Clostridium perfringens. The activity of the lipase preparations and the neuraminidase preparation isolated from Clostridium perfringens appeared to be caused by proteolytic enzyme contamination. Enzyme preparations that proved ineffecitve in rendering the line-10 cells sensitive to killing by antibody and guinea pig complement included DNase, RNase, beta-glucuronidase type 6A or type B10, hyaluronidase type V or type VI, and pectinesterase.     

Identification of a tumor-associated antigen of the guinea pig L2C leukemia by using syngeneic antisera.

Inbred strain 2 guinea pigs immunized with L2C leukemia cells produced antibodies to L2C cells detected by 125I-protein A assay. L2C-associated tumor antigens were reacted with syngeneic antisera and analyzed by immunoprecipitation and SDS-polyacrylamide gel electrophoresis. These sera recognized idiotypic determinants on surface IgM molecules of L2C cells but did not recognize any determinants on normal strain 2 spleen cells. Thus, determinants on IgM molecular act as tumor-associated antigens in the L2C system and can be detected by syngeneic sera.     

Detection of antibodies bound to tumor cell surface antigens with radioiodinated Staphylococcus aureus protein A (SPA)

Summary Staphylococcal protein A (SPA) is known to bind to the Fc portion of certain subclasses of IgG. On the basis of this property, radioiodinated SPA (¹²⁵I-SPA) was used to detect antibodies reacting with surface antigens of tumor cells. Target cells derived from an osteosarcoma growing in C3H/fHeJ mice and antiserum to this tumor prepared in female Hartley guineapigs were used to establish optimum conditions for the assay. Similar optimum conditions were also determined for human melanoma target cells. Target cells were plated at a concentration of either 3×10⁵ cells per well or 1×10⁵ cells per well in Microtest II plates, and allowed to form semiconfluent monolayers for 24–48 h respectively. Target cells thus prepared were treated with antiserum and then with ¹²⁵I-SPA. A minimum of 30 min incubation time was found to be optimal for the antigen-antibody reaction. The quantity of ¹²⁵I-SPA bound to antisera-treated target cells was found to depend on the time of incubation with ¹²⁵I-SPA and on the concentration of SPA used. Longer incubation times and increasing concentrations of SPA resulted in greater amounts of ¹²⁵I-SPA being bound to antiserum treated target cells. This assay was employed for the detection of antibodies in the sera of two melanoma patients and two colon carcinoma patients. The results of absorption analysis suggest that the antibody activity in the sera of the melanoma patients may be of four different specificities: (a) autoantibodies, (b) alloantibodies, (c) antibodies reacting with common, cross-reacting melanoma-associated antigens, and (d) antibodies reacting with unique antigens specific for autologous melanoma cells.     

Novel monoclonal antibodies identify antigenic determinants unique to cellular senescence.

Normal human diploid fibroblasts exhibit a limited lifespan in vitro and are used as a model to study in vivo aging. Monoclonal antibodies were generated against partially purified surface membranes from human diploid fibroblasts at the end of their lifespan (senescent). Three hybridomas were isolated that secreted antibodies reacting to cellular determinants expressed specifically on senescent human fibroblasts of different origin, including neonatal foreskin, embryonic lung, and adult skin punch biopsy, but not expressed on matched young cells. The antibodies did not bind to immortal human cells and normal young cells made reversibly nondividing, indicating the antigens are not expressed in cells that are not senescent. The antibodies identified senescent cells in a mixed cell population and expression of the senescent cell antigens correlated strongly with the cells inability to synthesize DNA at the onset of senescence. The antigens appeared to be cell surface or extracellular matrix associated, and the epitopes were destroyed by mild trypsin treatment. Western analysis indicated all three antibodies reacted with fibronectin. Though the antigenic determinants on the fibronectin molecule were not accessible in the intact young cell, the epitopes were present in fibronectin extracted from both senescent and young cells, as well as purified human plasma fibronectin. These antibodies and the senescent specific expression of the antigens provide powerful tools to investigate the mechanisms leading to in vitro senescence. This may enable us to investigate directly the relationship between cellular aging and aging of the individual.