H2-O inhibits presentation of bacterial superantigens, but not endogenous self antigens.

H2-O/HLA-DO are MHC class II accessory molecules that modulate exogenous Ag presentation. Most class II accessory molecules are expressed in all professional APC; however, H2-O is only expressed in B cells and medullary thymic epithelial cells. Because B cells present exogenous Ags and superantigens (SAgs), and medullary thymic epithelial cells are specialized APC for self Ags during negative selection in the thymus, we have hypothesized that H2-O might play a role in MHC class II-restricted SAg and self Ag presentation. In this study, we demonstrate that H2-O expression inhibits presentation of the bacterial SAgs staphylococcal enterotoxins A and B to four SAg-reactive T hybridoma cells. In contrast, H2-O has no effect on presentation of endogenous self Ags, as measured by tumorigenicity in vivo and Ag presentation to three self Ag-specific T hybridoma cells. Additional experiments suggest that H2-O inhibits presentation of exogenous Ags by both newly synthesized and recycling MHC class II molecules. These data suggest H2-O may have a physiological role in tolerance induction and SAg-mediated toxic shock.

Mouse 4T1 breast tumor model.

The 4T1 mammary carcinoma is a transplantable tumor cell line that is highly tumorigenic and invasive and, unlike most tumor models, can spontaneously metastasize from the primary tumor in the mammary gland to multiple distant sites including lymph nodes, blood, liver, lung, brain, and bone The 4T1 tumor has several characteristics that make it a suitable experimental animal model for human mammary cancer. First, tumor cells are easily transplanted into the mammary gland so that the primary tumor grows in the anatomically correct site, as described in this unit. Second, as in human breast cancer, 4T1 metastatic disease develops spontaneously from the primary tumor. Also, the progressive spread of 4T1 metastases to the draining lymph nodes and other organs is very similar to that of human mammary cancer. In this unit, a protocol describes surgical removal of the primary tumor, so that metastatic disease can be studied in an animal setting comparable to the clinical situation where the primary tumor is surgically removed, and metastatic foci remain intact. Another advantage of 4T1 is its resistance to 6-thioguanine. This property enables precise quantitation of metastatic cells, even when they are disseminated and at sub-microscopic levels in distant organs, as described here.

Mouse SaI sarcoma tumor model.

SaI is an A/J-derived (H-2K(k)D(d)) transplantable mouse fibrosarcoma that produces a solid tumor when inoculated subcutaneously, intradermally, or intramuscularly, and an ascites tumor when inoculated intraperitoneally into syngeneic mice. This unit describes the establishment of primary solid SaI/N tumor, the establishment of SaI ascites tumor, and the removal for sampling of in vivo-passaged SaI ascites tumor cells in mice.

Cutting edge: STAT6-deficient mice have enhanced tumor immunity to primary and metastatic mammary carcinoma.

STAT4 and STAT6 are essential for the development of CD4(+) Th1 and Th2 development, respectively. Tumor immunologists have hypothesized that Th1 cells are critical in tumor immunity because they facilitate differentiation of CD8(+) T cells, which are potent anti-tumor effectors. We have used STAT4(-/-) and STAT6(-/-) mice to test this hypothesis. BALB/c and knockout mice were challenged in the mammary gland with the highly malignant and spontaneously metastatic BALB/c-derived 4T1 mammary carcinoma. Primary tumor growth and metastatic disease are reduced in STAT6(-/-) mice relative to BALB/c and STAT4(-/-) mice. Ab depletions demonstrate that the effect is mediated by CD8(+) T cells, and immunized STAT6(-/-) mice have higher levels of 4T1-specific CTL than BALB/c or STAT4(-/-) mice. Surprisingly, Th1 or Th2 cells are not involved, because CD4 depletion does not diminish the anti-tumor effect. Therefore, deletion of the STAT6 gene facilitates development of potent anti-tumor immunity via a CD4(+)-independent pathway.

Tumor cells present MHC class II-restricted nuclear and mitochondrial antigens and are the predominant antigen presenting cells in vivo.

MHC class II-restricted tumor Ags presented by class II(+) tumor cells identified to date are derived from proteins expressed in the cytoplasm or plasma membrane of tumor cells. It is unclear whether MHC class II(+) tumor cells present class II-restricted epitopes derived from other intracellular compartments, such as nuclei and/or mitochondria, and whether class II(+) tumor cells directly present Ag in vivo. To address these questions, a model Ag, hen egg lysozyme, was targeted to various subcellular compartments of mouse sarcoma cells, and the resulting cells were tested for presentation of three lysozyme epitopes in vitro and for presentation of nuclear Ag in vivo. In in vitro studies, Ags localized to all tested compartments (nuclei, cytoplasm, mitochondria, and endoplasmic reticulum) are presented in the absence invariant chain and H-2M. Coexpression of invariant chain and H-2M inhibit presentation of some, but not all, of the epitopes. In vivo studies demonstrate that class II(+) tumor cells, and not host-derived cells, are the predominant APC for class II-restricted nuclear Ags. Because class II(+) tumor cells are effective APC in vivo and probably present novel tumor Ag epitopes not presented by host-derived APC, their inclusion in cancer vaccines may enhance activation of tumor-reactive CD4(+) T cells.

Cooperativity of Staphylococcal aureus enterotoxin B superantigen, major histocompatibility complex class II, and CD80 for immunotherapy of advanced spontaneous metastases in a clinically relevant postoperative mouse breast cancer model.

One of the leading causes of death for women is metastatic breast cancer. Because most animal tumors do not accurately model clinical metastatic disease, the development of effective therapies has progressed slowly. In this study, we establish the poorly immunogenic mouse 4T1 mammary carcinoma as a postsurgical animal model. 4T1 growth characteristics parallel highly invasive human metastatic mammary carcinoma and, at the time of surgery, the extent of disease is comparable with human stage IV breast cancer. Progress in understanding the immune response has led to innovative immune-based anticancer therapies. Here, we test in this postsurgical model, a novel cell-based vaccine, combining MHC class II, CD80(B7.1), and SEB superantigen. Effective treatment of tumor-bearing mice with this immunotherapy requires expression of all three molecules. Mean survival time is extended from 5-7.5 weeks for control-treated mice to 6-10.5 weeks for therapy-treated mice. Increased survival is accompanied by a maximum of 100-fold decrease in clonogenic lung metastases. These therapeutic effects are particularly noteworthy because: (a) the postoperative model demonstrates that early metastases responsible for morbidity are established by 2 weeks after tumor inoculation with 7 x 10(3) parental 4T1 cells into the mammary gland; (b) the immunotherapy is started 4 weeks after tumor inoculation when the mice contain extensive, pre-established, disseminated metastases; and (c) CD4+ and CD8+ T cells are required for the effect.

Immunotherapy with vaccines combining MHC class II/CD80+ tumor cells with interleukin-12 reduces established metastatic disease and stimulates immune effectors and monokine induced by interferon gamma.

Because they are difficult to treat, animal models of widespread, established metastatic cancer are rarely used to test novel immunotherapies. Two such mouse models are used in this report to demonstrate the therapeutic efficacy and to probe the mechanisms of a novel combination immunotherapy consisting of the cytokine interleukin-12 (IL-12) combined with a previously described vaccine based on MHC class II, CD80-expressing cells. BALB/c mice with 3-week established primary 4T1 mammary carcinomas up to 6 mm in diameter and with extensive, spontaneous lung metastases show a significant reduction in lung metastases following a 3-week course of immunotherapy consisting of weekly injections of the cell-based vaccine plus injections of IL-12 three times per week. C57BL/6 mice with 7-day established intravenous B16 melF10 lung metastases show a similar response following immunotherapy with IL-12 plus a vaccine based on B16 MHC class II, CD80-expressing cells. In both systems the combination therapy of cells plus IL-12 is more effective than IL-12 or the cellular vaccine alone, although, in the 4T1 system, optimal activity does not require MHC class II and CD80 expression in the vaccine cells. The cell-based vaccines were originally designed to activate tumor-specific CD4+ T lymphocytes specifically and thereby provide helper activity to tumor-cytotoxic CD8+ T cells, and IL-12 was added to the therapy to facilitate T helper type 1 lymphocyte (Th1) differentiation. In vivo depletion experiments for CD4+ and CD8+ T cells and natural killer (NK) cells and tumor challenge experiments in beige/nude/XID immunodeficient mice demonstrate that the therapeutic effect is not exclusively dependent on a single cell population, suggesting that T and NK cells are acting together to optimize the response. IL-12 may also be enhancing the immunotherapy via induction of the chemokine Mig (monokine induced by interferon gamma), because reverse PCR experiments demonstrate that Mig is present in the lungs of mice receiving therapy and is most likely synthesized by the tumor cells. These results demonstrate that the combination therapy of systemic IL-12 and a cell-based vaccine is an effective agent for the treatment of advanced, disseminated metastatic cancers in experimental mouse models and that multiple effector cell populations and anti-angiostatic factors are likely to mediate the effect.

MHC class II presentation of endogenous tumor antigen by cellular vaccines depends on the endocytic pathway but not H2-M.

We have developed cell-based cancer vaccines that activate anti-tumor immunity by directly presenting endogenously synthesized tumor antigens to CD4+ T helper lymphocytes via MHC class II molecules. The vaccines are non-conventional antigen-presenting cells because they express MHC class II, do not express invariant chain or H-2M, and preferentially present endogenous antigen. To further improve therapeutic efficacy we have studied the intracellular trafficking pathway of MHC class II molecules in the vaccines using endoplasmic reticulumlocalized lysozyme as a model antigen. Experiments using endocytic and cytosolic pathway inhibitors (chloroquine, primaquine, and brefeldin A) and protease inhibitors (lactacystin, LLnL, E64, and leupeptin) indicate antigen presentation depends on the endocytic pathway, although antigen degradation is not mediated by endosomal or proteasomal proteases. Because H2-M facilitates presentation of exogenous antigen via the endocytic pathway, we investigated whether transfection of vaccine cells with H-2M could potentiate endogenous antigen presentation. In contrast to its role in conventional antigen presentation, H-2M had no effect on endogenous antigen presentation by vaccine cells or on vaccine efficacy. These results suggest that antigen/MHC class II complexes in the vaccines may follow a novel route for processing and presentation and may produce a repertoire of class II-restricted peptides different from those presented by professional APC. The therapeutic efficacy of the vaccines, therefore, may reside in their ability to present novel tumor peptides, consequently activating tumor-specific CD4+ T cells that would not otherwise be activated.

Cell-based vaccines for the stimulation of immunity to metastatic cancers.

We are developing vaccines for inducing immunity to metastatic cancers. Although primary tumors are frequently cured by surgery, chemotherapy, or radiation therapy, metastatic lesions often do not respond to these treatments or proliferate after conventional therapy is terminated. Vaccine therapy for established metastases as well as prophylactic vaccine treatment to prevent outgrowth of latent metastatic tumor cells would therefore be beneficial. Our goal is to activate CD4+ and CD8+ T lymphocytes; however, we have focused on activating tumor-specific CD4+ T-helper lymphocytes because of their pivotal role as regulatory cells and in the generation of long-term immunological memory. The vaccines are based on the premise that tumor cells express potentially immunogenic antigens that could be targeted for T-cell activation, and that if appropriately genetically modified, tumor cells could be antigen presenting cells for these antigens. To facilitate direct antigen presentation to CD4+ T cells, tumor cells have been transfected with syngeneic major histocompatibility complex class II, co-stimulatory molecule, and/or superantigen genes. In vivo studies in three mouse tumor models demonstrate that vaccination protects against future challenge with wild-type tumor, cures some solid primary tumors, reduces established metastatic disease, and extends mean survival time. Antigen presentation studies demonstrate that in vivo vaccine efficacy is directly related to in vitro antigen presentation activity. The relevance of antigen presentation activity of the vaccines is further confirmed by in vivo studies demonstrating that during the immunization process, the vaccines directly present tumor-encoded antigens to CD4+ T lymphocytes. Adaptation of these vaccines for the treatment of human metastatic cancers is discussed.

Class II-transfected tumor cells directly present endogenous antigen to CD4+ T cells in vitro and are APCs for tumor-encoded antigens in vivo.

We have previously demonstrated that class II-transfected tumor cells are very effective immunogens that protect against wild-type primary and metastatic tumor and, if supertransfected with genes encoding co-stimulatory molecules, are immunotherapeutic agents that successfully treat mice with established solid tumor. These results are consistent with our hypothesis that the class II-transfected tumor cells act as antigen-presenting cells (APCs) that directly activate tumor-specific CD4+ T cells; however, direct data supporting this hypothesis are lacking. In the present study, we test this hypothesis using class II-transfected tumor cells supertransfected with the hen egg lysozyme gene as a surrogate tumor antigen. In vitro antigen presentation assays demonstrate that class II-transfected tumor cells present to CD4+ T cells endogenously encoded tumor antigen, provided they do not co-express the class II-associated invariant chain. In vivo experiments using genetically marked tumor cells and host APCs demonstrate that both class II-transfected tumor cells and host cells are APCs for tumor-encoded antigens, although tumor cells appear to dominate the response. These results support the hypothesis that the immunogenicity and therapeutic value of class II-transfected tumor cells stem from their ability to function as APCs for tumor-encoded antigens and directly activate tumor-specific CD4+ T lymphocytes.

Tumor antigen presentation: changing the rules.

Cell-based tumor vaccines have been developed on the basis of the hypothesis that tumor cells can be genetically modified to present antigen to T lymphocytes directly. Contrary to expectations, cross-priming is the predominant pathway for activation of tumor-specific CD8+ T cells, while direct presentation of antigen dominates activation of tumor-specific CD4+ T cells. These results pose interesting paradoxes for the generation of immune responses, and have definite implications for the development of anti-cancer vaccines.

MHC class II-transfected tumor cells directly present antigen to tumor-specific CD4+ T lymphocytes.

We have developed and shown to be efficacious an immunotherapeutic strategy to enhance the generation of tumor-specific CD4+ T helper lymphocytes. The approach uses autologous tumor cells genetically modified to express syngeneic MHC class II genes as cell-based immunogens and is based on the hypothesis that tumor cells directly present tumor Ags to CD4+ T cells. Since the conventional pathway for CD4+ T cell activation is indirect via professional APC, induction of immunity following immunization with class II-transfected tumor cells was examined in bone marrow chimeric mice. Both tumor and host-derived cells are APC for tumor Ags, suggesting that the efficacy of tumor cell vaccines can be significantly improved by genetic modifications that enhance tumor cell Ag presentation.

Reduction of established spontaneous mammary carcinoma metastases following immunotherapy with major histocompatibility complex class II and B7.1 cell-based tumor vaccines.

For many cancer patients, removal of primary tumor is curative; however, if metastatic lesions exist and are not responsive to treatment, survival is limited. Although immunotherapy is actively being tested in animal models against primary tumors and experimental metastases (i.v. induced), very few studies have examined immunotherapy of spontaneous, established metastatic disease. The shortage of such studies can be attributed to the paucity of adequate animal models and to the concern that multiple metastatic lesions may be more resistant to immunotherapy than a localized primary tumor. Here, we use the BALB/c-derived mouse mammary carcinoma, 4T1, and show that this tumor very closely models human breast cancer in its immunogenicity, metastatic properties, and growth characteristics. Therapy studies demonstrate that treatment of mice with established primary and metastatic disease with MHC class II and B7.1-transfected tumor cells reduces or eliminates established spontaneous metastases but has no impact on primary tumor growth. These studies indicate that cell-based vaccines targeting the activation of CD4+ and CD8+ T cells may be effective agents for the treatment of malignancies, such as breast cancer, where the primary tumor is curable by conventional methods, but metastatic lesions remain refractile to current treatment modalities.

Major histocompatibility complex class II-transfected tumor cells present endogenous antigen and are potent inducers of tumor-specific immunity.

We have developed an immunotherapy in which tumor cells transfected with syngeneic major histocompatibility complex (MHC) class II genes are cell-based vaccines for the treatment of established tumor and metastatic disease. If this strategy is to be used clinically, convenient methods for generating class II+ tumor cells are necessary. Interferon-gamma treatment or transduction of the class II transactivator (CIITA) gene induces class II expression but also up-regulates the class II-associated accessory molecules, invariant chain (Ii) and DM. To determine if interferon-gamma treatment and CIITA transduction are potential immunotherapies, we assessed the tumorigenicity of sarcoma cells expressing combinations of class II, Ii, and DM. Since we hypothesized that class II-transfected tumor cells not coexpressing Ii and DM present endogenously encoded tumor peptides, we have assessed the transfectants for antigen presentation activity to MHC class II-restricted antigen-specific CD4(+) T cells. Tumor challenge studies demonstrate that tumor cells expressing class II without coexpression of Ii or Ii plus DM are highly immunogenic and preferentially present endogenous antigens, while tumors coexpressing class II with Ii or Ii plus DM are not effective immunogens. Because tumor rejection correlates with expression of class II without coexpression of Ii and DM, the most efficacious vaccines will express MHC class II without coexpression of Ii and DM and will preferentially present endogenous antigen.

Single amino acid mutations in the murine MHC class II A beta cytoplasmic domain abrogate antigen presentation.

Class II MHC molecules are heterodimeric transmembrane glycoproteins that function in the presentation of Ag to CD4+ T cells. Deletion of the cytoplasmic domains of the murine class II A alpha- and A beta-chains has previously been shown to diminish Ag presentation and abrogate rejection of class II-transfected tumor cells. To examine the contributions of individual amino acid residues of the A beta cytoplasmic domain to Ag presentation and tumor rejection, we have produced a series of cell lines expressing A beta class II molecules with site-directed mutations. An A beta(k) cDNA was constructed with mutations in the five conserved amino acid residues, Q224, K225, L235, L236, and Q237 (delta5). In addition, cDNA were produced in which alanine was individually substituted for A beta(k) cytoplasmic domain residues 224 through 237 or doubly substituted at residues G226 and P227 or L235 and L236. These mutant cDNAs were individually cotransfected with wild-type A alpha cDNA into the class II-negative M12.C3 B lymphoma and Sal sarcoma cell lines. As was previously reported for transfectants lacking the entire A beta(k) cytoplasmic domain, the delta5 M12.C3 transfectant could not effectively present Ag to an autoreactive Ak-restricted T cell hybrid, and the delta5 Sal transfectant was not rejected when inoculated into syngeneic hosts. A finer analysis revealed that alteration of the individual residue Q224 or the two residues G226 and P227 abrogated Ag presentation in vitro, while mutation of G226 diminished tumor rejection in vivo. Thus, the function of the A beta cytoplasmic domain in Ag presentation both in vitro and in vivo can be disturbed by mutation of single amino acid residues.

Rejection of MHC class II-transfected tumor cells requires induction of tumor-encoded B7-1 and/or B7-2 costimulatory molecules.

Many tumor cells that have been transfected with genes encoding B7 costimulatory molecules become effective cellular vaccines against wild-type tumor. The improved immunity is dependent on newly induced tumor-specific CD8+ and/or CD4+ T cells and presumably occurs because the B7 transfectants provide the requisite second signal for activation of T cells in conjunction with tumor cell-presented MHC class I/tumor peptide and/or MHC class II/tumor peptide complexes, respectively. Since B7 expression is such a potent enhancer of tumor immunity, and yet some tumors are immunogenic in the absence of B7 transfection, we have used class I+ class-II-transfected tumors to investigate whether costimulatory molecules are also involved in rejection of immunogenic, non-B7-transfected tumor. Blocking studies with B7 mAbs demonstrate that induction of tumor immunity in naive mice requires B7-1 and/or B7-2 expression, while experiments with tumor-primed mice indicate that once antitumor immunity is established, expression of B7 is not necessary. Flow cytometry analyses demonstrate that costimulatory molecules are expressed by the tumor cells via an in vivo induction process. Experiments with class II genes with truncated cytoplasmic tails indicate that the cytoplasmic region of the tumor-expressed class II heterodimer is involved in induction of B7. We therefore conclude that for this class I+ class II-transfected tumor, generation of tumor immunity requires induction of tumor cell-encoded B7 molecules that are mediated by the cytoplasmic region of the transfected class II heterodimer.

Expression of MHC Class II and B7-1 and B7-2 costimulatory molecules accompanies tumor rejection and reduces the metastatic potential of tumor cells.

Mouse tumor cells transfected with syngeneic MHC class II genes are highly immunogenic in the autologous host, and induce a potent tumor-specific immunity against wild type tumor. Previous studies with sarcoma tumor cells expressing transfected class II gene products with truncated cytoplasmic domains suggested that during the process of tumor rejection costimulatory molecules are induced on the tumor cells, contributing to the cells' ability to stimulate immunity. In the present study we directly demonstrate that tumor cells containing full-length class II heterodimers are induced to express B7-1 and B7-2 costimulatory molecules during the rejection process. In contrast, tumor cells expressing class II heterodimers truncated for their cytoplasmic tails are not induced to express B7-1 and/or B7-2. Blocking the interaction of the induced costimulatory molecules with their corresponding receptors on T cells prevents tumor rejection. These results support the hypothesis that the cytoplasmic domain of the MHC class II molecule is involved in induction of costimulatory molecule expression, perhaps via intracellular signalling pathways. Because class II, B7 transfected tumor cells are such effective immunogens against ascites and solid tumors, they have also been tested in metastatic disease. K1735 and B16BL6 mouse melanomas, when transfected with syngeneic MHC class II and B7-1 genes, are significantly less metastatic than parental cells, and immunization with the transfectants protects against subsequent challenge with wild type tumor.

Major histocompatibility complex class II+B7-1+ tumor cells are potent vaccines for stimulating tumor rejection in tumor-bearing mice.

Mice carrying large established major histocompatibility complex (MHC) class 1+ sarcoma tumors can be successfully treated by immunization with genetically engineered sarcoma cells transfected with syngeneic MHC class II plus B7-1 genes. This approach is significantly more effective than previously described strategies using cytokine- or B7-transduced tumor cells which are only effective against smaller tumor loads, and which cannot mediate regression of longer-term established tumors. The most efficient tumor rejection occurs if both the class II and B7-1 molecules are coexpressed on the same tumor cell. Immunity induced by immunization with class II+B7-1(+)-transfected sarcoma cells involves CD4+ and CD8+ T cells, suggesting that the increased effectiveness of the transfectants is due to their ability to activate both of these T cell populations.

Tumor immunotherapy: the tumor cell as an antigen-presenting cell.

Increased knowledge in basic immunology has led to a variety of innovative and imaginative approaches for tumor-specific immunotherapy. One of these approaches is based on the premise that tumor cells do not normally stimulate an effective tumor-specific immune response, because they do not efficiently present tumor antigens to the relevant lymphocytes. To overcome inadequate antigen presentation, it has been hypothesized that tumor cells can be genetically engineered to present tumor peptides directly to T lymphocytes.