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. 2021 Jul 9;12(5):1543–1565. doi: 10.1016/j.jcmgh.2021.06.023

Figure 4.

Figure 4

TLS-associated B cells provide significant help in priming the antitumor response. (A) IF staining for TLSs (upper left panel) and DC-LAMP (upper right panel) of sequential human PDAC sections. Plot (bottom panel) showing the significant correlation of DC-LAMP+ cells with TLS density. Spearman r = 0.83, P = .0001. (B) Dendritic cells (DC-Lamp+) colocalization with B cells (CD20+) in human TLSs. (C) Dendritic cells (CD11c+) colocalization with B cells (B220+) in KPC TLSs. (D) Absence of dendritic cells (CD11c+) colocalization with B cells (B220+) in orthotopic TLSs. (E) Timeline and gating strategy of in vitro co-culture of BMDCs and B cells from healthy spleen, KPC spleen, and KPC tumors to assess dendritic cell activation by flow cytometric analysis. (F) Representative overlay histograms and representative dot plots of flow cytometry analysis for CD86 expression on dendritic cells after 48 hours of co-culture with B cells from KPC spleen and tumor and healthy spleen B cells. No co-culture (media only) as negative control. The FMO (fluorescence minus 1) for CD86 was used to define the gate. (G) Quantification of expression of CD86 on dendritic cells after 48 of co-culture, and no co-culture (media only) as negative control. One-way analysis of variance and Bonferroni's posttest: ∗∗∗P < .001. (H) KPC hematoxylin and eosin section demonstrating relationship of TLSs (box) to tumor tissue, confirmed by staining on sequential sections for CD3+ (green)/B220+ (red)/CD21+ (white) TLSs (lower left panel), and juxtaposition of CD11c+ dendritic (green)/B220+ B (red) cells (upper right panel), costaining demonstrating CD11c+ (green)/CD86+ (red) dendritic cells (middle right panel), and granzyme B+ (green)/CD8+ (red) T cells (bottom right panel) with respective boxes highlighted on the right and arrowheads pointing to appropriate cell types. (I) Plot showing the significant correlation of CD8+ T cells with TLS density in KPC mice. Empty circles represent TLS+ KPC tumors, bold circles represent TLS– KPC tumors. Spearman r = 0.76, P = .04. (J) Difference of CD8+ CD3+ T cell infiltration in TLS+ vs TLS– KPC tumors. Two-samples Kolmogorov-Smirnov test: ∗P = .0286. Empty circles represent TLS+ PDAC patients, bold circles represent TLS– PDAC patients. Scale bars: (A) 250 μm, (B–D) 50 μm; (H) 20 μm for IF images, 100 μm for hematoxylin and eosin.