Anti-GD2 synergizes with CD47 blockade to mediate tumor eradication.

The disialoganglioside GD2 is overexpressed on several solid tumors, and monoclonal antibodies targeting GD2 have substantially improved outcomes for children with high-risk neuroblastoma. However, approximately 40% of patients with neuroblastoma still relapse, and anti-GD2 has not mediated significant clinical activity in any other GD2+ malignancy. Macrophages are important mediators of anti-tumor immunity, but tumors resist macrophage phagocytosis through expression of the checkpoint molecule CD47, a so-called 'Don't eat me' signal. In this study, we establish potent synergy for the combination of anti-GD2 and anti-CD47 in syngeneic and xenograft mouse models of neuroblastoma, where the combination eradicates tumors, as well as osteosarcoma and small-cell lung cancer, where the combination significantly reduces tumor burden and extends survival. This synergy is driven by two GD2-specific factors that reorient the balance of macrophage activity. Ligation of GD2 on tumor cells (a) causes upregulation of surface calreticulin, a pro-phagocytic 'Eat me' signal that primes cells for removal and (b) interrupts the interaction of GD2 with its newly identified ligand, the inhibitory immunoreceptor Siglec-7. This work credentials the combination of anti-GD2 and anti-CD47 for clinical translation and suggests that CD47 blockade will be most efficacious in combination with monoclonal antibodies that alter additional pro- and anti-phagocytic signals within the tumor microenvironment.

Evorpacept alone and in combination with pembrolizumab or trastuzumab in patients with advanced solid tumours (ASPEN-01): a first-in-human, open-label, multicentre, phase 1 dose-escalation and dose-expansion study.

BACKGROUND: Both innate and adaptive immune responses are important components of anticancer immunity. The CD47-SIRPα interaction could represent an important pathway used by tumour cells to evade immune surveillance. We aimed to evaluate the safety, pharmacokinetics, pharmacodynamics, and anticancer activity of evorpacept (also known as ALX148), a high-affinity CD47-blocking protein with an inactive IgG Fc region in patients with solid tumours. METHODS: We did a first-in-human, open-label, multicentre, phase 1 dose-escalation and dose-expansion study at nine hospitals and one clinic in the USA and Korea. Eligible patients for the dose-escalation and safety lead-in phases were aged 18 years or older with histological or cytological diagnosis of advanced or metastatic solid tumours with no available standard therapy, measurable or unmeasurable disease according to the Response Evaluation Criteria in Solid Tumors version 1.1, and an Eastern Cooperative Oncology Group performance status score of 0 or 1. In the dose-escalation phase, which used a 3 + 3 design, patients received intravenous evorpacept at either 0·3, 1, 3, or 10 mg/kg once per week in 21-day cycles, or 30 mg/kg once every other week in 28-day cycles. In the safety lead-in phase, patients were given the maximum tolerable dose of evorpacept from the dose-escalation phase plus either intravenous pembrolizumab (200 mg administered once every 3 weeks) or intravenous trastuzumab (8 mg/kg loading dose followed by 6 mg/kg once every 3 weeks). In the dose-expansion phase, additional patients aged 18 years or older with second-line or later-line advanced malignancies were enrolled into three parallel cohorts: those with head and neck squamous cell carcinoma (HNSCC) and those with non-small-cell lung cancer (NSCLC) were given the maximum tolerated dose of evorpacept plus intravenous pembrolizumab (200 mg administered once every 3 weeks), and patients with HER2-positive gastric or gastroesophageal junction cancer were given the maximum tolerated dose of evorpacept plus intravenous trastuzumab (8 mg/kg loading dose followed by 6 mg/kg once every 3 weeks) until disease progression, voluntary withdrawal from the study, or unacceptable toxicity. The primary endpoint was the maximum tolerated dose of evorpacept administered as a single agent and in combination with pembrolizumab or trastuzumab, measured by the occurrence of dose-limiting toxicities during the first cycle, and was assessed in all patients who had received at least one dose of evorpacept. Secondary outcomes included the safety, tolerability, and antitumour activity of evorpacept, alone or in combination with pembrolizumab or trastuzumab. The primary outcome, safety, and tolerability were assessed in all patients who had received at least one dose of evorpacept, and antitumour activity was assessed in those who recieved at least one dose of study treatment and underwent at least one post-baseline tumor assessment. This trial is registered with ClinicalTrials.gov, NCT03013218. FINDINGS: Between March 6, 2017, and Feb 21, 2019, 110 patients received single-agent evorpacept (n=28), evorpacept plus pembrolizumab (n=52), or evorpacept plus trastuzumab (n=30), and were included in the safety analysis. Median follow-up was 29·1 months (95% CI not calculable [NC]-NC) in the single-agent cohort, 27·0 months (25·1-28·8) in the evorpacept plus pembrolizumab cohort, and 32·7 months (27·0-32·7) in the evorpacept plus trastuzumab cohort. Two (7%) dose-limiting toxicities in the first cycle were reported in patients who received single-agent evorpacept; neutropenia with an associated infection in one patient with gastroesophageal junction cancer who received 3 mg/kg once per week, and thrombocytopenia with associated bleeding in one patient with pancreatic cancer who received 30 mg/kg once every other week. No maximum tolerated dose was reached; the maximum administered doses were 10 mg/kg once per week or 30 mg/kg once every other week. The 10 mg/kg once per week dose was used in the expansion cohorts in combination with pembrolizumab or trastuzumab. The most common grade 3 or worse treatment-related adverse events were thrombocytopenia with single-agent evorpacept (two [7%] patients) and evorpacept plus pembrolizumab (two [4%]), and thrombocytopenia (two [7%]) and neutropenia (two [7%]) with evorpacept plus trastuzumab. In patients who received single-agent evorpacept, four treatment-related serious adverse events were reported. Five serious treatment-related adverse events related to evorpacept plus pembrolizumab were reported, and one serious adverse event related to evorpacept plus trastuzumab was reported. In response-evaluable patients in the dose-escalation phase (n=15) receiving single-agent evorpacept once per week, four (27%) had a best overall response of stable disease (two received 0·3 mg/kg, one received 3 mg/kg, and one received 10 mg/kg); in the 11 patients who received single-agent evorpacept at the highest dose of 30 mg/kg once every other week, two (18%) had stable disease. In the dose-expansion cohort, overall responses were recorded in four (20·0%; 95% CI 5·7-43·7) of 20 patients with HNSCC who received evorpacept plus pembrolizumab, in one (5·0%; 0·1-24·9) of 20 patients with NSCLC who received evorpacept plus pembrolizumab, and in four (21·1%; 6·1-45·6) of 19 patients with gastric or gastroesophageal junction cancer who received evorpacept plus trastuzumab. INTERPRETATION: The safety findings support the use of evorpacept in combination with pembrolizumab or trastuzumab for patients with advanced solid tumours. Preliminary antitumour activity results support future investigation of evorpacept combined with pembrolizumab or trastuzumab in patients with HNSCC, gastric or gastroesophageal junction cancer, and NSCLC. FUNDING: ALX Oncology.

Targeting the myeloid checkpoint receptor SIRPα potentiates innate and adaptive immune responses to promote anti-tumor activity.

BACKGROUND: Signal regulatory protein α (SIRPα) is a myeloid-lineage inhibitory receptor that restricts innate immunity through engagement of its cell surface ligand CD47. Blockade of the CD47-SIRPα interaction synergizes with tumor-specific antibodies and T-cell checkpoint inhibitors by promoting myeloid-mediated antitumor functions leading to the induction of adaptive immunity. Inhibition of the CD47-SIRPα interaction has focused predominantly on targeting CD47, which is expressed ubiquitously and contributes to the accelerated blood clearance of anti-CD47 therapeutics. Targeting SIRPα, which is myeloid-restricted, may provide a differential pharmacokinetic, safety, and efficacy profile; however, SIRPα polymorphisms and lack of pan-allelic and species cross-reactive agents have limited the clinical translation of antibodies against SIRPα. Here, we report the development of humanized AB21 (hAB21), a pan-allelic anti-SIRPα antibody that binds human, cynomolgus monkey, and mouse SIRPα alleles with high affinity and blocks the interaction with CD47. METHODS: Human macrophages derived from donors with various SIRPα v1 and v2 allelic status were used to assess the ability of hAB21 to enhance phagocytosis. HAB21_IgG subclasses were evaluated for targeted depletion of peripheral blood mononuclear cells, phagocytosis and in vivo efficacy in xenograft models. Combination therapy with anti-PD1/anti-PD-L1 in several syngeneic models was performed. Immunophenotyping of tissues from MC38 tumor-bearing mice treated with AB21 and anti-PD-1 was evaluated. PK, PD and tolerability of hAB21 were evaluated in cynomolgus monkeys. RESULTS: SIRPα blockade with hAB21 promoted macrophage-mediated antibody-dependent phagocytosis of tumor cells in vitro and improved responses to rituximab in the Raji human tumor xenograft mouse model. Combined with PD-1/PD-L1 blockade, AB21 improved response rates by facilitating monocyte activation, dendritic cell activation, and T cell effector functions resulting in long term, durable antitumor immunity. In cynomolgus monkeys, hAB21 has a half-life of 5.3 days at 10 mg/kg and complete target occupancy with no hematological toxicity or adverse findings at doses up to 30 mg/kg. CONCLUSIONS: The in vitro and in vivo antitumor activity of hAB21 broadly recapitulates that of CD47 targeted therapies despite differences in ligand expression, binding partners, and function, validating the CD47-SIRPα axis as a fundamental myeloid checkpoint pathway and its blockade as promising therapeutic intervention for treatment of human malignancies.

Preclinical Efficacy and Safety Comparison of CD3 Bispecific and ADC Modalities Targeting BCMA for the Treatment of Multiple Myeloma.

The restricted expression pattern of B-cell maturation antigen (BCMA) makes it an ideal tumor-associated antigen (TAA) for the treatment of myeloma. BCMA has been targeted by both CD3 bispecific antibody and antibody-drug conjugate (ADC) modalities, but a true comparison of modalities has yet to be performed. Here we utilized a single BCMA antibody to develop and characterize both a CD3 bispecific and 2 ADC formats (cleavable and noncleavable) and compared activity both in vitro and in vivo with the aim of generating an optimal therapeutic. Antibody affinity, but not epitope was influential in drug activity and hence a high-affinity BCMA antibody was selected. Both the bispecific and ADCs were potent in vitro and in vivo, causing dose-dependent cell killing of myeloma cell lines and tumor regression in orthotopic myeloma xenograft models. Primary patient cells were effectively lysed by both CD3 bispecific and ADCs, with the bispecific demonstrating improved potency, maximal cell killing, and consistency across patients. Safety was evaluated in cynomolgus monkey toxicity studies and both modalities were active based on on-target elimination of B lineage cells. Distinct nonclinical toxicity profiles were seen for the bispecific and ADC modalities. When taken together, results from this comparison of BCMA CD3 bispecific and ADC modalities suggest better efficacy and an improved toxicity profile might be achieved with the bispecific modality. This led to the advancement of a bispecific candidate into phase I clinical trials.

Discovery of high affinity, pan-allelic, and pan-mammalian reactive antibodies against the myeloid checkpoint receptor SIRPα.

Targeting the CD47-signal-regulatory protein α (SIRPα) pathway represents a novel therapeutic approach to enhance anti-cancer immunity by promoting both innate and adaptive immune responses. Unlike CD47, which is expressed ubiquitously, SIRPα expression is mainly restricted to myeloid cells and neurons. Therefore, compared to CD47-targeted therapies, targeting SIRPα may result in differential safety and efficacy profiles, potentially enabling lower effective doses and improved pharmacokinetics and pharmacodynamics. The development of effective SIRPα antagonists is restricted by polymorphisms within the CD47-binding domain of SIRPα, necessitating pan-allele reactive anti-SIRPα antibodies for therapeutic intervention in diverse patient populations. We immunized wild-type and human antibody transgenic chickens with a multi-allele and multi-species SIRPα regimen in order to discover pan-allelic and pan-mammalian reactive anti-SIRPα antibodies suitable for clinical translation. A total of 200 antibodies were isolated and screened for SIRPα reactivity from which approximately 70 antibodies with diverse SIRPα binding profiles, sequence families, and epitopes were selected for further characterization. A subset of anti-SIRPα antibodies bound to both human SIRPα v1 and v2 alleles with high affinity ranging from low nanomolar to picomolar, potently antagonized the CD47/SIRPα interaction, and potentiated macrophage-mediated antibody-dependent cellular phagocytosis in vitro. X-ray crystal structures of five anti-SIRPα antigen-binding fragments, each with unique epitopes, in complex with SIRPα (PDB codes 6NMV, 6NMU, 6NMT, 6NMS, and 6NMR) are reported. Furthermore, some of the anti-SIRPα antibodies cross-react with cynomolgus SIRPα and various mouse SIRPα alleles (BALB/c, NOD, BL/6), which can facilitate preclinical to clinical development. These properties provide an attractive rationale to advance the development of these anti-SIRPα antibodies as a novel therapy for advanced malignancies. Abbreviations: ADCC: antibody-dependent cellular cytotoxicity; ADCP: antibody-dependent cellular phagocytosis; CFSE: carboxyfluorescein succinimidyl ester; Fab: fragment antigen binding; Fc: fragment crystallizable; FcγR: Fcγ receptor; Ig: immunoglobulin; IND: investigational new drug; MDM⊘: monocyte-derived macrophage; NOD: non-obese diabetic; scFv: single chain fragment variable; SCID: severe combined immunodeficiency; SIRP: signal-regulatory protein.

Preclinical Evaluation of Allogeneic CAR T Cells Targeting BCMA for the Treatment of Multiple Myeloma.

Clinical success of autologous CD19-directed chimeric antigen receptor T cells (CAR Ts) in acute lymphoblastic leukemia and non-Hodgkin lymphoma suggests that CAR Ts may be a promising therapy for hematological malignancies, including multiple myeloma. However, autologous CAR T therapies have limitations that may impact clinical use, including lengthy vein-to-vein time and manufacturing constraints. Allogeneic CAR T (AlloCAR T) therapies may overcome these innate limitations of autologous CAR T therapies. Unlike autologous cell therapies, AlloCAR T therapies employ healthy donor T cells that are isolated in a manufacturing facility, engineered to express CARs with specificity for a tumor-associated antigen, and modified using gene-editing technology to limit T cell receptor (TCR)-mediated immune responses. Here, transcription activator-like effector nuclease (TALEN) gene editing of B cell maturation antigen (BCMA) CAR Ts was used to confer lymphodepletion resistance and reduced graft-versus-host disease (GvHD) potential. The safety profile of allogeneic BCMA CAR Ts was further enhanced by incorporating a CD20 mimotope-based intra-CAR off switch enabling effective CAR T elimination in the presence of rituximab. Allogeneic BCMA CAR Ts induced sustained antitumor responses in mice supplemented with human cytokines, and, most importantly, maintained their phenotype and potency after scale-up manufacturing. This novel off-the-shelf allogeneic BCMA CAR T product is a promising candidate for clinical evaluation.

ALX148 blocks CD47 and enhances innate and adaptive antitumor immunity with a favorable safety profile.

CD47 is a widely expressed cell surface protein that functions as an immune checkpoint in cancer. When expressed by tumor cells, CD47 can bind SIRPα on myeloid cells, leading to suppression of tumor cell phagocytosis and other innate immune functions. CD47-SIRPα signaling has also been implicated in the suppression of adaptive antitumor responses, but the relevant cellular functions have yet to be elucidated. Therapeutic blockade of the CD47 pathway may stimulate antitumor immunity and improve cancer therapy. To this end, a novel CD47-blocking molecule, ALX148, was generated by fusing a modified SIRPα D1 domain to an inactive human IgG1 Fc. ALX148 binds CD47 from multiple species with high affinity, inhibits wild type SIRPα binding, and enhances phagocytosis of tumor cells by macrophages. ALX148 has no effect on normal human blood cells in vitro or on blood cell parameters in rodent and non-human primate studies. Across several murine tumor xenograft models, ALX148 enhanced the antitumor activity of different targeted antitumor antibodies. Additionally, ALX148 enhanced the antitumor activity of multiple immunotherapeutic antibodies in syngeneic tumor models. These studies revealed that CD47 blockade with ALX148 induces multiple responses that bridge innate and adaptive immunity. ALX148 stimulates antitumor properties of innate immune cells by promoting dendritic cell activation, macrophage phagocytosis, and a shift of tumor-associated macrophages toward an inflammatory phenotype. ALX148 also stimulated the antitumor properties of adaptive immune cells, causing increased T cell effector function, pro-inflammatory cytokine production, and a reduction in the number of suppressive cells within the tumor microenvironment. Taken together, these results show that ALX148 binds and blocks CD47 with high affinity, induces a broad antitumor immune response, and has a favorable safety profile.

Long-term clinical effect of group cognitive behavioral therapy for insomnia: a case series study.

OBJECTIVE: Cognitive-behavioral therapy for insomnia (CBT-I) is recognized as the first-choice intervention for insomnia. One of the best-known advantages of CBT-I in comparison with pharmacotherapy is its long-term effect. However, only few studies have assessed its benefits with follow-up periods of longer than three years. In this clinical case series study we aimed to describe the long-term effects of group CBT-I after a mean 7.8 ± 1.6 years of follow-up (range 4-10 years). METHODS: A total of 292 insomnia disorder (ID) patients were consecutively enrolled at the Sleep Disorders Center of San Raffaele Hospital, Milan; 123 patients (82 (66.7%) females and 41 (33.3%) males, mean age 40.59 ± 11.89 years) completed the follow-up evaluation within a range of 4-10 years. RESULTS: In the 258 patients who completed the treatment, insomnia severity index (ISI) total score improved significantly as well as all variables of the sleep diaries. Using ISI as the primary outcome, we demonstrated that the effect of CBT-I is maintained up to 10 years after the end of treatment. Furthermore, we found that patients that used only CBT-I techniques to deal with relapses were the ones with better outcomes, in particular compared to the patients that re-used medications. CONCLUSION: To the best of our knowledge this is the longest follow-up evaluation in the literature, both for group and individual CBT-I. These findings have an important clinical implication both suggesting and confirming that CBT-I can be considered the treatment of choice for insomnia.

Immunoglobulin class-switched B cells form an active immune axis between CNS and periphery in multiple sclerosis.

In multiple sclerosis (MS), lymphocyte--in particular B cell--transit between the central nervous system (CNS) and periphery may contribute to the maintenance of active disease. Clonally related B cells exist in the cerebrospinal fluid (CSF) and peripheral blood (PB) of MS patients; however, it remains unclear which subpopulations of the highly diverse peripheral B cell compartment share antigen specificity with intrathecal B cell repertoires and whether their antigen stimulation occurs on both sides of the blood-brain barrier. To address these questions, we combined flow cytometric sorting of PB B cell subsets with deep immune repertoire sequencing of CSF and PB B cells. Immunoglobulin (IgM and IgG) heavy chain variable (VH) region repertoires of five PB B cell subsets from MS patients were compared with their CSF Ig-VH transcriptomes. In six of eight patients, we identified peripheral CD27(+)IgD(-) memory B cells, CD27(hi)CD38(hi) plasma cells/plasmablasts, or CD27(-)IgD(-) B cells that had an immune connection to the CNS compartment. Pinpointing Ig class-switched B cells as key component of the immune axis thought to contribute to ongoing MS disease activity strengthens the rationale of current B cell-targeting therapeutic strategies and may lead to more targeted approaches.

The restricted DH gene reading frame usage in the expressed human antibody repertoire is selected based upon its amino acid content.

The Ab repertoire is not uniform. Some variable, diversity, and joining genes are used more frequently than others. Nonuniform usage can result from the rearrangement process, or from selection. To study how the Ab repertoire is selected, we analyzed one part of diversity generation that cannot be driven by the rearrangement mechanism: the reading frame usage of DH genes. We have used two high-throughput sequencing methodologies, multiple subjects and advanced algorithms to measure the DH reading frame usage in the human Ab repertoire. In most DH genes, a single reading frame is used predominantly, and inverted reading frames are practically never observed. The choice of a single DH reading frame is not limited to a single position of the DH gene. Rather, each DH gene participates in rearrangements of differing CDR3 lengths, restricted to multiples of three. In nonproductive rearrangements, there is practically no reading frame bias, but there is still a striking absence of inversions. Biases in DH reading frame usage are more pronounced, but also exhibit greater interindividual variation, in IgG(+) and IgA(+) than in IgM(+) B cells. These results suggest that there are two developmental checkpoints of DH reading frame selection. The first occurs during VDJ recombination, when inverted DH genes are usually avoided. The second checkpoint occurs after rearrangement, once the BCR is expressed. The second checkpoint implies that DH reading frames are subjected to differential selection. Following these checkpoints, clonal selection induces a host-specific DH reading frame usage bias.

B cell exchange across the blood-brain barrier in multiple sclerosis.

In multiple sclerosis (MS) pathogenic B cells likely act on both sides of the blood-brain barrier (BBB). However, it is unclear whether antigen-experienced B cells are shared between the CNS and the peripheral blood (PB) compartments. We applied deep repertoire sequencing of IgG heavy chain variable region genes (IgG-VH) in paired cerebrospinal fluid and PB samples from patients with MS and other neurological diseases to identify related B cells that are common to both compartments. For the first time to our knowledge, we found that a restricted pool of clonally related B cells participated in robust bidirectional exchange across the BBB. Some clusters of related IgG-VH appeared to have undergone active diversification primarily in the CNS, while others have undergone active diversification in the periphery or in both compartments in parallel. B cells are strong candidates for autoimmune effector cells in MS, and these findings suggest that CNS-directed autoimmunity may be triggered and supported on both sides of the BBB. These data also provide a powerful approach to identify and monitor B cells in the PB that correspond to clonally amplified populations in the CNS in MS and other inflammatory states.

Naive antibody gene-segment frequencies are heritable and unaltered by chronic lymphocyte ablation.

A diverse antibody repertoire is essential for an effective adaptive immune response to novel molecular surfaces. Although past studies have observed common patterns of V-segment use, as well as variation in V-segment use between individuals, the relative contributions to variance from genetics, disease, age, and environment have remained unclear. Using high-throughput sequence analysis of monozygotic twins, we show that variation in naive V(H) and D(H) segment use is strongly determined by an individual's germ-line genetic background. The inherited segment-use profiles are resilient to differential environmental exposure, disease processes, and chronic lymphocyte depletion therapy. Signatures of the inherited profiles were observed in class switched germ-line use of each individual. However, despite heritable segment use, the rearranged complementarity-determining region-H3 repertoires remained highly specific to the individual. As it has been previously demonstrated that certain V-segments exhibit biased representation in autoimmunity, lymphoma, and viral infection, we anticipate our findings may provide a unique mechanism for stratifying individual risk profiles in specific diseases.

Forced expression of cyclin-dependent kinase 6 confers resistance of pro-B acute lymphocytic leukemia to Gleevec treatment.

The gene encoding c-ABL, a nonreceptor protein tyrosine kinase, is involved in a chromosomal translocation resulting in expression of a BCR-Abl fusion protein that causes most chronic myelogenous and some acute lymphocytic leukemias (CML and ALL) in humans. The Abelson murine leukemia virus (A-MuLV) expresses an alternative form of c-Abl, v-Abl, that transforms murine pro-B cells, resulting in acute leukemia and providing an experimental model for human disease. Gleevec (STI571) inhibits the Abl kinase and has shown great utility against CML and ALL in humans, although its usefulness is limited by acquired resistance. Since STI571 is active against A-MuLV-transformed cells in vitro, we performed a retroviral cDNA library screen for genes that confer resistance to apoptosis induced by STI571. We found that forced expression of Cdk6 promotes continued cell division and decreased apoptosis of leukemic cells. We then determined that the transcription factor E2A negatively regulates Cdk6 transcription in leukemic pro-B cells and that the v-Abl kinase stimulates Cdk6 expression via an extracellular signal-regulated kinase 1-dependent pathway. Finally, we show that the cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitor PD0332991 can act synergistically with STI571 to enhance leukemic cell death, suggesting a potential role for CDK6 inhibitors in the treatment of STI571-resistant CML or ALL.

Blimp-1/Prdm1 alternative promoter usage during mouse development and plasma cell differentiation.

The zinc-finger PR domain transcriptional repressor Blimp-1/Prdm1 plays essential roles in primordial germ cell specification, placental, heart, and forelimb development, plasma cell differentiation, and T-cell homeostasis. The present experiments demonstrate that the mouse Prdm1 gene has three alternative promoter regions. All three alternative first exons splice directly to exon 3, containing the translational start codon. To examine possible cell-type-specific functional activities in vivo, we generated targeted deletions that selectively eliminate two of these transcriptional start sites. Remarkably, mice lacking the previously described first exon develop normally and are fertile. However, this region contains NF-kappaB binding sites, and as shown here, NF-kappaB signaling is required for Prdm1 induction. Thus, mutant B cells fail to express Prdm1 in response to lipopolysaccharide stimulation and lack the ability to become antibody-secreting cells. An alternative distal promoter located approximately 70 kb upstream, giving rise to transcripts strongly expressed in the yolk sac, is dispensable. Thus, the deletion of exon 1B has no noticeable effect on expression levels in the embryo or adult tissues. Collectively, these experiments provide insight into the organization of the Prdm1 gene and demonstrate that NF-kappaB is a key mediator of Prdm1 expression.

Mechanisms controlling expression of the RAG locus during lymphocyte development.

Recombination activating genes (RAG)1 and RAG2 are expressed in developing B and T lymphocytes and are required for the rearrangement of antigen receptor genes. In turn, RAG expression is regulated by the products of these assembled immunoglobulin (Ig) and T cell receptor (TCR) genes. Upon successful assembly of Ig genes, the antigen receptor is expressed on the immature B cell surface and tested for autoreactivity leading to either maintenance or inactivation of RAG expression. Successful assembly of TCR genes is followed by surface TCR expression and testing for its ability to interact with self-MHC, which if appropriate leads to the inactivation of RAG expression. Recent studies in B and T lymphocytes demonstrate that the reduction in RAG expression at the immature B and double-positive (DP) T cell stages is mediated through tonic (foreign antigen independent) receptor signaling. In B cells, tonic signaling activates PI(3)K and Akt kinases, which phosphorylate and lead to the cytoplasmic sequestration of FoxO proteins, the key transcriptional activators of RAG expression. In T cells, tonic signaling activates Abl and Erk kinases, leading to the transcriptional inactivation of RAGs.

Cognitive behavioral therapy for insomnia enhances depression outcome in patients with comorbid major depressive disorder and insomnia.

STUDY OBJECTIVE: Insomnia impacts the course of major depressive disorder (MDD), hinders response to treatment, and increases risk for depressive relapse. This study is an initial evaluation of adding cognitive behavioral therapy for insomnia (CBTI) to the antidepressant medication escitalopram (EsCIT) in individuals with both disorders. DESIGN AND SETTING: A randomized, controlled, pilot study in a single academic medical center. PARTICIPANTS: 30 individuals (61% female, mean age 35 +/- 18) with MDD and insomnia. INTERVENTIONS: EsCIT and 7 individual therapy sessions of CBTI or CTRL (quasi-desensitization). MEASUREMENTS AND RESULTS: Depression was assessed with the HRSD17 and the depression portion of the SCID, administered by raters masked to treatment assignment, at baseline and after 2, 4, 6, 8, and 12 weeks of treatment. The primary outcome was remission of MDD at study exit, which required both an HRSD17 score < or =7 and absence of the 2 core symptoms of MDD. Sleep was assessed with the insomnia severity index (ISI), daily sleep diaries, and actigraphy. EsCIT + CBTI resulted in a higher rate of remission of depression (61.5%) than EsCIT + CTRL (33.3%). EsCIT + CBTI was also associated with a greater remission from insomnia (50.0%) than EsCIT + CTRL (7.7%) and larger improvement in all diary and actigraphy measures of sleep, except for total sleep time. CONCLUSIONS: This pilot study provides evidence that augmenting an antidepressant medication with a brief, symptom focused, cognitive-behavioral therapy for insomnia is promising for individuals with MDD and comorbid insomnia in terms of alleviating both depression and insomnia.

Who is at risk for dropout from group cognitive-behavior therapy for insomnia?

OBJECTIVE: The aim of the present study was to identify characteristics of patients who are at risk for dropout from a seven-session group cognitive-behavior therapy for insomnia (CBT-I) in a clinical setting using the receiver operating characteristic curve (ROC) approach. METHODS: Two separate ROC analyses were conducted using predictor variables taken from questionnaire packets and sleep diaries collected at baseline including age, gender, Beck Depression Inventory (BDI), Morningness-Eveningness Questionnaire, Beliefs and Attitudes about Sleep, use of sleep medication, sleep onset latency, wake time after sleep onset, and total sleep time (TST). RESULTS: The first ROC analysis was conducted on the entire sample of 528 patients with treatment completion vs. dropout (noncompletion) as the outcome variable. No significant predictor variables were found in this analysis. The second ROC analysis was conducted on the 211 patients who did not complete treatment with early termination (prior to fourth session) vs. late termination (at or after fourth session) as the outcome variable. The results revealed that patients who reported an average baseline TST <3.65 h were at greatest risk for early termination. Sixty percent of patients in this group terminated early compared to 9.3% of patients with TST > or =3.65 h. Among patients with TST > or =3.65 h, 22% of those with BDI scores > or =16 were early dropouts compared to 4.3% of those who reported BDI <16. CONCLUSION: These findings indicate that short sleep duration and elevated symptoms of depression at baseline are associated with increased risk of early termination from CBT-I.

Patient expectations and therapeutic alliance as predictors of outcome in group cognitive-behavioral therapy for insomnia.

Despite growing evidence for the efficacy of cognitive-behavioral therapy for insomnia (CBT-I), few data exist on the relation between process and outcome for this treatment. Drawing on interpersonal theory and the broader psychotherapy literature, this study examined the contribution of patient expectations and the therapeutic alliance to outcomes in group CBT-I. For patients with low early treatment expectations for improvement, those perceiving the therapist as higher in affiliation had greater reduction in sleep problems. Perceiving the therapist as critically confrontive was generally associated with less treatment satisfaction, and particularly so for those individuals who came to treatment with high expectations for improvement. Critical confrontation also differentiated dropouts from continuers, with dropouts experiencing their therapist as more critically confrontive.

Characteristics of insomniacs with self-reported morning and evening chronotypes.

STUDY OBJECTIVES: This study examines the relevance of self-reported morning and evening chronotypes in treatment-seeking insomniacs presenting to a tertiary sleep clinic setting. DESIGN: Using a cross-sectional design, patients were categorized as morning, intermediate, and evening chronotypes based upon scores on the Morningness-Eveningness Composite Scale (MECS). Group comparisons were made on self-report measures of nocturnal sleep, sleep period variability, and waking correlates and consequences of insomnia. SETTING: Sleep disorders clinic PATIENTS: The sample consisted of 312 patients who presented to a group cognitive-behavior therapy for insomnia (CBT-I) at the sleep clinic. MEASUREMENTS AND RESULTS: Participants completed the MECS, Beck Depression Inventory (BDI), Dysfunctional Beliefs and Attitudes about Sleep Scale (DBAS), and one week of sleep diary prior to treatment. Even after adjusting for total wake time as an index of insomnia severity, differences between the three chronotypes were present on several measures. Compared to the morning and intermediate types, evening types reported more total sleep time, more time in bed, greater variability in the time out of bed, and higher levels of distress on the DBAS and BDI. CONCLUSIONS: These results indicate that insomniacs presenting to a sleep specialist who endorse an evening chronotype report sleep/wake irregularities and waking distress greater than expected in association with the level of insomnia severity. These factors may serve to perpetuate the insomnia disorder and might be particularly important to consider when treating this subgroup of insomniacs.

Bcl-6 mediates the germinal center B cell phenotype and lymphomagenesis through transcriptional repression of the DNA-damage sensor ATR.

Antibody specificity and diversity is generated in B cells during germinal center maturation through clonal expansion while they undergo class-switch recombination and somatic hypermutation. Here we demonstrate that the transcriptional repressor Bcl-6 mediates this phenotype by directly repressing ATR in centroblasts and lymphoma cells. ATR is critical in replication and DNA damage-sensing checkpoints. Bcl-6 allowed B cells to evade ATR-mediated checkpoints and attenuated the response of the B cells to exogenous DNA damage. Repression of ATR was necessary and sufficient for those Bcl-6 activities. CD40 signaling 'rescued' B cells from those effects by disrupting the Bcl-6 transcription-repression complex on the promoter of the gene encoding ATR. Our data demonstrate a transcriptional regulatory loop whereby Bcl-6 mediates the centroblast phenotype through transient silencing of ATR.