A candidate antibody drug for prevention of malaria.

Over 75% of malaria-attributable deaths occur in children under the age of 5 years. However, the first malaria vaccine recommended by the World Health Organization (WHO) for pediatric use, RTS,S/AS01 (Mosquirix), has modest efficacy. Complementary strategies, including monoclonal antibodies, will be important in efforts to eradicate malaria. Here we characterize the circulating B cell repertoires of 45 RTS,S/AS01 vaccinees and discover monoclonal antibodies for development as potential therapeutics. We generated >28,000 antibody sequences and tested 481 antibodies for binding activity and 125 antibodies for antimalaria activity in vivo. Through these analyses we identified correlations suggesting that sequences in Plasmodium falciparum circumsporozoite protein, the target antigen in RTS,S/AS01, may induce immunodominant antibody responses that limit more protective, but subdominant, responses. Using binding studies, mouse malaria models, biomanufacturing assessments and protein stability assays, we selected AB-000224 and AB-007088 for advancement as a clinical lead and backup. We engineered the variable domains (Fv) of both antibodies to enable low-cost manufacturing at scale for distribution to pediatric populations, in alignment with WHO's preferred product guidelines. The engineered clone with the optimal manufacturing and drug property profile, MAM01, was advanced into clinical development.

Mobilization of innate and adaptive antitumor immune responses by the RNP-targeting antibody ATRC-101.

Immunotherapy approaches focusing on T cells have provided breakthroughs in treating solid tumors. However, there remains an opportunity to drive anticancer immune responses via other cell types, particularly myeloid cells. ATRC-101 was identified via a target-agnostic process evaluating antibodies produced by the plasmablast population of B cells in a patient with non-small cell lung cancer experiencing an antitumor immune response during treatment with checkpoint inhibitor therapy. Here, we describe the target, antitumor activity in preclinical models, and data supporting a mechanism of action of ATRC-101. Immunohistochemistry studies demonstrated tumor-selective binding of ATRC-101 to multiple nonautologous tumor tissues. In biochemical analyses, ATRC-101 appears to target an extracellular, tumor-specific ribonucleoprotein (RNP) complex. In syngeneic murine models, ATRC-101 demonstrated robust antitumor activity and evidence of immune memory following rechallenge of cured mice with fresh tumor cells. ATRC-101 increased the relative abundance of conventional dendritic cell (cDC) type 1 cells in the blood within 24 h of dosing, increased CD8+ T cells and natural killer cells in blood and tumor over time, decreased cDC type 2 cells in the blood, and decreased monocytic myeloid-derived suppressor cells in the tumor. Cellular stress, including that induced by chemotherapy, increased the amount of ATRC-101 target in tumor cells, and ATRC-101 combined with doxorubicin enhanced efficacy compared with either agent alone. Taken together, these data demonstrate that ATRC-101 drives tumor destruction in preclinical models by targeting a tumor-specific RNP complex leading to activation of innate and adaptive immune responses.

Salmonella Typhi Vi capsule prime-boost vaccination induces convergent and functional antibody responses.

Vaccine development to prevent Salmonella Typhi infections has accelerated over the past decade, resulting in licensure of new vaccines, which use the Vi polysaccharide (Vi PS) of the bacterium conjugated to an unrelated carrier protein as the active component. Antibodies elicited by these vaccines are important for mediating protection against typhoid fever. However, the characteristics of protective and functional Vi antibodies are unknown. In this study, we investigated the human antibody repertoire, avidity maturation, epitope specificity, and function after immunization with a single dose of Vi-tetanus toxoid conjugate vaccine (Vi-TT) and after a booster with plain Vi PS (Vi-PS). The Vi-TT prime induced an IgG1-dominant response, whereas the Vi-TT prime followed by the Vi-PS boost induced IgG1 and IgG2 antibody production. B cells from recipients who received both prime and boost showed evidence of convergence, with shared V gene usage and CDR3 characteristics. The detected Vi antibodies showed heterogeneous avidity ranging from 10 µM to 500 pM, with no evidence of affinity maturation after the boost. Vi-specific antibodies mediated Fc effector functions, which correlated with antibody dissociation kinetics but not with association kinetics. We identified antibodies induced by prime and boost vaccines that recognized subdominant epitopes, indicated by binding to the de­O-acetylated Vi backbone. These antibodies also mediated Fc-dependent functions, such as complement deposition and monocyte phagocytosis. Defining strategies on how to broaden epitope targeting for S. Typhi Vi and enriching for antibody Fc functions that protect against typhoid fever will advance the design of high-efficacy Vi vaccines for protection across diverse populations.

Lipid Rafts Interaction of the ARID3A Transcription Factor with EZRIN and G-Actin Regulates B-Cell Receptor Signaling.

Several diseases originate via dysregulation of the actin cytoskeleton. The ARID3A/Bright transcription factor has also been implicated in malignancies, primarily those derived from hematopoietic lineages. Previously, we demonstrated that ARID3A shuttles between the nucleus and the plasma membrane, where it localizes within lipid rafts. There it interacts with components of the B-cell receptor (BCR) to reduce its ability to transmit downstream signaling. We demonstrate here that a direct component of ARID3A-regulated BCR signal strength is cortical actin. ARID3A interacts with actin exclusively within lipid rafts via the actin-binding protein EZRIN, which confines unstimulated BCRs within lipid rafts. BCR ligation discharges the ARID3A-EZRIN complex from lipid rafts, allowing the BCR to initiate downstream signaling events. The ARID3A-EZRIN interaction occurs almost exclusively within unpolymerized G-actin, where EZRIN interacts with the multifunctional ARID3A REKLES domain. These observations provide a mechanism by which a transcription factor directly regulates BCR signaling via linkage to the actin cytoskeleton with consequences for B-cell-related neoplasia.

Distinct clonal evolution of B-cells in HIV controllers with neutralizing antibody breadth.

A minor subset of individuals infected with HIV-1 develop antibody neutralization breadth during the natural course of the infection, often linked to chronic, high-level viremia. Despite significant efforts, vaccination strategies have been unable to induce similar neutralization breadth and the mechanisms underlying neutralizing antibody induction remain largely elusive. Broadly neutralizing antibody responses can also be found in individuals who control HIV to low and even undetectable plasma levels in the absence of antiretroviral therapy, suggesting that high antigen exposure is not a strict requirement for neutralization breadth. We therefore performed an analysis of paired heavy and light chain B-cell receptor (BCR) repertoires in 12,591 HIV-1 envelope-specific single memory B-cells to determine alterations in the BCR immunoglobulin gene repertoire and B-cell clonal expansions that associate with neutralizing antibody breadth in 22 HIV controllers. We found that the frequency of genomic mutations in IGHV and IGLV was directly correlated with serum neutralization breadth. The repertoire of the most mutated antibodies was dominated by a small number of large clones with evolutionary signatures suggesting that these clones had reached peak affinity maturation. These data demonstrate that even in the setting of low plasma HIV antigenemia, similar to what a vaccine can potentially achieve, BCR selection for extended somatic hypermutation and clonal evolution can occur in some individuals suggesting that host-specific factors might be involved that could be targeted with future vaccine strategies.

Convergent antibody evolution and clonotype expansion following influenza virus vaccination.

Recent advances in high-throughput single cell sequencing have opened up new avenues into the investigation of B cell receptor (BCR) repertoires. In this study, PBMCs were collected from 17 human participants vaccinated with the split-inactivated influenza virus vaccine during the 2016-2017 influenza season. A combination of Immune Repertoire Capture (IRCTM) technology and IgG sequencing was performed on ~7,800 plasmablast (PB) cells and preferential IgG heavy-light chain pairings were investigated. In some participants, a single expanded clonotype accounted for ~22% of their PB BCR repertoire. Approximately 60% (10/17) of participants experienced convergent evolution, possessing public PBs that were elicited independently in multiple participants. Binding profiles of one private and three public PBs confirmed they were all subtype-specific, cross-reactive hemagglutinin (HA) head-directed antibodies. Collectively, this high-resolution antibody repertoire analysis demonstrated the impact evolution can have on BCRs in response to influenza virus vaccination, which can guide future universal influenza prophylactic approaches.

Potent antibody lineage against malaria transmission elicited by human vaccination with Pfs25.

Transmission-blocking vaccines have the potential to be key contributors to malaria elimination. Such vaccines elicit antibodies that inhibit parasites during their development in Anopheles mosquitoes, thus breaking the cycle of transmission. To date, characterization of humoral responses to Plasmodium falciparum transmission-blocking vaccine candidate Pfs25 has largely been conducted in pre-clinical models. Here, we present molecular analyses of human antibody responses generated in a clinical trial evaluating Pfs25 vaccination. From a collection of monoclonal antibodies with transmission-blocking activity, we identify the most potent transmission-blocking antibody yet described against Pfs25; 2544. The interactions of 2544 and three other antibodies with Pfs25 are analyzed by crystallography to understand structural requirements for elicitation of human transmission-blocking responses. Our analyses provide insights into Pfs25 immunogenicity and epitope potency, and detail an affinity maturation pathway for a potent transmission-blocking antibody in humans. Our findings can be employed to guide the design of improved malaria transmission-blocking vaccines.

Pneumatic compression device does not show effective thromboprophylaxis following total knee arthroplasty in a low incidence population.

INTRODUCTION: Purpose of this study was to assess whether the intermittent pneumatic compression (IPC) device would be an effective prophylaxis for deep vein thrombosis (DVT) following total knee arthroplasty (TKA) in a low incidence population. HYPOTHESIS: The mechanical thromboprophylaxis could reduce the incidence of DVT compared to non-prophylaxis group and would have similar efficacy as the chemoprophylaxis following TKA in a low DVT incidence population. MATERIALS AND METHODS: From January 2009 to June 2016, 1259 elective primary TKA with preoperative diagnosis of primary osteoarthritis in a single institute were enrolled. Patients were divided into three groups: those who were managed with chemoprophylaxis (CPX group, 414 cases), with mechanical prophylaxis (IPC group, 425 cases), or without pharmacological and mechanical prophylaxis (control group, 420 cases). All patients underwent preoperative ultrasonography and computed tomographic venography on postoperative day 6 to assess development of DVT. The incidence of overall, proximal, symptomatic DVT and symptomatic pulmonary embolism (PE) were compared among the groups. Major and minor bleeding complications were also evaluated. RESULTS: The incidence of overall DVT was 14.8% in control group, 6.3% in CPX group and 11.3% in IPC group respectively and CPX group showed significantly lower incidence than other two groups (p<0.001). The incidence of proximal DVT was 1.9% in control group, 0.7% in CPX group and 0.9% in IPC group respectively (p>0.05). The incidence of symptomatic DVT was 0.7% in control group, 0% in CPX group and 0.7% in IPC group respectively (p>0.05). There was no case of symptomatic PE diagnosed during hospital stay in all patients. DISCUSSION: Single use of IPC device could not reach significant level of DVT prophylaxis compared to control group and only chemoprophylaxis showed significantly reduce the incidence of overall DVT following TKA. Single use of IPC device does not show effective thromboprophylaxis in a low DVT incidence population. LEVEL OF EVIDENCE: III, case control study.

Identification of Near-Pan-neutralizing Antibodies against HIV-1 by Deconvolution of Plasma Humoral Responses.

Anti-HIV-1 envelope broadly neutralizing monoclonal antibodies (bNAbs) isolated from memory B cells may not fully represent HIV-1-neutralizing profiles measured in plasma. Accordingly, we characterized near-pan-neutralizing antibodies extracted directly from the plasma of two "elite neutralizers." Circulating anti-gp120 polyclonal antibodies were deconvoluted using proteomics to guide lineage analysis of bone marrow plasma cells. In both subjects, a single lineage of anti-CD4-binding site (CD4bs) antibodies explained the plasma-neutralizing activity. Importantly, members of these lineages potently neutralized 89%-100% of a multi-tier 117 pseudovirus panel, closely matching the specificity and breadth of the circulating antibodies. X-ray crystallographic analysis of one monoclonal, N49P7, suggested a unique ability to bypass the CD4bs Phe43 cavity, while reaching deep into highly conserved residues of Layer 3 of the gp120 inner domain, likely explaining its extreme potency and breadth. Further direct analyses of plasma anti-HIV-1 bNAbs should provide new insights for developing antibody-based antiviral agents and vaccines.

Non-progressing cancer patients have persistent B cell responses expressing shared antibody paratopes that target public tumor antigens.

There is significant debate regarding whether B cells and their antibodies contribute to effective anti-cancer immune responses. Here we show that patients with metastatic but non-progressing melanoma, lung adenocarcinoma, or renal cell carcinoma exhibited increased levels of blood plasmablasts. We used a cell-barcoding technology to sequence their plasmablast antibody repertoires, revealing clonal families of affinity matured B cells that exhibit progressive class switching and persistence over time. Anti-CTLA4 and other treatments were associated with further increases in somatic hypermutation and clonal family size. Recombinant antibodies from clonal families bound non-autologous tumor tissue and cell lines, and families possessing immunoglobulin paratope sequence motifs shared across patients exhibited increased rates of binding. We identified antibodies that caused regression of, and durable immunity toward, heterologous syngeneic tumors in mice. Our findings demonstrate convergent functional anti-tumor antibody responses targeting public tumor antigens, and provide an approach to identify antibodies with diagnostic or therapeutic utility.

Competitive Promoter-Associated Matrix Attachment Region Binding of the Arid3a and Cux1 Transcription Factors.

Arid3a/Bright/Dril1 is a B cell-specific transactivator that regulates immunoglobulin heavy chain (IgH) gene transcription by binding promoter and enhancer-associated matrix attachment regions (MARs) within the IgH gene locus. Promoter MAR-mediated Arid3a transactivation is antagonized by direct competition of MAR binding by Cux1/CDP-a ubiquitously expressed repressor originally termed NF-µNR. We report that the NF-µNR complex includes Arid3a in B cells but not in non-B cells through mobility shift assays. The binding activity of NF-µNR and Arid3a in B cells is reciprocally altered during the cell division cycle and by the B cell mitogen lipopolysaccharide LPS. LPS treatment had no effect on Arid3a localization but increased its total abundance within the nucleus and cytoplasm. We show that this increased level of Arid3a is capable of displacing Cux from the MARs to facilitate IgH gene transcription. Finally, we showed that the MARs (termed Bf150 and Tx125) associated with the VH1 rearranged variable region expressed in the S107 murine plasmacytoma, can repress reporter gene transcription in non-B cells and that they can relieve the repression mediated by Eµ enhancer in B cells. These results have significant implications for early human development and demonstrate that MARs in IgH locus, NF-µNR and Arid3a regulate IgH gene expression in a concerted fashion. This paves the way for future studies examining the misregulation of this pathway in pediatric disease.

Medial Open-Wedge High Tibial Osteotomy May Adversely Affect the Patellofemoral Joint.

PURPOSE: To evaluate 2-stage arthroscopic findings of the patellofemoral joint before and after medial open-wedge high tibial osteotomy (HTO) and verify whether the patellofemoral joint would be influenced by medial open-wedge HTO. METHODS: We prospectively reviewed 114 cases of medial open-wedge HTO for the treatment of osteoarthritis with a varus knee. First-look arthroscopy was performed during HTO. The mean age at the time of HTO was 56.34 ± 5.4 years (range, 40-69 years). Second-look arthroscopy was performed concomitantly with plate removal at an average of 26.1 ± 6.0 months (range, 21.6-32.0 months) after HTO. We assessed the patellofemoral joint using the International Cartilage Repair Society (ICRS) grading system by first- and second-look arthroscopy and compared it before and after HTO. Postoperative anterior knee pain was also evaluated. RESULTS: Compared with first-look findings, second-look arthroscopic ICRS grading was changed as follows: In terms of the patella, 89 cases (78.1%) were not progressed whereas 25 cases (21.9%) were progressed. In terms of the femoral trochlea, 67 cases (58.8%) were not progressed whereas 47 cases (41.2%) were progressed. There was significant progression of the ICRS grade of the patella (P = .001) and femoral trochlea (P < .001) compared with first-look arthroscopic findings. The incidence of postoperative anterior knee pain was 11.4% (13 cases), and it was related to the ICRS grade of the patellofemoral joint at the time of second-look arthroscopy (P < .001 for patella and P < .001 for trochlea). CONCLUSIONS: This study showed that the patellofemoral joint might be adversely affected by medial open-wedge HTO. Although the incidence of postoperative anterior knee pain was low, it was clinically correlated with patellofemoral arthritis. LEVEL OF EVIDENCE: Level IV, therapeutic case series.

Genetic requirement for Mycl and efficacy of RNA Pol I inhibition in mouse models of small cell lung cancer.

Small cell lung cancer (SCLC) is a devastating neuroendocrine carcinoma. MYCL (L-Myc) is frequently amplified in human SCLC, but its roles in SCLC progression are poorly understood. We isolated preneoplastic neuroendocrine cells from a mouse model of SCLC and found that ectopic expression of L-Myc, c-Myc, or N-Myc conferred tumor-forming capacity. We focused on L-Myc, which promoted pre-rRNA synthesis and transcriptional programs associated with ribosomal biogenesis. Deletion of Mycl in two genetically engineered models of SCLC resulted in strong suppression of SCLC. The high degree of suppression suggested that L-Myc may constitute a therapeutic target for a broad subset of SCLC. We then used an RNA polymerase I inhibitor to target rRNA synthesis in an autochthonous Rb/p53-deleted mouse SCLC model and found significant tumor inhibition. These data reveal that activation of RNA polymerase I by L-Myc and other MYC family proteins provides an axis of vulnerability for this recalcitrant cancer.

Are Korean Patients Different from Other Ethnic Groups in Total Knee Arthroplasty?

Most of the implants used for total knee arthroplasty (TKA) in Asian patients have been produced based on anthropometry of Western people. Since anatomic features and life styles are different between Western and Eastern people, there would be ethnic differences in terms of conformity of implants to the patient's anatomy or clinical results after TKA. Therefore, surgeons in Asia are particularly interested in related surgical techniques and implant designs used in TKA for improved clinical results and patient satisfaction. In this review, we investigated the anthropometric differences of Koreans from Westerners. Koreans are of shorter stature, less weight, and smaller skeletal structure and have a higher incidence of constitutional varus alignment of the lower extremity. Moreover, compared to Westerner TKA populations, the proportion of female patients was large and primary osteoarthritis was prevalent in preoperative diagnosis in Korean TKA patients. Culturally, Koreans have life styles that demand high flexion positions of the knee such as squatting, kneeling, and cross-legged sitting. Although there were no notable differences in the complication and revision rates following TKA between Westerners and Koreans, the incidence of postoperative deep vein thrombosis and pulmonary thromboembolism was lower in Koreans than Westerners. We hope that further research on implant designs and more interest in TKA will improve outcomes in Korean patients.

Is Single-Radius Design Better for Quadriceps Recovery in Total Knee Arthroplasty?

PURPOSE: Although single-radius (SR) designs have a theoretical advantage in quadriceps recovery following total knee arthroplasty (TKA), there has been a paucity of objective evaluation studies. MATERIALS AND METHODS: One hundred and twenty minimally invasive TKAs were prospectively randomized by a single surgeon into 2 groups: SR design TKA group and multi-radius design TKA group. Quadriceps force and power were assessed using a dynamometer, and clinical data were investigated preoperatively and 6 weeks, 3 months, 6 months and 1 year postoperatively. RESULTS: There were no differences between two groups in quadriceps recovery and clinical results throughout the follow-up period. Furthermore, the proportion of patients whose postoperative quadriceps force and power reached preoperative level was similar in both groups. CONCLUSIONS: Femoral component design itself would not significantly influence quadriceps recovery after TKA.

The prognostic landscape of genes and infiltrating immune cells across human cancers.

Molecular profiles of tumors and tumor-associated cells hold great promise as biomarkers of clinical outcomes. However, existing data sets are fragmented and difficult to analyze systematically. Here we present a pan-cancer resource and meta-analysis of expression signatures from ∼18,000 human tumors with overall survival outcomes across 39 malignancies. By using this resource, we identified a forkhead box MI (FOXM1) regulatory network as a major predictor of adverse outcomes, and we found that expression of favorably prognostic genes, including KLRB1 (encoding CD161), largely reflect tumor-associated leukocytes. By applying CIBERSORT, a computational approach for inferring leukocyte representation in bulk tumor transcriptomes, we identified complex associations between 22 distinct leukocyte subsets and cancer survival. For example, tumor-associated neutrophil and plasma cell signatures emerged as significant but opposite predictors of survival for diverse solid tumors, including breast and lung adenocarcinomas. This resource and associated analytical tools (http://precog.stanford.edu) may help delineate prognostic genes and leukocyte subsets within and across cancers, shed light on the impact of tumor heterogeneity on cancer outcomes, and facilitate the discovery of biomarkers and therapeutic targets.

The CD47-signal regulatory protein alpha (SIRPa) interaction is a therapeutic target for human solid tumors.

CD47, a "don't eat me" signal for phagocytic cells, is expressed on the surface of all human solid tumor cells. Analysis of patient tumor and matched adjacent normal (nontumor) tissue revealed that CD47 is overexpressed on cancer cells. CD47 mRNA expression levels correlated with a decreased probability of survival for multiple types of cancer. CD47 is a ligand for SIRPα, a protein expressed on macrophages and dendritic cells. In vitro, blockade of CD47 signaling using targeted monoclonal antibodies enabled macrophage phagocytosis of tumor cells that were otherwise protected. Administration of anti-CD47 antibodies inhibited tumor growth in orthotopic immunodeficient mouse xenotransplantation models established with patient tumor cells and increased the survival of the mice over time. Anti-CD47 antibody therapy initiated on larger tumors inhibited tumor growth and prevented or treated metastasis, but initiation of the therapy on smaller tumors was potentially curative. The safety and efficacy of targeting CD47 was further tested and validated in immune competent hosts using an orthotopic mouse breast cancer model. These results suggest all human solid tumor cells require CD47 expression to suppress phagocytic innate immune surveillance and elimination. These data, taken together with similar findings with other human neoplasms, show that CD47 is a commonly expressed molecule on all cancers, its function to block phagocytosis is known, and blockade of its function leads to tumor cell phagocytosis and elimination. CD47 is therefore a validated target for cancer therapies.

Signalling of the BCR is regulated by a lipid rafts-localised transcription factor, Bright.

Regulation of BCR signalling strength is crucial for B-cell development and function. Bright is a B-cell-restricted factor that complexes with Bruton's tyrosine kinase (Btk) and its substrate, transcription initiation factor-I (TFII-I), to activate immunoglobulin heavy chain gene transcription in the nucleus. Here we show that a palmitoylated pool of Bright is diverted to lipid rafts of resting B cells where it associates with signalosome components. After BCR ligation, Bright transiently interacts with sumoylation enzymes, blocks calcium flux and phosphorylation of Btk and TFII-I and is then discharged from lipid rafts as a Sumo-I-modified form. The resulting lipid raft concentration of Bright contributes to the signalling threshold of B cells, as their sensitivity to BCR stimulation decreases as the levels of Bright increase. Bright regulates signalling independent of its role in IgH transcription, as shown by specific dominant-negative titration of rafts-specific forms. This study identifies a BCR tuning mechanism in lipid rafts that is regulated by differential post-translational modification of a transcription factor with implications for B-cell tolerance and autoimmunity.

REKLES is an ARID3-restricted multifunctional domain.

Bright/Dril1/ARID3a is a B cell-specific, matrix association (or attachment) region-binding transcriptional regulator of immunoglobulin heavy chain genes and of E2F1-dependent cell cycle progression. Bright contains a central DNA binding domain termed ARID (AT-rich interacting domain) and a C-terminal region termed REKLES (for a conserved amino acid motif). The ARID domain has been identified in seven highly conserved families of metazoan proteins (ARID1-5 and JARID1-2), whereas REKLES is found only in the ARID3 subfamily (composed of Bright/ARID3a, Bdp/ARID3b, and Bright-like/ARID3c). REKLES consists of two subdomains: a modestly conserved N-terminal REKLESalpha and a highly conserved (among ARID3 orthologous proteins) C-terminal REKLESbeta. Previously we showed that Bright undergoes nucleocytoplasmic shuttling and that REKLESalpha and -beta were required, respectively, for nuclear import and Crm1-dependent nuclear export. Here we show that Bright further requires REKLESbeta for self-association or paralogue association and for nuclear matrix targeting. REK-LES promotes and regulates the extent of Bright multimerization, which occurs in the absence or presence of target DNA and is necessary for specific DNA binding. REKLESbeta-mediated interaction of Bright with Bdp, which localizes strictly to the nucleus, traps Bright within the nucleus via neutralization of its nuclear export activity. These results identify REKLES as a multifunctional domain that has co-evolved with and regulates functional properties of the ARID3 DNA binding domain.

A regulated nucleocytoplasmic shuttle contributes to Bright's function as a transcriptional activator of immunoglobulin genes.

Bright/ARID3a has been implicated in mitogen- and growth factor-induced up-regulation of immunoglobulin heavy-chain (IgH) genes and in E2F1-dependent G1/S cell cycle progression. For IgH transactivation, Bright binds to nuclear matrix association regions upstream of certain variable region promoters and flanking the IgH intronic enhancer. While Bright protein was previously shown to reside within the nuclear matrix, we show here that a significant amount of Bright resides in the cytoplasm of normal and transformed B cells. Leptomycin B, chromosome region maintenance 1 (CRM1) overexpression, and heterokaryon experiments indicate that Bright actively shuttles between the nucleus and the cytoplasm in a CRM1-dependent manner. We mapped the functional nuclear localization signal to the N-terminal region of REKLES, a domain conserved within ARID3 paralogues. Residues within the C terminus of REKLES contain its nuclear export signal, whose regulation is primarily responsible for Bright shuttling. Growth factor depletion and cell synchronization experiments indicated that Bright shuttling during S phase of the cell cycle leads to an increase in its nuclear abundance. Finally, we show that shuttle-incompetent Bright point mutants, even if sequestered within the nucleus, are incapable of transactivating an IgH reporter gene. Therefore, regulation of Bright's cellular localization appears to be required for its function.