Fig 2: TLSs are highly organized structures in OSCC tissues and appear as clusters of B-cell follicles surrounded by T-cell zones and HEVs. a Mature TLS, similar to lymph node; CD20+ B cells form a follicular structure. The CD3+ T-cell zone is located near the follicle, and HEV are diffusely distributed around the follicle. b Immature TLS; CD20+ B cells do not form any follicular structure within the PNAd+ HEV area. CD3+ T cells were more diffusely distributed than mature TLSs. c Representative multiplex IHC images showing immune cells of immature TLS and mature TLS samples by simultaneous staining of B cells (CD20, yellow), T cells (CD3, red), mature dendritic cells (DC-LAMP, green), high endothelial venules (PNAd, cyan) and the nuclear stain DAPI (blue) (×200, original magnification). d TLS grading in OSCC tissues. Grade 0, no TLS present; grade 1, immature TLS; grade 2, mature TLS. Scale bar, 500 µm in “4X” pictures and 200 µm in “10X” pictures

Fig 3: Characterization of tertiary lymphoid structures in breast carcinoma. Immunohistochemical detection of the indicated antigens in serial sections of breast carcinoma with extensive immune cell infiltration. Tertiary lymphoid structures with germinal center formation were detected by A) CD20+ B lymphocyte follicle comprising a network of B) CD21+ FDC, with C) CD4+ and D) CD8+ T lymphocytes in the T cell zone. E) Bcl6+ germinal center B lymphocytes and Tfh cells were also observed within the B cell follicle with surrounding F) PNAd + HEV like vessels. Positively stained antigens are shown by brown DAB staining. Magnification 100X.

Fig 4: Lymphoma growth disrupts CCL21 migration cues on FRCs(A) Microscopic analysis of the FRC network (Ccl19RFP) in LN sections. Bottom: examples of FRC sphericity (Ccl19RFP segmentation).(B) Quantification of the FRC density in LN sections of Ccl19RFP mice. n = 7–10 mice per group.(C) Representative images of CCL21 expression in LNs sections of Ccl19RFP mice and gradients of the fluorescence intensity over the distance.(D) CCL21 distribution around and within HEVs (PNAd+) and FRCs (Ccl19RFP).(E) Microscopic analysis of CCL21 (MFI) within masked Ccl19RFP (FRCs) and PNAd+ (HEVs). Control n = 5, tumor n = 7.(F) Immunohistochemistry of CCL21 expression in controls and LNs from diseased tg. Eµ-Myc (n = 7) and Cd19-TAg mice (n = 11); box indicates magnified area. Bar graph depicts percentage of LNs with preserved (pos.) or lost (neg.) CCL21 expression.(G) Proportions of mesenchymal cell subsets (all Ccl19RFP), CD21/CD35+ follicular DCs (FDCs); MadCAM-1+ marginal reticular cells (MRCs); SCA-1+ pericytic reticular cells (PRCs); and T-zone reticular cells (TRCs). n = 4 mice per group.(H and I) FCM quantification of CCL21+ (n = 6–7 mice per group) (H) and PDPN+ expression (n = 7–8 mice per group) (I) in LNs of Ccl19RFP mice.(J and K) Exemplary images (J) and analysis (K) of aSMA (MFI) and CCL21 (integrated density) in a FRC line cultured under static conditions or with laminar flow.Scale bars, 20 µm (A, D, and J) and 100 µm (C and F). Mean and SEM are indicated. Data points represent individual cells (K) or mice (B, E, and G–I). A Mann-Whitney U-test was applied. *p < 0.05; **p < 0.01; ***p < 0.005.

Fig 5: Lymphoma progression is accompanied by degeneration of the reticular conduit system(A) Representative LN section with low tumor (Eµ-Myc cellsCFP), stained for PNAd and the basal lamina of the conduit system (COL-IV).(B) Representative images of FITC-dextran (10 kDa, sc injected) in draining LNs.(C) Quantification of FITC-dextran MFI in whole-LN sections and LN parenchyma (excluding template volume of the Ccl19RFP FRC network). n = 5 mice per group.(D) Left: representative images of FITC-dextran (10 kDa, sc injected) within conduits (masked by the Ccl19RFP FRC network). Right: quantification of FITC-dextran area within masked Ccl19RFP FRC network. n = 6 mice per group.(E) Representative images of COL-IV and COL-I within the FRC network (Ccl19RFP template).(F) Quantification of COL-IV and COL-I MFI within FRC network (Ccl19RFP template). n = 5 mice per group.(G) Heatmap depicts enrichment scores (ESs) and p value (p) of the indicated Gene Ontology (GO) pathways.(H) DLBCL gene expression heterogeneity discovered by unsupervised signal dissection (SDCM). ECM and collagen-related genes are overrepresented, and signature expression is associated with patient’s outcome. All significantly correlated probe sets are depicted (462 correlated probe sets corresponding to 299 genes and 2 anti-correlated probe sets/genes from 54,675 measured in total). Right, outcome is depicted as progressive course (progr.) and non-progressive course (censored) for n = 498 cases.Boxes indicate magnified areas (A, B, and D). Scale bars, 100 µm (A and B) and 20 µm (D and E). Mean and SEM are indicated. All data points represent individual mice (C–F). All statistics were calculated with Mann-Whitney U-test. *p < 0.05; **p < 0.01.