Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Glucocorticoids regulate TCR-induced elevation of CD4: functional implications.

doi: 10.4049/jimmunol.164.12.6213

Figure Lengend Snippet: FIGURE 1. CD4 expression increases after T cell activation indepen- dently of the stimulus used to activate CD41 T cells. A, Cells were acti- vated with either plate-bound anti-TCR or anti-CD3 mAb (1 mg/well) or the soluble stimulants SEA (1 mg/ml) and Con A (1.5 mg/ml). Cells were cultured for 24–120 h and analyzed by double-immunofluorescence stain- ing with PE-conjugated anti-CD25 mAb and FITC-conjugated anti-CD4 mAb. Unrelated PE- or FITC-conjugated IgG1 mAb were used as controls. Results (representative of four to six independent experiments) are ex- pressed as the mean fluorescence intensity (MFI). Mean CD4 fluorescence of freshly isolated cells was 580 vs 9 for mAb control. Similar results were obtained after staining with OX35 (data not shown). B, FACS plot of SEA-activated CD41 T cells (as shown in A) after 24 h (solid lines) or 96 h (dashed lines) days of incubation. The histograms on the left show control staining with unrelated FITC-conjugated IgG1 mAb. C, CD4 expression by CD251 vs CD252, SEA-treated CD41 T cells (as described in A) after 24–72 h of culture.

Article Snippet: For flow cytometry, FITC-conjugated mouse anti-rat TCRab IgG1 mAb, FITC-conjugated mouse anti-rat CD4 IgG1 mAb (clone W3/25, which recognizes an epitope of domain 1 of CD4), FITCconjugated mouse anti-rat CD8a IgG1 mAb (clone OX8), PE-conjugated anti-CD4 mAb (W3/25), PE-conjugated anti-CD8a mAb (OX8), and PEconjugated mouse anti-rat CD25 IgG1 mAb (clone OX39) were purchased from Serotec.

Techniques: Expressing, Activation Assay, Cell Culture, Staining, Isolation, Control, Incubation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Glucocorticoids regulate TCR-induced elevation of CD4: functional implications.

doi: 10.4049/jimmunol.164.12.6213

Figure Lengend Snippet: FIGURE 6. Activation-induced TCR down-regulation is not affected by CORT. A, Cells were activated as described in Fig. 1 and cultured for 24–120 h. TCR expression was analyzed by double-immunofluorescence staining with PE-conjugated anti-CD4 mAb and FITC-conjugated anti- TCR mAb. Unrelated PE- or FITC-conjugated IgG1 mAb were used as controls. Results (representative of four independent experiments) are ex- pressed as the percent MFI of TCR expression by fresh cells (5100%; MFI 5 389.8; MFI for mAb control, 8). B, Cells were incubated in the absence or the presence of CORT (1026 M) and were simultaneously ac- tivated with plate-bound anti-TCR mAb (1 mg/well). Cell culture and TCR analysis were performed as described in A.

Article Snippet: For flow cytometry, FITC-conjugated mouse anti-rat TCRab IgG1 mAb, FITC-conjugated mouse anti-rat CD4 IgG1 mAb (clone W3/25, which recognizes an epitope of domain 1 of CD4), FITCconjugated mouse anti-rat CD8a IgG1 mAb (clone OX8), PE-conjugated anti-CD4 mAb (W3/25), PE-conjugated anti-CD8a mAb (OX8), and PEconjugated mouse anti-rat CD25 IgG1 mAb (clone OX39) were purchased from Serotec.

Techniques: Activation Assay, Cell Culture, Expressing, Staining, Control, Incubation

Journal: NPJ Precision Oncology

Article Title: Clock pathway inhibitor overcomes tumor immune-exclusion via regulation of fibrocyte differentiation

doi: 10.1038/s41698-025-01066-6

Figure Lengend Snippet: A Double staining of CD8 and αSMA in mouse various subcutaneous tumors. Scale bar, 200 μm. The quantitative evaluation of sections from each tumor stained for B αSMA ( n = 12 fields from four mice per group) and C CD8 ( n = 12 fields from four mice per group). D The correlation between the number of CD8 + T cells and αSMA + area in each mouse tumor tissue. E Double staining of collagen 1 and CD45 to detect fibrocytes in mouse various subcutaneous tumors. Scale bar, 100 μm. F The quantitative evaluation of sections from each tumor stained for fibrocytes ( n = 12 fields from four mice per group). G The correlation between the number of fibrocytes and αSMA + area in each mouse tumor tissue. The correlation was estimated by Spearman’s correlation and a linear regression analysis (the best-fit line is indicated). Representative images of tumor sections resected from non-small cell lung carcinoma (NSCLC) patients stained to detect αSMA + cancer-associated fibroblasts ( H , αSMA + FAP + ) and fibrocytes ( I , CD45 + FSP-1 + ). Scale bar, 100 μm. J Quantitative evaluation of tumor-infiltrating fibrocytes (CD45 + FSP-1 + ) in NSCLC tumor tissue stained in Fig. 1H and I ( n = 50 patients). K Representative images of resected NSCLC tissue stained to detect CD8 + T cells and EpCAM + tumor cells. Scale bar, 100 μm. L Quantitative evaluation of tumor-infiltrating fibrocytes (CD45 + FSP-1 + ) in each NSCLC group divided by the immune phenotypes of tumor-infiltrating CD8 + T cells (inflamed, desert, and exclusion). * P < 0.05 by a one-way ANOVA. All data are shown as the mean ± s.e.m.

Article Snippet: CD8 + and CD4 + T cells, macrophages, and Tregs were identified using a rat anti-CD8a monoclonal antibody (1:150, 53-6.7; BD Pharmingen, Franklin Lakes, NJ, USA), rat anti-mouse CD4 (1:150, H129.19; BD Pharmingen), rat anti-mouse F4/80 monoclonal antibody (1:100 dilution, CI:A3-1; Abcam, Cambridge, MA, USA), and rabbit anti-Foxp3 polyclonal antibody (1:400 dilution; Novus Biologicals, Centennial, CO, USA), respectively.

Techniques: Double Staining, Staining

Journal: NPJ Precision Oncology

Article Title: Clock pathway inhibitor overcomes tumor immune-exclusion via regulation of fibrocyte differentiation

doi: 10.1038/s41698-025-01066-6

Figure Lengend Snippet: A The evaluation of the tumor volume of AB1-HA-bearing balb/c nude mice treated with KL001 from 5 days after tumor cell injection ( n = 6 per group). B Double staining of CD8 and αSMA in AB1-HA tumors from balb/c mice treated with or without KL001 (studied in Fig. ). Scale bar, 200 μm. C Representative images and D quantitative evaluation of sections from KL001-treated AB1-HA tumors stained for CD8, CD4, Foxp3, and F4/80 ( n = 15 fields from five mice per group). Scale bar, 200 μm. E Quantitative evaluation of sections from CLK8-treated AB1-HA tumors stained for CD8, CD4, Foxp3 ( n = 15 fields from five mice per group). * P < 0.05 by the Mann-Whitney U test. All data are shown as the mean ± s.e.m.

Article Snippet: CD8 + and CD4 + T cells, macrophages, and Tregs were identified using a rat anti-CD8a monoclonal antibody (1:150, 53-6.7; BD Pharmingen, Franklin Lakes, NJ, USA), rat anti-mouse CD4 (1:150, H129.19; BD Pharmingen), rat anti-mouse F4/80 monoclonal antibody (1:100 dilution, CI:A3-1; Abcam, Cambridge, MA, USA), and rabbit anti-Foxp3 polyclonal antibody (1:400 dilution; Novus Biologicals, Centennial, CO, USA), respectively.

Techniques: Injection, Double Staining, Staining, MANN-WHITNEY

Journal: NPJ Precision Oncology

Article Title: Clock pathway inhibitor overcomes tumor immune-exclusion via regulation of fibrocyte differentiation

doi: 10.1038/s41698-025-01066-6

Figure Lengend Snippet: A The evaluation of the tumor volume and B the representative image of tumor tissue of LLC-bearing mice treated with KL001 and/or αPD-L1 Ab (n = 7 per group). * P < 0.05 by a one-way ANOVA. C Representative images and D the quantitative evaluation of sections from LLC tumors stained for αSMA ( n = 15 fields from five mice per group). The tumors were harvested at day 21 from each group studied in Fig. 6A. Scale bar, 200 μm. Representative images and the quantitative evaluation of sections from LLC tumors stained for collagen 1 + CD45 + fibrocytes ( E, F ), CD8 + T cells ( G, H ) and CD4 + T cells ( I, J ). Scale bar, 100 μm. * P < 0.05 by a one-way ANOVA. K The evaluation of the tumor volume of AB1-HA-bearing mice treated with KL001 and/or αCTLA-4 Ab ( n = 6 per group). * P < 0.05 by a one-way ANOVA. The quantitative evaluation of sections from AB1-HA tumors ( n = 15 fields from five mice per group) stained for CD8 ( L ), CD4 ( M ), and Foxp3 ( N ). The tumors were harvested at day 18 from each group studied in Fig. 6K. * P < 0.05 by a one-way ANOVA. All data are shown as the mean ± s.e.m.

Article Snippet: CD8 + and CD4 + T cells, macrophages, and Tregs were identified using a rat anti-CD8a monoclonal antibody (1:150, 53-6.7; BD Pharmingen, Franklin Lakes, NJ, USA), rat anti-mouse CD4 (1:150, H129.19; BD Pharmingen), rat anti-mouse F4/80 monoclonal antibody (1:100 dilution, CI:A3-1; Abcam, Cambridge, MA, USA), and rabbit anti-Foxp3 polyclonal antibody (1:400 dilution; Novus Biologicals, Centennial, CO, USA), respectively.

Techniques: Staining

Journal: Cell reports

Article Title: Loss of PBAF promotes expansion and effector differentiation of CD8 + T cells during chronic viral infection and cancer

doi: 10.1016/j.celrep.2023.112649

Figure Lengend Snippet: (A) Mixed bone marrow (MBM) chimeric mice reconstituted with CD45.1 CD8α −/− and wild-type CD45.2 or Arid2 −/− CD45.2 were infected with LCMV clone 13 (Cl13) and analyzed on day 21 p.i. (B) Representative flow plots showing the frequency of GP33 + splenic CD8 + T cells in MBM chimera mice. (C–G) Summary data showing the proportion, subset distribution, and total number of GP33 + splenic CD8 + T cells in MBM chimera mice. (H) Summary data displaying the per cell expression (geometric mean fluorescence intensity [gMFI]) of PD-1, LAG3, and TIM3 in GP33 + splenic CD8 + T cells. (I) Representative flow plots and summary data showing the proportion of IFN-γ + CD8 + T cells. (J) Representative flow plots and summary data showing gMFI of granzyme B in GP33 + splenic CD8 + T cells. (K) Summary data showing the gMFI of TCF1, T-bet, Eomes, and TOX in GP33 + splenic CD8 + T cells. (L) Scatterplot displaying viral load in the sera. Summary data (mean ± SEM) are pooled from 2 experiments with at least 3 mice/group/experiment. Data are representative of two independent experiments. *p < 0.05, **p < 0.01, ***p < 0.0001.

Article Snippet: BV480 Rat Anti-Mouse CD8a , BDbiosciences , Cat# 566096, RRID:AB_2739500.

Techniques: Infection, Expressing, Fluorescence

Journal: Cell reports

Article Title: Loss of PBAF promotes expansion and effector differentiation of CD8 + T cells during chronic viral infection and cancer

doi: 10.1016/j.celrep.2023.112649

Figure Lengend Snippet: (A) CD45.1 + P14 + CD8 + T cells transduced with sgCtrl and Arid2 or Pbrm1 guide RNA were adoptively transferred into CD45.2 + C57BL/6 mice that were subsequently infected with LCMV Cl13. (B) Representative flow plots showing the frequency of control and Arid2 - or Pbrm1 -deleted CD8 + T cells on days 8 and 21. (C and D) Proportion and frequency of CD45.1 + cells in PBMCs on days 8, 15, and 21. (E–H) Frequency and absolute numbers of CD45.1 + cells in lymph nodes and spleen on day 21 p.i. (I) PCA plot of bulk RNA-seq of sgCtrl and sgArid2. (J) PCA plot of bulk RNA-seq of sgCtrl and sgPbrm1. (K) GSEA showing pathways significantly up- or downregulated in chronically infected Pbrm1-deficient P14 T cells. (L and M) Enrichment plots showing representative gene sets identified in the GSEA of Pbrm1-deficient P14 T cells. Summary data (mean ± SEM) in (C), (D), (G), and (H) are pooled from at least 2 independent experiments with at least 3 mice/group per experiment. (E) and (F) are from one independent experiment with at least 4 mice/group. Data in (B)–(D) are representative of three independent experiments. (I–M) Four replicates were included in each condition. *p < 0.05, **p < 0.01, ***p < 0.0001. Illustration created with BioRender.com .

Article Snippet: BV480 Rat Anti-Mouse CD8a , BDbiosciences , Cat# 566096, RRID:AB_2739500.

Techniques: Transduction, Infection, Control, RNA Sequencing

Journal: Cell reports

Article Title: Loss of PBAF promotes expansion and effector differentiation of CD8 + T cells during chronic viral infection and cancer

doi: 10.1016/j.celrep.2023.112649

Figure Lengend Snippet: (A) Representative flow plots and summary data showing the frequency of the three subsets of control and Arid2 - or Pbrm1 -deleted CD45.1 + CD8 + T cells 21 days p.i. (B and C) Representative and summary plot displaying the frequency and numbers of the three subsets within control or Arid2 and Pbrm1 guide RNA (gRNA) transduced P14 CD8 + T cells. (D and E) Summary data showing gMFI of LAG3, Tim3, PD-1, 2B4, KLRD1, KLRG1, TIGIT, TCF1, Tbet, Eomes, and TOX in CD45.1 + CD8 T cells. (F) Plot displaying viral load in the sera from experimental mice reciving control or Arid2 - or Pbrm1 -deleted T cells. (G) Bulk RNA-seq GSEA results showing pathways significantly up- or downregulated in Pbrm1-deficient P14 T cells during LCMV Cl13 infection. (H and I) Enrichment plots showing representative gene sets identified in the GSEA of Pbrm1-deficient P14 T cells. (J) Heatmap of IFN pathway-related genes with differential expression between control and Pbrm1-deficient conditions. Summary data (mean ± SEM) are pooled from at least 2 independent experiments with at least 4 mice/group per experiment. (F) is from one independent experiment with at least 4 mice/group. Data are representative of three independent experiments. (G–J) Four replicates were included in each condition. *p < 0.05, **p < 0.01, ***p < 0.0001.

Article Snippet: BV480 Rat Anti-Mouse CD8a , BDbiosciences , Cat# 566096, RRID:AB_2739500.

Techniques: Control, RNA Sequencing, Infection, Quantitative Proteomics

Journal: Cell reports

Article Title: Loss of PBAF promotes expansion and effector differentiation of CD8 + T cells during chronic viral infection and cancer

doi: 10.1016/j.celrep.2023.112649

Figure Lengend Snippet: (A) Experimental design for multimodal analysis of the same cell with both single-nucleus RNA (snRNA) and scATAC-seq following chronic infection with LCMV Cl13. (B) Weighted nearest-neighbor uniform manifold approximation and projection (wnnUMAP) plot of splenic GP33 + CD8 + T cells from three mice from 21 days post-LCMV Cl13 infection. Each dot represents one cell, and cells are colored by cluster identity. (C) Bar graph showing cluster distribution frequencies of each sample. (D) Heatmap of differentially expressed genes for sgCtrl, sgArid2, and sgPbrm1 conditions for each cluster with K-means clustering. (E) Heatmap of differentially expressed chromatin regions for sgCtrl, sgArid2, and sgPbrm1 conditions for each cluster with K-means clustering. (F) Module scores of the top 100 differentially expressed genes from previously identified exhausted splenic CD8 + T cells for sgCtrl, sgArid2, and sgPbrm1 in the progenitor cluster. (G) Module scores of the top 100 differentially expressed genes from previously identified effector splenic CD8 + T cells for sgCtrl, sgArid2, and sgPbrm1 in the progenitor cluster. (F and G) p values determined by two-sided Wilcoxon rank-sum test. *p < 0.05, **p < 0.01, ***p < 0.0001. (H and I) Dot plot showing expression of proliferation markers. Dot size denotes the number of cells with a particular gene expressed, and intensity of dot color indicates the expression level of RNA expression (H) and transcription start site (TSS) accessibility (I).

Article Snippet: BV480 Rat Anti-Mouse CD8a , BDbiosciences , Cat# 566096, RRID:AB_2739500.

Techniques: Infection, Expressing, RNA Expression

Journal: Cell reports

Article Title: Loss of PBAF promotes expansion and effector differentiation of CD8 + T cells during chronic viral infection and cancer

doi: 10.1016/j.celrep.2023.112649

Figure Lengend Snippet: (A) Experimental design. (B) Tumor growth curves; of note, summary data are from (n = 9) sgCtrl transduced cell recipients and (n = 13) sg Arid2 transduced cell recipients. sgCtrl transduced cell recipient mice that did not developed >10% of tumor volume (n = 1) were excluded from the analysis. (C and D) Summary plot showing the frequency of CD45.1 + CD8 + T cells in the tumor and draining lymph node at day 8 post-ACT. (E) FlowJo analysis showing summary data showing the per-cell expression (gMFI) of LAG3, PD1, and CXCR6 in CD45.1 + CD8 + T cells. (F-J) The phenotype of CD45.1 + CD8 + T cells in the tumor was assessed by high-dimensional spectral flow cytometry on 12 markers using Cytobank . (F and G) Unbiased UMAP displaying clusters identified four clusters following concatenation of CD45.1 + CD8 T cells: (n = 6) sgCtrl transduced cells and (n = 6) sg Arid2 transduced cells. (H and I) Frequency of the three populations displayed using Population Sunburst. (J) Heatmap showing the mean expression of each marker in gated population. Data (mean ± SEM) in (C) and (E) are pooled and are from at least 3 mice/group/experiment and are representative of at least 3 independent experiments. (D) is from one independent experiment with 4 mice/group. Heatmaps were generated using hyperbolic arcsine (arcsinh) transformation against the mean expression of the combined concatenated samples. *p < 0.05, **p < 0.01, ***p < 0.0001.

Article Snippet: BV480 Rat Anti-Mouse CD8a , BDbiosciences , Cat# 566096, RRID:AB_2739500.

Techniques: Expressing, Flow Cytometry, Marker, Generated, Transformation Assay

Journal: Cell reports

Article Title: Loss of PBAF promotes expansion and effector differentiation of CD8 + T cells during chronic viral infection and cancer

doi: 10.1016/j.celrep.2023.112649

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: BV480 Rat Anti-Mouse CD8a , BDbiosciences , Cat# 566096, RRID:AB_2739500.

Techniques: Marker, Recombinant, Virus, Cell Isolation, Gene Expression, Infection, Software

Journal: Biology of blood and marrow transplantation : journal of the American Society for Blood and Marrow Transplantation

Article Title: Functional divergence of antigen-specific T-lymphocyte responses in syngeneic graft-versus-host disease.

doi: 10.1016/j.bbmt.2004.05.003

Figure Lengend Snippet: Figure 2. Flow cytometric selection of CLIP antigen-specific T-cell subsets. Splenic T lymphocytes harvested on day 14 after cessation of cyclosporine treatment were subjected to 3-color immunofluorescence. The cells were stained with the biotinylated soluble MHC class II immunoglobulin construct loaded with the N- or C-terminal CLIP variants (counterstained with CyChrome/streptavidin) and with fluorescein isothiocyanate (FITC)–and phycoerythrin (PE)–conjugated antibodies to CD8 and CD25, respectively. The percentage of cells staining with the construct is shown in the histogram; the dot plot displays CD8 and CD25 expression of the cells reacting with the peptide-loaded construct.

Article Snippet: The shaded area, however, repr etween N- and C-terminal flanking domain dependence. eric molecule loaded with unrelated peptides was [ B & M T neffective in isolating CLIP-specific T cells [23]. low cytometric multicolor analysis was used to isoate the antigen-specific T cells and to confirm expresion of the CD4, CD8, and CD25 cell-surface markrs (phycoerythrin- or fluorescein isothiocyanate– onjugated mouse anti-rat CD8, anti-CD4, and CD25 onoclonal antibodies; Serotec, Harlan Bioproducts or Science, Indianapolis, IN).

Techniques: Selection, Staining, Construct, Expressing