Figure 1.

Immunologically active TLSs are present in a fraction of chemo-naïve human PDAC. (A) Colocalization of T cells (CD3⁺), B cells (CD20⁺), and FDCs (CD21⁺) with high endothelial venules (peripheral node addressin positive [PNAd⁺]) in dense, compact lymphoid aggregates on consecutive sections defining human TLSs. (B) Human PDAC section stained for CD20 (magenta), CD21 (green), PNAd (red), and DAPI (blue) (upper panel). Sequential section with in situ hybridization of CXCL13 (red) RNA-scope probe in human PDAC patient (lower panel). (C) Frequency of TLSs in human PDAC (n = 56 TMAs, n = 31 full sections). (D) TLS density (expressed as number of CD3⁺CD20⁺CD21⁺ clusters/mm²) in a cohort of human PDAC (n = 17). The dotted line represents cutoff for identification of TLS⁺ (empty circles) and TLS^– (bold circles) PDAC patients. (E) Plot showing the significant correlation of CD20⁺ B cell density with TLS density. The dotted line represents the cutoff of minimal B cell density needed to induce ectopic lymphoneogenesis. Empty circles represent TLS⁺ patients, bold circles represent TLS^– patients. Spearman r = 0.84, P = .0001. (F) Distribution of TLS stages in patients with different TLS densities (TLS maturation). (G) Human PDAC full section stained for CD3⁺ T cells, CD20⁺ B cells, and FDCs, using modified immunohistochemistry stripping and reprobing protocol. Fourth (bottom) panel represents pseudo-color immunofluorescence image of the same sections, showing a cell phenotype map (immune, stromal, and tumor cells) using different colors to better depict spatial distribution. The boxes identify subsequent adjacent panels (I–V). Scale bar: 1000 μm. (I) Higher magnification of a representative area of presence of scattered T cells, with absence of B cells and FDCs. (II) Higher magnification of a representative area of cluster of T cells, with absence of B cells and FDCs. (III) Higher magnification of a representative area of sparse conglomerates of T cells and sparse B cells. FDCs are absent. (IV) Higher magnification of a representative area where T and B cells are clustered but not organized in distinct zones. FDCs are absent (early TLSs).(V) Higher magnification of TLSs, where a cloud of T cells surrounds a core of B cells that includes a FDC network. Those TLSs can show absence or presence of a germinal center; therefore, they are celled primary follicle-like TLSs and secondary follicle-like TLSs, respectively. Each data point represents an individual patient and lines represent median and interquartile range. Empty circles represent TLS⁺ tumors, bold circles represent TLS^– tumors.