The p110δ subunit of phosphoinositide 3-kinase (PI(3)K) is selectively expressed in leukocytes and is critical for lymphocyte biology. Here we report three different germline, heterozygous, gain-of-function mutations in the PIK3CD gene encoding p110δ in fourteen patients from seven families. These patients presented with sinopulmonary infections, lymphadenopathy, nodular lymphoid hyperplasia and CMV and/or EBV viremia. Strikingly, naïve and central memory T cells were severely deficient, while senescent effector T cells were over-represented. In vitro, patient T cells exhibited increased phosphorylation of Akt and hyperactivation of mTOR, enhanced glucose uptake and terminal effector differentiation. Importantly, treatment with rapamycin to inhibit mTOR activity in vivo partially restored naïve T cells, largely rescued the in vitro T cell defects, and improved clinical course.

Anterior Gradient 2 (AGR2) is a protein disulfide isomerase that plays important roles in diverse processes in multiple cell lineages as a developmental regulator, survival factor and susceptibility gene for inflammatory bowel disease. Here, we show using germline and inducible Agr2−/− mice that Agr2 plays important roles in intestinal homeostasis. Agr2−/− intestine has decreased goblet cell Mucin 2, dramatic expansion of the Paneth cell compartment, abnormal Paneth cell localization, elevated endoplasmic reticulum (ER) stress, severe terminal ileitis and colitis. Cell culture experiments show that Agr2 expression is induced by ER stress, and that siRNA knockdown of Agr2 increases ER stress response. These studies implicate Agr2 in intestinal homeostasis and ER stress and suggest a role in the etiology of inflammatory bowel disease.

Humans and mice deficient in the adaptor protein SAP (Sh2d1a) have a major defect in humoral immunity, due to lack of T cell help for B cells. The role of SAP in this process is incompletely understood. We found that deletion of receptor Ly108 (Slamf6) in CD4+ T cells reversed the Sh2d1a−/− phenotype, eliminating the SAP requirement for germinal centers. This potent negative signaling by Ly108 required immunotyrosine switch motifs (ITSMs) and SHP-1 recruitment, resulting in high amounts of SHP-1 at the T:B synapse, limiting T:B adhesion. Ly108 negative signaling was not only important in CD4+ T cells, as we found that NKT cell differentiation was substantially restored in Slamf6−/−Sh2d1a−/− mice. The ability of SAP to regulate both positive and negative signals in T cells can explain the severity of SAP-deficiency and highlights the importance of SAP and SHP-1 competition for Ly108 ITSM binding as a rheostat for the magnitude of T cell help to B cells.

SUMMARY X-linked lymphoproliferative syndrome, characterized by fatal responses to Epstein-Barr virus infection, is caused by mutations affecting the adaptor SAP, which links SLAM family receptors to downstream signaling. Although cytotoxic defects in SAP-deficient T cells are documented, the mechanism remains unclear. We show that SAP-deficient murine CD8+ T cells exhibited normal cytotoxicity against fibrosarcoma targets, yet had impaired adhesion to and killing of B cell and low avidity T cell targets. SAP-deficient cytotoxic lymphocytes showed specific defects in immunological synapse organization with these targets, resulting in inefficient actin clearance. In the absence of SAP, signaling through the SLAM family members Ly108 and 2B4 resulted in increased recruitment of the SHP-1 phosphatase, associated with altered SHP-1 localization and decreased activation of Src kinases at the synapse. Hence, SAP and SLAM receptors regulate positive and negative signals required for organizing the T:B cell synapse and setting thresholds for cytotoxicity against distinct cellular targets.

Initially discovered as an estrogen-responsive gene in breast cancer cell lines, anterior gradient 2 (AGR2) is a developmentally regulated gene belonging to the protein disulfide isomerase (PDI) gene family. Developmentally, AGR2 is expressed in the mammary gland in an estrogen-dependent manner, and AGR2 knockout and overexpression mouse models indicate that the gene promotes lobuloalveolar development by stimulating cell proliferation. Although AGR2 overexpression alone seems insufficient for breast tumorigenesis in mice, several lines of investigations suggest that AGR2 promotes breast tumorigenesis. Overexpression of AGR2 in several breast cancer cell lines increases cell survival in clonogenic assays and cell proliferation, whereas AGR2 loss of function leads to decreased cell cycle progression and cell death. In addition, AGR2 was shown to promote metastasis of breast epithelial cells in an in vivo metastasis assay. As a PDI, AGR2 is thought to be involved in the unfolded protein response that alleviates endoplasmic reticulum stress. Since cancer has to overcome proteotoxic stress due to excess protein production, AGR2 may be one of many pro-survival factors recruited to assist in protein folding or degradation or both. When AGR2 is secreted, it plays a role in cellular adhesion and dissemination of metastatic tumor cells. In breast cancer, AGR2 expression is associated with estrogen receptor (ER)-positive tumors; its overexpression is a predictor of poor prognosis. The AGR2 gene is directly targeted by ER-alpha, which is preferentially bound in tumors with poor outcome. Whereas aromatase inhibitor therapy decreases AGR2 expression, tamoxifen acts as an agonist of AGR2 expression in ER-positive tumors, perhaps contributing to tamoxifen resistance. AGR2 is also overexpressed in a subset of ER-negative tumors. Furthermore, AGR2 expression is associated with the dissemination of metastatic breast cancer cells and can be used as a marker to identify circulating tumor cells and metastatic cells in sentinel lymph nodes. In conclusion, AGR2 is a promising drug target in breast cancer and may serve as a useful prognostic indicator as well as a marker of breast cancer metastasis.

Pollination in species with dry stigmas begins with the hydration of desiccated pollen grains on the stigma, a highly regulated process involving the proteins and lipids of the pollen coat and stigma cuticle. Self-incompatible species of the Brassicaceae block pollen hydration, and while the early signaling steps of the self-incompatibility response are well studied, the precise mechanisms controlling pollen hydration are poorly understood. Both lipids and proteins are important for hydration; loss of pollen coat lipids and proteins results in defective or delayed hydration on the stigma surface. Here, we examine the role of the pollen coat protein extracellular lipase 4 (EXL4), in the initial steps of pollination, namely hydration on the stigma. We identify a mutant allele, exl4-1, that shows a reduced rate of pollen hydration. exl4-1 pollen is normal with respect to pollen morphology and the downstream steps in pollination, including pollen tube germination, growth, and fertilization of ovules. However, owing to the delay in hydration, exl4-1 pollen is at a disadvantage when competed with wild-type pollen. EXL4 also functions in combination with GRP17 to promote the initiation of hydration. EXL4 is similar to GDSL lipases, and we show that it functions in hydrolyzing ester bonds. We report a previously unknown function for EXL4, an abundant pollen coat protein, in promoting pollen hydration on the stigma. Our results indicate that changes in lipid composition at the pollen-stigma interface, possibly mediated by EXLs, are required for efficient pollination in species with dry stigmas.

Syntaxin 11 (STX11) controls vesicular trafficking and is a key player in exocytosis. SinceStx11 mutations are causally associated with a familial hemophagocytic lymphohistiocytosis, we wanted to clarify whether STX11 is functionally important for key immune cell populations. This was studied in primary cells obtained from newly generated Stx11 −/− mice. Our data revealed that STX11 is not only widely expressed in different immune cells, but also induced upon LPS or IFN-γ treatment. However, Stx11 deficiency does not affect macrophage phagocytic function and cytokine secretion, mast cell activation, or antigen presentation by DCs. Instead, STX11 selectively controls lymphocyte cytotoxicity in NK and activated CD8 + T cells and degranulation in neutrophils. Stx11 −/− NK cells and CTLs show impaired degranulation, despite a comparable activation, maturation and expression of the complex-forming partners MUNC18-2 and VTI1B. In addition, Stx11 −/− CTLs and NK cells produce abnormal levels of IFN-γ. Since functional reconstitution rescues the defective phenotype of Stx11 −/− CTLs, we suggest a direct, specific and key role of STX11 in controlling lymphocyte cytotoxicity, cytokine production and secretion. Finally, we show that these mice are a very useful tool for dissecting the role of STX11 in vesicular trafficking and secretion. Keywords: CTL r Cytokines r Exocytosis r N-ethylmaleimide-sensitive factor attachment protein receptorSee accompanying Commentary by Lopez and Voskoboinik.Additional supporting information may be found in the online version of this article at the publisher's web-site IntroductionSyntaxin 11 (STX11) belongs to the family of N-ethylmaleimidesensitive factor attachment protein receptor (SNARE) proteins Correspondence: Prof. Silvia Bulfone-Paus e-mail: sbulfone@fz-borstel.de and is involved in intracellular membrane trafficking events by interacting with other family members or by associating with membranes by palmitoylation of cysteine residues [1][2][3]. STX11 is expressed in a wide variety of cells including human monocytes/macrophages, neutrophils, B cells, NK cells and CD8 + T cells [2][3][4][5][6][7][8][9]. STX11 is enriched in immune tissues such as thymus, spleen C 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu Eur. J. Immunol. 2013. 43: 194-208 Immunomodulation 195 Values represent the mean ± SEM percentage of cell subsets in the spleen (n = 8) and lymph nodes (WT n = 7; Stx11 −/− n = 6) and are the summary of three experiments performed.and lymph nodes, but lower levels of protein are detectable also in heart, kidney and liver [2]. STX11 localizes mainly in the vesicular tubular clusters, an organelle complex between the endoplasmic reticulum and the Golgi, and displays, in addition, a spotted staining pattern throughout the cell periphery [2,9]. While STX11 has been recognized to act as a negative regulator of both phagocytosis and intracellular trafficking between endosomes, lysosomes and the outer membrane in macrophages [6,9], in human NK cells and CTLs, ST...

Background-Familial haemophagocytic lymphohistiocytosis (FHL) is a rare immune deficiency with uncontrolled inflammation; the clinical course usually starts within the first years of life, and is usually fatal unless promptly treated and then cured with haematopoietic stem cell transplant. FHL is caused by genetic mutations resulting in defective cell cytotoxicity; three disease related genes have been identified to date: perforin, Munc13-4 and syntaxin-11. A fourth gene, STXBP2, has been identified very recently as responsible for a defect in Munc18-2 in FHL-5.