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

Figure 3.

Figure 3

Evidence of active TLSs in human PDAC, and presence of germinal centers in human and KPC tumors but not in orthotopic tumors. (A) BCL6+ and AID+ (activation-induced deaminase) germinal centers (GCs) presence within B cell (CD20+) core in human PDAC. (B) TLS density in GC+/– patients (n = 17). (C) GL7+ GCs within B cell (B220+) core in KPC murine model of PDAC. (D) Absence of GL7+ GCs in orthotopic PDAC mice. (E) Pseudo-multicolor immunophenotype of fully formed TLSs in human PDAC showing clustering of CD4+, CD8+, and TIA+ T cells with exclusion of Foxp3+ cells around a compact CD20+ core with BCL6+ B cells and a network of FDCs (FDCs, CD21+) cells. Presence of DC-LAMP+ mature DCs within TLSs, but not CD68+ macrophages. Inset shows hematoxylin and eosin stain of same section. Adjacent small panels show original images of same section stained using chromogenic immunohistochemistry detection with several immune markers and overlay of pseudo-colored images obtained using ImageJ. Inset in pseudo-color images shows a zoomed-in view. (F) Staining for GrB CD8 T cells in a patient with TLSs (upper panels), a patient with lymphoid aggregate (TLS– patient, middle panels), and a patient without TLSs (bottom panels). The first panel shows immunofluorescence staining for TLSs. The second panel shows immunohistochemistry for CD8 of sequential section. The third panel shows same section, stripped and reprobed for granzyme B (arrowheads) with inset showing magnified area. The fourth panel shows correspondent pseudo-color immunofluorescence image. (G) Difference of CD8+ CD3+ T cell infiltration in TLS+ vs TLS– KPC tumors. Two-samples Kolmogorov-Smirnov test: ∗P = .0286. (H) 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. Empty circles represent TLS+ PDAC tumors, bold circles represent TLS– PDAC tumors. Two-sample Kolmogorov-Smirnov test: (B) ∗P = .0014 and (G) Spearman r = 0.76, P = .04. Scale bar: (A–D) 50 μm, (E) 200 μm, (F) 100 μm.