Human B cell in vitro assays
Frozen human peripheral blood mononuclear cells (PBMCs) from healthy donors or individuals with SLE (AllCells) were treated with lanraplenib or dimethyl sulfoxide (DMSO) before stimulation. To measure B cell survival, B cells were treated with human BAFF (R&D Systems, Inc., Minneapolis MN) or RP10; viability was measured using RealTime-Glo™ reagent (Promega Corporation, Madison WI). To measure B cell activation, B cells were stimulated with 20 µg/mL antihuman F(ab’)2 immunoglobulin M (IgM) (SouthernBiotech, Birmingham, AL) for 16 hours; activation was measured by expression of CD69 and CD86 (BD Biosciences, San Jose, CA) measured by fluorescence-activated cell sorting (FACS) (FACSCanto™ II, BD Biosciences).
Naïve B cells were isolated from freshly collected PBMCs (AllCells) by negative selection (STEMCELL Technologies). They were then treated with lanraplenib or DMSO before stimulation with a stimulation cocktail of F(ab’)2 antihuman immunoglobulin D (IgD) (SouthernBiotech), IL-2 and IL-10 (BioLegend®, San Diego, CA), and human MEGACD40L (Enzo Life Sciences, Inc., Farmingdale, NY) for 7 days. Cells were stained for expression of CD3, CD19, and CD27 (BioLegend) before analysis on an LSRFortessa™ X-20 (BD Biosciences). Supernatant antibody concentration was measured using a human antibody isotyping 7-plex immunoassay kit (Invitrogen ProcartaPlex, Carlsbad, CA) read on a FLEXMAP 3D® (Luminex Corporation, Austin, TX).
NZB/W study: Ethical statement
This basic study design and animal usage was approved by Bolder BioPATH’s Institutional Animal Care and Use Committee (IACUC) for compliance with regulations prior to study initiation (IACUC Protocol #BBP-005).
NZB/W study: Study design
Mice were housed at Bolder BioPATH until enrollment in the study at 28 weeks. Mice were assigned to one of 4 groups of 15 mice each (1 positive, 1 negative control group, 2 experimental groups) by distributing mice by body weight and proteinuria equivalently across groups. All mice were bled by retro-orbital sinus, at ages 28, 34, and 40 weeks for plasma for anti–dsDNA antibody analysis (Alpha Diagnostic) and at 29, 31, and 35 weeks for PK plasma. Urine was collected from all mice weekly to measure proteinuria by dipstick measurement. Body weight was measured weekly. Food consumption was measured daily. An overview of the study design is shown in Figure 2.
NZB/W study: Experimental procedures
Animals were housed 3–5/cage in filtered, pie-shaped polycarbonate cages (Animal Care Systems, Inc.) with wood chip bedding and suspended food and water bottles. Animal care including room, cage, and equipment sanitation conformed to the guidelines cited in the Guide for the Care and Use of Laboratory Animals (Guide, 2011) and the applicable standard operating procedures of Bolder BioPATH. During the acclimation and study periods, animals were housed in a laboratory environment with temperatures ranging 67–76°F and relative humidity of 30%–70%. Automatic timers provided 12 hours of light and 12 hours of dark. Animals were allowed access ad libitum to Harlan Teklad Rodent Chow and fresh municipal tap water. Mice were dosed orally with lanraplenib (0.075% or 0.25%) or vehicle-administered ad libitum in chow. Positive controls were treated with cyclophosphamide (5 mg/kg) administered daily intraperitoneally.
NZB/W study: Experimental animals
Female NZBWF1/J mice were purchased from The Jackson Laboratory (Bar Harbor, ME) at 20 weeks of age; additional female mice (n=15) that were 14 weeks old on arrival at study week 37 were obtained and used as predisease FACS analysis controls. Mice weighed from 30-55 grams from arrival at Bolder BioPATH until study initiation. At the end of study, mice were anesthetized with Isoflurane (VetOne) and bled by cardiac puncture for terminal plasma, serum, and whole blood. Spleens and kidneys were also harvested and weighed immediately. Spleens were processed and immunophenotyped by flow cytometry. Kidneys were fixed in 10% neutral buffered formalin before being embedded into paraffin. Kidney sections were stained with hematoxylin and eosin for histologic analysis. For mice that died or were euthanized prior to study termination, terminal clinical scores were carried through to study termination for analysis purpose.
Serum cytokine concentration
Serum cytokine concentrations were measured with a murine cytokine and chemokine 36-plex immunoassay (Invitrogen ProcartaPlex). Samples were analyzed using a FLEXMAP 3D (Luminex Corporation). Measurements of concentrations of proinflammatory cytokines were analyzed.
Splenocyte preparation and staining for flow cytometry
Cells were stained for FcR blocking antibodies (BioLegend) before staining with Bcl6, CD3, CD4, CD8, CD11b, CD11c, CD19, CD23, CD44, CD45R/B220, CD62L, CD93, CD138, CD172a, CXCR5, IgM, MHCII (BioLegend), and IgD (Invitrogen) in 4 separate panels. Stained samples were analyzed on a FACSCelesta, an LSR II, or an LSRFortessa X-20 (BD Biosciences). Data were analyzed using FlowJo software.
RNA extraction and gene expression analysis
Total RNA was extracted from formalin fixed paraffin-embedded scrolls of mouse kidney (Acepix Bioscience, Inc., Hayward, CA). The expression of 93 target genes and 3 housekeeping genes (RPL19, RPL0, and ACTB) were analyzed with standard Taqman assays using Fluidigm Biomark RT-qPCR (bioanalytic and single-cell core at UT Health at San Antonio). Cycle threshold (Ct) data were collected by GE 96x96 Standard v2 software. Ct values for each transcript were normalized using the geometric mean of ACTB, RPL19, and RPLP0 Ct values. P values were calculated by Mann-Whitney U test. Nominal P values are presented.
Kidney H&E histopathology measurements
20 glomeruli per sample were measured to determine mean glomerular diameter and crescent score. Glomerular diameter was scored as follows: 0: >65 µm; 0.5: 60-70 µm; 1: 71-80 µm; 2: 81-90 µm; 3: 91-100 µm; 4: 101-110 µm; 5: >110 µm. Crescent percentage was scored as follows: 0: no crescents; 0.5: single glomerulus with crescent; 1: 2-4% of glomeruli with crescents; 2: 5-10% of glomeruli with crescents; 3: 11-25% of glomeruli with crescents; 4: 25-50% of glomeruli with crescents; 5: >50% of glomeruli with crescents. Protein cast severity was scored as followed: 0: normal tubules; 0.5: affects 1-2% of cortex; 1: affects 3-10% of cortex; 2: affects 11-25% of cortex; 3: affects 26-50% of cortex; 4: affects 51-75% of cortex; 5: affects >75% of cortex. Interstitial inflammation was scored as follows: 0: no inflammation; 0.5: small focal area of mononuclear cells in pelvis only; 1: occasional small focal accumulation of mononuclear cells affecting >10% of total interstitium; 2: multifocal mononuclear infiltrates affecting 10-25% of cortex area; 3: multifocal mononuclear infiltrates affecting 26-50% of cortex area; 4: multifocal mononuclear infiltrates affecting 51-75% of cortex area; 5: diffuse mononuclear infiltration affecting 75-100% of cortex area. Vasclitis was scored as follows: 0: no vasculitis; 0.5: one vessel with minimal perivascular infiltrate; 1: 1-2 foci of perivascular infiltrates; 2: 3-4 foci of perivascular infiltrate without necrosis; 3: 5-6 foci of perivascular infiltrate with or without necrosis of vessel wall; 4: 7-8 foci of perivascular infiltrate with or without necrosis of vessel wall; 5: >8 foci of perivascular infiltrate with extensive necrosis. Summed scores represent sum of each of 5 individually measured criteria above.
IgG staining and scoring
Kidney sections were stained with Alexa Fluor 647 conjugated anti-mouse IgG antibody (Cell Signaling Technology®, 4410S) and DAPI. IgG deposition in glomeruli was scored into 4 grades based on the amount of fluorescence signal observed in each glomerulus: Grade 1: <25% deposit; Grade 2: 25%–49% deposit; Grade 3: 50%–74% deposit; Grade 4: ≥75% deposit. Approximately 200 glomeruli were scored from each mouse.
Statistics were calculated in Prism (GraphPad Software Inc.) unless otherwise specified. Pairwise comparisons were made by testing for normal distribution of data. Data normality was tested by D’Agnostine-Pearson omnibus normality test. Normally distributed data were analyzed by ANOVA and non-normally distributed data were analyzed by a Kruskal-Wallis test. ANOVA was corrected for multiple comparisons by the Holm-Sidak method; Kruskal-Wallis was corrected with a Dunn’s multiple comparison test. For analyses with samples below the limit of detection, the limit of detection was used as the quantified value. Effective concentration (EC)50 values were calculated using a 4-parameter variable slope nonlinear regression fitted by least squares. Nonlinear best fit regressions were calculated using GraphPad Prism.