Journal: Frontiers in Genetics

Article Title: Activation of CCL21-GPR174/CCR7 on cardiac fibroblasts underlies myocardial ischemia/reperfusion injury

doi: 10.3389/fgene.2022.946524

Figure Lengend Snippet: mRNA and protein expression of key genes and related receptors. (A) mRNA expression of CCL21, XCR1, CXCL13, CASR, and EDN1. (B) Protein expression of CCL21, CXCL13, and XCR1. (C) Protein expression of GPR174/CCR7 (the receptors of CCL21) and CXCR5 (the receptors of CXCL13). n = 3–6. * p < 0.05, ** p < 0.01 vs. the sham group. I/R, ischemia reperfusion.

Article Snippet: The primary antibodies were as follows: goat anti-CCL21 (1:1000; R&D Systems, Minneapolis, MN), rabbit anti-XCR1 (1:1000; Novus Biologicals, Littleton, CO), goat anti-CXCL13 (1:1000; R&D Systems, Minneapolis, MN), goat anti-CCR7 (1:1000; Novus Biologicals, Littleton, CO), rabbit anti-GPR174 (1:1000; Biorbyt, Cambridge, United Kingdom), rabbit anti-CXCR5 (1:1000; Absin, Shanghai, China), and rabbit anti-β-tubulin (1:5000; Cell Signaling Technology, Beverly, MA).

Techniques: Expressing

Journal: iScience

Article Title: Tertiary lymphoid structure and decreased CD8 + T cell infiltration in minimally invasive adenocarcinoma

doi: 10.1016/j.isci.2022.103883

Figure Lengend Snippet:

Article Snippet: Rabbit anti-CXCR5 monoclonal antibody (D6L3C) , CST , Cat#: 72172S.

Techniques: Recombinant, Software

Journal: iScience

Article Title: Tertiary lymphoid structure and decreased CD8 + T cell infiltration in minimally invasive adenocarcinoma

doi: 10.1016/j.isci.2022.103883

Figure Lengend Snippet: TLS formation in MIA tumors (A) The schema of TLS formation. The first stage is B & T cell aggregation. The second stage is follicle formation with T-zone and B-zone. The third stage is ectopic germinal center formation with formation with follicular dendritic cell in light-zone. (B) Hierarchical clustering of Z score normalized gene expression of B cell infiltration markers (CD19 and CD20), follicle phenotype markers (CXCR5 and CXCL13), and lymphocyte homing signal markers (CCR7 and CCL19). Color of the top bar represents tumor (red) or adjacent normal (blue) tissue. Correlations between expression levels from these six TLS markers and immune cell abundance corrected by tumor purity are shown in the right bar. p Values are calculated using Spearman correlation; ∗∗∗p < 0.001; ∗∗p < 0.01; ∗p < 0.05; ns p > 0.05. (C) Typical observation field of MIA six-plex TLS panel staining. The merged-channel multispectral image was captured after 5-cycle TSA staining and DAPI counterstain, revealing a classic lymphoid structure residing in MIA stromal tissue. Single channels are shown for the area bounded by the white rectangle. Fake colors are assigned as DAPI: blue, CD8: yellow, CXCR5: pink, CD4: green, CD20: red, CD35: cyan. Tfh cells are highlighted with white arrows (lower right corner images). (D–G) Paired comparisons of CD20 + B cell density, CD4 + T cell density (E), CD35 + follicle dendritic cell density (F), and CD8 + T cell density (G) between non-follicle (Non-Fol) and TLS areas

Article Snippet: Rabbit anti-CXCR5 monoclonal antibody (D6L3C) , CST , Cat#: 72172S.

Techniques: Expressing, Staining

Journal: iScience

Article Title: Tertiary lymphoid structure and decreased CD8 + T cell infiltration in minimally invasive adenocarcinoma

doi: 10.1016/j.isci.2022.103883

Figure Lengend Snippet: Treg recruitment as potential mechanism of decreased CD8 + T cell infiltration in tertiary lymphoid structures (A) Comparison of immune suppressive cell infiltration estimated from XCELL between MIA tumor and adjacent normal tissues. p values are calculated using Mann-Whitney U test; ∗∗∗p < 0.001; ∗∗p < 0.01; ∗p < 0.05; ns p > 0.05. (B) Plot of Tfr/Tfh marker ratio vs CD8 + T cell infiltration. FoxP3, PDCD1, and IL2RA are markers of Tfr cells, while BCL6, ICOS, and IL7R are markers of Tfh cells. See text. p values are calculated using Spearman correlation test; ∗∗∗p < 0.001; ∗∗p < 0.01; ∗p < 0.05; ns p > 0.05. (C) Typical observation field of MIA 7-plex panel staining for Tfr and Tfh. Merged multispectral image was captured after 6-cycle TSA staining and DAPI counterstain and reveals a mature TLS architecture. Single channels are shown for the area bounded by the white rectangle. Fake colors are assigned as DAPI: blue, CD8: yellow, CXCR5: pink, CD4: green, FOXP3: red, BCL6: cyan and CD3: orange. Sub-populations of CD4 + T cells are highlighted by arrows (Tfr: red, Treg: purple, Th: green and Tfh: cyan). (D) Paired comparison of CD8 + T cell density between non-follicle (Non-Fol) and TLS areas. (E) Paired comparison of ratio of regulatory T cells to helper T cells between TLS and non-follicle areas. (F) Comparison of CD8 + T cell density between groups with high and low Tfr/Tfh ratio

Article Snippet: Rabbit anti-CXCR5 monoclonal antibody (D6L3C) , CST , Cat#: 72172S.

Techniques: MANN-WHITNEY, Marker, Staining

Journal: iScience

Article Title: Tertiary lymphoid structure and decreased CD8 + T cell infiltration in minimally invasive adenocarcinoma

doi: 10.1016/j.isci.2022.103883

Figure Lengend Snippet:

Article Snippet: Rabbit anti-CXCR5 monoclonal antibody (D6L3C) , CST , Cat#: 72172S.

Techniques: Recombinant, Software

Journal: Nature Communications

Article Title: Dysregulation of T FH -B-T RM lymphocyte cooperation is associated with unfavorable anti-PD-1 responses in EGFR -mutant lung cancer

doi: 10.1038/s41467-021-26362-0

Figure Lengend Snippet: a The dot plot summarizing the cell–cell interactions among CD8-C2 (T RM -like), B, and CD4-C9 (T FH -like) cells by CellPhoneDB analysis. Only significant ligand–receptor interactions are presented here. The size of the circle indicates the statistical significance, and the color gradient indicates the mean expression value of gene pairs. b Representative multiplexed immunofluorescence (IF) of TLS-like lesion in EGFR-WT ( n = 14) (upper) (scale bar 100 μm: left, scale bars 50 μm: right magnified) and EGFR-MT ( n = 14) (lower) (scale bar 100 μm: left, scale bars 50 μm: right magnified) tumors. Summarized graph for the area of TLS-like lesions in EGFR-WT ( n = 5) and EGFR-MT ( n = 4) tumors for single-cell analysis (** p ≤ 0.005, two-sided test) (upper panel) and in EGFR-WT ( n = 9) and EGFR-MT ( n = 10) tumors from independent patients for validation (* p ≤ 0.05, two-sided test) (lower panel). c Representative multiplexed IF of CD4, CXCL13, CD103, CD8, CXCR5, and CD20 in tumor specimens from EGFR-WT ( n = 14) (left) (scale bar 100 μm) and EGFR-MT ( n = 14) (right) (scale bar 100 μm) patients. d Summarized graph for the numbers of each cell type in total TLS-like lesions in EGFR-WT ( n = 5) and EGFR-MT ( n = 4) patients. The number of CD4 + , CXCL13 + CD4 + T cells, CD8 + T cells, CD103 + CD8 + T cells are significantly higher in EGFR-WT than EGFR-MT (* p ≤ 0.05, ** p ≤ 0.005, two-sided test). e Summarized graph for the numbers of each cell type in total TLS-like lesions in EGFR-WT ( n = 9) and EGFR-MT ( n = 10) tumors from independent patients for validation. Consistently, the number of CD4 + , CXCL13 + CD4 + T cells, CD8 + T cells, CD103 + CD8 + T cells were significantly higher in EGFR-WT than EGFR-MT (** p ≤ 0.005, *** p ≤ 0.0005, two-sided test). f An illustrative summary of the proposed model for T FH –B–T RM lymphocyte cooperation in TLS for antitumor immunity in lung cancer. Data are represented as mean ± SEM. Source data are provided as a Source Data file.

Article Snippet: To evaluate the CXCL13 + CD4, CXCR5 + CD20, CD103 + CD8, we performed another set of multiplexed IF using primary antibodies against the following antigens: for CD4 (ab133616, Abcam, Cambridge, UK, 1:200), CD8 (MCA1817, Bio-Rad, Hercules, CA, USA, 1:300), CXCL13 (PA5-47035, Invitrogen, Massachusetts, USA, 1:40), CXCR5 (72172 S, Cell Signaling Technology, Massachusetts, USA, 1:200), CD20 (ab9475, Abcam, Cambridge, UK, 1:100), and CD103 (ab129202, Abcam, Cambridge, UK, 1:500).

Techniques: Expressing, Immunofluorescence, Single-cell Analysis

Journal: Cancer Medicine

Article Title: Enhanced humoral immunity in breast cancer patients with high serum concentration of anti‐HER2 autoantibody

doi: 10.1002/cam4.3742

Figure Lengend Snippet: Tumor‐infiltrating CD20‐positive ICs, IGKC‐positive ICs, and CXCL13‐positive ICs, and follicular CD4‐positive ICs in the regional axillary lymph nodes in the high HER2‐AAb group and the low HER2‐AAb group. (A–F) Representative photographs of IHC staining for CD20 (A and B), IGKC (C and D), and CXCL13 (E and F) of the tumors with large cell counts (A, C and E) and the tumors with small cell counts (B, D and F). (G–J) Representative photographs of IHC staining for CD4 (brown) and CXCR5 (red) of the regional axillary lymph nodes with large cell counts (G and I) and with small cell counts (H and J). Images (I) and (J) are the high power fields of the images (G) and (H), respectively. Original magnification, ×20 (A–H) and ×40 (I and J). (K–M) Counts of ICs positive for CD20 (K), IGKC (L), and CXCL13 (M) in the high HER2‐AAb group and the low HER2‐AAb group. (N) Counts of CD4‐positive ICs in CXCR5‐positive B‐cell follicles (follicular CD4 + ) of the regional axillary lymph nodes in the high HER2‐AAb group and the low HER2‐AAb group. Medians are shown by horizontal dotted lines. p , Mann–Whitney U test. Scale bar, 100 µm (A–H) and 50 µm (I and J)

Article Snippet: The following primary antibodies were used: polyclonal anti‐Kappa Light Chain (1:200, Invitrogen), anti‐CD20 (L26, 1:50, Abcam), anti‐HLA Class 1 ABC (EMR8‐5, 1:100, Abcam), anti‐CD8 (C8/144B, 1:100, Dako), anti‐FOXP3 (236A/E7, 1:100, Abcam), anti‐CXCR5 (D6L3C, 1:200, Cell Signaling Technology), anti‐CD4 (4B12, 1:80, Dako), polyclonal anti‐CXCL13 (1:100, Invitrogen), and SP142‐compatible anti‐PD‐L1 (28‐8, 1:400, Abcam).

Techniques: Immunohistochemistry, MANN-WHITNEY

Journal: Science Advances

Article Title: A highly potent lymphatic system–targeting nanoparticle cyclosporine prevents glomerulonephritis in mouse model of lupus

doi: 10.1126/sciadv.abb3900

Figure Lengend Snippet: ( A ) Histological images showing that renal damage in the untreated MRL- lpr appeared to have a strong correlation with lymphocyte infiltration. Both CD20 + and CD3 + cells were enriched in the glomeruli (CD20 and CD3) and the interstitial space (CD3) ( n = 15 to 30 micrographs from at least three individuals; scale bars, 50 μm for first two rows of images; scale bars, 200 μm for third row of images). ( B ) Graphical analyses of CD20 + cells and ( C ) CD3 + cells in the histological images. ( D ) We observed elevated transcript levels of CXCR4 and ( E ) CXCR5 in the untreated MRL- lpr mice ( n = 5 to 6 individuals). ( F ) Renal histology also showed significant up-regulation of CXCR4 (brown) among infiltrating leukocytes and endogenous expression in the proximal tubules. CXCR5 + population (red) appeared to decorate the migrating front of infiltrating leukocytes. (scale bars, 200 μm for all images). Representative micrographs are shown. Data are represented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. Statistics were conducted against the MRL- lpr group. Comparisons were made with one-way ANOVA, followed by Tukey’s multiple comparisons test.

Article Snippet: Immunohistochemistry and immunofluorescence antibodies for CD20 (polyclonal) and IgG (polyclonal, Alexa Fluor 488) were purchased from Thermo Fisher Scientific (Waltham, MA); antibodies for WT1 (D8175), CXCR4 (D4Z7W), and CXCR5 (D6L3C) were purchased from Cell Signaling Technology (Danvers, MA); antibody for CD3 (polyclonal) was purchased from Agilent (Santa Clara, CA); and antibody for nephrin (polyclonal) was purchased from R&D Systems (Minneapolis, MN).

Techniques: Expressing