Activation-Induced Cytidine Deaminase Impacts the Primary Antibody Repertoire in Naive Mice.

Genetic and environmental cues shape the evolution of the B cell Ig repertoire. Activation-induced cytidine deaminase (AID) is essential to generating Ig diversity through isotype class switching and somatic mutations, which then directly influence clonal selection. Impaired B cell development in AID-knockout mice has made it difficult to study Ig diversification in an aging repertoire. Therefore, in this report, we used a novel inducible AID-knockout mouse model and discovered that deleting AID in adult mice caused spontaneous germinal center formation. Deep sequencing of the IgH repertoire revealed that Ab diversification begins early in life and evolves over time. Our data suggest that activated B cells form germinal centers at steady state and facilitate continuous diversification of the B cell repertoire. In support, we identified shared B cell lineages that were class switched and showed age-dependent rates of mutation. Our data provide novel context to the genesis of the B cell repertoire that may benefit the understanding of autoimmunity and the strength of an immune response to infection.

Treg specialization and functions beyond immune suppression.

The actions of the immune system are finely tuned, involving complex communication and coordination between diverse immune and non-immune cells across the tissues of the body. A healthy immune system requires a precise balance between immunity and tolerance. Regulatory T cells (Tregs) have long been appreciated as one of the master regulators of this balance; their importance is underscored by the autoimmunity that develops in mice and humans when Tregs are missing or dysfunctional. In addition to the immunoregulatory roles of Tregs in suppressing autoimmunity and inflammation via control of adaptive and innate immune responses, several non-immune modulatory functions of Tregs have been identified in recent years. In this review, we have highlighted the growing literature on the action of Tregs in metabolism, stem cell maintenance, tissue repair, and angiogenesis. Alongside Tregs' immune suppressive role, these non-suppressive activities comprise a key function of Tregs in regulating health and disease. As Tregs receive increasing attention as therapeutic targets, understanding their non-canonical functions may become an important feature of Treg-directed interventions.

Tpl2 kinase regulates inflammation but not tumorigenesis in mice.

Tumor progression locus 2 (Tpl2, gene name MAP3K8), a mitogen-activated protein kinase, is widely expressed in immune and non-immune cells to integrate tumor necrosis factor (TNF), toll-like receptors (TLRs), and interleukin-1 (IL1) receptor signaling to regulate inflammatory response. Given its central role in inflammatory response, Tpl2 is an attractive small molecule drug target. However, the role of Tpl2 as an oncogene or tumor suppressor gene remains controversial, and its function outside immune cells is not understood. We therefore utilized a Tpl2 kinase dead (Tpl2-KD) mouse model in an 18-month aging study to further elucidate Tpl2 effects on lifespan and chronic disease. Histopathological studies revealed the incidence and severity of spontaneous tumors and non-neoplastic lesions were comparable between wild type and Tpl2-KD mice. The only finding was that male Tpl2-KD mice had higher bodyweight and an increased incidence of liver steatosis, suggesting a sex-specific role for Tpl2 in hepatic lipid metabolism. In conclusion, loss of Tpl2 kinase activity did not lead to increased tumorigenesis over aging in mice but affected likely alterations in lipid metabolism in male animals.

Paired Immunoglobulin-like Type 2 Receptor Alpha G78R variant alters ligand binding and confers protection to Alzheimer's disease.

Paired Immunoglobulin-like Type 2 Receptor Alpha (PILRA) is a cell surface inhibitory receptor that recognizes specific O-glycosylated proteins and is expressed on various innate immune cell types including microglia. We show here that a common missense variant (G78R, rs1859788) of PILRA is the likely causal allele for the confirmed Alzheimer's disease risk locus at 7q21 (rs1476679). The G78R variant alters the interaction of residues essential for sialic acid engagement, resulting in >50% reduced binding for several PILRA ligands including a novel ligand, complement component 4A, and herpes simplex virus 1 (HSV-1) glycoprotein B. PILRA is an entry receptor for HSV-1 via glycoprotein B, and macrophages derived from R78 homozygous donors showed significantly decreased levels of HSV-1 infection at several multiplicities of infection compared to homozygous G78 macrophages. We propose that PILRA G78R protects individuals from Alzheimer's disease risk via reduced inhibitory signaling in microglia and reduced microglial infection during HSV-1 recurrence.

AID stabilizes stem-cell phenotype by removing epigenetic memory of pluripotency genes.

The activation-induced cytidine deaminase (AID; also known as AICDA) enzyme is required for somatic hypermutation and class switch recombination at the immunoglobulin locus. In germinal-centre B cells, AID is highly expressed, and has an inherent mutator activity that helps generate antibody diversity. However, AID may also regulate gene expression epigenetically by directly deaminating 5-methylcytosine in concert with base-excision repair to exchange cytosine. This pathway promotes gene demethylation, thereby removing epigenetic memory. For example, AID promotes active demethylation of the genome in primordial germ cells. However, different studies have suggested either a requirement or a lack of function for AID in promoting pluripotency in somatic nuclei after fusion with embryonic stem cells. Here we tested directly whether AID regulates epigenetic memory by comparing the relative ability of cells lacking AID to reprogram from a differentiated murine cell type to an induced pluripotent stem cell. We show that Aid-null cells are transiently hyper-responsive to the reprogramming process. Although they initiate expression of pluripotency genes, they fail to stabilize in the pluripotent state. The genome of Aid-null cells remains hypermethylated in reprogramming cells, and hypermethylated genes associated with pluripotency fail to be stably upregulated, including many MYC target genes. Recent studies identified a late step of reprogramming associated with methylation status, and implicated a secondary set of pluripotency network components. AID regulates this late step, removing epigenetic memory to stabilize the pluripotent state.

TPL2 kinase action and control of inflammation.

Tumor progression locus 2 (TPL2, also known as COT or MAP3K8) is a mitogen-activated protein kinase kinase (MAP3K) activated downstream of TNFαR, IL1R, TLR, CD40, IL17R, and some GPCRs. TPL2 regulates the MEK1/2 and ERK1/2 pathways to regulate a cascade of inflammatory responses. In parallel to this, TPL2 also activates p38α and p38δ to drive the production of various inflammatory mediators in neutrophils. We discuss the implications of this finding in the context of various inflammatory diseases.

IgH class switching exploits a general property of two DNA breaks to be joined in cis over long chromosomal distances.

Antibody class switch recombination (CSR) in B lymphocytes joins two DNA double-strand breaks (DSBs) lying 100-200 kb apart within switch (S) regions in the immunoglobulin heavy-chain locus (IgH). CSR-activated B lymphocytes generate multiple S-region DSBs in the donor Sµ and in a downstream acceptor S region, with a DSB in Sµ being joined to a DSB in the acceptor S region at sufficient frequency to drive CSR in a large fraction of activated B cells. Such frequent joining of widely separated CSR DSBs could be promoted by IgH-specific or B-cell-specific processes or by general aspects of chromosome architecture and DSB repair. Previously, we found that B cells with two yeast I-SceI endonuclease targets in place of Sγ1 undergo I-SceI-dependent class switching from IgM to IgG1 at 5-10% of normal levels. Now, we report that B cells in which Sγ1 is replaced with a 28 I-SceI target array, designed to increase I-SceI DSB frequency, undergo I-SceI-dependent class switching at almost normal levels. High-throughput genome-wide translocation sequencing revealed that I-SceI-generated DSBs introduced in cis at Sµ and Sγ1 sites are joined together in T cells at levels similar to those of B cells. Such high joining levels also occurred between I-SceI-generated DSBs within c-myc and I-SceI- or CRISPR/Cas9-generated DSBs 100 kb downstream within Pvt1 in B cells or fibroblasts, respectively. We suggest that CSR exploits a general propensity of intrachromosomal DSBs separated by several hundred kilobases to be frequently joined together and discuss the relevance of this finding for recurrent interstitial deletions in cancer.

PILRα negatively regulates mouse inflammatory arthritis.

Paired Ig-like type 2 receptor (PILR)α inhibitory receptor and its counterpart PILRß activating receptor are coexpressed on myeloid cells. In this article, we report that PILRα, but not PILRß, is elevated in human rheumatoid arthritis synovial tissue and correlates with inflammatory cell infiltration. Pilrα(-/-) mice produce more pathogenic cytokines during inflammation and are prone to enhanced autoimmune arthritis. Correspondingly, engaging PILRα with anti-PILRα mAb ameliorates inflammation in mouse arthritis models and suppresses the production of proinflammatory cytokines. Our studies suggest that PILRα mediates an important inhibitory pathway that can dampen inflammatory responses.

Polyclonal hyper-IgE mouse model reveals mechanistic insights into antibody class switch recombination.

Preceding antibody constant regions are switch (S) regions varying in length and repeat density that are targets of activation-induced cytidine deaminase. We asked how participating S regions influence each other to orchestrate rearrangements at the IgH locus by engineering mice in which the weakest S region, Sε, is replaced with prominent recombination hotspot Sµ. These mice produce copious polyclonal IgE upon challenge, providing a platform to study IgE biology and therapeutic interventions. The insertion enhances ε germ-line transcript levels, shows a preference for direct vs. sequential switching, and reduces intraswitch recombination events at native Sµ. These results suggest that the sufficiency of Sµ to mediate IgH rearrangements may be influenced by context-dependent cues.

Mechanisms promoting translocations in editing and switching peripheral B cells.

Variable, diversity and joining gene segment (V(D)J) recombination assembles immunoglobulin heavy or light chain (IgH or IgL) variable region exons in developing bone marrow B cells, whereas class switch recombination (CSR) exchanges IgH constant region exons in peripheral B cells. Both processes use directed DNA double-strand breaks (DSBs) repaired by non-homologous end-joining (NHEJ). Errors in either V(D)J recombination or CSR can initiate chromosomal translocations, including oncogenic IgH locus (Igh) to c-myc (also known as Myc) translocations of peripheral B cell lymphomas. Collaboration between these processes has also been proposed to initiate translocations. However, the occurrence of V(D)J recombination in peripheral B cells is controversial. Here we show that activated NHEJ-deficient splenic B cells accumulate V(D)J-recombination-associated breaks at the lambda IgL locus (Igl), as well as CSR-associated Igh breaks, often in the same cell. Moreover, Igl and Igh breaks are frequently joined to form translocations, a phenomenon associated with specific Igh-Igl co-localization. Igh and c-myc also co-localize in these cells; correspondingly, the introduction of frequent c-myc DSBs robustly promotes Igh-c-myc translocations. Our studies show peripheral B cells that attempt secondary V(D)J recombination, and determine a role for mechanistic factors in promoting recurrent translocations in tumours.

Design, synthesis and structure-activity relationships of a novel class of sulfonylpyridine inhibitors of Interleukin-2 inducible T-cell kinase (ITK).

Starting from benzylpyrimidine 2, molecular modeling and X-ray crystallography were used to design highly potent inhibitors of Interleukin-2 inducible T-cell kinase (ITK). Sulfonylpyridine 4i showed sub-nanomolar affinity against ITK, was selective versus Lck and its activity in the Jurkat cell-based assay was greatly improved over 2.

Evolutionarily conserved paired immunoglobulin-like receptor α (PILRα) domain mediates its interaction with diverse sialylated ligands.

Paired immunoglobulin-like receptor (PILR) α is an inhibitory receptor that recognizes several ligands, including mouse CD99, PILR-associating neural protein, and Herpes simplex virus-1 glycoprotein B. The physiological function(s) of interactions between PILRα and its cellular ligands are not well understood, as are the molecular determinants of PILRα/ligand interactions. To address these uncertainties, we sought to identify additional PILRα ligands and further define the molecular basis for PILRα/ligand interactions. Here, we identify two novel PILRα binding partners, neuronal differentiation and proliferation factor-1 (NPDC1), and collectin-12 (COLEC12). We find that sialylated O-glycans on these novel PILRα ligands, and on known PILRα ligands, are compulsory for PILRα binding. Sialylation-dependent ligand recognition is also a property of SIGLEC1, a member of the sialic acid-binding Ig-like lectins. SIGLEC1 Ig domain shares ∼22% sequence identity with PILRα, an identity that includes a conserved arginine localized to position 97 in mouse and human SIGLEC1, position 133 in mouse PILRα and position 126 in human PILRα. We observe that PILRα/ligand interactions require conserved PILRα Arg-133 (mouse) and Arg-126 (human), in correspondence with a previously reported requirement for SIGLEC1 Arg-197 in SIGLEC1/ligand interactions. Homology modeling identifies striking similarities between PILRα and SIGLEC1 ligand binding pockets as well as at least one set of distinctive interactions in the galactoxyl-binding site. Binding studies suggest that PILRα recognizes a complex ligand domain involving both sialic acid and protein motif(s). Thus, PILRα is evolved to engage multiple ligands with common molecular determinants to modulate myeloid cell functions in anatomical settings where PILRα ligands are expressed.

IgH class switching and translocations use a robust non-classical end-joining pathway.

Immunoglobulin variable region exons are assembled in developing B cells by V(D)J recombination. Once mature, these cells undergo class-switch recombination (CSR) when activated by antigen. CSR changes the heavy chain constant region exons (Ch) expressed with a given variable region exon from Cmu to a downstream Ch (for example, Cgamma, Cepsilon or Calpha), thereby switching expression from IgM to IgG, IgE or IgA. Both V(D)J recombination and CSR involve the introduction of DNA double-strand breaks and their repair by means of end joining. For CSR, double-strand breaks are introduced into switch regions that flank Cmu and a downstream Ch, followed by fusion of the broken switch regions. In mammalian cells, the 'classical' non-homologous end joining (C-NHEJ) pathway repairs both general DNA double-strand breaks and programmed double-strand breaks generated by V(D)J recombination. C-NHEJ, as observed during V(D)J recombination, joins ends that lack homology to form 'direct' joins, and also joins ends with several base-pair homologies to form microhomology joins. CSR joins also display direct and microhomology joins, and CSR has been suggested to use C-NHEJ. Xrcc4 and DNA ligase IV (Lig4), which cooperatively catalyse the ligation step of C-NHEJ, are the most specific C-NHEJ factors; they are absolutely required for V(D)J recombination and have no known functions other than C-NHEJ. Here we assess whether C-NHEJ is also critical for CSR by assaying CSR in Xrcc4- or Lig4-deficient mouse B cells. C-NHEJ indeed catalyses CSR joins, because C-NHEJ-deficient B cells had decreased CSR and substantial levels of IgH locus (immunoglobulin heavy chain, encoded by Igh) chromosomal breaks. However, an alternative end-joining pathway, which is markedly biased towards microhomology joins, supports CSR at unexpectedly robust levels in C-NHEJ-deficient B cells. In the absence of C-NHEJ, this alternative end-joining pathway also frequently joins Igh locus breaks to other chromosomes to generate translocations.

Increased targeting of donor switch region and IgE in Sgamma1-deficient B cells.

Ab class switch recombination involves a recombination between two repetitive DNA sequences known as switch (S) regions that vary in length, content, and density of the repeats. Abs expressed by B cells are diversified by somatic hypermutation and class switch recombination. Both class switch recombination and somatic hypermutation are initiated by activation-induced cytidine deaminase (AID), which preferentially recognizes certain hot spots that are far more enriched in the S regions. We found that removal of the largest S region, Sgamma1 (10 kb), in mice can result in the accumulation of mutations and short-range intra-S recombination in the donor Smu region. Furthermore, elevated levels of IgE were detected in trinitrophenol-OVA-immunized mice and in anti-CD40 plus IL-4-stimulated B cells in vitro. We propose that AID availability and targeting in part might be regulated by its DNA substrate. Thus, prominently transcribed S regions, such as Sgamma1, might provide a sufficient sink for AID protein to titrate away AID from other accessible sites within or outside the Ig locus.

Differential utilization of T cell receptor TCR alpha/TCR delta locus variable region gene segments is mediated by accessibility.

T cell receptor (TCR) variable region exons are assembled from germline V, (D), and J gene segments, each of which is flanked by recombination signal (RS) sequences that are composed of a conserved heptamer, a spacer of 12 or 23 bp, and a characteristic nonamer. V(D)J recombination only occurs between V, D, and J segments flanked by RS sequences that contain, respectively, 12(12-RS)- and 23(23-RS)-bp spacers (12/23 rule). Additional mechanisms can restrict joining of 12/23 RS matched segments beyond the 12/23 rule (B12/23). The TCRdelta locus is contained within the TCRalpha locus; TCRalpha variable region exons are encoded by TRAV and TRAJ segments and those of TCRdelta by TRDV, TRDD, and TRDJ segments. On the basis of the 12/23 rule, both TRAV and TRDV gene segments are compatible to rearrange with TRDD gene segments; however, TRAV-to-TRDD joins are not observed in vivo. Absence of TRAV-to-TRDD rearrangement might be explained either by B12/23 restriction or by differential accessibility of the TRDV versus TRAV gene segments for rearrangement to TRDD. We used in vitro substrate analysis to reveal that both TRAV and TRDV 23-RSs mediate rearrangements to the 5'TRDD1 12-RS, demonstrating that B12/23 restriction does not explain these rearrangement biases. However, targeted replacement of TRDD1 and its 12-RSs with TRAJ38 and its 12-RS showed that TRDV gene segments rearrange with the ectopic TRAJ38, whereas TRAV segments do not. Our results demonstrate that sorting of TRAV and TRDV gene segments is determined by differential locus accessibility during T cell development.

DNA targets of AID evolutionary link between antibody somatic hypermutation and class switch recombination.

As part of the adaptive immune response, B cells alter their functional immunoglobulin (Ig) receptor genes through somatic hypermutation (SHM) and/or class switch recombination (CSR) via processes that are initiated by activation induced cytidine deaminase (AID). These genetic modifications are targeted at specific sequences known as Variable (V) and Switch (S) regions. Here, we analyze and review the properties and function of AID target sequences across species and compare them with non-Ig sequences, including known translocation hotspots. We describe properties of the S sequences, and discuss species and isotypic differences among S regions. Common properties of SHM and CSR target sequences suggest that evolution of S regions might involve the duplication and selection of SHM hotspots.

Kinase inhibition in autoimmunity and inflammation.

Despite recent advances in the treatment of autoimmune and inflammatory diseases, unmet medical needs in some areas still exist. One of the main therapeutic approaches to alleviate dysregulated inflammation has been to target the activity of kinases that regulate production of inflammatory mediators. Small-molecule kinase inhibitors have the potential for broad efficacy, convenience and tissue penetrance, and thus often offer important advantages over biologics. However, designing kinase inhibitors with target selectivity and minimal off-target effects can be challenging. Nevertheless, immense progress has been made in advancing kinase inhibitors with desirable drug-like properties into the clinic, including inhibitors of JAKs, IRAK4, RIPKs, BTK, SYK and TPL2. This Review will address the latest discoveries around kinase inhibitors with an emphasis on clinically validated autoimmunity and inflammatory pathways.

Discovery of Potent Benzolactam IRAK4 Inhibitors with Robust in Vivo Activity.

IRAK4 kinase activity transduces signaling from multiple IL-1Rs and TLRs to regulate cytokines and chemokines implicated in inflammatory diseases. As such, there is high interest in identifying selective IRAK4 inhibitors for the treatment of these disorders. We previously reported the discovery of potent and selective dihydrobenzofuran inhibitors of IRAK4. Subsequent studies, however, showed inconsistent inhibition in disease-relevant pharmacodynamic models. Herein, we describe application of a human whole blood assay to the discovery of a series of benzolactam IRAK4 inhibitors. We identified potent molecule 19 that achieves robust in vivo inhibition of cytokines relevant to human disease.

The kinase IRAK4 promotes endosomal TLR and immune complex signaling in B cells and plasmacytoid dendritic cells.

The dysregulation of multiple signaling pathways, including those through endosomal Toll-like receptors (TLRs), Fc gamma receptors (FcγR), and antigen receptors in B cells (BCR), promote an autoinflammatory loop in systemic lupus erythematosus (SLE). Here, we used selective small-molecule inhibitors to assess the regulatory roles of interleukin-1 receptor (IL-1R)-associated kinase 4 (IRAK4) and Bruton's tyrosine kinase (BTK) in these pathways. The inhibition of IRAK4 repressed SLE immune complex- and TLR7-mediated activation of human plasmacytoid dendritic cells (pDCs). Correspondingly, the expression of interferon (IFN)-responsive genes (IRGs) in cells and in mice was positively regulated by the kinase activity of IRAK4. Both IRAK4 and BTK inhibition reduced the TLR7-mediated differentiation of human memory B cells into plasmablasts. TLR7-dependent inflammatory responses were differentially regulated by IRAK4 and BTK by cell type: In pDCs, IRAK4 positively regulated NF-κB and MAPK signaling, whereas in B cells, NF-κB and MAPK pathways were regulated by both BTK and IRAK4. In the pristane-induced lupus mouse model, inhibition of IRAK4 reduced the expression of IRGs during disease onset. Mice engineered to express kinase-deficient IRAK4 were protected from both chemical (pristane-induced) and genetic (NZB/W_F1 hybrid) models of lupus development. Our findings suggest that kinase inhibitors of IRAK4 might be a therapeutic in patients with SLE.

CSF1R Ligands IL-34 and CSF1 Are Differentially Required for Microglia Development and Maintenance in White and Gray Matter Brain Regions.

Microglia are specialized brain macrophages that play numerous roles in tissue homeostasis and response to injury. Colony stimulating factor 1 receptor (CSF1R) is a receptor tyrosine kinase required for the development, maintenance, and proliferation of microglia. Here we show that in adult mice peripheral dosing of function-blocking antibodies to the two known ligands of CSF1R, CSF1, and IL-34, can deplete microglia differentially in white and gray matter regions of the brain, respectively. The regional patterns of depletion correspond to the differential expression of CSF1 and IL-34. In addition, we show that while CSF1 is required to establish microglia in the developing embryo, both CSF1 and IL-34 are required beginning in early postnatal development. These results not only clarify the roles of CSF1 and IL-34 in microglia maintenance, but also suggest that signaling through these two ligands might support distinct sub-populations of microglia, an insight that may impact drug development for neurodegenerative and other diseases.