Immunoglobulins (Igs) are the soluble mediators of antigenic recognition. An Ig that is capable of specifically binding an antigen is termed an antibody. Antibodies are of critical importance in immuno-oncology and a cornerstone of cancer therapeutics. As such, in this chapter, we describe the central aspects of antibody structure, explain the genetic basis of antibody diversity, and summarize mechanisms underlying antibody function. We proceed to a practical review of therapeutic antibodies and their applications.

PDF file - 7618K, S1. Analysis of the genetic interaction between 53BP1 and ATM in organismal growth and development. S2. Analysis of ploidy in Trp53bp1-/-/Atm-/- lymphomas. S3. Analysis of Trp53bp1-/-/Atm-/- thymic lymphomas by TCRα/δ locus FISH. S4. SKY analysis of two Trp53bp1-/-/Atm-/- lymphomas. S5. Cell cycle analysis of resting lymphocytes after IR. S6. Aberrant Vd2-Dd1/Dd2-Jd1 coding junctions are not detected in Trp53bp1-/-/Atm-/- mice. S7. Aberrant Vd5-Dd2 Recombination Signal Junctions are not detected in Trp53bp1-/-/Atm-/- mice. S8. Histograms showing the frequency distribution of deletions from each coding end (A-D) and signal end (E-F) analyzed. S9. Cell cycle analysis of activated (cycling) lymphocytes after IR. S10. Analysis of genomic stability in splenic B or T cells treated with olaparib. S11. Analysis of the G2/M checkpoint. α-CD40/IL-4-activated B cells were harvested one hour after exposure to 2 Gy of IR, stained with a FITC-labeled antibody to phospho(P)-histone H3 (Ser10) and propidium iodide (PI) and analyzed by flow cytometry. S12. A second primer set detects deletions at hybrid V(D)J recombination junctions in Trp53bp1-/-/Atm-/- thymic DNA from 7 day-old mice. Table S1. Mendelian ratios in liveborn mice from 53BP1+/-/ATM+/- intercrosses (n=19 litters) Table S2. Analysis of genomic stability in HU-treated B lymphocytes deficient for 53BP1 and/or ATM. Table S3. Analysis of genomic stability in α-CD40+IL-4activated B lymphocytes deficient for 53BP1 and/or ATM. Table S4. Analysis of genomic stability in α-CD40/IL-4 activated B cells deficient for 53BP1 and/or ATM. Metaphase spreads were obtained 24 hr after IR and stained with a telomere probe. Table S5. Analysis of genomic stability in LPS-activated B cells deficient for 53BP1 and/or ATM. Metaphases were obtained 24 hr after exposure to IR and stained with a telomere probe. Table S6. Analysis of genomic stability in T lymphocytes deficient for 53BP1 and/or ATM.

BackgroundSome clinical features of severe COVID-19 represent blood vessel damage induced by activation of host immune responses initiated by the coronavirus SARS-CoV-2. We hypothesized autoantibodies against angiotensin-converting enzyme 2 (ACE2), the SARS-CoV-2 receptor expressed on vascular endothelium, are generated during COVID-19 and are of mechanistic importance.MethodsIn an opportunity sample of 118 COVID-19 inpatients, autoantibodies recognizing ACE2 were detected by ELISA. Binding properties of anti-ACE2 IgM were analyzed via biolayer interferometry. Effects of anti-ACE2 IgM on complement activation and endothelial function were demonstrated in a tissue-engineered pulmonary microvessel model.ResultsAnti-ACE2 IgM (not IgG) autoantibodies were associated with severe COVID-19 and found in 18/66 (27.2%) patients with severe disease compared with 2/52 (3.8%) of patients with moderate disease (OR 9.38, 95% CI 2.38-42.0; P = 0.0009). Anti-ACE2 IgM autoantibodies were rare (2/50) in non-COVID-19 ventilated patients with acute respiratory distress syndrome. Unexpectedly, ACE2-reactive IgM autoantibodies in COVID-19 did not undergo class-switching to IgG and had apparent KD values of 5.6-21.7 nM, indicating they are T cell independent. Anti-ACE2 IgMs activated complement and initiated complement-binding and functional changes in endothelial cells in microvessels, suggesting they contribute to the angiocentric pathology of COVID-19.ConclusionWe identify anti-ACE2 IgM as a mechanism-based biomarker strongly associated with severe clinical outcomes in SARS-CoV-2 infection, which has therapeutic implications.FUNDINGBill & Melinda Gates Foundation, Gates Philanthropy Partners, Donald B. and Dorothy L. Stabler Foundation, and Jerome L. Greene Foundation; NIH R01 AR073208, R01 AR069569, Institutional Research and Academic Career Development Award (5K12GM123914-03), National Heart, Lung, and Blood Institute R21HL145216, and Division of Intramural Research, National Institute of Allergy and Infectious Diseases; National Science Foundation Graduate Research Fellowship (DGE1746891).

B cells have been implicated as central regulators of immune responses in settings as diverse as mammalian pregnancy, mucosal tolerance, chronic infection states, autoimmunity, and the tumor microenvironment. Despite the established importance of B cells in these environments, the mechanisms by which B cells are maintained in these contexts remain undefined. Here, we report that IDO1 pathway inhibition with D-1-methyl-tryptophan (D-1MT) and linrodostat significantly decreases tumor infiltrating B (TIL-B) cells in a preclinical model of melanoma. Single cell RNA sequencing (scRNAseq) of murine melanoma demonstrate TIL-B cells are heterogeneous but primarily express markers consistent with an immune stimulatory phenotype. D-1MT decreases splenic B cells and bone marrow derived B cell precursors in tumor-bearing mice, suggesting that IDO1 pathway inhibition impedes B cell maturation. D-1MT decreases intratumoral myeloid derived suppressor cells (MDSCs), which are essential for maintenance of TIL-B cells. Unlike D-1MT, genetic deletion of tumor Ido1 does not impact TIL-B or MDSC numbers. In human solid tumors, intratumoral IDO1 expression consistently associates with high expression of a pan-B cell gene signature, and in patients with melanoma, scRNAseq analysis of tumor samples revealed most TIL-B cells express IDO1. Collectively, our data reveal the impact of pharmacologic IDO1 inhibition on B cells, which may have therapeutic implications for patients with solid tumors by informing the design of future oncology clinical trials.

Abstract Immunotherapy has revolutionized cancer treatment by improving survival in many cancer subtypes. While tumor infiltrating B (TIL-B) cells correlate with response to immunotherapy in selected solid tumors, they portend resistance to BRAF inhibitors in BRAF mutant melanoma. Thus, the mechanisms underlying TIL-B cell function within the tumor microenvironment have remained elusive. Here, we discovered that inhibition of the indoleamine 2,3 dioxygenase-1 (IDO1) pathway with D-1-methyl-tryptophan (D-1MT) markedly decreases TIL-B cells in a preclinical model of melanoma. Single cell RNA sequencing (scRNAseq) of murine melanoma demonstrate TIL-B cells are heterogeneous: while some B cells express markers consistent with an immune stimulatory phenotype, others express markers consistent with immune inhibitory function. D-1MT decreased splenic B cells and bone marrow derived B cell precursors in tumor bearing mice, suggesting that IDO1 pathway inhibition impedes B cell maturation. In four distinct human cancer subtypes, intratumoral IDO1 expression consistently correlates with high expression of a pan-B cell gene signature. Further, analysis of scRNAseq data from a cohort of patients with melanoma revealed that most TIL-B cells express IDO1. In mice, D-1MT also decreases intratumoral myeloid derived suppressor cells (MDSCs), which are essential for maintenance of TIL-B cells. Surprisingly, we found that D-1MT enhanced anti-tumor effects of the BRAF inhibitor, vemurafenib, in a preclinical model of BRAF mutant melanoma, suggesting that targeting B cells in this setting may be beneficial. Together, our data reveal a novel paradigm for the IDO1 pathway in regulating TIL-B cells, and uncovered IDO1 as a potential targetable mechanism of resistance to BRAF inhibition in melanoma. Citation Format: Burles Avner Johnson, Adam K. Aragaki, Donna M. Williams, Ophelia Rogers, Li Luo, Lingling Xian, Lionel Chia, Noah M. Hahn, Stephen Desiderio, Theodore S. Johnson, David J. McConkey, Linda M.S. Resar. The indoleamine 2,3 dioxygenase-1 pathway drives intratumoral B cell maintenance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2765.

V(D)J recombination is initiated by the recombination-activating gene protein (RAG) recombinase, consisting of RAG-1 and RAG-2 subunits. The susceptibility of gene segments to cleavage by RAG is associated with gene transcription and with epigenetic marks characteristic of active chromatin, including histone H3 trimethylated at lysine 4 (H3K4me3). Binding of H3K4me3 by a plant homeodomain (PHD) in RAG-2 induces conformational changes in RAG-1, allosterically stimulating substrate binding and catalysis. To better understand the path of allostery from the RAG-2 PHD finger to RAG-1, here we employed phylogenetic substitution. We observed that a chimeric RAG-2 protein in which the mouse PHD finger is replaced by the corresponding domain from the shark Chiloscyllium punctatum binds H3K4me3 but fails to transmit an allosteric signal, indicating that binding of H3K4me3 by RAG-2 is insufficient to support recombination. By substituting residues in the C. punctatum PHD with the corresponding residues in the mouse PHD and testing for rescue of allostery, we demonstrate that H3K4me3 binding and transmission of an allosteric signal to RAG-1 are separable functions of the RAG-2 PHD finger.

T and B cells are the two known lineages of adaptive immune cells. Here, we describe a previously unknown lymphocyte that is a dual expresser (DE) of TCR and BCR and key lineage markers of both B and T cells. In type 1 diabetes (T1D), DEs are predominated by one clonotype that encodes a potent CD4 T cell autoantigen in its antigen binding site. Molecular dynamics simulations revealed that this peptide has an optimal binding register for diabetogenic HLA-DQ8. In concordance, a synthetic version of the peptide forms stable DQ8 complexes and potently stimulates autoreactive CD4 T cells from T1D patients, but not healthy controls. Moreover, mAbs bearing this clonotype are autoreactive against CD4 T cells and inhibit insulin tetramer binding to CD4 T cells. Thus, compartmentalization of adaptive immune cells into T and B cells is not absolute, and violators of this paradigm are likely key drivers of autoimmune diseases.

Background. Rheumatoid arthritis (RA) is a systemic autoimmune disease associated with immune dysregulation and increased risk of infections. The presence of autoantibodies and immunoglobulin abnormalities indicates B-cell and antibody-secreting cell (ASC) dysfunction. We hypothesize that soluble factors associated with B-cell and ASC activity are decreased in RA patients and that this is linked to higher susceptibility to infections. Methods. Using the Johns Hopkins Arthritis Cohort and Biorepository, we contrasted serum protein levels of soluble factors involved in B-cell activation (CD40, CD40L) and B-cell/ASC homing (CXCL10, CXCL11, and CXCL13) or survival (BAFF, APRIL, TACI, and BCMA) in 10 healthy subjects and 23 adult RA patients (aged 24-65 years). We subdivided RA patients into those with (n=17) and those without infections (n=6) within a 2-year period. In order to reduce the effect of RA treatment, we only included patients receiving methotrexate monotherapy or no RA treatments at baseline. Soluble serum protein levels of B-cell/ASC factors were quantified by multiplex immunoassays. Results. We identified that (1) serum levels of soluble BCMA, APRIL, CD40, and CD40L were significantly decreased in RA patients relative to healthy individuals; (2) serum soluble BCMA, predominantly released by ASC, correlated with serum concentrations of class-switched immunoglobulins, IgG and IgA; and (3) RA patients with a history of infections had significantly lower soluble BCMA levels compared with healthy donors and with RA patients without infections. Conclusions. Our study using soluble factors linked to B-cell/ASC activation and survival suggests that there is a paucity of ASC in a subset of RA patients and that this may be linked to altered antibody production and increased risk of infections. Further delineating the link between ASC and infection susceptibility in RA may optimize disease management and provide novel insights into disease pathogenesis that are susceptible to intervention.

V(D)J recombination, the process by which antigen receptor genes are assembled from discrete DNA segments during lymphoid development, is responsible for the generation of the primary immune repertoire. Errors in V(D)J recombination have been implicated in the pathogenesis of lymphoid malignancies, including follicular lymphoma, MALT lymphoma and mantle cell lymphoma. V(D)J recombination is initiated by a specialized transposase, RAG, consisting of RAG-1 and RAG-2 subunits. RAG mobilizes participating gene segments in a site-specific fashion by cleaving DNA at conserved recombination signal sequences. The accessibility of these sequences to RAG is subject to locus- and developmental stage-specific control by mechanisms that are as yet poorly understood. Elucidation of these mechanisms is fundamental to our understanding of the off-target events linking RAG activity to tumorigenesis. The susceptibility of gene segments to cleavage by RAG is associated with histone modifications characteristic of active chromatin, including trimethylation of histone H3 at lysine 4 (H3K4me3). RAG-2 contains a plant homeodomain (PHD) finger that binds specifically to H3K4me3. Disruption of this PHD finger impairs V(D)J recombination in vivo. Peptides bearing H3K4me3 stimulate substrate binding and catalysis of DNA cleavage by RAG. This stimulation is dependent on an intact PHD finger, suggesting that H3K4me3 is an allosteric activator of the V(D)J recombinase. Indeed, binding of H3K4me3 to the RAG-2 PHD induces dynamic conformational changes in RAG-1. Because substrate binding and catalysis are functions of RAG-1, information regarding occupancy of the RAG-2 PHD must be transmitted to the RAG-1 subunit. To understand how the recognition of active chromatin is coupled to the binding and cleavage of recombination signal sequences, we sought to trace the path of allostery from the RAG-2 PHD finger to RAG-1. Our strategy has been: (1) to generate chimeric RAG-2 proteins in which the mouse PHD finger is replaced by the PHD finger of a phylogenetically distant RAG-2; (2) to identify chimeric RAG-2 proteins that are capable of binding H3K4me3 but incapable of allosteric activation; (3) to systematically back-mutate residues in the foreign PHD to the mouse sequence; and (4) to identify back-mutations that rescue allosteric activation. A chimeric RAG-2 protein in which the mouse PHD finger is replaced by the corresponding domain from the bamboo shark, C. punctatum, fails to support V(D)J recombination in vivo. This chimeric protein retains the ability to bind H3K4me3 but engagement of H3K4me3 does not result in allosteric activation, suggesting that the allosteric interface of the PHD finger is disrupted. The amino acid sequence differences between mouse and C. punctatum form several clusters, located on the opposite side of the PHD from the H3K4me3 binding site. Each of these clusters in the C. punctatum PHD finger was mutated to the mouse sequence and the corresponding back-mutated chimeric RAG-2 proteins were tested for their ability to support V(D)J recombination. Strikingly, mutation of one such cluster, corresponding to residues 425 - 429, 431 and 433 of mouse RAG-2, was sufficient to rescue recombination activity to the level of wild-type. Taken together, our observations indicate that the binding of H3K4me3 by RAG-2 is itself insufficient to support recombination; rather, information regarding the engagement of H3K4me3 must be transmitted allosterically. Moreover, our mutational analysis has identified a putative allosteric surface within the PHD finger and distinct from the H3K4me3 binding site that is responsible for transmitting the allosteric signal. The requirement for allosteric activation by H3K4me3 may play a role in defining patterns of RAG-mediated DNA cleavage during normal development and in the generation of lymphoid malignancies.

The stromal signals that promote B lymphopoiesis remain poorly understood. Hedgehog (Hh) signaling promotes B lymphopoiesis in a non-cell-autonomous fashion in vitro, and depletion of the Hh effector Smoothened (Smo) from stromal cells is associated with the loss of osteoblastoid markers. These observations suggested that Hh signaling in the osteoblastoid lineage promotes B lymphopoiesis in vivo. To test this, we employed a mouse model for conditional ablation of Smo in the osteoblastoid lineage. Depletion of Smo from osteoblastoid cells is associated with profound and selective reductions in the number and proportion of bone marrow B-lymphoid progenitors. Upon partial bone marrow ablation, mutant animals exhibit delayed repopulation of the B-lymphoid compartment after the early lymphoid progenitor stage. Primary osteoblasts from mutant mice are defective in supporting B lymphopoiesis in vitro, whereas hematopoietic progenitors from mutant mice exhibit normal differentiation. We conclude that efficient B lymphopoiesis in vivo is dependent on the maintenance of Hh signaling in the osteoblastoid lineage.

Accessibility of antigen receptor loci to RAG is correlated with the presence of H3K4me3, which binds to a plant homeodomain (PHD) in the RAG-2 subunit and promotes V(D)J recombination. A point mutation in the PHD, W453A, eliminates binding of H3K4me3 and impairs recombination. The debilitating effect of the W453A mutation is ameliorated by second-site mutations that locate an inhibitory domain in the interval from residues 352 through 405 of RAG-2. Disruption of the inhibitory domain stimulates V(D)J recombination within extrachromosomal substrates and at endogenous antigen receptor loci. Association of RAG-1 and RAG-2 with chromatin at the IgH locus in B cell progenitors is dependent on recognition of H3K4me3 by the PHD. Strikingly, disruption of the inhibitory domain permits association of RAG with the IgH locus in the absence of H3K4me3 binding. Thus, the inhibitory domain acts as a gate that prohibits RAG from accessing the IgH locus unless RAG-2 is engaged by H3K4me3.

The mechanisms that drive T cell aging are not understood. We report that children and adult telomerase mutation carriers with short telomere length (TL) develop a T cell immunodeficiency that can manifest in the absence of bone marrow failure and causes life-threatening opportunistic infections. Mutation carriers shared T cell-aging phenotypes seen in adults 5 decades older, including depleted naive T cells, increased apoptosis, and restricted T cell repertoire. T cell receptor excision circles (TRECs) were also undetectable or low, suggesting that newborn screening may identify individuals with germline telomere maintenance defects. Telomerase-null mice with short TL showed defects throughout T cell development, including increased apoptosis of stimulated thymocytes, their intrathymic precursors, in addition to depleted hematopoietic reserves. When we examined the transcriptional programs of T cells from telomerase mutation carriers, we found they diverged from older adults with normal TL. Short telomere T cells upregulated DNA damage and intrinsic apoptosis pathways, while older adult T cells upregulated extrinsic apoptosis pathways and programmed cell death 1 (PD-1) expression. T cells from mice with short TL also showed an active DNA-damage response, in contrast with old WT mice, despite their shared propensity to apoptosis. Our data suggest there are TL-dependent and TL-independent mechanisms that differentially contribute to distinct molecular programs of T cell apoptosis with aging.

V(D) J recombination is initiated by the recombination-activating gene (RAG) recombinase, consisting of RAG-1 and RAG-2 subunits. The susceptibility of gene segments to cleavage by RAG is associated with histone modifications characteristic of active chromatin, including trimethylation of histone H3 at lysine 4 (H3K4me3). Binding of H3K4me3 by a plant homeodomain (PHD) in RAG-2 stimulates substrate binding and catalysis, which are functions of RAG-1. This has suggested an allosteric mechanism in which information regarding occupancy of the RAG-2 PHD is transmitted to RAG-1. To determinewhether the conformational distribution of RAG is altered by H3K4me3, we mapped changes in solvent accessibility of cysteine thiols by differential isotopic chemical footprinting. Binding of H3K4me3 to the RAG-2 PHD induces conformational changes in RAG-1 within a DNA-binding domain and in the ZnH2 domain, which acts as a scaffold for the catalytic center. Thus, engagement of H3K4me3 by the RAG-2 PHD is associated with dynamic conformational changes in RAG-1, consistent with allosteric control by active chromatin.

V(D)J recombination, the process by which antigen receptor genes are assembled, is implicated in the pathogenesis of lymphoid malignancies. Rearrangement is initiated by the RAG recombinase, a heterotetrameric complex consisting of two subunits each of RAG-1 and RAG-2, which cleaves participating DNA segments at conserved signal sequences. The susceptibility of gene segments to undergo recombination is positively correlated with active chromatin marks, such as tri-methylation of histone H3 at lysine 4 (H3K4me3). RAG-2 interacts directly with H3K4me3 through a plant homeodomain (PHD), and this interaction is required for efficient V(D)J recombination in vivo. Genetic and biochemical evidence suggest that H3K4me3 stimulates substrate binding and DNA cleavage by RAG through an allosteric mechanism. This predicted that H3K4me3 binding induces conformational changes in RAG-2 that are transmitted to RAG-1. To determine whether the conformation of the RAG complex is altered by H3K4me3, we mapped changes in solvent accessibility of cysteine thiols using pulse-alkylation mass spectrometry. Binding of H3K4me3 to the RAG-2 PHD induces conformational changes in RAG-1 within the DNA-binding domains and in the ZnH2 domain, which comprises part of the scaffold for the catalytic center. Our results demonstrate that engagement of the RAG-2 PHD induces dynamic conformational changes in the RAG-1 catalytic subunit, consistent with a model of allosteric control of RAG by active chromatin.

The assembly of immunoglobulin genes occurs in ordered waves during B cell development. The heavy chain ( Igh ) locus generally recombines first, and each allele has at most one chance to undergo a productive rearrangement. Subsequently, at the κ ( Igk ) locus, individual alleles can undergo sequential rounds of rearrangement, permitting different light-heavy chain combinations to be tested until a functional, nonself-reactive immunoglobulin is produced. The two waves of recombination are separated by a checkpoint governed by the pre–B cell receptor (pre–BCR), which enforces allelic exclusion at the Igh locus, triggers proliferation, and promotes Igk rearrangement. This raises the question: how do we mitigate the genomic damage that might occur if DNA cleavage and cell cycle entry were initiated simultaneously? In this issue, Bednarski et al. suggest a solution: an unexpected mechanism by which RAG-induced DNA double-strand breaks (DSBs) suppress pre–BCR signaling. RAG-induced DSBs trigger a signalling pathway that culminates in the suppression of pre–BCR signals by the transcriptional repressor SPIC.

V(D)J recombination is initiated by a specialized transposase consisting of the subunits RAG-1 and RAG-2. The susceptibility of gene segments to DNA cleavage by the V(D)J recombinase is correlated with epigenetic modifications characteristic of active chromatin, including trimethylation of histone H3 on lysine 4 (H3K4me3). Engagement of H3K4me3 by a plant homeodomain (PHD) in RAG-2 promotes recombination in vivo and stimulates DNA cleavage by RAG in vitro. We now show that H3K4me3 acts allosterically at the PHD finger to relieve autoinhibition imposed by a separate domain within RAG-2. Disruption of this autoinhibitory domain was associated with constitutive increases in recombination frequency, DNA cleavage activity, substrate binding affinity, and catalytic rate, thus mimicking the stimulatory effects of H3K4me3. Our observations support a model in which allosteric control of RAG is enforced by an autoinhibitory domain whose action is relieved by engagement of active chromatin.

The landscape of human phosphorylation networks has not been systematically explored, representing vast, unchartered territories within cellular signaling networks. Although a large number of in vivo phosphorylated residues have been identified by mass spectrometry (MS)‐based approaches, assigning the upstream kinases to these residues requires biochemical analysis of kinase‐substrate relationships (KSRs). Here, we developed a new strategy, called CEASAR, based on functional protein microarrays and bioinformatics to experimentally identify substrates for 289 unique kinases, resulting in 3656 high‐quality KSRs. We then generated consensus phosphorylation motifs for each of the kinases and integrated this information, along with information about in vivo phosphorylation sites determined by MS, to construct a high‐resolution map of phosphorylation networks that connects 230 kinases to 2591 in vivo phosphorylation sites in 652 substrates. The value of this data set is demonstrated through the discovery of a new role for PKA downstream of Btk (Bruton's tyrosine kinase) during B‐cell receptor signaling. Overall, these studies provide global insights into kinase‐mediated signaling pathways and promise to advance our understanding of cellular signaling processes in humans.

Invariant natural killer T cells have a distinct developmental pathway from conventional αβ T cells. Here we demonstrate that the transcriptional repressor NKAP is required for invariant natural killer T cell but not conventional T cell development. In CD4-cre NKAP conditional knockout mice, invariant natural killer T cell development is blocked at the double-positive stage. This cell-intrinsic block is not due to decreased survival or failure to rearrange the invariant Vα14-Jα18 T cell receptor-α chain, but is rescued by overexpression of a rec-Vα14-Jα18 transgene at the double-positive stage, thus defining a role for NKAP in selection into the invariant natural killer T cell lineage. Importantly, deletion of the NKAP-associated protein histone deacetylase 3 causes a similar block in the invariant natural killer T cell development, indicating that NKAP and histone deacetylase 3 functionally interact to control invariant natural killer T cell development.

Abstract The DNA damage response (DDR) factors ataxia telangiectasia mutated (ATM) and p53 binding protein 1 (53BP1) function as tumor suppressors in humans and mice, but the significance of their mutual interaction to the suppression of oncogenic translocations in vivo has not been investigated. To address this question, the phenotypes of compound mutant mice lacking 53BP1 and ATM (Trp53bp1−/−/Atm−/−), relative to single mutants, were examined. These analyses revealed that loss of 53BP1 markedly decreased the latency of T-lineage lymphomas driven by RAG-dependent oncogenic translocations in Atm−/− mice (average survival, 14 and 23 weeks for Trp53bp1−/−/Atm−/− and Atm−/− mice, respectively). Mechanistically, 53BP1 deficiency aggravated the deleterious effect of ATM deficiency on nonhomologous end-joining (NHEJ)—mediated double-strand break repair. Analysis of V(D)J recombinase-mediated coding joints and signal joints in Trp53bp1−/−/Atm−/− primary thymocytes is, however, consistent with canonical NHEJ-mediated repair. Together, these findings indicate that the greater NHEJ defect in the double mutant mice resulted from decreased efficiency of rejoining rather than switching to an alternative NHEJ-mediated repair mechanism. Complementary analyses of irradiated primary cells indicated that defects in cell-cycle checkpoints subsequently function to amplify the NHEJ defect, resulting in more frequent chromosomal breaks and translocations in double mutant cells throughout the cell cycle. Finally, it was determined that 53BP1 is dispensable for the formation of RAG-mediated hybrid joints in Atm−/− thymocytes but is required to suppress large deletions in a subset of hybrid joints. Implications: The current study uncovers novel ATM-independent functions for 53BP1 in the suppression of oncogenic translocations and in radioprotection. Visual Overview: http://mcr.aacrjournals.org/content/11/10/1223/F1.large.jpg. Mol Cancer Res; 11(10); 1223–34. ©2013 AACR.

The transcription factor Stat3 is an activator of systemic inflammatory genes. Two isoforms of Stat3 are generated by alternative splicing, Stat3α and Stat3β. The β isoform lacks the transactivation domain but retains other functions, including dimerization and DNA binding. Stat3β-deficient mice exhibit elevated expression of systemic inflammatory genes and are hyperresponsive to lipopolysaccharide, suggesting that Stat3β functions predominantly as a suppressor of systemic inflammation. To test whether Stat3β deficiency would provoke pathologic effects associated with chronic inflammation, we asked whether selective removal of Stat3β would exacerbate the development of atherosclerosis in apolipoprotein E-deficient mice. In apoE−/−Stat3β−/− mice atherosclerotic plaque formation was significantly enhanced relative to apoE−/−Stat3β+/+ controls. The ability of Stat3β deficiency to promote atherosclerosis was more pronounced in female mice, but could be unmasked in males by feeding a high fat diet. Infiltrating macrophages were not increased in aortas of apoE−/−Stat3β−/− mice. In contrast, the proportion of pro-inflammatory TH17 cells was significantly elevated in aortic infiltrates from apoE−/−Stat3β−/− mice, relative to paired apoE−/−Stat3β+/+ littermates. These observations indicate that Stat3β can suppress pathologic sequelae associated with chronic inflammation. Our findings further suggest that in Stat3β-deficient mice the unopposed action of Stat3α may enhance atherogenesis in part by promoting differentiation of TH17 cells.