The human genome contains approximately 20 thousand protein-coding genes 1 , but the size of the collection of adaptive immune system antigen receptors generated by recombination of gene segments with non-templated junctional additions (on B cells) is orders of magnitude larger and unknown. It is not established whether individuals possess unique (private) repertoires or significant components of shared (public) repertoires. Here we sequenced the recombined and expressed B cell receptor gene repertoire in several individuals at unprecedented depth to determine the size of an individual repertoire and the extent of shared repertoire between individuals. The experiments revealed that each individual's circulating repertoire contained between 9 and 17 million B cell clonotypes. The three individuals studied possessed many shared clonotypes, including 1 to 6% B cell heavy chain clonotypes shared between two subjects (0.3% shared by all three) or 20 to 34% of λ or κ light chains shared between two subjects (16 or 22% λ or κ shared by all three). Some of the B cell clonotypes had thousands of clones (somatic variants) within the clonotype lineage. While some of these shared lineages might be driven by exposure to common antigens, prior foreign antigen exposure was not the only force shaping the shared repertoires, as we also identified shared clonotypes present in both human cord blood samples and in all adult repertoires. The unexpectedly high prevalence of shared clonotypes in B cell Reprints and permissions information is available at www.nature.com/reprints.

G protein-coupled receptor ligand-dependent transactivation of growth factor receptors has been implicated in human cancer cell proliferation, migration, and cell survival. For example, prostaglandin E 2 (PGE2)-induced transactivation of the EGF receptor (EGFR) in colorectal carcinoma cells is mediated by means of a c-Src-dependent mechanism and regulates cell proliferation and migration. Recent evidence indicates that ␤-arrestin 1 may act as an important mediator in G protein-coupled receptor-induced activation of c-Src. Whether ␤-arrestin 1 serves a functional role in these events is, however, unknown. We investigated the effects of PGE 2 on colorectal cancer cells expressing WT and mutant ␤-arrestin 1. Here we report that PGE 2 induces the association of a prostaglandin E receptor 4͞␤-arrestin 1͞c-Src signaling complex resulting in the transactivation of the EGFR and downstream Akt (PKB) signaling. The interaction of ␤-arrestin 1 and c-Src is critical for the regulation of colorectal carcinoma cell migration in vitro as well as metastatic spread of disease to the liver in vivo. These results show that the prostaglandin E͞␤-arrestin 1͞c-Src signaling complex is a crucial step in PGE2-mediated transactivation of the EGFR and may play a pivotal role in tumor metastasis. Furthermore, our data implicate a functional role for ␤-arrestin 1 as a mediator of cellular migration and metastasis. metastasis ͉ prostaglandin E2 ͉ c-Src ͉ EGF receptor ͉ prostaglandin E receptor G protein-coupled receptors (GPCRs) comprise the largest known family of plasma membrane receptors and consist of a seven-transmembrane-spanning region f lanked by an extracellular N terminus and an intracellular C terminus. Upon ligand binding, these receptors couple to heterotrimeric G proteins (G␣-and G␤␥-subunits) and catalyze the exchange of GDP for GTP, thus initiating a multitude of signaling events into the cell. These include the classical activation of phosholipases (phosholipases A, C, and D), protein kinases (PKA and PKC), and lipid kinases (phosphatidylinositol 3-kinase) as well as increased intracellular calcium levels. The desensitization of GPCRs occurs through a multistep process. GPCR kinases are recruited to the receptor by liberated ␤␥-subunits and phosphorylate the receptor on the cytoplasmic tail and intracellular loops. This phosphorylation event triggers the association of arrestin, which then traffics the receptors to clathrin-coated pits for endocytosis (1).Prostaglandins (PG) are important bioactive lipids which exert their effects through the activation of specific GPCRs as well as members of the peroxisome proliferator-activated receptor family. For example, PGE 2 is the ligand for four prostaglandin E (EP) receptor isoforms termed EP1, EP2, EP3, and EP4. Stimulation of these receptors elicits different intracellular responses (2). The stimulation of the EP1 receptor induces an increase in intracellular calcium by means of the activation of phospholipase C. EP2 and EP4 receptors couple to G␣s proteins, which generate incre...

Background Necrotising enterocolitis (NEC) is the most common gastrointestinal emergency in premature infants. Immaturity of gastrointestinal immune regulation may predispose preterm infants to NEC as FOXP3 T regulatory cells (Treg) are critical for intestinal immune homoeostasis. Objective To investigate the hypothesis that abnormal developmental regulation of lamina propria Treg would define premature infants with NEC. Design Lamina propria mononuclear cell populations from surgically resected ileum from 18 patients with NEC and 30 gestational age-matched non-NEC surgical controls were prospectively isolated. Polychromatic flow cytometry was performed to phenotype and analyse lamina propria T cell populations. The cytokine gene expression profile in NEC tissue was compared with that of non-NEC controls. Results The total number of Treg, CD4, or CD8 T cells in each ileum section was independent of gestational age, age or postmenstrual age and similar between patients with NEC and controls. In contrast, the ratio of Treg to CD4 T cells or Treg to CD8 T cells was significantly lower in NEC ileum than in infants without NEC (medians 2.9% vs 6.6%, p=0.001 and medians 6.6% vs 25.9%, p<0.001, respectively). For any given number of CD4 or CD8 T cells, Treg were, on average, 60% lower in NEC ileum than in controls. NEC tissue cytokine gene expression profiles were characteristic of inhibited Treg development or function. Treg/CD4 and Treg/CD8 ratios recovered between initial resection for NEC and reanastomosis. Conclusion The proportion of lamina propria Treg is significantly reduced in the ileum of premature infants with NEC and may contribute to the excessive inflammatory state of this disease.

Respiratory syncytial virus (RSV) remains a major human pathogen, infecting the majority of infants before age two and causing re-infection throughout life. Despite decades of RSV research, there is no licensed RSV vaccine. Most candidate vaccines studied to date have incorporated the RSV fusion (F) surface glycoprotein, because the sequence of F is highly conserved among strains of RSV. To better define the human B cell response to RSV F, we isolated from a single donor 13 new neutralizing human monoclonal antibodies (mAbs) that recognize the RSV F protein in the pre-fusion conformation. Epitope binning studies showed that the majority of neutralizing mAbs targeted a new antigenic site on the globular head domain of F, designated here antigenic site VIII, which occupies an intermediate position between the previously defined major antigenic sites II and site Ø. Antibodies to site VIII competed for binding with antibodies to both of those adjacent neutralizing sites. The new mAbs exhibited unusual breadth for pre-fusion F-specific antibodies, cross-reacting with F proteins from both RSV subgroups A and B viruses. We solved the X-ray crystal structure of one site VIII mAb, hRSV90, in complex with pre-fusion RSV F protein. The structure revealed a large footprint of interaction for hRSV90 on RSV F, in which the heavy chain and light chain both have specific interactions mediating binding to site VIII, the heavy chain overlaps with site Ø, and the light chain interacts partially with site II.