Journal: Science translational medicine

Article Title: The anti-CD40L monoclonal antibody AT-1501 promotes islet and kidney allograft survival and function in nonhuman primates

doi: 10.1126/scitranslmed.adf6376

Figure Lengend Snippet: (A) CD40L binding potency of AT-1501 (cyan) and Hu5C8 (orange) was determined by ELISA with recombinant human CD40L (rhuCD40L). Abatacept (purple), a variant of CTLA4-Ig that interferes with the CD28-CD80/86 costimulatory pathway, was used as a negative control. A450, absorbance at 450nm. (B) AT-1501 and Hu5C8 binding to human FcγRI, FcγRIIa, FcγRIIIa, and FcγRIIIb was measured by ELISA. (C to F) PAC-1 expression was measured in platelets from healthy controls incubated with soluble CD40L alone (C), Hu5C8 and sCD40L (D), Hu5C8 F(Ab’)2 lacking an Fc region and sCD40L (E), or AT-1501 and sCD40L (F). Blue tracings represent expression of PAC-1 following incubation with 5μg/mL sCD40L alone and red tracings represent expression of PAC-1 following incubation with the indicated combinations. (G) Platelet aggregation was measured in vitro using platelets incubated with the indicated combinations.

Article Snippet: ELISAs were run with Hu5C8 (ProSci, XP-5263BT), an anti-CD40L antibody, as a positive control.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Recombinant, Variant Assay, Negative Control, Expressing, Incubation, In Vitro

Journal: Vaccines

Article Title: Tracking B Cell Memory to SARS-CoV-2 Using Rare Cell Analysis System

doi: 10.3390/vaccines11040735

Figure Lengend Snippet: In vitro culture system to differentiate spike-RBD-specific cells into antibody secreting cells. ( A – C ) Concentration of neutralizing anti-spike-RBD IgG antibodies in culture supernatant over time from PBMC stimulated with R848, IL-2, IL-21, sCD40, and WT-RBD. ( D ) Total neutralizing anti-spike-RBD IgG concentrations in culture supernatant after 3 days of stimulation for each time point. The number of subjects (n) is shown below the graph. IU indicates the international units. The error bar indicates that the median and interquartile range. Statistics were calculated using the non-parametric Mann–Whitney test. The dotted line indicated the limit of detection (LOD) for the assay. ** p < 0.01; **** p < 0.0001; ns, no significant difference.

Article Snippet: Cryopreserved PBMCs were cultured at a density of 1 × 10 6 cells/mL and stimulated with R848 (1 μg/mL; MabTech, Human IgG ELISpot kit, 3850-2A), IL-2 (10 ng/mL; MabTech, Nacka Strand, Sweden, Human IgG ELISpot kit, 3850-2A), IL-21 (100 ng/mL; PeproTech, 200-21), sCD40 ligand (1 μg/mL; PeproTech, 310-02), and SARS-CoV-2 spike-RBD (1 μg/mL; R&D system) and then maintained in RPMI 1640 (Gibco; Thermo Fisher Scientific, Waltham, MA, USA, 61870036) containing 10% FBS, and 1% penicillin/streptomycin (Gibco; Thermo Fisher Scientific, 10378016) for 3 days.

Techniques: In Vitro, Concentration Assay, MANN-WHITNEY

Journal: Vaccines

Article Title: Tracking B Cell Memory to SARS-CoV-2 Using Rare Cell Analysis System

doi: 10.3390/vaccines11040735

Figure Lengend Snippet: Spike-RBD-specific cells induced by SARS-CoV-2 vaccination over time. ( A ) Timeline for blood draws for analysis of nAbs in plasma, the secretion from stimulated PBMCs, or cellular identification with the MiSelect R II. T4 was 4 weeks after a 3rd vaccination, while T5 was a blood draw 6 months later. ( B ) Concentration of neutralizing anti-spike-RBD IgG antibodies in plasma samples from vaccinated subjects over time. ( C ) Total number of spike-RBD-specific MBCs in PBMC samples from all vaccinated subjects for each time point. ( D ) Concentration of neutralizing anti-spike-RBD IgG antibodies in culture supernatant over time from PBMC stimulated with R848, IL-2, IL-21, sCD40, and WT-RBD. The error bar indicates the median. Statistics were calculated using the non-parametric Mann–Whitney test. The dotted line indicated the limit of detection (LOD) for the assay. * p < 0.05; ** p < 0.01; *** p < 0.001; ns, no significant difference.

Article Snippet: Cryopreserved PBMCs were cultured at a density of 1 × 10 6 cells/mL and stimulated with R848 (1 μg/mL; MabTech, Human IgG ELISpot kit, 3850-2A), IL-2 (10 ng/mL; MabTech, Nacka Strand, Sweden, Human IgG ELISpot kit, 3850-2A), IL-21 (100 ng/mL; PeproTech, 200-21), sCD40 ligand (1 μg/mL; PeproTech, 310-02), and SARS-CoV-2 spike-RBD (1 μg/mL; R&D system) and then maintained in RPMI 1640 (Gibco; Thermo Fisher Scientific, Waltham, MA, USA, 61870036) containing 10% FBS, and 1% penicillin/streptomycin (Gibco; Thermo Fisher Scientific, 10378016) for 3 days.

Techniques: Concentration Assay, MANN-WHITNEY