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. Author manuscript; available in PMC: 2023 Feb 5.
Published in final edited form as: Sci Immunol. 2022 Aug 5;7(74):eabl3995. doi: 10.1126/sciimmunol.abl3995

Fig. 4. Effects of epitope position on human primary CAR-T targeting CD22.

Fig. 4

(A) Schematics of CAR activation by membrane-proximal versus membrane-distal epitopes. LEFT: CARs bind membrane proximal epitopes to create a narrow intermembrane space that excludes phosphatase CD45 and induces CAR activation. Right: CARs recognize membrane-distal epitopes, which cannot form a close-contact zone to exclude CD45 effectively.

(B) Schematics of the binding sites of two scFvs, m971 and RFB4, that recognize CD22. CD22 contains seven extracellular immunoglobulin domains. RFB4 recognizes distal epitopes on Domain 3 whereas m971 recognizes proximal epitopes on Domain 7. A short version of CD22 was constructed by removing Domain 5–7 so that the epitopes on Domain 3 become membrane proximal. An mCherry tag replaced the intracellular part of both the full-length and short CD22.

(C) Expression of the full-length or short extracellular domain of CD22 in KU812 cells, as detected by western blot.

(D) Human primary T cells were purified from the peripheral blood of anonymous healthy donors, expanded, and engineered with lentivirus to express RFB4 CARs. CAR expression was evaluated by flow cytometry.

(E) Conjugation of RFB4 CAR-T cells with KU812 cells expressing the full-length (FL) or short CD22. Human primary T cells expressing RFB4 CAR were co-cultured with KU812 cells expressing FL or short CD22 extracellular domain (E:T =1:1) for 30 min before being imaged by confocal microscopy. Data were presented as mean with SD (Unpaired T test, p>0.05(ns)). N=3 co-culture experiments.

(F) Effects of epitope position on CD45 exclusion from the RFB4 CAR synapses. Primary T cells expressing RFB4 CAR were co-cultured with KU812 cells expressing the FL or short CD22 (E:T =1:1) for 30 min, followed by staining with an anti-CD45 antibody. Imaging was acquired by confocal microscopy. Data were presented as mean with SD (Mann-Whitney U test, p<0.01 (**)). N=50 synapses.

(G) ERK phosphorylation in RFB4 CAR-T cells engaged with the full-length or short CD22. Primary RFB4 CAR-T cells were cocultured with KU812 cells (E:T =1:1) for 10 minutes. ERK phosphorylation was assessed by flow cytometry. Data were presented as mean with SD (Unpaired T test, p<0.01 (**), p<0.05 (*)). N=3 co-culture experiments.

(H) Cytotoxicity of RFB4 CAR-T cells to cells expressing the full-length or short CD22. Primary RFB4 CAR T cells were co-cultured with FL or short CD22 cells (E:T =5:1) for 24 hours, followed by flow cytometry analysis. Data were presented as mean with SD (Unpaired T test, p<0.05 (*), p<0.01 (**)). N=3 co-culture experiments.

(I-J) TNFα and IFNγ production in RFB4 CAR-T cells. The co-cultured medium from (H) was used for the measurement of IFNγ and TNFα by ELISA. Data were presented as mean with SD (Unpaired T test, p>0.05 (ns), p<0.05 (*), p<0.01 (**), p<0.0001 (****)). N=3 co-culture experiments.