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Background. Sequential B cell targeted immunotherapy with BAFF antagonism (belimumab) and B cell depletion (rituximab) and may enhance B cell targeting in ANCA-associated vasculitis (AAV) through several mechanisms. Study Design. COMBIVAS is a randomised, double blind, placebo-controlled trial designed to assess the mechanistic effects of sequential therapy of belimumab and rituximab in patients with active PR3-AAV. The recruitment target is 30 patients who meet the criteria for inclusion in the per protocol analysis. Thirty-six participants have been randomised to one of two treatment groups in a 1:1 ratio; either rituximab plus belimumab or rituximab plus placebo (both groups with the same tapering corticosteroid regimen) and recruitment is now closed (final patient enrolled March 2021). For each patient, the trial will last for two years comprising a 12-month treatment period followed by a 12-month follow-up period. Participants. Participants have been recruited from five of seven UK trial sites. Eligibility criteria were age ³18 years, a diagnosis of AAV with active disease (newly diagnosed or relapsing disease), along with a concurrent positive test for PR3-ANCA by ELISA. Interventions. Rituximab 1000mg was administered by intravenous infusions on Day 8 and Day 22. Weekly subcutaneous injections of 200mg belimumab or placebo were initiated a week before rituximab on Day 1 and then weekly through to Week 51. All participants received a relatively low prednisolone (20 mg/day) starting dose from Day 1 followed by a protocol-specified corticosteroid taper aiming for complete cessation by 3 months. Outcomes. The primary endpoint of this study is time to PR3 ANCA negativity. Key secondary outcomes include change from baseline in naïve, transitional, memory, plasmablast B cell subsets (by flow cytometry) in blood at Months 3, 12, 18 and 24, time to clinical remission, time to relapse, and incidence of serious adverse events. Exploratory biomarker assessments include assessment of B cell receptor clonality, B cell and T cell functional assays, whole blood transcriptomic analysis and urinary lymphocyte and proteomic analysis. Inguinal lymph node and nasal mucosal biopsies have been performed on a subgroup of patients at baseline and Month 3. Discussion. This experimental medicine study provides a unique opportunity to gain detailed insights into the immunological mechanisms of belimumab-rituximab sequential therapy across multiple body compartments in the setting of AAV. Trial status. The final patient was recruited in March 2021. Last subject last visit will be March 2023. ClinicalTrials.gov Identifier: NCT03967925, May 30, 2019.

Ultrasound has previously been demonstrated to non-invasively cause tissue disruption. Small animal studies have demonstrated that this effect can be enhanced by contrast microbubbles and has the potential to be clinically beneficial in techniques such as targeted drug delivery or enhancing liquid biopsies when a physical biopsy may be inappropriate. Cavitating microbubbles in close proximity to cells increases membrane permeability, allowing small intracellular molecules to leak into the extracellular space. This study sought to establish whether cavitating microbubbles could liberate cell-specific miRNAs, augmenting biomarker detection for non-invasive liquid biopsies. Insonating human polarized renal proximal tubular epithelial cells (RPTECs), in the presence of SonoVue microbubbles, revealed that cellular health could be maintained while achieving the release of miRNAs, miR-21, miR-30e, miR-192 and miR-194 (respectively, 10.9-fold, 7.17-fold, 5.95-fold and 5.36-fold). To examine the mechanism of release, RPTECs expressing enhanced green fluorescent protein were generated and the protein successfully liberated. Cell polarization, cellular phenotype and cell viability after sonoporation were measured by a number of techniques. Ultrastructural studies using electron microscopy showed gap-junction disruption and pore formation on cellular surfaces. These studies revealed that cell-specific miRNAs can be non-specifically liberated from RPTECs by sonoporation without a significant decrease in cell viability.

Introduction Biclonal lymphoid disorders, when two distinct lymphoproliferative disorders (LPD) co-exist, are rare (incidence of 1.4%) and associated with a poor prognosis. NOTCH1 mutations occur in 10% of CLL at diagnosis, associated with a short disease-free interval and increased risk of Richter's transformation. We hypothesised that the incidence of NOTCH1 mutations in CLL with a second LPD may be increased, because the mutation occurs early in leukaemogenesis, permitting clonal divergence. Methods We identified 19 patients with biclonal LPD at diagnosis: 11 with CLL and a second LPD (group A) and 8 with a second distinct CLL (group B). NOTCH1 mutation analysis was performed and clinical outcome investigated. Results Ten of 19 (52%) were NOTCH1 mutated: 5 in group A (45%) and 5 in group B (62.5%) with a favourable clinical outcome observed among this cohort with 28.7 (range 1-99) months of follow-up. Conclusion In conclusion, we identified a significant (52%) incidence of NOTCH1 mutations in CLL in the context of biclonal LPD, associated with an indolent clinical course.

Small noncoding RNA, microRNAs (miRNAs), are emerging as important modulators in the pathogenesis of kidney disease, with potential as biomarkers of kidney disease onset, progression or possibly outcome from treatment. Bulk tissue small RNA-Sequencing (sRNA-seq) and microarrays are widely used to identify dysregulated miRNA expression but are limited by the lack of precision regarding the cellular origin of the miRNA. In this study, we performed cell-specific sRNA-seq on tubular cells, endothelial cells, fibroblasts and macrophages isolated from the injured and repairing kidneys of the murine reversible unilateral ureteric obstruction model. We devised an unbiased bioinformatics pipeline to define the miRNA enrichment within these cell populations, constructing a miRNA catalogue of injury and repair. Our analysis reveals that a significant proportion of cell-specific miRNAs in healthy animals were no longer specific following injury. We then apply this knowledge of the relative cell specificity of miRNAs to deconvolute bulk miRNA expression changes in kidney disease from murine models and human renal disease kidney samples comprised of mixed cell types. Finally, we use our data-driven approach to rationally select and demonstrate macrophage enriched miR-16-5p and miR-18a as promising urinary biomarkers of acute kidney injury in renal transplant recipients.

Background Elevated B lymphocyte stimulator (BLyS) levels in patients with systemic lupus erythematosus (SLE) correlate positively with disease activity; BLyS expression is directly linked to interferon (IFN) pathway activation. This post hoc meta-analysis of BLISS-52 and BLISS-76 explored the relationship between baseline BLyS mRNA/protein levels and/or type 1 IFN-inducible gene signature (IFN-1) and responses to the BLyS-targeting monoclonal antibody belimumab in SLE. Methods In BLISS-52 and BLISS-76, patients with autoantibody-positive SLE and a SELENA-SLEDAI score ≥ 6 and receiving stable standard SLE therapy were randomised to intravenous belimumab 10 mg/kg or placebo, plus standard of care (SoC), for 52 or 76 weeks. For this post hoc meta-analysis, patients with an appropriate mRNA sample were stratified by BLyS mRNA expression (tertiles: high/medium/low; revised quantiles: high/low), IFN-1 mRNA expression (high/low) and BLyS protein level (high/low). Co-primary endpoints were correlation between baseline BLyS and IFN-1 mRNA levels and SLE Responder Index (SRI)4 response at week 52 within BLyS/IFN-1 subgroups. Secondary endpoints included time to first severe SELENA-SLEDAI Flare Index (SFI) flare. Results Of 554 patients included in this analysis, 281 had received belimumab and 273 had received placebo. Baseline BLyS and IFN-1 mRNA levels were highly correlated (Spearman’s rank correlation coefficient 0.7799; 95% confidence interval [CI] 0.7451, 0.8106; p p = 0.0153), high BLyS mRNA quantile (OR 1.58; 95% CI 1.02, 2.44; p = 0.0402), high IFN-1 mRNA (OR 1.58; 95% CI: 1.08, 2.31; p = 0.0186) and high BLyS protein (OR 3.57; 95% CI 1.63, 7.83; p = 0.0015) subgroups only. The risk of severe SFI flare was significantly lower with belimumab than placebo in the high BLyS mRNA quantile (hazard ratio [HR] 0.59; 95% CI 0.36, 0.97; p = 0.0371) and high BLyS protein (HR 0.39; 95% CI 0.19, 0.79; p = 0.0090) subgroups. Conclusions This post hoc meta-analysis demonstrated a tendency towards improved response to add-on intravenous belimumab 10 mg/kg versus SoC alone in patients with high baseline BLyS protein and IFN-1 mRNA levels and medium/high BLyS mRNA levels.

Summary Background B cells produce alloantibodies and activate alloreactive T cells, negatively affecting kidney transplant survival. By contrast, regulatory B cells are associated with transplant tolerance. Immunotherapies are needed that inhibit B-cell effector function, including antibody secretion, while sparing regulators and minimising infection risk. B lymphocyte stimulator (BLyS) is a cytokine that promotes B-cell activation and has not previously been targeted in kidney transplant recipients. We aimed to determine the safety and activity of an anti-BLyS antibody, belimumab, in addition to standard-of-care immunosuppression in adult kidney transplant recipients. We used an experimental medicine study design with multiple secondary and exploratory endpoints to gain further insight into the effect of belimumab on the generation of de-novo IgG and on the regulatory B-cell compartment. Methods We undertook a double-blind, randomised, placebo-controlled phase 2 trial of belimumab, in addition to standard-of-care immunosuppression (basiliximab, mycophenolate mofetil, tacrolimus, and prednisolone) at two centres, Addenbrooke's Hospital, Cambridge, UK, and Guy's and St Thomas' Hospital, London, UK. Participants were eligible if they were aged 18–75 years and receiving a kidney transplant and were planned to receive standard-of-care immunosuppression. Participants were randomly assigned (1:1) to receive either intravenous belimumab 10 mg per kg bodyweight or placebo, given at day 0, 14, and 28, and then every 4 weeks for a total of seven infusions. The co-primary endpoints were safety and change in the concentration of naive B cells from baseline to week 24, both of which were analysed in all patients who received a transplant and at least one dose of drug or placebo (the modified intention-to-treat [mITT] population). This trial has been completed and is registered with ClinicalTrials.gov, NCT01536379, and EudraCT, 2011–006215–56. Findings Between Sept 13, 2013, and Feb 8, 2015, of 303 patients assessed for eligibility, 28 kidney transplant recipients were randomly assigned to receive belimumab (n=14) or placebo (n=14). 25 patients (12 [86%] patients assigned to the belimumab group and 13 [93%] patients assigned to the placebo group) received a transplant and were included in the mITT population. We observed similar proportions of adverse events in the belimumab and placebo groups, including serious infections (one [8%] of 12 in the belimumab group and five [38%] of 13 in the placebo group during the 6-month on-treatment phase; and none in the belimumab group and two [15%] in the placebo group during the 6-month follow-up). In the on-treatment phase, one patient in the placebo group died because of fatal myocardial infarction and acute cardiac failure. The co-primary endpoint of a reduction in naive B cells from baseline to week 24 was not met. Treatment with belimumab did not significantly reduce the number of naive B cells from baseline to week 24 (adjusted mean difference between the belimumab and placebo treatment groups −34·4 cells per μL, 95% CI −109·5 to 40·7). Interpretation Belimumab might be a useful adjunct to standard-of-care immunosuppression in renal transplantation, with no major increased risk of infection and potential beneficial effects on humoral alloimmunity. Funding GlaxoSmithKline.

BackgroundImmunosuppressant drugs reduce proteinuria and anti-phospholipase A2 receptor autoantibodies (PLA2R-Ab) in primary membranous nephropathy (PMN) with varying success and associated toxicities. This study aimed to evaluate the effect of belimumab on proteinuria and PLA2R-Ab in participants with PMN.MethodsIn this prospective, open-label, experimental medicine study, 14 participants with PMN and persistent nephrotic-range proteinuria received up to 2 years belimumab monotherapy (10 mg/kg, every 4 weeks). Changes in proteinuria (urinary protein:creatinine ratio), PLA2R-Ab, albumin, cholesterol, B-cell subsets and pharmacokinetics were analysed during treatment and up to 6 months after treatment.ResultsEleven participants completed to the primary endpoint (Week 28) and nine participants completed the study. In the intention-to-treat population population, baseline proteinuria of 724 mg/mmol [95% confidence interval (CI) 579–906] decreased to 498 mg/mmol (95% CI 383–649) and 130 mg/mmol (95% CI 54–312) at Weeks 28 and 104, respectively, with changes statistically significant from Week 36 (n = 11, P = 0.047). PLA2R-Ab decreased from 174 RU/mL (95% CI 79–384) at baseline to 46 RU/mL (95% CI 16–132) and 4 RU/mL (95% CI 2–6) at Weeks 28 and 104, respectively, becoming statistically significant by Week 12 (n = 13, P = 0.02). Nine participants achieved partial (n = 8) or complete (n = 1) remission. Participants with abnormal albumin and/or cholesterol at baseline gained normal/near normal levels by the last follow-up. Adverse events were consistent with those expected in this population.ConclusionsBelimumab treatment in participants with PMN can reduce PLA2R-Ab and subsequently proteinuria, important preludes to remission induction.

Background GPA and MPA (related types of ANCA-associated vasculitis [AAV]) are organ-/life-threatening systemic vasculitides. B cells and increased circulating BLyS (a B cell survival factor) are implicated in AAV pathogenesis, suggesting a role for BLyS in these vasculitides. BEL is an anti-BLyS human immunoglobulin (Ig) G1λ monoclonal antibody. Objectives Examine the effects of BEL plus azathioprine (AZA), on biomarkers in patients with AAV for remission maintenance following standard induction (IND) with glucocorticoids (GC) and CYC or RTX. Methods This double-blind, placebo-controlled, multicentre study (BEL115466/NCT01663623) randomised (1:1) patients (≥18 years) with new/relapsing AAV following IND (oral or IV CYC or RTX), to AZA 2 mg/kg/day and oral GC, plus IV BEL 10 mg/kg or PBO (Days 0, 14, 28 and every 28 days until completion). Remission was defined as Birmingham Vasculitis Activity Score=0, plus GC ≤10 mg/day. The study was truncated after initiation (n~300 to~100) due to revised AAV standard of care (SoC) affecting recruitment. Biomarker endpoints (serum Igs, B cells and ANCA [anti-MPO/PR3]) were measured at baseline (BL) and thereafter. Summaries by IR were post hoc; no analyses were performed. Results At BL, RTX patients had B cell counts≤LLQ. CYC patients had notably low BL B cell counts: the lowest were in oral-CYC patients. Circulating memory B cells (CD20+/CD27+) increased rapidly with BEL, then gradually returned to BL (CYC); no major changes occurred with PBO. BEL had no impact on the proportion of naive CD20 +CD27− B cells vs PBO, post CYC IND. The number of RTX patients with quantifiable data was low; partial reconstitution occurred in a minority of patients (PBO 2/13; BEL 4/14) and did not translate into vasculitis relapses. Overall Ig levels declined more noticeably with BEL vs PBO; suggesting BEL affects antibody-secreting cells. ANCA+patients were similar between groups (PBO 32/50; BEL 30/49). No trends in change in ANCA status over time occurred, regardless of IR. Individual patient data showed no apparent trends between ANCA titres and AAV activity. Conclusions Choice of IR for active AAV affects B cell dynamics. BEL pharmacodynamic effects occurred in patients with AAV receiving SoC. Given the small sample size and high variability, data must be interpreted with caution. Acknowledgements Study funded by GSK. Sam Halliwell, PhD, Fishawack Indicia Ltd, UK, provided editorial assistance funded by GSK. Disclosure of Interest D. Jayne Grant/research support from: GSK, Consultant for: GSK, D. Blockmans: None declared, R. Luqmani Grant/research support from: Arthritis Research UK, GSK, MRC, UCSF/OIF, Canadian Institutes of Health Research and The Vasculitis Foundation, Consultant for: Roche, GSK, Medpace and MedImmune, B. Ji Shareholder of: GSK, Employee of: GSK, Y. Green Shareholder of: GSK, Employee of: GSK, L. Hall Shareholder of: GSK, Employee of: GSK, D. Roth Shareholder of: GSK, Employee of: GSK, R. Henderson Shareholder of: GSK, Employee of: GSK, P. Merkel Grant/research support from: Actelion, Bristol-Myers Squibb, CaridianBCT, Celgene, ChemoCentryx, Genentech/Roche, GSK, Kypha, MedImmune/AstraZeneca, American College of Rheumatology, European League Against Rheumatism, National Institutes of Health (NHLBI, NIAMS, NIAID, NCATS, ORDR), US Food and Drug Administration, The Patient-Centred Outcomes Research Institute and The Vasculitis Foundation, Consultant for: Actelion, Alexion, Boston Pharm., Bristol-Myers Squibb, ChemoCentryx, Genzyme/Sanofi, GSK, Genentech/Roche, InflaRx, PrincipioBio, Proteon and Seattle Genetics

Primary immune thrombocytopenia is an autoimmune disorder in which platelet destruction is a consequence of both B- and T-cell dysregulation. Flow cytometry was used to further characterize the B- and T-cell compartments in a cross-sectional cohort of 26 immune thrombocytopenia patients including antiplatelet antibody positive (n=14) and negative (n=12) patients exposed to a range of therapies, and a cohort of matched healthy volunteers. Markers for B-cell activating factor and its receptors, relevant B-cell activation markers (CD95 and CD21) and markers for CD4(+) T-cell subsets, including circulating T-follicular helper-like cells, were included. Our results indicate that an expanded population of CD95(+) naive B cells correlated with disease activity in immune thrombocytopenia patients regardless of treatment status. A population of CD21-naive B cells was specifically expanded in autoantibody-positive immune thrombocytopenia patients. Furthermore, the B-cell maturation antigen, a receptor for B-cell activating factor, was consistently and strongly up-regulated on plasmablasts from immune thrombocytopenia patients. These observations have parallels in other autoantibody-mediated diseases and suggest that loss of peripheral tolerance in naive B cells may be an important component of immune thrombocytopenia pathogenesis. Moreover, the B-cell maturation antigen represents a potential target for plasma cell directed therapies in immune thrombocytopenia.

Neutrophils are the first immune cells to migrate into infected tissue sites. Therefore an important step in the initiation of an immune response is the synthesis of the neutrophil-recruiting chemokines. In this in vivo study in mice, we show that resident tissue macrophages are the source of the major neutrophil chemoattractants, KC and MIP-2. Synthesis of these chemokines is rapidly regulated at the transcriptional level by signaling through TLR2, TLR3, and TLR4 that have diverse specificities for pathogens. The major and alternative TLR signaling pathways are characterized by the adaptor proteins MyD88 or TRIF, respectively. KC and MIP-2 are both produced by signaling through MyD88. However MIP-2, but not KC, is also synthesized through the TRIF adaptor protein, identifying it as a new product of this alternative pathway. Use of both pathways by TLR4 ensures maximal levels of KC and MIP-2 that lead to robust neutrophil recruitment. However the MIP-2 generated exclusively by the TRIF pathway is still sufficient to cause an influx of neutrophils. In summary we show that TLR signaling by tissue macrophages directly controls the synthesis of neutrophil-attracting chemokines that are essential for the earliest recruitment step in the innate immune response to microbial challenge.

SummaryThe integrin leukocyte function-associated antigen-1 (LFA-1) is important in the promotion of B cell adhesion, thereby facilitating immunological synapse (IS) formation and B cell activation. Despite this significance, the associated signaling mechanisms regulating LFA-1 activation remain elusive. Here, we show that both isoforms of the small GTPase Rac expressed by primary B cells, Rac1 and Rac2, were activated rapidly downstream of Src-family kinases, guanine-nucleotide exchange factors Vav1 and Vav2, and phosphoinositide-3 kinase (PI3K) after BCR engagement. We identify Rac2, but not Rac1, as critical for B cell adhesion to intercellular adhesion molecule-1 (ICAM-1) and IS formation. Furthermore, B cells expressing constitutively active Rac2 are highly adhesive. We observe that Rac2-deficient B cells exhibit lower amounts of Rap1-GTP and severe actin polymerization defects, identifying a potential mechanism underlying their behavior. We postulate that this critical role for Rac2 in mediating B cell adhesion and IS formation might apply in all lymphocytes.

The T lymphocyte (or T cell) has classically been perceived to be a passive circular cell attaching to other cells or fibrils of the extracellular matrix when its integrins become activated. We now understand that the modus operandi of the T cell is migration. These cells are proving to be impressively fast migrators, clocking up basal speeds of approximately 10-15 microm/min which makes them amongst the fastest movers recorded to date. Therefore, migration is the business of the T cell and in this review we will discuss how its motility is regulated and what functions this activity makes possible.

Early neutrophil entry into an inflammatory site is thought to mediate a chemokine switch, inducing subsequent monocyte recruitment through the regulation of monocyte chemoattractant protein-1 (MCP-1) release. As the murine monocyte is poorly characterized and difficult to identify, there has been little examination of either its early recruitment in inflammatory models or of the factors that influence its early migration. The phenotyping of rapidly recruited inflammatory leukocytes with 7/4 and Gr-1 monoclonal antibodies (mAbs) identifies 2 distinct populations, which we characterize as murine monocytes and neutrophils. Monocytes migrate in the first 2 hours of inflammation making use of α4β1 but not of Mac-1 or lymphocyte function-associated antigen-1 (LFA-1) integrins. Early migration is dependent on MCP-1, but neither MCP-1 release nor monocyte recruitment is affected by the reduced neutrophil migration seen in LFA-1-/- mice. Endogenous peritoneal macrophages and mesothelial cells lining the peritoneum contain MCP-1, which is released following thioglycollate stimulation. The murine monocyte therefore responds rapidly to chemokines produced in situ by tissue cells at the site of inflammation with no requirement for prior influx of neutrophils. (Blood. 2003;102:328-335)

Biclonal lymphoid disorders (BLD) are a rare (1.4%) but distinct entity in which Chronic lymphocytic leukaemia (CLL) co-exists with a second lymphoproliferative disorder (LPD) and are poorly understood in terms of pathogenesis and clinical impact. The NOTCH1 pathway is involved in cell proliferation, cell cycle progression and is anti-apoptotic, with mutations of the regulatory PEST domain causing constitutive up-regulation of the pathway. NOTCH1 mutations occur early in CLL leukaemogenesis and are found in 10% of poor prognosis CLL at diagnosis as well as being associated with 50% of Richter's transformation (both Hodgkin's and non-Hodgkin's subtype). We hypothesised that NOTCH1 mutations may be implicated in BLD pathogenesis both because they occur early in leukaemogenesis when clonal divergence is possible and because of its association with Hodgkin's Richter's, which is biologically distinct from CLL. We identified 19 patients with BLD at initial presentation, confirmed by flow cytometry and/or molecular analysis of the Immunoglobulin heavy (IgH) and light (IgL) chain genes between 2008 and 2015. This cohort consisted of 14 males and 5 females with a mean age of 72 (range 47-90) years. BLD was defined as follows: (1) co-existence of cells with different immunophenotypes such as a CD5+/CD19+ and CD5-/CD10- population confirming the co-existence of 2 diagnoses, (2) demonstration of both kappa (κ) and lambda (λ) light chain restriction on a CD5+/CD19+ population and (3) CD5+/CD19+ population without demonstrable surface light chains but with >2 clonal gene rearrangements of their IgH and IgL loci. All 19 cases were tested for a c.7544-7545delCT resulting in truncation of the regulatory C-terminal PEST domain of the NOTCH1 gene by PCR. Eleven of 19 patients had CLL co-existing with a different LPD as follows: Mantle Cell Lymphoma (MCL) (2), Hairy Cell Leukaemia (HCL) (2) and Marginal Zone Lymphoma (7). Of this subgroup 5 of 11 cases (45%) had a NOTCH1 mutation. Four samples in our cohort displayed dual κ and λ light chain expression detected by flow cytometry and confirmed molecularly by identifying 2 clonally distinct populations of LPD and 3 samples (75%) had a NOTCH1 mutation. Four samples were identified with >90% CD5+/CD19+ expression but no monoclonality by flow cytometry, but with 2 distinct populations identified by IgH and IgL rearrangements and of this subgroup 2 (50%) had NOTCH1 mutations. Clinical data was available on 13/19 patients, of whom 11 patients had Binet A and 2 Binet B disease. No patient has required CLL-directed treatment, 2 patients have died of other causes and 11 remain under surveillance. Three of 9 patients with a second LPD have required treatment: both patients with HCL received cladribine for pancytopaenia and a patient with MCL was treated with Rituximab and Bendamustine. Overall, 10 of the 19 (52%) patients with BLD had a NOTCH1 mutation at diagnosis, which is higher than the 10% reported in CLL literature. The absence of any germinal centre cell derived lymphoma in the cohort of patients with additional LPD is intriguing and needs confirmation in other series. The prognostic implications of BLD and NOTCH1 mutations are not possible to assess in this small cohort, but accurate characterization of BLD aids therapeutic decision-making as in the case of concurrent CLL and HCL. Light chain restriction by flow cytometry in a mature B-cell population confirms monoclonality and suggests malignancy, however dual κ and λ expressing CLL may give a polyclonal pattern resulting in failure to make a CLL diagnosis; therefore awareness of this rare subtype of CLL is important. In summary we have added a further association between NOTCH1 mutations and CLL associated disorders which currently includes: poor prognosis CLL at diagnosis, Hodgkin's and non Hodgkin's Richter's transformation and most recently BLD. Increased sensitivity of NOTCH1 detection by next generation sequencing techniques may provide a further insight into the evolutionary origin and pathogenesis of BLD and further elucidate the enigmatic role of NOTCH1. Disclosures Crotty: BMS, Takeda, Novartis, Janssen, Roche: Honoraria.

Background Belimumab is a B-lymphocyte stimulator (BLyS)-specific inhibitor indicated for the treatment of adult patients with active, autoantibody-positive, systemic lupus erythematosus. Belimumab has also been shown to reduce anti-phospholipase A2 receptor (PLA2R) autoantibodies and proteinuria in a mechanistic study in primary membranous nephropathy (PMN) 1 . Here we report an interim analysis of B and T cell phenotypes in PMN subjects treated with belimumab to further elucidate the mechanism of action in autoantibody mediated disease. Objectives To characterize changes in B and T lymphocyte phenotypes in PMN patients treated with belimumab, as a comparison with known pharmacodynamic (PD) effects in SLE 2 , and to assess correlation with anti-PLA2R autoantibody. Methods 14 anti-PLA2R autoantibody positive PMN patients received 10mg/kg iv belimumab every 4 wks (or 2 wks) for up to 100 wks. 11 of these completed at least 28 wks treatment. Whole blood was collected at baseline, Wks 8, 16 and 28 for analysis of B and T-cell phenotypes by flow cytometry. Serum levels of anti-PLA2R antibodies were measured by ELISA (Euroimmun). Statistical analysis to identify PD-induced changes in lymphocyte subsets was carried out by paired t-test (2 tailed) against baseline. Correlations between anti-PLA2R antibody and B and T-cell subsets, either at a specific time point or in the linearised trend over time, were calculated using Pearson9s correlation coefficient (r). Results There was a significant reduction (-63%, p=0.002) in absolute numbers of naive B cells (CD19+ CD27-) at Wk 8 which was sustained through to Wk 28. There was a significant increase in absolute numbers of memory B cells (CD19+ CD27+) at Wk 8 (70%, p=0.023) which increased further through to Wk 28. Plasmablasts (or SLE subset, CD19lo CD38hi CD27hi) showed a trend at Wk 8 to decrease (-64%, p=0.20) but with high variance and low cell numbers. In addition, the expression (mean fluorescence intensity) of BAFF-R (B-cell activating factor receptor) on B-memory cells increased through Wk 28, while expression of TACI (transmembrane activator and calcium modulator and cyclophylin ligand interactor) decreased. Activated cells as identified by surface CD95 expression, decreased as a proportion of B-memory cells through Wk 28. No clear trend was seen in T-regulatory or T-activated cells. There was a significant negative correlation between decrease in absolute numbers of naive B-cells and immunological efficacy defined as level of anti-PLA2R antibody remaining at Wk 28 (r=-0.87, p=0.0005). Correlations were also seen between antibody and changes in proportion of B-memory CD95+ cells. Conclusions This mechanistic trial of belimumab in PMN has enabled a novel analysis of PD endpoints, confirming but also extending the PD effects previously reported in studies of SLE. Changes in B-cell subsets were associated with improvements in levels of autoantibody in PMN which in turn correlates with proteinuria. Further analysis of longer term anti-PLA2R antibody kinetics as related to early changes in B and T cell phenotypes may also be informative. References Willcocks L, Barrett C, Brenchley P et al. Nephrology Dialysis Transplantation. 2015; 30 (Supplement 3): iii32–iii33. Stohl W, Hiepe F, Latinis KM et al. Arthritis & Rheumatism. 2012;64(7):2328–2337. Acknowledgement This study was funded by GSK (study sponsor) (BEL116472, NCT01610492). Disclosure of Interest C. Barrett Shareholder of: GlaxoSmithKline, Employee of: GlaxoSmithKline, R. Henderson Shareholder of: GlaxoSmithKline, Employee of: GlaxoSmithKline, N. Galwey Shareholder of: GlaxoSmithKline, Employee of: GlaxoSmithKline, S. Flint Grant/research support from: GlaxoSmithKline, A. Gibson Shareholder of: GlaxoSmithKline, Employee of: GlaxoSmithKline, C. Savage Shareholder of: GlaxoSmithKline, Employee of: GlaxoSmithKline

The generation of reactive oxygen species (ROS) by the nicotinamide adenine dinucleotide phosphate oxidase is an important mechanism by which neutrophils kill pathogens. The oxidase is composed of a membrane-bound cytochrome and 4 soluble proteins (p67phox, p40phox, p47phox, and GTP-Rac). These components form an active complex at the correct time and subcellular location through a series of incompletely understood mutual interactions, regulated, in part, by GTP/GDP exchange on Rac, protein phosphorylation, and binding to lipid messengers. We have used a variety of assays to follow the spatiotemporal assembly of the oxidase in genetically engineered primary mouse neutrophils, during phagocytosis of both serum- and immunoglobulin G-opsonized targets. The oxidase assembles directly on serum-Staphylococcus aureus–containing phagosomes within seconds of phagosome formation; this process is only partially dependent (∼ 30%) on PtdIns3P binding to p40phox, but totally dependent on Rac1/2 binding to p67phox. In contrast, in response to immunoglobulin G-targets, the oxidase first assembles on a tubulovesicular compartment that develops at sites of granule fusion to the base of the emerging phagosome; oxidase assembly and activation is highly dependent on both PtdIns3P-p40phox and Rac2-p67phox interactions and delivery to the phagosome is regulated by Rab27a. These results define a novel pathway for oxidase assembly downstream of FcR-activation.

Rac1 and Rac2 GTPases transduce signals from multiple receptors leading to cell migration, adhesion, proliferation, and survival. In the absence of Rac1 and Rac2, B cell development is arrested at an IgD− transitional B cell stage that we term transitional type 0 (T0). We show that T0 cells cannot enter the white pulp of the spleen until they mature into the T1 and T2 stages, and that this entry into the white pulp requires integrin and chemokine receptor signaling and is required for cell survival. In the absence of Rac1 and Rac2, transitional B cells are unable to migrate in response to chemokines and cannot enter the splenic white pulp. We propose that loss of Rac1 and Rac2 causes arrest at the T0 stage at least in part because transitional B cells need to migrate into the white pulp to receive survival signals. Finally, we show that in the absence of Syk, a kinase that transduces B cell antigen receptor signals required for positive selection, development is arrested at the same T0 stage, with transitional B cells excluded from the white pulp. Thus, these studies identify a novel developmental checkpoint that coincides with B cell positive selection.

This chapter describes methods for the analysis of B and T lymphocyte development in mice deficient in Rac1 and/or Rac2 GTPases. The development of both B and T cells is critically dependent on transition through checkpoints monitoring for correct rearrangement of antigen receptor genes. Progression through these checkpoints depends on signaling from the antigen receptors. In addition, signals from cytokine, chemokine, Notch, and death receptors play important roles in the survival, proliferation, and migration of developing lymphocytes. Analysis of these processes in mice deficient in these GTPases can illuminate their roles in transducing signals from these different receptors.

Rac GTPases are believed to contribute to migration in leukocytes by transducing signals from cell surface receptors to the actin and microtubule cytoskeletons. Mammals have three closely related Rac isoforms, Rac1, Rac2 and Rac3, and it is widely assumed that cell migration requires the activity of these Rac GTPases. We have previously shown that Rac1-null mouse macrophages have altered cell shape and reduced membrane ruffling but normal migration speed. Here we investigate the behaviour of macrophages lacking Rac2 (Rac2–/–) or Rac1 and Rac2 (Rac1/2–/–). Rac2–/– macrophages have reduced F-actin levels and lack podosomes, which are integrin-based adhesion sites, and their migration speed is similar to or slightly slower than wild-type macrophages, depending on the substrate. Unexpectedly, Rac1/2–/– macrophages, which do not express Rac1, Rac2 or Rac3, migrate at a similar speed to wild-type macrophages on a variety of substrates and perform chemotaxis normally, although their morphology and mode of migration is altered. However, Rac1–/– and Rac1/2–/– but not Rac2–/– macrophages are impaired in their ability to invade through Matrigel. Together, these data show that Rac1 and Rac2 have distinct roles in regulating cell morphology, migration and invasion, but are not essential for macrophage migration or chemotaxis.

The role of Rac family proteins in platelet spreading on matrix proteins under static and flow conditions has been investigated by using Rac-deficient platelets. Murine platelets form filopodia and undergo limited spreading on fibrinogen independent of Rac1 and Rac2. In the presence of thrombin, marked lamellipodia formation is observed on fibrinogen, which is abrogated in the absence of Rac1. However, Rac1 is not required for thrombin-induced aggregation or elevation of F-actin levels. Formation of lamellipodia on collagen and laminin is also Rac1-dependent. Analysis of platelet adhesion dynamics on collagen under flow conditions in vitro revealed that Rac1 is required for platelet aggregate stability at arterial rates of shear, as evidenced by a dramatic increase in platelet embolization. Furthermore, studies employing intravital microscopy demonstrated that Rac1 plays a critical role in the development of stable thrombi at sites of vascular injury in vivo. Thus, our data demonstrated that Rac1 is essential for lamellipodia formation in platelets and indicated that Rac1 is required for aggregate integrity leading to thrombus formation under physiologically relevant levels of shear both in vitro and in vivo.

Summary(1) A compound frequently noted in a chromatographic study of urinary amino acids has now been isolated from the urines of two cancer patients and identified as beta-aminoisobutyric acid (BAIB). (2) In a group of 140 apparently healthy subjects, 52 individuals showed detectable urinary levels of BAIB (>0.15 millimole per liter), and three of these showed concentrations over 1 millimole per liter. Individual control subjects studied at intervals for periods up to 21/2 years showed little fluctuation in BAIB excretion. (3) In individual patients with neoplastic disease, the urinary BAIB level occasionally varied markedly and appeared to bear some relationship to the neoplastic process. Such variations are illustrated by a report of a case of chronic myelogenous leukemia. (4) Theoretical considerations pointed to a 5-methylpyrimidine as a likely precursor of BAIB, and evidence favoring this view was obtained by rat experiments in which BAIB excretion was produced by a diet containing large amounts of ...