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.

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.

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.

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.

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.

Beta 2M-/- knockout mice contain low levels of CD8+ cytotoxic T lymphocyte that mediate specific tumor rejection.

C57BL/6 mice with a disrupted beta 2M gene (beta 2M-/- mice) express very low levels of MHC class I molecules and are deficient for CD8+ T lymphocytes. Because CD8+ T cells are thought to be a principle effector cell in tumor rejection, we have assessed the ability of beta 2M-/- mice to respond to tumors. beta 2M-/- knockout mice were challenged with seven independent MHC allogeneic and syngeneic tumors. The beta 2M-/- mice responded very similarly to their CD8+ beta 2M+/- littermates in that they rejected high dose challenges of 4/5 allogeneic tumors and were susceptible to 3/3 syngeneic tumors. In vivo depletion of CD4+ or CD8+ cells from the beta 2M-/- mice resulted in susceptibility to allogeneic tumor. The apparent requirement for CD8+ cells for tumor immunity was corroborated by in vitro assays in which depletion of CD8+ but not CD4+ T cells eliminated tumor-specific CTL activity. mAb blocking studies in which target tumor cells were incubated with MHC class I-specific mAb demonstrated that the tumor-specific CD8+ activity was MHC class I restricted. beta 2M-/- mice therefore contain very small quantities of potent, CD8+ T cells that are capable of rejecting large challenges of allogeneic tumor cells.

Invariant chain alters the malignant phenotype of MHC class II+ tumor cells.

T lymphocytes usually recognize endogenously encoded Ag in the context of MHC class I molecules, whereas exogenous Ag is usually presented by MHC class II molecules. In vitro studies in model systems suggest that presentation of endogenous Ag by class II molecules is inhibited by the association of class II with its invariant chain (Ii). In the present study we test this hypothesis in an in vivo system in which endogenously encoded tumor peptides are presented by tumor cell MHC class II molecules. In this system, transfection of syngeneic MHC class II genes (Aak and Abk) into a highly malignant, Ii negative, mouse tumor (SaI sarcoma) produces an immunogenic tumor (SaI/Ak) that is rejected by the autologous host. The class II+ transfectants also effectively immunize autologous A/J mice against a subsequent challenge of wild-type class II- tumor cells. We have hypothesized that the SaI/Ak transfectants induce protective immunity because they function as APC for endogenously synthesized tumor peptides, and thereby stimulate tumor-specific Th cells, by-passing the need for professional APC. To test the role of Ii as an inhibitor of presentation of endogenous peptides, SaI/Ak tumor cells were supertransfected with Ii gene (SaI/Ak/Ii cells), and the tumorigenicity of the resulting cells determined. Nine SaI/Ak/Ii clones were tested, and their malignancy compared with that of SaI/Ak and SaI cells. Seven of the nine class II+/Ii+ tumor cells are more malignant than class II+/Ii- tumor cells in autologous A/J mice. Expression of Ii therefore restores the malignant phenotype, presumably by preventing presentation of endogenously synthesized tumor peptides. Ii therefore regulates Ag presentation and can be a critical parameter for in vivo tumor immunity.

Abrogation of tumorigenicity by MHC class II antigen expression requires the cytoplasmic domain of the class II molecule.

Transfection of syngeneic MHC class II genes into the lethal mouse SaI tumor abrogates the malignancy of the tumor in the autologous host, and protects the host against subsequent challenges with the wild type class II- tumor. We have hypothesized that the transfectants induce protective immunity by functioning as APC for tumor peptides, and stimulating tumor-specific Th cells. Recent in vitro studies suggest that Ag presentation by class II-restricted APC requires the cytoplasmic domain of the class II molecule, and may involve intracellular signaling via the cytoplasmic domain. To determine if the class II cytoplasmic domain is required for enhanced tumor-specific immunity, SaI mouse sarcoma cells were transfected with syngeneic Aak and Abk genes with truncated cytoplasmic domains. These transfectants are as malignant as wild type class II- SaI cells in autologous A/J mice. Stimulation of tumor-specific immunity by class II+ tumor cells is therefore dependent on the class II cytoplasmic region, and may involve intracellular signaling events.

Influence of major histocompatibility complex class I, class II and TLA genes on tumor rejection.

T lymphocytes recognize antigen associated with MHC class I and/or class II gene products. Recognition of malignant cells is therefore dependent on presentation of tumor associated antigen(s) by MHC molecules. We have studied immunity to tumors that have down-regulated class I expression. These studies demonstrate a requirement for class I antigens, but suggest that additional factors may also be required for tumor-specific immunity. The MHC also encodes TLA class I antigens, whose function is unknown. Our studies suggest that these molecules function is unknown. Our studies suggest that T lymphocytes, specifically in tumor cells that do not express H-2K or H-2D moieties. Other studies are aimed at improving tumor-specific Th cell generation by producing class II+ tumor cells. The success of these experiments indicates that this approach may be a potentially useful immunotherapy.

Tumor-specific immunity can be enhanced by transfection of tumor cells with syngeneic MHC-class-II genes or allogeneic MHC-class-I genes.

Mouse Sal sarcoma cells are lethal in the autologous A/J (KkDd) host. In order to improve the immune response to the Sal tumor, Sal cells have been transfected with syngeneic MHC-class-II or allogeneic MHC-class-I genes. MHC-class-II transfectants are uniformly rejected by the autologous host and immunization with them protects against subsequent Sal challenge. The improved immunity is probably the result of enhanced generation of tumor-specific Th cells. We hypothesize that class-II tumor cells trigger an improved Th-cell response because they directly present Sal tumor antigens in the context of class-II molecules to Th cells, by-passing professional APC. Studies by others have demonstrated that antigen presentation requires an intracellular signal transmitted by the cytoplasmic domain of the APC class-II molecule. Sal cells expressing class-II antigens with truncated cytoplasmic domains are as malignant as wild-type Sal cells. These experiments therefore support the role of tumor-cell class-II molecules as antigen presentation elements, and demonstrate the requirement for intact class-II molecules for tumor protection. Sal cells have also been transfected with allogeneic MHC-class-I genes. Although Kb-transfected cells are not rejected by A/J mice, Db-transfected Sal cells and Kb- plus Db-transfected cells are rejected. The Db transfectants effectively immunize A/J mice against subsequent Sal challenge. These experiments demonstrate that expression of certain allogeneic MHC-class-I genes can lead to tumor-specific immunity, and that such transfectants can protect against challenges of wild-type tumor cells. Transfection of tumor cells with syngeneic MHC-class-II or allogeneic MHC-class-I genes may therefore be a potential strategy for improving tumor-specific immunity in the autologous host.

Transfection and expression of syngeneic H-2 genes does not reduce malignancy of H-2 negative teratocarcinoma cells in the autologous host.

Rejection of the MHC class I negative 402AX teratocarcinoma is accompanied by induction of tumor cell-encoded H-2K and H-2D antigens by the genetically resistant host. To determine whether MHC antigen expression is required for 402AX rejection, we have prepared H-2Db-transfected 402AX cells (402AX/Db). Transfectants express high levels of H-2Db, most of which is not associated with beta 2-microglobulin. MHC syngeneic and allogeneic mice susceptible to 402AX are resistant to 402AX/Db, suggesting that MHC class I antigen expression is required for tumor rejection. Autologous 129 hosts, however, are susceptible to 402AX/Db. 402AX cells transfected with the H-2Kb gene (402AX/Kb) are also lethal in the autologous 129/J host, but rejected by MHC syngeneic and allogeneic mice. Non-129 strain 402AX-susceptible mice pre-immunized with 402AX/Db or simultaneously challenged with 402AX/Db plus 402AX are immune to 402AX. Mice immunized with 402AX/Db produce MHC class I induction factor. 402AX/Db and 402AX cells are lysed equally by natural killer cells, indicating that in 402AX cells the expression of class I antigens is unrelated to NK susceptibility. These studies confirm the requirement for class I expression in 402AX immunity, but demonstrate that in the autologous host immunity requires additional factors beyond class I antigen expression.

Transfection of major histocompatibility complex class I and class II genes causes tumour rejection.

Many human and mouse tumours do not express MHC class II antigens and have reduced levels of class I antigens. Because of the requirement for class I and/or class II antigen for antigen presentation to Th and Tc cells, these phenotypes may enable tumour cells to 'escape' the host's immune response. Experiments presented here are designed to assess the role of MHC class I and class II antigens in tumour immunity, and to overcome the MHC class I- or class II-negative phenotype. When transfected with the syngeneic H-2Db gene, the MHC antigen-negative 402AX teratocarcinoma expresses high levels of H-2Db antigen. 402AX/Db cells are rejected by MHC allogeneic and some MHC syngeneic 402AX-susceptible mice, however the fully syngeneic strain of origin (129) remains tumour-susceptible. Induction of MHC class I gene products on class I antigen-negative embryonal carcinoma cells therefore increases tumour immunogenicity in some hosts, but not in the fully syngeneic mouse. In an attempt to enhance antigen presentation of tumour-associated antigens to Th cells, MHC class I antigen-positive SaI (KkDd) sarcoma cells were transfected with syngeneic A alpha k and A beta k genes to generate Iak-expressing tumour cells. SaI/Ak cells are efficiently rejected by syngeneic A/J (KkDd) mice, while untransfected SaI cells are lethal. Induction of MHC class II antigen expression on the class I antigen-positive SaI sarcoma therefore completely abrogates malignancy.

Embryonal carcinoma cells express Qa and Tla class I genes of the major histocompatibility complex.

The murine major histocompatibility complex encodes H-2K and H-2D transplantation antigens and other class I-like proteins called Qa and Tla molecules; the functions of the Qa/Tla molecules are not known. That they may participate in embryonic cell-cell interactions and/or play a role in immune responses against tumors has been speculated. We have studied two murine embryonal carcinoma tumors, 402AX and PCC4, that are rejected in vivo immunologically, although they do not express H-2K or H-2D antigens. Transplantation studies with these cells suggest that rejection is mediated by class-I-like major histocompatibility complex antigens. As a first step in evaluating Qa/Tla function(s), we have characterized expression of class I-like genes and proteins in 402AX and PCC4 cells. Northern (RNA) blot hybridizations, polymerase chain reaction studies, and cDNA cloning experiments demonstrate that EC lines transcribe genes allelic to the Tla region gene "37", Qa-2 region gene "Q7", and another, previously uncharacterized, class I-like gene. Immunoprecipitation studies show that the embryonal carcinoma tumor cells contain low levels of beta 2-microglobulin expressed in association with non-H-2K, non-H-2D class I-like proteins.

Allogeneic H-2 antigen expression is insufficient for tumor rejection.

Murine A strain (KkDdLd) sarcoma I (SaI) tumor cells have been transfected with a cloned H-2Kb gene. The resulting clones (SKB clones) stably express high levels of a molecule that is serologically and biochemically indistinguishable from the H-2Kb antigen. SKB clones are not susceptible to cytotoxic T lymphocyte-mediated lysis by H-2Kb-specific bulk, cloned, or H-2Kb-restricted lymphocytic choriomeningitis virus-specific effectors. Survival times of A/J and B10.A mice challenged i.p. with the H-2Kb-expressing transfectants and the parental SaI cells are similar, suggesting that the presence of an allogeneic major histocompatibility complex class I antigen on the surface of this tumor line is insufficient for tumor rejection.