Elena Ambrosino

Cross-Regulation between Type I and Type II NKT Cells in Regulating Tumor Immunity: A New Immunoregulatory Axis

Negative immunoregulation is a major barrier to successful cancer immunotherapy. The NKT cell is known to be one such regulator. In this study we explored the roles of and interaction between the classical type I NKT cell and the poorly understood type II NKT cell in the regulation of tumor immunity. Selective stimulation of type II NKT cells suppressed immunosurveillance, whereas stimulation of type I NKT cells protected against tumor growth even when responses were relatively skewed toward Th2 cytokines. When both were stimulated simultaneously, type II NKT cells appeared to suppress the activation in vitro and protective effect in vivo of type I NKT cells. In the absence of type I, suppression by type II NKT cells increased, suggesting that type I cells reduce the suppressive effect of type II NKT cells. Thus, in tumor immunity type I and type II NKT cells have opposite and counteractive roles and define a new immunoregulatory axis. Alteration of the balance between the protective type I and the suppressive type II NKT cell may be exploited for therapeutic intervention in cancer.

Cure of Mammary Carcinomas in Her-2 Transgenic Mice through Sequential Stimulation of Innate (Neoadjuvant Interleukin-12) and Adaptive (DNA Vaccine Electroporation) Immunity

Purpose: Whereas neoadjuvant therapy is emerging as a treatment option in early primary breast cancer, no data are available on the use of antiangiogenic and immunomodulatory agents in a neoadjuvant setting. In a model of Her-2 spontaneous mammary cancer, we investigated the efficacy of neoadjuvant interleukin 12 (IL-12) followed by “immune-surgery” of the residual tumor. Experimental Design: Female BALB/c mice transgenic for the rat Her-2 oncogene inexorably develop invasive carcinomas in all their mammary glands by the 23rd week of age. Mice with multifocal in situ carcinomas received four weekly i.p. injections of 100 ng IL-12 followed by a 3-week rest. This course was given four times. A few mice additionally received DNA plasmids encoding portions of the Her-2 receptor electroporated through transcutaneous electric pulses. Results: The protection elicited by IL-12 in combination with two DNA vaccine electroporations kept 63% of mice tumor-free. Complete protection of all 1-year-old mice was achieved when IL-12-treated mice received four vaccine electroporations. Pathologic findings, in vitro tests, and the results from immunization of both IFN-γ and immunoglobulin gene knockout transgenic mice and of adoptive transfer experiments all show that IL-12 augments the B- and T-cell response elicited by vaccination and slightly decreases the number of regulatory T cells. In addition, IL-12 strongly inhibits tumor angiogenesis. Conclusions: In Her-2 transgenic mice, IL-12 impairs tumor progression and triggers innate immunity so markedly that DNA vaccination becomes effective at late points in time when it is ineffective on its own.

Delicate Balance among Three Types of T Cells in Concurrent Regulation of Tumor Immunity

The nature of the regulatory cell types that dominate in any given tumor is not understood at present. Here, we addressed this question for regulatory T cells (Treg) and type II natural killer T (NKT) cells in syngeneic models of colorectal and renal cancer. In mice with both type I and II NKT cells, or in mice with neither type of NKT cell, Treg depletion was sufficient to protect against tumor outgrowth. Surprisingly, in mice lacking only type I NKT cells, Treg blockade was insufficient for protection. Thus, we hypothesized that type II NKT cells may be neutralized by type I NKT cells, leaving Tregs as the primary suppressor, whereas in mice lacking type I NKT cells, unopposed type II NKT cells could suppress tumor immunity even when Tregs were blocked. We confirmed this hypothesis in 3 ways by reconstituting type I NKT cells as well as selectively blocking or activating type II NKT cells with antibody or the agonist sulfatide, respectively. In this manner, we showed that blockade of both type II NKT cells and Tregs is necessary to abrogate suppression of tumor immunity, but a third cell, the type I NKT cell, determines the balance between these regulatory mechanisms. As patients with cancer often have deficient type I NKT cell function, managing this delicate balance among 3 T-cell subsets may be critical for the success of immunotherapy for human cancer.

Erbb2 DNA Vaccine Combined with Regulatory T Cell Deletion Enhances Antibody Response and Reveals Latent Low-Avidity T Cells: Potential and Limits of Its Therapeutic Efficacy

Rat (r)Erbb2 transgenic BALB-neuT mice genetically predestined to develop multiple invasive carcinomas allow an assessment of the potential of a vaccine against the stages of cancer progression. Because of rErbb2 expression in the thymus and its overexpression in the mammary gland, CD8+ T cell clones reacting at high avidity with dominant rErbb2 epitopes are deleted in these mice. In BALB-neuT mice with diffuse and invasive in situ lesions and almost palpable carcinomas, a temporary regulatory T cells depletion combined with anti-rErbb2 vaccine markedly enhanced the anti-rErbb2 Ab response and allowed the expansion of latent pools of low-avidity CD8+ T cells bearing TCRs repertoire reacting with the rErbb2 dominant peptide. This combination of a higher Ab response and activation of a low-avidity cytotoxic response persistently blocked tumor progression at stages in which the vaccine alone was ineffective. However, when diffuse and invasive microscopic cancers become almost palpable, this combination was no longer able to secure a significant extension of mice survival.

Regulation of tumor immunity: the role of NKT cells

Background: Tumor immunosurveillance is a part of the dynamic process of interaction between abnormal cells and the host immune system. Tumor immunosurveillance is actively and continuously regulated in both positive and negative ways. Natural killer T (NKT) cells are cells that have been shown to play a role in both positive and negative regulation of tumor immunosurveillance. Recent studies suggest that NKT cells are a heterogeneous cell population with multiple subsets with distinct functions. Objective: This review discusses the functions of those NKT cell subsets in regulating tumor immunity and potential interactions or counter-regulation among the NKT cell subsets. Method: Selected literature is reviewed. Conclusion: Manipulation of the balance among those subsets may provide new modes of intervention for tumor immunotherapy.

Timely DNA Vaccine Combined with Systemic IL-12 Prevents Parotid Carcinomas before a Dominant-Negative p53 Makes Their Growth Independent of HER-2/neu Expression

Double transgenic mice overexpressing the transforming rat HER-2/neu oncogene and the mutated p53, with both dominant-negative and a gain-of-function properties, display early aggressive and metastasizing parotid tumors. Multiple acinar and ductal hyperplasia foci overexpressing the HER-2/neu gene product are evident at wk 5 and progress to poorly differentiated carcinoma by wk 7. Mice die before wk 18 with invasive carcinomas and multiple metastases that no longer express HER-2/neu. A combination of repeated electroporations of plasmids coding for the extracellular and transmembrane domains of the rat HER-2/neu receptor with systemic IL-12 administrations started when the parotids that present diffuse hyperplasia protected all female and 50% of the male mice until the close of the experiment at wk 40. This combined treatment began when multifocal in situ carcinomas that were already present cured 33% of the females and 25% of the males. The most prominent immunologic features associated with the antitumor protection were the production of high titers of anti-HER-2/neu Abs and the nonappearance of cell-mediated cytotoxic reactivity. In conclusion, anti-HER-2/neu vaccination combined with systemic IL-12 control parotid carcinomas as far as p53 mutation makes their growth independent of HER-2/neu expression.

Abstract 4746: Type I NKT cells mediate anti-tumor immunity through a TNF-α- and NOS-dependent pathway when stimulated with β-ManCer

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL CD1d-restricted type I NKT cells have been shown to play a critical role in the induction of tumor immunity by producing IFN-γ, which activates effector arms such as NK cells and CD8+ T cells. It has been considered that IFN-γ is necessary for type I NKT cell-induced tumor immunity. In this study, we show that type I NKT cells can induce strong tumor immunity through an IFN-γ-independent pathway when activated with a new type of agonistic antigen, β-mannosylceramide (β-ManCer). The protection induced by β-ManCer treatment was completely abrogated in type I NKT cell deficient Jα18 KO mice. With the observations that in vitro activation of type I NKT cells by β-ManCer was blocked by anti-CD1d blocking monoclonal antibody and that β-ManCer-loaded CD1d dimers bind to type I NKT cells and stimulate type I NKT cell hybridomas in an APC-free system, we concluded that recognition of CD1d presented β-ManCer by a TCR of type I NKT cells is necessary for the protection. Surprisingly β-ManCer-induced protection was not abrogated in IFN-γ deficient mice, which is in contrast with the protection induced by a prototypic agonist α-galactosylceramide (α-GalCer). Instead, β-ManCer-induced protection was dependent on nitric oxide synthase (NOS) and TNF-α since blockade of either completely abrogated the protection while the blockade did not affect protection induced by α-GalCer. The conclusion that NKT cells activated with β-ManCer utilize a different pathway from that used by the cells activated with α-GalCer to enhance tumor immunity was confirmed with the observation that the combination of both antigens synergistically enhanced tumor immunity when suboptimal doses of each were used. These data demonstrate that NKT cells mediate tumor immunity through a TNF-α- and NOS-dependent pathway when stimulated with β-ManCer and that β-ManCer represents a new class of type I NKT cell agonist. Since we observed that human type I NKT cells also can be activated by β-ManCer, β-ManCer may provide a new intervention against cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4746. doi:10.1158/1538-7445.AM2011-4746