Additional file 3 of Homogeneously high expression of CD32b makes it a potential target for CAR-T therapy for chronic lymphocytic leukemia
Creators: name: Wang, Guoling
name: Sun, Xiaolei
name: Zuo, Shiyu
name: Li, Chuo
name: Niu, Qing
name: Xia, Yonghui
name: Meng, Yuan
name: Liu, Min
name: Fang, Zihao
name: Yang, Xi
name: Jiang, Yanyu
name: Wang, Sheng
name: Cui, Haidong
name: Huang, Huifang
name: Jiang, Erlie
name: Zhou, Dongming
name: Deng, Qi
name: Pan, Jing
name: Feng, Xiaoming
Publisher: name: figshare
Year: 2021
Subjects: subject: Biochemistry
subject: Space Science
subject: Medicine
subject: Cell Biology
subject: Molecular Biology
subject: Biotechnology
subject: Immunology
subject: FOS: Clinical medicine
subject: Biological Sciences not elsewhere classified
subject: Developmental Biology
subject: Cancer
subject: Hematology
subject: Infectious Diseases
subject: FOS: Health sciences

description: Additional file 3: Fig. S1. CD32 expresses higher than other antigens on primary CLL samples. a Flow cytometric analysis of surface expression of CD32, CD19, CD20, CD22, ROR1, FcμR and CD23 in 4 CLL samples. CLL cells were gated as CD19+CD5+ cells. b Site density comparison between CD32 and CD19 in CLL patients (n = 41). c Quantification of mRNA transcripts from Raji cells, THP-1 cells and leukemic cells of 2 CLL patients by RNA sequencing. d 293T cells were genetically modified to express CD32a and CD32b; cells (unmodified 293T, CD32a+ 293T and CD32b+ 293T) were stained with 2B6-scFv-Flag Ab, 2108-scFv-Flag Ab and anti-CD32 mAb (clone FUN-2). Fig. S2. CD32b CAR-T against Raji cells in vitro and in vivo. a Flow cytometry analysis of CAR expression in T cells following lentiviral transduction. Left, control T cells; middle, T cells transduced with 2B6bbz; right, T cells transduced with 2108bbz. CARs were detected by CD32b-His followed by an anti-His-APC second antibody stain. b Flow cytometric analysis of surface expression of CD32b on the B-cell leukemia cell lines Mec-1 and Raji. c Antigen-specific cytokine production in response to CD32b+ Raji cells. 2B6bbz and control T cells were incubated with Raji cells (2 × 104) respectively for 24 h in E: T ratio of 1:1. The various proteins in the culture supernatant were detected using the bead-based “LEGENDplex multi-analyte assay.” d Representative flow cytometric plot and flow gating strategy of peripheral blood from Raji-NSG mice 15 days after receiving 2B6bbz or control T cells. Fig. S3. CD32b CAR-T had potent cytotoxicity to primary CLL. a Flow cytometry analysis of CAR expression in T cells following lentiviral transduction. Left, control T cells; left-center, T cells transduced with 2B6bbz; right-center, T cells transduced with 2108bbz; right, T cells transduced with CD19 CAR. CARs were detected by CD32b-His/CD19-Fc followed by an anti-His-APC/anti-Fc second antibody stain. b Specific cytotoxicity of 2B6bbz, 2108bbz or control T cells after coculture with primary CLL cells for 36 h at the indicated E:T ratios; c Antigen-specific cytokine production of 2B6bbz, CD19 CAR-T and control T cells in response to 24 h co-culturing with primary CLL cells. Fig. S4. 2B6bbz T against primary CLL cells in vivo. a Flow cytometric analysis of tumor percentage in peripheral blood before T cells infusion. b Representative flow cytometry plot of bone marrow, spleen and peripheral blood, from CLL-NSG mice after receiving CAR-T cells for 18 days. c Treatment response of 2B6bbz and CD19 CAR-T cells against mice transplanted with different antigen density primary CLL cells. d Flow cytometric analysis of CD32 and CD19 expression on CLL cells from peripheral blood of NSG mice before and at 7 days after CAR-T infusion. e Quantification of the percentage of CLL cells in peripheral blood, bone marrow, spleen and liver from CC and NC CLL-NSG mice after receiving CAR-T cells for 18 days. f Number of CAR-T cells in peripheral blood, bone marrow, spleen and liver from CC and NC CLL-NSG mice after receiving CAR-T cells for 18 days. Fig. S5. mRNA expression profile of fcgr2b and fcgr2c in normal human tissues at mRNA level according to publicly available database (BioGPS: fcgr2b, http://biogps.org/#goto=genereport&amp;id=2213 ; fcgr2c, http://biogps.org/#goto=genereport&amp;id=9103 )