Glioma immunotherapy by IL-21 gene-modified cells or by recombinant IL-21 involves antibody responses.

Most tumors of the central nervous system, especially glioblastoma, are refractory to treatment and invariably lethal. The aim of this study was to assess the ability of different interleukins (IL), IL-2, IL-12 and IL-21, produced by transduced glioma cells to activate an immune response and trigger intracranial tumor rejection. Such experiments were performed by the use of a slow-growing clone of GL261 (GL D2-60) that was used as orthotopic glioma model. Using GL D2-60-transduced cells, all cytokines elicited an immune response against the tumor. Most notably 100% of the animals receiving a primary implant of IL-21-transduced cells rejected the implant, and 76% of these animals survived to a subsequent rechallenge with GL261 parental cells, while the other transduced cytokine genes were not as effective. Rejection responses were also obtained by admixing wild-type tumor cells with IL-21-producing GL D2-60 cells, indicating a local bystander effect of IL-21. More importantly, IL-21-secreting GL D2-60 cells or 1 microg of rIL-21 protein stereotactically injected into established GL D2-60 tumors were able to trigger glioblastoma rejection in 90 and 77% of mice, respectively. Again most of these mice survived to GL261 rechallenge. Immune mice showed antibody responses to glioma antigens, predominantly involving IgG2a and IgG2b isotypes, which mediated complement- or cell-dependent glioma cell lysis. Antibody responses were crucial for glioma immunotherapy by IL-21-secreting GL D2-60 cells, as immunotherapy was uneffective in syngeneic microMT B-cell-deficient mice. These results suggest that IL-21 should be considered as a suitable candidate for glioma immunotherapy by local delivery.

CIITA-induced MHC class II expression in mammary adenocarcinoma leads to a Th1 polarization of the tumor microenvironment, tumor rejection, and specific antitumor memory.

PURPOSE: We have shown previously that the MHC class II-negative murine TS/A adenocarcinoma is rejected in vivo if induced to express MHC class II molecules by transfection of the MHC class II transactivator CIITA. In this study, we explored the immunologic basis of tumor rejection and the correlation between histopathology of tumor tissue and immune rejection. EXPERIMENTAL DESIGN: Stable TS/A-CIITA transfectants were generated and injected into mice. In vivo cell depletion, immunohistochemistry of tumor tissues, and immune functional assays were done to assess the cellular and immunologic basis of rejection. RESULTS: Ninety-two percent of mice injected with TS/A-CIITA rejected the tumor and were completely resistant to challenge with parental TS/A. Only CD4+ and CD8+ cells were required for rejection. The tumor microenvironment in TS/A-CIITA-injected mice changed dramatically when compared with the TS/A parental-injected mice. Rapid infiltration with CD4+ T cells followed by dendritic cells, CD8+ T cells, and granulocytes was observed. Importantly, TS/A-CIITA cells could act as antigen-presenting cells because they process and present nominal antigens to CD4+ T cells. Tumor-specific CD4+ T cells of TS/A-CIITA-injected mice had the functional characteristics of Th1 cells and produced IFN-gamma and this was relevant for generation and maintenance of protective antitumor response, because IFN-gamma knockout mice were no longer rejecting TS/A-CIITA tumor cells. CONCLUSION: CIITA-dependent MHC class II expression confers to TS/A tumor cells the capacity to act as a protective vaccine against the tumor by triggering tumor antigen presentation to T helper cells, antitumor polarization of cellular and soluble components of the tumor microenvironment, and establishment of antitumor immune memory.

CD25+ regulatory T cell depletion augments immunotherapy of micrometastases by an IL-21-secreting cellular vaccine.

IL-21 is an IL-2-like cytokine, signaling through a specific IL-21R and the IL-2R gamma-chain. Because the TS/A mammary adenocarcinoma cells genetically modified to secrete IL-21 (TS/A-IL-21) are strongly immunogenic in syngeneic mice, we analyzed their application as vaccine. In mice bearing TS/A-parental cell (pc) micrometastases, vaccination with irradiated TS/A-IL-21 cells significantly increased the animal life span, but cured only 17% of mice. Spleen cells from cured mice developed CTL activity and produced IFN-gamma in response to stimulation by the AH1 epitope of the gp70env Ag of TS/A-pc. We tested whether the low therapeutic outcome might be due to CD4+CD25+ regulatory T cells (Treg) present in TS/A-pc tumors and draining lymph nodes and whether IL-21 had any effect on these cells. Indeed, CD4+CD25+ cells suppressed IFN-gamma production by splenocytes from immune mice in response to stimulation by the AH1 peptide. Low concentrations of IL-21 (10 ng/ml) failed to reverse the inhibitory activity of CD4+CD25+ cells in an allogeneic MLR, whereas 60 ng/ml rIL-21 partially restored responder T cell proliferation. IL-21R expression on CD25- lymphocytes suggested that IL-21 could be more effective in mice depleted of CD25+ cells. Depletion of Treg cells by a single dose of anti-CD25 mAb combined with TS/A-IL-21 cell vaccine cured >70% of mice bearing micrometastases, whereas anti-CD25 mAb treatment alone had no effect. Successful combined immunotherapy required NK cells, CD8+ T cells, and IFN-gamma. In conclusion, immunotherapy of micrometastases by an IL-21-based cellular vaccine is strongly potentiated by CD25+ cell depletion.

Inhibition of mammary carcinoma development in HER-2/neu transgenic mice through induction of autoimmunity by xenogeneic DNA vaccination.

Plasmid DNA vectors encoding the full-length (VR1012/HER-2-FL) or only the extracellular and transmembrane domains (VR1012/HER-2-ECD-TM) of human (h) HER-2/neu proto-oncogene were used to vaccinate HER-2/neu transgenic mice (N202) engineered to overexpress the rat (r) neu proto-oncogene product (r-p185(neu)). Both the full-length and the deleted vaccines were significantly (P = 0.0001 and P = 0.06, respectively) more active than the empty vector (VR1012/EV) in preventing and delaying HER-2/neu-driven mammary carcinogenesis. A low-level intratumoral infiltrate of dendritic cells, macrophages, CD8 T cells and polymorphonuclear granulocytes in association with low-level cytokine production was observed, which was not detected in tumors from control mice. Morphologic analyses showed that vaccination with VR1012/HER-2-FL or ECD-TM also efficiently hampered the development of terminal ductal lobular units (TDLU). Analyses of sera from vaccinated mice revealed high titers of antihuman HER-2/neu antibodies, which correlated with the delayed time of tumor onset (P = 0.002). These antibodies did not cross-react with r-p185(neu). Nontransgenic mice treated with the vaccines produced autoreactive antibodies targeting mouse (m)-p185(neu) and showed impaired function of the lactating mammary gland and accelerated involution of the gland after weaning. Together, these data indicate that xenogeneic DNA immunization breaks tolerance against the endogenous m-p185(neu), impairing the development of mammary TDLU in which m-p185(neu) expression is concentrated. The reduction in the number of TDLU decreases the number of glandular structures available for r-p185(neu)-dependent mammary carcinogenesis, resulting in a significant inhibition of mammary carcinoma development.

Immunoprevention of HER-2/neu transgenic mammary carcinoma through an interleukin 12-engineered allogeneic cell vaccine.

This study evaluated the ability of cytokine-engineered allogeneic (H-2(q)) HER-2/neu-positive cells to prevent tumor development in mammary cancer-prone virgin female BALB/c (H-2(d)) mice transgenic for the transforming rat HER-2/neu oncogene (BALB-neuT mice). Repeated vaccinations with cells engineered to release interleukin (IL)-2, IL-12, IL-15, or IFN-gamma showed that IL-12-engineered cell vaccines had the most powerful immunopreventive activity, with >80% of 1-year-old BALB-neuT mice free of tumors. On the contrary all of the untreated mice and all of the mice vaccinated with IL-12-engineered cells lacking either HER-2/neu or allogeneic antigens developed mammary carcinomas within 22 or 33 weeks, respectively. Whole mount, histology, immunohistochemistry, and gene expression profile analysis showed that vaccination with IL-12-engineered cells maintained 26-week mammary glands free of neoplastic growth, with a gene expression profile that clustered with that of untreated preneoplastic glands. The IL-12-engineered cell vaccine elicited a high production of IFN-gamma and IL-4 and a strong anti-HER-2/neu antibody response. Immune protection was lost or markedly impaired in BALB-neuT mice lacking IFN-gamma or antibody production, respectively. The protection afforded by the IL-12-engineered cell vaccine was equal to that provided by the systemic administration of recombinant IL-12 in combination with HER-2/neu H-2(q) cell vaccine. However, IL-12-engineered cell vaccine induced much lower circulating IL-12 and IFN-gamma, and therefore lower potential side effects and systemic toxicity.

IL-21 induces tumor rejection by specific CTL and IFN-gamma-dependent CXC chemokines in syngeneic mice.

IL-21 is an immune-stimulatory four alpha helix cytokine produced by activated T cells. To study the in vivo antitumor activities of IL-21, TS/A murine mammary adenocarcinoma cells were genetically modified to secrete IL-21 (TS/A-IL-21). These cells developed small tumors that were subsequently rejected by 90% of s.c. injected syngeneic mice. Five days after injection, TS/A-IL-21 tumors showed numerous infiltrating granulocytes, NK cells, and to a lesser extent CD8(+) T cells, along with the expression of TNF-alpha, IFN-gamma, and endothelial adhesion molecules ICAM-1 and VCAM-1. At day 7, CD8(+) and CD4(+) T cells increased together with IFN-gamma, and the CXC chemokines IFN-gamma-inducible protein 10, monokine induced by IFN-gamma, and IFN-inducible T cell alpha-chemoattractant. The TS/A-IL-21 tumor displayed a disrupted vascular network with abortive sprouting and signs of endothelial cell damage. In vivo depletion experiments by specific Abs showed that rejection of TS/A-IL-21 cells required CD8(+) T lymphocytes and granulocytes. When injected in IFN-gamma-deficient mice, TS/A-IL-21 cells formed tumors that regressed in only 29% of animals, indicating a role for IFN-gamma in IL-21-mediated antitumor response, but also the existence of IFN-gamma-independent effects. Most immunocompetent mice rejecting TS/A-IL-21 cells developed protective immunity against TS/A-pc (75%) and against the antigenically related C26 colon carcinoma cells (61%), as indicated by rechallenge experiments. A specific CTL response against the gp70-env protein of an endogenous murine retrovirus coexpressed by TS/A and C26 cells was detected in mice rejecting TS/A-IL-21 cells. These data suggest that IL-21 represents a suitable adjuvant in inducing specific CTL responses.

Tumor cells engineered with IL-12 and IL-15 genes induce protective antibody responses in nude mice.

IL-12 and IL-15 stimulate T, B, and NK cell functions through independent mechanisms, and cooperative effects of these cytokines have been reported. The human MHC class I-negative small cell lung cancer cell line, N592, genetically engineered to secrete IL-15, N592/IL-15, showed a reduced tumor growth rate, while N592 cells engineered with IL-12, N592/IL-12, grew similarly to the wild-type N592, N592 parental cells (N592pc), in nude mice. However, N592 cells coexpressing both cytokines, N592/IL-12/IL-15 cells, were completely rejected by 100% of nude mice. Here we show that 60% of nude mice rejecting N592/IL-12/IL-15 cells were resistant to N592pc rechallenge. SCID mice rejected N592/IL-12/IL-15 cells, but did not develop resistance to N592pc rechallenge, suggesting a role of Ab responses. Among nude mice rejecting N592/IL-12/IL-15 cells, those developing resistance to N592pc rechallenge had significantly higher titers of anti-N592 IgG2b Abs than nonresistant nude mice. Induction of an Ig class switch in nude mice was related to the expression of IFN-gamma and CD40 ligand in the draining lymph nodes. An IgG2b, anti-N592 mAb, derived from N592/IL-12/IL-15-immunized nude mice splenocytes induced significant protection against N592pc, while an IgM mAb was ineffective. The protective IgG2b mAb, but not the IgM mAb, triggered Ab-dependent cell-mediated cytotoxicity by nude mouse splenocytes against N592pc. These data indicate that IL-12 and IL-15 synergistically trigger innate, immunity-mediated, anti-tumor effects, resulting in cytotoxic IgG Ab responses in T cell-deficient mice. Protective Ab responses may relate to both direct actions of IL-12 and IL-15 on B cells and to the activation of an innate immunity-B cell cross-talk.

Tumor rejection by gene transfer of the MHC class II transactivator in murine mammary adenocarcinoma cells.

The murine mammary adenocarcinoma cell line TS/A is a highly malignant MHC class II-negative tumor. We show that transfection of TS/A cells with the MHC class II transactivator CIITA renders them MHC class II-positive and highly immunogenic in vivo. These cells were fully rejected by 51% of syngeneic recipients and had a significantly lower growth rate in the remaining 49% of animals. This directly correlated to the amount of MHC class II molecules expressed in the transfected tumor. Tumor rejecting animals were protected against rechallenge with the parental TS/A tumor. The rejection required CD4(+) and CD8(+) T cells. CD4(+) T cells were fundamental in the priming phase of the antitumor response. CTL-specific for a peptide of the envelope gp70 of an endogenous ecotropic retrovirus were identified and explained the specificity of the effector mechanism of rejection against the TS/A and the antigenically related C26 carcinoma cells but not against the unrelated gp70-negative syngeneic fibrosarcoma F1F cells. This is the first example of successful tumor vaccination by genetic transfer of CIITA. These results open the way to a possible use of CIITA for increasing both the inducing and the effector phase of the antitumor immune response.

IFN-gamma-independent synergistic effects of IL-12 and IL-15 induce anti-tumor immune responses in syngeneic mice.

IL-15 and IL-12 display anti-tumor activity in different models and IFN-gamma has been reported as a secondary mediator of both IL-12 and IL-15 effects. TS/A murine adenocarcinoma cells were engineered to secrete IL-12, IL-15 or both cytokines. TS/A cells secreting IL-15 (TS/A-IL-15) displayed a reduced tumorigenicity (50%) when implanted subcutaneously in syngeneic mice, while both TS/A-IL-12 and TS/A-IL-12/IL-15 were rejected by 100% of animals. In contrast, TS/A-IL-15 and TS/A-IL-12 were tumorigenic in syngeneic IFN-gamma knockout mice (100% and >90% of take rate, respectively), but TS/A-IL-12/IL-15 were completely rejected by 90% of these mice. All IFN-gamma-deficient mice rejecting TS/A-IL-12/IL-15 developed protective immunity against wild-type TS/A, as indicated by re-challenge experiments. Immunohistochemical analysis of the TS/A-IL-12/IL-15 tumor rejection area in IFN-gamma-deficient mice showed a marked reactive cell infiltration constituted of CD8+ cells, granulocytes, NK cells, macrophages and dendritic cells associated with the expression of IL-1beta, TNF-alpha, GM-CSF, MCP-1 and MIP-2. In vivo depletion experiments showed that rejection of TS/A-IL-12/IL-15 cells required CD8+ T lymphocytes and also involved granulocytes, while CD4+ and NK cells played a minor role. These data show IFN-gamma-independent synergistic anti-tumor effects of IL-12 and IL-15, involving CD8+ cells and secondary chemokines and cytokines, such as TNF-alpha.