IntroductionImmune-mediated disorders often present as a combination of destructive tissue damage and variable systemic organ manifestations based on an overwhelming T-and/or B-cell reaction. There has been great interest recently in targeting the interleukin-2 (IL-2) pathway by specific immunosuppressive drugs and the use of CD4 ϩ CD25 ϩ Foxp3 ϩ regulatory T cells (Tregs) for the treatment of such inflammatory conditions. 1,2 To specifically modulate aberrant immune responses such as acute graft-versus-host disease (aGVHD), allograft rejection, or autoimmune disorders by immunosuppressive therapy, while sparing Treg activity, a more detailed insight into the biologic differences between Tconv and Tregs is crucial. One critical requirement for Treg function and expansion in preventing aGVHD is calcineurin-dependent IL-2 production, 3 which is affected by the immunosuppressant cyclosporin A (CSA). In light of the finding that the interaction between NFATc and Foxp3 is required for the suppressive effects of Treg cell 4,5 interference with nuclear factor of activated T cells (NFAT) by CSA may also contribute to the observed effects of CSA on Treg biology. We and others have shown that rapamycin (RAPA) but not CSA allows for Treg expansion and function in mice 3,6,7 and in humans. 8,9 In contrast to CSA, RAPA inhibits mammalian target of rapamycin (mTOR) pathway activity, which is downstream of IL-2/phosphatidylinositol 3-kinase (PI3K) signaling. 10 This pathway is negatively regulated by PTEN (phosphatase and tensin homolog deleted on chromosome 10), a phosphoinositol 3,4,5-triphosphatase that catalyzes the reverse reaction of PI3K. 11 Other pathways that are activated in response to IL-2 signaling that may be preferentially used when mTOR is inhibited include the Janus tyrosine kinase (JAK)/signal transducer and activator of transcription (STAT) and the mitogen-activated protein kinase (MAPK) pathway. Recent data have demonstrated an essential role for IL-2 receptor (R)␤-dependent STAT5 signaling for the development of Tregs. 12 Here we describe the in vivo impact of mTOR inhibition on expansion, migration, Foxp3 expression, and activity of Tregs after allogeneic hematopoietic cell transplantation (aHCT). From a mechanistic standpoint, we studied the differential impact of RAPA on Tconv and Tregs with respect to PI3K/mTOR and STAT5 pathway activity. Methods MiceC57BL/6 (H-2k b , Thy-1.2), FVB (H-2k q ), BALB/c (H-2k d , Thy-1.2), and C57BL/6 PTEN tm1Hwu mice were purchased from the Jackson Laboratory (Bar Harbor, ME) or Charles River Laboratories (Wilmington, MA). C57BL/6 Lck-Cre mice were a kind gift from Dr J. Crabtree (Stanford University, Stanford, CA). Mice were used between 6 and 12 weeks of age. For personal use only. on May 9, 2018. by guest www.bloodjournal.org From Only sex-matched combinations were used for transplant experiments. The luciferase-expressing (luc ϩ ) transgenic FVB/N L2G85 were described previously 13 and backcrossed for more than 10 generations to the C57BL/6 background. All animal prot...

Colonization of the human stomach by Helicobacter pylori is an important risk factor for development of gastric cancer. The H. pylori cag pathogenicity island (cag PAI) encodes components of a type IV secretion system (T4SS) that translocates the bacterial oncoprotein CagA into gastric epithelial cells, and CagL is a specialized component of the cag T4SS that binds the host receptor α5β1 integrin. Here, we utilized a mass spectrometry-based approach to reveal co-purification of CagL, CagI (another integrin-binding protein), and CagH (a protein with weak sequence similarity to CagL). These three proteins are encoded by contiguous genes in the cag PAI, and are detectable on the bacterial surface. All three proteins are required for CagA translocation into host cells and H. pylori-induced IL-8 secretion by gastric epithelial cells; however, these proteins are not homologous to components of T4SSs in other bacterial species. Scanning electron microscopy analysis reveals that these proteins are involved in the formation of pili at the interface between H. pylori and gastric epithelial cells. ΔcagI and ΔcagL mutant strains fail to form pili, whereas a ΔcagH mutant strain exhibits a hyperpiliated phenotype and produces pili that are elongated and thickened compared to those of the wild-type strain. This suggests that pilus dimensions are regulated by CagH. A conserved C-terminal hexapeptide motif is present in CagH, CagI, and CagL. Deletion of these motifs results in abrogation of CagA translocation and IL-8 induction, and the C-terminal motifs of CagI and CagL are required for formation of pili. In summary, these results indicate that CagH, CagI, and CagL are components of a T4SS subassembly involved in pilus biogenesis, and highlight the important role played by unique constituents of the H. pylori cag T4SS.

Gastric adenocarcinoma is strongly associated with Helicobacter pylori infection; however, most infected persons never develop this malignancy. H. pylori strains harboring the cag pathogenicity island (cag + ), which encodes CagA and a type IV secretion system (T4SS), induce more severe disease outcomes. H. pylori infection is also associated with iron deficiency, which similarly augments gastric cancer risk. To define the influence of iron deficiency on microbial virulence in gastric carcinogenesis, Mongolian gerbils were maintained on iron-depleted diets and infected with an oncogenic H. pylori cag + strain. Iron depletion accelerated the development of H. pylori-induced premalignant and malignant lesions in a cagA-dependent manner. H. pylori strains harvested from iron-depleted gerbils or grown under iron-limiting conditions exhibited enhanced virulence and induction of inflammatory factors. Further, in a human population at high risk for gastric cancer, H. pylori strains isolated from patients with the lowest ferritin levels induced more robust proinflammatory responses compared with strains isolated from patients with the highest ferritin levels, irrespective of histologic status. These data demonstrate that iron deficiency enhances H. pylori virulence and represents a measurable biomarker to identify populations of infected persons at high risk for gastric cancer.

Persistent colonization of the human stomach with Helicobacter pylori is a risk factor for gastric adenocarcinoma, and H. pyloriinduced carcinogenesis is dependent on the actions of a bacterial oncoprotein known as CagA. Epidemiological studies have shown that high dietary salt intake is also a risk factor for gastric cancer. To investigate the effects of a high-salt diet, we infected Mongolian gerbils with a wild-type (WT) cagA ؉ H. pylori strain or an isogenic cagA mutant strain and maintained the animals on a regular diet or a high-salt diet. At 4 months postinfection, gastric adenocarcinoma was detected in 100% of the WT-infected/highsalt-diet animals, 58% of WT-infected/regular-diet animals, and none of the animals infected with the cagA mutant strain (P < 0.0001). Among animals infected with the WT strain, those fed a high-salt diet had more severe gastric inflammation, higher gastric pH, increased parietal cell loss, increased gastric expression of interleukin 1␤ (IL-1␤), and decreased gastric expression of hepcidin and hydrogen potassium ATPase (H,K-ATPase) compared to those on a regular diet. Previous studies have detected upregulation of CagA synthesis in response to increased salt concentrations in the bacterial culture medium, and, concordant with the in vitro results, we detected increased cagA transcription in vivo in animals fed a high-salt diet compared to those on a regular diet. Animals infected with the cagA mutant strain had low levels of gastric inflammation and did not develop hypochlorhydria. These results indicate that a high-salt diet potentiates the carcinogenic effects of cagA ؉ H. pylori strains.

Peroxisome proliferator–activated receptor (PPAR)α is a nuclear receptor that mediates gender differences in lipid metabolism. PPARα also functions to control inflammatory responses by repressing the activity of nuclear factor κB (NF-κB) and c-jun in immune cells. Because PPARα is situated at the crossroads of gender and immune regulation, we hypothesized that this gene may mediate sex differences in the development of T cell–mediated autoimmune disease. We show that PPARα is more abundant in male as compared with female CD4+ cells and that its expression is sensitive to androgen levels. Genetic ablation of this gene selectively removed the brake on NF-κB and c-jun activity in male T lymphocytes, resulting in higher production of interferon γ and tumor necrosis factor (but not interleukin 17), and lower production of T helper (Th)2 cytokines. Upon induction of experimental autoimmune encephalomyelitis, male but not female PPARα−/− mice developed more severe clinical signs that were restricted to the acute phase of disease. These results suggest that males are less prone to develop Th1-mediated autoimmunity because they have higher T cell expression of PPARα.