Journal: bioRxiv

Article Title: A CD40-Targeted IL-21 Fusokine Enables Rapid Generation of Human IL-10⁺Granzyme B⁺ Regulatory B Cells

doi: 10.64898/2026.01.12.699061

Figure Lengend Snippet: (A) Amino acid sequence of the CD40scFv–IL-21 fusion protein (441 amino acids). The signal peptide is shown in red, the CD40-specific scFv in blue, the linker region in orange, and the IL-21 domain in black. A C-terminal spacer containing a TEV protease recognition site (ENLYFQG), followed by an additional spacer and an 8×His affinity tag, is included to facilitate purification and optional tag removal. (B) Representative FPLC chromatogram of Ni–NTA affinity purification. Conditioned medium from stable transduced HEK293T cells was pH-adjusted and loaded onto a 5 mL Ni–NTA column equilibrated in binding buffer. Elution was performed over 10 column bed volumes using a gradient to 100% buffer B. The dark shaded grey region indicates the pooled fractions containing purified CD40scFv–IL-21. (C) Ponceau S staining and Western blot analysis of purified CD40scFv–IL-21. The fusion protein was detected using an anti-human IL-21 antibody. Lane M, molecular weight markers; lane 1, recombinant human IL-21 (200 ng); lane 2, purified CD40scFv–IL-21; lane 3, conditioned media from transduced 293T cells. The expected molecular weight is 45.4 kDa. (D) Predicted three-dimensional structure of CD40scFv–IL-21 generated from the amino acid sequence using the Phyre2 protein modeling server .

Article Snippet: Human embryonic kidney 293T cells stably expressing the CD40scFv–IL-21 fusion protein (ATCC CRL-3216) were cultured in FreeStyleTM 293 Expression Medium (Thermo Fisher Scientific, Cat. #12338-018) under suspension conditions.

Techniques: Sequencing, Purification, Affinity Purification, Binding Assay, Staining, Western Blot, Molecular Weight, Recombinant, Generated

Journal: bioRxiv

Article Title: A CD40-Targeted IL-21 Fusokine Enables Rapid Generation of Human IL-10⁺Granzyme B⁺ Regulatory B Cells

doi: 10.64898/2026.01.12.699061

Figure Lengend Snippet: Human peripheral blood B cells were stimulated as indicated with CD40scFv–IL-21, CpG, or control conditions. Lane assignments were as follows: 1, unstimulated control; 2, CpG; 3, CpG, CD40 ligand (CD40L) and IL-21; 4, CpG and CD40scFv–IL-21; 5, CpG and IL-21. Whole-cell lysates were analyzed by immunoblotting for phosphorylated NF-κB p65 (p-NF-κB) and downstream signaling intermediates. IL-21–dependent signaling was preserved under these conditions. GAPDH and β-actin was used as a loading control. Representative blots from independent experiments are shown. Blots shown are representative of more than four independent experiments performed using B cells isolated from independent human blood donors.

Article Snippet: Human embryonic kidney 293T cells stably expressing the CD40scFv–IL-21 fusion protein (ATCC CRL-3216) were cultured in FreeStyleTM 293 Expression Medium (Thermo Fisher Scientific, Cat. #12338-018) under suspension conditions.

Techniques: Control, Western Blot, Isolation

Journal: bioRxiv

Article Title: A CD40-Targeted IL-21 Fusokine Enables Rapid Generation of Human IL-10⁺Granzyme B⁺ Regulatory B Cells

doi: 10.64898/2026.01.12.699061

Figure Lengend Snippet: (A) Principal component analysis (PCA) of RNA-seq data from purified human peripheral blood B cells cultured under four conditions: unstimulated B cells, CpG-stimulated B cells (B-CpG), CD40scFv–IL-21–stimulated B cells (B-ScFv), and B cells stimulated with the combination of CD40scFv–IL-21 and CpG (Bregs). PCA demonstrates clear separation among conditions, indicating distinct global transcriptional states induced by single versus combined stimulation. (B) Venn diagram showing the number of genes upregulated (log₂ fold change > 1) relative to unstimulated B cells in B-CpG, B-ScFv, and Bregs. The overlap highlights both shared and condition-specific transcriptional responses, with Bregs exhibiting a distinct set of upregulated genes not observed with single-stimulus activation. (C) Volcano plot depicting differential gene expression between Bregs and B-CpG cells. The x-axis represents log₂ fold change (Bregs vs B-CpG), and the y-axis represents −log₁₀ adjusted p-value. Genes significantly upregulated in Bregs include regulatory and effector-associated transcripts such as IL10, GZMB, CD274 (PD-L1), PDCD1LG2 (PD-L2) , and EBI3 , whereas CpG-associated inflammatory and chemokine-related genes are less prominent under combined stimulation. (D) Heat map showing expression of selected regulatory, checkpoint, transcriptional, and chemokine-associated genes comparing B-CpG and Bregs across three independent donors (D312, D919, D936). Expression values are displayed as log₂(FPKM + 1) and visualized using a red–blue diverging color scale, with red indicating higher relative expression and blue indicating lower expression. The heat map reveals coordinated upregulation of regulatory and effector-associated genes ( IL10, GZMB, CD274, PDCD1LG2, EBI3, PRDM1, IRF4 ) in Bregs, whereas B-CpG cells show relatively higher expression of chemokines such as CCL3 and CCL4 .

Article Snippet: Human embryonic kidney 293T cells stably expressing the CD40scFv–IL-21 fusion protein (ATCC CRL-3216) were cultured in FreeStyleTM 293 Expression Medium (Thermo Fisher Scientific, Cat. #12338-018) under suspension conditions.

Techniques: RNA Sequencing, Purification, Cell Culture, Activation Assay, Gene Expression, Expressing

Journal: bioRxiv

Article Title: A CD40-Targeted IL-21 Fusokine Enables Rapid Generation of Human IL-10⁺Granzyme B⁺ Regulatory B Cells

doi: 10.64898/2026.01.12.699061

Figure Lengend Snippet: NOD-SCID-IL2Rγnull (NSG) mice were sublethally irradiated and intravenously injected with 6 × 10⁶ human T cells either alone (T cells only) or premixed with autologous IL-10⁺GzmB⁺ regulatory B cells (Bregs) generated ex vivo using CD40ScFv–IL-21 and CpG stimulation. Mice were monitored longitudinally, and overall survival was used as the primary endpoint to assess T cell–mediated disease progression. Mice receiving T cells alone exhibited progressive mortality consistent with xenogeneic T cell–driven pathology, whereas co-administration of Bregs significantly delayed disease onset and improved survival. Survival curves were generated using the Kaplan–Meier method, and statistical significance was determined using the log-rank (Mantel–Cox) test. Data are representative of at least three independent experiments with n = 5 mice per group.

Article Snippet: Human embryonic kidney 293T cells stably expressing the CD40scFv–IL-21 fusion protein (ATCC CRL-3216) were cultured in FreeStyleTM 293 Expression Medium (Thermo Fisher Scientific, Cat. #12338-018) under suspension conditions.

Techniques: Irradiation, Injection, Generated, Ex Vivo, Biomarker Discovery