Complement receptor 2-negative (CR2/CD21

Protein tyrosine phosphatase nonreceptor type 22 (PTPN22) gene polymorphisms are associated with many autoimmune diseases. The major risk allele encodes an R620W amino acid change that alters B cell receptor (BCR) signaling involved in the regulation of central B cell tolerance. To assess whether this PTPN22 risk allele affects the removal of developing autoreactive B cells, we tested by ELISA the reactivity of recombinant antibodies isolated from single B cells from asymptomatic healthy individuals carrying one or two PTPN22 risk allele(s) encoding the PTPN22 R620W variant. We found that new emigrant/transitional and mature naive B cells from carriers of this PTPN22 risk allele contained high frequencies of autoreactive clones compared with those from non-carriers, revealing defective central and peripheral B cell tolerance checkpoints. Hence, a single PTPN22 risk allele has a dominant effect on altering autoreactive B cell counterselection before any onset of autoimmunity. In addition, gene array experiments analyzing mature naive B cells displaying PTPN22 risk allele(s) revealed that the association strength of PTPN22 for autoimmunity may be due not only to the impaired removal of autoreactive B cells but also to the upregulation of genes such as CD40, TRAF1, and IRF5, which encode proteins that promote B cell activation and have been identified as susceptibility genes associated with autoimmune diseases. These data demonstrate that early B cell tolerance defects in autoimmunity can result from specific polymorphisms and precede the onset of disease.

SUMMARY Most autoreactive B cells are normally counterselected during early B cell development. To determine whether Toll-like receptors (TLRs) regulate the removal of autoreactive B lymphocytes, we tested the reactivity of recombinant antibodies from single B cells isolated from patients deficient for IRAK-4, and MyD88, whose cells do not respond to TLRs except TLR3 and from UNC-93B-deficient patients whose cells are irresponsive to TLR3, TLR7, TLR8 and TLR9. All patients suffered from defective central and peripheral B cell tolerance checkpoints resulting in the accumulation of large numbers of autoreactive mature naïve B cells in their blood. Hence, TLR7, TLR8, and TLR9 may normally prevent the recruitment of developing autoreactive B cells in healthy donors. Paradoxically, IRAK-4-, MyD88- and UNC-93B-deficient patients do not display autoreactive antibodies in their serum nor develop autoimmune diseases revealing that IRAK-4/MyD88/UNC-93B pathways blockade is likely to thwart the development of autoimmunity in humans.

Hyper-IgM (HIGM) syndromes are primary immunodeficiencies characterized by defects of class switch recombination and somatic hypermutation. HIGM patients who carry mutations in the CD40-ligand (CD40L) gene expressed by CD4+ T cells suffer from recurrent infections and often develop autoimmune disorders. To investigate the impact of CD40L–CD40 interactions on human B cell tolerance, we tested by ELISA the reactivity of recombinant antibodies isolated from single B cells from three CD40L-deficient patients. Antibody characteristics and reactivity from CD40L-deficient new emigrant B cells were similar to those from healthy donors, suggesting that CD40L–CD40 interactions do not regulate central B cell tolerance. In contrast, mature naive B cells from CD40L-deficient patients expressed a high proportion of autoreactive antibodies, including antinuclear antibodies. Thus, CD40L–CD40 interactions are essential for peripheral B cell tolerance. In addition, a patient with the bare lymphocyte syndrome who could not express MHC class II molecules failed to counterselect autoreactive mature naive B cells, suggesting that peripheral B cell tolerance also depends on major histocompatibility complex (MHC) class II–T cell receptor (TCR) interactions. The decreased frequency of MHC class II–restricted CD4+ regulatory T cells in CD40L-deficient patients suggests that these T cells may mediate peripheral B cell tolerance through CD40L–CD40 and MHC class II–TCR interactions.

The human VH4-34 gene segment encodes intrinsically self-reactive antibodies that recognize I/i carbohydrates. Schickel et al. show that these self-reactive clones may represent an innate-like B cell population specialized in the containment of commensal bacteria when gut barriers are breached.

Fc␥RIIB are low-affinity receptors for IgG that contain an immunoreceptor tyrosine-based inhibition motif (ITIM) and inhibit immunoreceptor tyrosine-based activation motif (ITAM)-dependent cell activation. When coaggregated with ITAM-bearing receptors, Fc␥RIIB become tyrosyl-phosphorylated and recruit the Src homology 2 (SH2) domain-containing inositol 5-phosphatases SHIP1 and SHIP2, which mediate inhibition. The Fc␥RIIB ITIM was proposed to be necessary and sufficient for recruiting SHIP1/2. We show here that a second tyrosine-containing motif in the intracytoplasmic domain of Fc␥RIIB is required for SHIP1/2 to be coprecipitated with the receptor. This motif functions as a docking site for the SH2 domain-containing adapters Grb2 and Grap. These adapters interact via their C-terminal SH3 domain with SHIP1/2 to form a stable receptor-phosphatase-adapter trimolecular complex. Both Grb2 and Grap are required for an optimal coprecipitation of SHIP with Fc␥RIIB, but one adapter is sufficient for the phosphatase to coprecipitate in a detectable manner with the receptors. In addition to facilitating the recruitment of SHIPs, the second tyrosinebased motif may confer upon Fc␥RIIB the properties of scaffold proteins capable of altering the composition and stability of the signaling complexes generated following receptor engagement. (5), i.e. by all receptors containing immunoreceptor tyrosine-based activation motifs. Fc␥RIIB must be coaggregated with activating receptors via IgG immune complexes in order to exert their inhibitory effects (2). The in vivo relevance of the regulatory properties of Fc␥RIIB was ascertained in Fc␥RIIB-deficient mice. Fc␥RIIB Ϫ/Ϫ mice were shown to mount enhanced antibody responses (6), exhibit enhanced IgG-and IgE-induced anaphylactic reactions (7), be hypersensitive to collagen-induced arthritis (8, 9), and develop spontaneous systemic lupus erythematosus in the C57BL/6 background (10). Fc␥RIIB are therefore likely to play major roles in the prevention of autoimmune diseases, allergies, and other inflammatory diseases.The regulatory properties of Fc␥RIIB were shown to depend on the presence of an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its intracytoplasmic domain. This motif was defined as a tyrosine residue that is followed at position Tyr ϩ 3 and preceded at position Tyr Ϫ 2 by hydrophobic amino acids (11). The ITIM tyrosine becomes phosphorylated by a Src family protein tyrosine kinase upon the coaggregation of inhibitory receptors with activating receptors (12), providing a docking site for SH2 domain-containing cytosolic phosphatases (13). Because of the presence of a leucine residue at position Tyr ϩ 2 (14), Fc␥RIIB were shown to recruit selectively the single SH2 domain-containing inositol 5Ј-phosphatases SHIP1 (15, 16) and/or SHIP2 (17). These phosphatases dephosphorylate 5Ј-phosphate groups in 3Ј-phosphorylated inositols and phosphatidylinositides (18, 19) among which phosphatidylinositol 3,4,5-triphosphate (20, 21), generated by phosphatidylinositol 3-kinase duri...

The transmembrane adapter linker for activation of T cells (LAT) is thought to couple immunoreceptors to intracellular signaling pathways. In mice, its intracytoplasmic domain contains nine tyrosines which, when phosphorylated upon receptor aggregation, recruit Src-homology 2 domain-containing cytosolic enzymes and adapters. The four distal tyrosines are critical for both TCR and FcεRI signaling. Unexpectedly, knock-in mice expressing LAT with a point mutation of the first or of the last three of these tyrosines exhibited an abnormal T cell development characterized by a massive expansion of TH2-like αβ or γδ T cells, respectively. This phenotype suggests that, besides positive signals, LAT might support negative signals that normally regulate terminal T cell differentiation and proliferation. We investigated here whether LAT might similarly regulate mast cell activation, by generating not only positive but also negative signals, following FcR engagement. To this end, we examined IgE- and/or IgG-induced secretory and intracellular responses of mast cells derived from knock-in mice expressing LAT with combinations of tyrosine mutations (Y136F, Y(175, 195, 235)F, or Y(136, 175, 195, 235)F). A systematic comparison of pairs of mutants enabled us to dissect the respective roles played by the five proximal and the four distal tyrosines. We found that LAT tyrosines differentially contribute to exocytosis and cytokine secretion and differentially regulate biological responses of mucosal- and serosal-type mast cells. We also found that, indeed, both positive and negative signals may emanate from distinct tyrosines in LAT, whose integration modulates mast cell secretory responses.

Murine FcgammaRIIB were demonstrated to recruit SH2 domain-containing inositol 5-phosphatases (SHIP1/2), when their ITIM is tyrosyl-phosphorylated upon co-aggregation with BCR, and SHIP1 to account for FcgammaRIIB-dependent negative regulation of murine B cell activation. Although human FcgammaRIIB share the same ITIM as murine FcgammaRIIB and similarly inhibit human B cell activation, which among the four known SH2 domain-containing (tyrosine or inositol) phosphatases is/are recruited by human FcgammaRIIB is unclear. Our recent finding that, besides the ITIM, a second tyrosine-based motif is mandatory for murine FcgammaRIIB to recruit SHIP1 challenged the possibility that human FcgammaRIIB recruit this phosphatase. Human FcgammaRIIB indeed lack this motif. Using an experimental model which enabled us to compare human FcgammaRIIB and murine FcgammaRIIB under strictly controlled conditions, we show that SHIP1 is recruited to the intracytoplasmic domain of human FcgammaRIIB and inhibits the same biological responses and intracellular signals as when recruited by murine FcgammaRIIB. Identical results were observed in murine and in human B cells. We demonstrate that SHIP is necessary for human FcgammaRIIB to inhibit BCR signaling, and cannot be replaced by SHP-1 or SHP-2. Although it contains no tyrosine, the C-terminal segment of human FcgammaRIIB was as mandatory as the tyrosine-containing C-terminal segment of murine FcgammaRIIB for SHIP1 to be recruited to the ITIM. This segment, however, did not recruit the adapters Grb2/Grap which were demonstrated to stabilize the recruitment of SHIP1 to the ITIM in murine FcgammaRIIB.