Fig. 2: B cells expressed LTα upon αCD40 stimulation and were required for TLS formation.

All panels except (d–g) show data from GL261 tumor-bearing mice treated with rIgG2a or αCD40. a–c Immunofluorescent staining of therapeutic rat antibodies (αRat) in TLS co-stained for a, b B220 and c CD11b and CD11c. Images are representative of three mice. Arrows: cells positive for αRat. White square areas are magnified to the right in b, c. Scale bars: 50 µm. d Representative images of murine CD19⁺ splenic B cells stimulated in vitro with rIgG2a or αCD40 antibodies at indicated time points. e–g Gene expression of Lta, Ltb, and Tnfsf14 in B cells shown in d. n = 4 independent experiments. One-way ANOVA with Dunnett’s correction for multiple comparison. In e p_{(rIgG2a 6h vs. αCD40 48h)} = 0.0017, p_{(rIgG2a 6h vs. αCD40 72h)} = 0.005, p_{(αCD40 6h vs. αCD40 48h)} = 0.0012, p_{(αCD40 6h vs. αCD40 72h)} = 0.0035. In f p_{(αCD40 6h vs. αCD40 72h)} = 0.0223. In g p_{(rIgG2a 6h vs. αCD40 48h)} = 0.0439, p_{(αCD40 6h vs. αCD40 48h)} = 0.0105, p_{(αCD40 6h vs. αCD40 72h)} = 0.0169. h–k Gene expression of Lta and Ltb in CD19⁺B220⁺ B cells sorted from h, i spleen and j, k cranial lymph nodes. h, i n(rIgG2a) = 4 mice, n(αCD40) = 5 mice. j, k n = 7 mice/group. h p = 0.0285, j p = 0.0452. Two-tailed t-test, *p < 0.05. l Representative plot of quantifications shown in m, n. m Quantification of CD19⁺B220⁺ brain-infiltrating B cells as a percentage of CD45⁺ cells at day 20. n(rIgG2a) = 8 mice; n(αCD40) = 7 mice. n Quantification of CD19⁺B220⁺ brain-infiltrating B cells as a percentage of CD45⁺ cells at day 25. n = 8 mice/group. p = 0.0003. m, n Two-tailed t-test. o Quantification of CD19⁺B220⁺ B cells as a percentage of CD45⁺ cells in the blood of tumor-bearing mice treated with rIgG2a or αCD40 antibodies, with (+) or without (−) B cell depletion with an αCD20 antibody. n = 4 mice/group. All indicated p-values are p < 0.0001. One-way ANOVA with Tukey’s correction for multiple comparison. p Quantification of the number of dense CD45⁺B220⁺ clusters per 80 µm-thick section in αCD40-treated tumor-bearing brains with (+) or without (−) B cell depletion with an αCD20 antibody. n(αCD40) = 8 mice, n(αCD20 + αCD40) = 5 mice. *p = 0.0234. Two-tailed t-test. h–n Black circle indicates rat IgG2a (rIgG2a), red square indicates agonistic CD40 antibodies (αCD40). o, p Black circle indicates rIgG2a, black square indicates rIgG2a + αCD20, red circle indicates αCD40, red square indicates αCD40 + αCD20. For all graphs in this figure, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Bars: mean ± SEM. arb. units = arbitrary units. Source data are provided as a Source Data file.