Progressor Tumors Research Articles

Type I interferon activates MHC class I-dressed CD11b+ conventional dendritic cells to promote protective anti-tumor CD8+ T cell immunity

Tumor-infiltrating dendritic cells (DCs) assume varied functional states that impact anti-tumor immunity. To delineate the DC states associated with productive anti-tumor Tcell immunity, we compared spontaneously regressing and progressing tumors. Tumor-reactive CD8+ Tcell responses in Batf3-/- mice lacking type 1 DCs (DC1s) were lost in progressor tumors but preserved in regressor tumors. Transcriptional profiling of intra-tumoral DCs within regressor tumors revealed an activation state of CD11b+ conventional DCs (DC2s) characterized by expression of interferon (IFN)-stimulated genes (ISGs) (ISG+ DCs). ISG+ DC-activated CD8+ Tcells exvivo comparably to DC1. Unlike cross-presenting DC1, ISG+ DCs acquired and presented intact tumor-derived peptide-major histocompatibility complex class I (MHC class I) complexes. Constitutive type I IFN production by regressor tumors drove the ISG+ DC state, and activation of MHC class I-dressed ISG+ DCs by exogenous IFN-β rescued anti-tumor immunity against progressor tumors in Batf3-/- mice. The ISG+ DC gene signature is detectable in human tumors. Engaging this functional DC state may present an approach for the treatment of human disease.

Bronchioloalveolar invasion in non-small cell lung cancer is associated with expression of transforming growth factor-β1

BackgroundAdenocarcinoma in situ (AIS) and minimally invasive adenocarcinoma (MIA) with fibrous stromal invasion are newly introduced subtypes of small lung adenocarcinoma. AIS is a small localized adenocarcinoma in which growth is restricted to neoplastic cells along preexisting alveolar structures without fibrous stromal invasion. In MIA, by contrast, tumor cells have infiltrated the myofibroblastic stroma. Transforming growth factor (TGF)-β is known to be produced by progressor tumors, and excessive TGF-β contributes to a pathological excess of tissue fibrosis. TGF-β1 is the most abundant isoform, and its expression is a key event fostering tumor invasion and metastasis. We therefore analyzed the relationship between TGF-β1 expression and clinicopathological microinvasion in patients with small lung adenocarcinoma.MethodsThe study participants were 45 patients who underwent curative surgery for AIS and MIA 3 cm or less in size. Those tumors were assessed based on immunohistochemical staining using anti-TGF-β1 antibody. The TGF-β1 status was assessed immunohistochemically using the Allred 8-unit system.ResultsThe rates of TGF-β1 positivity in the AIS and MIA groups were 27.3% and 65.2%, respectively (P <0.05). The median of Allred score was 0.5 (range 0–5) in the AIS group and 3.0 (range 0–6) in the MIA group (P = 0.0017).ConclusionsWe suggest that TGF-β1 expression is likely to be significantly stronger in patients with MIA than in those with AIS, and the increased expression may be associated with minimal invasion and infiltration of the myofibroblastic stroma.

IL-17D mediated cancer rejection (162.26)

Abstract Immune system interaction with cancers can either enhance or prevent tumor formation. Cytokine secretion by immune infiltrates and tumor cells are important in regulating this process. IL-17A, a pro-inflammatory member of the IL-17 family, has been shown to have conflicting results in regards to cancer progression or regression. Other members of the IL-17 family, such as IL-17D, have no known roles in immune responses to cancer, or endogenous function. Our lab possesses matched methylcholanthrene (MCA)-induced tumor cell lines that will continuously grow (Progressors) or reject (Regressors) after transplantation into syngeneic WT mice. The regresssor cell lines can grow in immune-deficient RAG2-/- mice, indicating that their rejection in WT mice requires an intact immune system. Our lab has used these cell lines to identify differential cytokine expression profiles from microarray analysis. We have obtained data that shows IL-17D transcript and protein are significantly overexpressed in regressor cell lines when compared to progressor cell lines. Overexpression of IL-7D in progressor tumor cell lines leads to their rejection in WT mice in a NK cell dependent manner. We hypothesize that IL-17D expression in regressor cell lines leads to immune-mediated tumor rejection by recruiting NK cells that polarize M1 macrophages in the tumor microenvironment.

Inhibition of dendritic cell migration by transforming growth factor-β1 increases tumor-draining lymph node metastasis

BackgroundTransforming growth factor (TGF)-β is known to be produced by progressor tumors and to immobilize dendritic cells (DCs) within those tumors. Moreover, although TGF-β1 has been shown to promote tumor progression, there is still no direct, in vivo evidence as to whether TGF-β1 is able to directly induce distant metastasis.MethodsTo address that issue and investigate the mechanism by which TGF-β1 suppresses DC activity, we subdermally inoculated mouse ears with squamous cell carcinoma cells stably expressing TGF-β1 or empty vector (mock).ResultsThe numbers of DCs within lymph nodes draining the resultant TGF-β1-expressing tumors was significantly lower than within nodes draining tumors not expressing TGF-β1. We then injected fluorescently labeled bone marrow-derived dendritic cells into the tumors, and subsequent analysis confirmed that the tumors were the source of the DCs within the tumor-draining lymph nodes, and that there were significantly fewer immature DCs within the nodes draining TGF-β1-expressing tumors than within nodes draining tumors not expressing TGF-β1. In addition, 14 days after tumor cell inoculation, lymph node metastasis occurred more frequently in mice inoculated with TGF-β1 transfectants than in those inoculated with the mock transfectants.ConclusionsThese findings provide new evidence that tumor-derived TGF-β1 inhibits migration of DCs from tumors to their draining lymph nodes, and this immunosuppressive effect of TGF-β1 increases the likelihood of metastasis in the affected nodes.

The Immune-Modulating Cytokine and Endogenous Alarmin Interleukin-33 Is Upregulated in Skin Exposed to Inflammatory UVB Radiation

The cellular and molecular mechanisms by which UV radiation modulates inflammation and immunity while simultaneously maintaining skin homeostasis is complex and not completely understood. Similar to the effects of UV, IL-33 has potent immune-modulating properties that are mediated by the downstream induction of cytokines and chemokines. We have discovered that exposure of mice in vivo or human skin samples ex vivo to inflammatory doses of UVB induced IL-33 expression within the epidermal and dermal skin layers. Using a combination of murine cell lines and primary human cells, we demonstrate that both UV and the oxidized lipid platelet activating factor induce IL-33 expression in keratinocytes and dermal fibroblasts. Highlighting the significance of these results, we found that administering IL-33 to mice in vivo suppressed the induction of Th1-mediated contact hypersensitivity responses. This may have consequences for skin cancer growth because UV-induced squamous cell carcinomas that evade immunological destruction were found to express significantly higher levels of IL-33. Finally, we demonstrate that dermal mast cells and skin-infiltrating neutrophils closely associate with UV-induced IL-33-expressing fibroblasts. Our results therefore identify and support a role for IL-33 as an important early danger signal produced in response to inflammation-inducing UV radiation.

Intratumoral IL-12 Gene Therapy Results in the Crosspriming of Tc1 Cells Reactive Against Tumor-associated Stromal Antigens

HLA-A2 transgenic mice bearing established HLA-A2(neg) B16 melanomas were effectively treated by intratumoral (i.t.) injection of syngeneic dendritic cells (DCs) transduced to express high levels of interleukin (IL)-12, resulting in CD8(+) T cell-dependent antitumor protection. In this model, HLA-A2-restricted CD8(+) T cells do not directly recognize tumor cells and therapeutic benefit was associated with the crosspriming of HLA-A2-restricted type-1 CD8(+) T cells reactive against antigens expressed by stromal cells [i.e., pericytes and vascular endothelial cells (VEC)]. IL-12 gene therapy-induced CD8(+) T cells directly recognized HLA-A2(+) pericytes and VEC flow-sorted from B16 tumor lesions based on interferon (IFN)-γ secretion and translocation of the lytic granule-associated molecule CD107 to the T cell surface after coculture with these target cells. In contrast, these CD8(+) T effector cells failed to recognize pericytes/VEC isolated from the kidneys of tumor-bearing HHD mice. The tumor-associated stromal antigen (TASA)-derived peptides studied are evolutionarily conserved and could be recognized by CD8(+) T cells harvested from the blood of HLA-A2(+) normal donors or melanoma patients after in vitro stimulation. These TASA and their derivative peptides may prove useful in vaccine formulations against solid cancers, as well as, in the immune monitoring of HLA-A2(+) cancer patients receiving therapeutic interventions, such as IL-12 gene therapy.

Abstract 1929: CD4+ and CD8+ neu-reactive T cells are sensitized by breast cancer stem-like cells (BCSC) in tolerized Her-2/neu transgenic mice and can mediate tumor regression upon adoptive transfer

Abstract There is growing evidence that a variety of tumors including breast cancer may be driven by a small subset of cancer stem/progenitor cells that exhibit chemotherapeutic resistance and cause tumor relapse. The eradication of this small subset of cells might improve therapeutic outcome. However, it is unknown whether breast cancer stem/progenitor cells are susceptible to T-cell mediated immunotherapy. We used defined serum-free conditions to establish primary neupos BCSC lines. Spontaneous breast tumors arising in neu-N transgenic mice were passaged in vivo to generate progressor lines and tumors were harvested and prepared as a single cell suspension. Paired aliquots of tumor cells were cultured in either serum-free medium supplemented with EGF and bFGF to derive BCSC sub-lines, or alternatively in standard serum-containing medium to generate mouse mammary carcinoma (MMC) sub-lines. The BCSC cells display morphologic characteristics of breast cancer stem/progenitor cells, such as non-attached growth in spheroid colonies, and high expression of CD44. We vaccinated neu-N transgenic mice with 2×107 irradiated MMC10-BCSC or its paired cell line MMC10-AD and harvested vaccine-draining lymph nodes 9 days later. Antigen-primed CD62Llow cells were isolated using MACS beads and CD62LlowCD4+ or CD62LlowCD8+ T cells were additionally purified and stimulated with anti-CD3/IL-2/IL-7 for two stimulation cycles. Adoptive transfer of 3×107 CD62Llow CD4+ cells from MMC10-BCSC-draining LNs effectively eradicated pulmonary metastases (p&amp;lt;0.001). By contrast, 3×107 CD62Llow CD4+ T cells from MMC10-AD-draining LNs was sub-therapeutic (p&amp;gt;0.05 vs. control). In an independent experiment, adoptive transfer of 3×107 CD8+ T cells sensitized by MMC10-BCSCs mediated the regression of established MMC10 pulmonary metastasis (p&amp;lt;0.001), whereas 6×107 CD8+ cells primed by MMC10-AD was ineffective. More importantly, adoptive transfer of 4 × 107 in vitro-activated CD8+ cells combined with 2×107 CD4+ cells derived from LNs draining MMC10-BCSCs are effective against the parental MMC10 line as well as an independently derived neupos line MMC-1. Also, transfer of either 3×107 CD4+ or CD8+ T cells from LNs draining MMC10-BCSCs effectively mediated regression of established 3-day neupos MMC4 pulmonary metastasis. In conclusion, irradiated whole tumor cell vaccines derived from neupos BCSCs can prime T-cells in neu-tolerant hosts. Although this immune response is insufficient to prevent progressive tumor growth, highly activated effector function and large numbers of CD4+ and CD8+ T cells can be generated through in vitro activation with anti-CD3/IL-2/IL-7 stimulation. Either CD4+ or CD8+ T cells are cross-protective against other neu-positive tumor metastases indicating that this approach provides MHC class I and II immune responses. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1929.

Comparative Analysis of Regulatory and Effector T Cells in Progressively Growing versus Rejecting Tumors of Similar Origins

Although regulatory T cells (Tregs) have been detected in clinically apparent and experimentally induced tumors, the significance of their presence is obscured because past studies examined late-stage tumors that had formed in immunocompetent hosts and thus had evolved mechanisms to escape immunologic recognition and/or elimination. Herein, we report the first comparative analysis of the antitumor response to 3'-methylcholanthrene-induced tumors, which either grow progressively (progressor tumors) or are rejected by the immune system (regressor tumors). Surprisingly, we found that both progressor and regressor tumors harbored proliferating (i.e., activated) Foxp3+CD25+Tregs. However, progressor tumors contained a higher percentage of Tregs in the lymphocyte subset versus regressor tumors. The Tregs in progressor tumors were derived from peripheral CD25+ natural Tregs, accumulated early after tumor challenge and were actively proliferating, suggesting that progressor tumors recruited and/or activated endogenous Tregs as a mechanism of escaping immune destruction. To explore whether Tregs directly contributed to the progressive growth phenotype of progressor tumors, we monitored tumor outgrowth in naive wild-type recipients pretreated with either a control monoclonal antibody (mAb) or a depleting CD25-specific mAb. In mice predepleted of CD25+ cells, the tumors that subsequently developed displayed an increased accumulation of proliferating CD8+ T cells and were rejected. These results show that, although Tregs are activated in both regressor and progressor tumors, the ratio of regulatory to effector T cells is critical in determining whether the host successfully rejects the tumor or eventually succumbs to tumor outgrowth.

Transforming growth factor-beta produced by progressor tumors inhibits, while IL-10 produced by regressor tumors enhances, Langerhans cell migration from skin.

The induction of epidermal immunity depends on activation of local dendritic cells (DC), Langerhans cells (LC), to migrate from the skin to local lymph nodes and mature into potent immunostimulatory cells. We have previously shown that progressor skin tumors, which evade immunological destruction, prevent contact sensitizer-induced LC migration from the skin to draining lymph nodes. In contrast, regressor tumors, which evoke protective immunity, did not inhibit DC mobilization. In this study we utilized the skin explant model to determine the factors produced by skin tumors which regulate LC migration from the skin. Supernatants from two progressor squamous cell carcinoma lines both inhibited LC migration, whereas supernatants from two regressor squamous cell carcinoma lines both enhanced LC mobilization. Transforming growth factor (TGF)-beta1 inhibited, while IL-10 enhanced, LC migration from cultured skin. Both reduced the ability of LC to mature into potent allostimulators. Antibody neutralization identified that TGF-beta1 produced by the progressor tumor was responsible for inhibition of LC migration, while IL-10 produced by the regressor tumor enhanced LC mobilization. Thus these studies show that skin tumors influence DC mobilization from tumors by production of cytokines, and that TGF-beta1 is one factor produced by tumors which can immobilize LC and keep them in an immature form. This is likely to be an important mechanism of tumor escape from the immune system as progressor tumors inhibited, while regressor tumors enhanced DC mobilization.

Enhancement of tumorigenicity and invasion capacity of rat mammary adenocarcinoma cells by epidermal growth factor and transforming growth factor-beta.

We have studied the effects of growth factors and cytokines on the tumorigenicity and invasion capacity of tumor cells by using regressor and progressor tumor cell lines (ER‐1 and ERpP, respectively) derived from an SHR rat mammary adenocarcinoma. ER‐1 cells regress spontaneously whereas ERpP cells show invasive growth and high metastasis to lung and other organs in syngeneic SHR rats. When ER‐1 cells were pretreated with either epidermal growth factor (EGF) or transforming growth factor‐β (TGF‐β) for 24 h in vitro, and intraperitoneally transplanted into SHR rats, they grew and killed the host, whereas ER‐1 cells pretreated with tumor necrosis factor‐α did not. Tumorigenicity and invasion capacity of ERpP cells were also enhanced by treatment with EGF and TGF‐β. The ER‐1 cells pretreated with EGF, once grown in vivo, had acquired irreversible tumorigenicity and invasion capacity without requiring further EGF treatment, and the enhanced malignancy was irreversible. These findings suggest that growth factors play an important role in acquisition of malignancy of tumor cells.

In vivo T-cell activation by staphylococcal enterotoxin B prevents outgrowth of a malignant tumor.

Treatment of T cells with staphylococcal enterotoxins in vitro is known to activate T cells in a subset restricted manner based on beta-chain variable region (V beta) gene expression. In particular, staphylococcal enterotoxin B (SEB) activates T cells bearing V beta 7 or V beta 8. We examined the ability of SEB to activate T cells in vivo. Treatment of C3H mice with doses of SEB ranging from 5 to 250 micrograms resulted in a dose-dependent activation of V beta 8+ T cells as reflected by increased interleukin 2 receptor (IL-2R) expression, proliferation to exogenous IL-2 and allogeneic cells, and production of gamma interferon. SEB also caused proliferation of the CD8+ subset of V beta 8+ cells in vivo. Thus, T-cell activation by SEB in vivo appears to be specific since V beta 2+ cells (non-SEB reactive) did not show increases in IL-2R expression similar to those seen with V beta 8+ cells nor did they proliferate. We then studied the ability of these activated cells to potentiate the immune response to a malignant progressor tumor. Treatment of C3H mice with 50 micrograms of SEB at the time of inoculation with tumor fragments resulted in a statistically significant decrease in the frequency of tumor outgrowth. These data demonstrate that treatment of C3H mice with SEB results in specific activation of V beta 8+ cells in vivo and that these activated cells are capable of preventing the outgrowth of a malignant tumor.

Animals bearing malignant grafts reject normal grafts that express through gene transfer the same antigen.

Breaking the state of immunological unresponsiveness of tumor-bearing individuals to cancer is a prerequisite for active or passive tumor-specific immunotherapy. To study this problem the immunogenic MHC class I antigen, K216 was transfected into a progressor tumor. The transfected tumors were regularly rejected by normal mice but grew progressively in mice bearing nontransfected tumors. In addition, transgenic mice were derived to obtain normal cells and tissues expressing the same K216 gene product. Normal mice rejected K216-positive normal or malignant tissue grafts and generated K216-specific CTL in vitro and in vivo in response to these challenges. In contrast, mice bearing nontransfected tumors, though rejecting K216-positive nonmalignant tissue grafts, did not reject K216-positive tumors nor generate K216-specific CTL in response to K216-positive tumor cells. Mice bearing K216-positive tumors also rejected the nonmalignant K216-positive tissue grafts, but this in vivo response failed to lead to rejection of the simultaneously present tumor graft expressing the same antigen; in fact, immunity had no measurable effect whatsoever on tumor size or incidence and caused no selection for antigen loss variants. Taken together, the present findings suggest that transfer of expression of a target antigen into nonmalignant cells provides a way for obtaining effective stimulation of antigen-specific CTL in tumor-bearing mice, but that additional manipulations will be required to cause immunological rejection of established tumors.

Mechanism of tumor rejection in anti-CD3 monoclonal antibody-treated mice.

The present study was undertaken to determine the mechanism of tumor rejection in mice treated with low dose anti-CD3 mAb. It was found that treated mice developed nonrestricted antitumor cytolytic spleen cells of the Thy-1+, asialo GM-1+, CD4-, CD8- phenotype. Although these cells might play a role in immunopotentiating some immune responses, in vivo depletion studies using anti-asialo GM-1 mAb demonstrated that these cells were not involved in the rejection of the progressor tumor, 1591-PRO4L, by anti-CD3 mAb-treated mice. Mice treated with anti-CD3 did develop lasting tumor specific immunity as demonstrated by their ability to reject PRO4L on tumor rechallenge while being unable to reject an unrelated UV-induced tumor. The specificity of this memory implicated T cells in the response to PRO4L in anti-CD3-treated mice. Using in vivo T cell subset depletion of anti-CD3-treated animals, it was shown that both CD4+ and CD8+ T cells are required for anti-CD3-induced tumor rejection. The CD4+ cells provide helper function and are only required in the early rejection period, whereas CD8+ cells are required throughout the immune response. In fact, examination of rejecting tumors from treated animals revealed the presence of tumor-specific CD8+ cytolytic T cells capable of cytolysis immediately after removal from the rejecting PRO4L tumor. Thus, in vivo treatment with anti-CD3 mAb likely results in the pan-stimulation of the entire T cell population, which enhances the generation of specific CD8+ T cells, which then eliminate the tumor.

RELATIONSHIP BETWEEN INDUCIBLE H-2 EXPRESSION AND THE IMMUNOGENICITY OF MURINE SKIN NEOPLASMS

Our objective was to determine the relationship between major histocompatibility complex class I molecule expression and the tumorigenic properties of cutaneous neoplasms induced by ultraviolet radiation or chemical carcinogens. All tumors tested were found to express low constitutive levels of MHC class I molecules in vitro as determined by indirect immunofluorescence and flow cytometry. Those tumors capable of growth in UVR-exposed but not in normal recipients (regressors) were found to express enhanced levels of H-2Kk following incubation in the presence of gamma-IFN. In contrast, only one of the tumors that were capable of growth in normal recipients (progressors) exhibited more than moderate enhancement of H-2Kk expression in response to gamma-IFN. Analysis of tumor variants obtained by conversion of a UVR-induced regressor tumor to the progressor phenotype by passage through sublethally gamma-irradiated hosts, or the generation of regressor tumors by mutagen exposure of a benz [A] pyrene (BAP) induced progressor tumor, further supported the direct relationship between tumor immunogenicity in vivo and the capacity to elevate H-2Kk expression in response to gamma-IFN. No correlation existed between H-2Dk expression by the tumors and their transplantation phenotype. Furthermore, we failed to observe MHC class II expression by any of the tumors tested. Finally, the growth rate of a regressor tumor implanted into UVR-exposed hosts was significantly reduced if the tumor was pretreated with gamma-IFN in vitro prior to inoculation. This result suggests that UVR-exposed animals may be deficient in their ability to enhance the expression of MHC class I molecules on developing tumors. This alteration may, in part, account for the state of tumor susceptibility caused by UVR exposure.

In vivo administration of anti-CD3 prevents malignant progressor tumor growth.

Malignant progressor tumors are only weakly immunogenic and can evade host recognition and rejection. One approach to therapy involves activation of the host antitumor cellular effector mechanisms. Since monoclonal antibodies to CD3 (anti-CD3) can activate T cells in vitro, an attempt was made to determine if tumor immunity could be achieved by the administration of anti-CD3 in vivo. T lymphocytes from mice injected with anti-CD3 showed increased interleukin-2 receptor (IL-2R) expression, increased proliferation to recombinant IL-2 (rIL-2), and enhanced reactivity in both an allogeneic mixed lymphocyte reaction and a mixed lymphocyte tumor culture. Malignant tumor growth in treated mice was also examined. The anti-CD3 treatment prevented tumor outgrowth that would have killed untreated animals and also stimulated an in vivo response against a malignant progressor tumor providing lasting tumor immunity.

Role of humoral immunity in progressive and regressive and metastatic growth of the canine transmissible venereal sarcoma.

Canine transmissible venereal sarcoma (CTVS) is a contagious neoplasm which regresses spontaneously in adult dogs but metastasizes and kills puppies transplanted with the neoplasm at a very young age. Immunofluorescence studies showed that 30 +/- 14% of cells from steady-state and 22 +/- 7% of cells from regressing tumors had membrane-bound antibodies which could be eluted out with warm washes at 24 degrees C, whereas the cells from progressor tumors had very little such antibody (6 +/- 6%). Time-course kinetics of anti-CTVS antibodies in the serum of tumor-bearing dogs did not correlate well with tumor volume, however, the presence of such antibodies in adult dogs (47 +/- 13%) but absence (0%) in the puppies with tumor metastasis suggested the importance of antibodies in resistance to metastasis.

Protective immunity to progressive tumors can be induced by antigen presented on regressor tumors.

Immunization of animals with 1591-RE tumor cells, a highly immunogenic UV-induced epithelia cell tumor from C3H/HeN mice, that were haptenated with trinitrophenol (TNP) leads to protective immunity against a challenge of TNP-haptenated 3152-PRO tumor cells, a progressive highly malignant. MCA-induced fibrosarcoma from syngeneic mice. Animals that rejected TNP-1591-RE and subsequently TNP-3152-PRO tumor cells showed increased tumor-specific resistance to another challenge of 3152-PRO tumor cells, even when these fibrosarcoma cells had not been haptenated with TNP. Induction of protection required the presence of TNP-hapten groups on both 1591-RE and 3152-PRO during the initial immunization, and could be induced by immunization with other haptenated syngeneic highly immunogenic regressor tumor lines. In addition, TNP-haptenated progressor variants of the 1591-RE were ineffective in generating protection, suggesting that the immunogenicity of the haptenated tumor used for the initial immunization was a determining factor in whether or not protective immunity against the highly malignant tumor was later generated. Protection required at least two T cell types: a Lyt-1-2+ T cells, and a Lyt-1+2- T cell that also expressed I-J determinants and was Vicia villosa lectin adherent, suggesting it was not a classical helper T cell. These results suggest that presentation of a hapten by highly immunogenic tumor cells can lead to enhanced protective immunity to poorly immunogenic noncross-reactive tumors that co-express the same hapten, and rejection of these haptenated poorly immunogenic tumors leads to enhanced protection against a subsequent challenge of the same, but not noncross-reactive progressor tumors.

Identification of a unique tumor antigen as rejection antigen by molecular cloning and gene transfer.

Tumor-specific transplantation antigens are antigens that can lead to complete immunological destruction of a transplanted cancer by the syngeneic host. When such antigens are expressed on cancers induced by chemical or physical carcinogens, then they are usually unique, i.e., antigenically different for each independently induced tumor. In this study, we show that the product of a gene encoding a novel MHC class I molecule and isolated from the murine UV light-induced regressor tumor 1591 represents one such unique tumor-specific transplantation antigen that causes tumor rejection. The major evidence comes from our finding that 1591 progressor variants regularly lost the gene encoding this antigen that is expressed in the parental tumor that regresses in normal mice; furthermore, reintroduction of this gene into a 1591 progressor variant by DNA transfection caused the progressor variant to regress in normal immunocompetent mice. Thus, the progressor tumor reverted to the parental regressor phenotype following transfection. Consistent with the conclusion that the expression of the novel MHC class I gene following transfection was responsible for the regressor phenotype is also our finding that a variant of the transfected tumor that had lost expression of the transfected gene resumed its progressive growth behavior. Finally, we show that the molecule encoded by the novel class I gene is specifically recognized by a syngeneic tumor-specific cytolytic T cell clone that we have previously shown to select in vitro for progressor variants from the parental regressor tumor cell line. It remains to be determined to what extent unique tumor-specific rejection antigens of other highly immunogenic regressor tumors are encoded by novel MHC class I genes and whether these genes represent germline mutations or somatic mutations caused by the carcinogen treatment.

Identification of a unique tumor-specific antigen as a novel class I major histocompatibility molecule.

Cancers induced by physical or chemical carcinogens express tumor-specific antigens that are uniquely specific for any given tumor; therefore, there is a seemingly endless variety of these unique antigens. We have studied a UV-induced fibrosarcoma, designated 1591, to elucidate the obscure molecular nature and genetic origins of unique tumor-specific antigens. A monoclonal antibody raised against syngeneic 1591 tumor cells has unique tumor specificity. This tumor-specific monoclonal antibody precipitated from the tumor a 45-kDa molecule associated with a 12-kDa molecule having the pI of beta2-microglobulin. This and other evidence indicated that the 1591 tumor expresses a novel class I molecule. A 1591 variant selected for the absence of binding to the monoclonal antibody lacked the novel class I MHC molecule as well as reactivity with cytotoxic T lymphocytes specific for the 1591 tumor. Furthermore, tumor cells bearing the antigen are rejected while variants that have lost the antigen grow progressively. Fourteen of 14 host-selected progressor tumor variants lost reactivity with the monoclonal antibody and provided further evidence that this novel class I molecule is a transplantation antigen on the parental 1591 tumor required for immune rejection. The identification of a unique tumor-specific antigen as a novel class I major histocompatibility complex gene product allows us to search for the possible genetic mechanisms involved and to explore further the role such molecules play in tumor immunity and malignancy.

Pecking order among tumor-specific antigens.

The ultraviolet light-induced fibrosarcoma 1591 is regularly rejected upon transplantation into young syngeneic mice; in rare instances, however, this tumor grows progressively and the tumors that develop are then heritably stable variant progressor tumors (1591-PRO tumors). In this study, we have induced transplantation resistance to 1591-PRO tumors and determined which antigens were recognized by mice that rejected these progressor tumors. We found that cytolytic T cells of such mice recognized a 1591-specific antigen that was present not only on all the independently derived 1591-PRO tumors but also on the parental regressor tumor (1591-RE). However, the cytolytic immune response of mice that rejected 1591-RE lysed 1591-RE tumor cells but not 1591-PRO tumor cells. Thus, the 1591-RE tumor seemed to express two antigens that were specific for tumors of the 1591 lineage, one that was lost and a second that was retained by 1591-PRO tumor cells. Mice challenged with 1591-R# tumor cells mounted a response to only one of the tumor-specific antigens which was therefore "immunodominant" over the other "immunorecessive" antigen. This immunorecessive antigen became the target of the immune response once the immunodominant antigen was lost. This "pecking order" interfered with the simultaneous recognition of two tumor-specific antigens and this mechanism may favor immune escape.