Pancreatic Cancer Chemotherapy Is Potentiated by Induction of Tertiary Lymphoid Structures in Mice

Abstract

Background and aims: The presence of tertiary lymphoid structures (TLSs) may confer survival benefit to patients with pancreatic ductal adenocarcinoma (PDAC), in an otherwise immunologically inert malignancy. Yet, the precise role in PDAC has not been elucidated. Here, we aim to investigate the structure and role of TLSs in human and murine pancreatic cancer.

Methods: Multicolor immunofluorescence and immunohistochemistry were used to fully characterize TLSs in human and murine (transgenic [KPC (Kras^G12D, p53^R172H, Pdx-1-Cre)] and orthotopic) pancreatic cancer. An orthotopic murine model was developed to study the development of TLSs and the effect of the combined chemotherapy and immunotherapy on tumor growth.

Results: Mature, functional TLSs are not ubiquitous in human PDAC and KPC murine cancers and are absent in the orthotopic murine model. TLS formation can be induced in the orthotopic model of PDAC after intratumoral injection of lymphoid chemokines (CXCL13/CCL21). Coadministration of systemic chemotherapy (gemcitabine) and intratumoral lymphoid chemokines into orthotopic tumors altered immune cell infiltration ,facilitating TLS induction and potentiating antitumor activity of chemotherapy. This resulted in significant tumor reduction, an effect not achieved by either treatment alone. Antitumor activity seen after TLS induction is associated with B cell-mediated dendritic cell activation.

Conclusions: This study provides supportive evidence that TLS induction may potentiate the antitumor activity of chemotherapy in a murine model of PDAC. A detailed understanding of TLS kinetics and their induction, owing to multiple host and tumor factors, may help design personalized therapies harnessing the potential of immune-oncology.

Keywords: B Cells; Dendritic Cells; Orthotopic; T Cells; Transgenic Mice.

Graphical abstract

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.

Figure 2

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.

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.

Figure 4

TLS-associated B cells provide significant help in priming the antitumor response. (A) IF staining for TLSs (upper left panel) and DC-LAMP (upper right panel) of sequential human PDAC sections. Plot (bottom panel) showing the significant correlation of DC-LAMP⁺ cells with TLS density. Spearman r = 0.83, P = .0001. (B) Dendritic cells (DC-Lamp⁺) colocalization with B cells (CD20⁺) in human TLSs. (C) Dendritic cells (CD11c⁺) colocalization with B cells (B220⁺) in KPC TLSs. (D) Absence of dendritic cells (CD11c⁺) colocalization with B cells (B220⁺) in orthotopic TLSs. (E) Timeline and gating strategy of in vitro co-culture of BMDCs and B cells from healthy spleen, KPC spleen, and KPC tumors to assess dendritic cell activation by flow cytometric analysis. (F) Representative overlay histograms and representative dot plots of flow cytometry analysis for CD86 expression on dendritic cells after 48 hours of co-culture with B cells from KPC spleen and tumor and healthy spleen B cells. No co-culture (media only) as negative control. The FMO (fluorescence minus 1) for CD86 was used to define the gate. (G) Quantification of expression of CD86 on dendritic cells after 48 of co-culture, and no co-culture (media only) as negative control. One-way analysis of variance and Bonferroni's posttest: ∗∗∗P < .001. (H) KPC hematoxylin and eosin section demonstrating relationship of TLSs (box) to tumor tissue, confirmed by staining on sequential sections for CD3⁺ (green)/B220⁺ (red)/CD21⁺ (white) TLSs (lower left panel), and juxtaposition of CD11c⁺ dendritic (green)/B220⁺ B (red) cells (upper right panel), costaining demonstrating CD11c⁺ (green)/CD86⁺ (red) dendritic cells (middle right panel), and granzyme B⁺ (green)/CD8⁺ (red) T cells (bottom right panel) with respective boxes highlighted on the right and arrowheads pointing to appropriate cell types. (I) 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. Spearman r = 0.76, P = .04. (J) Difference of CD8⁺ CD3⁺ T cell infiltration in TLS⁺ vs TLS^– KPC tumors. Two-samples Kolmogorov-Smirnov test: ∗P = .0286. Empty circles represent TLS⁺ PDAC patients, bold circles represent TLS^– PDAC patients. Scale bars: (A) 250 μm, (B–D) 50 μm; (H) 20 μm for IF images, 100 μm for hematoxylin and eosin.

Figure 5

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.

Figure 6

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.

Figure 7

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.

Figure 8

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.

Figure 9

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 .

Figure 10

Combination of chemotherapy and immune activation within intratumoral TLSs improves survival in the orthotopic model of PDAC. (A) Schema of administration of gemcitabine in combination with intratumoral injection of lymphoid chemokines CXCL13 and CCL21 in orthotopic mice (C+G) (n = 10) for the lifespan of the mice. Controls were represented by intraperitoneal and intratumoral injections of PBS for the control group (S) (n = 10), intraperitoneal PBS injection and intratumoral chemokines injection for the chemokine-alone group (C) (n = 10), and intraperitoneal gemcitabine injections and intratumoral PBS injections for the gemcitabine group (G) (n = 10). (B) Tumor volume change with baseline at day 15 (100%) up to day 29 when all mice were available for measurements (before endpoint). (C) Kaplan-Meier survival curve for the 4 treatment arms. (C) Kaplan-Meier survival curve for subgroup of mice treated with chemotherapy with or without chemokines analyzed for presence or absence of TLSs. (B) Linear mixed-effect models, with Dunnett’s adjustment for multiple comparison, (C, D) Log-rank (Mantel-Cox) test. ∗P < .05; ∗∗P < .01; ∗∗∗∗P < .0001.