The Interaction between AID and CIB1 Is Nonessential for Antibody Gene Diversification by Gene Conversion or Class Switch Recombination

Activation-induced deaminase (AID) initiates somatic hypermutation, gene conversion and class switch recombination by deaminating variable and switch region DNA cytidines to uridines. AID is predominantly cytoplasmic and must enter the nuclear compartment to initiate these distinct antibody gene diversification reactions. Nuclear AID is relatively short-lived, as it is efficiently exported by a CRM1-dependent mechanism and it is susceptible to proteasome-dependent degradation. To help shed light on mechanisms of post-translational regulation, a yeast-based screen was performed to identify AID-interacting proteins. The calcium and integrin binding protein CIB1 was identified by sequencing and the interaction was confirmed by immunoprecipitation experiments. The AID/CIB1 resisted DNase and RNase treatment, and it is therefore unlikely to be mediated by nucleic acid. The requirement for CIB1 in AID-mediated antibody gene diversification reactions was assessed in CIB1-deficient DT40 cells and in knockout mice, but immunoglobulin gene conversion and class switch recombination appeared normal. The DT40 system was also used to show that CIB1 over-expression has no effect on gene conversion and that AID-EGFP subcellular localization is normal. These combined data demonstrate that CIB1 is not required for AID to mediate antibody gene diversification processes. It remains possible that CIB1 has an alternative, a redundant or a subtle non-limiting regulatory role in AID biology

HOIL1 regulates group 2 innate lymphoid cell numbers and type 2 inflammation in the small intestine

Patients with mutations in HOIL1 experience a complex immune disorder including intestinal inflammation. To investigate the role of HOIL1 in regulating intestinal inflammation, we employed a mouse model of partial HOIL1 deficiency. The ileum of HOIL1-deficient mice displayed features of type 2 inflammation including tuft cell and goblet cell hyperplasia, and elevated expression of Il13, Il5 and Il25 mRNA. Inflammation persisted in the absence of T and B cells, and bone marrow chimeric mice revealed a requirement for HOIL1 expression in radiation-resistant cells to regulate inflammation. Although disruption of IL-4 receptor alpha (IL4Rα) signaling on intestinal epithelial cells ameliorated tuft and goblet cell hyperplasia, expression of Il5 and Il13 mRNA remained elevated. KLRG

AID can restrict L1 retrotransposition suggesting a dual role in innate and adaptive immunity

Retrotransposons make up over 40% of the mammalian genome. Some copies are still capable of mobilizing and new insertions promote genetic variation. Several members of the APOBEC3 family of DNA cytosine deaminases function to limit the replication of a variety of retroelements, such as the long-terminal repeat (LTR)-containing MusD and Ty1 elements, and that of the non-LTR retrotransposons, L1 and Alu. However, the APOBEC3 genes are limited to mammalian lineages, whereas retrotransposons are far more widespread. This raises the question of what cellular factors control retroelement transposition in species that lack APOBEC3 genes. A strong phylogenetic case can be made that an ancestral activation-induced deaminase (AID)-like gene duplicated and diverged to root the APOBEC3 lineage in mammals. Therefore, we tested the hypothesis that present-day AID proteins possess anti-retroelement activity. We found that AID can inhibit the retrotransposition of L1 through a DNA deamination-independent mechanism. This mechanism may manifest in the cytoplasmic compartment co- or posttranslationally. Together with evidence for AID expression in the ovary, our data combined to suggest that AID has innate immune functions in addition to its integral roles in creating antibody diversity

Immunodeficiency, auto-inflammation and amylopectinosis in humans with inherited HOIL-1 and LUBAC deficiency

We report the clinical description and molecular dissection of a new fatal human inherited disorder characterized by chronic auto-inflammation, invasive bacterial infections and muscular amylopectinosis. Patients from two kindreds carried biallelic loss-of-expression and loss-of-function mutations in HOIL1, a component the linear ubiquitination chain assembly complex (LUBAC). These mutations resulted in impairment of LUBAC stability. NF-κB activation in response to interleukin-1β (IL-1β) was compromised in the patients’ fibroblasts. By contrast, the patients’ mononuclear leukocytes, particularly monocytes, were hyperresponsive to IL-1β. The consequences of human HOIL-1 and LUBAC deficiencies for IL-1β responses thus differed between cell types, consistent with the unique association of auto-inflammation and immunodeficiency in these patients. These data suggest that LUBAC regulates NF-κB-dependent IL-1β responses differently in different cell types

CIB1 over-expression in DT40.

<p><b>A</b>. An IGC fluctuation analysis showing the percentage of surface Ig-positive cells in subclone cultures over-expressing human (h) or chicken (c) CIB1. Each X represents data from an individual subclone and the labeled horizontal bars report the medians for each data set. <b>B</b>. CIB1 over-expression confirmed by immunoblotting. Loading was controlled by stripping and re-probing the blot with an α-tubulin antibody.</p

AID localization in CIB^−/− DT40 cells.

<p><b>A</b>. AID-EGFP localization in CIB1^+/+ DT40. <b>B</b>. AID-EGFP localization in CIB1^−/− DT40. Images were taken using a 40× objective and the scale bars indicate 10 µm.</p

CIB1 is dispensable for CSR in mice.

<p><b>A</b>. Relative levels of each antibody isotype in sera from CIB1^+/+ or CIB1^−/− mice as measured by ELISA. The CIB1^−/− data were normalized to the mean antibody levels in sera from wildtype (WT) littermates (arbitrarily set to 1 for comparison). Each X represents data from an independent animal and the horizontal bars and labels report the median values (n = 3 for CIB^+/+ and n = 6 for CIB^−/−). <b>B</b>. IgM to IgG1 CSR <i>ex vivo</i>. B-cells were isolated from the spleens of CIB1^+/+ or CIB1^−/− mice, cultured for 4 days in the presence of LPS and IL-4, and analyzed by α-IgG1-PE labeling and flow cytometry. Each X represents data from an independent animal and the horizontal bars and labels report the median values. <b>C</b>. Images of hematoxylin and eosin stained sections of spleen isolated from CIB1^+/+ and CIB1^−/− mice. Scale bars indicate 500 µm.</p

AID interacts with the calcium and integrin binding protein 1.

<p><b>A</b>. myc-CIB1 co-IPs AID-EGFP but not EGFP only (left and right lanes, respectively). A myc-specific monoclonal antibody was used to precipitate complexes, and AID-GFP was detected with an α-GFP polyclonal antibody. <b>B</b>. AID-EGFP co-IPs myc-CIB1 in a DNase I- and RNase A-resistant manner. An α-GFP monoclonal antibody was used to precipitate AID-GFP, and myc-CIB1 was detected with an α-myc monoclonal antibody. <b>C</b>. AID-STZ pulls down endogenous CIB1 from HEK-293T cell extracts. IgG Sepharose was used to pull-down STZ complexes, and CIB1 was detected using an α-CIB1 polyclonal antibody. AID-STZ and GFP-STZ were detected with an α-strep antibody. A low level of non-specific background was observed in the vicinity of AID-STZ. For cell lysate (input) control blots, two panels are shown because GFP-STZ is expressed over 100-fold better than AID-STZ. A quantification of the input versus pull-down signal indicated that 1% of cellular CIB1 can be pulled-down with AID complexes when IgG sepharose beads are limiting.</p