Fig 1: Combination of chemotherapy and immune activation within intratumoral TLSs is necessary for antitumor activity. (A) Schema of short-term administration of gemcitabine in combination or not with intratumoral injection of lymphoid chemokines CXCL13 and CCL21 in orthotopic mice. (B) TLS density after coinjection of gemcitabine and chemokine (C+G) compared with appropriate controls (PBS [S], chemokine [C], or gemcitabine [G] alone. (C) Flow cytometric analysis of CD45+ immune cells in PBS-treated (S) (n = 7) gemcitabine-treated (G) (n = 9) mice, chemokine-treated (C) (n = 6), and C+G-treated (n = 8) mice. Flow cytometric analysis of lymphoid and myeloid immune cells per gram of tumor tissue after chemokine and/or chemotherapy injection. (D) B cells (CD19+), (E) CD3+ T cells, (F) CD8+ T cells, (G) CD4+ T cells, (H) dendritic cells (CD11c+), (I) myeloid cells (CD11b+), (J) MDSC Ly6GC+ subset, (K) macrophages (F4/80+, MHC-II+). (L) Tumor volume in C+G-coinjected mice and appropriate controls (chemokines [C], or gemcitabine [G] alone). Each data point represents 1 mouse (S n = 7, C n = 6, G n = 9, C+G n = 9). Kolmogorov-Smirnov test. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001.
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: Differential immune cell infiltration after chemokine injection in orthotopic murine tumors. Flow cytometric analysis, in lymphoid aggregate negative tumors from PBS-treated (n = 6) and TLS+ CXCL13/CCL21-treated (n = 6) mice, of (A) CD19+ B cells out of CD45+ cells, (B) CD3+ T cells out of CD45+ cells, (C) CD8+ cytotoxic T cells out of CD3+ T cells, (D) CD4+ helper T cells out of CD3+ T cells, (E) FOXP3+ regulatory T cells out of CD4+CD3+ T cells, (F) CD11b+ myeloid cells out of CD45+ cells, and (G) tumor weight in LA–tumors from PBS-treated (n = 6) and TLS+ CXCL13/CCL21–treated (n = 6) mice. Each data point represents 1 mouse. Two-sample Kolmogorov-Smirnov test: ∗P < .05 and ∗∗P < .01. ns, not significant.
Fig 4: Coinjection of CXCL13 and CCL21 into orthotopic tumors. (A) Correlation of CD19+ B cell density with TLS density in orthotopic pancreatic tumors. The dotted line represents the cutoff of minimal B cell density needed to induce lymphoneogenesis. Pink circles represent lymphoid aggregate (LA)– PBS-treated mice, red circles represent LA+ PBS-treated mice, blue circles represent TLS+ CXCL13/CCL21-treated mice, empty blue circles represent stress-induced LA+ CXCL13/CCL21-treated mice. Spearman r = 0.79, P < .0001. (B–G) Flow cytometric analysis, in LA+/– tumors from PBS-treated mice (n = 12) and TLS+/– tumors from CXCL13/CCL21-treated mice (n = 16), of (B) CD19+ B cells out of CD45+ cells, (C) CD3+ T cells out of CD45+ cells, (D) CD4+ helper T cells out of CD3+ T cells, (E) CD8+ cytotoxic T cells out of CD3+ T cells, (F) FOXP3+ regulatory T cells out of CD4+CD3+ T cells, and (G) CD11b+ myeloid cells out of CD45+ cells. The mean percentage of CD19+ cells out of CD45+ cells within the PBS-treated group used to identify stress-induced lymphoid aggregates from potential chemokine-induced TLSs (dotted line in A). (H) Tumor weight in CXCL13/CCL21 intratumoral single or coinjected compared with PBS-treated mice. Each data point represents 1 mouse. Empty circles in CXCL13/CCL21 treated mice represent stress-induced lymphoid aggregates. Two-sample Kolmogorov-Smirnov test. ∗P < .05. ns, not significant.
Fig 5: Coinjection of CXCL13 and CCL21 into orthotopic tumors, and administration of intraperitoneal gemcitabine. (A–D) Representative immunofluorescence images of aggregates or TLSs as detected by the presence of B220 (green), and CD21 (red) with DAPI (blue) staining nuclei in (A) PBS-treated, (B) chemokine-treated, (C) gemcitabine-treated, (D) and C+G-treated mice. (E, F) IF staining of gemcitabine- and C+G-treated mice for granzyme B (green) and CD3 (red) T cells. Insets represent a zoomed-in view of the aggregate. (G) Quantification of the granzyme B+ T cells in gemcitabine alone (G) and in combination with C+G-treated mice. Two-samples Kolmogorov-Smirnov test: ∗∗P < .01. Scale bar, (A–D) 50 μm, (E, F) 250μm .
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