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Bispecific T-cell engager (BiTE®) molecules recruit T cells to cancer cells through CD3ε binding, independently of T-cell receptor (TCR) specificity. Whereas physiological T-cell activation is dependent on signal 1 (TCR engagement) and signal 2 (co-stimulation), BiTE molecule-mediated T-cell activation occurs without additional co-stimulation. As co-stimulatory and inhibitory molecules modulate the strength and nature of T-cell responses, we studied the impact of the expression profile of those molecules on target cells for BiTE molecule-mediated T-cell activation in the context of acute myeloid leukemia (AML). Accordingly, we created a novel in vitro model system using murine Ba/F3 cells transduced with human CD33 ± CD86 ± PD-L1. T-cell fitness was assessed by T-cell function assays in co-cultures and immune synapse formation by applying a CD33 BiTE molecule (AMG 330). Using our cell-based model platform, we found that the expression of positive co-stimulatory molecules on target cells markedly enhanced BiTE molecule-mediated T-cell activation. The initiation and stability of the immune synapse between T cells and target cells were significantly increased through the expression of CD86 on target cells. By contrast, the co-inhibitory molecule PD-L1 impaired the stability of BiTE molecule-induced immune synapses and subsequent T-cell responses. We validated our findings in primary T-cell-AML co-cultures, demonstrating a PD-L1-mediated reduction in redirected T-cell activation. The addition of the immunomodulatory drug (IMiD) lenalidomide to co-cultures led to stabilization of immune synapses and improved subsequent T-cell responses. We conclude that target cells modulate CD33 BiTE molecule-dependent T-cell activation and hence, combinatorial strategies might contribute to enhanced efficacy.

Background: SWAY was a cross-sectional survey that assessed the global impact and treatment of sickle cell disease (SCD) (James et al. ASH 2019). SCD puts patients at risk of multiple complications driven by vaso-occlusion and hemolytic anemia. Vaso-occlusive crises (VOCs) are the hallmark of SCD and can require healthcare attention. VOC frequency may be reduced by HU (Charache et al. N Engl J Med 1995). Aims: We assessed self-reported symptoms and quality of life (QoL) indicators for patients who reported using HU at the time of SWAY versus patients who did not, and we collected data on all treatments reported by SCD patients, by geographical region. Data were also collected regarding historical patient-reported use of HU prior to SWAY but these are not included here. Methods: SWAY was completed between April and October 2019 by SCD patients from 16 countries across 6 regions. A limitation is that Asia and South America were represented by single countries (India and Brazil, respectively). SWAY was completed by proxy (parent/guardian/caregiver) for patients aged 6-11 years and could be optionally self-completed by patients aged ≥12 years. Opinions were captured using a 1-7 Likert scale for some questions (5-7 indicated high satisfaction/impact/agreement). SWAY did not assess treatment outcomes. Results: Of 2145 patients, 652 (30%) reported receiving HU at the time of SWAY (56% female; 50% aged 6-25 years); 1493 patients reported not receiving HU at the time of SWAY (51% female; 59% aged 6-25 years). The number of patients reporting HU use varied regionally (Table). Patients who reported using HU also reported a lower VOC burden than patients who did not report using HU at the time of SWAY (median: 3 vs 4 VOCs in the 12 months before SWAY, respectively). However, considering other symptoms commonly experienced in the month prior to SWAY, a greater proportion of patients who reported using HU experienced these symptoms than patients who did not report using HU, except for headache and poor appetite, which were experienced by a lower proportion of patients who reported HU use (Figure). Similar proportions of patients reported that SCD had a high impact (Likert scale 5-7) on emotional wellbeing (61% [reported HU use at time of SWAY] vs 59% [did not report HU use at time of SWAY]) and daily activities (39% vs 40%, respectively). Overall, when including dietary supplements, the most common treatment reported at the time of SWAY in all regions except the Middle East was folic acid. Common treatments varied regionally when excluding supplements (Table). Top treatment goals for patients in all regions were to improve QoL and prevent SCD worsening. Treatment satisfaction (range: 57-92%) was highest in Asia (Table). Over 70% of patients wanted alternatives to their ongoing pain medications in all regions, except Asia (44%). Discussion: The proportion of patients reporting HU use at the time of SWAY was variable, but relatively low; HU was not in the top 3 treatments for Africa, Europe or North America. HU use was lowest in Africa, where no patients reported receiving HU at the time of the survey, which probably reflects high relative cost and poor access. Our regional analysis showed that many patients take supplements, such as folic acid, rather than HU. This may be indicative of limited alternatives to HU and reflects the differing global costs and availability of both medication and monitoring blood tests. Low reported use of HU may also reflect patients' concerns about side effects or reluctance to take daily medication. It should be noted that some patients may be familiar with 'hydroxycarbamide' as a name for HU and might not have recognized the term 'hydroxyurea' on the survey. Data from SWAY were not validated by medical records and depended on patients' recall. Patients reporting HU use at the time of SWAY had a lower VOC burden than patients not reporting HU use. QoL indicators were similar for the 2 groups, but the overall symptom burden was higher for patients who reported HU use compared with those who did not. However, the 2 groups were not matched for pre-treatment disease burden and no information was collected regarding adherence or duration of previous HU use. Overall, although many patients reported treatment satisfaction, many wanted alternative pain management therapies. A wider range of treatment options are needed to reduce SCD symptoms and improve QoL, ultimately helping patients achieve their treatment goals. Disclosures El Rassi: Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Cyclerion: Research Funding; Pfizer: Research Funding; Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees; Bluebird bio: Membership on an entity's Board of Directors or advisory committees. James:Sickle Cell Society: Current Employment; Novartis: Honoraria. Andemariam:Terumo BCT: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sanofi Genzyme: Consultancy, Membership on an entity's Board of Directors or advisory committees; Guidepoint: Honoraria; Accordant: Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; NovoNordisk: Consultancy, Membership on an entity's Board of Directors or advisory committees; Imara: Research Funding; Hemanext: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; CHNCT: Consultancy; Global Blood Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Vertex: Honoraria; bluebird bio: Consultancy, Membership on an entity's Board of Directors or advisory committees; Emmaus: Membership on an entity's Board of Directors or advisory committees; CRISPR/Vertex: Consultancy, Membership on an entity's Board of Directors or advisory committees; Cyclerion: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees. Inusa:Bluebird bio: Research Funding; AstraZeneca: Honoraria, Other: Steering committee participation, Research Funding, Speakers Bureau; Global Blood Therapeutics: Honoraria, Other: Steering committee participation, Research Funding, Speakers Bureau; Vertex: Research Funding; Novartis: Honoraria, Other: Steering committee participation, Research Funding, Speakers Bureau. Francis-Gibson:Sickle Cell Disease Association of America: Current Employment. Nero:Novartis: Consultancy; Bluebird bio: Consultancy. Minniti:Global Blood Therapeutics: Consultancy, Research Funding; Emmaus: Consultancy, Research Funding; Roche: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; TauTona: Consultancy, Research Funding; Bluebird bio: Consultancy, Research Funding; CLS Bering: Consultancy. Trimnell:Novartis: Consultancy; Cyclerion: Consultancy; Global Blood Therapeutics: Consultancy. Abboud:Eli Lilly: Research Funding; Modus Pharmaceuticals: Research Funding; Crispr Therapeutics: Membership on an entity's Board of Directors or advisory committees; Amgen: Other: Travel support; Novartis: Consultancy, Honoraria, Research Funding; Novo Nordisk: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Global Blood Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AstraZeneca: Membership on an entity's Board of Directors or advisory committees, Research Funding. Arlet:Novartis: Consultancy, Honoraria. Colombatti:Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Addmedica: Membership on an entity's Board of Directors or advisory committees. de Montalembert:Addmedica: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bluebird bio: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Vertex: Honoraria, Membership on an entity's Board of Directors or advisory committees. Jain:Thalassemia and Sickle Cell Society: Other: Chief Medical Research Officer and Secretary. Jastaniah:Novartis: Consultancy, Honoraria. Nur:Novartis: Consultancy. Ramscar:Novartis Pharma AG: Current Employment. Bailey:Novartis: Other: Employee of Adelphi Real World, which has received consultancy fees from Novartis; Adelphi Real World: Current Employment. Rajkovic-Hooley:Adelphi Real World: Current Employment; Novartis: Other: Employee of Adelphi Real World, which has received consultancy fees from Novartis. Osunkwo:Global Blood Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Acceleron: Membership on an entity's Board of Directors or advisory committees; Data and Safety Monitoring Board (DSMB) membership for Micella Biopharma: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; FORMA Therapeutics: Membership on an entity's Board of Directors or advisory committees; Health Resources and Services Administration (HRSA): Research Funding; Patient Centered Outcomes Research Institute (PCORI): Research Funding; Terumo: Consultancy; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Background: Sickle cell disease (SCD) is associated with many clinical complications, with vaso-occlusive crises (VOCs) being a hallmark of the disease. SCD-related complications are largely driven by vaso-occlusion and hemolytic anemia, and can lead to end-organ damage and early death. Analyses of SWAY, a cross-sectional survey, highlighted a substantial global impact of SCD on patients' quality of life (QoL) (James et al. ASH 2019; Osunkwo et al. ASH 2019). However, understanding how the burden of disease differs for pts of different ages could help improve management of SCD over a pt's lifespan. Aim: To assess, using data from SWAY, whether symptoms (excluding VOCs, as previously analyzed by Osunkwo et al. EHA 2020), treatment goals and the perceived impact of SCD were different for pts of different ages. Methods: Between April and October 2019, 2145 SCD pts aged ≥6 years participated in SWAY. The survey was completed by proxy (parent/caregiver/guardian) for pts aged 6-11 years and could be optionally self-completed by pts aged ≥12 years. Opinions were captured using a 1-7 Likert scale for some questions (5-7 indicated high satisfaction/impact/agreement). SWAY was not designed to assess treatment outcomes; all analyses are descriptive. Age groups were not matched and pts were not followed over time. Results: To understand how the most dominant symptoms of SCD differ for pts of different ages, the top 5 most commonly reported symptoms, stratified by age, were analyzed (Figure 1). Fatigue and bone aches were consistently reported, and the proportion of pts reporting them trended towards increasing with age. Furthermore, when asked which symptoms they most wanted to be resolved, fatigue was ranked in the top 3 by 40.7% of pts. Anxiety was a dominant symptom for pts aged 19-25, 36-45 and 46-50 years, whereas low mood was a dominant symptom for pts aged 19-50 and ≥60 years. Poor appetite was a dominant symptom for pts aged 6-16 years. Breathing issues were a dominant symptom for pts aged 6-18, 46-50 and ≥60 years, whereas vision issues were a dominant symptom for pts aged 51-59 years only. Insomnia was dominant for pts aged 46-50. The treatment goals that pts ranked as the most important were similar across age groups. Pts consistently included improving QoL, preventing worsening of SCD, reducing the number of VOCs and improving overall symptoms in their top 3, although the proportions of pts reporting these varied across age groups (Figure 2). Unsurprisingly, 23.0% of pts aged 12-16 years ranked increasing the ability to attend school in their top 3 goals. For pts aged 46-50 and 51-59 years, 27.5% and 20.7%, respectively, ranked reducing fatigue in their top 3 treatment goals. The highest proportions of pts reporting a high impact of SCD on their emotional wellbeing were aged 46-50 years (74.5%) and ≥60 years (73.9%), compared with 59.6% of all pts. Similarly, the highest proportions of pts reporting a high impact of SCD on daily activities were aged 46-50 years (51.0%) and ≥60 years (60.9%), compared with 38.1% of all pts. Limitations: These findings are based on pt and proxy reports, with potential parental bias being introduced for pediatric patients. There were variations in sample sizes, which was most noticeable for patients aged ≥46 years. Discussion: Fatigue and bone aches were consistently reported as dominant symptoms for all ages. Other dominant symptoms that were not consistent across age groups were anxiety, low mood, poor appetite, breathing issues and vision issues. In addition to the 36-45 years and 46-50 years groups, anxiety was a dominant symptom for pts aged 19-25 years. Along with other changes that could occur at this age, a recent transition from pediatric to adult care may contribute to anxiety being a dominant symptom; however, any direct relationship between anxiety and transitioning between care systems requires further investigation. The consistent reporting of low mood among adults, but not pediatrics, may reflect the increasing burden of disease that occurs with age. This is supported by higher proportions of pts aged ≥46 years versus Improving QoL was consistently ranked the most important treatment goal for pts. This emphasizes, from the pt's perspective, the need for further improvements in the management of SCD. Disclosures Colombatti: Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Addmedica: Membership on an entity's Board of Directors or advisory committees. James:Sickle Cell Society: Current Employment; Novartis: Honoraria. Andemariam:Hemanext: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; CHNCT: Consultancy; CRISPR/Vertex: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Cyclerion: Consultancy, Membership on an entity's Board of Directors or advisory committees; Accordant: Membership on an entity's Board of Directors or advisory committees; Guidepoint: Honoraria; Global Blood Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; NovoNordisk: Consultancy, Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Terumo BCT: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sanofi Genzyme: Consultancy, Membership on an entity's Board of Directors or advisory committees; Imara: Research Funding; Vertex: Honoraria; Emmaus: Membership on an entity's Board of Directors or advisory committees; bluebird bio: Consultancy, Membership on an entity's Board of Directors or advisory committees. Inusa:Vertex: Research Funding; Bluebird bio: Research Funding; AstraZeneca: Honoraria, Other: Steering committee participation, Research Funding, Speakers Bureau; Global Blood Therapeutics: Honoraria, Other: Steering committee participation, Research Funding, Speakers Bureau; Novartis: Honoraria, Other: Steering committee participation, Research Funding, Speakers Bureau. El Rassi:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Cyclerion: Research Funding; Pfizer: Research Funding; Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees; Bluebird bio: Membership on an entity's Board of Directors or advisory committees. Francis-Gibson:Sickle Cell Disease Association of America: Current Employment. Nero:Bluebird bio: Consultancy; Novartis: Consultancy. Minniti:Global Blood Therapeutics: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Roche: Consultancy, Research Funding; TauTona: Consultancy, Research Funding; Bluebird bio: Consultancy, Research Funding; Emmaus: Consultancy, Research Funding; CLS Bering: Consultancy. Trimnell:Novartis: Consultancy; Cyclerion: Consultancy; Global Blood Therapeutics: Consultancy. Abboud:Amgen: Other: Travel support; Novo Nordisk: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Crispr Therapeutics: Membership on an entity's Board of Directors or advisory committees; Global Blood Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AstraZeneca: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Eli Lilly: Research Funding; Modus Pharmaceuticals: Research Funding. Arlet:Novartis: Consultancy, Honoraria. de Montalembert:Addmedica: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bluebird bio: Honoraria, Membership on an entity's Board of Directors or advisory committees; Vertex: Honoraria, Membership on an entity's Board of Directors or advisory committees. Jain:Thalassemia and Sickle Cell Society: Other: Chief Medical Research Officer and Secretary. Jastaniah:Novartis: Consultancy, Honoraria. Nur:Novartis: Consultancy. Ramscar:Novartis Pharma AG: Current Employment. Bailey:Novartis: Other: Employee of Adelphi Real World, which has received consultancy fees from Novartis; Adelphi Real World: Current Employment. Rajkovic-Hooley:Adelphi Real World: Current Employment; Novartis: Other: Employee of Adelphi Real World, which has received consultancy fees from Novartis. Osunkwo:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Terumo: Consultancy; Global Blood Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Acceleron: Membership on an entity's Board of Directors or advisory committees; FORMA Therapeutics: Membership on an entity's Board of Directors or advisory committees; Health Resources and Services Administration (HRSA): Research Funding; Patient Centered Outcomes Research Institute (PCORI): Research Funding; Data and Safety Monitoring Board (DSMB) membership for Micella Biopharma: Membership on an entity's Board of Directors or advisory committees.

Zhang et al. identify human IL-2Rβ deficiency as a cause of severe immune dysregulation. The hypomorphic gene mutations reveal variable IL-2Rβ expression and function between different lymphocyte subsets as a means of selectively modulating immune responses., Interleukin-2, which conveys essential signals for immunity, operates through a heterotrimeric receptor. Here we identify human interleukin-2 receptor (IL-2R) β chain (IL2RB) gene defects as a cause of life-threatening immune dysregulation. We report three homozygous mutations in the IL2RB gene of eight individuals from four consanguineous families that cause disease by distinct mechanisms. Nearly all patients presented with autoantibodies, hypergammaglobulinemia, bowel inflammation, dermatological abnormalities, lymphadenopathy, and cytomegalovirus disease. Patient T lymphocytes lacked surface expression of IL-2Rβ and were unable to respond to IL-2 stimulation. By contrast, natural killer cells retained partial IL-2Rβ expression and function. IL-2Rβ loss of function was recapitulated in a recombinant system in which IL2RB mutations caused reduced surface expression and IL-2 binding. Stem cell transplant ameliorated clinical symptoms in one patient; forced expression of wild-type IL-2Rβ also increased the IL-2 responsiveness of patient T lymphocytes in vitro. Insights from these patients can inform the development of IL-2–based therapeutics for immunological diseases and cancer.

T cells discriminate between healthy and infected cells with remarkable sensitivity when mounting an immune response, which is hypothesized to depend on T cells combining stimuli from multiple antigen‐presenting cell interactions into a more potent response. To quantify the capacity for T cells to accomplish this, we have developed an antigen receptor that is optically tunable within cell conjugates, providing control over the duration, and intensity of intracellular T‐cell signaling. We observe limited persistence within the T‐cell intracellular network on disruption of receptor input, with signals dissipating entirely in ~15 min, and directly show sustained proximal receptor signaling is required to maintain gene transcription. T cells thus primarily accumulate the outputs of gene expression rather than integrate discrete intracellular signals. Engineering optical control in a clinically relevant chimeric antigen receptor (CAR), we show that this limited signal persistence can be exploited to increase CAR‐T cell activation threefold using pulsatile stimulation. Our results are likely to apply more generally to the signaling dynamics of other cellular networks., An optically controllable chimeric antigen receptor is developed for modulating T cell signaling dynamics. Limited signal persistence is found at key nodes in the intracellular network, constraining the window over which T cells can integrate discrete activation signals.

Sickle cell disease (SCD) is a genetic disorder, characterized by hemolytic anemia and vaso‐occlusive crises (VOCs). Data on the global SCD impact on quality of life (QoL) from the patient viewpoint are limited. The international Sickle Cell World Assessment Survey (SWAY) aimed to provide insights into patient‐reported impact of SCD on QoL. This cross‐sectional survey of SCD patients enrolled by healthcare professionals and advocacy groups assessed disease impact on daily life, education and work, symptoms, treatment goals, and disease management. Opinions were captured using a Likert scale of 1‐7 for some questions; 5‐7 indicated “high severity/impact.” Two thousand one hundred and forty five patients (mean age 24.7 years [standard deviation (SD) = 13.1], 39% ≤18 years, 52% female) were surveyed from 16 countries (six geographical regions). A substantial proportion of patients reported that SCD caused a high negative impact on emotions (60%) and school achievement (51%) and a reduction in work hours (53%). A mean of 5.3 VOCs (SD = 6.8) was reported over the 12 months prior to survey (median 3.0 [interquartile range 2.0‐6.0]); 24% were managed at home and 76% required healthcare services. Other than VOCs, fatigue was the most commonly reported symptom in the month before survey (65%), graded “high severity” by 67% of patients. Depression and anxiety were reported by 39% and 38% of patients, respectively. The most common patient treatment goal was improving QoL (55%). Findings from SWAY reaffirm that SCD confers a significant burden on patients, epitomized by the high impact on patientsʼ QoL and emotional wellbeing, and the high prevalence of self‐reported VOCs and other symptoms.

SUMMARYT cells discriminate between healthy and infected cells with remarkable sensitivity when mounting an immune response. It has been hypothesized that this efficient detection requires combining signals from discrete antigen-presenting cell interactions into a more potent response, requiring T cells to maintain a ‘memory’ of previous encounters. To quantify the magnitude of this phenomenon, we have developed an antigen receptor that is both optically and chemically tunable, providing control over the initiation, duration, and intensity of intracellular T-cell signaling within physiological cell conjugates. We observe very limited persistence within the T cell intracellular network on disruption of receptor input, with signals dissipating entirely in ~15 minutes, and directly confirm that sustained proximal receptor signaling is required to maintain active gene transcription. Our data suggests that T cells are largely incapable of integrating discrete antigen receptor signals but instead simply accumulate the output of gene expression. By engineering optical control in a clinically relevant chimeric antigen receptor, we show that this limited signal persistence can be exploited to increase the activation of primary T cells by ~3-fold by using pulsatile stimulation. Our results are likely to apply more generally to the signaling dynamics of other cellular networks.

Current materials used for in vitro 3D cell culture are often limited by their poor similarity to human tissue, batch-to-batch variability and complexity of composition and manufacture. Here, we present a “blank slate” culture environment based on a self-assembling peptide gel free from matrix motifs. The gel can be customised by incorporating matrix components selected to match the target tissue, with independent control of mechanical properties. Therefore the matrix components are restricted to those specifically added, or those synthesised by encapsulated cells. The flexible 3D culture platform provides full control over biochemical and physical properties, allowing the impact of biochemical composition and tissue mechanics to be separately evaluated in vitro. Here, we demonstrate that the peptide gels support the growth of a range of cells including human induced pluripotent stem cells and human cancer cell lines. Furthermore, we present proof-of-concept that the peptide gels can be used to build disease-relevant models. Controlling the peptide gelator concentration allows peptide gel stiffness to be matched to normal breast (1 kPa), with higher stiffness favouring the viability of breast cancer cells over normal breast cells. In parallel, the peptide gels may be modified with matrix components relevant to human breast, such as collagen I and hyaluronan. The choice and concentration of these additions affect the size, shape and organisation of breast epithelial cell structures formed in co-culture with fibroblasts. This system therefore provides a means of unravelling the individual influences of matrix, mechanical properties and cell-cell interactions in cancer and other diseases.

Introduction: The Sickle Cell World Assessment Survey (SWAY) was a cross-sectional survey to assess the global impact and treatment of sickle cell disease (SCD). Complications of SCD can lead to significant negative effects on patient (pt) quality of life. Recurrent vaso-occlusive crises (VOCs) are one of the most common SCD complications and can lead to poor quality of life and chronic organ damage. SCD manifestations can start as early as the first year of life. The implications of SCD on a child's life can be far reaching and may affect education, the global impact of which has not been well described. Here, we assess data from SWAY to better understand the impact of SCD on education among pediatric pts in the US vs other high-income countries (HIC) and low/middle-income countries (LMIC). Methods: SWAY included individuals aged ≥6 years with a diagnosis of SCD. The survey was completed by proxy (parent/caregiver/guardian) for pts aged 6-11 years and could be optionally self-completed by pts aged ≥12 years. The survey consisted of 7 ratings-based (Likert scale) questions focused on education, where a score of 5, 6, or 7 indicated increasing levels of agreement. Pediatric pts were defined as those aged Results: Among the 769 pediatric pts participating in SWAY, there were 77 US respondents to the educational survey (mean age, 12 y), 200 HIC respondents (mean age, 13 y), and 492 LMIC respondents (mean age, 12 y, [one respondent did not provide an age]). Pediatric pts in all groups reported that SCD adversely impacted their education. Of the US respondents, 51%, 45%, and 52% agreed that SCD negatively impacted performance on school tests, overall performance at school, and school attendance, respectively. This was a higher rate of agreement for these statements than that reported by pediatric pts from other HIC (25%, 23%, 36%) and LMIC (37%, 41%, 50%). The US respondents also agreed that SCD negatively affected performance on homework (45%), caused them to repeat a year or class (42%), lowered interest in school (36%), and limited educational progression (35%). Again, this was a higher rate of agreement than that reported by pediatric pts from other HIC (26%, 14%, 19%, 20%) and LMIC (37%, 32%, 34%, 29%). Interestingly, the largest differences in reported school impact occurred between the US and HIC, where the US respondents showed nearly two-fold higher agreement for all statements except for reduced attendance. Conversely, there were only minor differences between respondents from the US and LMIC. Full results are presented in the Figure. Conclusions: A higher proportion of pediatric pts in the US reported a negative impact of SCD on schooling compared with those in HIC and LMIC. These results were unexpected but align strongly with the emerging evidence that social determinants prevalent in the US lends itself away from the benefits of living in a resource-rich nation. Figure 1 Figure 1. Disclosures James: GBT: Honoraria; Novartis: Honoraria. Francis-Gibson: Global Alliance of SCD Organizations: Membership on an entity's Board of Directors or advisory committees; Sickle Cell Disease Association of America: Current Employment; Alliance for Regenerative Medicine Foundation for Cell and Gene Medicine: Membership on an entity's Board of Directors or advisory committees; ASH: Membership on an entity's Board of Directors or advisory committees; Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees. Minniti: GBT: Consultancy, Research Funding; Novartis: Consultancy, Honoraria; NovoNordisk: Consultancy, Honoraria; Roche: Consultancy, Honoraria. Paulose: Novartis Pharmaceuticals Corporation: Current Employment. Bailey: Novartis Pharmaceuticals: Other: I am an employee of Adelphi Real World, which received payment from Novartis Pharmaceuticals for this research. Rajkovic-Hooley: Novartis Pharmaceuticals: Other: I am an employee of Adelphi Real World, which received payment from Novartis Pharmaceuticals for this research. Osunkwo: Forma Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Health and Services Administration: Research Funding; Patient Centered Outcomes Research Instituted: Research Funding; Micella Biopharma: Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Chiesi: Consultancy; Emmaus: Consultancy; Cyclerion: Consultancy; Acceleron: Consultancy; Global Blood Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Terumo: Consultancy.

Background: SCD is an inherited blood disorder that for many patients (pts) has a high clinical burden, results in poor quality of life (QoL), and reduces life expectancy. Gaining a deeper understanding of pt and HCP experiences of SCD is important to improve pt management. Aims: SWAY was a cross-sectional survey that assessed pt and HCP experiences of SCD. Here we focus on the experiences of HCPs from various regions on SCD symptoms and complications, impact of SCD on QoL, treatment goals and treatment satisfaction. Methods: SWAY was developed by international SCD expert physicians, pt advocates and Novartis. HCPs completed the survey between Apr and Oct 2019. Eligible HCPs had qualified in their primary specialty by 2014 and were managing ≥10 SCD pts at the time of survey (≥5 pts per HCP in Canada; ≥2 pts in the Netherlands). Responses to questions on how much SCD impacts pt QoL, and on HCP treatment satisfaction, were ranked on a Likert scale (1-7, where 1=not at all/strongly dissatisfied, 7=a great deal/strongly satisfied; 5-7 indicated high impact/satisfaction). The data reflect only the experiences of the surveyed HCPs in each region (recruited by Adelphi Real World fieldwork). A limitation is that Asia and South America (SA) were represented by single countries (India and Brazil, respectively). Results: SWAY was completed by 365 HCPs from 6 regions (Table). In all regions HCPs recognized the prevalence of acute and chronic pain, however acute pain was reported less frequently by HCPs in Africa than in other regions (Table). Acute chest syndrome and joint issues were among the top 5 most frequently mentioned complications by HCPs in all regions. Globally, HCPs recognized the high impact of SCD symptoms and complications on pt QoL and the high negative impact of SCD on pt emotional wellbeing (Likert score 5-7 reported by 79-100% and 71-97%, respectively). Fewer HCPs in the Middle East (ME) reported a high impact of SCD on physical and sexual activity, compared with HCPs in other regions. Around 40% of HCPs in the ME and Asia thought SCD has a high impact on daily activities, compared with 79-90% of HCPs in other regions. In Asia, fewer HCPs reported that SCD has a high impact on pts' education and ability to maintain a job compared with HCPs in other regions (Figure). Hydroxyurea (HU) was among the top 3 most common therapies ever initiated and was the therapy most likely to be initiated in any age group by HCPs in almost all regions. In Africa, the most common therapy ever initiated and the therapy most likely to be initiated in any age group was opioids (Table). Fewer HCPs in North America (NA; 32%) and SA (27%) were highly satisfied with current SCD treatments, compared with HCPs in other regions (46-72%). The main reason for dissatisfaction was limited treatment options in all regions except Asia, where HCPs said they were unable to reach their treatment goals with current therapies. Improving pts' QoL was among the top 3 treatment goals for 51-84% of HCPs across all regions. For HCPs in NA and the ME, the most important goal when treating vaso-occlusive crises was to improve QoL; in SA, Europe and Asia it was to avoid organ damage; and in Africa it was to eliminate pain completely. Discussion: The top 5 most frequent SCD symptoms and complications that HCPs reported were similar across all regions. There were regional differences in HCP experiences of how SCD impacts aspects of pts' daily life, with fewer HCPs in the ME reporting a high impact on physical and sexual activity, and fewer HCPs in Asia and the ME reporting a high impact on daily activities compared with other regions. This may be due to cultural variations, with pts in these regions being less comfortable discussing these topics with HCPs. There was a difference in the reported impact of SCD on school and work between HCPs in Asia and other regions, which could be due to varying expectations regarding school/work productivity. HU was one of the top 3 most common treatments ever initiated by HCPs for pts of any age, except in Africa, which may be due to an educational knowledge gap about HU, high cost, or poor access in this region. HCPs in almost all regions, except Asia, were dissatisfied with current SCD treatments because of limited therapeutic options, indicating a global unmet need for additional treatment choices. Improving QoL was the most important treatment goal for HCPs in all regions, demonstrating the high negative impact that SCD has on pt QoL and the ongoing need for methods to address this. Figure 1 Figure 1. Disclosures Osunkwo: Novartis Pharmaceuticals: Consultancy, Honoraria, Speakers Bureau; FORMA Therapeutics: Consultancy; Global Blood Therapeutics: Consultancy, Speakers Bureau; Chiesi: Consultancy; Acceleron: Consultancy; Cyclerion: Consultancy; Emmaus: Consultancy. Minniti: Roche: Consultancy, Honoraria; NovoNordisk: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; GBT: Consultancy, Research Funding. Nur: Roche: Speakers Bureau; Celgene: Speakers Bureau; Novartis: Research Funding, Speakers Bureau. Nero: Global Blood Therapeutics: Consultancy; Editas Medicine: Consultancy; bluebird bio: Consultancy; Novartis: Consultancy. Colombatti: Global Blood Therapeutics: Research Funding; Addmedica: Consultancy; Forma Therapeutics: Consultancy; Novartis: Consultancy; NovoNordisk: Consultancy; BlueBirdBio: Consultancy; Global Blood Therapeutics: Consultancy; BlueBirdBio: Research Funding. de Montalembert: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Addmedica: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; bluebird bio: Membership on an entity's Board of Directors or advisory committees; Vertex: Membership on an entity's Board of Directors or advisory committees. Abboud: Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Research Support and Advisory Board, Research Funding; AstraZeneca: Membership on an entity's Board of Directors or advisory committees, Other: Reserach support and advisory board , Research Funding; GBT: Other: Research Support, Research Funding; Vertex Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: DSMB. Arlet: Addmedica: Research Funding; Pfizer: Honoraria; Novartis company: Consultancy, Honoraria, Research Funding. Jastaniah: Novartis: Consultancy, Honoraria, Research Funding. Pita: GLOBAL ALLIANCE OF SCD: Membership on an entity's Board of Directors or advisory committees; LUA VERMELHA SCD ASSOCIATION: Membership on an entity's Board of Directors or advisory committees. Francis-Gibson: Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees; Sickle Cell Disease Association of America: Current Employment; ASH: Membership on an entity's Board of Directors or advisory committees; Alliance for Regenerative Medicine Foundation for Cell and Gene Medicine: Membership on an entity's Board of Directors or advisory committees; Global Alliance of SCD Organizations: Membership on an entity's Board of Directors or advisory committees. Trimnell: Novartis: Consultancy; Cyclerion: Consultancy; Global Blood Therapeutics: Consultancy. DeBonnett: Novartis Pharmaceuticals Corporation: Current Employment. Bailey: Novartis Pharmaceuticals: Other: I am an employee of Adelphi Real World, which received payment from Novartis Pharmaceuticals for this research. Rajkovic-Hooley: Novartis Pharmaceuticals: Other: I am an employee of Adelphi Real World, which received payment from Novartis Pharmaceuticals for this research. James: GBT: Honoraria; Novartis: Honoraria.

How G protein-coupled receptors are assembled is a matter of considerable interest owing in large part to their remarkable pharmacological importance. For determining receptor stoichiometry, resonance energy transfer-based methods offer considerable advantages insofar as they provide the necessary spatial resolution, and because measurements can be made in situ, relatively easily. This chapter describes three complementary stoichiometric assays that rely on measurements of bioluminescence resonance energy transfer. These quantitative approaches make it possible to identify true protein-protein interactions from non-specific associations that inevitably result from constraining proteins in cellular membranes. In our experience, concordant data obtained in two or more of these assays, benchmarked with suitable controls, strongly predict receptor stoichiometry.

How G protein-coupled receptors are assembled is a matter of considerable interest owing in large part to their remarkable pharmacological importance. For determining receptor stoichiometry, resonance energy transfer-based methods offer considerable advantages insofar as they provide the necessary spatial resolution, and because measurements can be made in situ, relatively easily. This chapter describes three complementary stoichiometric assays that rely on measurements of bioluminescence resonance energy transfer. These quantitative approaches make it possible to identify true protein-protein interactions from non-specific associations that inevitably result from constraining proteins in cellular membranes. In our experience, concordant data obtained in two or more of these assays, benchmarked with suitable controls, strongly predict receptor stoichiometry.

Interleukin-2, which conveys essential signals for effective immunity and immunological tolerance, operates through a heterotrimeric receptor comprised of α, β and γ chains. Genetic deficiency of the α or γ chain causes debilitating disease. Here we identify human interleukin-2 receptor (IL-2R) β chain (CD122) gene defects as a cause of life-threatening dysregulation of immunity and peripheral tolerance. We report homozygous mutations in the human IL-2Rβ gene from four consanguineous families, comprising either recessive missense mutations in five children or an early stop-gain mutation in two deceased fetuses and a premature neonate. All patients surviving to childhood presented with autoantibodies, hypergammaglobulinemia, bowel inflammation, and dermatological abnormalities, as well as cytomegalovirus disease in most cases. Patient T lymphocytes lacked surface expression of IL-2Rβ and were unable to normally respond to high-dose IL-2 stimulation. By contrast, patient natural killer (NK) cells retained partial IL-2Rβ expression and cytotoxic function. IL-2Rβ loss of function was recapitulated in a recombinant system, in which endoplasmic reticulum sequestration was revealed as the mechanism by which certain missense mutations cause disease. Hematopoietic stem cell transplant resulted in resolution of clinical symptoms in one patient. The hypomorphic nature of this disease highlights the significance of variable IL-2Rβ expression in different lymphocyte subsets as a means of modulating immune function. Insights from these patients can inform the development of IL-2-based therapeutics for immunological diseases and cancer.

The T-cell antigen receptor (TCR) recognizes peptides from pathogenic proteins bound in the MHC molecule. To convert this binding event into downstream signaling, the TCR has phosphorylatable intracellular motifs (ITAMs) that act as docking sites for ZAP70, a cytoplasmic tyrosine kinase. Uniquely, the TCR employs 10 ITAMs to transduce pMHC binding to the cell interior. Why this multivalency is required at the mechanistic level remains unclear. Using synthetic, drug-inducible receptor/ligand pairs, we find that greater ITAM multiplicity primarily enhances the efficacy of these engineered receptors, by increasing the efficiency with which ligand binding is converted into an intracellular signal. This manifests as an increase in the fraction of cells that become activated, rather than directly amplifying the intracellular signal and a more synchronous initiation of proximal signaling. Exploiting these findings, we show that the potency and selectivity of chimeric antigen receptors targeted against cancer can be substantially enhanced by modulating the number of encoded ITAMs.

LCK is a tyrosine kinase that is essential for initiating T-cell antigen receptor (TCR) signaling. A complete understanding of LCK function is constrained by a paucity of methods to quantitatively study its function within live cells. To address this limitation, we generated LCK*, in which a key active-site lysine is replaced by a photocaged equivalent, using genetic code expansion. This strategy enabled fine temporal and spatial control over kinase activity, thus allowing us to quantify phosphorylation kinetics in situ using biochemical and imaging approaches. We find that autophosphorylation of the LCK active-site loop is indispensable for its catalytic activity and that LCK can stimulate its own activation by adopting a more open conformation, which can be modulated by point mutations. We then show that CD4 and CD8, T-cell coreceptors, can enhance LCK activity, thereby helping to explain their effect in physiological TCR signaling. Our approach also provides general insights into SRC-family kinase dynamics.

T cells are an essential cell type of our adaptive immune system, helping to both detect and eliminate pathogens that may have infected our bodies. This essential function is mediated by the T cell antigen receptor complex (TCR) expressed at the plasma membrane, which can interrogate the intracellular state of host cells by scanning for pathogen-derived peptides presented on the surface of host cells. For us to remain healthy, T cells must be able to respond extremely robustly to ensure a resolution of the diseased state, even when there are only a few TCR ligands expressed on the infected cells. In PNAS, Feng et al. (1) show that exertion of force on the TCR during ligand binding is required for the extraordinary potency toward ligand binding observed in vivo. The ligand for the TCR is a pathogen-derived peptide bound within the MHC protein (pMHC). In contrast to receptors that bind soluble ligands, the pMHC ligand is a membrane protein expressed at the surface of antigen-presenting cells. This inherently leads to tension, or a pulling force, being generated between the two cells upon TCR/pMHC engagement. This is compounded by the relatively small height of the receptor complex (∼13 nm) compared with the apposition of two cells (∼100 nm) normally restricted by the glycocalyx surrounding cells, driving the two membranes away from their natural resting state. The forces typically found for biologically relevant processes are in the low pico-Newton (pN; 10−12 N) range. Experimentally manipulating proteins with forces of this magnitude can be achieved using a variety of biophysical methods (2, 3). In the current study, Feng et al. (1) used an optical trap (4), also known as tweezers, to apply a force on the TCR complex in a direct and quantitative manner. This was achieved by trapping a polystyrene … [↵][1]1Email: jj400{at}cam.ac.uk. [1]: #xref-corresp-1-1

Background: Sickle cell disease (SCD) is a multi-system disease affecting millions of people. The disease is characterized by vaso-occlusive crises (VOCs), resulting in acute and chronic pain, end-organ damage and life-threatening complications. The symptoms experienced by patients, caused largely by multi-cellular adhesion leading to vaso-occlusion and vasculopathy, significantly affect their quality of life (QoL) and ability to work/study. Understanding the effect of SCD on patients' daily lives can inform management of the disease and improve patients' QoL. Aims: To assess the impact of SCD on patients' daily lives, including physical symptoms, emotional wellbeing and economic burden, based on the worldwide SWAY survey. Methods: SWAY is an ongoing multi-country, cross-sectional survey of SCD patients, caregivers and treating physicians. Data are collected using self-completed or proxy surveys and categories include demographics, symptoms and impact of disease (physical, emotional and financial) and need for a caregiver. Where relevant, questions include a 7-point severity scale for each statement, with a score of 5 to 7 indicating 'high severity/impact'. Patient enrollment is via treating physicians and patient association groups. The enrollment target is approximately 2000 patients. Results: To date, 1513 SCD patients (48% male, mean age 24.1 years, 63% HbSS and 30% SC disease) have been surveyed from countries across the world, including the USA (365), UK (299), Nigeria (264), Ghana (255), Brazil (160) and Germany (91). The most commonly experienced symptoms of SCD were bone aches (63%), difficulty concentrating (49%), difficulty gaining weight (49%) and joint stiffness (41%). For these symptoms, 67%, 53%, 70% and 59% of patients rated them as 'high severity' on the 7-point scale, respectively. Background pain was present on an average of 2.9 days per week (standard deviation [SD]=2.2). Commonly experienced complications of SCD include fever (65%), joint issues (55%) and infections (55%). Patients reported a total of 7829 VOCs, with a mean of 5.2 VOCs per patient (SD=5.07) in the 12 months before survey completion; 38% of these VOCs resulted in overnight hospitalization, 24% were managed at home, 19% were treated in the emergency room and 19% of patients sought medical assistance in the community. A high impact on emotional wellbeing was reported by 61% of patients. The highest impact was caused by frustration with symptoms (60%), worry about disease worsening (59%) and worry about family/friends/children who care for them (54%). Only 42% of patients have received professional emotional support (eg psychiatrist, psychologist, counseling), but 67% reported a desire to receive this support. A high impact on household daily activities (eg food preparation, housework, childcare) and on family or social life was reported by 39% and 43% of patients, respectively, with 34% stating a high impact in terms of sexual desire/activity and 35% a high impact on relationships with spouse/partner. Physical activity was also affected in patients with SCD, with 60% reporting they avoid intense physical activity and 30% avoiding even mild physical activity, most commonly because of concerns about pain, exhaustion and dehydration (58%, 57% and 50%, respectively). Of the patients surveyed, 32% were employed, 8% not working but seeking employment, 6% not working and not seeking employment, 36% students, 1% retired, 13% on disability pay and 1% long-term sick leave. Employed patients reported SCD had a high impact on their ability to work, with 57% reducing their hours and 47% considering leaving their job. An average of 6.3 hours missed from work was reported in the 7 days prior to survey completion due to SCD. Half of patients believe that their income would be higher if they did not have SCD, 60% reported often missing school in the past and 52% felt that their disease has had a negative impact on their academic achievements. Conclusions: The acute and chronic symptoms associated with SCD have a substantial impact on patients' daily lives, including emotional and physical wellbeing, relationships and school/work. In addition, the acute symptoms associated with VOCs occur several times a year, resulting in frequent hospitalizations. Improved management of SCD would enhance patients' quality of life both at home and at school/work, likely leading to improved emotional wellbeing and a positive economic effect. Disclosures Osunkwo: Micella Biopharma: Other: DSMB Member ; Novartis: Consultancy, Speakers Bureau; Pfizer: Consultancy; Terumo: Speakers Bureau. Andemariam:Bluebird Bio: Membership on an entity's Board of Directors or advisory committees; Global Blood Therapeutics: Other: DSMB Member; Imara: Research Funding; NovoNordisk: Membership on an entity's Board of Directors or advisory committees; Sanofi Genzyme: Membership on an entity's Board of Directors or advisory committees; Terumo BCT: Membership on an entity's Board of Directors or advisory committees; Cyclerion: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Emmaus: Membership on an entity's Board of Directors or advisory committees; New Health Sciences: Membership on an entity's Board of Directors or advisory committees; Community Health Network of Connecticut: Consultancy. El-Rassi:Novartis Pharmaceuticals: Research Funding. Francis-Gibson:Sickle Cell Disease Association of America: Employment. Nero:Novartis: Consultancy. Minniti:Doris Duke Foundation: Research Funding. Trimnell:Global Blood Therapeutics: Consultancy; Novartis: Consultancy; Cyclerion: Consultancy. Abboud:AstraZeneca: Membership on an entity's Board of Directors or advisory committees, Research Funding; CRSPR Therapeutics: Membership on an entity's Board of Directors or advisory committees; Modus: Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Eli Lilly: Research Funding; GBT: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Other: Travel support; Novo Nordisk: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Colombatti:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Addmedica: Membership on an entity's Board of Directors or advisory committees; Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees. de Montalembert:AddMedica: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; bluebird bio, Inc: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Jastaniah:Novartis Pharmaceuticals: Consultancy, Honoraria. Nur:Novartis Pharmaceuticals: Consultancy. DeBonnett:Novartis Pharmaceuticals Corporation: Employment. James:Sickle Cell Society: Employment; Novartis: Honoraria.

Background: Sickle cell disease (SCD) is a complex genetic disorder affecting mainly people of African origin. A hallmark of SCD is vaso-occlusive crisis (VOC) - this event is acutely painful and the primary cause of hospitalization in SCD patients. VOC can lead to life-threatening complications such as acute chest syndrome. SCD is also associated with chronic complications including pulmonary hypertension, damage to organs and shortened life expectancy. Therefore, effective management and treatment strategies are essential to reduce burden of illness and ensure high quality of life for the patient. Aim: To survey the treatment and management strategies used by patients with SCD, and to determine patient satisfaction levels. Methods: SWAY is an ongoing multi-country, cross-sectional survey of SCD patients, caregivers and treating physicians. The survey, conducted online and in print, includes 6 categories: demographics, symptoms, impact of disease and use of a caregiver, impact on work and finances, disease management/treatment approaches, and patient-physician relationship. Where relevant, questions include a 7-point severity scale for each statement; a score of 5−7 indicates 'high severity/impact'. Patient enrollment is via treating physicians and patient association groups. The enrollment target is approximately 2000 patients. Results: To date, 1513 SCD patients (48% male, mean age 24.1 years, 63% HbSS and 30% HbSC disease) have been surveyed from 11 countries across North and South America, Europe and Africa. When considering the main person responsible for SCD treatment and management, patients primarily reported management by an SCD specialist (59% of patients) or GP/family doctor (20%). Most patients were satisfied with the frequency of interaction with their doctor (78%) and reported they are confident they are being assessed and treated properly (66%; based on high-impact scores 5−7). Accordingly, 60% of patients (scoring 5−7) reported sharing the same goals for SCD management and treatment as their doctor. The most common treatment goals for patients are to improve quality of life (80% of patients), prevent SCD worsening (59%), improve long-term survival (42%) and improve overall symptoms (40%). Patients reported receiving ongoing treatment with folic acid (58%), antibiotics (37%), anti-inflammatories (37%), over-the-counter pain medication (37%), opioids (35%), hydroxyurea (23%), blood transfusions (10%) and L-glutamine (4%). Having surgery or a medical procedure to manage their SCD was reported by 47% of patients, with gall bladder removal (16%), port placement (15%) and splenectomy (11%) being the most frequent. Although 63% of patients (scoring 5−7) indicated satisfaction with their treatment received to manage their SCD, 75% of patients (scoring 5−7) agreed they would like an alternative treatment to their current pain management medication, and 67% of patients would like additional professional emotional support. In the 12 months before survey completion, 7829 VOCs were reported (mean of 5.2 VOCs per patient); 8% of patients experienced 0 VOCs, 51% experienced 1−4 VOCs and 40% experienced ≥5 VOCs. Of these, 38% of VOCs led to overnight hospitalization, 24% were managed at home and 19% were treated in the emergency room. The main reasons that patients chose to manage their VOCs at home include a previous poor experience at hospital (40%), the opinion that medical assistance was not required (28%), the perception that medical professionals do not understand SCD (27%) and the cost of hospital treatment (22%; 41% of patients have no health insurance). Patients who self-managed their VOCs primarily did so with rest/sleep (73%), by drinking fluids (72%) and with opioid-based analgesia (58%). Conclusions: This interim analysis of the SWAY survey suggests that although many patients report satisfaction with their current level of management and treatment, there is still a need for additional healthcare support and alternative treatments. Underlining this, many patients experiencing VOCs do not seek medical assistance despite the potential for life-threatening complications, and >75% of patients surveyed are not receiving hydroxyurea even though the majority are cared for by SCD specialists. Further data collection and analysis will highlight any geographic differences in SCD management strategies and help identify any region-specific unmet patient needs. Disclosures James: Novartis: Honoraria; Sickle Cell Society: Employment. Andemariam:NovoNordisk: Membership on an entity's Board of Directors or advisory committees; New Health Sciences: Membership on an entity's Board of Directors or advisory committees; Emmaus: Membership on an entity's Board of Directors or advisory committees; Cyclerion: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Imara: Research Funding; Global Blood Therapeutics: Other: DSMB Member; Community Health Network of Connecticut: Consultancy; Bluebird Bio: Membership on an entity's Board of Directors or advisory committees; Sanofi Genzyme: Membership on an entity's Board of Directors or advisory committees; Terumo BCT: Membership on an entity's Board of Directors or advisory committees. El-Rassi:Novartis Pharmaceuticals: Research Funding. Francis-Gibson:Sickle Cell Disease Association of America: Employment. Nero:Novartis: Consultancy. Minniti:Doris Duke Foundation: Research Funding. Trimnell:Novartis: Consultancy; Global Blood Therapeutics: Consultancy; Cyclerion: Consultancy. Abboud:Novo Nordisk: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Modus: Research Funding; CRSPR Therapeutics: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Research Funding; Amgen: Other: Travel support; Eli Lilly: Research Funding; AstraZeneca: Membership on an entity's Board of Directors or advisory committees, Research Funding; GBT: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Colombatti:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Addmedica: Membership on an entity's Board of Directors or advisory committees; Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees. de Montalembert:AddMedica: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; bluebird bio, Inc: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Jastaniah:Novartis: Consultancy, Honoraria. Nur:Novartis: Consultancy. DeBonnett:Novartis Pharmaceuticals Corporation: Employment. Osunkwo:Micella Biopharma: Other: DSMB Member ; Terumo: Speakers Bureau; Pfizer: Consultancy; Novartis: Consultancy, Speakers Bureau.

The platelet receptor CLEC-2 binds to the snake venom toxin rhodocytin and the tumor cell surface protein podoplanin. Binding of either of these ligands promotes phosphorylation of a single tyrosine residue in the YXXL motif in the intracellular domain of CLEC-2. Phosphorylation of this tyrosine initiates binding of spleen tyrosine kinase (Syk) and triggers further downstream signaling events and ultimately potent platelet activation and aggregation. However, it is unclear how a single YXXL motif can interact efficiently with Syk, which usually recognizes two tandem YXXL repeats presented as an immunoreceptor tyrosine-based activation motif (ITAM). Using bioluminescence resonance energy transfer, coimmunopreciptitation, recombinant protein expression and analytical gel filtration chromatography, surface plasmon resonance, Western blotting, multiangle light scattering (MALS), and analytical ultracentrifugation, we show that CLEC-2 exists as a non-disulfide-linked homodimer which could allow each Syk molecule to interact with two YXXL motifs, one from each CLEC-2 monomer.

Understanding the component stoichiometry of the T cell antigen receptor (TCR) triggering apparatus is essential for building realistic models of signal initiation. Recent studies suggesting that the TCR and other signaling-associated proteins are preclustered on resting T cells relied on measurements of the behavior of membrane proteins at interfaces with functionalized glass surfaces. Using fluorescence recovery after photobleaching, we show that, compared with the apical surface, the mobility of TCRs is significantly reduced at Jurkat T cell/glass interfaces, in a signaling-sensitive manner. Using two biophysical approaches that mitigate these effects, bioluminescence resonance energy transfer and two-color coincidence detection microscopy, we show that, within the uncertainty of the methods, the membrane components of the TCR triggering apparatus, i.e. the TCR complex, MHC molecules, CD4/Lck and CD45, are exclusively monovalent or monomeric in human T cell lines, implying that TCR triggering depends only on the kinetics of TCR/pMHC interactions. These analyses also showed that constraining proteins to two dimensions at the cell surface greatly enhances random interactions versus those between the membrane and the cytoplasm. Simulations of TCR-pMHC complex formation based on these findings suggest how unclustered TCR triggering-associated proteins might nevertheless be capable of generating complex signaling outputs via the differential recruitment of cytosolic effectors to the cell membrane.

The T cell receptor (TCR) expressed on most T cells is a protein complex consisting of TCRαβ heterodimers that bind antigen and cluster of differentiation (CD) 3εδ, εγ, and ζζ dimers that initiate signaling. A long-standing controversy concerns whether there is one, or more than one, αβ heterodimer per complex. We used a form of single-molecule spectroscopy to investigate this question on live T cell hybridomas. The method relies on detecting coincident fluorescence from single molecules labeled with two different fluorophores, as the molecules diffuse through a confocal volume. The fraction of events that are coincident above the statistical background is defined as the “association quotient,” Q . In control experiments, Q was significantly higher for cells incubated with wheat germ agglutinin dual-labeled with Alexa488 and Alexa647 than for cells incubated with singly labeled wheat germ agglutinin. Similarly, cells expressing the homodimer, CD28, gave larger values of Q than cells expressing the monomer, CD86, when incubated with mixtures of Alexa488- and Alexa647-labeled antibody Fab fragments. T cell hybridomas incubated with mixtures of anti-TCRβ Fab fragments labeled with each fluorophore gave a Q value indistinguishable from the Q value for CD86, indicating that the dominant form of the TCR comprises single αβ heterodimers. The values of Q obtained for CD86 and the TCR were low but nonzero, suggesting that there is transient or nonrandom confinement, or diffuse clustering of molecules at the T cell surface. This general method for analyzing the subunit composition of protein complexes could be extended to other cell surface or intracellular complexes, and other living cells.

Triggering of the T cell receptor initiates a signaling cascade resulting in the activation of the T cell. These signals are integrated alongside those resulting from the triggering of other receptors whose function is to modulate the overall response. CD5 is an immunotyrosine-based inhibition motif-bearing receptor that antagonizes the overt T cell receptor activation response by recruiting inhibitory intracellular mediators such as SHP-1, RasGAP, or Cbl. We now propose that the inhibitory effects of CD5 are also mediated by a parallel pathway that functions at the level of inhibition of Fyn, a kinase generally associated with T cell receptor-mediated activation. After CD5 ligation, phosphorylation of the negative regulatory tyrosine (Tyr(531)) of Fyn increases, and this correlates with a substantial reduction in the kinase activity of Fyn and a profound inhibition of ZAP-70 activation. The effect requires the last 23 amino acids of the cytoplasmic domain of the receptor, strongly implying the involvement of a new CD5-interacting signaling or adaptor protein. Furthermore, we show that upon CD5 ligation there is a profound shift in its distribution from the bulk fluid phase to the lipid raft environment, where it associates with Fyn, Lck, and PAG. We suggest that the relocation of CD5, which we also show is capable of forming homodimers, to the proximity of raft-resident molecules enables CD5 to inhibit membrane proximal signaling by controlling the phosphorylation and activity of Fyn, possibly by interfering with the disassembly of C-terminal Src kinase (Csk)-PAG-Fyn complexes during T cell activation.

Nicotine improves cognitive enhancement and there are indications that neurodegenerative (age-related) cognitive disorders could be treated with nicotine-based drugs. The zebra finch is a well-recognized model to study cognitive functioning; hence this model could be used to study the effects of nicotine in neurodegenerative cognitive disorders. However, nicotine's in vivo physiological and behavioral effects have never been studied in the zebra finch. Here we present the first in vivo nicotine study in zebra finches. We evaluated the dose-response effects of nicotine on locomotor activity, song production, food intake and body weight. A liquid chromatography tandem mass spectrometry method was developed and validated for quantification of nicotine and cotinine in feces. The subcutaneous nicotine drug regiment (0.054-0.54mg/kg) induced physiologically significant values of nicotine and cotinine. The mid (0.18mg/kg) and high (0.54mg/kg) dose of nicotine promoted the development and expression of a sensitized response of song production and locomotor activity. Food intake and body weight were not affected following nicotine exposure. In conclusion, the zebra finch can be used as an innovative animal model not only in nicotine-related research studying cognitive functioning, but also in studies examining nicotine dependence and addictive mechanisms.

The lymphatic endothelial hyaluronan (HA) receptor Lyve-1 is a member of the Link protein superfamily most similar to the leukocyte HA receptor CD44. However, the structure of Lyve-1 and the nature of its interaction with ligand are obscure. Here we present new evidence that Lyve-1 is functionally distinct from CD44. Using truncation mutagenesis we confirm that Lyve-1 in common with CD44 contains an extended HA-binding unit, comprising elements flanking the N and C termini of the consensus lectin-like Link module, bridged by a third conserved disulfide linkage that is critical for HA binding. In addition, we identify six essential residues Tyr-87, Ile-97, Arg-99, Asn-103, Lys-105, and Lys-108 that define a compact HA-binding surface on Lyve-1, encompassing the epitope for an adhesion-blocking monoclonal antibody 3A, in an analogous position to the HA-binding surface in CD44. The overtly electrostatic character of HA binding in Lyve-1 and its sensitivity to ionic strength (IC(50) of 150 mm NaCl) contrast markedly with CD44 (IC(50) > 2 m NaCl) in which HA binding is mediated by hydrogen bonding and hydrophobic interactions. In addition, unlike the extended Link module in CD44, which binds HA efficiently when expressed as a soluble monomer (K(d) = 65.7 mum), that of Lyve-1 requires artificial dimerization, although the full ectodomain is active as a monomer (K(d) = 35.6 mum). Finally, full-length Lyve-1 did not form stable dimers in binding-competent 293T transfectants when assessed using bioluminescent resonance energy transfer. These results reveal that elements additional to the extended Link module are required to stabilize HA binding in Lyve-1 and indicate important structural and functional differences with CD44.

The Lewis (LEW) strain of rat appears more sensitive to nicotine than other strains in self administration, conditioned place preference, and drug discrimination behavioral studies. The present study sought to further evaluate the behavioral effects of chronic nicotine treatment in the LEW strain by assessing spontaneous activity, which has consistently revealed sensitization to chronic nicotine administration in Sprague Dawley (SD) rats. High active and low active male and female LEW rats (N = 8 per group) were treated twice daily with either nicotine (0.4 mg/kg, sc) or vehicle for 14 consecutive days. Regardless of baseline activity level or sex, spontaneous activity was significantly decreased, compared to saline-treated rats, after a single nicotine injection. However, spontaneous activity increased in both low and high activity rats (both sexes) over the two-weeks of nicotine administration to levels that were significantly higher than saline-treated rats. Based on these findings, acute and chronic nicotine administration had greater suppressive and enhancing effects on spontaneous activity in LEW rats compared to other strains of rats previously studied. These results further clarify the behavioral sensitivity of the LEW strain of rat to nicotine exposure and lend credence to the role of genetics in the individual susceptibility to nicotine dependence.

Ferroportin is a multi-transmembrane glycoprotein that mediates iron export from cells. Mutations in ferroportin are linked to type IV hemochromatosis, a dominantly inherited disorder of iron metabolism. Multimers of ferroportin, whose existence may relate to the dominant inheritance pattern of disease, have been detected in some studies but not others. We looked for evidence of multimerization in several different types of experiment. We assayed the maturation of mutant and wild-type ferroportin and found that loss-of-function mutants had a reduced half-life but did not alter the stability of coexpressed wild-type. Using bioluminescence resonance energy transfer analysis, we tested how mature wild-type ferroportin behaved in intact live cell membranes. Ferroportin-ferroportin interactions gave the very low acceptor/donor ratio-independent energy transfer levels characteristic of random protein-protein interactions, consistent with ferroportin behaving as a monomer. Consistent with these experiments, we were unable to detect a dominant negative functional effect of mutant ferroportin on wild-type, even when expression of wild-type protein was titrated to low levels. These data suggest that dominantly inherited ferroportin disease does not result from the direct action of a mutated protein inhibiting a wild-type protein within multimers. We propose other possible mechanisms of disease.

Acute behavioral tolerance to nicotine is a well-established phenomenon in animals, although previous drug discrimination studies have suggested that subpopulations of rats may exist that fail to exhibit acute tolerance to nicotine. The present study sought to determine whether these findings might extend to the conditioned taste aversion (CTA) paradigm. In experiment 1, rats were administered 0.8 mg/kg s.c. nicotine or vehicle before a pairing session with saccharin, and then administered either 0.4 mg/kg nicotine or vehicle immediately afterward. Rats treated with vehicle pre-session and then nicotine post-session developed a significant CTA to nicotine, whereas rats treated with 0.8 mg/kg nicotine s.c. pre-session failed to develop a CTA to nicotine. In experiment 2, rats were identified as exhibiting or failing to exhibit acute tolerance to nicotine in a drug discrimination paradigm. Rats that produced acute nicotine tolerance failed to develop a CTA after 24 h, but did so after 48 h, while rats that did not produce nicotine tolerance exhibited CTA after 24 h, but not after 48 h. These findings indicate that acute behavioral tolerance to nicotine extends to its aversion effects. Drug Dev Res 68:522–528, 2007. © 2008 Wiley-Liss, Inc.

Individuals vary in their susceptibility to nicotine addiction. However, there is little evidence that behavioral sensitivity to nicotine is dependent upon the functional state of nicotinic cholinergic receptors (nAChRs).This study aims to determine the relationship between in vivo behavioral desensitization and in vitro desensitization of nAChR function.Male Sprague-Dawley rats trained to discriminate nicotine were tested for development of acute behavioral tolerance. The rats were injected with nicotine (0.4 mg/kg free base, s.c.), tested for nicotine discrimination for 2 min, then injected with the same dose of nicotine 90, 180, and 270 min after the first injection and tested for nicotine discrimination after each injection. Susceptibility of nAChRs of individual rats to desensitization was assessed by use of the (86)Rb(+) efflux assay using synaptosomes prepared from the "thalamus," which included the hypothalamus and midbrain as well as the thalamic nuclei. To desensitize nAChRs, synaptsosomes were superfused with low concentrations of nicotine (5, 10, 20, and 30 nM) before stimulation of (86)Rb(+) efflux with nicotine (10 muM).The slopes of the behavioral desensitization were plotted as a function of the decline of nicotine-stimulated (86)Rb(+) efflux after in vitro desensitization. A significant correlation was observed between the in vitro desensitization of thalamic (86)Rb(+) efflux and the extent of behavioral desensitization of individual rats.These findings are consistent with the idea that production of acute behavioral tolerance by nicotine is related to its ability to induce nAChR desensitization at the cellular level.

The rapid and selective regulation of a target protein within living cells that contain closely related family members is an outstanding challenge. Here we introduce genetically directed bioorthogonal ligand tethering (BOLT) and demonstrate selective inhibition (iBOLT) of protein function. In iBOLT, inhibitor-conjugate/target protein pairs are created where the target protein contains a genetically encoded unnatural amino acid with bioorthogonal reactivity and the inhibitor conjugate contains a complementary bioorthogonal group. iBOLT enables the first rapid and specific inhibition of MEK isozymes, and introducing photoisomerizable linkers in the inhibitor conjugate enables reversible, optical regulation of protein activity (photo-BOLT) in live mammalian cells. We demonstrate that a pan kinase inhibitor conjugate allows selective and rapid inhibition of the lymphocyte specific kinase, indicating the modularity and scalability of BOLT. We anticipate that BOLT will enable the rapid and selective regulation of diverse proteins for which no selective small molecule ligands exist.

This article reviews publisher and vendor use of library advisory boards. While they are not new to the information industry, library advisory boards have assumed new importance. From the industry perspective, library advisory boards provide valuable market feedback concerning products and services. For librarians, membership on library advisory boards is an opportunity to learn about new products, provide feedback on existing products, and better understand the information marketplace. Recent literature is reviewed and a number of publishers and vendors are surveyed. The uses and organization of advisory boards and their value to both companies and the library community are presented.

Nicotine tolerance is often accompanied by an upregulation of brain area nicotinic acetylcholine receptors (nAChRs) in both animal and human subjects. This upregulation has been hypothesized to result from repeated or prolonged exposures of these receptors to nicotine. To explore this further, this study examined the level of nAChR desensitization following acute and repeated nicotine administration in the male Lewis rat. Nicotine-stimulated 86 Rb + efflux was measured in synaptosomes prepared from the frontal cortex, hippocampus, striatum, and thalamus. Analysis of receptor functionality was achieved by calculating area-under-the-curve (AUC) for nicotine-induced fractional 86 Rb + efflux. Nicotine-stimulated 86 Rb + efflux from all brain regions was significantly less in rats that received an acute injection of 0.4 mg/kg nicotine (s.c.) 15 min prior to dissection compared to control rats. This decrease in nAChR functional status was also observed in rats treated with 1 day or 14 days of twice-daily nicotine administration. These results are consistent with the concept that acute exposure to nicotine induces rapid desensitization of nAChRs. In addition, following repeated exposure to nicotine, nAChRs did not become tolerant to the loss in receptor function that occurs after an initial nicotine administration. Overall, these data suggest that neuronal adaptations underlying nicotine tolerance may begin upon initial exposure then persist following repeated exposures.

Individuals vary in their susceptibility to nicotine addiction. However, there is little evidence that behavioral sensitivity to nicotine is dependent upon the functional state of nicotinic cholinergic receptors (nAChRs).To determine the relationship between in vivo pharmacological desensitization (in other words, acute tolerance) and brain regional nAChR function.Male Sprague-Dawley rats, trained to discriminate nicotine (0.4 mg/kg free base) from saline in a two-lever drug discrimination task, were tested for the development of acute tolerance. Rats were injected with 0.4 mg/kg nicotine, tested for nicotine discrimination for 2 min, then injected with the same dose of nicotine 90 min, 180 min, and 270 min after the first injection and tested for nicotine discrimination after each injection. These subjects were separated into two groups, desensitizers (DZ) and nondesensitizers (NDZ), based upon performance in the repetitive dosing drug discrimination paradigm. The sensitivity of nAChRs in specific brain regions of these two groups was assessed by the use of an 86Rb+ efflux assay using synaptosomes prepared from the frontal cortex, hippocampus, striatum, and "thalamus," which included the midbrain and hypothalamus as well as the thalamus.The nicotine-induced increase in 86Rb+ efflux was significantly greater in NDZ as compared to DZ in the "thalamus." There was no statistically significant difference in the effects of nicotine in the frontal cortex, hippocampus, and striatum of these two groups. A significant correlation was observed between thalamic 86Rb+ efflux and the rate of behavioral desensitization of individual rats.These findings are consistent with the concept that the production of acute tolerance by nicotine in vivo correlates directly with its ability to induce nAChR desensitization at the cellular level.

Individual and strain variability in the effects of nicotine suggests the involvement of a genetic component in nicotinic cholinergic receptor (nAChR) function, which may help explain nicotine's variable behavioral and pharmacological effects in different individuals. The present study evaluated differential responses to the discriminative stimulus (DS) and rewarding properties of nicotine in Lewis (LEW) and Fischer-344 (F-344) rats. Drug discrimination (DD) data suggest that the LEW rat is more sensitive to nicotine as LEW rats acquired the nicotine discrimination at a dose of 0.4 mg/kg, whereas F-344 rats acquired the dose of 0.9 mg/kg (all nicotine doses expressed as free base). Similarly, LEW rats exhibited nicotine-conditioned place preference (CPP) at 0.6 mg/kg, whereas the F-344 rats did not. Subsequent testing with a higher dose (0.9 mg/kg) failed to maintain the nicotine-CPP in the LEW rats. Conversely, nicotine-place preference in the F-344 rats was not changed at the higher dose. Taken together, these results suggest potential differences of sensitivities in LEW and F-344 rats to the rewarding and discriminative stimulus (DS) properties of nicotine. These findings support previous research by demonstrating that the F-344 rat is less sensitive to nicotine compared to the LEW rat.

Colorimetric test strip assays are a convenient and inexpensive means for the determination of cotinine in human urine because they can be performed in a nonlaboratory environment using a trained technician. Four hundred human urine samples were separated into four categories: (1) heavy smokers (>20 cigarettes smoked per day), (2) light smokers (

The interaction, binding, and colocalization of two or more molecules in living cells are essential aspects of many biological molecular processes, and single-molecule technologies for investigating these processes in live cells, if successfully developed, would become very powerful tools. Here, we developed simultaneous, dual-color, single fluorescent molecule colocalization imaging, to quantitatively detect the colocalization of two species of individual molecules. We first established a method for spatially correcting the two full images synchronously obtained in two different colors, and then for overlaying them with an accuracy of 13 nm. By further assessing the precision of the position determination, and the signal/noise and signal/background ratios, we found that two single molecules in dual color can be colocalized to within 64-100 nm (68-90% detectability) in the membrane of cells for GFP and Alexa633. The detectability of true colocalization at the molecular level and the erroneous inclusion of incidental approaches of two molecules as colocalization have to be compromised at different levels in each experiment, depending on its purpose. This technique was successfully demonstrated in living cells in culture, monitoring colocalization of single molecules of E-cadherin fused with GFP diffusing in the plasma membrane with single molecules of Alexa633 conjugated to anti-E-cadherin Fab externally added to the culture medium. This work established a benchmark for monitoring the colocalization of two single molecules, which can be applied to wide ranges of studies for molecular interactions, both at the levels of single molecules and collections of molecules.

The prototypic nicotinic acetylcholinergic receptor (nAChR) agonist, nicotine, is one of the primary psychoactive ingredients of tobacco. This review examines the effects of nicotine and similar compounds in nicotine discrimination procedures, a paradigm in which the subjective effects of nicotine can be modeled in animals. Results of these studies have shown that the discriminative stimulus effects of nicotine are shared by nicotine analogues and by the novel nicotinic agonists, epibatidine and ABT-418, and can be antagonized by the nAChR channel blocker, mecamylamine. Individual and strain differences in sensitivity to the discriminative stimulus effects of nicotine have been noted, suggesting the possibility of genetic differences in the functioning of nACh receptors or related processes. In human smokers, individual variability is also observed ; however, the degree of within smoker (but between situation) variability far exceeds between smoker variability. Further research into the ways in which situational factors affect the discriminative stimulus effects of nicotine is needed.

Bispecific antibodies that retarget cytotoxic T cell activity to kill cancer cells are currently under clinical evaluation. However, the molecular mechanism for how CD3-bispecific antibodies 'trigger' intracellular T cell signaling is not known. We demonstrate that bispecific antibodies invoke an equivalent biophysical mechanism of TCR triggering as that observed for the TCR/pMHC interaction, including target clustering and exclusion of CD45 phosphatase from the synapse. The dimensions of the target molecule play a key role in the efficiency of the synapse formation. However, we demonstrate that rational epitope selection can overcome the spatial inhibition caused by target molecules with a large extracellular domain and result in efficient synapse formation and highly potent T cell triggering. With this insight, we developed a novel T-cell dependent bispecific (TDB) antibody, anti-FcRH5/CD3 TDB, targeting the B cell lineage marker FcRH5 for multiple myeloma. Anti-FcRH5/CD3 TDB demonstrated cytotoxicity against human plasma cells and patient derived myeloma tumor cells at picomolar doses. Very low target expression level is sufficient to induce anti-FcRH5/CD3 TDB mediated killing, indicating broad activity in multiple myeloma where the prevalence of FcRH5 expression is 100%. In primates, anti-FcRH5/CD3 treatment resulted in complete depletion of tissue B cells and bone marrow plasma cells. Anti-FcRH5/CD3 TDB induces immunosuppressive feedback signaling, including PD1 up-regulation, which can be overcome by PD-L1 antibodies. These data demonstrate the potential for the anti-FcRH5/CD3 TDB, alone or in combination with inhibition of PD1/PDL1 signaling in the treatment of multiple myeloma and other B-cell malignancies. Disclosures Li: Genentech: Employment. Stagg:Genentech: Employment. Johnston:Genentech: Employment. DiCara:Genentech: Employment. Clark:Genentech: Employment. Cook:Genentech: Employment. Slaga:Genentech: Employment. Nakamura:Genentech: Employment. Luke:Genentech: Employment. Sukumaran:Genentech: Employment. Luis:Genentech: Employment. Ye:Genentech: Employment. Sumiyoshi:Genentech: Employment. Danilenko:Genentech: Employment. Lee:Genentech: Employment. Totpal:Genentech: Employment. Ellerman:Genentech: Employment. Hötzel:Genentech: Employment. Junttila:Genentech: Employment.