Jessica J. O'Konek

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.

β-Mannosylceramide Activates Type I Natural Killer T Cells to Induce Tumor Immunity without Inducing Long-Term Functional Anergy

Purpose: Most studies characterizing antitumor properties of invariant natural killer T (iNKT) cells have used the agonist, α-galactosylceramide (α-GalCer). However, α-GalCer induces strong, long-lasting anergy of iNKT cells, which could be a major detriment for clinical therapy. A novel iNKT cell agonist, β-mannosylceramide (β-ManCer), induces strong antitumor immunity through a mechanism distinct from that of α-GalCer. The objective of this study was to determine whether β-ManCer induces anergy of iNKT cells. Experimental Design: Induction of anergy was determined by ex vivo analysis of splenocytes from mice pretreated with iNKT cell agonists as well as in the CT26 lung metastasis in vivo tumor model. Results: β-ManCer activated iNKT cells without inducing long-term anergy. The transience of anergy induction correlated with a shortened duration of PD-1 upregulation on iNKT cells activated with β-ManCer, compared with α-GalCer. Moreover, whereas mice pretreated with α-GalCer were unable to respond to a second glycolipid stimulation to induce tumor protection for up to 2 months, mice pretreated with β-ManCer were protected from tumors by a second stimulation equivalently to vehicle-treated mice. Conclusions: The lack of long-term functional anergy induced by β-ManCer, which allows for a second dose to still give therapeutic benefit, suggests the strong potential for this iNKT cell agonist to succeed in settings where α-GalCer has failed. Clin Cancer Res; 19(16); 4404–11. ©2013 AACR.

Nanoemulsion adjuvant–driven redirection of TH2 immunity inhibits allergic reactions in murine models of peanut allergy

Background: Immunotherapy for food allergies involves progressive increased exposures to food that result in desensitization to food allergens in some subjects but not tolerance to the food. Therefore new approaches to suppress allergic immunity to food are necessary. Previously, we demonstrated that intranasal immunization with a nanoemulsion (NE) adjuvant induces robust mucosal antibody and TH17‐polarized immunity, as well as systemic TH1‐biased cellular immunity with suppression of pre‐existing TH2‐biased immunity. Objective: We hypothesized that immunization with food in conjunction with the nanoemulsion adjuvant could lead to modulation of allergic reactions in food allergy by altering pre‐existing allergic immunity and enhancing mucosal immunity. Methods: Mice were sensitized to peanut with aluminum hydroxide or cholera toxin. The animals were then administered 3 monthly intranasal immunizations with peanut in the nanoemulsion adjuvant or saline. Mice were then challenged with peanut to examine allergen reactivity. Results: The NE intranasal immunizations resulted in marked decreases in TH2 cytokine, IgG1, and IgE levels, whereas TH1 and mucosal TH17 immune responses were increased. After allergen challenge, these mice showed significant reductions in allergic hypersensitivity. Additionally, the NE immunizations significantly increased antigen‐specific IL‐10 production and regulatory T‐cell counts, and the protection induced by NE was dependent in part on IL‐10. Control animals immunized with intranasal peanut in saline had no modulation of their allergic response. Conclusions: NE adjuvant–mediated induction of mucosal TH17 and systemic TH1‐biased immunity can suppress TH2‐mediated allergy through multiple mechanisms and protect against anaphylaxis. These results suggest the potential therapeutic utility of this approach in the setting of food allergy.

Intranasal nanoemulsion-based inactivated respiratory syncytial virus vaccines protect against viral challenge in cotton rats

Respiratory Syncytial Virus is a leading cause of bronchiolitis and pneumonia in infants, the elderly and individuals with compromised immune systems. Despite decades of research, there is currently no available vaccine for RSV. Our group has previously demonstrated that intranasal immunization of mice with RSV inactivated by and adjuvanted with W805EC nanoemulsion elicits robust humoral and cellular immune responses, resulting in protection against RSV infection. This protection was achieved without the induction of airway hyper-reactivity or a Th2-skewed immune response. The cotton rat Sigmodon hispidus has been used for years as an excellent small animal model of RSV disease. Thus, we extended these rodent studies to the more permissive cotton rat model. Intranasal immunization of the nanoemulsion-adjuvanted RSV vaccines induced high antibody titers and a robust Th1-skewed cellular response. Importantly, vaccination provided sterilizing cross-protective immunity against a heterologous RSV challenge and did not induce marked or severe histological effects or eosinophilia in the lung after viral challenge. Overall, these data demonstrate that nanoemulsion-formulated whole RSV vaccines are both safe and effective for immunization in multiple animal models.

Abstract 460: Delicate balance among three types of T cells in concurrent regulation of tumor immunity.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DCnnVarious immunosuppressive cells, such as Tregs and type II NKT cells, mediate the immune regulation of cancer. Many studies have demonstrated the importance of regulatory cells; however, it is still not clear why different suppressive cells play a dominant role in different models. Here, we examined the relative role of two suppressors - Tregs and type II NKT cells – in a subcutaneous CT26 tumor model in three strains of mice: wild-type, NKT cell-deficient CD1dKO mice and Jα18KO, which lack type I NKT cells but still retain type II NKT cells. Tumors grew in all three strains, but Treg blockade by anti-CD25 mAb (PC61) led to tumor rejection in WT and CD1dKO but not in Jα18KO mice, suggesting that in Jα18KO mice, Tregs are not necessary for immune suppression. We hypothesized that cross regulation between type I and type II NKT cells in wild-type mice leaves Treg cells as primary suppressors, whereas in mice lacking type I NKT cells, unopposed type II NKT cells can suppress tumor immunity even when Tregs are blocked. We confirmed this hypothesis in PC61-treated Jα18KO mice by blocking type II NKT cells using anti-CD1d mAb (1B1) or by adoptive transfer of type I NKT cells combined with Treg blockade. These results support our hypothesis that it is necessary to block both suppressors, type II NKT cells and Tregs, in order to achieve protection. Also, activation of type II NKT cells by sulfatide in PC61-treated wild-type mice abrogated the protective effect of Treg blockade, indicating that shifting the balance between the two types of NKT cells toward immunosuppressive type II NKT cell dominance suppresses tumor immunity even in the absence of Tregs. We conclude that both Tregs and type II NKT cells can concurrently suppress tumor immunity and the balance between these is controlled in part by a third T cell, the type I NKT cell, regulating the regulators. Finally as cancer patients often have deficient type I NKT cell function somewhat like the Jα18KO mice, managing this delicate balance among three T cells may be critical for the success of immunotherapy of human cancer.nnCitation Format: Liat Izhak, Elena Ambrosino, Jessica J. OKonek, Shingo Kato, Stanley T. Parish, Zheng Xia, David Venzon, Jay A. Berzofsky, Masaki Terabe. Delicate balance among three types of T cells in concurrent regulation of tumor immunity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 460. doi:10.1158/1538-7445.AM2013-460

Abstract 1556: α-mannosylceramide, a new NKT cell agonist, induces tumor immunity without anergy induction in NKT cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, ILnnNKT cells are CD1d-restricted T cells recognizing lipid antigens. When NKT cells are activated with a prototypic antigen, α-galactosylceramide (α-GalCer), they induce strong tumor immunity. Strong preclinical evidence for α-GalCers ability to induce tumor immunity through NKT cell-activation led to translation into therapy of cancer patients. However, so far minimal success has been achieved in patients. We reasoned that having an α-linked sugar moiety in the structure of α-GalCer can be a target for anti-α-linked galactose natural antibodies, which are a cause of acute xenograft rejection, and which might abrogate the biological activity of α-GalCer. After testing multiple glycosylceramides with potential agonistic activity for NKT cells, we discovered that α-mannosylceramide (α-ManCer) has strong activity to induce tumor immunity through a TNF-α- and NOS-dependent mechanism that is distinct from the IFN-γ dependent protection induced by α-GalCer. Another downside of α-GalCer treatment is induction of long-lasting anergy in activated NKT cells. Since we did not observe massive cytokine production with α-ManCer, in contrast to α-GalCer, we asked whether this new compound activates NKT cells without inducing long-lasting anergy in vivo. In contrast to α-GalCer, α-ManCer did not induce long-lasting anergy of activated NKT cells. This was correlated with the observation that α-ManCer induced only a short term expression of PD-1 on activated NKT cells, which has been reported to be responsible for α-GalCer-induced long-term anergy. Furthermore, while prior treatment with α-GalCer abrogated the protective effect of either α-GalCer or α-ManCer against CT26 colon carcinoma tumor challenge for at least two months, the pre-treatment with α-ManCer had no effect on the protection induced by either NKT cell agonist. Since α-ManCer can activate human NKT cells, this new NKT cell agonist may provide a better opportunity for NKT cell targeted immunotherapy in cancer.nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1556. doi:1538-7445.AM2012-1556

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, FLnnCD1d-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.nnCitation 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

1033. A Novel Mechanism of Synergistic Cytoxicity between Prodrugs, 5-FC and GCV in Double Suicide Gene Therapy

The simultaneous use of CD/5-FC and HSV-TK/GCV suicide gene protocols has resulted in enhanced antitumor activity in cultured tumor cell lines as well as in mouse models. It has been suggested that the CD/5-FC-mediated decrease in dTTP leads to lower thymidine levels and decreased competition for GCV phosphorylation by HSV-TK. In this study, we demonstrate that concurrent addition of prodrugs, 5-FC and GCV, was less efficacious than sequential addition of 5-FC followed by GCV treatment in human DU145 prostate carcinoma cells infected with an adenovirus containing a CD/HSV-TK fusion gene. The level of radiolabeled GCV-TP in cells treated simultaneously with 200 μg/ml 5-FC and 1.0μM GCV for 24 hr was 188 pmol/106 cells compared to 143 pmol/106 cells in cells treated with GCV alone. However, if the cells were pretreated 24 hr with 5-FC and then incubated 24 hr with GCV, GCV-TP levels were 2.6-fold higher compared to GCV treatment alone. [H3]GCV incorporation into DNA was also elevated more than 2-fold in pretreated cells compared to simultaneous addition. In addition, compared to GCV treatment alone, sequential addition of 5-FC and GCV resulted in an additional 60% growth inhibition, whereas only 15% inhibition was observed when both drugs were given together. We measured the effect of 5-FC pre-incubation on endogenous deoxynucleotide concentrations by incubating DU145 cells transduced with the CD/HSV-TK adenovirus with 200 μg/ml 5-FC over a 24 hr period. As expected, cellular dTTP levels were gradually depleted over the 24 hr incubation (from 82 to 11 pmol/106 cells). However, the level of dGTP also decreased (from 9 to 1 pmol/106 cells) concurrently with the dTTP pool decrease over this time period. This reduction of dTTP and dGTP concentrations was not observed when CD/HSV-TK-expressing DU145 cells were treated over 24 hr with both 5-FC and GCV simultaneously. The results from these experiments allowed us to propose and test a novel hypothesis for the synergistic interaction between CD/5-FC and HSV-TK/GCV treatments. We suggest that the CD/5-FC-mediated reduction of dTTP concentrations results in concurrent decrease of dGTP levels due to allosteric regulation of ribonucleotide reductase. Since dGTP is the endogenous competitor of GCV-TP, a depleted dGTP pool at the time of GCV addition can result in increased GCV incorporation into DNA and cell kill. In fact, including increasing amounts of deoxyguanosine during the 5-FC pre-incubation reverses the depletion of endogenous dGTP, reduces the amount of GCV incorporated into DNA and negates the GCV-mediated cytotoxicity associated with 5-FC pre-incubation in DU145 cells expressing CD and HSV-TK. Understanding this mechanistic interaction between these two enzyme/prodrug systems may help recognize better strategies for creating more efficacious clinical protocols.