In vitro induction of tumor-specific immunity. II. Activation of cytotoxic lymphocytes to murine oncofetal antigens.

The presence of oncofetal antigens (OFA) on a wide variety of murine tumor cells was demonstrated to a totally in vitro system of cellular immunity. Nonimmune spleen lymphocytes were cocultivated with irradiated syngeneic fetal liver cells and, at various times after initiation of culture, were tested for the presence of cytotoxic lymphocytes (CL) by 51Cr-release assay with labeled tumor target cells. Significant cytotoxic activity was regularly detected after such culture, whereas only minor levels appeared in control cultures of spleen lymphocytes with irradiated syngeneic spleen cells. Specificity of the reaction was assessed by inhibition tests in which nonlabeled cells were admixed to the CL and 51Cr-labeled tumor targets. Fetal liver cells gave significant inhibition; however, no inhibition was found with adult spleen cells. Various tumor types gave inhibition, and fibrosarcomas were more effective than plasmacytomas or lymphomas. The results suggested that all tumor types tested possess such OFA, as well as their unique or virus-associated, tumor-associated transplantation antigens, and that the in vitro system permits a more active response to the tumor-associated OFA than that observed in in vivo studies.     

In vitro induction of tumour-specific immunity V. Detection of common antigenic determinatnts of murine fibrosarcomas.

Two 3-methylcholanthrene and a spontaneous BALB/c fibrosarcoma were examined for tumour-associated antigens (TAA) by in vivo and in vitro induction of tumour-immune responses. When BALB/c mice were immunized to these fibrosarcomas by surgical tumour removal, cross-reacting tumour-associated transplantation antigens (TATA) were detected on all 3 tumours. Cytotoxic effector cells (CL) were then induced in vitro by co-culture of BALB/c spleen cells with the spontaneous, or one of the carcinogen-induced fibrosarcomas. These CL were shown to be cytotoxic T cells (Tc) and to be directed against cross-reacting TAA on all 3 tumours, by two in vitro 51Cr-release assay systems, direct 51Cr-release cytotoxicity and cellular competitive inhibition of 51Cr release. Further studies demonstrated that the fibrosarcoma TAA involved in in vitro induction of Tc were not present on normal adult or foetal tissues. A secondary cytotoxic response was also detected in vitro when spleen cells from mice immunized to a carcinogen-induced fibrosarcoma were tested. The patterns of cross-reactivity detected by the in vivo and primary in vitro tumour-immune responses suggested that the TAA detected in vivo (TATA) were not identical to the TAA detected in vitro.     

Immunogenicity, antigenicity and mechanisms of tumor rejection of mineral-oil-induced plasmacytomas in syngeneic BALB/c mice

Transplantation immunity studies were conducted to determine whether mineral-oil-induced plasmacytomas of syngeneic BALB/c mice possess tumor-associated transplantation antigens (TATAs). In a series of experiments to determine whether TATA could be detected, it was established that the optimum immunization regimen against TATA was obtained by immunizing syngeneic hosts with an intradermal inoculation of viable plasmacytoma cells, followed by therapeutic drug-induced tumor regression with aniline mustard. This immunization regimen induced transplantation immunity not only to the homologous tumor employed for immunization, but was effective in demonstrating some cross-protection when mice were rechallenged with other plasmacytomas. Thus, definite cross-reactivity of antigens was observed between some plasmacytomas, but others shown to possess TATA did not share antigens. These, therefore, may possess distinct TATA (s). Further, studies with Winn assays employing cells from BALB/c mice immune to plasmacytoma cells support the idea that cell-mediated immune functions are responsible for syngeneic plasmacytoma rejection. Specificity studies in subsequent Winn assays demonstrated that the kill was directed against plasmacytoma cells and not against TATA (s) of an MLV-induced Moloney leukemia and SV40 virus-induced tumor cells. The nature of the TATA (s) detected is not known, but they probably represent new “cell-associated” antigens acquired during malignant transformation. Finally, the selective removal of the thymus-dependent cell component of the immune lymphocyte population with anti-theta serum and complement eliminated the tumor-neutralizing properties of the immune cells in the Winn assay.     

In vitro induction of tumor-specific immunity by highly purified VSV grown in SV40-transformed cells and tumor glycolipids incorporated into liposomes

Cytotoxic effector lymphocytes (CL) were induced by in vitro immunization of spleen cells from normal Syrian hamsters to syngeneic tumor cells, either SV40-transformed (EH-SV) or spontaneously transformed (EH-N). The lymphocyte reactivity was measured in a direct 51Cr release cytotoxicity assay performed with EH-SV- and EH-N-labelled targets. A specific cytotoxic effect against tumor cells carrying the sensitizing antigens was observed. Cytotoxic effector lymphocytes were also induced by in vitro immunization of hamster spleen cells to highly purified vesicular stomatitis virus (VSV) grown either in syngeneic SV40-transformed fibroblasts or in "normal" fibroblasts. Purified virus possessing an intact envelope or virus subparticles devoid of their glycoprotein spikes stimulated the cellular immune responses against host tumor antigens present within the viral envelope. Cytotoxicity assays have revealed two tumor-specific antigens (TSA), one induced by SV40 and present in SV40-transformed cell lines and the other present in "normal" cells. CL were also induced by in vitro sensitization of spleen cells from normal hamsters to liposomes containing the polar glycolipid fraction from EH-SV and/or EH-N cells. A specific cytotoxic effect against tumor cells that have supplied the glycolipid extract was observed, suggesting specific recognition of glycolipid antigens characteristic for each tumor line. This study supports the view that surface glycolipids act as tumor-specific antigens implicated in the destruction of SV40-induced tumors in Syrian hamsters.     

Cell-mediated immunity against particulate and solubilized tumor-associated antigens of murine plasmacytomas detected by macrophage migration inhibition assays

Cell-mediated immunity (CMI), as detected by the agarose microdroplet macrophage migration inhibition (MMI) assay, was investigated using peritoneal exudate cells (PEC) of BALB/c mice and several crude membrane (CM) and solubilized preparations of murine plasmacytomas. The MMI assay was quite sensitive and detected inhibition of macrophage migration as low as picogram quantities of CM, NP40 detergent- and papain-solubilized preparations (CS) of ADJ-PCS and LPC-1 plasmacytomas. The data were highly reproducible from one experiment to the next with the same or different lots of the CM or solubilized extracts. Specificity studies demonstrated that ADJ-PCS and LPC-1 plasmacytomas expressed cross-reactive tumor-associated antigens (TAA) as detected by MMI and confirmed by tumor challenge and Winn neutralization experiments. No cross-reactivity was observed with similar extracts prepared from an unrelated syngeneic simian virus 40 (SV40)-induced sarcoma. The inhibition of macrophage migration observed was mediated by culture supernatants generated from the mixture of plasmacytoma-immune spleen cells with antigens and then assayed in an indirect MMI assay on normal PEC. The agarose microdroplet MMI assay appeared to be a rapid and sensitive method to measure TAA recognition and to monitor TAA isolation and solubilization with minimum numbers of immune cells.     

Use of IL-2 gene transfer in local immunotherapy of cancer.

Insertion of functional interleukin-2 (IL-2) gene into a plasmacytoma cell line X63-Ag8.653 substantially reduced tumorigenicity of the resulting cloned cells, designated as X63-m-IL-2. Peritumoral administration of the X63-m-IL-2 cells, producing constitutively large quantities of IL-2, resulted in regressions of established X63-Ag8.653 plasmacytomas growing in the peritoneal cavity of syngeneic mice. In vitro activation of BALB/c spleen cells by co-culture with X63-m-IL-2 cells or their supernatants gave rise to cytotoxic lymphocytes with lymphokine-activated killer (LAK) activity against syngeneic X63-Ag8.653 plasmacytoma and other tumor targets. In contrast, peritumoral administration of X63-Ag8.653 cells carrying an inserted interleukin-4 (IL-4) gene (designated X63-m-IL-4 cells) and producing constitutively large quantities of IL-4 did not result in a therapeutic effect. Moreover, the admixture of the X63-m-IL-4 and X63-m-IL-2 cells substantially diminished the X63-m-IL-2 cell-mediated therapeutic effect. Similarly, IL-4-containing supernatants generated from X63-m-IL-4 cell cultures substantially diminished LAK activation by X63-m-IL-2 cell produced supernatants.     

Specific cytotoxic lymphocytes against syngeneic tumors are generated in culture in the presence of syngeneic, but not xenogeneic, serum.

Experiments were performed to test the effect of xenogeneic (fetal calf) serum (FCS) , as compared to syngeneic mouse serum (SMS) on the generation in culture of specific cytotoxic lymphocytes (CL) against syngeneic tumors. Sensitization in FCS against 3LL tumor cells resulted in CL cross-reacting with B-16 tumor cells and vice versa. Anti-syngeneic fibroblast CL also cross-reacted with 3LL. Such cross-reactivities were shown to be derived from CL directed against FCS determinants. In contrast, sensitization in the presence of SMS resulted in CL directed against tumor-specific antigens. Anti-3LL generated in SMS lysed 3LL targets but not B-16, and anti-B-16 lysed B-16 but not 3LL. The two types of CL had two distinct reactivities in vivo. Anti-3LL CL generated in FCS enhanced tumor growth in vivo, whereas anti-3LL CL generated in SMS had an inhibiting effect on the growth of tumor cells. These results indicate that the application of syngeneic serum during in vitro sensitization against syngeneic tumors may open up new possibilities for the analysis of tumor-specific antigens and for eliciting specific immune reactions against such antigens.     

Induction of protective antitumor immune response against a murine plasmacytoma not curable by alkylating-drug treatment

Immunization with viable tumor cells followed by subsequent administration of glutaraldehyde-treated tumor cells induced a protective antitumor immune response in the host toward the alkylating-drug resistant RPC-5 plasmacytoma. This was proven by resistance to challenge with RPC-5 tumor cells, neutralization in Winn tests, by effectiveness of combined chemotherapy with melphalan plus immunotherapy with spleen cells from RPC-5 immunized mice and in vitro by cytotoxicity tests. The specificity of the immune response was ascertained in vivo by comparison with the response toward MOPC-315 plasmacytoma. However, in vitro cytotoxicity tests revealed the occurrence of shared antigens between the RPC-5 and MOPC-315 tumor cells. It is concluded that the ineffectiveness of alkylating-drug treatment toward the RPC-5 tumor is not due to the inability of this tumor to induce a specific antitumor immune response, and that cross-antigenic relationship as revealed by in vitro cytotoxicity tests does not necessarily reflect cross-protection between various plasmacytomas.     

Secondary in vitro generation of cytolytic T-lymphocytes (CTL's) in the murine sarcoma virus system. Virus-specific CTL induction across the H-2 barrier.

Following in vitro stimulation of murine sarcoma virus Moloney isolate (M-MuSV)-immune spleen cells with syngeneic antigenically related Moloney leukemia cells, highly efficient cytotoxic T-lymphocytes (CTL's) were generated. The cytotoxic effect was directed only against H-2-compatible target cells bearing M-MuSV tumor-associated antigens (TAA). However, in a cold target competition assay a weak but detectable capacity to block CTL activity was also obtained when allogeneic Moloney leukemia cells were added. Moreover, when M-MuSV-immune spleen cells from mice inoculated with virus 14 days previously were stimulated by allogeneic Moloney leukemia cells, a strong cytotoxic effect toward syngeneic and allogeneic tumor cells bearing M-MuSV TAA was elicited.     

Development of resistance to MOPC-315 plasmacytoma after intralesional and intraperitoneal melphalan therapy of tumor-bearing BALB/c mice. II. Enhancement of in vitro cell-mediated cytotoxicity by combined chemotherapy-immunotherapy

As previously reported, tumor-bearing BALB/c mice can be cured by split-course melphalan therapy, with 40-60% of the treated animals developing resistance to subsequent challenge with viable MOPC-315. The present study deals with the identification of effector-cytotoxic cells that may be developed as a result of chemotherapy-induced tumor regression and their possible potentiation by active, specific immunization with melphalan- and glutaraldehyde-treated MOPC-315 plasmacytoma cells. The cytotoxic potential of spleen-derived lymphocytes in treated animals could be demonstrated only after in vitro sensitization against mitomycin-treated MOPC-315 cells. Lymphocyte-mediated cytotoxicity, as measured against syngeneic 51Cr-labeled MOPC-315, could be detected in melphalan-cured animals and was significantly enhanced by active immunization as compared to the cytotoxicity seen in normal and tumor-bearing mice. With the use of M109 syngeneic, unrelated tumor cells as control targets in the assay, no cytotoxicity was detected. Macrophage cytotoxicity was not significantly enhanced in any of the treatment groups described, with these assays performed 6-8 weeks following treatment and cure. When in vitro killing of MOPC-315 targets was tested with the use of peritoneal macrophages harvested shortly following cure of ascitic tumor by ip injected melphalan, the cytotoxic response was significantly enhanced. In conclusion, following chemotherapy-mediated cure of established MOPC-315 tumors, splenic lymphocytes exhibited enhanced antitumor cytotoxicity, which was further augmented by active immunization. Macrophage activation, as measured by direct cytotoxicity against MOPC-315 targets, was found to occur locally and early following the event of melphalan-induced tumor regression.     

Studies of lymphocyte stimulation by intact tumor-cell and solubilized tumor antigen.

BALB/c mice were rendered immune to syngeneic SV40-induced sarcoma by subcutaneous injection of mKSA-TU5 tissue-culture adapted cells. Spleen cells from immune mice were examined for tumor-cell neutralization in the Winn assay as well as in in vitro lymphocyte stimulation assays. A microculture (200 mul) lymphocyte stimulation (LS) assay utilizing immune spleen cells was employed with mixed lymphocyte/tumor-cell cultures (MLTC) and the papain crude soluble (CS) extracts from mKSA-TU5 cells. Specificity in the LS assay was determined using spleen cells from mice immune to other syngeneic tumors and by soluble antigenic preparation of normal BALB/c spleen cells. The Winn assay studies demonstrated that spleen cells from mKSA-sensitized mice neutralized mKSA tumor cells and this was corroborated by their resistance to direct tumor challenge. Positive lymphocyte transformation responses in MLTC were observed when mKSA-TU5-sensitized spleen cells were mixed with mitocycin-C-treated intact tumor cells or when papain-solubilized antigens of mKSA cells were employed, but not with non-immune spleen cells or with a soluble antigen from normal cells. Papain-solubilized antigen preparations employed in in vitro assays also immunized against challenge with mKSA tumor cells. Specificity of these lymphocyte transformation reactions was demonstrated with non-sensitized lymphoid cells or lymphoid cells from mice sensitized with a syngeneic Kirsten virus-induced respond. Thus, mKSA tumor surface antigens were recognized on intact tumor cells or with microgram quantities of papain-solubilized extracts from these tumor cells. We believe the lymphocyte stimulation assay affords a method for demonstrating the presence of tumor-specific antigen and for monitoring further purification procedures.     

Specific chromosome translocation in pristane-induced plasmacytomas of NZB mice

The karyotypes of 3 pristane (2,6,10,14-tetramethyl-pentadecane)-induced NZB mouse plasmacytomas producing IgA (plasmacytoma TEPC-3660), IgG3K (plasmacytoma TEPC-6583), and IgG2aK plus IgG3K (plasmacytoma TEPC-6906) were studied in early transplantation generations by the combined Hoechst 33258-quinacrine mustard banding method. The two IgG producers (TEPC-6583 and TEPC-6906) showed diploid chromosome number, whereas the IgA producer (TEPC-3660) showed hypotetraploid chromosome number. Banding analysis revealed that these 3 plasmacytomas consistently had the t(12;15) translocation with the specific breakpoints at 12F2 and 15D2. Taken together with the previous observation of t(12;15) in plasmacytomas of BALB/c and C3H mice, these results suggest that the t(12;15) is specific for murine plasmacytomas and that the putative gene(s) on the t(12;15) may participate in the genesis of murine plasmacytomas.     

Ability of delayed-type hypersensitivity reactions to distinguish tumor-associated antigens and histocompatibility antigens in soluble extracts from murine fibrosarcomas.

The footpad swelling (FPS) test for delayed-type hypersensitivity in the mouse was evaluated for its ability to measure both tumor-associated antigens (TAA) and histocompatibility (H) antigens solubilized from methylcholanthrene (MCA) induced fibrosarcomas of C57B1/6 (B6) mice. Tests for TAA were performed in mice immune to syngeneic tumors while H-antigens were assayed in mice immunized with skin allografts. FPS was most intense in B6 mice challenged with TAA from the immunizing B6 tumor, but also occurred in response to cross-reactive TAA solubilized from another B6 fibrosarcoma. Tests for tumor-associated H-antigens in allograft immune mice were strongly positive in response to donor/recipient H-antigen differences and proved sensitive to shared third-party H-antigen differences. Comparison of soluble antigens from the same tumor maintained in vitro and in vivo revealed that, while both TAA and H-antigens could be detected in preparations from the in vivo tumor line, only TAA and not H-antigens could be detected by RPS in extracts prepared from the in vitro tumor line. These experiments have demonstrated that the mouse FPS test can distinguish both TAA and H-antigen specificities persent in the same complex mixture of tumor-cell antigens.     

Effects of presensitization in vivo on cell-mediated responses to embryonic antigens in vitro

Lymphoid cells specifically reactive with antigens shared by rat bowel carcinomas and mid-term embryo cells were generated by in vitro culture on monolayers of embryo cells. Spleen cells from WF females were cultured for 5 days on monolayers of syngeneic embryo cells or adult cells and assayed for cytotoxic activity on syngeneic embryo, bowel carcinoma, or adult fibroblast target cells in microcytotoxicity and 51Cr-release assays. After culture on embryo monolayers, spleen cells were cytotoxic for embryo and tumor but not for adult fibroblast target cells. Enhanced cytotoxicity was recorded when the spleen cells were cultured from females after interstrain (WF X BN) pregnancy rather than from virgin females. In contrast, previous intrastrain (WF X WF) pregnancy appeared to depress the generation of spleen cells cytotoxic for target cells bearing embryonic antigens.     

Immunoregulation of Murine Plasmacytoma I. Generation of Anomalous Killer Cells in Vitro by Cocultivation with MOPC 104E

Murine plasmacytoma MOPC 104E-KI81 is a tissue culture cell line of MOPC 104E derived from BALB/c mice. MOPC 104E-KI81 implanted subcutaneously in syngeneic normal mice regresses spontaneously after an initial growth of about 10 mm. Mice that regressed tumors or mice immunized intraperitoneally with mitomycin C-treated MOPC 104E-KI81 myeloma could reject subsequent challenge of viable KI81 myeloma cells. In contrast to euthymic mice, T-cell-deficient athymic nude mice developed subcutaneous tumors after challenge and died from progressive tumor growth, suggesting the critical role of T cells in tumor regression. In vitro induction of cytotoxic cells was used to define the immunologic mechanism by which the host can suppress tumor growth. Spleen cells from immune mice did not show cytolytic activity in ⁵¹Cr release cytotoxicity assay, but showed inhibitory action of tumor proliferation in vitro at an effector cell to target cell ratio of 500:1 in a [³H]thymidine incorporation assay. To determine if cytotoxicity could be induced against MOPC 104E-KI81 cells, in vitro sensitizing cultures were studied. We have demonstrated that normal BALB/c spleen cells became cytotoxic against MOPC 104E-KI81 cells after 5 days cultivation with mitomycin C-treated stimulator cells at an optimal responder to stimulator cell ratio of 5:1. Treatment of anti-Thy-1.2 serum plus complement abolished cytotoxic activity of effector cells. Cytotoxic cells lysed not only MOPC 104E-KI81 cells used for stimulation but also H-2^k osteosarcoma cells. It was concluded that Thy-1.2-positive cytotoxic cells with nonspecific anomalous reactivity could be induced in murine plasmacytoma-stimulating cultures.     

Kinetics of responses to tumor antigens and mouse mammary tumor virus in BALB/cCrgl and BALB/cfC3H mice

Spleen cells from BALB/cCrgl mice responded to murine mammary tumor virus (MuMTV) in cell-mediated immune assays at higher levels than did the spleen cells from the syngeneic BALB/cfC3H mice. Implantation in BALB/cCrgl with a chemically induced mammary tumor and in BALB/cfCrgl mice with spontaneous mammary tumors (SMT) arising in the same strain resulted in sensitization of these animals to the antigens of their tumors. Reactivities peaked 3 weeks after transplantation, whereas no positive reactions could be detected when tumors reached maximum size. A kinetic study with the use of MuMTV antigen(s) showed that the responses of lymphocytes from BALB/cfC3H with SMT followed the same pattern as that obtained with tumor antigens, which indicated that this might be a de novo sensitization. In sharp contrast, a steady type of response to MuMTV was observed with the spleen cells from BALB/cCrgl mice; i.e., the levels of responsiveness to MuMTV did not significantly vary at any stage of tumor development. In vivo studies explored the possible relevance of the in vitro cell-mediated immunity to the host defenses. MuMTV-expressing mammary tumor cells were implanted in BALB/cCrgl and BALB/cfC3H mice. The total incidence of tumors was significantly reduced and a delay occurred in their time of appearance in BALB/cCrgl mice in relation to BALB/cfC3H animals. Thus the in vitro reactivity to MuMTV antigen(s) in BALB/cCrgl mice was found to be coincidental with a degree of protection against the development of MuMTV-expressing mammary tumors.     

Effector and enhancing lymphoid cells in plasmacytoma-bearing mice. II. Dynamic changes during tumor progression

The Winn assay was used for the study of effector (tumor-inhibiting) and enhancing (tumor-promoting) lymphoid cells in BALB/c mice bearing MOPC-104E plasmacytomas. Kinetic studies with thymus, lymphnode, spleen and bone-marrow cells revealed that spleen, lymph node and to a lesser extent bone-marrow cells from 7- and 10-day tumor-bearers inhibited MOPC-104E growth, while at day 13 all three cell populations enhanced tumor growth. However, at day 16 strong tumor inhibition was observed again by spleen cells and to a lesser extent by lymph-node cells and thymocytes. Peritoneal cells from normal and tumor-bearing (7 and 10 days) animals enhanced tumor growth significantly. Separation of spleen cells on nylon wool columns showed that at 10 days the effector cells were T lymphocytes, but at a later stage (35 days) a different effector mechanism was present in the spleen. Treatment of MOPC-104E recipients with carrageenan or silica had little influence on tumor growth, but marked tumor inhibition occurred after lethal irradiation and bone-marrow reconstitution. This latter observation, together with the finding that bone-marrow, lymph node and peritoneal cells from normal donors enhanced tumor growth inseveral experiments, suggests that this plasmacytoma, like hormone-dependent tumors, requires lymphocyte-derived growth factors.     

Effect of heat treatment on tumor cells and antitumor effector cells

We examined the effects of heat treatment on tumor cells and antitumor effector cells in order to investigate the combined effects of hyperthermia and immunotherapy including adoptive immunotherapy. Plasmacytoma MOPC104E syngeneic to BALB/c mice was used as the tumor cell line, while fresh spleen cells immunized to MOPC104E (IM-FSC) and interleukin-2-cultured lymphocytes induced in vitro from IM-FSC (CL) were used as the effector cells. Tumor cells or effector cells were heat-treated in a water bath at 42 degrees C for 30 or 60 min. Tumor cells heat-treated at 42 degrees C for 30 min grew temporarily and then regressed in the tumor transfer test, whereas untreated tumor cells showed no regression under any conditions. Furthermore, fresh spleen cells of mice inoculated with heat-treated tumor cells from regressed tumors showed marked tumor-neutralizing activity. The antitumor effects of CL were markedly inhibited by heat treatment according to the results of the tumor-neutralizing test and the 51Cr release assay, whereas heat treatment had little influence on the antitumor activity of IM-FSC. However, the neutralizing activity of effectors and the killing activity of CL against heat-treated tumor cells were both markedly augmented, since the susceptibility of the tumor cells to the antitumor effector cells was augmented by heat treatment. These results suggest that heat treatment of tumor cells augments the antitumor effects of IM-FSC and CL, hence we speculate that hyperthermia augments the effects of immunotherapy including adoptive immunotherapy.     

Loss of marrow allograft resistance in mice with transplanted methylcholanthrene-induced sarcomas.

To determine if the effector cells responsible for allogeneic marrow stem cell rejections were suppressed in mice with tumors, C57BL/6 (B6) mice were inoculated with 3-methylcholanthrene (MCA)-induced sarcoma cells. When the tumor reached 2.0--2.5 cm in diameter, these mice and control B6 and (BALB/c times A)F1 (CAF1) uninoculated animals were irradiated and given BALB/c marrow cells in the first of a two-step "stem cell rescue" experiment. Four days later, spleen cells of the primary hosts were reinoculated into irradiated CAF1 secondary hosts compatible with BALB/c marrow cells and immunized against B6 antigens. Splenic uptake (percent) of 125I-5-iodo-2'-deoxyuridine 5 days after spleen cell regrafting was used as a measure of cell proliferation and reflected growth of the stem cells in the primary hosts. BALB/c stem cells grew as well in B6 mice with tumors as in CAF1 primary hosts but were rejected by B6 controls. Seeding efficiency of BALB/c stem cells 6 hours after infusion of marrow cells and growth of syngeneic B6 stem cells were enhanced twofold in spleens of tumor-bearing B6 mice. To exclude the possibility that enhanced seeding resulted in greater survival of allogeneic stem cells, more DBA/2 marrow cells were infused into control B6 primary hosts than into tumor-bearing B6 and control DBA/2 mice. Control B6 mice resisted growth of even 7.5 times 10(6) DBA/2 marrow cells, whereas B6 tumor bearers allowed growth of 2.5 times 10(6) cells. No "suppressor cells" capable of inhibiting marrow cell allograft reactions were detected in spleens of tumor-bearing mice. Thus transplanted syngeneic MCA-induced sarcomas abrogated the ability of mice to reject allogeneic marrow stem cells.     

Some characteristics of the cyclophosphamide-induced immunopotentiating cells in the spleen of mice bearing a large MOPC-315 tumor

We have shown previously (Ye, Q-W., and Mokyr, M. B. Cancer Res., 44: 3873-3879, 1984) that, following low-dose cyclophosphamide (CY) therapy (15 mg/kg) of mice bearing a large s.c. MOPC-315 tumor and extensive metastases, T-cell-dependent immunopotentiating activity appears in their hitherto immunosuppressive Sephadex G-10-adherent spleen cell population. Here we show that the CY-induced immunopotentiating T-cells express the Lyt 1, Lyt 2, and L3T4 phenotypes. The phenotype of the immunopotentiating T-cells was deduced from our observations that depletion of Lyt 1+, Lyt 2+, or L3T4+ cells from the Sephadex G-10-adherent spleen cell population of CY-treated tumor bearers abolished the ability of the adherent cells to enhance the generation of antitumor cytotoxicity when added to the in vitro immunization culture of normal spleen cells. Moreover, admixture of a Sephadex G-10-adherent cell population depleted of Lyt 2+ cells with a Sephadex G-10-adherent cell population depleted of L3T4+ cells failed to restore the immunopotentiating activity, indicating that T-cells that are apparently expressing simultaneously the Lyt 2 and L3T4 antigens are required for the exertion of the CY-induced immunopotentiating activity. The CY-induced immunopotentiating T-cells from MOPC-315 tumor bearers brought about the appearance of enhanced antitumor cytotoxicity not only against the MOPC-315 tumor cells, but also against two other syngeneic plasmacytomas, with surface immunoglobulin of a different class and antigenic specificity than the MOPC-315 tumor cells, as well as against a variant MOPC-315 tumor line which lacks surface immunoglobulin. The CY-induced immunopotentiating T-cells did not enhance the appearance of antitumor cytotoxicity against a syngeneic (WEHI 22.1) or an allogeneic (EL4) tumor of T-cell origin nor against the natural killer-sensitive YAC-1 cells. Thus, L3T4+, Lyt2+ T-cells from CY-treated MOPC-315 tumor bearers enhance the generation of antitumor cytotoxicity that is directed against plasmacytoma shared antigens other than immunoglobulins.