Regulation of inflammatory responses by the commensal microbiota.

It is well established that dysregulation of the interactions between the immune system and commensal bacteria is one factor that underpins the development and chronicity of a number of inflammatory diseases. Certain phyla of bacteria within the microbiota have been associated with 'health', but the mechanisms by which the presence of these bacteria supports a healthy environment are still being unravelled. Recent evidence indicates that one such mechanism involves the anti-inflammatory properties of fermentation products of fibre, short-chain fatty acids and their signalling through the G-protein coupled receptor GPR43. Recent findings also indicate that, even in health, bacterial communities harbour in the airways, indicating that direct exposure to bacterial products at this site may provide a further explanation for how commensal bacteria can regulate chronic airway inflammation.

New loci associated with birth weight identify genetic links between intrauterine growth and adult height and metabolism.

Birth weight within the normal range is associated with a variety of adult-onset diseases, but the mechanisms behind these associations are poorly understood. Previous genome-wide association studies of birth weight identified a variant in the ADCY5 gene associated both with birth weight and type 2 diabetes and a second variant, near CCNL1, with no obvious link to adult traits. In an expanded genome-wide association meta-analysis and follow-up study of birth weight (of up to 69,308 individuals of European descent from 43 studies), we have now extended the number of loci associated at genome-wide significance to 7, accounting for a similar proportion of variance as maternal smoking. Five of the loci are known to be associated with other phenotypes: ADCY5 and CDKAL1 with type 2 diabetes, ADRB1 with adult blood pressure and HMGA2 and LCORL with adult height. Our findings highlight genetic links between fetal growth and postnatal growth and metabolism.

The SOS screen in Arabidopsis: a search for functions involved in DNA metabolism.

The SOS screen, as originally described by Perkins et al. (1999) [7], was setup with the aim of identifying Arabidopsis functions that might potentially be involved in the DNA metabolism. Such functions, when expressed in bacteria, are prone to disturb replication and thus trigger the SOS response. Consistently, expression of AtRAD51 and AtDMC1 induced the SOS response in bacteria, even affecting E. coli viability. 100 SOS-inducing cDNAs were isolated from a cDNA library constructed from an Arabidopsis cell suspension that was found to highly express meiotic genes. A large proportion of these SOS(+) candidates are clearly related to the DNA metabolism, others could be involved in the RNA metabolism, while the remaining cDNAs encode either totally unknown proteins or proteins that were considered as irrelevant. Seven SOS(+) candidate genes are induced following gamma irradiation. The in planta function of several of the SOS-inducing clones was investigated using T-DNA insertional mutants or RNA interference. Only one SOS(+) candidate, among those examined, exhibited a defined phenotype: silenced plants for DUT1 were sensitive to 5-fluoro-uracil (5FU), as is the case of the leaky dut-1 mutant in E. coli that are affected in dUTPase activity. dUTPase is essential to prevent uracil incorporation in the course of DNA replication.

Molecular and therapeutic characterization of anti-ectodysplasin A receptor (EDAR) agonist monoclonal antibodies.

The TNF family ligand ectodysplasin A (EDA) and its receptor EDAR are required for proper development of skin appendages such as hair, teeth, and eccrine sweat glands. Loss of function mutations in the Eda gene cause X-linked hypohidrotic ectodermal dysplasia (XLHED), a condition that can be ameliorated in mice and dogs by timely administration of recombinant EDA. In this study, several agonist anti-EDAR monoclonal antibodies were generated that cross-react with the extracellular domains of human, dog, rat, mouse, and chicken EDAR. Their half-life in adult mice was about 11 days. They induced tail hair and sweat gland formation when administered to newborn EDA-deficient Tabby mice, with an EC(50) of 0.1 to 0.7 mg/kg. Divalency was necessary and sufficient for this therapeutic activity. Only some antibodies were also agonists in an in vitro surrogate activity assay based on the activation of the apoptotic Fas pathway. Activity in this assay correlated with small dissociation constants. When administered in utero in mice or at birth in dogs, agonist antibodies reverted several ectodermal dysplasia features, including tooth morphology. These antibodies are therefore predicted to efficiently trigger EDAR signaling in many vertebrate species and will be particularly suited for long term treatments.

Cross-presenting human gammadelta T cells induce robust CD8+ alphabeta T cell responses.

Gammadelta T cells are implicated in host defense against microbes and tumors but their mode of function remains largely unresolved. Here, we have investigated the ability of activated human Vgamma9Vdelta2(+) T cells (termed gammadelta T-APCs) to cross-present microbial and tumor antigens to CD8(+) alphabeta T cells. Although this process is thought to be mediated best by DCs, adoptive transfer of ex vivo antigen-loaded, human DCs during immunotherapy of cancer patients has shown limited success. We report that gammadelta T-APCs take up and process soluble proteins and induce proliferation, target cell killing and cytokine production responses in antigen-experienced and naïve CD8(+) alphabeta T cells. Induction of APC functions in Vgamma9Vdelta2(+) T cells was accompanied by the up-regulation of costimulatory and MHC class I molecules. In contrast, the functional predominance of the immunoproteasome was a characteristic of gammadelta T cells irrespective of their state of activation. Gammadelta T-APCs were more efficient in antigen cross-presentation than monocyte-derived DCs, which is in contrast to the strong induction of CD4(+) alphabeta T cell responses by both types of APCs. Our study reveals unexpected properties of human gammadelta T-APCs in the induction of CD8(+) alphabeta T effector cells, and justifies their further exploration in immunotherapy research.

Selective requirement for c-Myc at an early stage of V(alpha)14i NKT cell development.

Valpha14 invariant (Valpha14i) NKT cells are a subset of regulatory T cells that utilize a semi-invariant TCR to recognize glycolipids associated with monomorphic CD1d molecules. During development in the thymus, CD4(+)CD8(+) Valpha14i NKT precursors recognizing endogenous CD1d-associated glycolipids on other CD4(+)CD8(+) thymocytes are selected to undergo a maturation program involving sequential expression of CD44 and NK-related markers such as NK1.1. The molecular requirements for Valpha14i NKT cell maturation, particularly at early developmental stages, remain poorly understood. In this study, we show that CD4-Cre-mediated T cell-specific inactivation of c-Myc, a broadly expressed transcription factor with a wide range of biological activities, selectively impairs Valpha14i NKT cell development without perturbing the development of conventional T cells. In the absence of c-Myc, Valpha14i NKT cell precursors are blocked at an immature CD44(low)NK1.1(-) stage in a cell autonomous fashion. Residual c-Myc-deficient immature Valpha14i NKT cells appear to proliferate normally, cannot be rescued by transgenic expression of BCL-2, and exhibit characteristic features of immature Valpha14i NKT cells such as high levels of preformed IL-4 mRNA and the transcription factor promyelocytic leukemia zinc finger. Collectively our data identify c-Myc as a critical transcription factor that selectively acts early in Valpha14i NKT cell development to promote progression beyond the CD44(low)NK1.1(-) precursor stage.

Exhaustion of tumor-specific CD8⁺ T cells in metastases from melanoma patients.

In chronic viral infections, CD8⁺ T cells become functionally deficient and display multiple molecular alterations. In contrast, only little is known of self- and tumor-specific CD8⁺ T cells from mice and humans. Here we determined molecular profiles of tumor-specific CD8⁺ T cells from melanoma patients. In peripheral blood from patients vaccinated with CpG and the melanoma antigen Melan-A/MART-1 peptide, we found functional effector T cell populations, with only small but nevertheless significant differences in T cells specific for persistent herpesviruses (EBV and CMV). In contrast, Melan-A/MART-1-specific T cells isolated from metastases from patients with melanoma expressed a large variety of genes associated with T cell exhaustion. The identified exhaustion profile revealed extended molecular alterations. Our data demonstrate a remarkable coexistence of effector cells in circulation and exhausted cells in the tumor environment. Functional T cell impairment is mediated by inhibitory receptors and further molecular pathways, which represent potential targets for cancer therapy.

Immunogenicity of the carcinoembryonic antigen derived peptide 694 in HLA-A2 healthy donors and colorectal carcinoma patients.

Carcinoembryonic antigen (CEACAM5) is commonly overexpressed in human colon cancer. Several antigenic peptides recognized by cytolytic CD8+ T-cells have been identified and used in colon cancer phase-I vaccination clinical trials. The HLA-A*0201-binding CEA(694-702) peptide was recently isolated from acid eluted MHC-I associated peptides from a human colon tumor cell line. However, the immunogenicity of this peptide in humans remains unknown. We found that the peptide CEA(694-702) binds weakly to HLA-A*0201 molecules and is ineffective at inducing specific CD8+ T-cell responses in healthy donors. Immunogenic-altered peptide ligands with increased affinity for HLA-A*0201 were identified. Importantly, the elicited cytolytic T lymphocyte (CTL) lines and clones cross-reacted with the wild-type CEA(694-702) peptide. Tumor cells expressing CEA were recognized in a peptide and HLA-A*0201 restricted fashion, but high-CEA expression levels appear to be required for CTL recognition. Finally, CEA-specific T-cell precursors could be readily expanded by in vitro stimulation of peripheral blood mononuclear cell (PBMC) from colon cancer patients with altered CEA peptide. However, the CEA-specific CD8+ T-cell clones derived from cancer patients revealed low-functional avidity and impaired tumor-cell recognition. Together, using T-cells to demonstrate the processing and presentation of the peptide CEA694-702, we were able to corroborate its presentation by tumor cells. However, the low avidity of the specific CTLs generated from cancer patients as well as the high-antigen expression levels required for CTL recognition pose serious concerns for the use of CEA694-702 in cancer immunotherapy.

Ly49D-mediated ITAM signaling in immature thymocytes impairs development by bypassing the pre-TCR checkpoint.

Activating and inhibitory NK receptors regulate the development and effector functions of NK cells via their ITAM and ITIM motifs, which recruit protein tyrosine kinases and phosphatases, respectively. In the T cell lineage, inhibitory Ly49 receptors are expressed by a subset of activated T cells and by CD1d-restricted NKT cells, but virtually no expression of activating Ly49 receptors is observed. Using mice transgenic for the activating receptor Ly49D and its associated ITAM signaling DAP12 chain, we show in this article that Ly49D-mediated ITAM signaling in immature thymocytes impairs development due to a block in maturation from the double negative (DN) to double positive (DP) stages. A large proportion of Ly49D/DAP12 transgenic thymocytes were able to bypass the pre-TCR checkpoint at the DN3 stage, leading to the appearance of unusual populations of DN4 and DP cells that lacked expression of intracellular (ic) TCRβ-chain. High levels of CD5 were expressed on ic TCRβ(-) DN and DP thymocytes from Ly49D/DAP12 transgenic mice, further suggesting that Ly49D-mediated ITAM signaling mimics physiological ITAM signaling via the pre-TCR. We also observed unusual ic TCRβ(-) single positive thymocytes with an immature CD24(high) phenotype that were not found in the periphery. Importantly, thymocyte development was completely rescued by expression of an Ly49A transgene in Ly49D/DAP12 transgenic mice, indicating that Ly49A-mediated ITIM signaling can fully counteract ITAM signaling via Ly49D/DAP12. Collectively, our data indicate that inappropriate ITAM signaling by activating NK receptors on immature thymocytes can subvert T cell development by bypassing the pre-TCR checkpoint.

RANKL expression, function, and therapeutic targeting in multiple myeloma and chronic lymphocytic leukemia.

Bone destruction is a prominent feature of multiple myeloma, but conflicting data exist on the expression and pathophysiologic involvement of the bone remodeling ligand RANKL in this disease and the potential therapeutic benefits of its targeted inhibition. Here, we show that RANKL is expressed by primary multiple myeloma and chronic lymphocytic leukemia (CLL) cells, whereas release of soluble RANKL was observed exclusively with multiple myeloma cells and was strongly influenced by posttranscriptional/posttranslational regulation. Signaling via RANKL into multiple myeloma and CLL cells induced release of cytokines involved in disease pathophysiology. Both the effects of RANKL on osteoclastogenesis and cytokine production by malignant cells could be blocked by disruption of RANK-RANKL interaction with denosumab. As we aimed to combine neutralization of RANKL with induction of antibody-dependent cellular cytotoxicity of natural killer (NK) cells against RANKL-expressing malignant cells and as denosumab does not stimulate NK reactivity, we generated RANK-Fc fusion proteins with modified Fc moieties. The latter displayed similar capacity compared with denosumab to neutralize the effects of RANKL on osteoclastogenesis in vitro, but also potently stimulated NK cell reactivity against primary RANKL-expressing malignant B cells, which was dependent on their engineered affinity to CD16. Our findings introduce Fc-optimized RANK-Ig fusion proteins as attractive tools to neutralize the detrimental function of RANKL while at the same time potently stimulating NK cell antitumor immunity.

Role of the GNOM gene in Arabidopsis apical-basal patterning--From mutant phenotype to cellular mechanism of protein action.

How the apical-basal axis of polarity is established in embryogenesis is still a mystery in plant development. This axis appeared specifically compromised by mutations in the Arabidopsis GNOM gene. Surprisingly, GNOM encodes an ARF guanine-nucleotide exchange factor (ARF-GEF) that regulates the formation of vesicles in membrane trafficking. In-depth functional analysis of GNOM and its closest relative, GNOM-LIKE 1 (GNL1), has provided a mechanistic explanation for the development-specific role of a seemingly mundane trafficking regulator. The current model proposes that GNOM is specifically involved in the endosomal recycling of the auxin-efflux carrier PIN1 to the basal plasma membrane in provascular cells, which in turn is required for the accumulation of the plant hormone auxin at the future root pole through polar auxin transport. Thus, the analysis of GNOM highlights the importance of cell-biological processes for a mechanistic understanding of development.

Peroxisome proliferator-activated receptor gamma activation is required for maintenance of innate antimicrobial immunity in the colon.

Crohn's disease (CD), a major form of human inflammatory bowel disease, is characterized by primary immunodeficiencies. The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma) is essential for intestinal homeostasis in response to both dietary- and microbiota-derived signals. Its role in host defense remains unknown, however. We show that PPARgamma functions as an antimicrobial factor by maintaining constitutive epithelial expression of a subset of beta-defensin in the colon, which includes mDefB10 in mice and DEFB1 in humans. Colonic mucosa of Ppargamma mutant animals shows defective killing of several major components of the intestinal microbiota, including Candida albicans, Bacteroides fragilis, Enterococcus faecalis, and Escherichia coli. Neutralization of the colicidal activity using an anti-mDefB10 blocking antibody was effective in a PPARgamma-dependent manner. A functional promoter variant that is required for DEFB1 expression confers strong protection against Crohn's colitis and ileocolitis (odds ratio, 0.559; P = 0.018). Consistently, colonic involvement in CD is specifically linked to reduced expression of DEFB1 independent of inflammation. These findings support the development of PPARgamma-targeting therapeutic and/or nutritional approaches to prevent colonic inflammation by restoring antimicrobial immunity in CD.

A natural structural variant of the mouse TCR beta-chain displays intrinsic receptor function and antigen specificity.

The Cbeta0 alternate cassette exon is located between the Jbeta1 and Cbeta1 genes in the mouse TCR beta-locus. In T cells with a VDJbeta1 rearrangement, the Cbeta0 exon may be included in TCRbeta transcripts (herein called TCRbeta-Cbeta0 transcripts), potentially inserting an additional 24 aa between the V and C domains of the TCR beta-chain. These TCRbeta splice isoforms may be differentially regulated after Ag activation, because we detected TCRbeta-Cbeta0 transcripts in a high proportion (>60%) of immature and mature T cells having VDJbeta1 rearrangements but found a substantially reduced frequency (<35%) of TCRbeta-Cbeta0 expression among CD8 T cells selected by Ag in vivo. To study the potential activity of the TCRbeta-Cbeta0 splice variant, we cloned full-length TCR cDNAs by single-cell RT-PCR into retroviral expression vectors. We found that the TCRbeta-Cbeta0 splice isoform can function during an early stage of T cell development normally dependent on TCR beta-chain expression. We also demonstrate that T hybridoma-derived cells expressing a TCRbeta-Cbeta0 isoform together with the clonally associated TCR alpha-chain recognize the same cognate peptide-MHC ligand as the corresponding normal alphabetaTCR. This maintenance of receptor function and specificity upon insertion of the Cbeta0 peptide cassette signifies a remarkable adaptability for the TCR beta-chain, and our findings open the possibility that this splice isoform may function in vivo.

Positive impact of inhibitory Ly49 receptor-MHC class I interaction on NK cell development.

NK cells can kill MHC-different or MHC-deficient but not syngeneic MHC-expressing target cells. This MHC class I-specific tolerance is acquired during NK cell development. MHC recognition by murine NK cells largely depends on clonally distributed Ly49 family receptors, which inhibit NK cell function upon ligand engagement. We investigated whether these receptors play a role for the development of NK cells and provide evidence that the expression of a Ly49 receptor transgene on developing NK cells endowed these cells with a significant developmental advantage over NK cells lacking such a receptor, but only if the relevant MHC ligand was present in the environment. The data suggest that the transgenic Ly49 receptor accelerates and/or rescues the development of NK cells which would otherwise fail to acquire sufficient numbers of self-MHC-specific receptors. Interestingly, the positive effect on NK cell development is most prominent when the MHC ligand is simultaneously present on both hemopoietic and nonhemopoietic cells. These findings correlate with functional data showing that MHC class I ligand on all cells is required to generate functionally mature NK cells capable of reacting to cells lacking the respective MHC ligand. We conclude that the engagement of inhibitory MHC receptors during NK cell development provides signals that are important for further NK cell differentiation and/or maturation.

TNF deficiency fails to protect BAFF transgenic mice against autoimmunity and reveals a predisposition to B cell lymphoma.

TNF is well characterized as a mediator of inflammatory responses. TNF also facilitates organization of secondary lymphoid organs, particularly B cell follicles and germinal centers, a hallmark of T-dependent Ab responses. TNF also mediates defense against tumors. We examined the role of TNF in the development of inflammatory autoimmune disorders resembling systemic lupus erythematosus and Sjögren's syndrome induced by excess B cell-activating factor belonging to the TNF family (BAFF), by generating BAFF-transgenic (Tg) mice lacking TNF. TNF(-/-) BAFF-Tg mice resembled TNF(-/-) mice, in that they lacked B cell follicles, follicular dendritic cells, and germinal centers, and have impaired responses to T-dependent Ags. Nevertheless, TNF(-/-) BAFF-Tg mice developed autoimmune disorders similar to that of BAFF-Tg mice. Disease in TNF(-/-) BAFF-Tg mice correlates with the expansion of transitional type 2 and marginal zone B cell populations and enhanced T-independent immune responses. TNF deficiency in BAFF-Tg mice also led to a surprisingly high incidence of B cell lymphomas (&gt;35%), which most likely resulted from the combined effects of BAFF promotion of neoplastic B cell survival, coupled with lack of protective antitumor defense by TNF. Thus, TNF appears to be dispensable for BAFF-mediated autoimmune disorders and may, in fact, counter any proneoplastic effects of high levels of BAFF in diseases such as Sjögren's syndrome, systemic lupus erythematosus, and rheumatoid arthritis.