Journal: iScience

Article Title: A combinatorial CAAR-T cell strategy to eliminate factor VIII inhibitors in preclinical models of hemophilia A

doi: 10.1016/j.isci.2026.114924

Figure Lengend Snippet: Construction of CAAR-T cells targeting FVIII inhibitors and their functional characterization (A) Schematic diagram of FVIII proteolytic fragments. The domain structure of the FVIII protein after physiological cleavage is shown, illustrating its division into a heavy chain (A1-A2) and light chain (A3-C1-C2). (B) Design of chimeric autoantibody receptor constructs. Two CAAR constructs were generated using the A2 or C1 domains of FVIII as target antigens. Each CAAR consists of the FVIII-derived domain fused to CD28 transmembrane and co-stimulatory regions, CD3ζ signaling domain, and a P2A self-cleaving peptide linked to GFP for expression tracking. (C) Flow cytometric analysis of CAAR expression on human T cells. Top, GFP reporter was used to assess transduction efficiency of A2-CAAR and C1-CAAR constructs, with both showing high GFP + proportions compared to control T cells. Bottom, HA-tag detection was used to confirm surface expression of CAAR constructs; the HA-tag was inserted between the A2 or C1 extracellular domain and the CD28 transmembrane region within the CAAR plasmid. (D) Characterization of engineered K562 target cells. BOIIB2- and RHD5-expressing K562 cells were sorted by flow cytometry based on strong mCherry fluorescence, achieving >97% purity in both lines. (E) Cytotoxic activity of CAAR-T cells against antigen-expressing target cells. Cytotoxicity assays were performed using LDH release to evaluate the lytic activity of A2-CAAR T and C1-CAAR T cells at various effector-to-target (E:T) ratios (0.3:1, 1:1, and 3:1). A2-CAAR T cells were tested against BOIIB2 targets, and C1-CAAR T cells against RHD5 targets. UTD were used as controls. Results are shown as mean ± SEM ( n = 4). Experiments were independently repeated 3 times. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. (F) IFN-γ secretion by CAAR-T cells upon target recognition. ELISA quantification of IFN-γ released by CAAR-T cells co-cultured with their respective antigen-expressing targets at an E:T ratio of 3:1. Both A2-CAAR T and C1-CAAR T cells showed significantly increased cytokine release compared to UTD controls. Results are shown as mean ± SEM ( n = 4). Experiments were independently repeated 3 times. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. (G) Degranulation activity of CAAR-T cells assessed by CD107α expression. Flow cytometry analysis of CD107α surface mobilization in CAAR-T cells after 4 h co-culture with target cells at an E:T ratio of 3:1. Quantification and mean fluorescence intensity (MFI) demonstrates elevated degranulation in both A2-CAAR and C1-CAAR groups compared to UTD controls. Results are shown as mean ± SEM ( n = 4). Experiments were independently repeated 2 times. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: Human IFN-γ(Interferon Gamma) ELISA Kit , Elabscience , Cat#E-EL-H0108.

Techniques: Functional Assay, Construct, Generated, Derivative Assay, Expressing, Transduction, Control, Plasmid Preparation, Flow Cytometry, Fluorescence, Activity Assay, Enzyme-linked Immunosorbent Assay, Cell Culture, Co-Culture Assay

Journal: iScience

Article Title: A combinatorial CAAR-T cell strategy to eliminate factor VIII inhibitors in preclinical models of hemophilia A

doi: 10.1016/j.isci.2026.114924

Figure Lengend Snippet: In vivo evaluation of CAAR-T cells targeting FVIII inhibitors in a hemophilia A mouse model (A) Schematic of the FVIII inhibitor mouse model and CAAR-T cell treatment timeline. FVIII-deficient mice were immunized subcutaneously with recombinant FVIII (rFVIII and 10 IU) once per week for 3 consecutive weeks (day −21, −15, and −8) to induce anti-FVIII inhibitor formation. On day 0, mice received adoptive transfer of CAAR-T cells. CD45.1 + donor mice were used as the T cell source for tracking transferred cells. (B) Design of chimeric autoantibody receptor constructs. Two CAAR constructs were generated using the A2 or C1 domains of FVIII as target antigens. Each CAAR consists of the FVIII-derived domain fused to mice CD28 transmembrane and co-stimulatory regions, mice CD3ζ signaling domain, and a P2A self-cleaving peptide linked to GFP for expression tracking. (C) Measurement of FVIII inhibitor titers in serum following CAAR-T therapy. Inhibitor titers were measured by ELISA over time. Compared to UTD, both A2-CAAR and C1-CAAR treatments significantly reduced FVIII inhibitor levels in serum starting from day 0 and continuing through day 70. The ELISA detection threshold was defined based on the IgG levels of unimmunized F8KO mice, all of which were undetectable. n = 5 per group; experiments were independently repeated 3 times. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. (D) Detection of CAAR-T cell persistence in the spleen. Immunohistochemical staining of splenocytes harvested on day 35 post-transfer revealed the presence of CD45.1 + donor CAAR-T cells in mice treated with A2-CAAR and C1-CAAR, but not in the UTD group (brown DAB signal). HA-tag staining indicates the presence of infused CAAR-T cells in the spleen. Scale bars, 50 μm. (E) Schematic illustration of the FVIII re-challenge model. F8 knockout mice were first immunized subcutaneously with 10 IU rFVIII on day −21 to induce inhibitor formation, followed by infusion of CAAR-T cells on day 0. On day 14, a second dose of rFVIII (10 IU) was administered to assess the durability of immune suppression. (F) Quantification of FVIII inhibitor levels over time. On day 14, mice received an FVIII re-challenge, and plasma inhibitor levels were measured on day 35. Mice treated with combined C1 + A2 CAAR-T cells showed significantly reduced inhibitor titers compared to the A2-CAAR T alone group, indicating enhanced suppression of anti-FVIII immunity. n = 5 per group; experiments were independently repeated 2 times. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. (G) CAART therapy reduces splenic cells that secrete FVIII inhibitors in hemophilic mice. After establishing the hemophilia mouse model, mice were randomized to receive an infusion of UTD or CAART. On day 21 post-infusion, splenocytes were collected and antigen-specific ELISpot assays (FVIII-coated plates) were performed to detect cells secreting FVIII inhibitors. (H) Bethesda assay quantification of inhibitory anti-FVIII antibody titers in plasma 21 days post-infusion. Groups: UTD, A2-CAART, C1-CAART, and A2+C1-CAART. Data are shown as mean ± SD. Significance: ns, not significant; ∗ p < 0.05; ∗∗∗ p < 0.001. (I) Representative flow cytometry plots of splenocytes collected on day 35 post-infusion. The percentages of CD45.1 + CD44 + cells, indicating persisting donor-derived CAAR-T memory T cells in the spleen, are shown for each group.

Article Snippet: Human IFN-γ(Interferon Gamma) ELISA Kit , Elabscience , Cat#E-EL-H0108.

Techniques: In Vivo, Recombinant, Adoptive Transfer Assay, Construct, Generated, Derivative Assay, Expressing, Enzyme-linked Immunosorbent Assay, Immunohistochemical staining, Staining, Knock-Out, Clinical Proteomics, Enzyme-linked Immunospot, Flow Cytometry

Journal: medRxiv

Article Title: CD19 CAR T-cell (BY19) therapy for Pediatric and Adult Patients with Relapsed or Refractory B-Cell Neoplasms in Belarus: Phase 1 trial

doi: 10.1101/2025.08.20.25333782

Figure Lengend Snippet: A) CAR expression of transduced T-cells. Extracellular modules of BY19 and 28Z were directly stained with biotin-labeled CD19 protein and AlexaFluor 647-conjugated streptavidin, and tEGFR reporter was stained with AlexaFluor 488-conjugated cetuximab biosimilar. MOCK - untransduced cells; B) CAR expression normalized to tEGFR. BY19 and 28Z MFI levels were normalized to the MFI levels of tEGFR reporter protein expression; C) CAR mediated cytotoxic activity of T-cells in 24h co-cultivation assay. Fold expansion of target cells in short term co-cultivation assay with BY19 and 28Z CAR T-cells at E:T ratio 1:1. Data represent Mean ± SD. MOCK - co-culture of target cells with untransduced T-cells. Null - culture of target cells alone; D) ELISA-based quantification of IFN γ production. Supernatants after 24h co-culture of engineered CAR T-cells with Raji or CII target cells. Data represent means ± SD; E) Control of Raji cells and proliferation of CAR T cells in rechallenge assay. AUC of target and CAR T cells in five rounds of rechallenge assay. MOCK - co-culture of target cells with untransduced T-cells; F) Control of CII cells and proliferation of CAR T cells in rechallenge assay. AUC of target and CAR T cells in five rounds of rechallenge assay. MOCK - co-culture of target cells with untransduced T-cells.

Article Snippet: CAR-T culture supernatants were assessed for IFNγ production after 24 h of co-cultivation with target cells using an ELISA kit (ElabScience, E-UNEL-H0069), following the manufacturer’s instructions.

Techniques: Expressing, Staining, Labeling, Activity Assay, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Control

Journal: Frontiers in Immunology

Article Title: Influenza A Virus PA Antagonizes Interferon-β by Interacting with Interferon Regulatory Factor 3

doi: 10.3389/fimmu.2017.01051

Figure Lengend Snippet: Inhibition of SEV-induced interferon-β (IFN-β) expression by PA. 293T cells in 12-well plates were transfected with 0.5 µg of Flag-tagged pdm/09 PA, F9PA, H5N1PA, H7N9PA, or an empty vector, together with 0.3 µg of IFN-β-luc and 0.02 µg of RL-TK. After 24 h, cells were infected with SEV or transfected with 200 ng of Poly(I:C) for 8 h and lysed for luciferase assay (A) . A549 cells in 6-well plates were transfected with indicated quantities of Flag-tagged pdm/09 PA or empty vector. After 24 h, cells were infected with SEV or left uninfected for 8 h. The supernatants were harvested for IFN-β ELISA (C) . The cells were harvested, and total RNAs were extracted for detecting the expression levels of IFN-β (B) , CXCL-10 (D) , ISG-15 (E) , and ISG-56 (F) by real-time q-PCR. 293T cells were transfected with increasing quantities of Flag-tagged pdm/09 PA for 24 h and then treated with human IFN-β for 1 h or left untreated. Then, cells were lysed for Western blot using anti-STAT1 and anti-phospho STAT1 antibodies (G) . The bars represent the SEs of the means, based on three experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 [as determined by Student’s t -test (A,C) or by one-way ANOVA (B,D–F) ].

Article Snippet: To measure IFN-β secretion, A549 cells were transfected with an empty vector or plasmids with Flag-tagged pdm/09 PA. After 24 h, the cells were left uninfected or were infected with SEV for 8 h. The supernatants were harvested for an ELISA using a human IFN-β ELISA kit (Elabscience, Wuhan, China) according to the manufacturer’s instructions.

Techniques: Inhibition, Expressing, Transfection, Plasmid Preparation, Infection, Luciferase, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Cell reports. Medicine

Article Title: Targeting neoadjuvant chemotherapy-induced metabolic reprogramming in pancreatic cancer promotes anti-tumor immunity and chemo-response.

doi: 10.1016/j.xcrm.2023.101234

Figure Lengend Snippet: Figure 6. Targeting CD36 synergistically promoted AG-mediated killing of PDAC in preclinical models (A) Visual presentation of subcutaneous xenograft murine PDAC tumor models (C57 mice) for each group. (B) Measurement of tumor volumes showed CD36 blockage synergistically promoted AG-mediated killing of PDAC in subcutaneous xenograft murine PDAC tumor models. (C) Measurement of tumor weights showed CD36 blockage synergistically promoted AG-mediated killing of PDAC in subcutaneous xenograft murine PDAC tumor models (n = 5). (D) Representative IHC staining showed Ki67 expression in subcutaneous xenografts treated with different regimens. (E) t-Distributed stochastic neighbor embedding (TSNE) analyses showed the clustering for CD36+ CD8+ T cells and GZMB+ CD8+ T cells. (F) Flow cytometry revealed that more CD8+ T cells infiltrated PDAC with NAC, while the percentage of CD36+ CD8+ T cells also increased (n = 5) (mean with standard deviation). (G) ELISA results showed the combination of AG and CD36 blockade significantly improved IFN-g and tumor necrosis factor a (TNF-a) levels intratumorally (n = 5). (H) Representative image of orthotopic murine models of PDAC. (I) Kaplan-Meier curve revealed the combination of CD36 blockade and AG significantly prolonged the survival interval of mice that received orthotopic PDAC cell transplantation (n = 10). Circle or square referred to a happened event (death or censored). Censored event means the mice is still alive at the time point that we ended follow-up. (J) CD36 blockade synergistically with AG regimens optimally narrowed the PDAC tumor size in a humanized PDX model (n = 10). (K) Representative IHC staining image of CD36-high and -low PDAC. (L) Kaplan-Meier curve showed increased CD36 expression predicted worse prognosis of PDAC patients with adjuvant AG chemotherapy. The statistical sig- nificance shown in this figure was detected using t test.

Article Snippet: The ELISA kits used in the present study were as follows: Human IFN-gamma ELISA Kit (absin, abs510007); Human IL-2 ELISA Kit (Boster, EK0397); Mouse TNF alpha ELISA Kit (absin, abs520010) and Mouse IFN- gamma ELISA Kit (absin, abs520007).

Techniques: Immunohistochemistry, Expressing, Flow Cytometry, Standard Deviation, Enzyme-linked Immunosorbent Assay, Transplantation Assay, Adjuvant