Distinct effects of DNA-PKcs and Artemis inactivation on signal joint formation in vivo.

The assembly of functional immune receptor genes via V(D)J recombination in developing lymphocytes generates DNA double-stranded breaks intermediates that are repaired by non-homologous end joining (NHEJ). This repair pathway requires the sequential recruitment and activation onto coding and signal DNA ends of several proteins, including the DNA-dependent protein kinase and the nuclease Artemis. Artemis activity, triggered by the DNA-dependent protein kinase, is necessary to process the genes hairpin-sealed coding ends but appears dispensable for the ligation of the reciprocal phosphorylated, blunt-ended signal ends into a signal joint. The DNA-dependent protein kinase is however present on signal ends and could potentially recruit and activate Artemis during signal joint formation. To determine whether Artemis plays a role during the resolution of signal ends during V(D)J recombination, we analyzed the structure of signal joints generated in developing thymocytes during the rearrangement of T cell receptor genes in wild type mice and mice mutated for NHEJ factors. These joints exhibit junctional diversity resulting from N nucleotide polymerization by the terminal nucleotidyl transferase and nucleotide loss from one or both of the signal ends before they are ligated. Our results show that Artemis participates in the repair of signal ends in vivo. Furthermore, our results also show that while the DNA-dependent protein kinase complex protects signal ends from processing, including deletions, Artemis seems on the opposite to promote their accessibility to modifying enzymes. In addition, these data suggest that Artemis might be the nuclease responsible for nucleotide loss from signal ends during the repair process.

XIAP deficiency in humans causes an X-linked lymphoproliferative syndrome.

The homeostasis of the immune response requires tight regulation of the proliferation and apoptosis of activated lymphocytes. In humans, defects in immune homeostasis result in lymphoproliferation disorders including autoimmunity, haemophagocytic lymphohystiocytosis and lymphomas. The X-linked lymphoproliferative syndrome (XLP) is a rare, inherited immunodeficiency that is characterized by lymphohystiocytosis, hypogammaglobulinaemia and lymphomas, and that usually develops in response to infection with Epstein-Barr virus (EBV). Mutations in the signalling lymphocyte activation molecule (SLAM)-associated protein SAP, a signalling adaptor molecule, underlie 60% of cases of familial XLP. Here, we identify mutations in the gene that encodes the X-linked inhibitor-of-apoptosis XIAP (also termed BIRC4) in patients with XLP from three families without mutations in SAP. These mutations lead to defective expression of XIAP. We show that apoptosis of lymphocytes from XIAP-deficient patients is enhanced in response to various stimuli including the T-cell antigen receptor (TCR)-CD3 complex, the death receptor CD95 (also termed Fas or Apo-1) and the TNF-associated apoptosis-inducing ligand receptor (TRAIL-R). We also found that XIAP-deficient patients, like SAP-deficient patients, have low numbers of natural killer T-lymphocytes (NKT cells), indicating that XIAP is required for the survival and/or differentiation of NKT cells. The observation that XIAP-deficiency and SAP-deficiency are both associated with a defect in NKT cells strengthens the hypothesis that NKT cells have a key role in the immune response to EBV. Furthermore, by identifying an XLP immunodeficiency that is caused by mutations in XIAP, we show that XIAP is a potent regulator of lymphocyte homeostasis in vivo.

Autoantigen, innate immunity, and T cells cooperate to break B cell tolerance during bacterial infection.

Autoantibody production during infections is considered to result from nonspecific activation of low-affinity autoreactive B cells. Whether this can lead to autoimmune disease remains uncertain. We show that chronic infection by Borrelia burgdorferi of Tg animals expressing human rheumatoid factor (RF) B cells (of low or intermediate affinities) in the absence or in the constitutive presence of the autoantigen (represented here by chimeric IgG with human constant region) breaks their state of immunological ignorance, leading to the production of RFs. Surprisingly, this production was more pronounced in intermediate-affinity RF Tg mice co-expressing the autoantigen. This overproduction was mediated by immune complexes and involved synergistic signaling between the B cell receptor and Toll-like receptors and T cell help. These findings indicate that chronic infection can activate autoreactive B cells with significant affinity and creates conditions that can drive them to differentiate into memory cells. Such cells may have some physiological yet undetermined role, but in autoimmune-prone individuals, this scenario may initiate autoimmunity.

B cell positive selection by soluble self-antigen.

It is well established that autoreactive B cells undergo negative selection. This stands in paradox with the high frequency of so-called natural autoreactive B cells producing low affinity polyreactive autoantibodies with recurrent specificities, suggesting that these B cells are selected on the basis of their autoreactivity. We previously described two transgenic mouse lines (with and without IgD) producing a human natural autoantibody (nAAb) that binds ssDNA and human Fcgamma. In the absence of human IgG, nAAb-transgenic B cells develop normally. By crossing these mice with animals expressing knockin chimeric IgG with the human Fcgamma, we now show that the constitutive expression of chimeric IgG promotes the increase of nAAb-expressing B cells. This positive selection is critically dependent on the presence of IgD, occurs in the spleen, and concerns all mature B cell subsets, with a relative preferential enrichment of marginal zone B cells. These data support the view that soluble self-Ags can result in positive clonal selection.

A role for membrane IgD in the tolerance of pathological human rheumatoid factor B cells.

Under non-autoimmune conditions, rheumatoid factor (RF) B cells coexist peacefully with their antigen (IgG), or can be transiently activated during secondary immune responses because they can present xenoantigens to specific T cells captured in immune complex form. Such a situation should lead to affinity maturation of RF B cells and potentially dangerous production of high-affinity RF. We used two lines of transgenic mice expressing a somatically mutated pathological human RF in presence (IgM and IgD) or in absence (IgM only) of surface IgD, and confirm that RF B cell tolerance can result from an antigen-induced specific, but incomplete, deletion of naive RF B cells after antigen encounter. This deletion mainly concerns immature, transitional B cells. On the contrary, mature, IgM- and IgD-expressing RF B cells are resistant to such a deletion. These IgM and IgD RF B cells are functional and activable through both B cell receptor dependent (anti-IgM) and independent (LPS) pathways, but they are not fully responsive to human IgG either in vivo or in vitro. Taken together, these results suggest that another mechanism could be involved in the silencing of mature naive IgM and IgD RF B cells.