Autophagy inhibition promotes defective neosynthesized proteins storage in ALIS, and induces redirection toward proteasome processing and MHCI-restricted presentation.

A significant portion of newly synthesized protein fails to fold properly and is quickly degraded. These defective ribosomal products (DRiPs) are substrates for the ubiquitin-proteasome system (UPS) and give rise to a large fraction of peptides presented by major histocompatibility complex class I molecules (MHCI). Here, we showed that DRiPs are also autophagy substrates, which accumulate upon autophagy inhibition in aggresome-like-induced structures (ALIS). Aggregation is critically depending on p62/SQSTM1, but occurs in the absence of activation of the NRF2 signaling axis and transcriptional regulation of p62/SQSTM1. We demonstrated that autophagy-targeted DRiPs can become UPS substrates and give rise to MHCI presented peptides upon autophagy inhibition. We further demonstrated that autophagy targeting of DRiPs is controlled by NBR1, but not p62/SQSTM1, CHIP or BAG-1. Active autophagy therefore directly modulates MHCI presentation by constantly degrading endogenous defective neosynthesized antigens, which are submitted to at least two distinct quality control mechanisms.

MHC class II stabilization at the surface of human dendritic cells is the result of maturation-dependent MARCH I down-regulation.

In response to Toll-like receptor ligands, dendritic cells (DCs) dramatically enhance their antigen presentation capacity by stabilizing at the cell-surface MHC II molecules. We demonstrate here that, in human monocyte-derived DCs, the RING-CH ubiquitin E3 ligase, membrane-associated RING-CH I (MARCH I), promotes the ubiquitination of the HLA-DR beta-chain. Thus, in nonactivated DCs, MARCH I induces the surface internalization of mature HLA-DR complexes, therefore reducing their stability and levels. We further demonstrate that the maturation-dependent down-regulation of MARCH I is a key event in MHC class II up-regulation at the surface of LPS-activated DCs. MARCH I is, therefore, a major regulator of HLA-DR traffic, and its loss contributes to the acquisition of the potent immunostimulatory properties of mature human DCs.

Clinical, immunologic and genetic analysis of 29 patients with autosomal recessive hyper-IgM syndrome due to Activation-Induced Cytidine Deaminase deficiency.

Mutations of the Activation-Induced Cytidine Deaminase (AID) gene have been found in patients with autosomal recessive hyper-IgM (HIGM) syndrome type 2. We retrospectively analyzed clinical, immunologic and genetic characteristics of 29 patients from 22 families with AID deficiency. Patients' median age at diagnosis and at last evaluation was 4.9 years (range: 0 to 53) and 14.2 years (range: 2.7 to 63), respectively. Most patients had suffered from recurrent and severe infections, however, intravenous immunoglobulin (IVIG) replacement therapy resulted in a dramatic decrease in the number of infections. Lymphoid hyperplasia developed in 22 patients and persisted in 7 at last follow-up. It is striking to note that six patients developed autoimmune or inflammatory disorders including diabetes mellitus, polyarthritis, autoimmune hepatitis, hemolytic anemia, immune thrombocytopenia, Crohn's disease and chronic uveitis. Fifteen distinct AID mutations were found but there was no significant genotype-phenotype correlation. In conclusion, AID-deficient patients are prone to infections and lymphoid hyperplasia, which may be prevented by early-onset IVIG replacement, but also to autoimmune and inflammatory disorders.

Human uracil-DNA glycosylase deficiency associated with profoundly impaired immunoglobulin class-switch recombination.

Activation-induced cytidine deaminase (AID) is a 'master molecule' in immunoglobulin (Ig) class-switch recombination (CSR) and somatic hypermutation (SHM) generation, AID deficiencies are associated with hyper-IgM phenotypes in humans and mice. We show here that recessive mutations of the gene encoding uracil-DNA glycosylase (UNG) are associated with profound impairment in CSR at a DNA precleavage step and with a partial disturbance of the SHM pattern in three patients with hyper-IgM syndrome. Together with the finding that nuclear UNG expression was induced in activated B cells, these data support a model of CSR and SHM in which AID deaminates cytosine into uracil in targeted DNA (immunoglobulin switch or variable regions), followed by uracil removal by UNG.

The block in immunoglobulin class switch recombination caused by activation-induced cytidine deaminase deficiency occurs prior to the generation of DNA double strand breaks in switch mu region.

Affinity maturation of the Ab repertoire in germinal centers leads to the selection of high affinity Abs with selected heavy chain constant regions. Ab maturation involves two modifications of the Ig genes, i.e., somatic hypermutation and class switch recombination. The mechanisms of these two processes are not fully understood. As shown by the somatic hypermutation and class switch recombination-deficient phenotype of activation-induced cytidine deaminase (AID)-deficient patients (hyperIgM type 2 syndrome) and mice, both processes require the AID molecule. Somatic DNA modifications require DNA breaks, which, at least for class switch recombination, lead to dsDNA breaks. By using a ligation-mediated PCR, it was found that class switch recombination-induced dsDNA breaks in S mu switch regions were less frequent in AID-deficient B cells than in AID-proficient B cells, thus indicating that AID acts upstream of DNA break induction.

AID mutant analyses indicate requirement for class-switch-specific cofactors.

Activation-induced cytidine deaminase (AID) is the essential and sole B cell-specific factor required for class-switch recombination (CSR) and somatic hypermutation (SHM). However, it is not known how AID differentially regulates these two independent events. Involvement of several cofactors interacting with AID has been indicated by scattered distribution of loss-of-function point mutations and evolutionary conservation of the entire 198-amino-acid protein. Here, we report that human AID mutant proteins with insertions, replacements or truncations in the C-terminal region retained strong SHM activity but almost completely lost CSR activity. These results indicate that AID requires interaction with a cofactor(s) specific to CSR.

Hyper-IgM syndrome type 4 with a B lymphocyte-intrinsic selective deficiency in Ig class-switch recombination.

Hyper-IgM syndrome (HIGM) is a heterogeneous condition characterized by impaired Ig class-switch recombination (CSR). The molecular defects that have so far been associated with this syndrome - which affect the CD40 ligand in HIGM type 1 (HIGM1), CD40 in HIGM3, and activation-induced cytidine deaminase (AID) in HIGM2 - do not account for all cases. We investigated the clinical and immunological characteristics of 15 patients with an unidentified form of HIGM. Although the clinical manifestations were similar to those observed in HIGM2, these patients exhibited a slightly milder HIGM syndrome with residual IgG production. We found that B cell CSR was intrinsically impaired. However, the generation of somatic hypermutations was observed in the variable region of the Ig heavy chain gene, as in control B lymphocytes. In vitro studies showed that the molecular defect responsible for this new HIGM entity (HIGM4) occurs downstream of the AID activity, as the AID gene was induced normally and AID-induced DNA double-strand breaks in the switch micro region of the Ig heavy chain locus were detected during CSR as normal. Thus, HIGM4 is probably the consequence of a selective defect either in a CSR-specific factor of the DNA repair machinery or in survival signals delivered to switched B cells.