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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 naive 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 naive 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.

Summary The mammalian (mechanistic) target of rapamycin (mTOR) regulates critical immune processes that remain incompletely defined. Interest in mTOR inhibitor drugs is heightened by recent demonstrations that the mTOR inhibitor rapamycin extends lifespan and healthspan in mice. Rapamycin or related analogues (rapalogues) also mitigate age‐related debilities including increasing antigen‐specific immunity, improving vaccine responses in elderly humans, and treating cancers and autoimmunity, suggesting important new clinical applications. Nonetheless, immune toxicity concerns for long‐term mTOR inhibition, particularly immunosuppression, persist. Although mTOR is pivotal to fundamental, important immune pathways, little is reported on immune effects of mTOR inhibition in lifespan or healthspan extension, or with chronic mTOR inhibitor use. We comprehensively analyzed immune effects of rapamycin as used in lifespan extension studies. Gene expression profiling found many and novel changes in genes affecting differentiation, function, homeostasis, exhaustion, cell death, and inflammation in distinct T‐ and B‐lymphocyte and myeloid cell subpopulations. Immune functions relevant to aging and inflammation, and to cancer and infections, and innate lymphoid cell effects were validated in vitro and in vivo. Rapamycin markedly prolonged lifespan and healthspan in cancer‐ and infection‐prone mice supporting disease mitigation as a mechanism for mTOR suppression‐mediated longevity extension. It modestly altered gut metagenomes, and some metagenomic effects were linked to immune outcomes. Our data show novel mTOR inhibitor immune effects meriting further studies in relation to longevity and healthspan extension.

Mutation of a single copy of the adenomatous polyposis coli (APC) gene results in familial adenomatous polyposis (FAP), which confers an extremely high risk for colon cancer. ApcMin/+ mice exhibit multiple intestinal neoplasia (MIN) that causes anemia and death from bleeding by 6 months. Mechanistic target of rapamycin complex 1 (mTORC1) inhibitors were shown to improve ApcMin/+ mouse survival when administered by oral gavage or added directly to the chow, but these mice still died from neoplasia well short of a natural life span. The National Institute of Aging Intervention Testing Program showed that enterically targeted rapamycin (eRapa) extended life span for wild-type genetically heterogeneous mice in part by inhibiting age-associated cancer. We hypothesized that eRapa would be effective in preventing neoplasia and extend survival of ApcMin/+ mice. We show that eRapa improved survival of ApcMin/+ mice in a dose-dependent manner. Remarkably, and in contrast to previous reports, most of the ApcMin/+ mice fed 42 parts per million eRapa lived beyond the median life span reported for wild-type syngeneic mice. Furthermore, chronic eRapa did not cause detrimental immune effects in mouse models of cancer, infection, or autoimmunity, thus assuaging concerns that chronic rapamycin treatment suppresses immunity. Our studies suggest that a novel formulation (enteric targeting) of a well-known and widely used drug (rapamycin) can dramatically improve its efficacy in targeted settings. eRapa or other mTORC1 inhibitors could serve as effective cancer preventatives for people with FAP without suppressing the immune system, thus reducing the dependency on surgery as standard therapy. Cancer Prev Res; 7(1); 169–78. ©2013 AACR.

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+CD25hi 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. Cancer Res; 72(8); 2089–99. ©2012 AACR.

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.

mTOR regulates many important immune processes. Interest in immune effects of mTOR inhibitors is heightened by recent findings of ability to boost specific immunity and vaccine response in elderly humans, treat cancers and autoimmunity, and extend life and healthspan in mice, suggesting important new clinical applications. However, immune toxicity concerns for long-term mTOR inhibition, particularly immunosuppression, persist. We comprehensively analyzed immune effects of long-term rapamycin in mice. Gene expression profiling of purified T and B cells (CD4+PD1+, CD4+PD1-, CD8+PD1+, CD8+PD1-, B220+), CD11b+CD11c- myeloid cells and CD11c+ dendritic cells showed numerous, novel changes in genes affecting differentiation, function, homeostasis, migration, exhaustion, cell death and inflammation. Novel effects on Th9, Th22 and Tfh differentiation were seen as was improved function of exhausted PD1+ T cells (supported by human clinical data) and increased numbers and function of innate lymphoid cells. Immune functions relevant to cancer, infections, aging and inflammation, and innate lymphoid cell effects were validated in vitro and in vivo. eRapa prolonged life in RAG KO mice (without T or B cells) >12x and in IFN-γ KO mice >3x over wild type mice, supporting mTOR suppression immune effects mitigating disease as a mechanism for longevity extension. Metagenomic effects were linked to immune outcomes. Our data demonstrate novel mTOR immune effects meriting further investigations.

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

eRapa extends life in mice and cancer prevention could be a mechanism. Rapamycin inhibits mTOR, which has significant immune effects that are surprisingly unstudied in cancer therapy or prevention. We hypothesize that cancer prevention by eRapa is mediated by improved cancer immune surveillance, which we tested in the well-established DMBA/TPA carcinogen-induced skin cancer model. Mice got eRapa or control chow, and skin tumors were induced with DMBA/TPA over 24 weeks. eRapa reduced benign (p=.001) and malignant (p=.05) tumors in WT mice. T cells and IFN-γ mediate cancer immune surveillance. eRapa reduced skin tumors in βδ KO mice lacking all T cells (p=.04), but not in IFN-γ KO mice (p=.13), consistent with loss of beneficial eRapa-induced, non-T cell IFN-γ. In support, WT or IFN-γ KO T cell transfer into IFN-γ KO mice did not alter eRapa cancer prevention in DMBA/TPA. In WT mice on DMBA/TPA, eRapa increased IFN-γ-producing natural killer cells (p=.01) that could mediate skin cancer immune surveillance, and decreased CD34+CD49fint skin cancer stem cells (p=.01) and CXCR3+ T cells (p

Tregs blunt anti-tumor immunity, and their depletion effectively treats tumor in mouse cancer models. However, Treg depletion in human trials is only partially effective. In our phase II ovarian cancer trial, denileukin diftitox (DT) effected significant Treg depletion in blood and the tumor microenvironment but did not produce significant clinical efficacy. Interferon-α is not an effective ovarian cancer treatment, but we show here that it significantly augments immune and clinical DT-mediated Treg depletion efficacy in human ovarian cancer. In a mouse ovarian cancer model, DT modestly improved anti-tumor immunity and survival. Interferon-α alone did not alter Treg numbers or function, but boosted CD8+ T cell anti-tumor immunity. Interferon-α plus DT significantly prolonged mouse survival over either individual agent. Using type I IFNR-/- mice that cannot mediate interferon-α signals, we showed that interferon-α directly boosted CD8+ T cell function independent of CD4+ T cell help, and in combination with DT reduced Treg function (without further numerical reduction) through indirect effects on tumor microenvironmental dendritic cells. When three ovarian cancer patients failed DT alone, two experienced immunologic and clinical benefit by adding pegylated interferon-α2a, with manageable toxicities. These studies demonstrate novel immune and clinical interferon-α anti-cancer benefits that augment Treg depletion using FDA-approved agents for rapid clinical translation.

A widely applicable cancer prevention strategy would be a cost-effective approach to lessen the enormous burden of cancer. Enterically-released microencapsulated rapamycin (eRapa) extends lifespan in mice and delays or prevents cancer development, suggesting cancer prevention as a longevity mechanism. Rapamycin acts through inhibition of mechanistic target of rapamycin (mTOR), which has significant immune effects that are surprisingly little studied in cancer treatment or prevention. In naive mice, chronic eRapa significantly reduced Th1 (CXCR3 + ) cell numbers ( p = .03) and T cell interferon (IFN)- γ ( p = .04), increased Th17 (CCR6 + ) cell numbers ( p = .04) and T cell IL-17 ( p = .03), increased T cell IL-22 ( p = .02), and reduced immune suppressing regulatory T cells ( p βδ −/− and IFN- γ −/− mice (lacking all T cells or IFN- γ , respectively) were given eRapa or control, and skin tumors were induced with the carcinogen 7,12-dimethylbenz[a]anthracene (DMBA) followed by the inflammatory promoting agent 12-O-tetradecanoylphorbol-13-acetate (TPA). eRapa reduced papilloma size in WT BL6 mice ( p = .02) versus controls, the first demonstration that oral rapamycin prevents inflammation-mediated neoplasia. eRapa also significantly reduced papillomas in βδ −/− mice ( p = .04) and increased papillomas in IFN- γ −/− mice ( p = .05). These data are consistent with the concept that eRapa protects from carcinogen-induced dermal carcinogenesis through a non-T cell, IFN- γ -dependent mechanism, which we suspect includes natural killer cells. We have also shown that eRapa prevents cancer in a variety of mouse cancer models, in which we are further studying immune mechanisms. Immune contributions from mTOR inhibitors in cancer prevention (and treatment) require more attention.

Denileukin diftitox (DD) is an FDA-approved conjugate of human IL-2 + diphtheria toxin. It depletes Tregs and improves clinical and immune outcomes, but Treg generation can reduce efficacy. B7-H1 is an immune co-signaling molecule that promotes Treg generation and hinders anti-cancer immunity. αB7-H1 or DD each slowed tumor growth and improved anti-tumor immunity in B16 melanoma. DD reduced Treg numbers but αB7-H1 also reduced Treg suppression. In vitro, αB7-H1 reduced Treg generation from naïve CD4+CD25- T cells. We thus hypothesized that αB7-H1 would synergistically improve clinical efficacy of Treg depletion with DD by slowing regeneration or function of Tregs after depletion. We treated mice 7 days after B16 challenge when detectable tumors were present. DD+αB7-H1 significantly slower tumor growth at 25 days (avg tumor ~600 mm3 vs either single agent treatments (avg 1500-2000 mm3; p

We recently showed that B7-H1 blockade in mouse models for B16 melanoma and ID8 ovarian carcinoma worked better in females versus males through reduced regulatory T cell (Treg) function. We now show that B7-H1 blunts estrogen-mediated mTOR activation in differentiating Tregs as the mechanism. When we differentiated naïve CD4+ T cells into Tregs in vitro, estrogen reduced Treg numbers and function only if B7-H1 was blocked. The effect was equivalent in males and females. Treg depletion for cancer immunotherapy is promising but hampered by rapid Treg regeneration. Because estrogen signals reduced Treg generation when B7-H1 was blocked, we hypothesized that B7-H1 block during therapeutic Treg depletion would improve clinical efficacy of either individual approach, and that estrogen signals would further boost combination effects. We then showed that estrogen effects were through estrogen receptor beta, suggesting that estrogen receptor beta agonists (in human clinical trials with promising results) could boost Treg depletion with B7-H1 blockade as cancer immunotherapy. In a proof-of-concept experiment, in B16 melanoma challenge, estrogen improved tumor resistance in B7-H1 KO males but not WT males through reduced Treg function. B7-H1 blockade plus estrogen also improved anti-CTLA-4 effects, suggesting rational combinations of immune blockade, Treg depletion and estrogen signals as novel cancer immunotherapies.

Age is the biggest risk factor for cancer but most studies are in young hosts. We mitigated age-specific tumor-related immune dysfunction to develop immunotherapy for B16 melanoma effective in aged hosts. Young and aged CD4+CD25hi regulatory T cells (Tregs) had similar in vitro function and in vivo tumor-associated increases, but denileukin diftitox (DT)-mediated Treg depletion improved tumor-specific immunity and was clinically effective only in young mice. Aged mice had more basal and tumor-related myeloid derived suppressor cells (MDSC). Depleting MDSC with anti-Gr-1 Ab improved tumor-specific immunity and clinical response better in aged versus young mice. Treg depletion significantly increased MDSC in aged but not young mice, suggesting age-related Treg control of MDSC. In vitro, Treg from aged tumor bearing mice suppressed GM-CSF-induced bone marrow MDSC differentiation. Treg depletion + anti-Gr-1 Ab was more efficacious than anti-Gr-1 alone in aged B16-bearing mice but was no better than Treg depletion alone in young mice. In MC-38 colon cancer, DT and anti-Gr-1 Ab effects were similar in young and aged hosts. Thus aged anti-tumor immune effector cells are competent to combat tumor when tumor-associated immune dysfunction is appropriately mitigated, and age-related immune dysfunction is tumor-dependent. Tailoring immunotherapy to counter age-related, tumor-associated immune dysfunctions remarkably improves cancer immunotherapy for aged patients with specific tumors.

Sex-based immune differences generally include a greater autoimmune disease risk in females. Regulatory T cells (Tregs) control peripheral tolerance and defend against autoimmunity. B7-H1 is a co-signaling molecule with an incompletely understood role in peripheral tolerance. Tregs in female B7-H1-/- (KO) mice are poorly functional in vitro compared to male KO or wild type (WT). Transfer of KO male Tregs protected Rag1-/- mice from colitis in vivo but KO female Tregs did not. Differences did not depend on known B7-H1 ligands. CD4+CD25- T cells from KO females converted into phenotypically and functionally normal Tregs with IL-2+TGF-β in vitro, suggesting no significant cell-autonomous defects in Treg differentiation or function. Treg function in sexually immature KO females was equivalent to adult WT females, but Treg function in KO females declined by 8 weeks, just after estrus onset. Oöpherectomized mice receiving estrogen maintained defective Treg function, whereas mice receiving placebo had functional Tregs. B7-H1-mediated Treg functional defects significantly reduced female protection from anti-CTLA4 induced autoimmunity and resulted from altered B7-H1-dependent mTOR and PTEN signals. Dendritic cell B7-H1 corrected KO female Treg defects in vivo. These data demonstrate a previously unknown sex-dependent relationship between estrogen, B7-H1, mTOR signaling, and Treg function that corrected predict important sex-dependent differences to immune insults and immunotherapies.

Inhibitors of histone deacetylases (HDACIs) are a new generation of anticancer agents that selectively kill tumor cells. However, the molecular basis for their tumor selectivity is not well understood. we employed a genetic technique, named suppression of mortality by antisense rescue technique(SMART), to understand the molecular mechanism of apoptosis induced by HDACI and identify the genes that participate in this process. We identified several novel genes and demonstrated that HDACIs promote apoptosis through activation ubiquitin/proteasome by inducing ubiquitin B not ubiquitin C gene expression. According to degrading the mitochondria membrane protein Mcl-1 by activated ubiquitin/proteasome system, the mitochondria potential collapse and cell apoptosis. Further observation demonstrated that depletion of MCL-1 by RNA interference (RNAi) sensitizes Hela cells to TSA mediated mitochondria membrane potential collapse and apoptosis, and overexpression of MCL-1 confers apoptosis resistance.