Fig 2: Anti-murine CXCR5 CAR-T cells gain access to tumor cells in the B cell follicles and exert potent anti-lymphoma activity.a–e Congenic mice were injected with 2 × 106 primary Eµ-Tcl1 lymphoma cells 8 days later, the mice were irradiated (4 Gy) followed by i.v. administration of 2 × 107 CAR-T cells expressing mTurquoise2 24 h later. In two consecutive experiments mice were sacrificed either 24 h or 5 days (5d) thereafter and splenic CAR-T cells (mTurquoise2+/CD45.1+CD3+), Eµ-Tcl1 lymphoma cells (CD19+ CD5+/CD45.2+) and B cells (CD19+/CD45.1+) were analyzed (b) by immunohistology (Scale bars, 50 µm) and (c, d, e) by flow cytometry. Spleen sections were stained with an anti-CD21/CD35 antibody to indicate localization of FDC networks in the B cell follicle. Twenty-four-hour group n = 3, 5-day group n = 4, control n = 1 (no CAR-T cells). Additional histologies are shown in Supplementary Fig. 14. (f) Congenic mice (CD45.1+, Ly5.1, SJL) were injected with 2 × 106 primary Eµ-Tcl1 lymphoma cells and irradiated (4 Gy) 8 days later, followed by i.v. administration of 2 × 106 mCXCR5 or mSP6 (control) CAR-T cells the next day. g, h On day 21, splenocytes were analyzed by flow cytometry for Eµ-Tcl1 lymphoma cells (CD45.1-/CD19+CD5+), B cells (CD45.1+/CD3-/CD19+), CXCR5+ helper T cells (CD45.1+/CD3+/CD4+/CXCR5+) and transferred CAR T-cells (CD45.2+/CD4+/IgG+ and CD45.2+/CD8+/IgG+). mSP6 CAR group n = 5, mCXCR5 CAR group n = 6. P values are calculated for CXCR5 CAR-T cells compared to SP6 CAR-T cells by a two-tailed Mann–Whitney U test. Source data are provided as a Source Data file.

Fig 3: Anti-murine CXCR5 CAR T cells selectively delete CXCR5+ benign lymphocyte subsets without harming the host’s T cell immunocompetence.a Schematic representation of the anti-murine CXCR5-CD28 CAR (mCXCR5 CAR). SP signal peptide, VH variable heavy chain, L Whitlow linker, VL variable light chain, tm transmembrane region, ic intracellular, LTR long terminal repeat. b Murine splenic T cells were transduced with the mCXCR5 CAR or GFP, or left UT. CAR surface expression was detected by anti-IgG and anti-CD8 co-staining. Percentages of CD8+CAR+ and CD8-CAR+-T cells are indicated. c UT, mCXCR5 CAR- or GFP-transduced T cells (5 × 104) were cocultured for 24 h in a 1:1 ratio with murine Eµ-Tcl1 (CXCR5+), CXCR5-deficient Eµ-Tcl1, splenic B220+ B cells, CXCR5-deficient BW5147 T cell lymphoma cells, and CXCR5-transduced BW5147 cells. IFN? and IL2 in the supernatant were quantified by ELISA. Max, CAR-T cells and PMA/Ionomycin; Min, CAR-T cells only. Data of n = 2 independently transduced effector cells are shown. d, e C57BL/6 mice were sublethally (4 Gy) irradiated, followed by i.v. administration of mCXCR5 CAR- or GFP-transduced T cells 4–6 h later in two consecutive experiments: (i) 1 × 106 transduced T cells were transferred (n = 3 mice per group), treated mice and n = 2 naive mice were sacrificed at day 7; (ii) 2 × 106 transduced T cells were transferred (n = 6 mice per group). Three mice were sacrificed at day 14 and 20, respectively, naive animals served as controls (n = 4). Splenocytes were analyzed for (d) percentage of splenic CXCR5+B220+ B cells and of (e) CXCR5+CD4+ helper T cells among CD4 T cells. d, e Data of n = 2 experiments and n = 3–4 animals per treatment group and time point. P values are calculated for CXCR5 CAR-T cells compared to GFP-transduced T cells by an unpaired two-tailed Student’s t test. Further related data to these experiments are shown in Supplementary Fig. 13b and c. f, g Irradiated (4 Gy) B6 mice were treated with 2 × 106 mCXCR5 or mSP6 (control) CAR-T cells from congenic mice (CD45.1+, Ly5.1; SJL). 18 days later, blood samples were analyzed for (g) B cells (CD45.2+/CD3-/CD19+CXCR5+) and (h) CAR-T cells (CD45.2-/CD3+/IgG+) and the mice were immunized with 5 × 106 SV40 Tag+ 16.113 tumor cells (i.p.). i On day 27, antigen-specific T cells in the spleen were analyzed by flow cytometry using a PE-conjugated Kb-Tag IV dextramer (B220-/CD8+Db-Tag IV+). Bars represent means ± SEM. Dots represent individual mice. Treatment groups were n = 5, control groups were n = 3 (Ctr B6) or n = 4 (naive); P values are calculated for mCXCR5 CAR-T cells compared to mSP6 CAR-T cells by a two-tailed Mann–Whitney U test. Source data are provided as a Source Data file.