FOXP3 Mutations Research Articles

Deficiency of foxp3 regulatory T cells exacerbates autoimmune arthritis by altering the synovial proportions of CD4 T cells and dendritic cells.

BackgroundCD4+Fop3+ regulatory T cells (Tregs) are needed to maintain peripheral tolerance, but their role in the development of autoimmune arthritis is still debated. The present study was undertaken to investigate the mechanism by which Tregs influence autoimmune arthritis, using a mouse model entitled K/BxN.MethodsWe generated Treg-deficient K/BxNsf mice by congenically crossing K/BxN mice with Foxp3 mutant scurfy mice. The arthritic symptoms of the mice were clinically and histopathologically examined. The proportions and activation of CD4+ T cells and/or dendritic cells were assessed in the spleens, draining lymph nodes and synovial tissue of these mice.ResultsK/BxNsf mice exhibited earlier onset and more aggressive progression of arthritis than their K/BxN littermates. In particular, bone destruction associated with the influx of numerous RANKL+ cells into synovia was very prominent. They also contained more memory phenotype CD4+ T cells, more Th1 and Th2 cells, and fewer Th17 cells than their control counterparts. Plasmacytoid dendritic cells expressing high levels of CD86 and CD40 were elevated in the K/BxNsf synovia.ConclusionWe conclude that Tregs oppose the progression of arthritis by inhibiting the development of RANKL+ cells, homeostatically proliferating CD4+ T cells, Th1, Th2 and mature plasmacytoid dendritic cells, and by inhibiting their influx into joints.

Plasticity of T reg cells: Is reprogramming of T reg cells possible in the presence of FOXP3?

Regulatory T cells (T reg cells) are involved in self tolerance, immune homeostasis, prevention of autoimmunity, and suppression of immunity to pathogens or tumors. The forkhead transcription factor FOXP3 is essential for T reg-cell development and function as mutations in FOXP3 cause severe autoimmune diseases in mice and humans. Over the last years it has been postulated that FOXP3 expression in T reg prevents effector T-cell (T effector-cell) lineage commitment, yet several recent studies suggest that the co-existence of effector and regulatory T-cell programs can occur and might help to enable T reg cells with properties necessary to exert their function in peripheral tissues. Furthermore, downregulation of FOXP3 in the periphery might help T reg cells to lose suppressive functions and gain memory properties with specificity for self-antigens and an effector phenotype including the ability to produce IFN-γ and IL-17. This plasticity might have an impact on their reactivity towards autoimmunity as well as tumors or infections.

Identification of new FOXP3 mutations and prenatal diagnosis of IPEX syndrome

Molecular diagnosis and prenatal care of two pregnant women at risk of transmitting immunodysregulation, polyendocrinopathy, enteropathy X-linked (IPEX) syndrome. FOXP3 coding sequence and exon boundaries were analyzed in the two consultants and family members. Non-invasive sex determination and specific prenatal diagnosis was realized. Following sequence analysis a new FOXP3 mutation was identified in each consultant. Sex diagnosis realized by amplification of Y sequences from the plasma of the two mothers revealed a male and a female fetus, respectively. Prenatal diagnosis showed that the male fetus was unaffected. The baby is now born and healthy. Subsequent ultrasound examinations confirmed the sex in the second pregnancy but unfortunately led to the diagnosis of a 69,XXX triploidy. The pregnancy was thus interrupted. Two new FOXP3 mutations were identified and prenatal diagnosis could be proposed. Due to the rarity of the disease, clinical diagnosis is often considered with delay. Both patients reported here were already pregnant at the beginning of the genetic investigation and one had previously interrupted a male pregnancy for lack of diagnosis. When faced with children with severe refractory diarrhea, clinicians should entertain the possibility of IPEX.

Tregs and Human Atherothrombotic Diseases

Immunology has recently penetrated the field of atherothrombosis. A number of human and experimental studies have documented that both cellular and molecular immune effectors are involved at various stages of the pathological process.1 This has directed our attention toward the use of immunomodulatory strategies in atherothrombosis; these strategies are commonly used for the treatment of other chronic inflammatory diseases. Notably, the administration of immunosuppressive drugs is the most common approach for treating immune-mediated diseases. However, the long-term administration of non–antigen-specific agents that cannot distinguish between beneficial and destructive immune responses is a major drawback of this strategy. See accompanying articles on pages 1825 and 1832 Interesting alternatives consist of strategies aimed at potentiating the physiological regulatory mechanisms, if defective. In the field of atherothrombosis research, experimental studies have clearly shown that a defect in regulatory CD4+ T cells (Tregs) favors atherogenesis.2 Based on their potent immunosuppressive activity, the expansion of the Treg compartment by cell transfer or the administration of stimulating molecules can be envisaged as a new therapeutic strategy. These findings have provided a strong incentive to perform clinical studies with the aim of assessing whether a defective Treg compartment is associated with the progression of atherosclerotic disease and the occurrence of atherothrombotic manifestations in humans. Three clinical studies,3–5 in a limited number of patients with coronary artery disease, first reported that patients with acute coronary syndromes have fewer and/or inefficient Tregs in their blood. In the present issue of Arteriosclerosis, Thrombosis, and Vascular Biology , a larger clinical study6 reports interesting challenges to these findings; a second study7 explores the role of Tregs in stenotic and dilative pathological remodeling …

Reduced Expression of FOXP3 and Regulatory T-Cell Function in Severe Forms of Early-onset Autoimmune Enteropathy

Little is known about the pathophysiology of early onset forms of autoimmune enteropathy (AIE). AIE has been associated with mutations in FOXP3-a transcription factor that controls regulatory T-cell development and function. We analyzed the molecular basis of neonatal or early postnatal AIE using clinical, genetic, and functional immunological studies. Gastroenterological and immunological features were analyzed in 9 boys and 2 girls with AIE that began within the first 5 months of life. FOXP3 and IL2RA were genotyped in peripheral blood monocytes. FOXP3 messenger RNA and protein expression were analyzed using reverse-transcription polymerase chain reaction, flow cytometry, and confocal immunofluorescence of CD4(+) T cells. Regulatory T-cell function (CD4(+)CD25(+)) was assayed in coculture systems. AIE associated with extraintestinal autoimmunity was severe and life-threatening; all patients required total parenteral nutrition. Regulatory T cells from 7 patients had altered function and FOXP3 mutations that resulted in lost or reduced FOXP3 protein expression; 2 infants had reduced regulatory T-cell activity and reduced levels of FOXP3 protein, although we did not detect mutations in FOXP3 coding region, poly-A site, or promoter region (called FOXP3-dependent AIE). Two patients had a normal number of regulatory T cells that expressed normal levels of FOXP3 protein and normal regulatory activity in in vitro coculture assays (called FOXP3-independent AIE). No mutations in IL2RA were found. Most cases of AIE are associated with alterations in regulatory T-cell function; some, but not all, cases have mutations that affect FOXP3 expression levels. Further studies are needed to identify mechanisms of AIE pathogenesis.

Human FOXP3 and cancer

FOXP3 is a transcription factor necessary and sufficient for induction of the immunosuppressive functions in regulatory T lymphocytes. Its expression was first considered as specific of this cell type, but FOXP3 can also be transiently expressed in T-cell antigen receptor-activated human nonregulatory T cells. Recent data indicate that FOXP3 is also expressed by some nonlymphoid cells, in which it can repress various oncogenes that are restored following FOXP3 deletion or mutation. This review summarizes major advances in (1) the understanding of Foxp3 functions in human regulatory T cells, (2) the prognostic significance of Foxp3-expressing T cells in human malignancies and (3) the significance of Foxp3 expression in human tumor cells.

Foxp3 is a novel repressor of microglia activation

Forkhead transcription factor 3 (Foxp3) is critical for generating CD4(+)CD25(+) regulatory T cells. However, its role in microglia has not been identified. Here, we show that Foxp3 is expressed in microglia and is upregulated upon activation. In Foxp3 mutant mice (Foxp3(sf)), microglia release higher levels of inflammatory cytokines and mediators such as NO, MCP-1, CXCL10, and ROS upon liposaccharide treatment than the wild type, while TNF-alpha and IL-1 beta were not significantly different between wild and mutant microglial cells. In addition, Foxp3 silencing enhances inflammatory responses, suggesting that the major role of Foxp3 in microglia is that of a repressor of activation. Similarly, Foxp3 overexpression reduces inflammatory responses in microglia. We also demonstrate that Foxp3 interacts directly with NF-kappaB and modulates its transcriptional activities. These findings point to the importance of Foxp3 in NF-kappaB mediated inflammatory responses in microglia.

Autoantibodies in Scurfy Mice and IPEX Patients Recognize Keratin 14

Scurfy mice have a deletion in the Foxp3 gene, resulting in a failure to generate Foxp3(+) regulatory T cells, and they subsequently develop severe CD4(+) T-cell-mediated autoimmune inflammation. Multiple organs are involved, but the skin is one of the main organs affected. During the course of disease, Scurfy mice develop autoantibodies; however, the targeted antigens are unknown. In this study, we show that Scurfy mice develop autoantibodies directed against skin antigens. Using western blot analysis, we found that Scurfy serum reacted with proteins in total skin lysate, as well as in a keratinocyte lysate. Most of the Scurfy sera tested identified a major band at 50 kDa. Transfer of Scurfy CD4(+) T cells into nu/nu mice yielded autoantibodies with similar reactivity. Further analysis using 2D western blots, followed by peptide mass fingerprinting, identified several keratins as targets. To confirm this observation, we chose one of the identified targets, keratin 14, and prepared recombinant proteins encompassing the N-terminal, middle, and C-terminal portions of the keratin 14 protein. Scurfy serum predominantly recognized the C-terminal fragment. Sera from patients with immunodysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome, the human disease resulting from FOXP3 mutations, also recognized skin antigens, including keratin 14. Thus, the results of our study indicate that autoantibodies in Scurfy mice and patients with IPEX target keratins.

CARMA1 Regulation of Regulatory T Cell Development Involves Modulation of Interleukin-2 Receptor Signaling

T cell receptor-stimulated NF-kappaB activation requires CARMA1 and is negatively regulated by the deubiquitinase CYLD. Recent studies suggest that CARMA1 regulates regulatory T cell (Treg) development, although the role of NF-kappaB in this event is incompletely understood. We show that CYLD deficiency causes constitutive NF-kappaB activation in thymocytes, which is associated with enhanced frequency of Treg cells. The NF-kappaB activation in CYLD-deficient thymocytes is independent of CARMA1, because the NF-kappaB activation was also detected in CYLD/CARMA1 double knock-out thymocytes. Interestingly, although loss of CYLD causes NF-kappaB activation in the CARMA1-deficient thymocytes, the CYLD deficiency fails to rescue the defect of CARMA1 knock-out mice in Treg development. Furthermore, inhibition of canonical NF-kappaB by an IkappaBalpha transgene only partially inhibits Treg development. We demonstrate that CARMA1 regulates IL-2 receptor signaling and controls the IL-2-stimulated maturation of Treg precursors to mature Tregs. These results suggest that the role of CARMA1 in Treg regulation involves both NF-kappaB activation and IL-2 receptor signaling.

S2013 The Role of MicroRNA in the Pathogenesis Associated With Necrotizing Enterocolitis

IPEX Syndrome is a rare disorder presenting in the first few months of life and characterized by a triad of severe autoimmune enteropathy (AIE), endocrinopathy (usually Type I diabetes or thyroiditis), and dermatitis. In addition, patients frequently present with other autoimmune phenomena including hemolytic anemia, thrombocytopenia, neutropenia, hepatitis, and glomerulonephropathy. The disease has been linked to mutations in the FOXP3 gene, located on the X-chromosome (Xp11.23-Xp13.3). FOXP3 encodes a 48-kD protein that is a member of the forkhead/winged-helix family of transcription factors and is essential for development of CD4+CD25+ regulatory T-cells. We have evaluated a large cohort of patients with IPEX symptoms who were referred to our lab for FOXP3 gene sequencing. In this cohort, we have identified 26 different pathogenic FOXP3 mutations in 39 families. In addition, we have identified 2 mutations that are likely non-pathogenic, as they have been found in unaffected family members, or have been identified in the general population. We have also collected a large cohort of patients with the IPEX phenotype, who do not have a FOXP3 mutation and are therefore “IPEX-like”. Combining our mutations with those reported by others in the literature, there are now a total of 55 FOXP3 mutations identified in 84 families with IPEX syndrome. Of these, 16 are splicingmutations, 26missense, 8 frameshift (including 1 premature ATG, 2 alternative ATG, and 1 premature termination), 2 in-frame deletions, 1 complex(insertion/deletion/frameshift), and 2 polyadenylation site mutations. The majority of mutations are located in the forkhead DNA-binding domain of the protein (26/55 exons 9-11). The two most common mutations are a missense mutation in exon 11 (c.1150G>A) resulting in an Ala to Thr substitution at residue 384 (p.A384T) and a 3 nucleotide inframe deletion in Exon 7 leading to a single amino acid deletion (p.251del) in the leucine zipper. These mutations were found in 11 and 6 families respectively. The remainder of the mutations are equally distributed throughout the gene and affect all exons and include mutations in other important functional domains including the upstream proline-rich region (exon 1-4) and the leucine-zipper (exons 6-7). We also describe some basic genotypephenotype correlations that are beginning to emerge as a result of the identification of more FOXP3 genemutations in IPEX.We believe that by increasing our knowledge of the molecular basis of IPEX syndrome we will better understand how specific FOXP3 mutations impact regulatory T cell development which will aid our understanding of autoimmune diseases in general.

S2012 Novel Mutations and Clinical Features in Immune Dysregulation, Polyendocrinopathy, Enteropathy, X-Linked (IPEX) Syndrome

IPEX Syndrome is a rare disorder presenting in the first few months of life and characterized by a triad of severe autoimmune enteropathy (AIE), endocrinopathy (usually Type I diabetes or thyroiditis), and dermatitis. In addition, patients frequently present with other autoimmune phenomena including hemolytic anemia, thrombocytopenia, neutropenia, hepatitis, and glomerulonephropathy. The disease has been linked to mutations in the FOXP3 gene, located on the X-chromosome (Xp11.23-Xp13.3). FOXP3 encodes a 48-kD protein that is a member of the forkhead/winged-helix family of transcription factors and is essential for development of CD4+CD25+ regulatory T-cells. We have evaluated a large cohort of patients with IPEX symptoms who were referred to our lab for FOXP3 gene sequencing. In this cohort, we have identified 26 different pathogenic FOXP3 mutations in 39 families. In addition, we have identified 2 mutations that are likely non-pathogenic, as they have been found in unaffected family members, or have been identified in the general population. We have also collected a large cohort of patients with the IPEX phenotype, who do not have a FOXP3 mutation and are therefore “IPEX-like”. Combining our mutations with those reported by others in the literature, there are now a total of 55 FOXP3 mutations identified in 84 families with IPEX syndrome. Of these, 16 are splicingmutations, 26missense, 8 frameshift (including 1 premature ATG, 2 alternative ATG, and 1 premature termination), 2 in-frame deletions, 1 complex(insertion/deletion/frameshift), and 2 polyadenylation site mutations. The majority of mutations are located in the forkhead DNA-binding domain of the protein (26/55 exons 9-11). The two most common mutations are a missense mutation in exon 11 (c.1150G>A) resulting in an Ala to Thr substitution at residue 384 (p.A384T) and a 3 nucleotide inframe deletion in Exon 7 leading to a single amino acid deletion (p.251del) in the leucine zipper. These mutations were found in 11 and 6 families respectively. The remainder of the mutations are equally distributed throughout the gene and affect all exons and include mutations in other important functional domains including the upstream proline-rich region (exon 1-4) and the leucine-zipper (exons 6-7). We also describe some basic genotypephenotype correlations that are beginning to emerge as a result of the identification of more FOXP3 genemutations in IPEX.We believe that by increasing our knowledge of the molecular basis of IPEX syndrome we will better understand how specific FOXP3 mutations impact regulatory T cell development which will aid our understanding of autoimmune diseases in general.

Update in neonatal diabetes

Here we give context to new data on neonatal diabetes mellitus, a rare group of insulin-requiring monogenic forms of diabetes presenting at birth or shortly thereafter. Genetic studies are critical in the diagnosis and treatment of these patients. The most common causes of neonatal diabetes are activating mutations in the two protein subunits of the ATP-sensitive potassium channel. These are responsible for about half of all cases of permanent neonatal diabetes and some cases of transient neonatal diabetes. Identification of these mutations allows patients treated with insulin to be transferred to sulfonylureas, but associated conditions and other causes must be considered. Recent data suggest that neonatal diabetes is more common than previously thought, with variable presentations. Continued studies provide further evidence for amelioration of developmental and neurological dysfunction exhibited by a significant proportion of patients. Abnormalities of chromosome 6q24 remain the most common cause of transient neonatal diabetes. Other causes of neonatal diabetes being studied include mutations in proinsulin, FOXP3 mutations in immunodysregulation, polyendocrinopathy, enteropathy, X-linked syndrome, homozygous glucokinase mutations, and Wolcott-Rallinson/EIF2AK3 diabetes. We still have much to learn about the different forms of neonatal diabetes, their associated clinical features, and the optimization of therapy using a growing number of available therapeutic agents.

Wild-type FOXP3 is selectively active in CD4+CD25hi regulatory T cells of healthy female carriers of different FOXP3 mutations

AbstractForkhead box P3 (FOXP3) is constitutively expressed by CD4+CD25hi regulatory T cells (nTregs). Mutations of FOXP3 cause a severe autoimmune syndrome known as immune dysregulation polyendocrinopathy enteropathy X-linked, in which nTregs are absent or dysfunctional. Whether FOXP3 is essential for both differentiation and function of human nTreg cells remains to be demonstrated. Because FOXP3 is an X-linked gene subject to X-chromosome inactivation (XCI), we studied 9 healthy female carriers of FOXP3 mutations to investigate the role of wild-type (WT) versus mutated FOXP3 in different cell subsets. Analysis of active WT versus mutated (mut)–FOXP3 allele distribution revealed a random pattern of XCI in peripheral blood lymphocytes and in naive and memory CD4+T cells, whereas nTregs expressed only the active WT-FOXP3. These data demonstrate that expression of WT-FOXP3 is indispensable for the presence of a normal nTreg compartment and suggest that FOXP3 is not necessary for effector T-cell differentiation in humans.

X-linked Foxp3 (Scurfy) Mutation Dominantly Inhibits Submandibular Gland Development and Inflammation Respectively through Adaptive and Innate Immune Mechanisms

Scurfy (Foxp3(Sf)/Y), Il2(-/-), and Il2ralpha(-/-) mice are deficient in CD4(+)Foxp3(+) regulatory T cells (Treg), but only the latter two develop inflammation in the submandibular gland (SMG), a critical target of Sjögren's syndrome. In this study, we investigated the reason that SMG of Scurfy (Sf), Sf.Il2(-/-), Sf.Il2ralpha(-/-), and the long-lived Sf.Fas(lpr/lpr) mice remained free of inflammation, even though their lymph node cells induced SMG inflammation in Rag1(-/-) recipients. A strong correlation was observed between the development of the granular convoluted tubules (GCT) of the SMG in these mice and SMG resistance to inflammation. Moreover, GCT development in Sf.Rag1(-/-) mice was not impeded, indicating a role of adaptive immunity. In the Sf.Fas(lpr/lpr) mice, this block was linked to atrophy and inflammation in the accessory reproductive organs. Testosterone treatment restored GCT expression, but did not induce SMG inflammation, indicating GCT is not required for inflammation and additional mechanisms were controlling SMG inflammation. Conversely, oral application of LPS induced SMG inflammation, but not GCT expression. LPS treatment induced up-regulation of several chemokines in SMG with little effect on the chemokine receptors on CD4(+) T cells in Sf mice. Our study demonstrates that Sf mutation affects SMG development through adaptive immunity against accessory reproductive organs, and the manifestation of SMG inflammation in Sf mice is critically controlled through innate immunity.

FOXP3 germline polymorphisms are not associated with risk of breast cancer

Recent studies have characterized X-linked transcriptional regulator FOXP3 as a tumor suppressor gene involved in the pathogenesis of breast cancer. A high rate of somatic FOXP3 mutations in breast tumors and the consequent upregulation of ERBB2 (alias HER-2) make FOXP3 a good candidate gene for breast cancer susceptibility. By taking advantage of a highly conserved FOXP3 sequence, we genotyped three haplotype-tagging single-nucleotide polymorphisms (htSNPs) that cover 40 kb around the FOXP3 gene region in 1,529 cases and 1,528 controls from the large population-based Genetic Epidemiology Breast Cancer (GEBC) study in northern Israel. None of the FOXP3 htSNPs showed strong association with breast cancer risk. There was no evidence for significant heterogeneity by ethnicity (Ashkenazi Jews, Sephardic Jews, Arabs), first-degree family history of breast cancer, or age at diagnosis in stratified analysis. The analysis of reconstructed haplotypes based on the three FOXP3 htSNPs did not identify any haplotypes associated with breast cancer. Although FOXP3 is a biologically relevant gene in the pathogenesis of breast cancer, germline variation was not meaningfully associated with risk of the disease.

Regulation of multi-organ inflammation in the regulatory T cell-deficient scurfy mice

Scurfy mice display the most severe form of multi-organ inflammation due to total lack of the CD4+Foxp3+ regulatory T cells (Treg) resulted from a mutation of the X-linked transcription factor Foxp3. A large repertoire of Treg-suppressible, inflammation-inducing T cells was demonstrated by adoptive transfer experiments using Rag1-/- mice as recipients and by prolongation of lifespan through breeding with Faslpr/lpr mutant. Inflammation in the ear, eyes, skin, tail, salivary glands, lungs, stomach, pancreas, liver, small intestine, colon, skeletal muscle, and accessory reproductive organs are identified. Genetic and cellular regulations of specific organ inflammation are described. Sf mice may be useful for the identification of organ-specific antigens and Treg capable of suppressing inflammation in an organ-specific manner. Sf mice are also useful to determine the important inflammation process at the checkpoint after Treg regulation using genetic analysis through breeding.

Regulatory T-cell suppressor program co-opts transcription factor IRF4 to control T(H)2 responses.

In the course of infection or autoimmunity, particular transcription factors orchestrate the differentiation of T(H)1, T(H)2 or T(H)17 effector cells, the responses of which are limited by a distinct lineage of suppressive regulatory T cells (T(reg)). T(reg) cell differentiation and function are guided by the transcription factor Foxp3, and their deficiency due to mutations in Foxp3 results in aggressive fatal autoimmune disease associated with sharply augmented T(H)1 and T(H)2 cytokine production. Recent studies suggested that Foxp3 regulates the bulk of the Foxp3-dependent transcriptional program indirectly through a set of transcriptional regulators serving as direct Foxp3 targets. Here we show that in mouse T(reg) cells, high amounts of interferon regulatory factor-4 (IRF4), a transcription factor essential for T(H)2 effector cell differentiation, is dependent on Foxp3 expression. We proposed that IRF4 expression endows T(reg) cells with the ability to suppress T(H)2 responses. Indeed, ablation of a conditional Irf4 allele in T(reg) cells resulted in selective dysregulation of T(H)2 responses, IL4-dependent immunoglobulin isotype production, and tissue lesions with pronounced plasma cell infiltration, in contrast to the mononuclear-cell-dominated pathology typical of mice lacking T(reg) cells. Our results indicate that T(reg) cells use components of the transcriptional machinery, promoting a particular type of effector CD4(+) T cell differentiation, to efficiently restrain the corresponding type of the immune response.

Decreased FoxP3 gene expression in the nasal secretions from patients with allergic rhinitis

To determine whether patients with allergic rhinitis have altered FoxP3 gene expression and/or mutations. We collected nasal secretions from 14 volunteers (five of whom have allergic rhinitis) and five ENT allergy patients. Total RNA was isolated from these nasal secretions. The gene expression levels of FoxP3 were quantified by both semi-quantitative RT-PCR and real-time PCR using Actin as a housekeeping gene. The cDNA fragments amplified by RT-PCR were analyzed by DNA sequencing. We found that patients with allergic rhinitis had significantly lower FoxP3 mRNA compared to nonallergic controls (P < 0.01). In addition, we found a point mutation in the FoxP3 gene from a patient who not only has severe allergic rhinitis, but also has asthma. This mutation locates in a highly conserved region of FoxP3 gene and partially impaired FoxP3 functions. Our data indicate that either reduced FoxP3 gene expression or impaired FoxP3 functions are involved in the development of allergic disease in humans.

Index of Suspicion

Index of Suspicion

Abstract PL01-04: The role of regulatory T cells in K-Ras driven lung cancer

Abstract PL01-04 Background K-Ras mutations occur in up to 30% of human lung adenocarcinomas (Salgia and Skarin 1998) and occur almost exclusively in patients with a history of smoking. These mutations confer resistance to commonly used cytotoxic chemotherapies as well as targeted agents (Eberhard, Johnson et al. 2005). In preclinical mouse models of lung cancer, K-Ras mutations are necessary for tobacco carcinogen-driven lung tumorigenesis and are sufficient to cause lung adenocarcinomas in transgenic mice (Johnson, Mercer et al. 2001). Inflammation is characteristic of smoking-related lung cancer and preclinical models of K-Ras-driven lung tumorigenesis. Specific components of the inflammatory response such as regulatory T cells (Treg) regulate inflammation and are thought to contribute to tumorigenesis. Because inhibitors of mTOR such as the immunosuppressant rapamycin can prevent K-Ras mediated murine lung tumorigenesis (Wislez, Spencer et al. 2005; Granville, Warfel et al. 2007), we hypothesized that the anti-tumor effects of rapamycin might be related to alterations in lung-associated inflammation. Methods and Findings Lung tumorigenesis was studied in three different murine models that depend on mutant K-Ras; a tobacco carcinogen-driven model, a syngeneic inoculation model and a transgenic model. Splenic and lung-associated T cells were studied using flow cytometry and immunohistochemistry. Exposure of A/J mice to the tobacco-carcinogen NNK that causes K-Ras mutations (Belinsky, Devereux et al. 1989) tripled the fraction of lung-associated Treg prior to tumor development. Induction of lung tumors and induction of lung associated Foxp3+ cells by NNK was dose dependent. When administered to achieve trough levels comparable to that in humans, rapamycin prevented the induction of lung associated Foxp3+ cells by NNK, coincident with a 90% decrease in lung tumors. In A/J mice inoculated with syngeneic lung adenocarcinoma cells that were resistant to the anti-proliferative effects of rapamycin, rapamycin neither inhibited tumor growth nor decreased Treg. In contrast, depletion of Treg in inoculated mice using an anti-CD25 antibody prevented lung tumorigenesis by 80%. Transgenic mice that express mutant K-Ras (K-RasLA2) also develop lung tumors with infiltrating Foxp3+ cells, and when K-RasLA2 mice were crossed to genetically engineered mice that bear a loss of function mutation in Foxp3, lung tumorigenesis was inhibited by 75%. Conclusions These studies show that Treg are required for K-Ras-mediated lung tumorigenesis, and provide a strong rationale to clinically evaluate rapamycin or other agents that decrease Treg, to determine if decreasing Treg will inhibit K-Ras driven lung cancer. Citation Information: Cancer Prev Res 2008;1(7 Suppl):PL01-04.