Fig 2: Artificial induction of TLSs in orthotopic model of PDAC. (A) Ultrasound images of the pancreatic tumor of orthotopic mice preinjection (left panel), during injection (middle panel), and postinjection (right panel) and schema of intratumoral injection of lymphoid chemokines CXCL13 and CCL21 after development of orthotopic PDAC tumor. Red arrow: needle trajectory; red dotted line: tumor; red asterisk: spread of solution. Schema of experiment. (B) Proportion of CD3+, CD19+ CD11b+ and other immune cells out of total CD45+ cells after intratumor lymphoid chemokine injection as assessed by flow cytometric analysis (PBS n = 12, CXCL13/CCL21 n = 16, CXCL13 n = 4, CCL21 n = 4). (C) Representative immunofluorescence staining on sequential sections of lymphoid aggregates as detected by the presence of B cells (B220), the presence of T cells (CD3) (upper panels), and the near absence of FDCs (CD21) (lower panels) with DAPI staining nuclei in PBS (vehicle control) injected mice. (D) Representative immunofluorescence images of TLSs as detected by the presence of B cells (B220), T cells (CD3) (upper panels), and well-formed network of FDCs (CD21) (lower panels) with DAPI staining nuclei in dual chemokine-injected mice, showing mature TLSs. (E) Lymphoid aggregate and TLSs induction in CXCL13/CCL21 intratumoral coinjected mice. (PBS n = 12, CXCL13/CCL21 n = 16). (B) Kruskal-Wallis and (E) chi-square test. *P < .05, ****P < .0001. Scale bar: 50 µm. n.s, not significant.

Fig 3: Immunologically active TLSs are present in a fraction of KPC PDAC, similar to human PDAC, and are infrequent in orthotopic model. (A) Colocalization of T cells (CD3+), B cells (B220+), and FDCs (CD21+) in dense, compact lymphoid aggregates in KPC tumors defining murine TLSs. (B) Smaller and less defined conglomerates of T (CD3+) and B (B220+) cells in orthotopic model of PDAC on consecutive sections resembling TLSs when weak presence of FDCs (CD21+), named lymphoid aggregates, can be detected. (C) TLS frequency in KPC and orthotopic murine models of PDAC. (D) Density of TLSs in KPC and orthotopic murine models of PDAC. (E) Plot showing the significant correlation of CD20+ B cell density with TLS density in KPC murine model of PDAC. The dotted line represents the cutoff of minimal B cell density needed to induce lymphoneogenesis. Spearman r = 0.95, P = .0015. (F) Representative immunofluorescence images of sparse T cells (CD3+, green) and no B cells (B220+, red) and FDCs, with DAPI (blue) as a nuclear stain. (G) Representative immunofluorescence images in sequential sections of clustered T (CD3+, green) and B (B220+, red or green) cells without compartmentalization and without FDCs (CD21+, red) network. Each data point represents a mouse and lines represent median and interquartile range. Empty circles represent TLS+ murine tumors, bold circles represent TLS– murine tumors. (C) Chi-square test, (D) Mann-Whitney U test. *P < .05. Scale bar: 50 µm.

Fig 4: 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.

Fig 5: Analysis of B cell development in bone marrow and spleen.A. FACS analysis of BM B cells. BM cells from WT and RhoF KO mice (6–7 weeks old, n = 5, male: 40%) were stained with antibodies against CD43, B220, BP1, and CD24 and analyzed by flow cytometry. The number shows the percentage (mean ± standard deviation) of the indicated subpopulation within the parent population; Fraction A (germline pro-B cells), fraction B (DJ-rearranged pro-B cells), fraction C (Early pre-B cells), fraction D (Late pre-B cells), fraction E (newly formed B cells), and fraction F (follicular-type recirculating B cells). The figures are representatives of three independent experiments.B. FACS analysis of B cells from the spleen. Splenocytes from WT and RhoF KO mice (6–7 weeks old, n = 6, male: 33%) were stained with antibodies against CD23, CD21, and IgM and analyzed by flow cytometry. The number shows the percentage (mean ± standard deviation) of the indicated subpopulation within the parent population; T1 (Transitional 1), T2 (Transitional 2), Fo (mature follicular B cells), and MZ (MZ B cells).C. The number of B cell subsets in BM was calculated by multiplying the total number of viable (trypan blue negative) BM cells by the fraction of the target population in viable (7AAD negative) cells. The data are shown as mean ± standard deviation (6–7 weeks old, n = 5, male: 40%). White bars: WT. Black bars: RhoF KO.D. The number of B cell subsets in the spleen was also calculated by multiplying the total number of viable (trypan blue negative) splenocytes by the fraction of the target population in viable (7AAD negative) cells. The data were shown as mean ± standard deviation (6–7 weeks old, n = 6, male: 33%). White bars: WT. Black bars: RhoF KO. *: P < 0.05, WT vs. RhoF KOE. Immunohistostaining of CD169 (Green, MZ metallophilic macrophages) and B220 (Red, B cells) in WT (upper left panel) and RhoF KO (lower left panel) spleen. Scale bars: 100 µm. The widths of the MZ B cell region (B220-positive region outside CD169-positive cells) in the WT and RhoF KO mice are summarized (right panel, n = 32). Four MZ B cell regions per section were captured for four individual sections per mouse, and two mice per group were used for this experiment. Circles: WT. Crosses: RhoF KO. ***: P< 0.0001, WT vs. RhoF KO