Cross-reactive cellular and humoral immunity to carcinogen-induced chicken fibrosarcomas. II. Effect of prior immunization with transplantable tumors on primary tumor incidence

Primary carcinogen-induced (7,12-dimethyl-benz[a]anthracene; DMBA) tumor-bearing SC chickens (B2/B2) frequently showed antibodies in their sera which reacted with cells from their autochthonous tumors, chicken embryo fibroblasts (CEF), tumor cells from some transplantable tumor lines, and from approximately 10% of other primary tumors. Similar results were obtained by ELISA on glutaraldehyde-fixed cells and by immunofluorescence on viable cells. The serum antibody reactivity could be removed by absorption with CEF but not with non-cross-reacting primary tumor cells or a variety of normal tissues. Although sera from normal chickens never showed significant reactivity, a high percentage of sera from chickens that had been injected with DMBA but failed to develop detectable tumors showed antibody activity to a transplantable DMBA-induced tumor and to CEF. On the basis of previously established cross-reactivity patterns in protective immunity to transplantable carcinogen-induced fibrosarcomas, attempts were made to protect against chemical carcinogenesis by prior immunization with selected DMBA-induced transplantable tumors. Tumor-immune chickens showed a significant decrease in the development of tumors during the first 3 mo after injection of DMBA (p = 0.001) or methylcholanthrene (p = 0.033) when compared to controls. This resistance to tumor induction in immune chickens was correlated to the degree of tumor immunity to the immunizing tumor present 1 mo after carcinogen injection (p = 0.046). There was, however, no detectable difference in the incidence of tumors arising later than 3 mo after carcinogen injection. The reduction in tumor incidence in immune as compared to control chickens at 5 mo was therefore less striking than the reduction seen at 3 mo. Immunization with CEF and adjuvants or with adjuvants alone afforded no protection to tumor induction.     

Immunity to transplantable carcinogen induced fibrosarcomas in B2/B2 chickens. II. Macrophage dependency of adoptive T cell mediated tumor immunity.

The nature of immunity to transplantable chemically induced fibrosarcomas in SC (B2/B2) chicken was examined using Winn tests performed in the wing web. Immunity in spleen cell donors was induced by pretreatment with C. parvum or BCG followed by tumor cells + bacterial adjuvant in one and tumor cells alone in the other wing web. The T cells mediating the adoptive immunity were sensitive to anti-T + C, nylon wool nonadherent, mitomycin resistant and radiation (1000 R) sensitive. The adoptive immunity could not be expressed in heavily irradiated recipients or in hosts pretreated with trypan blue or silica. The host contribution could be reconstituted by iv injection of spleen or bone marrow cells from agamma-globulinemic (Aγ) unimmunized donors 2 days prior to Winn tests or by the local injection into wing webs of spleen cells or purified peripheral blood monocytes from Aγ donors. It was concluded that cooperation between immune donor T cells and normal monocytes of host origin mediated the inhibition of SCFS growth in Winn tests.     

Cross-reactive cellular and humoral immunity to carcinogen-induced chicken fibrosarcomas. I. Protective cross-immunity between transplantable tumor lines

Dimethylbenz[a]anthracene (DMBA)-induced transplantable fibrosarcomas in B2 homozygous chickens (SC line) grow progressively in normal chickens, but are rejected by chickens immunized previously with irradiated tumor cells and Corynebacterium parvum. Tumor-immune chickens resist challenge by the immunizing tumor lines as well as by some, but not all, fibrosarcoma lines. The pattern of cross-reactivity between four DMBA-induced transplantable tumor lines was examined in detail. Ability to reject a tumor challenge correlated very well (p less than 0.001) with the presence of delayed-type hypersensitivity (DTH) to that tumor. Immunization with one of two of the DMBA-induced lines tested also caused rejection of transplantable tumors developed from methylcholanthrene-induced and benzo(a)pyrene-induced primary fibrosarcomas. Although immunization with tumor caused DTH to chicken embryo fibroblasts (CEF), immunization with CEF failed to cause protective immunity or DTH to tumors. Presence of protective immunity, where tested, also correlated with the ability of spleen cells from immune donors to inhibit tumor growth in Winn tests. Humoral immunity exhibited even greater cross-reactivity than did cellular immunity. Distinct patterns of cross-reactivity were nevertheless observed with respect to the serum antibodies as detected in ELISA. Two of these patterns were also observed in several sera from primary tumor-bearing chickens, both including reactivity with CEF. Such reactivity was absent from normal chicken sera.     

Activation of antigen-specific suppressor T cells by the intravenous injection of soluble blood-stage malarial antigen

The regulation of delayed-type hypersensitivity (DTH) to soluble antigens derived from blood-stage parasites was investigated. DTH responses to soluble blood-stage malarial antigen were induced by subcutaneous (sc) sensitization in the flanks and elicited by ear challenge with the same antigen 6 days later. Adoptive transfer studies revealed that T cells of the L3T4+ phenotype were mediating this response. When a high dose of malarial antigen was injected intravenously (iv) prior to sc sensitization, immunosuppression of DTH resulted. The degree of immunosuppression was dependent on the dose of antigen injected iv and the time at which it was administered prior to sc sensitization. Immunosuppression was antigen-specific and mediated by Lyt-2+ splenic T cells.     

T-cell responses induced by the parenteral injection of antigen-modified syngeneic cells. III. Dissociation of primed cytolytic T-cell and efferent suppressor-T-cell activity following intravenous injection of trinitrophenol-modified syngeneic spleen cells

The parenteral injection of ligand-coupled syngeneic spleen cells has profound effects on immune responsiveness. In this regard, it was examined whether the primed in vitro trinitrophenol (TNP)-specific cytotoxic T-lymphocyte (CTL) responses observed in splenic T-cell populations from mice injected intravenously (iv) with syngeneic TNP-modified spleen cells (TNP-SC) are related to the efferent-acting suppressor-T-cell (Ts) activity observed in splenocytes from iv primed mice. Treatment of mice with cyclophosphamide, adult thymectomy, or monoclonal anti-I-J antiserum prior to the iv injection of TNP-SC was found to eliminate the ability of splenic Ts from these mice to suppress the passive transfer of delayed-type hypersensitivity (DTH) mediated by trinitrochlorobenzene-immune T cells. In contrast, spleen cells from these pretreated mice showed no impairment in the development of augmented TNP-specific CTL responses upon in vitro restimulation with TNP-SC. Separation of the two activities was also achieved in a kinetic analysis. It is concluded that specific enhancement of CTL responsiveness induced by the iv injection of TNP-SC is related to the expansion of a population prelytic Lyt 2+ CTL effector cells which does not appear to contain efferent-acting Lyt 2+ Ts active in suppressing DTH expression.     

Functional analysis of cloned macrophage hybridomas. VI. Differential ability to induce immunity or suppression

We previously screened a series of macrophage hybridomas derived from fusion of P388D1 (H-2d) tumor cells with CKB (H-2k) splenic adherent cells for their ability to induce I-J restricted Ts cell responses. One Ia+ macrophage clone (63) consistently induced Ag-specific, I-J-restricted Ts. To evaluate whether macrophage hybridoma 63 also induced delayed-type hypersensitivity (DTH) immunity, mice were immunized with hapten-coupled macrophage hybridoma cells. Hapten-coupled splenic adherent cells and control macrophage hybridomas induced significant primary DTH responses, whereas hapten-coupled macrophage 63 induced little or no immunity when injected into H-2 compatible hosts. However, macrophage hybridoma 63 specifically activated I-Ak, I-Ad, or I-Ed restricted T cell hybridomas/clones, in vitro in the presence of appropriate Ag. Three different strategies designed to eliminate suppressor cell activity were successfully used to demonstrate that hapten-coupled macrophage 63 could also induce in vivo immunity. First, after immunization with hapten-coupled macrophages, mice were treated with cyclophosphamide. Second, macrophage 63 was treated with anti-IJ idiotype antibody before 4-hydroxy-3-nitrophenyl acetyl hapten (NP) coupling. Finally, haptenated macrophages were injected into I-A compatible but I-J incompatible recipients. These protocols are known to inhibit the induction of Ts activity, thus these results indirectly suggest that there is stimultaneous generation of Ts activity in vivo. The latter hypothesis was tested in adoptive transfer experiments. Transfer of lymph node cells from NP-63 primed B10.BR (H-2k) mice induced immunity in naive 4R animals, whereas the same number of immune cells suppressed NP-induced DTH responses in 5R mice. The combined results indicate that a cloned macrophage line can activate both Th and Ts cells. Macrophages which induce Ts activity may be responsible for maintaining the balance of immunity vs suppression. The data support the hypothesis that IJ interacting molecules (IJ-IM) expressed on macrophages are critical for induction of suppressor cell activity.     

A syngeneic MLR induced in vivo results in T-cell mediated immune suppression

The proliferation of murine T lymphocytes in response to syngeneic Ia bearing non-T cells (syngeneic mixed lymphocyte reaction, SMLR) has been shown to generate regulatory T cells in vitro. An in vivo regulatory role has therefore been proposed for the SMLR. To study this role more directly, we examined the effects of repeated iv injection of mice with activated syngeneic B cells. Three such weekly injections induced a suppression of the plaque forming cell response to a subsequent injection of trinitrophenylated keyhole limpet hemocyanin (TNP-KLH). The suppression was transient and could not be maintained by additional injections of activated syngeneic B cells. The suppression was transferable to syngeneic recipients with splenic lymphocytes. Continued weekly iv injections of LPS induced blasts, as well as weekly intraperitoneal injections, caused enhancement rather than inhibition of the response to iv injected TNP-KLH. The enhancement was prevented by injection of anti-L3T4. Spleen cells from mice which had received three iv injections of activated syngeneic cells suppressed an in vitro secondary response to TNP-KLH by normal immune spleen cells. The cells responsible for the immune suppression were Thy 1.2+. The results indicate that repeated exposure to activated B cells causes activation of suppressor pathways but does not bring about a chronic state of immune suppression.     

Transfer of agammaglobulinemia in the chicken. I. Generation of suppressor activity by injection of bursa cells

Spleen cells from adult agammaglobulinemic (bursectomized) chickens taken 1 to 3 weeks after an injection of histocompatible bursa cells can inhibit the adoptive antibody response to B. abortus of normal spleen or bursa cells in irradiated recipients. Spleen cells from Aγ chickens not injected with bursa cells generally do not. Moreover, bursectomized chickens which have been reconstituted with spleen cells within the first week after hatching do not respond with suppressor cell formation upon bursa cell injection. This apparent “autoimmunization” with bursa cells induces suppressor T cells which are only minimally sensitive to treatment with mitomycin C or to 5000 R γ irradiation. The suppressor activity is neither induced nor potentiated by concanavalin A in vivo. It is much stronger in spleen than in thymus cells and appears to be macrophage independent and to require intact cells. The cell component which stimulates the suppressor activity is more pronounced on bursa than on spleen cells, and is at most present to a very limited extent on bone marrow, thymus, or peritoneal exudate cells. It is better represented in comparable cell numbers of Day 17 than of Day 14 embryonic bursa. The inducing cell component is present in the membrane fraction of disrupted bursa cells. Immunization with bursa cells from B locus histoincompatible chickens leads to suppressor activity against histocompatible bursa cells. Although the removal of Ig-bearing cells from bursa greatly diminishes its immunizing capacity, injection of serum IgM and IgG does not induce suppressor cells. It is suggested that tolerance to a B-cell antigen is lacking in adult Aγ chickens, resulting in an autoimmune response upon exposure to B cells. The B-cell antigen may be a cell surface-specific form of Ig, a complex of Ig and a membrane component, or a differentiation antigen which appears simultaneously with Ig during ontogeny.     

Immunity to transplantable carcinogen-induced fibrosarcomas in B2/B2 chickens. I. Use of bacterial adjuvants in induction of immunity demonstrable in Winn tests.

Tumor line specific immunity could be induced in chickens with the use of BCG or CP. Direct contact between bacteria and tumor cells was required for immunity induction, but succeeded only in animals which also had been previously injected with the same bacterial vaccine. While other methods such as iv injected live mitomycin treated tumor was capable of inducing immunity, only BCG or CP mediated immunity was capable of being adoptively transferred in the Winn assay. Bursectomized agamma-globulinemic chickens also produced strong transferable immunity. Crossreactivity between transplant lines was detectable upon rechallenge of immune donors but was not evident upon adoptive immunity transfer. Immunity was detectable in the spleen and in peripheral blood lymphocytes but could not be transferred by thymus cells.     

Immunity to carcinogen-induced transplantable fibrosarcoma in B2B2 chickens: V. Relationship to tumor cell-specific delayed hypersensitivity and serum antibody

Lasting immunity to the chemically induced (DMBA) fibrosarcomas (CHCT-NYU1, 2, and 4) of SC chickens ($\text{B2}\text{B2}$) can be obtained by injection of Corynebacterium parvum (CP)-primed chickens with tumor cells and CP in one wing and tumor cells alone in the other wing. The local delayed hypersensitivity (DH) reaction to CP in the wing inhibits local growth completely, whereas the tumor on the contralateral side shows transient growth. In the present studies, the development of tumor immunity was studied in detail by monitoring DH and antibody formation to the tumor cells and adoptive immunity with spleen cells in Winn tests. Injection of NYU1 cells alone in normal or CP-treated animals induced transient immunity in Winn tests in 50% of the animals, weak DH reactivity, and antibody detectable by immunofluorescence within the first 2 weeks. Chickens receiving both NYU1 cells and CP in one wing and NYU1 cells alone on the other side developed stronger DH reactions to the tumor cells and a higher incidence of immunity in Winn tests which was sustained throughout the period of observation. Antibody levels were similar to those of animals receiving tumor cells alone. In contrast, injection of CP and tumor cells on one side without a tumor challenge on the contralateral side did not induce detectable immunity in CP-primed chickens. Chickens immunized to NYU2 and 4 cells were also tested for DH reactivity and antibody formation. Studies on the cross-reactivity between the tumor lines showed that there was cross-reactivity at the humoral level while at the cellular level this was not apparent. However, animals immune to one tumor line rejected transplants of another tumor line. Observations on the antibody specificity(s) suggested that it was not directed against minor histocompatibility or avian sarcoma viral antigens. SC embryo fibroblasts could induce DH, and serum antibody induced by tumor cells usually reacted also with such embryo cells.     

Alloreactivity against IE-encoded antigens: evidence of the discrepancy between graft rejection and reactivity of IE-reactive T cells.

Participation of IE antigens (Ag) in immune response as the transplantation Ag was examined. IE- B10.A(4R)(4R; Kk, IAk, IE-, Db) mice could not reject skin graft from IE Ag alone-disparate B10.A(2R) (2R; Kk, IAk, IEk, Db) mice despite intravenous (iv) injection of 2R spleen cells (SC) before or after skin grafting, indicating that graft rejection could not be caused across IE Ag-barrier alone. Furthermore, 4R SC could not induce lethal graft-versus-host disease (GVHD) in supralethally (950 rad) irradiated 2R mice. On the other hand, infiltration of lymphoid cells was observed at the site of transplanted 2R skin in 4R mice. SC of 4R mice unprimed or primed with 2R skin or 2R SC showed the capability to proliferate in vitro in response to 2R Ag. In immunofluorescence analysis of lymph node cells (LNC) of 4R mice injected iv with 2R SC 7 days earlier, IE-reactive CD4+Vbeta 11+ T cells did not change in number, but slightly increased the expression of interleukin-2 receptor (IL-2R). In 2R mice irradiated with 670 rad and injected iv with 4R SC 7 days earlier, 4R-derived CD4+V beta 11+ T cells proliferated, changed to blastoid form, and showed a markedly increased expression of IL-2R. To further investigate the influence of IE alloantigens on transplantation immunity, IL-2 production and anti-class I CTL activity were assayed. The 4R SC capable of recognizing IEk and Dk Ag of B10.BR (Kk, IAk, IEk, Dk) generated levels of both IL-2 and CTL activities higher than those of 2R SC capable of recognizing Dk Ag alone. These results strongly suggest that IE alloantigens indirectly act as the transplantation Ag by the stimulation of IE-reactive CD4+ helper T cells resulting in the differentiation of class I-restricted CD8+ T cells.     

Recombinant cholera toxin B subunit activates dendritic cells and enhances antitumor immunity

Activation of dendritic cells (DC) is crucial for priming of cytotoxic T lymphocytes (CTL), which have a critical role in tumor immunity, and it is considered that adjuvants are necessary for activation of DC and for enhancement of cellular immunity. In this study, we examined an adjuvant capacity of recombinant cholera toxin B subunit (rCTB), which is non-toxic subunit of cholera toxin, on maturation of murine splenic DC. After the in vitro incubation of DC with rCTB, the expression of MHC class II and B7-2 on DC was upregulated and the secretion of IL-12 from DC was enhanced. In addition, larger DC with longer dendrites were observed. These data suggest that rCTB induced DC maturation. Subsequently, we examined the induction of tumor immunity by rCTB-treated DC by employing Meth A tumor cells in mice. Pretreatment with subcutaneous injection of rCTB-treated DC pulsed with Meth A tumor lysate inhibited the growth of the tumor cells depending on the number of DC. Moreover, intratumoral injection of rCTB-treated DC pulsed with tumor lysate had therapeutic effect against established Meth A tumor. Immunization with DC activated by rCTB and the tumor lysate increased number of CTL precursor recognizing Meth A tumor. The antitumor immune response was significantly inhibited in CD8+ T cell-depleted mice, although substantial antitumor effect was observed in CD4+ T cell-depleted mice. These results indicated that rCTB acts as an adjuvant to enhance antitumor immunity through DC maturation and that CD8+ T cells play a dominant role in the tumor immunity. Being considered to be safe, rCTB may be useful as an effective adjuvant to raise immunity for a tumor in clinical application.     

Asialo-GM1-positive T killer cells are generated in F1 mice injected with parental spleen cells

(C57BL/6 x DBA/2)F1 mice transplanted with parental C57BL/6 spleen cells become splenic chimeras, show donor antihost cytotoxic T cell activity, and lose their T cell-mediated, humoral, and natural immunity. Injection of anti-asialo-GM1 (ASGM1) into transplanted mice strongly suppresses splenic cytotoxic activity and causes a significant reduction of spleen cells expressing ASGM1, Thy-1, and Lyt-2. In vitro treatment of spleen cells from transplanted mice with antibody and complement shows that the cytotoxic effector cells are ASGM1+, Thy-1+, Lyt-2+, L3T4-, NK1.1-, and H-2d-, hence of donor origin. The cytotoxic effector cells are specific for H-2d targets and lack NK activity. In an attempt to explore whether in vivo elimination of the cytotoxic effector cells has any influence on splenic chimerism or humoral immunity, F1 mice injected with parental splenocytes were treated with anti-ASGM 1. Results show that this treatment eliminates a substantial proportion of cytotoxic effector cells but has no effect on splenic chimerism or restoration of humoral immunity. It therefore appears that cytotoxic effector cells are not primarily responsible for induction of chimerism or suppression of humoral immunity. In support of this injection of parental spleen cells with the nu/nu mutation induces killer cells in F1 mice but fails to induce splenic chimerism or immunosuppression. In contrast, injection of parental spleen cells with the bg/bg mutation generates both splenic chimerism and suppression of humoral immunity although their ability to generate cytotoxic effector cells in F1 hosts is seriously impaired and comparable to the cytotoxic potential of C57BL/6 nu/nu cells. It is concluded that the ASGM1 + cytotoxic T cells are not primarily responsible for splenic chimerism and suppression of humoral immunity and that the two effects are likely caused by parental cells with a different phenotype and function.     

Suppressor cells as an agent of immune facilitation. II. Adoptive transfer of passively induced enhancement of allografted tumors

CBA mice injected intravenously with CBA anti-A/J spleen cell antiserum, and challenged subcutaneously 24 hr later with A/J-derived sarcoma 1 (Sa 1) develop progressive tumors. “Normal” CBA mice (i.e., injected with normal CBA serum or noninjected) reject the allograft within 20 days. Spleen cells taken from mice 20–35 days after the injection of antiserum and Sa 1 challenge can specifically transfer the ability to enhance tumor growth when injected into 200 rad irradiated recipients. Spleen cells taken 8 days after antiserum and Sa 1 challenge cannot transfer suppression. The induction of suppressor cells requires both antiserum treatment and Sa 1 challenge. Serum from suppressed mice, and from control mice that are rejecting their tumors, can also transfer suppression but only when taken 20–35 days after treatment. The suppressor cells function most effectively when transferred at the time of tumor challenge, however, they also inhibit the rejection of Sa 1 when mixed with nylon-wool purified sensitized T cells. Suppressor cells are both nylon-wool nonadherent and adherent. Further purification of the column-enriched cells using antiimmunoglobulin or anti-Thy 1.2, plus complement, suggests that both T and B cells can suppress. The mixed lymphocyte response (MLR) of spleen cells from mice challenged only with Sa 1 is inhibited 8 days after challenge. A secondary response is obtained at 20–27 days. In contrast, the MLR from antiserum and Sa 1-treated mice 8 days after challenge resembles a secondary response. By 20–27 days mice with progressive tumors have only a primary-like response. In the present experimental situation, mitomycin-treated spleen cells from antiserum and Sa 1-treated mice cannot significantly inhibit the MLR of normal cells.     

Establishment of a tumor-specific immunotherapy model utilizing TNP-reactive helper T cell activity and its application to the autochthonous tumor system

Preinduction of potent hapten-reactive helper T cell activity and subsequent immunization with hapten-coupled syngeneic tumor cells result in enhanced induction of tumor-specific immunity through T-T cell collaboration between anti-hapten helper T cells and tumor-specific effector T cells. On the basis of this augmenting mechanism, a tumor-specific immunotherapy protocol was established in which a growing tumor regresses by utilizing a potent trinitrophenyl (TNP)-helper T cell activity. C3H/He mice were allowed to generate the amplified (more potent) TNP-helper T cell activity by skin painting with trinitrochlorobenzene (TNCB) after pretreatment with cyclophosphamide. Five weeks later, the mice were inoculated intradermally with syngeneic transplantable X5563 tumor cells. When TNCB was injected into X5563 tumor mass, an appreciable number of growing tumors, in the only group of C3H/He mice in which the amplified TNP-helper T cell activity had been generated were observed to regress (regressor mice). These regressor mice were shown to have acquired tumor-specific T cell-mediated immunity. Such immunity was more potent than that acquired in mice whose tumor was simply removed by surgical resection. These results indicate that in situ TNP haptenation of the tumor cells in TNP-primed mice can induce the enhanced tumor-specific immunity leading to the regression of a growing tumor. Most importantly, the present study further investigates the applicability of this TNP immunotherapy protocol to an autochthonous tumor system. The results demonstrate that an appreciable percent of growing methylcholanthrene-induced autochthonous tumors regressed by the above TNP immunotherapy protocol. Thus, the present model provides an effective maneuver for tumor-specific immunotherapy in syngeneic transplantable as well as autochthonous tumor systems.     

Chicken T lymphocyte clones with specificity for Eimeria tenella. I. Generation and functional characterization

T lymphocyte clones reacting specifically with the antigenic components of Eimeria tenella were generated from splenic lymphocytes of immunized chickens and were maintained for 12 to 14 wk in vitro. These T cell growth factor-dependent T lymphocyte clones from bursectomized and normal chickens proliferated in vitro when stimulated with antigens from different developmental stages of homologous but not heterologous species of the parasite. Specific proliferative responses of the cloned T cells showed an absolute requirement for antigen presentation by histocompatible antigen-presenting cells. Some of the T cell clones exhibited functionally discrete interactions with syngeneic primed B cells; 25% of the T cell clones from immunized normal chickens and 7% of those obtained from immunized bursectomized chickens showed antigen-dependent helper activity and induced specific antibody production by syngeneic primed B cells. Of the T cell clones from immunized normal chickens, 19% showed suppression of in vitro antibody production in comparison to 7% of those isolated from immunized bursectomized chickens. The frequency of cloned T cells with ability to induce cytotoxic activity in macrophages against the sporozoites of E. tenella was much higher in those isolated from bursectomized chickens (80%) than in those isolated from normal chickens. Because both bursectomized and normal chickens can be immunized by repeated infections, differences in the distribution among cloned T cells suggest different effector mechanisms of immunity against coccidiosis in these chickens. Lack of B cells seem to affect the development of T cell immunity as reflected by slower development of immunity and enhanced activation of cytotoxic T cell function.     

Dendritic cells charged with apoptotic tumor cells induce long-lived protective CD4+ and CD8+ T cell immunity against B16 melanoma

Dendritic cells (DCs) are potent APCs and attractive vectors for cancer immunotherapy. Using the B16 melanoma, a poorly immunogenic experimental tumor that expresses low levels of MHC class I products, we investigated whether DCs loaded ex vivo with apoptotic tumor cells could elicit combined CD4(+) and CD8(+) T cell dependent, long term immunity following injection into mice. The bone marrow-derived DCs underwent maturation during overnight coculture with apoptotic melanoma cells. Following injection, DCs migrated to the draining lymph nodes comparably to control DCs at a level corresponding to approximately 0.5% of the injected inoculum. Mice vaccinated with tumor-loaded DCs were protected against an intracutaneous challenge with B16, with 80% of the mice remaining tumor-free 12 wk after challenge. CD4(+) and CD8(+) T cells were efficiently primed in vaccinated animals, as evidenced by IFN-gamma secretion after in vitro stimulation with DCs loaded with apoptotic B16 or DCs pulsed with the naturally expressed melanoma Ag, tyrosinase-related protein 2. In addition, B16 melanoma cells were recognized by immune CD8(+) T cells in vitro, and cytolytic activity against tyrosinase-related protein 2(180-188)-pulsed target cells was observed in vivo. When either CD4(+) or CD8(+) T cells were depleted at the time of challenge, the protection was completely abrogated. Mice receiving a tumor challenge 10 wk after vaccination were also protected, consistent with the induction of tumor-specific memory. Therefore, DCs loaded with cells undergoing apoptotic death can prime melanoma-specific helper and CTLs and provide long term protection against a poorly immunogenic tumor in mice.     

Suppression of generation of concomitant antitumor immunity by passively transferred suppressor T cells from tumor-bearing donors

Summary Infusion of normal recipient mice with suppressor T cells from donors bearing a progressive Meth A fibrosarcoma results in a diminished capacity of the recipients to generate concomitant and postexcision antitumor immunity. The passive transfer of suppressor cells which prevented the generation of immunity to the Meth A fibrosarcoma did not affect the capacity of the recipients to reject an allogeneic tumor. The data provides direct evidence in support of the hypothesis that suppressor T cells, generated at later stages of growth of Meth A fibrosarcoma, function to down-regulate an already acquired mechanism of concomitant immunity.This work was supported by Grants CA-16642 and CA-17794 from the National Cancer Institute, Grant RR-05705 from the Division of Research Resources, NIH, and a grant from R. J. Reynolds Industries, Inc     

Characterization of cytostatic effector lymphocytes during the development of a syngeneic lymphosarcoma in C3H mice: use of monoclonal reagents to identify T-cell subsets

The development of the in vitro cytostatic capacity of splenic lymphocyte subpopulations from C3H mice carrying the syngeneic Gardner tumor was examined at different times after intramuscular tumor injection. Most mice died between 3 to 6 weeks after tumor injection, while some rejected their tumors or survived longer than 3 months. Cell separation procedures and monoclonal antibodies against T-cell subsets were used to identify the cells responsible in anti-tumor immunity. Cytostatic capacity against tumor cells developed in the T-cell enriched subpopulation of splenocytes 3 days after tumor injection and was partly abrogated by anti-Lyt-1. Effector function of Lyt-2+ T cells and B cells developed later and peaked at around 10 days after tumor injection. Another cell population with cytostatic capacity which was not blocked by anti-Lyt-1, anti-Lyt-2, or anti-Ly-5 was noted to develop early after tumor injection and lacked both T-cell and B-cell markers ("null"). This subpopulation was eluted with T cells from nylon wool columns and comprised up to 50% of the T-enriched fraction of splenocytes in later stages of tumor growth. An interesting characteristic of these "null" cells was susceptibility to T-cell suppression both in early and later stages of tumor growth except in regressor mice which lacked suppressor T cells. The cytostatic capacity of the "null" cells could be restored either by removal of Thy-1+ cells from the T-enriched fraction by panning, or the addition of anti-Thy-1 or F(ab')2 fragments of anti-Thy-1 to the lymphocyte-tumor reaction mixtures. Most mice examined after 10 days of tumor growth were immunosuppressed to varying degrees. Unseparated splenocytes from these mice were not cytostatic but removal of T cells allowed the B cells to exert their cytostatic capacity. A strong underlying B-cell cytostasis was shown to be present in long survivor mice even though their unseparated spleen cells were only weakly cytostatic. T cells did not play a role in the regression of tumors or long-term survival of tumor bearer mice. Splenocytes from regressor mice were strongly cytostatic, their anti-tumor activity residing in the "null" and B-cell populations.     

The therapeutic significance of concomitant antitumor immunity

It is shown that progressive growth of the SA1 sarcoma in its semisyngeneic AB6F1 host results in the generation of concomitant immunity to growth of a tumor challenge implant, and in the generation of T cells in the spleen capable, on passive transfer, of causing regression of an established tumor in gamma-irradiated recipients, but not in normal recipients. T cells that passively transferred concomitant immunity against an established tumor were first generated around day 6 of tumor growth, reached peak numbers on day 9, and slowly decreased in number thereafter. They were of the Ly-1-2+ phenotype, in that they were functionally eliminated by treatment with monoclonal anti-Ly-2 antibody and complement, but not by treatment with anti-Ly-1 antibody and complement. The paradoxical ability of T cells from a donor with a relatively large tumor to cause the regression of a tumor in sublethally gamma-irradiated recipients is explained with reference to the facts that the recipient tumor was only half as large as the donor tumor at the time of passive transfer, and that the recipient was incapable of generating suppressor T cells that would function to inhibit the expression of adoptive immunity.