Tumor-bearer Spleen Research Articles

IT-01 * PD1/PDL1 INTERACTIONS MEDIATE SUPPRESSION OF ANTI-TUMOR IMMUNE ACTIVITY IN GLIOMA

Recent work has demonstrated that the interaction of the Programed Death-1 (PD1) negative costimulatory molecule with its ligand, PDL1 in the tumor microenvironment, plays an immunoregulatory role that may prevent the development of a successful anti-tumor response. We hypothesized that dendritic cell vaccination (DCVax) would induce a compensatory upregulation of PDL1 within the tumor microenvironment as a result of increased tumor infiltrating lymphocytes (TILs). We also hypothesized that simultaneous blockade of the PD1/ PDL1 interaction would promote a successful antitumor response. To test this, immunocompetent C57BL/6 mice were implanted with GL261 glioma cells in the brain. Mice were then stratified into 4 treatment groups (Tx on days 3 and 13 post-implant): (1) Control; (2) IP murine anti-PD1 mAb (10mg/kg) only; (3) subcutaneous GL261 lysate-pulsed DCVax only; and (4) combination (anti-PD1 mAb plus DCVax). Overall survival was quantified. On post-implant days 15 and 20, mice were imaged using a Bruker 7T MR scanner to obtain anatomical, perfusion, and pH-weighted data. On post-implant days 16 and 21, mice were imaged with [18F]-FAC PET to evaluate the lymphocyte response. Tumor-bearing hemisphere, spleen, and lymph node (LN) leukocytes were then harvested, processed, and stained for FACS analysis. Median survival of the combination group was significantly greater than the DCVax or PD-1 treatment groups (p < 0.005). MRI showed decreased tumor volume and inflammation in the combination group. FACS analysis and [18F]-PET demonstrated significantly enhanced numbers of activated lymphocytes accumulating within the cervical LN and tumor in PD-1 mAb, DCVax, and combination treatment groups. Elevated PDL1 expression on tumor-associated APCs was also noted in the DCVax group, but specifically lower in the combination group. We propose that PDL1 is upregulated within the tumor in response to immunotherapy, but can be blocked to restore effective anti-tumor immune responses.

Transforming growth factor-beta-mediated down-regulation of antitumor cytotoxicity of spleen cells from MOPC-315 tumor-bearing mice engaged in tumor eradication following low-dose melphalan therapy.

We have previously shown that treatment of mice bearing a large MOPC-315 plasmacytoma with a low dose of the anticancer drug melphalan (L-phenylalanine mustard; L-PAM) results in the acquisition of a potent CD8+ T-cell-mediated anti-MOPC-315 cytotoxic T lymphocyte (CTL) activity by the hitherto immunosuppressed tumor bearers, and this immunity contributes to complete tumor eradication. In the studies presented here, we sought to determine how the acquisition of this antitumor immunity following low-dose chemotherapy is possible, in light of the report that MOPC-315 tumor cells produce transforming growth factor-beta (TGF-beta), an immunosuppressive cytokine that can down-regulate the generation of CTL responses. We found that the acquisition of CTL activity following low-dose L-PAM therapy is not due to a chemotherapy-induced decrease in the sensitivity of MOPC-315 tumor bearer spleen cells to TGF-beta-mediated inhibition of CTL generation. Moreover, even spleen cells from MOPC-315 tumor-bearing mice, which had received L-PAM therapy 7 days earlier and had acquired CTL activity in vivo, were sensitive to the inhibitory activity of TGF-beta upon culture for as little as 1 day, with or without stimulator tumor cells. However, the production of TGF-beta by MOPC-315 tumors decreased drastically as a consequence of the low-dose chemotherapy. Thus, the curative effectiveness of low-dose L-PAM therapy for MOPC-315 tumor-bearing mice may be due, at least in part, to a reduction in TGF-beta production that enables the development of tumor-eradicating immunity.

Development of splenic natural suppressor (NS) cells in ehrlich tumor‐bearing mice

Spleen cells from C57BL/6J mice bearing Ehrlich carcinoma growing as a solid tumor show progressive unresponsiveness to concanavalin A (Con A) and lipopolysaccharide (LPS) mitogens. This is accompanied by striking spleen enlargement with marked hematopoietic activity. Lymphoproliferative assays of normal spleen cells in co-culture with tumor-bearing spleen cells (TBSC) show that: (a) TBSC contain non-specific suppressor cells able to abrogate both Con A and LPS responses, or mixed lymphocyte reaction, of normal spleen cells and (b) suppression by TBSC is MHC-unrestricted, non-prostaglandin-mediated and greatly enhanced by Con A supernatants. Suppressor cells associated with TBSC are large, low-density cells without markers of mature B or T lymphocytes or of the mononuclear phagocyte system. Most appear to be asialo-GM1-negative, as suppression was only partially inhibited by treatment with anti-asialo-GM1 and complement. Since NK activity is lacking in TBSC, our data strongly suggest that these "null" suppressor cells are related to the natural suppressor (NS) cells found described in normal bone-marrow and neonatal spleens, or induced in adult spleens by total lymphoid irradiation, graft-vs.-host disease, or cyclophosphamide treatment.

In vivo and in vitro synergistic antitumor effect of interleukin-2-cultured tumor-bearer spleen cells and immune fresh spleen cells.

The synergistic antitumor effect of interleukin-2(IL-2)-cultured tumor-bearer spleen cells (cultured lymphocytes) and immune fresh spleen cells was examined. Tumor-bearer cultured lymphocytes were obtained by culturing BALB/c spleen cells from syngeneic MOPC104E-tumor-bearing mice for 11 days with crude IL-2 and a soluble tumor extract. These cultured lymphocytes had weak antitumor activity when transferred i.p. into tumor-bearing mice that had been inoculated i.p. with 10(5) tumor cells 5 days previously. Immune fresh spleen cells, obtained from mice in complete remission after the treatment with cyclophosphamide, also had weak antitumor activity when transferred at the same schedule. The cultured cells and the fresh cells, mixed together before transfer, significantly augmented the therapeutic effect. At least 1 x 10(7) tumor-bearer cultured lymphocytes and 4 x 10(7) immune cells were needed for the synergistic effect. A tumor-specific combination was needed for both cultured and fresh cells. The effective subpopulation of tumor-bearer cultured lymphocytes was a cytotoxic one from an Lyt2+ precursor, and that of the immune fresh spleen cells was noncytotoxic, Lyt1+ and Lyt2+ T-cells. A similar synergistic effect was also observed during in vitro coculture of tumor-bearer and immune cells. Cytotoxicity, as assessed by the 51Cr-release test, of tumor-bearer IL-2-cultured lymphocytes was maintained most effectively after 3 or 4 days of culture without IL-2 when the lymphocytes were cocultured with immune fresh spleen cells and tumor cells.

Treatment for unresectable hepatoma via selective hepatic arterial infusion of lymphokine-activated killer cells generated from autologous spleen cells

Nonspecific tumoricidal effectors, called lymphokine-activated killer (LAK) cells, can be induced from the tumor-bearer's spleen in vitro. The adoptive transfer of such LAK cells to a patient with unresectable hepatoma was performed in this study. About 2.4 X 10(9) LAK cells generated from the autologous spleen were adoptively transferred to the patient via hepatic arterial catheter. Signs of toxicity encountered with LAK cell infusions comprised chills and fever only. Chemical studies of hepatic, renal, and hematologic parameters were normal; pulmonary function studies revealed no changes after infusion. With the transfer of LAK cells, serum alpha-fetoprotein (AFP) levels were markedly decreased and ascitic fluid retention was transiently reduced. Though the therapeutic effect was transient, these trials offered hope for a new therapeutic approach to unresectable hepatoma. Further, the availability of large amounts of recombinant interleukin-2 (rIL-2) may now make widespread use of this approach possible.

Macrophage-mediated suppression of natural killer cell activity in mice bearing Lewis lung carcinoma.

The natural killer (NK) cell cytotoxic capacity of C57BL/6 mice with implantation of Lewis lung carcinoma (LLC) was quantitated during tumor growth. The NK activity became suppressed at 1 week of tumor growth and remained suppressed. The mechanisms for the suppression during the first 3 weeks of tumor growth included secretion of prostaglandin E2 (PGE2) by both the LLC tumor and host macrophages. With tumor growth, plasma PGE2 concentrations progressively increased. Oral administration of indomethacin to tumor-bearing mice prevented the rise in serum PGE2 concentrations and the suppression of NK activity. Cultured LLC cells and splenic macrophages isolated from mice during the first 3 weeks of tumor growth secreted increased amounts of PGE2. Macrophages from tumor-bearer spleen cells were shown to suppress NK activity. Depletion of these macrophages restored the NK activity, and addition of these macrophages to normal spleen cells resulted in an indomethacin-sensitive suppression of the NK response. The mechanisms of suppression in mice bearing large tumors were different than those observed with smaller tumors. With a large tumor burden, the plasma PGE2 concentrations declined. Indomethacin treatment did not prevent the suppression of NK activity, and depletion of splenic macrophages did not restore NK cytotoxicity.

The effect of indomethacin on the activation and effector function of suppressor cells from tumor-bearing mice.

The spleens of mice with large M-1 fibrosarcomas contain two populations of suppressor cells with the properties of macrophages and T cells. In this study, we tested the effect of indomethacin on suppressor cell activation and effector function. Neither the activation nor the effector function of the suppressor macrophages was inhibited by indomethacin, and the activity of suppressor macrophages correlated with the tumor size. In contrast, the treatment of tumor-bearing mice with indomethacin from the day of injection of tumor cells completely blocked the in vivo activation of suppressor T cells. Indomethacin did not, however, depress suppressor T cell activity if mice were treated only during the third week of tumor growth. The effector function of the suppressor T cells, as assessed in mixing assays, was partially blocked by indomethacin, while selective suppression by low-molecular-weight factors was completely blocked if indomethacin was present in the cultures. Furthermore, the in vitro activation of suppressor cells by soluble factors secreted by tumor-bearer spleen cells was completely blocked by indomethacin, and this inhibition was reversed by prostaglandin E1. These data are consistent with the hypothesis that prostaglandins are involved in the activation, but not the effector function, of tumor-activated suppressor T cells.

Cyclophosphamide-mediated enhancement of antitumor immune potential of immunosuppressed spleen cells from mice bearing a large MOPC-315 tumor

We have utilized 4-hydroperoxycyclophosphamide (4-HP-CY) as a probe for the immunomodulatory activity of the metabolites of cyclophosphamide (CY) since 4-HP-CY hydrolyzes spontaneously in aqueous solution to the same metabolites as those formed after in vivo conversion of CY by microsomal enzymes. Exposure of immunosuppressed MOPC-315 tumor bearer spleens to a low concentration of 4-HP-CY (0.1–3.0 μm) resulted in augmented antitumor immune potential. The level of antitumor immune potential exhibited by 4-HP-CY-treated tumor bearer spleen cells was not further augmented but was actually reduced by depletion of glass-adherent cells, a procedure which is effective in removing the cells known to have immunosuppressive activity (i.e. metastatic tumor cells and macrophages) from the spleen of untreated MOPC-315 tumor-bearing mice. In fact, 4-HP-CY was superior to depletion of glass-adherent cells in augmenting the antitumor immune potential of immunosuppressed tumor bearer spleen cells. When cells from the primary tumor nodule were incubated with a low concentration of 4-HP-CY which only marginally inhibited their proliferation, the drug completely abolished the suppressive activity of the cells for the in vitvo generation of antitumor cytotoxicity by normal spleen cells. Moreover, a high level of antitumor cytotoxicity developed when normal spleen cells were cultured vitro with 4-HP-CY-treated tumor cells at a wide range of ratios of spleen cells to tumor cells. Thus, in the MOPC-315 tumor model, metabolites of CY eliminate the inhibitory effectiveness of splenic suppressor cells and induce the appearance of immunopotentiating activity. The results obtained with 4-HP-CY in vitro provide support for the hypothesis that low-dose CY therapy of mice bearing a large MOPC-315 tumor leads to the appearance of augmented antitumor immune potential in their hitherto immunosuppressed spleen cells as a result of the in situ immunomodulatory effect of the drug on cells in the spleen.

Melphalan-mediated potentiation of antitumor immune responsiveness of immunosuppressed spleen cells from mice bearing a large MOPC-315 tumor.

Administration of a low dose of L-PAM (0.75 mg/kg) to mice bearing a large SC MOPC-315 tumor and extensive metastases led to the development of augmented antitumor immune potential in their hitherto immunosuppressed spleen cells. Such drug-induced potentiation of antitumor immune responsiveness appeared by day 2 after chemotherapy, and it could not be further enhanced but was actually reduced by depletion of glass-adherent cells, a procedure which is effective in depleting the cells known to have inhibitory activity (i.e., macrophages and metastatic tumor cells). To establish that L-PAM can lead to selective in situ abrogation of the inhibitory effectiveness of the splenic macrophages and metastatic tumor cells, we demonstrated that incubation of immunosuppressed tumor-bearer spleen cells with a low concentration of L-PAM in vitro also resulted in augmented antitumor immune potential that could not be further augmented by depletion of glass-adherent cells. L-PAM-mediated enhancement of the antitumor immune potential of immunosuppressed tumor bearer spleen cells was due at least in part to the effects of the drug on the splenic metastatic tumor cells. Isolated tumor cells treated with a low concentration of L-PAM were not only devoid of inhibitory activity for the primary in vitro antitumor immune response by normal spleen cells, but actually manifested a strong immunostimulatory capacity. Thus, L-PAM given at a low dose enhances the development of potent antitumor immunity which brings about the eradication of a large tumorigenic load that remains after the drug has been cleared from the circulation.

Suppression of antitumor immunity by macrophages in spleens of mice bearing a large MOPC-315 tumor.

We had shown previously that progression of MOPC-315 plasmacytoma growth is associated with an increase in the percentage of macrophages in the spleen as well as a decrease in the ability of tumor-bearer spleen cells to mount an antitumor cytotoxic response upon in vitro immunization. Here we provide evidence that macrophages in the MOPC-315 tumor-bearer spleen are responsible at least in part for the suppression of the generation of antitumor cytotoxicity. Accordingly, removal of most macrophages by depletion of phagocytic cells or Sephadex G-10-adherent cells from spleens of mice bearing a large tumor resulted in augmented antitumor immune potential. Also, Sephadex G-10-adherent spleen cells from tumor-bearing (but not normal) mice drastically suppressed the in vitro generation of antitumor cytotoxicity by normal spleen cells. The suppressive activity of these adherent cells did not reside in contaminating suppressor T cells, since it was not reduced by treatment with monoclonal anti-Thy 1.2 antibody plus complement. The Sephadex G-10-adherent cell population from the tumor-bearer spleen suppressed the in vitro generation of antitumor cytotoxicity against autochthonous tumor cells but not against allogeneic EL4 tumor cells, and hence the suppression was apparently specific. The suppressive activity of the Sephadex G-10-adherent cell population from tumor-bearer spleens was overcome by treatment of the tumor-bearing mice with a low curative dose of cyclophosphamide. This immunomodulatory effect of a low dose of the drug in overcoming the suppression mediated by the Sephadex G-10-adherent cell population enables the effector arm of the immune system of tumor-bearing mice to cooperate effectively with the drug's tumoricidal activity in tumor eradication.

Lymphoreticular cells isolated by centrifugal elutriation from a mammary adenocarcinoma. I. Characterization of an in situ lymphocyte suppressor population by surface markers and functional reactivity.

The procedure of centrifugal elutriation was evaluated as a means of purifying large numbers of in situ lymphocytes from enzymatically disaggregated mouse mammary tumors. The eluate obtained at a flow rate of 3.0 ml/min was optimal for high levels of lymphocyte recovery with low levels of contaminating tumor cells, polymorphonuclear leukocytes, and macrophages. The majority of the tumor infiltrating lymphocytes (90%) expressed the Thy 1.2 antigen, while less than 5% possessed surface immunoglobulin. Further analysis of the T lymphocyte population was accomplished by flow cytometric analysis of in situ lymphocytes stained with fluorescein-conjugated monoclonal anti-Lyt 2 antibodies. The results of such studies reveal an increase in the levels of Lyt 2+ lymphocytes within the in situ population. To determine whether these Lyt 2+ cells were functionally active as suppressor cells, the ISL1 were mixed with spleen cells from tumor bearers and then tested for their ability to respond to mitogen and TAA-induced blast transformation of tumor-bearer spleen cells. Removal of macrophages from ISL by Sephadex G-10 columns did not alter the suppression. Treatment with monoclonal anti-Lyt 1 antibody and complement did not affect the inhibition observed. However, treatment of ISL with anti-Lyt 2+ monoclonal antibody and complement resulted in the elimination of the suppressor cell activity. We concluded that within the tumor-infiltrating lymphoreticular cells there is a population of Thy 1.2+ Lyt 2.2+ lymphocytes responsible for the suppression of mitogen and tumor-antigen-induced blastogenesis.

Reactivity of subpopulations of mouse spleen cells to concanavalin A in vitro.

Subpopulations of spleen cells of mice bearing virus-induced mammary tumors were assessed for responsiveness to Concanavalin A (Con A) under in vitro xenogeneic and syngeneic culture conditions. When cultured with human A+ plasma (xenogeneic), spleen cells of normal, nontumor-bearing BALB/c mice exhibited a high degree of synthesis as compared to unstimulated cells in response to Con A. Spleen cells of mice inoculated with tumor exhibited response exceeding that of their normal counterparts by 59%. When normal BALB/c sera (syngeneic) was used as the supplement, no response of normal spleen cells was observed upon stimulation by Con A; however, spleen cells from tumour bearing individuals demonstrated heightened activity. Tumor-bearer spleen cell response to Con A of mice inoculated with 1 X 10(6) viable cells was biphasic. The peak of the first phase appeared as early as 10 days postinoculation and declined within 3 days after its appearance. The peak of the second phase appeared within 6 days following decline of the first and declined drastically within 3 days after its appearance. Coincidental with the onset of the second phase, the tumor became ulcerated, purulent, and necrotic. Both phases were predominantly T-cell mediated.

Suppressor cell activity in mice bearing a progressively growing Simian virus 40-induced sarcoma

General and cell-mediated immunity (CMI) were investigated in BALB/c mice bearing progressively growing Simian virus 40-induced (mKSA) sarcoma by means of the Winn tumor cell neutralization (WN), 125I isotopic footpad (IFP), lymphoproliferative (LP) and plaque-forming cell (PFC) assays. Correlation between depressed antitumor immunity and the IFP responses was observed in tumor-bearing (TB) mice. Depressed LP responses to both T- and B-cell mitogens were observed in both early and late stages of tumor growth. Results obtained with the PFC assay similarly demonstrated depressed humoral immunity to sheep red blood cells (SRBC). Suppressor cell activity was demonstrated in cocultivation experiments in which spleen cells of TB mice were mixed with normal spleen cells. Treatment of TB spleen cells by passage through Sephadex G-10 columns or incubation on plastic surfaces to deplete the adherent cells restored LP responses. Cocultivation of Sephadex G-10- or plastic-adherent cells from TB mice with normal spleen cells significantly reduced mitogen-induced LP responses of normal cells. Examination of cell surface markers indicated an increase in the proportion of spleen cells bearing γFc receptors, which correlated with progressive mKSA tumor growth. There was also a correlation between γFc receptor-bearing spleen cells and macrophages, as shown by nonspecific esterase staining. These results indicate that depressed LP and PFC responses and the appearance of suppressor cells in mKSA tumor-bearing mice parallel an impaired ability to recognize (IFP responses) and neutralize (WN responses) tumor cells.

Increased Susceptibility to Tumor Cell Immunosuppressive Effect in Tumor-Bearing Mice&lt;xref ref-type="fn" rid="FN2"&gt;2&lt;/xref&gt;

Compared to normal hosts, tumor-bearing BALB/c mice were more susceptible to the immunosuppressive effect of tumor cells. At least a tenfold increase was found in the susceptibility mediated by a population of radioresistant spleen adherent cells (AC). The experiments were performed by the study of the suppressive effect of tumor cells on the generation of cytotoxic T-cells in allogeneic mixed lymphocyte culture reactions and allogeneic mixed lymphocyte tumor cell culture reactions. Fewer tumor cells were needed to suppress the T-cell-mediated cytotoxic responses of tumor bearers compared to normal hosts. A normal spleen population could be made to react like the tumor-bearing host by first depletion of its normal macrophages and then reconstitution with spleen AC from tumor bearers. Conversely, reconstitution of the macrophage-depleted tumor bearer's spleen with normal spleen AC made the tumor bearer react like the normal host. Furthermore, tumor cells were needed to trigger the spleen AC of the tumor bearer to fully exert their effect.

Antibody Responses of Tumor-Bearing Mice to Their Own Tumors Captured and Perpetuated As Hybridomas

Abstract Experiments were performed to determine whether antibody-producing hybridomas could be derived by fusion of BALB/c P3X63Ag8 myeloma cells and spleen cells from C57BL/6 mice either immune to or bearing a syngeneic methylcholanthrene (MCA)-induced fibrosarcoma (M3). Mice were immunized by tumor amputation followed by injections of tumor cells, purified tumor cell membranes, or murine leukemia virus isolated from M3 cells (M3-MuLV). Tumor-bearer spleens were obtained at weekly intervals over a 6-week course of tumor bearing. Hybridoma cultures showing the best growth in selective HAT medium were screened by radioimmunoassay for antibody production against a) viable and glutaraldehyde-fixed cultured M3 cells, b) glutaraldehyde-fixed in vivo-passaged M3 cells, c) purified M3 cell membranes, and d) M3-MuLV. Stable antibody-producing cloned hybridomas were obtained only from fusions involving tumor-bearer spleen cells. One clone (2C2-A1) secretes an IgM antibody that binds well to purified M3-MuLV and M3 membranes, but only weakly to intact M3 cells. Another cloned line (3A5-L1) secretes an IgG1 antibody that binds to M3 cells but not to M3-MuLV. Further analysis of specificity by binding and absorption studies involving more than 30 other normal and transformed murine cell types indicates that 3A5-L1 antibody binds to some, but not all, MCA-induced murine fibrosarcomas. 3A5-L1 antibody demonstrates little, if any, binding to murine tumors of other histologic types or to normal tissues. The results demonstrate that it is possible to capture and perpetuate part of the tumor-bearing host's antibody repertoire in the form of hybridomas secreting monoclonal antibodies specific for tumor-associated antigens.

The development of in vitro and in vivo anti-tumor cytotoxicity in noncytotoxic, MOPC-315-tumor-bearer, spleen cells ?educated? in vitro with MOPC-315 tumor cells

Spleen cells from mice bearing various sizes of MOPC-315 plasmacytoma tumors were not cytotoxic in the 51Cr release assay or the local adoptive transfer assay. These noncytotoxic spleen cells became cytotoxic, however, upon in vitro cocultivation with MOPC-315 tumor cells. The maximal level of in vitro anti-tumor cytotoxicity (51Cr release) with in vitro “educated” tumor-bearer spleen cells was obtained on the fifth day of the cocultivation and was equal to or lower than the level of cytotoxicity seen with in vitro educated normal spleen cells. On the other hand, the level of in vivo anti-tumor cytotoxicity (Winn assay) achieved with tumor-bearer spleen cells educated in vitro was at least equal to, but usually greater than the level of anti-tumor cytotoxicity obtained with normal spleen cells educated in vitro. Thus, in vitro education can generate anti-tumor cytotoxicity in autochthonous lymphoid cells from tumor-bearing hosts. Such educated histocompatible cells should be useful for immunotherapy regimens that might be applicable to man.

Immunologic Responses to a Murine Mammary Adenocarcinoma: in Vitro Production of Specific Killer Cells Is Dependent on Active T Lymphocytes

Abstract The mode of production of specifically armed monocytic killer cells was investigated with the T1699 mammary adenocarcinoma in syngeneic DBA/2 mice. After overnight in vitro incubation of cells from the spleen but not from the lymph nodes, blood, or from the peritoneal cavity produced specific killer cells. The activation of spleen cells was inhibited by pretreatment with anti-ϑ serum and C; however, already activated specific killer cells were not sensitive to the same treatment. Removal of phagocytic cells did not significantly affect the cytotoxicity of the splenic killer cells whereas removal of rayon-wool adherent cells greatly reduced both the total cytotoxicity, and to a lesser extent, the cytotoxicity indices. Overnight co-cultivation of normal peritoneal-exudate cells with the lymph node cells from tumor-bearers, although neither class of cells alone was cytotoxic to T1699 cells in vitro, produced specific monocytic killer cells, through steps dependent on active T lymphocyte functions. Culture supernatants of tumor-bearer's spleen cells also contained factor(s) which induced cytotoxicity mediated by normal peritoneal-exudate cells against T1699 cells in vitro; and the production of the factor(s) was also inhibited by pretreatment of the spleen cells with anti-ϑ serum but not by anti-mouse IgG or anti-mouse whole γ-globulins serum and C.

Suppressor Cells in the Spleens of Tumor-Bearing Mice: Enrichment by Centrifugation on Hypaque-Ficoll and Characterization of the Suppressor Population

Spleen cells from mice bearing methylcholanthrene-induced sarcomas or a mammary adenocarcinoma suppressed the mitogen responses of normal spleen and lymph node cells. Lymph node cells from tumor bearers had no suppressive effects. Centrifugation of spleen cells layered on Hypaque-Ficoll (specific gravity of 1.08) produced a dense fraction which pelleted and a light fraction which was retained at the Hypaque-Ficoll-medium interface. Suppressive activity was not found in either fraction of normal spleen cells. In tumor-bearer spleen cells suppressor activity was greatly enriched in the light fraction. Treatment of the suppressor fraction with anti-theta or anti-Ig serum and complement did not remove suppressor activity. However, the suppressor cells were removed by passage through nylon wool or by carbonyl iron treatment. Also, the population which adhered to plastic Petri dishes contained the suppressor cell activity.

Specific anti-tumor responses of cultured immune spleen cells III. Further characterization of cells which synthesize factors with blocking and antiserum-dependent cellular cytotoxic (ADC) activities.

Spleen cells from BALB/c mice bearing syngeneic, methylcholanthrene-induced sarcomas were cultured in vitro. In agreement with previous observations, two tumor-specific activities could be demonstrated in the culture supernatants: the supernatants suppressed (blocked) specific lymph-node cell-mediated cytotoxicity directed against the respective neoplasm and they induced specific antibody-dependent-cellular cytotoxicity (ADC) mediated by lymph-node cells from non-immune mice. In the present study, the effects of specific depletion or enrichment of different celltypes on the ability to synthesize the blocking and ADC activities was explored. Spleen cells passed through columns of Sephadex G-10, which removed plasma cells and macrophages, no longer synthesized factors with ADC activity though synthesis of blocking factors continued. However, lysis of Thy-1-positive cells with antiserum and complement did abolish the synthesis of blocking factors. Spleen cells from tumor-bearing mice were then enriched for Thy-1-positive cells by selective removal of macrophages and plasma cells on G-10 columns and of immunoglobulin-bearing cells on anti-mouse immunoglobulin affinity columns. This T-cell-enriched population synthesized blocking factors and restored blocking factor synthesis in cultures depleted of Thy-1-positive cells. Simiarly enriched spleen cells from normal control mice did not synthesize tumor-specific blocking factors but partially restored synthesis of blocking factor in tumor-bearer spleen cultures depleted of Thy-1-positive cells.

Studies of Antigens Associated With Hodgkin's Disease

Abstract Following a previous preliminary report, this study was initiated to demonstate further evidence of tumor-associated antigens in Hodgkin’s disease and to determine if these antigens are found in other pathologic states. Tumor-associated antigens are now demonstrated by three techniques in 18 Hodgkin’s invoved spleens and are not found to be present in equal concentration in normal spleens. Using immunofluorescence techniques and absorbed tumor antisera, fluorescence is demonstrated in tumors but not in the normal region of the same tumor-bearing spleen or in normal spleens. Gel diffusion with absorbed tumor antisera revealed a common precipitin band in Hodgkin’s disease not present in normal spleens or spleens from other diseases. Studies with immunoelectrophoresis have demonstrated two antigens present in the tumors in the spleen. Eighteen out of 19 Hodgkin’s tumors from the spleen have such antigens identifiable by immunoelectrophoresis with concomitant negative normal splenic controls in each case. Five out of 18 spleens from other disease states shared one of the antigens. These data confirm the presence of common tumor-associated antigens in Hodgkin’s disease, and it is postulated that at least one antigen may be a host cell reactant substance.