Suzanne R. Thibodeaux

B7-H1-dependent sex-related differences in tumor immunity and immunotherapy responses

CD4+CD25+Foxp3+ regulatory T cells (Tregs) are immunopathogenic in cancers by impeding tumor-specific immunity. B7-homologue 1 (B7-H1) (CD274) is a cosignaling molecule with pleiotropic effects, including hindering antitumor immunity. In this study, we demonstrate sex-dependent, B7-H1–dependent differences in tumor immunity and response to immunotherapy in a hormone-independent cancer, murine B16 melanoma. Antitumor immunity was better in B7-H1−/− females versus males as a result of reduced regulatory T cell function in the B7-H1−/− females, and clinical response following B7-H1 blockade as tumor immunotherapy was significantly better in wild-type females than in males, owing to greater B7-H1 blockade-mediated reduction of Treg function in females. Wild-type female Tregs expressed significantly lower B7-H1 versus males but were insensitive to estrogen in vitro. Female B7-H1−/− Tregs were exquisitely sensitive to estrogen-mediated functional reduction in vitro, suggesting that B7-H1 effects occur before terminal Treg differentiation. Immune differences were independent of known B7-H1 ligands. Sex-dependent immune differences are seldom considered in designing immune therapy or interpreting immunotherapy treatment results. Our data demonstrate that sex is an important variable in tumor immunopathogenesis and immunotherapy responses through differential Treg function and B7-H1 signaling.

Mitigating Age-Related Immune Dysfunction Heightens the Efficacy of Tumor Immunotherapy in Aged Mice

Although cancer tends to affect the elderly, most preclinical studies are carried out in young subjects. In this study, we developed a melanoma-specific cancer immunotherapy that shows efficacy in aged but not young hosts by mitigating age-specific tumor-associated immune dysfunction. Both young and aged CD4(+)CD25(hi) regulatory T cells (Treg) exhibited equivalent in vitro T-cell suppression and tumor-associated augmentation in numbers. However, denileukin diftitox (DT)-mediated Treg depletion improved tumor-specific immunity and was clinically effective only in young mice. DT-mediated Treg depletion significantly increased myeloid-derived suppressor cell (MDSC) numbers in aged but not young mice, and MDSC depletion improved tumor-specific immunity and reduced tumor growth in aged mice. Combining Treg depletion with anti-Gr-1 antibody was immunologically and clinically more efficacious than anti-Gr-1 antibody alone in aged B16-bearing mice, similar to Treg depletion alone in young mice. In contrast, DT increased MDSCs in young and aged mice following MC-38 tumor challenge, although effects were greater in aged mice. Anti-Gr-1 boosted DT effects in young but not aged mice. Aged antitumor immune effector cells are therefore competent to combat tumor when underlying tumor-associated immune dysfunction is appropriately mitigated, but this dysfunction varies with tumor, thus also varying responses to immunotherapy. By tailoring immunotherapy to account for age-related tumor-associated immune dysfunctions, cancer immunotherapy for aged patients with specific tumors can be remarkably improved.

Aged regulatory T cells protect from autoimmune inflammation despite reduced STAT3 activation and decreased constraint of IL-17 producing T cells

Regulatory T cells (Tregs) are specialized CD4+ T lymphocytes helping defend against autoimmunity and inflammation. Although age is associated with increased inflammation and autoimmunity, few reports address age effects of immune regulation or auto‐aggressive T cells. We show here that young and aged naïve CD4+ T cells are equivalently auto‐aggressive in vivo in T cell‐driven autoimmune colitis. Young and aged CD4+ Tregs equally suppressed age‐matched T cell proliferation in vitro and controlled clinical and pathologic T cell‐driven autoimmune colitis, suggesting equivalent regulatory function. However, whereas young and aged CD4+ Tregs suppressed interferon (IFN)‐γ+ T cells equivalently in this model, aged CD4+ Tregs unexpectedly failed to restrain interleukin (IL)‐17+ T cells. Nonetheless, young and aged CD4+ Tregs equally restrained IL‐17+ T cells in vivo during acute inflammation, suggesting a chronic inflammation‐related defect in aged CD4+ Tregs. In support, aged Tregs expressed reduced STAT3 activation, a defect associated with poor IL‐17‐producing T cell restraint. Aged naïve mice had markedly increased programmed death (PD)‐1+ T cells, but these exhibited no significant auto‐aggressive or regulatory functions in T cell‐driven colitis. Young CD8+ CD122− T cells induce autoimmune bone marrow failure, but we show that aged CD8+ CD122− T cells do not. These data demonstrate no apparent age‐related increase in auto‐aggressive T cell behavior, but disclose previously unrecognized functional defects in aged CD4+ Tregs during chronic inflammation. IL‐17 can be inflammatory and contributes to certain autoimmune disorders. Reduced aged Treg function during chronic inflammation and reduced IL‐17 restraint could contribute to age‐related inflammation or autoimmunity.

Immune Therapy for Ovarian Cancer: Promise and Pitfalls

This review covers immune and anatomic considerations specific for ovarian cancer immune therapy. Prior approaches and potential reasons for lack of better efficacy are discussed, along with newer approaches in pre-clinical and early clinical development. Potential means for future investigations and means to integrate immune with other approaches are discussed.

TgMAPK1 is a Toxoplasma gondii MAP kinase that hijacks host MKK3 signals to regulate virulence and interferon-γ-mediated nitric oxide production.

The parasite Toxoplasma gondii controls tissue-specific nitric oxide (NO), thereby augmenting virulence and immunopathology through poorly-understood mechanisms. We now identify TgMAPK1, a Toxoplasma mitogen-activated protein kinase (MAPK), as a virulence factor regulating tissue-specific parasite burden by manipulating host interferon (IFN)-γ-mediated inducible nitric oxide synthase (iNOS). Toxoplasma with reduced TgMAPK1 expression (TgMAPK1(lo)) demonstrated that TgMAPK1 facilitates IFN-γ-driven p38 MAPK activation, reducing IFN-γ-generated NO in an MKK3-dependent manner, blunting IFN-γ-mediated parasite control. TgMAPK1(lo) infection in wild type mice produced ≥ten-fold lower parasite burden versus control parasites with normal TgMAPK1 expression (TgMAPK1(con)). Reduced parasite burdens persisted in IFN-γ KO mice, but equalized in normally iNOS-replete organs from iNOS KO mice. Parasite MAPKs are far less studied than other parasite kinases, but deserve additional attention as targets for immunotherapy and drug discovery.

A simple method to detect Toxoplasma gondii-specific cytotoxic T cells in vivo.

Cytotoxic T cells (CTLs) are an important component of adaptive immunity. The study of antigen-specific CTLs in vivo is desirable yet difficult. Identification of the class I-restricted peptide used by CTLs for target recognition is often required for detailed studies, but is generally not known for most antigens. Toxoplasma gondii is a medically important, obligate intracellular parasite and is often used as a model for studies of parasite immunology. No class I-restricted peptides for CTLs are known. We show here a new and convenient method to detect T. gondii-specific CTLs in vivo. We engineered T. gondii tachyzoites to express the model antigen ovalbumin, for which many useful reagents and transgenic mice are available. Using ovalbumin-transgenic T. gondii tachyzoites, antigen-specific CTLs were detected in vivo, and at much earlier time points post-infection than previously reported. This new method has several additional advantages over current methods to detect T. gondii-specific CTLs.

Abstract LB-259: Interferon-α enhances clinical benefits of regulatory T cell depletion in ovarian cancer through direct T cell effects and by inducing bystander IL-6

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Tregs hinder anti-tumor immunity, and depleting them treats cancers effectively in mice. By contrast, Treg depletion in human trials has limited efficacy. In a phase I trial of advanced stage carcinomas (breast, lung, melanoma, ovarian, bladder), we showed that the IL-2/diphtheria fusion toxin denileukin diftitox (DT) significantly depleted functional CD4+CD25hiFoxp3+ Tregs in blood with improved T cell function (increased Ki-67 and interferon-γ) with minimal effects on other blood mononuclear cells. One patient with stage IV ovarian carcinoma experienced significant reduction of metastatic tumor burden with denileukin diftitox at 12 μg/kg. Additional work showed that Treg depletion effects could last up to 4 weeks, and that weekly DT at 12 μg/kg eventually depleted anti-tumor and tumor-specific CD8+ T cells. Based on these data, we conducted a phase II clinical trial of epithelial ovarian carcinomas FIGO stage III-IV failing standard treatments, using DT as a single agent at 12 μg/kg by intravenous infusion every 3-4 weeks. In this trial, DT significantly depleted blood and tumor microenvironmental Tregs with only grade I-II toxicities, but with minimal clinical efficacy in 28 consecutive patients. This trial was halted for futility according to the Simon 2-stage design. Interferon-α alone does not treat ovarian cancer, but we now show that it significantly improves clinical and immune DT-mediated Treg depletion efficacy in human ovarian cancer. In the ID8 mouse ovarian carcinoma model, DT modestly increased survival and anti-tumor immunity. Interferon-α alone did not affect Treg numbers or function, but enhanced CD8+ T cell anti-tumor immunity. Interferon-α plus DT increased mouse survival significantly over either individual drug. In type I IFNR-/- mice unable to mediate interferon-α signals, interferon-α directly increased adoptively transferred IFNR+CD8+ T cell function independent of CD4+ T cell help. When combined with DT, IFN-α reduced Treg function without further numerical Treg reduction by indirect effects on tumor microenvironmental dendritic cells. In vitro studies identified IFN-α-driven dendritic cell IL-6 as a mechanism for reducing Treg function. When three ovarian cancer patients failed DT alone in the phase II trial, two had clinical and immune benefit by adding pegylated interferon-α2a in a separate trial, with acceptable toxicities. This trial was halted due to a lack of further DT. These studies demonstrate that DT depletes Tregs in distinct human carcinomas but is unlikely to be clinically effective as a single agent. We identified novel IFN-α mechanisms that improve Treg depletion effects using FDA-approved agents that can be rapidly translated. More selective Treg depletion agents and rationale combinations with other agents could improve clinical efficacy further. Citation Format: Suzanne Thibodeaux, Vincent Hurez, Shawna Wall, Srilakshmi Pandeswara, Benjamin Daniel, Aijie Liu, Lishi Sun, Leslie Wood, Weiping Zou, Tyler Curiel. Interferon-α enhances clinical benefits of regulatory T cell depletion in ovarian cancer through direct T cell effects and by inducing bystander IL-6. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-259. doi:10.1158/1538-7445.AM2014-LB-259

General Properties of Dendritic Cell Populations in Cancer

Various, distinct populations of dendritic cells (DC) populate different normal, quiescent tissues in differing amounts, where they subserve a variety of functions generally for the benefit of the host. In cancers, the types, relative proportions, and functions of DC are altered, often to the detriment of the host. This chapter reviews the general issues related to imbalanced DC populations and their causes and dysfunctional consequences in the tumor microenvironment. Potential applications of this knowledge to novel anti-cancer immunotherapy strategies are outlined. Human and mouse disease models suitable for the study of tumor microenvironmental DC are discussed.