Fig. 1: Drug induction of TLS and HEVs in subcutaneous tumors.

a, STING agonist ADU-S100 (2 μg intratumoral) and/or anti-LTβR agonistic antibody (100 μg, intraperitoneal (i.p.)) were administered to mice bearing subcutaneous KPC tumors as monotherapy or in combination as indicated. Immunohistochemistry of B cells (CD19, brown) and HEVs (MECA-79, magenta) shows numerous TLS induced by combination therapy (day 14). Scale bars, 1 mm (low magnification) and 100 μm (high magnification). b, CD19 and MECA-79 staining of TLS-rich human breast adenocarcinoma for comparison. Scale bars, 1 mm (low magnification) and 100 μm (high magnification). c, KPC tumor sections of different treatment groups were stained for CD19 and MECA-79, scanned for the whole tumor area, and the number of TLS and HEV endothelial cells (HEV-ECs) were quantified per tumor area (mm²) by Halo image analyses. The results of two independent experiments were combined to generate the graphs. A total of N = 17–19 tumors were analyzed. *P < 0.05, ***P < 0.001, ****P < 0.0001. d, Left: immunofluorescence confocal image (z-stack) of TLS induced by combination therapy. Scale bar, 100 μm. Right: the TLS area indicated by the arrow in the left image is shown in higher magnification (three-dimensional confocal image). Scale bar, 10 μm. Nuclear staining of germinal center/follicular cell transcription factor Bcl6 (green) is surrounded by B cell surface marker CD19 (white) or helper T cell marker CD4 (red), indicating germinal center B cells or T_FH cells, respectively. Arrows indicate T_FH cells found among the dense B cell cluster, demonstrating the intimate interaction between the T_FH cell surface (red) and the B cell surface (white). e, Proliferating B cells were detected in TLS by Ki-67 and CD19 double staining. Scale bar, 100 μm. f, Triple immunofluorescence staining of LTβR monotherapy-induced TLS for CD4, CD19 and CD23 (a follicular cell marker) shown in merged and individual color channels. Blue, DAPI. Scale bar, 100 μm. g, Combination therapy on KPC tumors grown in wild-type or T cell-deficient nude mice (C57BL/6 background) demonstrating that neither TLS nor HEVs formed in tumors in the absence of T cells. A representative image of 16 tumors is shown for each genotype. Scale bar, 100 μm. h, CD4⁺ T cells and/or CD8⁺ T cells are depleted in wild-type mice by i.p. injection of depleting antibodies one or two days before KPC tumor inoculation as described in Extended Data Fig. 3a. Isotype IgG was injected for the no-depletion control group. Alternatively, KPC tumors were grown in B cell-deficient CD79a knockout mice and treated as shown in a. The development of TLS and HEVs was studied in these mice after combination therapy. A representative image of six tumors is shown for each group. Scale bar, 100 μm. i, The number of TLS and HEV-ECs were quantified per tumor area (mm²). N = 6. P < 0.0001. Representatives of at least three independent experiments are shown in d–f. Data are presented as the mean ± s.e.m. and analyzed using one-way analysis of variance (ANOVA) with Tukey’s test for statistical significance. All replicates represent biological replicates. NS, not significant.