Journal: iScience

Article Title: Highly immunogenic DNA/LION nanocarrier vaccine potently activates lymph nodes inducing long-lasting immunity in macaques

doi: 10.1016/j.isci.2025.112232

Figure Lengend Snippet: DNA/LION vaccination induces germinal center activation (A) Proliferating LN cell subsets based on unsupervised t-SNE clustering of Ki67 + live cells (gray background) with overlaid manually gated CD4 Tfh (CD3 + CD4 + CXCR5 + PD-1 int ; light blue), CD4 GC-Tfh (CD4 + CD3 + CXCR5 + PD-1 high ; dark blue), CD8 Tfc1 (CD3 + CD8 + CXCR5 + PD-1 int/high [purple] with PD-1 high fraction [darker purple]), and GC-B (CD3 − CD20 + CXCR5 + CCR6 + Bcl-6 + ; green) cells. (B) Heatmap showing Log2FC estimate from GEE of LN subsets. p values from GEE summarized in the left columns with significant increase (red) and decrease (blue) with >2x and <2x upregulation are shown in respective shades. (C) Direct correlations between the changes (Log2FC D8 and D22 vs. D1) of the listed populations. Color and size of square represent Spearman R, and asterisks indicate spearman p < 0.05. (D) Representative pictures of activated CD4, CD8, and CD20 populations showing a B cell follicle (BCF), germinal center (GC), and T cell zone (TCZ) within an LN. Scale bars: 100 μm. In situ quantification of activated CD4 (CD3 + CD4 + Ki67 + ) and CD8 (CD3 + CD8a + CD4 - Ki67 + ) T cells within GC in LN per million nuclei. Combined data points from 7 animals and 3 controls. Number of analyzed GC: control, 144; D3/4, 46; D8, 155; D22, 219. p values from linear mixed effect model. Mean and SEM are shown. The p values are defined as ≤0.05, ∗; ≤0.01, ∗∗; ≤0.001, ∗∗∗; ≤0.0001,∗∗∗∗. (E) Heatmap showing Log2FC estimate from GEE of LN subsets +/− CXCR3. Statistical analysis as in (B). (F) Bubble plot summarizing Spearman correlations ( p < 0.05) between LN cell subset changes (log2FC, indicated by different bubble size) and the Log2FC of plasma cytokines/chemokines (D2 vs. D1). (G) Correlation plots summarize spearman correlations of GC cell subset frequencies and Ab levels to WA1 (left) and BA.4/5 (right) over time. Color and size of square represent Spearman R, and asterisks indicate p < 0.05.

Article Snippet: Mouse anti-human CD185 (CXCR5) Monoclonal Antibody (MU5UBEE), PE, e-Bioscience , Thermo Fisher Scientific , Cat#12-9185-42; RRID: AB_11219877.

Techniques: Activation Assay, In Situ, Control, Clinical Proteomics

Journal: iScience

Article Title: Highly immunogenic DNA/LION nanocarrier vaccine potently activates lymph nodes inducing long-lasting immunity in macaques

doi: 10.1016/j.isci.2025.112232

Figure Lengend Snippet: DNA/LION vaccination affects CD8 cell subpopulations (A) Pie charts depict the mean frequency in blood and LN of manually gated T EM (CD28 + CD95 + ), central memory (T CM; CD28 − CD95 + ), and naive (CD28 + CD95 − ) CD8 + T cell subsets and the circulating CXCR5 + PD-1 int/high CD8 cTfc1 and the two LN-associated CXCR5 + PD-1 int CD8 Tfc2 and CXCR5 + PD-1 high CD8 Tfc3 subsets. (B) Heatmap shows manually gated Log2FC estimates from GEE of Ki67 + and Ki67 + GzmB + T CM and T EM in blood and LN. p values from GEE are summarized in the left columns with significant increase (red) and decrease (blue) with >2x and <2x upregulation shown in respective shades. (C) Heatmap shows manually gated Log2FC of CD8 cTfc1 in blood and CD8 Tfc1 in LN. Statistical analysis as in (B). (D and E) Bubble plots summarizing Spearman correlation ( p = 0.05)of changes in cell populations (Log2FC D3/4vsD1; D8vsD1, D22vsD1; indicated by different bubble sizes) and changes of plasma cytokines/chemokines (log2FC, D2 vs. D1) for (D) T CM and T EM and (E) CD8 cTfc1 in blood and CD8 Tfc1 in LN.

Article Snippet: Mouse anti-human CD185 (CXCR5) Monoclonal Antibody (MU5UBEE), PE, e-Bioscience , Thermo Fisher Scientific , Cat#12-9185-42; RRID: AB_11219877.

Techniques: Clinical Proteomics

Journal: iScience

Article Title: Highly immunogenic DNA/LION nanocarrier vaccine potently activates lymph nodes inducing long-lasting immunity in macaques

doi: 10.1016/j.isci.2025.112232

Figure Lengend Snippet:

Article Snippet: Mouse anti-human CD185 (CXCR5) Monoclonal Antibody (MU5UBEE), PE, e-Bioscience , Thermo Fisher Scientific , Cat#12-9185-42; RRID: AB_11219877.

Techniques: Staining, Clinical Proteomics, Recombinant, Binding Assay, Biomarker Discovery, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Software

Journal: Biochemistry and Biophysics Reports

Article Title: BAFF induces CXCR5 expression during B cell differentiation in bone marrow

doi: 10.1016/j.bbrep.2023.101451

Figure Lengend Snippet: Suppressed Cxcr5 expression in bone marrow cells of BAFF-KO mice (A) The gene expression of Cd19, Bcl2, Igμ and Cxcr5 in the spleen of WT and BAFF-KO mice analyzed by qPCR. (B) The expression of Cd19, Bcl2, Igμ, Il7r and Cxcr5 in the bone marrow of WT and BAFF-KO mice analyzed by qPCR. Graphs show mean and SD of each group (n = 5). * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant by unpaired t -test.

Article Snippet: Single cell suspensions of the bone marrow and spleen cells in PBS with 1% HI-FBS were incubated with FITC-conjugated anti -IgM monoclonal antibody and PE-conjugated anti -CXCR5 monoclonal antibody (BD Pharmingen) for 1 h on ice and washed three times with PBS plus 1% HI-FBS.

Techniques: Expressing

Journal: Biochemistry and Biophysics Reports

Article Title: BAFF induces CXCR5 expression during B cell differentiation in bone marrow

doi: 10.1016/j.bbrep.2023.101451

Figure Lengend Snippet: Decreased CXCR5 expression of IgM + cell in bone marrow of BAFF-KO mice Bone marrow and spleen cells from WT and BAFF-KO mice were collected and stained with FITC-αIgM and PE-αCXCR5. (A) Representative profiles of flow cytometry for lymphocyte-gated bone marrow and spleen cells. The numbers in the graphs show the proportion of cells in each quadrant. (B) CXCR5 fluorescence intensity of IgM + cells from bone marrow and spleen (Black, WT; Blue, BAFF-KO). CXCR5 high IgM + bone marrow cells, which were detectable only in WT mice, are pointed by an arrow. (C, D) Total cell numbers of IgM + cells and IgM + CXCR5 + cells as well as the proportion of CXCR5 + cells in IgM + cells in bone marrow (C) and spleen (D) for both WT and BAFF-KO mice. (E) The proportion of CXCR5-expressing cells in IgM + cells of the bone marrow and spleen from WT and BAFF-KO mice represented as relative fold change to bone marrow or relative fold change to WT mice. (F, G) Geometric mean of CXCR5 fluorescence on IgM + cells and IgM + CXCR5 + cells, and IgM fluorescence on IgM + cells of the bone marrow (F) and spleen (G) in WT and BAFF-KO mice. Graphs show mean and SD of each group (n = 5). ** P < 0.01; *** P < 0.001; ns, not significant by either unpaired t -test or two-way ANOVA followed by Bonferroni multiple comparisons test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Single cell suspensions of the bone marrow and spleen cells in PBS with 1% HI-FBS were incubated with FITC-conjugated anti -IgM monoclonal antibody and PE-conjugated anti -CXCR5 monoclonal antibody (BD Pharmingen) for 1 h on ice and washed three times with PBS plus 1% HI-FBS.

Techniques: Expressing, Staining, Flow Cytometry, Fluorescence

Journal: Biochemistry and Biophysics Reports

Article Title: BAFF induces CXCR5 expression during B cell differentiation in bone marrow

doi: 10.1016/j.bbrep.2023.101451

Figure Lengend Snippet: Direct induction of CXCR5 expression in IgM + bone marrow cells from BAFF-KO mice by BAFF (A, B) Bone marrow cells from WT and BAFF-KO mice were cultured for 48 h in the presence of rmBAFF, rmIL-6, and/or rmTNF-α. Bone marrow cells were co-stained with FITC-αIgM and PE-αCXCR5, and lymphocyte-gated cells were analyzed by flow cytometry. (A) The proportion of CXCR5 + cells in IgM + cell. (B) The proportion of IgM + CXCR5 + cells in lymphocyte-gated cells. Graphs show mean and SD of each group (n = 5). Different letters on the bars in the graphs indicate statistical difference between groups. (C, D) Bone marrow cells from BAFF-KO mice were separated to CD19 − and CD19 + cell populations by MACS. CD19 + cells were cultured with rmBAFF for 48 h. Cells were co-stained with FITC-αIgM and PE-αCXCR5, and lymphocyte-gated cells were analyzed by flow cytometry. (C) Representative profiles of flow cytometry for total, CD19 − , and CD19 + bone marrow cells. The numbers in the graphs show the proportion of cells in the gated areas. (D) The proportions of IgM + cells and IgM + CXCR5 + cells in lymphocyte-gated CD19 + cells as well as the proportion of CXCR5 + cells in IgM + cells. Graphs show mean and SD of each group (n = 5). ** P < 0.01; *** P < 0.001; ns, not significant by unpaired t -test.

Article Snippet: Single cell suspensions of the bone marrow and spleen cells in PBS with 1% HI-FBS were incubated with FITC-conjugated anti -IgM monoclonal antibody and PE-conjugated anti -CXCR5 monoclonal antibody (BD Pharmingen) for 1 h on ice and washed three times with PBS plus 1% HI-FBS.

Techniques: Expressing, Cell Culture, Staining, Flow Cytometry

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: Gene Expression, 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: Comparison, 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