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ABSTRACTVici syndrome is a human inherited multi-system disorder caused by recessive mutations in EPG5, encoding the EPG5 protein that mediates the fusion of autophagosomes with lysosomes. Immunodeficiency characterized by lack of memory B cells and increased susceptibility to infection is an integral part of the condition, but the role of EPG5 in the immune system remains unknown. Here we show that EPG5 is indispensable for the transport of the TLR9 ligand CpG to the late endosomal-lysosomal compartment, and for TLR9-initiated signaling, a step essential for the survival of human memory B cells and their ultimate differentiation into plasma cells. Moreover, the predicted structure of EPG5 includes a membrane remodeling domain and a karyopherin-like domain, thus explaining its function as a carrier between separate vesicular compartments. Our findings indicate that EPG5, by controlling nucleic acids intracellular trafficking, links macroautophagy/autophagy to innate and adaptive immunity.

A young woman presenting respiratory infections, polyarthritis, severe neutropenia, and increased serum IgM was treated with Intravenous Immunoglobulin (IVIG) with good clinical and laboratory outcome followed by a loss of efficacy. The increased serum IgM associated to recurrent infections and autoimmune manifestations suggested the diagnosis of a hyper–IgM syndrome (HIGMs). The frequency of peripheral T cells, the expression of CD40 on the patients' B cells and CD40L on T cells and the Activation-Induced cytidine Deaminase (AID) and Uracyl-DNA glycosylase (UNG) at mRNA level was comparable to controls. In contrast, the frequency of B cells was one half of the healthy control and all cells showed an atypical phenotype. Although AID and UNG were normal, class-switch recombination was not very efficient because circulating switched memory were reduced and, once stimulated with CpG, generated less antibody-secreting cells than controls. An increase in serum B Lymphocytes stimulator (BLyS) was also found. The patient presented a peculiar clinical and immunological phenotype fitting for many aspects of both HIGM4 and Common Variable Immunodeficiency (CVID). These findings underline the need to better explore the complex link between these two diseases.

Transient hypogammaglobulinemia of infancy (THI) is a heterogenous disorder characterized by reduced serum IgG levels in early infancy. A putative diagnosis is initially made after exclusion of other causes of hypogammaglobulinemia while a definitive diagnosis of THI can only be made a posterioriin patients with normalization of IgG levels. The aim of this study is to characterize clinical and immunological features of children with an initial diagnosis of THI in correlation to natural outcome, and to assess predictive laboratory parameters of clinical evolution for this disorder. We prospectively analysed clinical and immunological characteristics of 77 THI children at initial diagnosis and of 57 patients at follow-up. Memory B cell subsets and in vitroimmunoglobulin production were evaluated. Seventy patients (91%) showed clinical symptoms. Patients suffered from infections (91%), allergies (47%) and autoimmune disease (4%). During follow-up 41/57 children (72%) normalized IgG values, mostly within 24 months of age (p<0.001), allowing the diagnosis of THI. The 16 children who did not normalize their IgG levels showed a higher frequency of severe infections and autoimmune disease (p<0.01). Moreover, they expressed a reduced frequency of IgM and switched memory B cells (p<0.01) and an inability to produce IgG in vitro(p<0.02). We conclude that most patients with an initial diagnosis of THI spontaneously recover within 24 months of age and have a benign clinical course, while a subgroup of children with undefined hypogammaglobulinemia share a clinical and immunological profile with other primary immunodeficiencies. Early recognition of children with hypogammaglobulinemia during infancy who are likely to suffer from permanent immunodeficiencies later in life would allow prompt and appropriate laboratory and clinical interventions.

Although adult murine B cells can be stimulated to proliferate by Igm receptor cross-linking, we and others have shown that these cells will undergo apoptosis in vitro in a dose-, time- and temperature-dependent manner with polyclonal but not monoclonal anti-IgM, To test the role of c-myc and cell cycle progression in B cell apoptosis, we examined normal, Sp6 anti-TNP lg and E micro-myc transgenic splenocytes for receptor-mediated apoptosis in vitro. In normal mice, both spontaneous and anti-IgM-induced programmed cell death were specifically blocked by antisense oligodeoxynucleotides for the c-myc proto-oncogene, whereas nonsense myc oligonucleotides and irrelevant oligonucleotides had only a minor effect. Similarly, TNP-dextran-induced apoptosis in Sp6 anti-TNF transgenics was inhibited by antisense c-myc. This effect was not due to the mitogenic effects of unmethylated CpG-containing sequences because ones lacking this motif, as well as methylated oligonucleotides containing this motif, prevented apoptosis, and mitogenic doses of lipopolysaccharide failed to inhibit anti-IgM-driven cell death. Importantly, antisense c-myc also prevented the anti-IgM-induced increase in myc protein species. Moreover, spontaneous apoptosis in vitro was exaggerated in E micro-myc transgenic B cells. To examine the role of CD45 in anti-IgM-induced apoptosis, we treated spleen cells from CD45 knockout mice, which do not proliferate with anti-IgM, and found that B cells from these underwent apoptosis normally despite the lack of entry into S. These data suggest that anti-IgM driven apoptosis does not require CD45. Rather, apoptosis may be due to an overexpression of myc protein in the absence of signals which can drive B cells productively into S, but the failure to proliferate normally is insufficient for apoptosis to occur.

We describe a novel mAb (F3) which reacts with a 65 kDa thymocyte surface protein, expressed on approximately 80% of thymocytes, referred to as F3Ag. In ontogeny, F3Ag expression begins in the CD4(-)CD8(-) double-negative (DN) CD25(+) population and is maintained through approximately 85% of the CD4(+)CD8(+) double-positive (DP) stage. DP cells with high TCR expression and CD4(+) single-positive (SP) cells are predominantly negative for F3Ag, whereas many CD8(+) SP thymocytes express F3Ag. F3Ag-DP thymocytes show a reduced expression of RAG-1 and RAG-2 compared with F3Ag+ DP cells. The shutdown of F3Ag expression during the DP stage is related to positive selection: mice deficient for MHC class I and class II molecules maintain F3Ag expression in almost all DP cells. Transgenic (tg) mice carrying TCR restricted for MHC class II show a more pronounced down-regulation of F3Ag in the DP compartment than normal mice, depending on the presence of a positively selecting MHC. The size of the F3Ag- DP subset is positively correlated with the efficacy of positive selection into the CD4(+) SP compartment. Because some CD8(+) SP cells express F3Ag, the relationship between F3Ag down-regulation and positive selection is less obvious in DP cells of mice carrying MHC class I-restricted tg TCR. However, in reaggregate thymic organ cultures, sorted F3Ag- DP cells differentiate into CD8(+) SP cells more rapidly than do F3Ag+ DP cells. Thus, after down-regulation in the DP stage, a proportion of CD8(+) SP cells appears to re-express F3Ag. In addition, the proportion of F3Ag-CD8(+) SP cells depends on the efficacy of positive selection into the CD8 lineage. Taken together, the regulation of the expression of the F3Ag appears to be associated with signals that control thymic repertoire selection.

Recent studies have shown that maturation of CD4-8- double negative (DN) thymocytes to the CD4+8+ double positive (DP) stage is dependent on expression of the T cell receptor (TCR)-beta polypeptide. The exact mechanism by which the TCR-beta chain regulates this maturation step remains unknown. Previous experiments had suggested that in the presence of some TCR+ thymocytes, additional DN thymocytes not expressing a TCR-beta chain may be recruited to mature to the DP stage. The recent demonstration of an immature TCR-beta-CD3 complex on early thymocytes lead to the alternative hypothesis that signal transduction through an immature TCR-CD3 complex may induce maturation to the DP stage. In the latter case, maturation to the DP stage would depend on the expression of TCR-beta-CD3 in the same cell. We examined these two hypotheses by studying the expression of the intra- and extracellular CD3 epsilon, CD3 zeta, and TCR-beta polypeptides in intrathymic subpopulations during embryogenesis. CD3 epsilon and CD3 zeta were expressed intracellularly 2 and 1 d, respectively, before intracellular expression of the TCR-beta chain, potentially allowing immediate surface expression of an immature TCR-beta-CD3 complex as soon as functional rearrangement of a TCR-beta gene locus has been accomplished. Calcium mobilization could be induced by stimulation with anti-CD3 epsilon mAb as soon as intracellular TCR-beta was detectable, suggesting that a functional TCR-beta-CD3 complex is indeed expressed on the surface of early thymocytes. From day 17 on, most cells were in the DP stage, and over 95% of the DP cells expressed on the TCR-beta chain intracellularly. At day 19 of gestation, extremely low concentrations of TCR-beta chain and CD3 epsilon were detectable on the cell surface of nearly all thymocytes previously thought to be TCR-CD3 negative. These findings strongly support the hypothesis that maturation to the DP stage depends on surface expression of and subsequent signal transduction through an immature TCR-beta-CD3 complex and suggest that maturation to the DP stage by recruitment, if it occurs at all, is of minor relevance.

CD45 expression is essential for immunoglobulin (Ig)-mediated B cell activation. Treatments with either anti-Ig or anti-CD45 suggest that CD45 may facilitate early signaling events such as calcium mobilization, and phosphoinositide hydrolyis as well as later events leading to transcription of genes such as c-myc. To examine the role of CD45 more extensively, CD45-deficient mice were generated by disruption of exon 6. Although normal numbers of B cells were found in peripheral lymphoid tissues, CD45-deficient cells failed to proliferate upon IgM crosslinking. In the present study, we demonstrate that the fraction of high buoyant density B cells is reduced while low buoyant density cells are increased. Moreover, there is a significant decline in the number of splenic B cells of the mature IgDhi, IgMlo phenotype. Although both the basal and anti-Ig-induced levels of phosphorylation of Ig-alpha and phospholipase C gamma 2 are indistinguishable from that observed in CD45+ control B cells, a major distinction was found in Ca2+ mobilization. While anti-Ig-induced mobilization of intracellular Ca2+ stores was normal, influx from extracellular sources was abrogated. This finding reveals a novel pathway of regulating B cell responses mediated by CD45.

B lymphocytes recognize antigen through membrane-bound antigen-receptors, membrane IgM and IgD (mIgM and mIgD). Binding to foreign antigens initiates a cascade of biochemical events that lead to activation and differentiation. In contrast, binding to self-antigens leads to death or to inactivation. It is commonly believed that the B cells acquire the ability to discriminate between self and nonself in the early phases of development. We report here that immature B cells, which have just emerged from the mIgMneg, B220pos pool, are not deleted upon binding of self-antigen. In vivo, developing B cells become sensitive to tolerance induction in a relatively late window of differentiation, when they are in transition from the immature (HSAbright, B220dull) to the mature (HSAdull, B220bright) stage. In the transitional B cells, early markers of differentiation such as Pgp1 (CD44) and ThB reach the highest level of expression, while the expression of CD23 and mIgD, late markers of differentiation, and expression of class II MHC, progressively increases. Most of the transitional B cells, but only few of the mature and of the immature B cells, express the fas antigen, while mature B cells, but not immature and transitional B cells, express bcl-2 protein. mIgM is present in low amounts in immature B cells, reaches the highest level of expression in transitional B cells and is down-regulated in mature resting B cells, where it is coexpressed with mIgD. The high expression of mIgM, the presence of the fas antigen and the absence of bcl-2 protein is compatible with the high sensitivity of transitional B cells to negative selection. In vitro, immature B cells die rapidly by apoptosis after cross-linking of mIgM. This result, combined with the resistance of immature B cells to elimination in vivo, suggests that early in development the stroma cell microenvironment modulates signals transduced through mIgM. The functional and phenotypic division of IgMpos bone marrow B cells in three compartments not only allows to define the target population of physiological processes like negative selection, but will also be a helpful tool for an accurate description of possible developmental blocks in mutant mice.