Cancer inflammation and regulatory T cells

Abstract

Chronic inflammation is essential for cancer growth and metastasis. It follows that factors reducing inflammation would abrogate cancer and restore tissue health. However, roles for anti-inflammatory CD41 regulatory cells (T_REG) in cancer are enigmatic and controversial. Our recent data reveal that T_REG may function in cancer similarly to inflammatory bowel disease or multiple sclerosis, whereby T_REG accumulate but lack potency to restore tissue homeostasis under inflammatory conditions. Interestingly, early life exposures to diverse environmental organisms reinforce a protective T_REG phenotype that inhibits cancer. In contrast, hygienic individuals with few exposures earlier in life suffer from a dysregulated T_REG feedback loop. Consequently, hygienic subjects have increased risk of malignancy later in life. This cancer condition is reversible by blocking underlying inflammation. Taken together, these data help explain increased inflammation-associated cancer rates in hygienic societies and identify targets to abrogate cancer and restore overall health.

The link between chronic inflammation and cancer has been recognized for many years.¹ However, the precise interaction between inflammatory cells and their elaborated factors and tumor cells remains unknown. Originally, inflammation was believed to be primarily a beneficial host response, representing the body’s fight against invading tumor cells. More recent data however suggest just the opposite: that inflammation may be the cause of some cancers and a powerful stimulus for tumor growth and invasion.^1–4 Indeed, there are numerous data supporting the association of chronic long-standing inflammation and increased risk of tumors of the gastrointestinal (GI) tract.^5–8 In addition, systemic non-steroidal anti-inflammatory drug (NSAID) use has been linked with a significant decrease in cancer of the bowel, lung, liver and prostate.⁹ However, therapies aimed at restoring homeostasis involve numerous challenges including immune suppression with increased risk of pathogenic infections, as well as removing beneficial antitumor effects of immune surveillance.

Balanced Activities of T Cells are Essential for Health

Overall health with ability to control inflammation and restore homeostasis requires adaptation to diverse challenges throughout life.^10,11 Pivotal roles for T lymphocytes in this process of homeostasis were first discovered in mouse models more than a decade ago.¹² A major component of immune tolerance and homeostasis is performed by CD4+ cells expressing CD25, namely regulatory T cells (T_REG). T_REG prevent immune disorders by suppressing proliferation of reactive T lymphocytes and other destructive immune responses.^11,13–15 Ability of CD4+ T_REG to offer protection from other destructive inflammatory sequelae has been convincingly demonstrated during inflammatory bowel disease (IBD) in humans and in mice.¹⁰ However, maintenance of lower bowel homeostasis actually also depends on differentiation and specialization of proinflammatory T-effector cells (T_EFF). Along with corresponding anti-inflammatory T_REG cells, the CD4+ subsets function together to allow for effective immune responses to diverse GI tract insults in naïve animals. In the bowel, prior exposures to bacteria appear to impart increased T_EFF and T_REG efficiency to downregulate subsequent infectious challenges and IBD.^11,14–19 T_REG functions to inhibit pathology have also been well characterized in autoimmune disorders such as multiple sclerosis (MS).²⁰ Mouse models of experimental allergic encephalitis (EAE) mimic MS in human patients and show that equilibrium between activities of proinflammatory T_EFF cells and anti-inflammatory T_REG is needed to sustain overall health. Taken together, T_REG are widely viewed as essential to maintain immune homeostasis and restore tissue and whole body health.

T_REG are generally regarded as a homogeneous population of cells. However, recent data suggest that the same transcriptional factors involved in T_EFF antigen specific polarization may also be involved in programming of matching T_REG.²¹ According to this paradigm, context-dependent T_REG suppress the corresponding type of host T_EFF responses, whether these may be dedicated to T helper (Th) type-1 for self or intracellular pathogens, to Th-2 for extracellular pathogens, to Th-17 for self or pathogens, or, finally, to lymphoid follicle stimulating events.²¹ Thus, T_REG primed toward diverse classes of organisms earlier in life will more efficiently downregulate corresponding inflammation later in life, whether that inflammation is driven by Th-1, Th-2 or Th-17 (Fig. 1). This raises the possibility that T_REG may be re-directed and applied therapeutically during conditions involving uncontrolled inflammation such as IBD, MS and some types of inflammation-associated cancer.

Figure 1.

Immunological profile is pivotal in cancer risk and outcome. Epidemiologic studies have shown that by midlife multiple preneoplastic lesions exist throughout the human body. However, only a few lesions will become cancerous. Data from mouse models suggest that interactions between infectious agents alter immunological profile and modulate cancer outcomes. Microbial exposures may either inhibit or exacerbate carcinogenesis by triggering immune cell activation or secretion of factors. The “hygiene hypothesis” theorizes that exposures to diverse microbiota reinforce innate immunity and T helper (Th)-1 host responses that lead to balanced and efficient immune responses later in life. By contrast, stringent hygiene conditions deprive the immune system of essential priming rendering T_REG cells ineffectual and readily directed toward a carcinogenic Th-17 host response.

Foxp3+ T_REG Cells Accumulate But Fail to Protect During Inflammatory Disorders

A large body of literature exists demonstrating the efficacy of T_REG to suppress inflammation in humans. However, an enigma arises: the presence of large numbers of T_REG cells does not assure anti-inflammatory potency, tissue restoration or health, even though properly functioning T_REG are clearly protective in humans and in mice. To address this paradox, molecular and cellular mechanisms for T_REG have been studied using animals models with antibody-mediated blockade or adoptive transfer of T_REG.²² One challenge to better understand T_REG biology has been the lack of T_REG identity markers. Transcription factor Foxp3 is critical for T_REG functions and therefore has been widely used for phenotyping and localization,²³ albeit with some limitations.

Although adoptive transfer of T_REG cures IBD in mice, Foxp3+ T_REG cells accumulate in colon and secondary lymphoid organs during IBD with low T_REG potency to restore tissue balance (Fig. 2).²⁴ This observation may be explained by insufficiently low levels of anti-inflammatory cytokine interleukin (IL)-10 in bowel that predispose to a break in immune tolerance. This undermines T_REG function and favors proinflammatory Th-17 T_EFF cells.^25,26 Likewise, in autoimmune disorders Viglietta et al.²⁷ found a significant decrease in functional efficacy of T_REG from blood of human patients with MS. Bettelli et al.²⁰ subsequently showed that Th-17 was crucial in the break in tolerance during MS. Large numbers of T_REG accumulate during MS but fail to suppress disease in humans or mice with chronically elevated levels of TNF-α and IL-6.²⁸

Under normal physiological conditions, T_REG responses are beneficial to the host to reinforce protective acute inflammation. Afterward, T_REG regain anti-inflammatory functions and restore tissue homeostasis and health.

Figure 2.

Reciprocal roles of Th-17 and T_REG cells emerge in seemingly diverse diseases: multiple sclerosis (MS), inflammatory bowel disease (IBD) and some types of cancer. Improvements in societal cleanliness have reduced many serious infections but led to an increase in allergies, asthma and autoimmune diseases including multiple sclerosis (MS) and inflammatory bowel disease (IBD). Research using mouse models with experimental allergic encephalitis (EAE), mimicking patients with MS, displays an interleukin (IL)-6-mediated shift in T cells toward pathogenic Th-17. EAE is remedied by downregulation of inflammation and restoration of properly functioning T_REG cells. Some types of inflammation-associated cancers such as colorectal carcinoma (CRC) demonstrate similar immunologic underpinnings. Most surprising were our discoveries that cancers lacking overt inflammation in extraintestinal sites, such as prostate and mammary tissue, display similar T_REG cell distribution and clinical responses after anti-inflammatory therapy. Broader relevancy of these findings is highlighted by observations that non-steroidal anti-inflammatory drugs (NSAIDs) lower the risk of many cancers including nor only CRC but also cancers of prostate, lung and breast in humans. Hygienic rearing increases risk of uncontrollable Th-17 host response.

Recent work from our laboratory found robust accumulations of Foxp3+ T_REG surrounding colonic polyps as well as rapidly growing mammary tumors in mice (Fig. 2).²⁹ In those mice, Foxp3+ cells accumulated in large numbers in the desmoplastic periphery and local lymph nodes, but failed to protect and instead exacerbated pathology. Blocking inflammation using anti-TNF-α antibody or by supplementation with gut bacteria-primed T_REG resulted in rapid tumor regression with commensurate reduction and redistribution of Foxp3+ T_REG to intratumoral locations.²⁹ Remarkably, Salama et al.³⁰ found that patients bearing colonic tumors with a high intratumoral density of FOXP3+ T_REG showed a significant improvement in survival. A better understanding of this process to harness potency of T_REG to restore normal tissue architecture and homeostasis may provide insight into novel therapies for inflammation- associated cancers.

IL-10-Dependent Functions of T_REG Are Pivotal at Environmental Interfaces Such as Colon, Lung and Skin

Although Foxp3+ T_REG are best known for ability to suppress T_EFF proliferation,^31,32 cytokine-dependent suppression by IL-10 and transforming growth factor (Tgf)-β also appear essential to reduce inflammation and facilitate wound repair. IL-10 production by T_REG serves to maintain epithelial normalcy in tissues such as GI tract and lungs,²² despite continual interactions with diverse infectious agents.^16,21 In addition, mice lacking IL-10 are also unable to sustain normal T_REG functions needed to mount tolerance.³³ Inter-related roles between T_REG, IL-10 and Tgf-β in vivo appear to be complexly interdependent and essential for epithelial integrity. Tgf-β normally induces functionally suppressive Foxp3+ T_REG from naive T cells in the periphery. However, under conditions of uncontrollable inflammation, IL-6 together with Tgf-β may instead inhibit T_REG and re-direct CD4+ cells toward a pathogenic Th-17 phenotype.²⁰ Likewise, uncontrollable elevations in other inflammatory cytokines such as IL-4 (Th-2) also block the generation of Tgf-β-induced T_REG and instead induce a population of T_EFF that produce IL-9; interestingly, the IL-9+ T cells demonstrate no regulatory properties despite producing abundant IL-10.³⁴ Instead, IL-9 subverts IL-10 antineoplastic functions. Thus, it appears that resident T cells—including resident T_REG—may be re-directed toward proinflammatory phenotypes for “rescue” whenever Th-1 or Th-2 fails to restore balance. Taken together, these data from animal models show that uncontrollable inflammation, as in the developing cancer microenvironment, may modulate T_REG function. This may help explain apparent T_REG accumulation without restoration of immune or epithelial homeostasis.

T_REG Are Sufficient to Protect from Inflammation-Associated Carcinogenesis

Relationships between GI tract bacteria and cancer have been studied using mutant mice mimicking human cancers. Recombinase activating gene 2 (Rag2)-deficient mice lack functional lymphocytes³⁵ and are highly susceptible to microbial infections resulting in IBD.^10,36–38 IBD eventually progresses to colorectal carcinoma (CRC) in those animals³⁷ (Fig. 3). One well-established bacterial etiology for IBD in mice is Helicobacter hepaticus³⁹— a bacterium also linked with carcinogenic inflammatory responses in both the liver and lower bowel of mice.³⁷ A closely related bacterial organism, Helicobacter pylori, is classified as a carcinogen by the World Health Organization (WHO) for stomach cancer in humans.^6–8 Taken together, H. hepaticus infection and innate immune inflammatory events were sufficient for carcinoma. However, strain-matched wild-type (WT) mice did not develop carcinoma after H. hepaticus infection, showing that effects of lymphocytes were sufficient to protect from cancer (Fig. 3).³⁷ In these models, IBD and carcinoma arose only when T_REG failed to suppress inflammation and restore epithelial health.

IL-10-dependent activities of T_REG cells were essential to maintain mucosal integrity in the lower bowel. IL-10 served to normalize oncogenic K-ras within epithelia.

Figure 3.

T_REG cells suppress deleterious immune-mediated pathology and afterward restore epithelial homeostasis. Similarities in roles for T_REG in inflammation and carcinogenesis emerge during bacteriatriggered inflammatory diseases of the gastrointestinal (GI) tract. Gastritis-associated stomach cancer in humans, for example, is caused by infection with Helicobacter pylori, which is classified as a carcinogen by the World Health Organization (WHO). Transgenic mouse models have been widely employed to mimic GI tract inflammation and cancer in humans. Rag2-deficient mice entirely lacking functional lymphocytes are highly susceptible to inflammation-associated carcinoma after infection with closely related bacteria, H. hepaticus. These data from mice indicated that innate immune inflammatory events were sufficient for carcinoma. However, wild-type animals did not develop carcinoma after H. hepaticus infection, showing that effects of lymphocytes were sufficient to protect from cancer. Adoptive transfer of highly purified T_REG cells was sufficient to prevent cancer in Rag−/− mice. Inflammatory bowel disease (IBD) and carcinoma arose only when T_REG failed to suppress inflammation and restore epithelial homeostasis.

Poutahidis et al.⁴⁰ demonstrated rapid reversibility of established neoplastic epithelial invasion. This work showed that an imbalance between IL-10, Tgf-β and IL-6 modulated T_REG phenotype and predisposed to colorectal malignancy. Adoptive transfer of T_REG collected from IL-10-deficient mice exacerbated neoplastic peritoneal invasion, whereas T_REG from WT mice induced tumor remission. This loss of protection may have been due to decreased potency of IL-10 secretion by T_REG, or to an inability to sustain T_REG suppressive identity in the absence of IL-10.^29,33,40 In any event, supplementation with exogenous IL-10 served to downregulate IL-6 and oncogenic K-ras expression within epithelial cells. Interestingly, administration of exogenous IL-10 in H. hepaticus-infected mice increased frequency of IFN-γ-bearing T cells (unpublished data), suggesting that IL-10 reinforced Th-1-mediated tolerance in these murine models. In addition, treatment with IL-10 also significantly reduced Gr-1+ 7/4+ (neutrophils) cells proven necessary for cancer, as neoplastic invasion was reversible using anti-Ly-6G (Gr-1) antibody.⁴¹ Taken together with earlier findings of Kullberg et al.,^25,29 we concluded that IL-10 protected against a bacteria-triggered IL-6-mediated Th-17 host response contributing to IBD-associated cancer development and growth.

T_REG Cells Suppressed Established Adenomatous Polyps—Even in Mice Lacking Overt IBD

In most human patients, CRC is not directly linked with IBD. Instead, sporadic CRC arises from intestinal adenomatous polyps that undergo a well-characterized series of genetic mutations⁴² rather than from IBD-associated premalignant foci.⁴³ Mice with a heterozygous mutation in Apc (Apc^Min/+) are predisposed to intestinal adenomas⁴⁴ mimicking 85% of sporadic colon cancers in patients. In humans, inactivation of the Apc gene and alterations in β-catenin and the wnt signaling pathway lead to cancer.^45–50 Amazingly, adoptive-transfer of highly purified CD4+CD25+ T_REG abrogated adenomatous intestinal polyps in aged Apc^Min/+ mice.^29,51–53 This was a T_REG-mediated effect, as transfer of purified T_EFF cells under the same conditions did not suppress polyps and instead exacerbated invasive adenocarcinoma within polyps.⁵³ T_REG under these conditions express IFN-γ but require IL-10 for antineoplastic efficacy. These data supported that purified T_REG may adopt hybrid phenotypes under conditions of uncontrollable inflammation.

Adoptive transfer of T_REG cells induced regression of established intestinal polyps. However, T_REG from “hygienic” donor animals failed to protect from cancer and instead increased IL-17 levels and carcinogenesis in tumor-prone tissues.

At least some of the antineoplastic benefits of T_REG are attributable to reduced inflammation. Although Min mice entirely lacked overt IBD, intestinal polyp growth required proinflammatory cytokine TNF-α to sustain oncogenic c-myc levels and tumor growth.^52,53 It is possible that T_REG or IL-10 act directly to restore c-myc expression to baseline levels. Attempts at dissecting requirements for T_REG utilized targeted depletion of CD25+ cells in young adult mice leading to increased bowel and prostate malignancy commensurate with increased levels of TNF-α, IL-6 and IL-9. Lesions lacked overt inflammation except for mast cells⁵⁴ that were previously proven essential in tumorigenesis in Apc mutant mice.^55,56 Mast cells may also be important systemically in regional lymph nodes following microbial infections, but subsequent coordination of lymphocyte progenitor and neutrophil recruitment and activation remain poorly understood (Fig. 4).^57–59

Figure 4.

Gut microbial infections modulate systemic immune events and outcome of carcinogenesis throughout the body. Helicobacter hepaticus colonizes the large intestine of C57BL/6 Apc^Min\− mice genetically prone to adenomatous intestinal polyps without causing overt typhlocolitis. Nonetheless, malignant transformation of adenomatous polyps is enhanced after infection with H. hepaticus, even in the absence of overt IBD. Surprisingly, H. hepaticus colonization in the lower bowel greatly accelerated carcinogenesis in extraintestinal tissues such as the mammary gland. The tumorigenic effect coincided with a generalized enlargement of the lymph nodes and an elevation of proinflammatory cytokines including TNF-α, interleukin (IL)-6 and IL-17. Interestingly, innate immunity was sufficient for this carcinogenic effect. Mast cells were a feature of intestinal polyps, as well as mammary and prostate carcinogenesis in murine models.

Gut Bacteria-Triggered T_REG Protect from Cancer in Extraintestinal Tissues

Surprisingly, earlier infection experiments with H. hepaticus in Min mice revealed that pathogenic gut bacteria significantly modulate carcinogenesis in extraintestinal tissues (Fig. 4). In those studies, H. hepaticus infection increased risk of mammary tumors in susceptible female Min mice when compared with uninfected counterparts. Mammary-associated lymph node enlargement after H. hepaticus infection may have been due to H. hepaticus-stimulated dendritic cells from either systemic activation or local exposure via teat mucosa. In this model, T cells from WT donors with earlier H. hepaticus infection had significantly increased potency to protect from not only adenomatous polyps but also mammary tumors.⁵² Based on our earlier data, T_REG may require IL-10 exposure for cancer suppressive functions. Alternatively, secretion of IL-10 by T_REG may be needed for epithelial balance. Kullberg et al.¹⁶ showed in vivo and in vitro that ability of T cells to produce IL-10 led to their enhanced anti-inflammatory potency after infection with H. hepaticus. Taken together, these data suggested that IL-10-dependent functions of T_REG limited inflammation, restored epithelia and subsequently protected from cancer.

Pathogenic gut microbial infections trigger an IL-6 and Th-17-associated break in immune tolerance resulting in IBD. Diverse microbial infections in youth stimulate protective immunity and tolerance and lower likelihood of IBD later in life.

Anti-inflammatory drugs significantly reduce risk of cancer in breast,²⁹ prostate,⁵⁴ lung and skin, even though these tissues may lack overt inflammatory disease. Supplementation with T_REG suppresses expression of COX-2⁵¹ previously linked with several cancers in humans.⁶⁰ Likewise, T_REG also normalizes downstream expression of the c-myc⁵³ oncogene previously linked with breast cancer in women.⁶¹ Indeed, uncontrolled inflammation increases relative risk for breast cancer in women.^62–67 One possible factor that lowers risk for breast cancer in women is prior exposures to microbial products found in soil, untreated water and unpasteurized foods. Exposure to these factors is widely believed to trigger innate immune responses that, in turn, encourage the development of a competent immune responses and subsequently promote constructive host responses to diverse inflammatory triggers during adulthood.^68–71 This is consistent with data from mouse models showing that gut bacteria-triggered T_REG are highly potent to suppress extraintestinal carcinogenesis.^52,54,72 In these studies protection from cancer was transferable between animals using purified T_REG from IL-10 competent donor mice. These data raise the possibility that T_REG may be modulated to protect from cancer.

Protective T_REG Functions Are Unified in Seemingly Divergent Disease Phenotypes: Allergies, Autoimmune Disease and Cancer

While improvements in societal cleanliness have reduced the incidence of many serious infections, with increased hygiene there has been a concomitant increase in ailments including autoimmune diseases and some types of cancers.^{5,11,14,29,73} The “hygiene hypothesis” model suggests beneficial effects as a result of exposure to infectious agents earlier in life; those individuals have fewer aberrant immune reactions such as allergies and asthma later in life (Fig. 5).⁷³ Recent evidence suggests survival benefit due to efficient induction of tolerance during infancy.⁷⁴ This raises the possibility that periparturient and early life exposures denied during modern sanitization may increase vulnerability to some diseases later in life. Selectively re-introducing less pathogenic forms of microorganisms may protect against inflammation-associated pathology later in life due to infectious or noninfectious causes (Fig. 5).

Hygienic individuals with a dysregulated IL-10 and T_REG feedback loop suffer from uncontrollable inflammation that redirects T_REG cells toward a T helper (Th)-17-driven procarcinogenic process.

Thus, efficient immune tolerance limits subsequent tissue damage^75,76 and prevents a host from “inflaming itself to death.” One possibility is that early life bacterial exposures result in more efficient IL-10-dependent T_EFF polarization toward Th-1 and robust immune tolerance.²¹ A naïve host with insufficient exposures earlier in life has weakened Th-1, uncontrolled TNF-α, subsequent upregulation of IL-1β and IL-6 and exuberant Th-17 host responses.^20,25,40 In this context, mice lacking IL-10 are unable to sustain Foxp3 expression and functional T_REG and unable to mount immune tolerance.³³ In modernized societies, hygienic individuals with a weakened IL-10 and T_REG feedback loop consequently suffer uncontrollable inflammatory disorders that re-direct T_REG toward a carcinogenic Th-17 phenotype. This cancer condition is readily reversible by blocking underlying inflammation.

IL-10 stimulated by bacteria and parasites serve to enhance protective immune balance and reinforce epithelial repair and health.

These hypotheses are supported by evidence that risks of inflammation-associated cancer, such as CRC, are increased in societies with rigorous hygiene practices.⁵ Murine models of colitis also support this “hygiene hypothesis” paradigm with dependency on IL-10 and T_REG^{16,17,40,52,72} consistent with the observations of Belkaid and Rouse.¹¹ Specifically, beneficial cancer-suppressing effects of microbial infections were dependent on IL-10^40,51,52,77 similar to proposed roles for T_REG in allergies and many types of autoimmune responses.¹⁴ This is displayed in mouse models of CRC whereby exogenous IL-10 administration is sufficient to restore T_REG anti-inflammatory and antineoplastic functions.^40,41 IL-10 arising after bacterial or parasitic challenges may serve to enhance protective immunity through-out the body via Th-1 versus Th-2.^{5,40,52,54,72} This process may involve antigenic crossreactivity as described with heterologous immunity.^78,79 A robust IFN-γ response may explain greater T_REG anticancer potency after gut bacterial infections in mice.^52,72 Antigen specificity in T cells may be targeted against bacteria, even in extraintestinal sites. Using this strategy lifelong protection from cancer may be achieved using probiotic, non-pathogenic or sterile bacterial antigens. Probiotic Lactobacillus reuteri was previously shown to restore GI homeostasis in mice and humans.^80,81

Figure 5.

Insufficient microbial exposures earlier in life predispose to uncontrollable inflammatory disorders later in life. Modern sanitization has reduced the incidence of many serious infections. However, individuals living in developed countries with more stringent hygiene practices also suffer from increased incidence of allergies, asthma, autoimmune disorders and some types of inflammation-associated cancer. This is due, at least in part, to dysregulated immune tolerance. Inability of T_REG to inhibit autoimmune diseases such as MS depends on levels of IL-6 that redirect toward a Th-17 host response. Likewise, an IL-10 deficiency during IBD leads to break in immune tolerance and a proinflammatory Th-17 response. Hygienic individuals with a weakened IL-10 and T_REG feedback loop suffer from uncontrollable inflammation that subsequently redirects T_REG cells toward a Th-17-driven procarcinogenic process. In contrast, gut microbial exposures earlier in life may reinforce Th-1 host responses and homeostasis. Taken together, these observations link the immune system, gastrointestinal infections and seemingly divergent downstream phenotypes: IBD, MS and other autoimmune disease and cancer.

Plasticity and Reciprocal Relationships Between T_REG and Th-17

The immune system is designed to recognize and destroy foreign pathogens while preserving immune tolerance to self. T_REG cells in particular, play an essential role in maintaining immunological tolerance. A primary peripheral mechanism to adapt to novel immune challenges involves the Th-17 host response. Normally functioning T_REG efficiently protect against tissue injury to self or microbes in mouse models. However, under overwhelming proinflammatory conditions, T_REG may be redirected toward Th-17 response. Erdman et al.²⁹ showed that prior microbial exposures reinforced immune tolerance conveyed by T_REG; in contrast, hygienic rearing of donor mice yielded T_REG that exacerbated IL-6 and IL-17 in target tissue. A key role for IL-17 is demonstrable using neutralization with anti-IL17A antibody to inhibit tumorigenesis in mice (data not shown), and this has been shown by others using Min mice.²⁶ Targeting underlying IL-6 or STAT3 confers improved prognosis in colonic^26,40,82 and breast malignancies (Fig. 4).^83,84

Reciprocal roles exist between T_REG and Th-17 cells. Chronic inflammatory conditions subvert T_REG cell functions, recruit Th-17 cells and factors, and increase risk for malignancy.

Discovery of Foxp3 as a key transcription factor of T_REG has provided new insights into T_REG biology and revealed unexpected features of this lineage.⁸⁵ Recent studies suggest that differences in the ability of T_REG to suppress cancer may be due to downregulation of Foxp3. In hygienic donors, uncontrollable inflammation increases risk that T_REG will lose regulatory activity and become ex-T_REG. Under these conditions, T_REG become capable of T_EFF activities including the production of IL-17 (during high-ambient levels of IL-6) or perhaps IL-9 (with high levels of IL-4). Likelihood of these ex-T_REG⁸⁵ increases in hygienic, immunologically naïve animals. There is mounting evidence that the Foxp3-deficient ex-T_REG survive with considerable biologic function and converts to Th-17 cells.

During health, Foxp3 appears to maintain the suppressor cell program that allows a relatively small subset of T_REG to efficiently preserve immune tolerance.⁸⁶ If T_REG lose Foxp3 expression under inflammatory conditions, then these cells can develop into T_EFF thus explaining massive peritumoral accumulations of Foxp3 cells with only rare Foxp3+ cells within actual tumor parenchyma.²⁹ If T_REG subsets based on T-bet or IRF4 may selectively suppress Th-1 and Th-2 effector subsets,^87,88 then early life exposures to diverse microbiota may favor robust Th-1 that subsequently reduces likelihood of Th-17 or Th-2 responses associated with pathology later in life.

Harnessing the Homeostatic Potency of T_REG to Prevent or Treat Cancer

Underlying commonalities in diseases such as IBD and MS suggest that therapeutic approaches exploiting T-cell plasticity may be beneficial for a wide variety of immune-mediated diseases. Downregulating destructive inflammatory processes may be more effective and less toxic than more traditional chemotherapeutic approaches to cancer (Fig. 6). These approaches could be used collectively or alone:

1. Directly stimulate immune tolerance using GI bacteria

   1. probiotic bacteria such as Lactobacillus reuteri

   2. apathogenic commensal microbiota

   3. synthetic, sterile microbial antigen preparations

2. Downregulate underlying inflammation

   1. directly block key proinflammatory factors: TNF-α, IL-6 or STAT3 (chemotherapeutic or dietary)

   2. stimulate IL-10 in GI tract: block inflammation, induce immune tolerance and restore epithelial oncogenes to baseline levels

3. Preferentially stimulate beneficial T_REG in vivo

   1. vitamin A derivatives, i.e., retinoic acid to induce T_REG

   2. vitamin D derivatives, i.e., to stimulate T_REG

4. Re-program T_REG ex vivo

   1. “Condition” naïve CD4+ cells ex vivo and afterward return to host

Figure 6.

Proposed strategies to reinforce protective T_REG functions to abolish cancer and restore overall health. Cancer immunotherapy strategies to date focus primarily on boosting the antitumoral immunological responses of the cancer patient. In that way, T_REG are viewed as an obstacle in tumor immunotherapy and a target for elimination. Recent data suggests that tumor survival and progression are instead vitally dependent on elevated and sustained local and systematic proinflammatory signaling. This raises the possibility that interrupting this proinflammatory loop of signals may be a more direct and constructive route to tumor eradication and restoration of overall health. Thus blocking TNF-α or the STAT3 inflammatory signaling pathway have been shown to rapidly and efficiently abrogate growing cancers in mouse models. Blocking these proinflammatory factors rapidly restores beneficial T_REG functions and epithelial homeostasis. An emerging concept of overall health is systemic benefit of gut microbiota to reinforce beneficial effects of IL-10-dependent anti-inflammatory T_REG cells. Adoptive-transfer studies in mice have shown the unsurpassed ability of gut bacteria-primed T_REG to rapidly restore homeostasis in tissues of the GI tract and also in extraintestinal sites. It was surprising that transfer of T_REG actually suppressed established carcinoma in mice. One therapeutic challenge will be to overcome redirection of T_REG in the proinflammatory tumor environment. Harnessing antineoplastic potential may involve ex vivo cultivation, expansion and stabilization of human T_REG for individualized therapies using autologous transfusions. Broader population-based reinforcement of T_REG could be achieved by dietary habits undertaken earlier in life including probiotics or other bacterialbased modalities, or supplementation with retinoic acid and vitamin D.

Using gut microbiota to enforce an anti-inflammatory T_REG phenotype serves to protect from a wide variety of immune-mediated diseases. Certain types of bacteria may uniquely coordinate the intestinal T-cell profile, and thus may be utilized to repair dysfunctional immunity arising from the bowel. Population-based strategies such as probiotic bacteria in foods⁸¹ may ultimately be used to reduce inflammation-associated cancer risk. Healthful benefits of routine yogurt consumption are well documented.

Directly targeting underlying inflammation would serve to block carcinogenic factors as well as to restore T_REG to a cancer-protective phenotype. Humans with elevated TNF-α levels have poor cancer outcomes,^1–3 and TNF-α is required for IBD-associated CRC and polyposis in mice.^{29,40,52,54,55} Highly efficacious monoclonal antibodies against TNF (infliximab) and TNF-binding fusion proteins (etanercept) have been routinely used in human patients with IBD and arthritis,^89,90 albeit with reduced protection from serious bacterial infections, highlighting importance of overall immune balance for health. Restoration of homeostasis through suppression of TNF-α and reinforcement of T_REG cells has been proposed for human patients suffering from IBD and other uncontrolled inflammatory disorders.⁸⁹

Enhanced nutrition or therapeutic delivery of vitamin derivatives may also be used to facilitate induction and recruitment of T_REG. Vitamin D promotes the differentiation of T_REG and inhibits the differentiation of Th-17 cells.⁹¹ Increased serum levels of vitamin D have been associated with a decreased risk of MS, an autoimmune disorder characterized by a break in tolerance involving upregulation of Th-17. Vitamin D not only favors the induction of T_REG but also enhances recruitment to inflammatory sites by modulating dendritic cells. A potent anti-inflammatory T_REG phenotype could also be induced in resident T cells by administration of retinoic acid that favors anti-inflammatory Foxp3+ as described for IBD.⁹²

Immediate benefits of T_REG could be achieved in humans using ex vivo stimulation with bacterial antigens to educate and tolerize CD4+ cells.⁹³ T_REG may then afterward be returned to a patient. Many unanswered questions remain to optimize this process for therapeutic purposes, in particular regarding T_REG identity and T_REG recruitment with polarization toward Th-1 rather than toward Th-17 later in life.

Conclusions

Mouse models that mimic diseases in humans provide evidence unifying immune cell biology in IBD, autoimmune diseases, asthma and cancer. Under normal physiological conditions, T_REG responses are beneficial to reinforce protective acute inflammation, and later regain protection from pathology.⁷² In immune-mediated diseases, normal homeostatic mechanisms become dysregulated leading to chronic inflammatory disease. During a break in immune tolerance, resident and recruited T cells are redirected toward a pro-inflammatory host response. This inducible proinflammatory condition creates a feedback loop involving STAT-3, IL-6 and IL-17 that further contributes to neoplastic invasion and metastases. Under these conditions, T_REG accumulate locally, are not beneficial, and instead contribute to cancer growth. Interestingly, IL-10-dependent activities in the GI tract impart sustained protection from aforementioned events. Taken together, these observations connect GI tract infections, the immune system, and diverse immune-mediated diseases including autoimmune diseases and cancer, and help explain paradoxical aspects of T_REG cell biology in these diseases.

Roles for T_REG cells in cancer appear contradictory on the surface.^94–97 We postulate that T_REG are beneficial—in fact, pivotal—in sustaining overall health and preventing cancer. Functional benefits of T_REG depend on the age and prior health status of the host, and are context within the cancer microenvironment. In immune-competent animals, T_REG constructively regulate inflammatory and epithelial growth factors that when uncontrolled lead to cancer growth. Thus, while it is true that T_REG accumulate in the vicinity of many types of tumors, T_REG also accumulate in large numbers during other uncontrolled inflammatory disease such as with IBD, but nonetheless may ultimately rescue pathology.^24,98 Conditions such as IBD are curable after restoring immune balance and relevant T_REG potency in mice. Under these conditions, T_REG redistribute within tumors with anti-neoplastic effect.^99–101 Likewise, supplementation with exogenous T_REG induces regression of inflammation-associated neoplastic conditions of colon, breast and prostate tissues in our mouse models.^40,52,53,72

The interplay of carcinogenic host factors is showcased in Apc^Min/+ mice that are genetically susceptible to tumors arising from disruptions of the wnt signaling pathway. In these animals, growing intestinal tumor burden coincides with significantly increased levels of inflammatory cytokines IL-9, IL-6 and IL-17 predisposing to cancer growth.⁵⁴ Accumulation of improperly functioning T_REG under these conditions exacerbates cancer growth. These events accelerate a feedback loop that hastens cancer growth. In Apc mutant mice thymic depletion is coincident with polypogenesis^102–104 may be a result and a cause of widespread immune dysfunction impeding T_REG and further elevating cancer risk. Supplementary CD4+ T_REG collected from immune competent donor animals would, at least initially, be untainted by high levels of inflammation and provide rescue of homeostasis. In support of this, experimental depletion of CD25+ cells in young Apc^Min/+ mice rapidly induces intestinal and prostate carcinogenesis.⁵⁴

The therapeutic strategy of blocking inflammation and redirecting T_REG would directly target and inhibit factors required to sustain cancer growth. Permanent health benefits could be achieved using combinations of diet, anti-inflammatory agents and probiotics. Ex vivo stimulation of T cells with bacterial antigens or other factors that stabilize T_REG phenotype may have utility to rescue individual cancer patients,⁹³ as demonstrated using adoptive transfer in murine models with IBD or CRC. Many unanswered questions remain to optimize this process, especially regarding T_REG identity and T_REG recruitment later in life with underlying immune instability. As this field evolves, knowledge of factors that promote terminal differentiation of T_REG may one day allow selective inhibition or exacerbation of polarized CD4+ T-cell responses in the clinics.

Abbreviations

CRC

colorectal cancer

EAE

experimental allergic encephalitis

IBD

inflammatory bowel disease

IFN

interferon

IL

interleukin

MS

multiple sclerosis

T_EFF

proinflammatory CD4+ effector T cell

Th

T helper

Tgf

transforming growth factor

TNF

tumor necrosis factor

T_REG

anti-inflammatory CD4+ regulatory T cell

Rag

recombinase activating gene (lacking functional lymphocytes)

wt

wild type (genotype)