Fig 2: BAFF activates the alternative NF-ĸB pathway in patient-derived HCL-v cells. (A) Bonna-12 cells or (B,C) HCL patient-derived cancer cells (Patients (‘PT’) 131 or 154) were stimulated with 250 ng/mL of recombinant human BAFF. Cytoplasmic and nuclear lysates (Bonna-12 and PT 131) or total cell lysate (PT 154) was harvested after 16 h. Activation of the nonclassical NF-ĸB pathway is measured by the accumulation of p52 (A–C). AKT and ERK pathway activation is measured in total cell lysate from PT 154 by the accumulation of p-AKT and p-ERK relative to the total protein. Band intensities were measured using ImageJ software. The Bonna-12 Western was performed three independent times, and Westerns using either patient samples were performed at least twice. (D) HCL-v patient cells (PT 131) were stimulated with 500 ng/mL with or without 15 µg/mL of BAFF-neutralizing antibody. After 16 h of incubation, cytoplasmic and nuclear protein lysates were harvested, and activation of the nonclassical NF-ĸB pathway was measured by p52 accumulation.

Fig 3: Overview of BAFF induced gene signatures in HCL-v. Cancer cells derived from splenocytes of an HCL patient (Patient 195) were stimulated with 250 ng/mL of recombinant human BAFF for 16 h (n = 4 control, n = 4 BAFF stimulated). RNA was extracted after incubation and normalized to 50 ng/µL. Sequencing reads were aligned to the human reference genome (GRCh38) using the STAR aligner. Differential genes were identified with a significance cutoff of q-value < 0.05 using Benjamini–Hochberg multiple testing correction. (A) Principal component analysis (PCA) shows clustering across quadruplicate samples in each group. (B) The volcano plot represents 1447 differentially expressed genes. (C) Cytokine–cytokine receptor interaction, MAPK signaling, NF-ĸB signaling, and pathways in cancer were among the most significantly upregulated processes in the pathway analysis. (D) The ribbon plot represents the most significantly upregulated genes per process.

Fig 4: BAFF protects patient-derived HCL-v cells from cladribine. Patient-derived HCL-v cells (Patient 058) were plated at 400,000 cells per well and pre-incubated with 250 ng/mL recombinant human BAFF three days prior to cladribine (cDa) treatment. On day 1, cells were treated with 250 nM cDa and re-stimulated with rhBAFF. Cells were stimulated with BAFF again on day 2. (A) Proliferation and (B) viability were measured by trypan blue staining. The control well received the solvent control (0.1% DMSO). A one-way ANOVA with Tukey’s multiple hypothesis correction was used to measure significance over time (upper). p-values of proliferation (A) were 0.0086 (day 4) and 0.0006 (day 5). p-values for viability (B) were 0.0096 (day 3) and 0.0260 (day 5). The experiment was performed with this patient sample n = 2 times. * p < 0.05, ** p < 0.01, *** p < 0.001.

Fig 5: Organoids derived from secondary lymphoid tissue can be used to evaluate engineered cell responses to HIV Env based immunogens.A Schematic of organoid culture steps, where cryopreserved cells from biopsied LNs or spleens can be resuspended in media containing BAFF andinsulin/selenium/transferrin supplement and cultured in transwells. Engineered cells can be added to organoids at the time of culture to evaluate survival and phenotype over time. In addition, organoids can be vaccinated on the first day of culture (day-0) to evaluate endogenous and engineered responses. B Viability of all single cells in organoid cultures at the indicated time points over a 14-day period (n = 2–4 where error bars are present). Error bars represent standard deviation from the mean. C Phenotype of organoid B cells on day 0 and 14 of culture for three representative animals (n = 2–3 replicates per animal; error bars represent standard deviation). D (From top to bottom) Fold expansion of B cell phenotypes over time in negative control organoid (top row), organoids vaccinated with 1 µg/ml MD39-ferritin and SMNP (second row), engineered cells cultured in organoids (third row), and engineered cells cultured in organoids and vaccinated with 1 µg/ml MD39-ferritin and SMNP (bottom row). E Quantification of VRC01 IgG secreted by engineered cells with or without MD39-ferritin vaccination, as determined by anti-P2A ELISA. Each symbol represents a different donor, n = 2 for each donor, and error bars represent standard deviation from the mean. LOD is the lower limit of detection.