Journal: Respiratory Research

Article Title: Attenuation of antigen-induced airway hyperresponsiveness and inflammation in CXCR3 knockout mice

doi: 10.1186/1465-9921-12-123

Figure Lengend Snippet: Effect of OVA sensitization and exposure on CD8+ and CD4+ T cell infiltration into airways in CXCR3 KO and WT mice . Top panel, representative histogram showing expression of CD4+ T cells and CD8+ T cells in BAL fluid. The data presented are from one representative of four independent experiments. Bottom panel, pooled data showing the percentage and aboslute number of CD4+ T cells and CD8+ T cells in BAL fluid, n = 4 separate experiments, *, p < 0.05 , **, p < 0.01.

Article Snippet: After washing, cells were then incubated with 50 μL of FITC-conjugated anti-CD4 Ab and PE-conjugated anti-CD8 Ab or control mouse IgG2b (BD PharMingen, San Diego, CA) for 1 hr on ice.

Techniques: Expressing

Journal: PLoS ONE

Article Title: Combined Stimulation of IL-2 and 4-1BB Receptors Augments the Antitumor Activity of E7 DNA Vaccines by Increasing Ag-Specific CTL Responses

doi: 10.1371/journal.pone.0083765

Figure Lengend Snippet: (A) Five tumor-cured mice treated with pE7+pIL-2+anti-4-1BB Abs (from Fig. 3B) and 2 tumor-cured mice treated with pE7+pIL-2+control Abs (from Fig. 3B) were re-challenged s.c. with 4 x 10 5 TC-1 cells per mouse at 120 days post-first treatment. The tumor size was measured over time, and the mean tumor size was recorded. The values and bars represent the mean tumor sizes and SD, respectively. The numbers in (/) indicate the number of mice showing tumor regression for 270 days post-re-challenge/the number of mice tested. (B,C,D) Three of the 5 mice that received tumor cell re-challenge and rejected their tumors (from Fig. 4A) were sacrificed at 270 days post-tumor re-challenge and the spleens were removed for immune cell isolation. The immune cells were tested for the level of the CD44 high CD8+ T cell population among CD8+ T cells (B,C) and for the level of IFN-γ induction (D). Fig. 4B shows one representative figure displaying the level of CD44 high CD8+ T cells. Fig. 4C shows the mean percentage of CD44 high CD8+ T cells in each group and SD. Fig. 4D shows IFN-γ levels after in vitro stimulation with either E7 or control Trp2 peptides. (E,F) In another set of mice from a tumor treatment study, 3 of 5 tumor-cured mice following treatment with E7 DNA+IL-2+anti-4-1BB were sacrificed at 120 days post-treatment and the spleens were removed for immune cell isolation. The immune cells were used for IFN-γ assays (E). The remaining two mice from Fig. 4E were re-challenged with TC-1 cells (2×10 5 cells/mouse) at 138 days post-treatment in parallel with naïve mice. The mice were sacrificed at 18 days post-re-challenge and the spleens were removed for an IFN-γ assay (F). (E,F) show the IFN-γ levels in each group. The values and bars represent the mean IFN-γ levels of each test group and SD. *p<0.05 using one-way ANOVA compared to naïve control.

Article Snippet: The isolated cells were incubated with APC-labeled anti-CD44 (BD) and PE-labeled anti-CD8 Abs (BD) for fluorescence-activated cell sorting (FACS) analysis.

Techniques: Cell Isolation, In Vitro

Journal: Scientific Reports

Article Title: Determining the immune environment of cutaneous T-cell lymphoma lesions through the assessment of lesional blood drops

doi: 10.1038/s41598-021-98804-0

Figure Lengend Snippet: Cell populations differ between peripheral and lesional blood in patients with mycosis fungoides (MF). ( a ) Flow cytometry analysis of CD4 + and CD8 + T cells from 2, 5, and 10 μL peripheral blood for the detection of T cells in small amounts of blood. The number in the upper right corner represents the number of CD4 + and CD8 + T cells. Mean (+ standard deviation) numbers of CD4 + and CD8 + cells (n = 3) are shown in the bar graph (right). ( b ) Lesional blood was collected during a skin biopsy. Peripheral and lesional blood was obtained from the patient’s arm and erythematous lesion, respectively, on the same day. The collected blood cells and serum were used for the subsequent experiments. ( c ) Flow cytometry of CD4 + and CD8 + T cells obtained from 5 μL of peripheral and lesional blood. Numbers of CD4 + and CD8 + T cells in 5 μL of peripheral and lesional blood are shown (right, n = 4). ( d ) Representative mass cytometry analysis of peripheral and lesional blood from a patient with MF through viSNE analysis using Cytobank. The color of each dot represents the immune cell subset. ( e ) The proportion of each cell population in peripheral and lesional blood obtained from patients with MF (n = 5) quantified by mass cytometry. A paired t -test was used for the statistical analysis. * P < 0.05.

Article Snippet: Skin specimens were cut from the tissue block in 4-μm sections and stained with hematoxylin and eosin, anti-CD4 antibodies (Ab; clone EPR6855, Abcam), anti-CD8 Ab (clone C8/144B, Abcam), or anti-CD45RO Ab (clone UCH-L1, Absolute Antibody).

Techniques: Flow Cytometry, Standard Deviation, Mass Cytometry

Journal: Scientific Reports

Article Title: Determining the immune environment of cutaneous T-cell lymphoma lesions through the assessment of lesional blood drops

doi: 10.1038/s41598-021-98804-0

Figure Lengend Snippet: CD8 + CD45RO + cells negatively correlate with the mSWAT score in patients with MF. ( a ) Typical clinical features of MF lesional skin with an mSWAT score of 22 (left) and an mSWAT score of 138 (right). ( b,c ) The percentage of CD4 + CD45RO + and CD8 + CD45RO + T cells in lesional blood was higher than that in peripheral blood. Paired t -test. ** P < 0.01. ( d,e ) Correlation of CD4 + CD45RO + T cells and CD8 + CD45RO + T cells in peripheral blood and lesional blood with the mSWAT score evaluated by Pearson correlation coefficient analysis (n = 14). ( f–i ) Representative immunofluorescence staining of CD4/8 (green) and CD45RO (red) in MF lesional skin. Scale bar = 100 μm. Correlation of CD4 + CD45RO + cells and CD8 + CD45RO + cells with mSWAT (n = 14) evaluated by Pearson correlation coefficient analysis. Quantification of the number of cells per visual field was performed using the Hybrid Cell Count BZ-H4C analyzer software. Data were statistically analyzed using the Pearson correlation test (2-tailed). j ) Results of multiplex chemokine bead assay using sera from peripheral and lesional blood (n = 14). Paired t -test. * P < 0.05, ** P < 0.01.

Article Snippet: Skin specimens were cut from the tissue block in 4-μm sections and stained with hematoxylin and eosin, anti-CD4 antibodies (Ab; clone EPR6855, Abcam), anti-CD8 Ab (clone C8/144B, Abcam), or anti-CD45RO Ab (clone UCH-L1, Absolute Antibody).

Techniques: Immunofluorescence, Staining, Cell Counting, Software, Multiplex Assay

Journal: Scientific Reports

Article Title: Determining the immune environment of cutaneous T-cell lymphoma lesions through the assessment of lesional blood drops

doi: 10.1038/s41598-021-98804-0

Figure Lengend Snippet: TCR repertoire of lesional blood is skewed, and expression of genes differs between lesional and peripheral blood. ( a ) Gene expression analysis through RNA-seq in CD4 + CD45RO + T cells of peripheral and lesional blood from 3 patients (Cases 12, 16, and 17). Scatter plots show the expression values of every annotated gene. Blue and red dots indicate significant upregulation of CD4 + CD45RO + T cells in peripheral and lesional blood, respectively. ( b ) Ten top-ranked terms from the Jensen Diseases library of Enrichr for genes upregulated in CD4 + CD45RO + T cells of lesional blood. ( c ) Circos plots of frequencies of Vα and Jα gene usage and combinations of productive sequences in CD4 + CD45RO + T cells of peripheral and lesional blood. The band widths represents the frequency of each VJ pair. ( d ) Diversity of the TCR repertoire in CD4 + CD45RO + T cells of peripheral and lesional blood was evaluated using the Shannon–Weaver Index. ( e ) RNA-seq in CD8 + CD45RO + T cells of peripheral and lesional blood from 3 patients (Cases 4, 18, and 19). ( f ) Ten top-ranked gene ontology terms of biologic processes for genes upregulated in CD8 + CD45RO + T cells of lesional blood. ( g ) Circos plots of CD8 + CD45RO + T cells of peripheral and lesional blood. ( h ) Shannon–Weaver Index of CD8 + CD45RO + T cells of peripheral and lesional blood.

Article Snippet: Skin specimens were cut from the tissue block in 4-μm sections and stained with hematoxylin and eosin, anti-CD4 antibodies (Ab; clone EPR6855, Abcam), anti-CD8 Ab (clone C8/144B, Abcam), or anti-CD45RO Ab (clone UCH-L1, Absolute Antibody).

Techniques: Expressing, RNA Sequencing Assay

Journal: Scientific Reports

Article Title: Determining the immune environment of cutaneous T-cell lymphoma lesions through the assessment of lesional blood drops

doi: 10.1038/s41598-021-98804-0

Figure Lengend Snippet: CD8 + CD45RO + TCR repertoire differs depending on the stage of MF. ( a,c,e ) Hematoxylin and eosin (HE) staining and immunofluorescence (IF) staining of 3 types of skin lesions (erythema, plaque, and tumor) from Case 10. CD8 (green) and CD45RO (red) are shown in the immunofluorescence staining image. Collagen is stained green, and red blood cells are stained red. Scale bar = 100 μm. ( b,d,f,g ) Circos plots of frequencies of Vα and Jα gene usage and combinations for CD8 + CD45RO + T cells in lesional blood from erythema, plaque, and tumor stages. The Circos plot at the bottom ( g ) is of cells obtained from peripheral blood. ( h–l ) Erythema and plaque of the same skin lesion from case 15. Same experiment as shown in panels ( a–g ).

Article Snippet: Skin specimens were cut from the tissue block in 4-μm sections and stained with hematoxylin and eosin, anti-CD4 antibodies (Ab; clone EPR6855, Abcam), anti-CD8 Ab (clone C8/144B, Abcam), or anti-CD45RO Ab (clone UCH-L1, Absolute Antibody).

Techniques: Staining, Immunofluorescence

Journal: Scientific Reports

Article Title: Determining the immune environment of cutaneous T-cell lymphoma lesions through the assessment of lesional blood drops

doi: 10.1038/s41598-021-98804-0

Figure Lengend Snippet: Patient background.

Article Snippet: Skin specimens were cut from the tissue block in 4-μm sections and stained with hematoxylin and eosin, anti-CD4 antibodies (Ab; clone EPR6855, Abcam), anti-CD8 Ab (clone C8/144B, Abcam), or anti-CD45RO Ab (clone UCH-L1, Absolute Antibody).

Techniques: