Journal: The Journal of Experimental Medicine

Article Title: Follicular dendritic cells help establish follicle identity and promote B cell retention in germinal centers

doi: 10.1084/jem.20111449

Figure Lengend Snippet: DTx-mediated selective FDC ablation disrupts B cell follicle architecture despite retention of CXCL13. Unimmunized CD21-DTR chimeras were control (Ctrl) or DTx treated 2 d before analysis of spleen (spl) and pLN sections. (A) Immunofluorescence analysis for BP3 + stromal cells, CD35 + FDCs, and IgD + follicular B cells. (B) In situ hybridization for Cxcl13 mRNA with serial sections stained for B220 and CD35 or for Cxcl13 and CD35. (C) Quantitative RT-PCR analysis for Cxcl13 , Cr1 (CD35), Mfge8 , and Tnsf13B (BAFF) mRNA in pLNs, spleen, and mLNs, normalized to Hprt mRNA. Data are representative of four experiments (mean ± SEM). Statistical analysis was performed with the two-tailed unpaired Student’s t test. *, P < 0.05; n.s., not significant. (D) Immunohistochemical analysis for CXCL13 and CD3 or B220 in serial sections of spleen and pLNs. Data in A, B, and D are representative of at least three experiments (at least two mice of each type per experiment). Bars: (A [left] and B [left and middle]) 200 µm; (A and D, right) 100 µm; (B [right] and D [left]) 50 µm.

Article Snippet: Cryosections of 7 μm were fixed and stained immunohistochemically as previously described ( ) with the following first antibodies: PE-conjugated anti-IgD (11-26c.2a; BD), biotin-conjugated anti-CD35 (8C12; BD), rat anti–mouse fibroblast (ER-TR7; Novus Biologicals), rabbit anti–mouse Collagen IV (Abcam), rat anti–mouse B220 (RA-6B2; BD), biotin-conjugated anti–T/B cell activation antigen or Ly77 (GL7; BD), biotin conjugated anti–mouse CD3e (145-2C11; BD), goat anti–mouse CXCL13 (R&D Systems), goat anti–mouse CCL21 (R&D Systems), rat anti-CD169 (Ser4; P. Crocker, University of Glasgow, Glasgow, Scotland, UK), rat anti–mouse madcam-1 (MECA-367; BD), rat anti–mouse FDC (FDC-M1; BD), and FITC-conjugated rat anti–mouse CD16/32 (FcγRII/III; UCSF Hybridoma Core).

Techniques: Control, Immunofluorescence, In Situ Hybridization, Staining, Quantitative RT-PCR, Two Tailed Test, Immunohistochemical staining

Journal: Nature Communications

Article Title: Single-cell and spatial transcriptome analyses reveal tertiary lymphoid structures linked to tumour progression and immunotherapy response in nasopharyngeal carcinoma

doi: 10.1038/s41467-024-52153-4

Figure Lengend Snippet: a Representative images of multiplex IHC staining of TLS in NPC tissue biopsy. Cells were coloured according to their staining with CD3 (green), CD4 (cyan), CD8 (orange), CD20 (red), and FAP-α (purple) proteins as indicated on top. Images are representative data from three independent NPC samples. Scale bars is 50 µm. b UMAP plots of cell clusters for B cells, CD4 + T cells, CD8 + T cells, and CAFs. Individual cell types were segregated from the major cell clusters identified in Supplementary Fig and subsequently clustered independently. Each dot represents a single cell, with colour coded according to the cell types or clusters as indicated in the legend to the right (comprising five, 16, 11, and five clusters for 97,360 B cells, 88,009 CD4 + T cells, 95,686 CD8 + T cells, and 1652 CAFs, respectively). c Heatmap plots of the normalised mean expression of TLS canonical marker genes (columns) for different cell clusters (rows) in B, CD4 + T, CD8 + T cells, and CAFs. Filled colours from blue to red represent scaled expression levels from low to high. d Representative images of Hematoxylin-Eosin staining (left) for TLSs and spatial feature plots of TLS-associated cell types (right) from the Stereo-seq (top; n = 3) and the Visium cohorts (bottom; n = 12). Dashed outlines indicate TLS areas on the H&E slides with scale bars of 0.1 mm and 300 μm. In spatial feature plots, the expression levels of the TLS-associated cluster signatures for each cell type (indicated on top) in each spot are filled with colours from black to yellow, representing scaled expression levels from low to high. e Box plots showing the signature scores of B lineage cells (Naïve_B, Memory_B, GC_B, and plasma cells), CD4_C8_CXCR5, CD8_C8_CXCL13, and iCAF_C2_CXCL13 in different tumour regions from NPC patients of the Stereo-seq (top; n = 9563) and the Visium cohorts (bottom; n = 21,753). Centre lines denote median values, and whiskers denote 1.5 × the interquartile range. P values are derived from two-sided student t-tests. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies included anti-CD20 (Abcam, Cat. no. ab9475, 1:50), anti-CD4 (ZSbio, Cat. no. ZA-0519), anti-CD8A (ZSbio, Cat. no. TA802079), anti-FAP-α (Abcam, Cat. no. ab207178, 1:200), anti-CXCL13 (NOVUS, Cat. no. NBP2-16041, 1:100; R&D systems, Cat. no. MAB8012, 1.7 µg/mL), anti-CASP3 (CST, Cat. no. 9664, 1:1000), anti-CD3 (ZSbio, Cat no. ZA-0503), anti-CD68 (ZSbio, Cat no. TA802952), anti-CD56 (ZSbio, Cat no. ZM-0057), anti-PanCK (ZSbio, Cat no. ZM-0069), anti-CXCR5 (Abcam, Cat. no. ab254415, 1:1000), anti-PD1 (ZSbio, Cat no. ZM-0381), anti-TIM3 (CST, Cat. no. 45208, 1:400), anti-CD70 (CST, Cat. no. 69209, 1:500), and anti-CD27 (Proteintech, Cat. no. 66308-1-lg, 1:1000).

Techniques: Multiplex Assay, Immunohistochemistry, Staining, Expressing, Marker, Derivative Assay

Journal: Nature Communications

Article Title: Single-cell and spatial transcriptome analyses reveal tertiary lymphoid structures linked to tumour progression and immunotherapy response in nasopharyngeal carcinoma

doi: 10.1038/s41467-024-52153-4

Figure Lengend Snippet: a Heatmap showing the normalised mean expression of cluster markers, complement, adhesion molecules, and chemokines (rows) for each CAF cluster (columns). Filled colours from blue to red represent scaled expression levels from low to high. b Bar plot of GSVA results showing the selected up-regulated signalling pathways enriched in iCAF_C2_CXCL13 CAFs. c Box plots showing the proportions of fibroblast clusters among all fibroblasts in NPC samples at different stages ( n = 16; removing samples without detectable malignant cells in the NPC tissue and samples with fewer than 100 CAFs). Centre lines denote median values, and whiskers denote 1.5 × the interquartile range. P values are derived from two-sided student t-tests. d Representative images for multiplex IHC staining of CXCL13 + CAFs and CXCR5 + B cells in NPC tissue biopsy. Cells were coloured according to their staining with CXCR5 (green), CD20 (cyan), FAP-α (orange), and CXCL13 (red) proteins as indicated on top. The green, cyan, orange, and red arrows indicated positive cells with the expression of CXCR5, CD20, FAP-α, and CXCL13 proteins in NPC tissue, respectively. Images are representative data from three independent NPC samples. Scale bars are 30 µm or 15 µm, respectively. e Dot plot showing selected ligand-receptor interactions between iCAF_C2_CXCL13 CAFs and B cells (GC_B, Memory_B, Naïve_B, and plasma cells) in NPC (red) or NPN (blue) samples. Ligand-receptor interactions are indicated in rows. The means of the average expression levels of two interacting molecules are indicated by the colour heatmap (bottom panel), with blue to red representing low to high expression. P values are obtained by one-sided permutation tests and indicated by circle size. f Spatial distribution of ligand-receptor interactions in representative NPC sections from the Stereo-seq (top panel) and the Visium cohorts (bottom panel). Each ligand and its corresponding receptor are indicated on top, with different colours for low and high intensities, as indicated at the right panel. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies included anti-CD20 (Abcam, Cat. no. ab9475, 1:50), anti-CD4 (ZSbio, Cat. no. ZA-0519), anti-CD8A (ZSbio, Cat. no. TA802079), anti-FAP-α (Abcam, Cat. no. ab207178, 1:200), anti-CXCL13 (NOVUS, Cat. no. NBP2-16041, 1:100; R&D systems, Cat. no. MAB8012, 1.7 µg/mL), anti-CASP3 (CST, Cat. no. 9664, 1:1000), anti-CD3 (ZSbio, Cat no. ZA-0503), anti-CD68 (ZSbio, Cat no. TA802952), anti-CD56 (ZSbio, Cat no. ZM-0057), anti-PanCK (ZSbio, Cat no. ZM-0069), anti-CXCR5 (Abcam, Cat. no. ab254415, 1:1000), anti-PD1 (ZSbio, Cat no. ZM-0381), anti-TIM3 (CST, Cat. no. 45208, 1:400), anti-CD70 (CST, Cat. no. 69209, 1:500), and anti-CD27 (Proteintech, Cat. no. 66308-1-lg, 1:1000).

Techniques: Expressing, Derivative Assay, Multiplex Assay, Immunohistochemistry, Staining

Journal: Nature Communications

Article Title: Single-cell and spatial transcriptome analyses reveal tertiary lymphoid structures linked to tumour progression and immunotherapy response in nasopharyngeal carcinoma

doi: 10.1038/s41467-024-52153-4

Figure Lengend Snippet: a Schematic diagram showing the B cell adhesion assay (left panel). Bar plots showing the capability of B cell adhesion to CAFs (right panel; n = 3). B cells were cocultured with either VCAM1 - CAFs, VCAM1 + CAFs, or VCAM1 + CAFs in combination with a VCAM1 neutralising antibody. The relative number (OD value) of B cells bound to CAFs were quantified using CCK8 assays. b Bar plot showing the relative expression level of CXCL13 in fibroblasts (left panel; n = 3). Fibroblasts were transduced with an empty vector or CXCL13 plasmid. Bar plot illustrates the migration capabilities of B cells under various co-culture conditions with fibroblasts (right panel; n = 11). The B cells were co-cultured with fibroblasts overexpressing either an empty vector (Con_EV), CXCL13 alone, or CXCL13 in combination with a CXCL13-specific neutralising antibody. c Schematic diagram showing the B cell co-culture assay (left panel). Bar plots showing IgG production of B cells without (grey) or with coculture of VCAM1 + (orange) or VCAM1 - (blue) CAFs (right panel; n = 3). The IgG levels in the cocultured supernatant were determined using ELISA. d Bar plot showing the relative expression level of TNFSF13B in fibroblasts (left panel; n = 3). Fibroblasts were transduced with an empty vector or TNFSF13B plasmid. Bar plot showing the IgG production of B cells co-cultured with fibroblasts overexpressing either an empty vector (Con_EV), TNFSF13B alone, or TNFSF13B in combination with a TNFSF13B-specific neutralising antibody (right panel; n = 4). IgG level in supernatant was determined using ELISA. P values are derived from two-sided student t-tests. The data is presented as mean ± SD. OE overexpression, Neu neutralising antibody, Con control, EV empty vector. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies included anti-CD20 (Abcam, Cat. no. ab9475, 1:50), anti-CD4 (ZSbio, Cat. no. ZA-0519), anti-CD8A (ZSbio, Cat. no. TA802079), anti-FAP-α (Abcam, Cat. no. ab207178, 1:200), anti-CXCL13 (NOVUS, Cat. no. NBP2-16041, 1:100; R&D systems, Cat. no. MAB8012, 1.7 µg/mL), anti-CASP3 (CST, Cat. no. 9664, 1:1000), anti-CD3 (ZSbio, Cat no. ZA-0503), anti-CD68 (ZSbio, Cat no. TA802952), anti-CD56 (ZSbio, Cat no. ZM-0057), anti-PanCK (ZSbio, Cat no. ZM-0069), anti-CXCR5 (Abcam, Cat. no. ab254415, 1:1000), anti-PD1 (ZSbio, Cat no. ZM-0381), anti-TIM3 (CST, Cat. no. 45208, 1:400), anti-CD70 (CST, Cat. no. 69209, 1:500), and anti-CD27 (Proteintech, Cat. no. 66308-1-lg, 1:1000).

Techniques: Cell Adhesion Assay, Expressing, Transduction, Plasmid Preparation, Migration, Co-Culture Assay, Cell Culture, Co-culture Assay, Enzyme-linked Immunosorbent Assay, Derivative Assay, Over Expression, Control

Journal: Nature Communications

Article Title: Single-cell and spatial transcriptome analyses reveal tertiary lymphoid structures linked to tumour progression and immunotherapy response in nasopharyngeal carcinoma

doi: 10.1038/s41467-024-52153-4

Figure Lengend Snippet: a Heatmap showing normalised mean expression of markers (naïve, effector, tissue resident, exhausted, hypoxia, activation, stem-like, toll-like receptor families, and chemokines) for each CD8 + T cell cluster. Colours from blue to red represent low to high scaled expression levels. b Representative images for multiplex IHC staining of CXCL13 + CD8 + T cells in NPC tissue biopsy. Cells coloured by PD1 (green), TIM3 (yellow), CD20 (orange), CD8 (purple), and CXCL13 (red). Arrows indicate cells expressing PD1 (green), TIM3 (yellow), CD20 (orange), CD8 (purple), and CXCL13 (red) in NPC tissue. Representative images from three independent NPC samples. Scale bars = 40 µm. c Pseudotime trajectories of CD8 + T cells (CD8_C1-C11; n = 41,736) with TCR information. Each dot represents a single cell, coloured by cluster label (top) or top 10 TCR clonotypes of CD8_C8_CXCL13 cells (bottom). d Scatter plots showing exhausted (left) and stem-like (right) scores of CD8 + T cells along pseudotime scores (x-axis) among clusters. Each dot represents a single cell, coloured by cluster label as in panel ( c ). e Box plots showing the percentage of CD8 + T cells sharing TCR clonotypes with top 10 TCR clonotypes of CD8_C8_CXCL13 cells across all CD8 + T cells. Coloured dots represent patients ( n = 3). Comparison made using a two-sided Kruskal-Wallis test across all clusters. f Representative spatial feature plots showing expression levels of CD8_C8_CXCL13 and CD8_C11_HAVCR2 signatures in each data spot from Stereo-seq (left; n = 9563) and Visium (right; n = 21,753) cohorts. Colours from black to yellow represent low to high scaled expression levels. Box plots show signature scores of CD8_C8_CXCL13 and CD8_C11_HAVCR2 in TLS, TCA, and stromal regions from Stereo-seq (left) and Visium (right) cohorts. Comparisons were made using a two-sided student t-test. g Pie charts showing contributions of ligand-receptor pairs among interactions between malignant and CD8_C8_CXCL13 cells (top) or CD8_C11_HAVCR2 cells (bottom) in NPC. Ligand-receptor pairs indicated on right panel with different colours. Percentages are shown in pies. In box plots, endpoints depict minimum and maximum values; centre lines denote median values; whiskers denote 1.5 × the interquartile range. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies included anti-CD20 (Abcam, Cat. no. ab9475, 1:50), anti-CD4 (ZSbio, Cat. no. ZA-0519), anti-CD8A (ZSbio, Cat. no. TA802079), anti-FAP-α (Abcam, Cat. no. ab207178, 1:200), anti-CXCL13 (NOVUS, Cat. no. NBP2-16041, 1:100; R&D systems, Cat. no. MAB8012, 1.7 µg/mL), anti-CASP3 (CST, Cat. no. 9664, 1:1000), anti-CD3 (ZSbio, Cat no. ZA-0503), anti-CD68 (ZSbio, Cat no. TA802952), anti-CD56 (ZSbio, Cat no. ZM-0057), anti-PanCK (ZSbio, Cat no. ZM-0069), anti-CXCR5 (Abcam, Cat. no. ab254415, 1:1000), anti-PD1 (ZSbio, Cat no. ZM-0381), anti-TIM3 (CST, Cat. no. 45208, 1:400), anti-CD70 (CST, Cat. no. 69209, 1:500), and anti-CD27 (Proteintech, Cat. no. 66308-1-lg, 1:1000).

Techniques: Expressing, Activation Assay, Multiplex Assay, Immunohistochemistry, Comparison

Journal: Nature Communications

Article Title: Single-cell and spatial transcriptome analyses reveal tertiary lymphoid structures linked to tumour progression and immunotherapy response in nasopharyngeal carcinoma

doi: 10.1038/s41467-024-52153-4

Figure Lengend Snippet: a Survival analysis of the signature scores for B lineage cells (Naïve_B, Memory_B, GC_B, and plasma cells), CD4_C8_CXCR5, CD8_C8_CXCL13, and iCAF_C2_CXCL13 in two NPC cohorts (Bulk-RNA-seq, n = 147; and Microarray, n = 150). P value (y-axis) and HR (x-axis) were calculated using two-sided cox tests and plotted in different colours for specific cell types annotated at the right. PFS, progression-free survival; OS, overall survival. b Heatmap showing the normalised signature scores for B lineage cells (Naïve_B, Memory_B, GC_B, and plasma cells), CD4_C8_CXCR5, CD8_C8_CXCL13, and iCAF_C2_CXCL13 (rows) in each sample (columns) from three NPC cohorts as indicated (source). Filled colours from blue to red represent scaled expression levels from low to high. Coloured bars on top of the heatmaps indicate metastasis, OS (0: dead; 1: alive) or PFS (0 or 1 represent with or without progression), and sample source. Samples were grouped using two modules with low or high TLS cell signature (TLS-CS). c Kaplan-Meier survival curves of the two NPC cohorts (Bulk-RNA-seq, n = 147; Microarray, n = 150) with patients stratified by two modules of low or high TLS-CS. Survival duration and probability are indicated at the x- and y-axis, respectively. P value and HR were calculated using a two-sided cox test. d Box plots showing the signature scores for B lineage cells (Naïve_B, Memory_B, GC_B, and plasma cells), CD4_C8_CXCR5, CD8_C8_CXCL13, and iCAF_C2_CXCL13 in the responders (R) and non-responders (NR) to ICB (toripalimab plus chemotherapy) treatment from the Visium cohort of NPC ( n = 12). Centre lines denote median values, and whiskers denote 1.5 × the interquartile range. P values are derived from two- sided student t-tests. **** P < 2.22×10 -16 . Source data are provided as a Source Data file.

Article Snippet: Primary antibodies included anti-CD20 (Abcam, Cat. no. ab9475, 1:50), anti-CD4 (ZSbio, Cat. no. ZA-0519), anti-CD8A (ZSbio, Cat. no. TA802079), anti-FAP-α (Abcam, Cat. no. ab207178, 1:200), anti-CXCL13 (NOVUS, Cat. no. NBP2-16041, 1:100; R&D systems, Cat. no. MAB8012, 1.7 µg/mL), anti-CASP3 (CST, Cat. no. 9664, 1:1000), anti-CD3 (ZSbio, Cat no. ZA-0503), anti-CD68 (ZSbio, Cat no. TA802952), anti-CD56 (ZSbio, Cat no. ZM-0057), anti-PanCK (ZSbio, Cat no. ZM-0069), anti-CXCR5 (Abcam, Cat. no. ab254415, 1:1000), anti-PD1 (ZSbio, Cat no. ZM-0381), anti-TIM3 (CST, Cat. no. 45208, 1:400), anti-CD70 (CST, Cat. no. 69209, 1:500), and anti-CD27 (Proteintech, Cat. no. 66308-1-lg, 1:1000).

Techniques: RNA Sequencing Assay, Microarray, Expressing, Derivative Assay

Journal: Nature materials

Article Title: Colony stimulating factor-1 receptor is a central component of the foreign body response to biomaterial implants in rodents and non-human primates.

doi: 10.1038/nmat4866

Figure Lengend Snippet: Figure 5 | CSF1R-dependent macrophages recruit fibrosis-potentiating B cells via CXCL13. a, CSF1R inhibition prevents the entire (innate and adaptive) immune response to implanted biomaterials. Phase contrast images showing that fibrosis, as compared with wild-type (WT) vehicle-treated control, was partially eliminated by CXCL13 neutralization, and completely eliminated with continuous CSF1R inhibition (inh.; 160 mg kg−1 body weight GW2580 s.c., daily) over a 14-day implant period. Scale bars, 2,000 µm. Fibrosis was reduced with CXCL13 neutralization by the same extent as that of the B cell knockout (B KO) shown in Fig. 3a. b, Flow analysis for responding macrophages, neutrophils and B cells dissociated directly from spheres (100 µl material in all cases) 14 days post-intraperitoneal (i.p.) implant, showing partial loss of cell presence with CXCL13 neutralization, and complete loss following CSF1R inhibition. c, Bright-field images showing that fibrosis, compared with vehicle control, was completely eliminated by both macrophage depletion (−Macrophages) and CSF1R inhibition. Scale bar, 1,000 µm. d, Flow analysis for responding host innate immune macrophages and neutrophils dissociated directly from spheres (100 µl of each material) 14 days post-i.p. implantation, showing the loss of immune adhesion with loss of fibrosis due to either macrophage depletion (−Macrphages) or CSF1R inhibition. e, NanoString expression analysis for all known cytokine and cytokine receptors (see Supplementary Fig. 18 for complete data set, excerpted here), showing similar unique factors increased across all material (hydrogel alginate, ceramic glass, and polymer polystyrene (PS)) groups. f, Confocal for DAPI (cellular nuclei), macrophage marker CD68 (green), B cell marker CD19 (magenta), fibrosis-associated myofibroblast marker alpha smooth muscle actin (αSMactin, myofibroblasts, red), overlay, and bright-field imaging, showing that CXCL13 neutralization resulted in loss of B cell recruitment. Scale bar, 200 µm. g, qPCR expression analysis of α-SMactin directly on retrieved spheres from vehicle-treated (WT), B KO, and CXCL13-neutralized WT mice, plotted relative to expression levels on spheres from WT mice. Statistical analysis: one-way ANOVA with Bonferroni multiple comparison correction ∗∗∗p<0.0001, versus vehicle. Error bars, mean ± s.e.m. n=5 mice per group. Experiments were repeated at least 2–3 times.

Article Snippet: To neutralize secreted CXCL13, anti-CXCL13 antibody (mAB470, R&D Systems) was injected i.p. in sterile 1× PBS at a dose of 100 μg per mouse, once every 3 days, starting 3 days prior to implantation as well.

Techniques: Inhibition, Control, Neutralization, Knock-Out, Expressing, Polymer, Marker, Imaging, Comparison

Journal: Nature materials

Article Title: Colony stimulating factor-1 receptor is a central component of the foreign body response to biomaterial implants in rodents and non-human primates.

doi: 10.1038/nmat4866

Figure Lengend Snippet: Figure 6 | Essential fibrotic cascade players are also increased in non-human primates. SLG20 (0.5 mm) hydrogel spheres were implanted into either the intraperitoneal (i.p.) or subcutaneous (s.c.) dorsal region of cynomolgus monkeys and retrieved by laparoscopy-guided tissue excision or biopsy punch after 28 days (ref. 27). a, Laparoscopy images taken on days 0 and 28 (initial implantation and retrieval from the i.p. space). b, H&E- and Masson’s trichrome-stained histological sections of excised i.p. omentum tissue at 28 days for non-fibrosed fat-laden (No material, Mock) or heavily collagen-deposited and sphere-embedded (Implanted) omental tissue. Scale bars, 400 µm. c, Confocal staining showing DAPI (cellular nuclei), innate immune macrophage marker CD68 (green), and fibrosis-associated activated myofibroblast alpha smooth muscle actin (αSMactin) staining (red), showing cellular infiltration around and fibrosis deposition on an embedded 500 µm alginate sphere. Inset: additional confocal for CSF1R (green), showing positive staining on not only more distant macrophages but also material-proximal and fused foreign body giant cells; both ×20 magnification. White scale bars: both 200 µm for each respective image. d, Flow analysis showing similar host innate immune macrophage (CD68+CD11b+, top right quadrants) and remaining neutrophil/myeloid (CD68−CD11b+, bottom right quadrants) cells across C57BL/6 mice and cynomolgus monkeys, dissociated directly from fibrosed spheres and adjacent fibrosed omentum tissue (as percent composition) 28 days post-i.p. implantation. While the prominence of CD11b seems to be inverted in C57BL/6 mice versus cynomolgus monkeys, population response percentages are similar 28 days post-i.p. implantation. e, NanoString analysis for immune markers and cytokines, originally identified in C57BL/6 mice (note: CD66b is used here as a neutrophil marker, as Ly6g/Gr1 does not exist in NHPs or humans). Significant increases are observed for macrophage markers, as well as CSF1R and CXCL13 in both i.p. and s.c. implant sites, as compared with mock (saline-injected) controls (there was no difference between s.c. and i.p. mock controls). N=2 for i.p.-implanted groups; N=4 for s.c. treatment groups. These experiments were performed once for s.c. and twice for i.p. delivery.

Article Snippet: To neutralize secreted CXCL13, anti-CXCL13 antibody (mAB470, R&D Systems) was injected i.p. in sterile 1× PBS at a dose of 100 μg per mouse, once every 3 days, starting 3 days prior to implantation as well.

Techniques: Staining, Marker, Saline, Injection