Journal: Journal of Gastrointestinal Oncology

Article Title: The targeted delivery of interleukin-12 to the carcinoembryonic antigen increases the intratumoral density of NK and CD8 + T cell in an immunocompetent mouse model of colorectal cancer

doi: 10.21037/jgo.2020.04.02

Figure Lengend Snippet: Therapy study and microscopic analysis of tumor-infiltrating lymphocytes. (A) Dose escalation study of Sm3E-mIL12 in mice bearing C51-CEA lesions intravenously injected every second day (arrows) (B) Therapeutic performance of Saline, 60 µg of KSF-mIL12 and 60 µg of Sm3E-mIL12 in C51-CEA-bearing BALB/c mice, injected every second day (arrows). Treatment started when subcutaneous tumors reached an approximate size of 100 mm3. Statistical differences were assessed between KSF-mIL12 (non-targeted product) and Sm3E-mIL12 (targeted product). ****, P<0.0001 (regular two-way ANOVA test with Bonferroni post-test). Data represent mean tumor volume and body weight change (± SEM). CR, complete response. n=5 mice per group. (C) ex vivo immunofluorescence analysis on C51-CEA sections, 24 h after the first and third injection(s) of Saline, 60 µg of KSF-mIL12 or 60 µg of Sm3E-mIL12. Markers specific for apoptotic cells (caspase-3), NK cells (NKp46), Tregs (Foxp3), CD4+ T cells (CD4), CD8+ T cells (CD8) were used (green). Blood vessels were stained with an anti-CD31 antibody (red). 20× magnification; scale bars =100 µm.

Article Snippet: Tumors were excised and embedded in cryoembedding medium (ThermoScientific) and the corresponding cryostat tissue sections (8–10 µm thickness) were stained using the following primary antibodies: goat anti-CD31 (R&D System; AF3628), rabbit anti-Foxp3 (Invitrogen; 7000914), rat anti-NKp46 antibody (BioLegend; 137602), rabbit anti-CD4 (Sino Biological; 50134-R001), rabbit anti-CD8 (Sino Biological; 50389-R208).

Techniques: Injection, Ex Vivo, Immunofluorescence, Staining

Journal: Cell reports

Article Title: Consumption of fish oil high-fat diet induces murine hair loss via epidermal fatty acid binding protein in skin macrophages

doi: 10.1016/j.celrep.2022.111804

Figure Lengend Snippet: (A) Representative hematoxylin and eosin staining of skin tissues from mice fed the LFD, fish oil HFD, or cocoa butter diet at different magnifications (red arrows: infiltrating inflammatory cells). Scale bars, 100 μm. (B and C) Flow cytometric analysis of immune cell phenotype in the dermis of mice on different diets for 3 months. A cell population with strong autofluorescence (red trapezoid gate) was specifically accumulated in the dermis of mice fed the fish oil HFD (B). Multichannel signals of the autofluorescent cells were analyzed using a BD Fortessa flow cytometer (C). (D–F) Autofluorescent cells in the dermis of fish oil HFD-fed mice were purified using a BD FACSAria II flow sorter and stained with a panel of metal-tagged CyTOF antibodies. Uniform manifold approximation and projection (UMAP) was used to visualize and identify immune cell populations in unsorted dermal cells (D) and sorted autofluorescent dermal cells (E). Individual surface marker signatures in the CyTOF panel are shown in (F). (G and H) Representative IHC images of F4/80 + macrophages (brown staining, G) and CD8 + T cells (brown staining, H) in the skin of mice fed the LFD, fish oil HFD, or cocoa butter HFD. Scale bars, 100 μm. See also . These in vitro experiments were repeated with at least three biological replicates.

Article Snippet: Rat monoclonal anti-mouse CD8a - 168Er , Fluidigm , Cat#3168003B; RRID:AB_2811241.

Techniques: Staining, Flow Cytometry, Purification, Marker, In Vitro

Journal: Cell reports

Article Title: Consumption of fish oil high-fat diet induces murine hair loss via epidermal fatty acid binding protein in skin macrophages

doi: 10.1016/j.celrep.2022.111804

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rat monoclonal anti-mouse CD8a - 168Er , Fluidigm , Cat#3168003B; RRID:AB_2811241.

Techniques: Purification, Recombinant, Activation Assay, SYBR Green Assay, Reverse Transcription, Detection Assay, Enzyme-linked Immunosorbent Assay, Selection, Software

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: bioRxiv

Article Title: Bone marrow mesenchymal stromal cells mediate cellular inflammation in HFpEF

doi: 10.1101/2025.03.28.645924

Figure Lengend Snippet: ( A ) Experimental outline of HFpEF in IFNɣ reporter mice (GREAT). ( B ) Flow cytometry gating for IFNɣ expression in CD8+ T cells from visceral adipose tissue. ( C ) Total and IFNɣ-producing CD8+ T cell numbers in visceral adipose tissue (VAT) in GREAT mice with HFpEF compared to GREAT control mice without HFpEF (n=8 mice per group, two-tailed Mann-Whitney test). ( D ) Experimental outline of CD8+ T cell depletion in mice with HFpEF. ( E ) Flow cytometry gating for CD8+ T cells in blood. ( F ) CD8+ T cell numbers in blood and VAT following CD8+ T cell depletion (n=6 per group, unpaired student’s t-test). ( G ) IFNɣ levels in blood in mice with HFpEF with and without CD8+ T cell depletion (n=6 per group, two-tailed Mann-Whitney test). ( H ) CXCL12 levels in the bone marrow plasma of mice with HFpEF with and without CD8+ T cell depletion (n=6 per group, unpaired student’s t-test). ( I ) Gating for blood monocytes in CD8+ depleted mice vs. isotype control mice. ( J ) Monocyte levels in the blood of CD8+ depleted mice vs. isotype controls (n=6 per group, unpaired student’s t-test).

Article Snippet: To deplete CD8+ T cells, mice were intraperitoneally (i.p.) injected with 200 µg of InVivo MAb anti-mouse CD8a (clone 2.43, BioXCell) or InVivo MAb rat IgG2b isotype control, anti-keyhole limpet hemocyanin (clone LTF-2, BioXCell), every 3 days throughout the course of the HFpEF protocol.

Techniques: Flow Cytometry, Expressing, Control, Two Tailed Test, MANN-WHITNEY

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Nitric oxide short-circuits interleukin-12-mediated tumor regression

doi: 10.1007/s00262-011-0998-2

Figure Lengend Snippet: IL-12 induction of iNOS in TAM. a Quantitative RT-PCR analysis of iNOS expression in tumors. Fine needle aspirates were obtained from pre- (day 0) and post-therapy (days 1, 2, 3, and 7) tumors, and the fold-change in iNOS as well as arginase mRNAs was quantified. *The increases in iNOS expression on days 1–7 were significant compared to day 0 (P ≤ 0.001), and day 2 iNOS expression was significantly higher than all other days (P ≤ 0.0029). The changes in arginase expression were not significant. Error bars = SD, n = 5–8 mice per time point. b Flow cytometric analysis of tumor-infiltrating myeloid cell subsets. Single-cell suspensions prepared from post-therapy day 2 tumors were analyzed for CD11b+ Gr1+ populations. Control tumors displayed an essentially identical profile (data not shown). c Analysis of intratumoral myeloid cell subsets for iNOS. The subsets identified in (b) were gated on and analyzed for membrane F4/80 and intracellular iNOS in control and IL-12-treated mice. Typical staining profiles for the P1 and P2/P3 populations are shown (Both P2 and P3 populations were negative for iNOS). P1 population could be separated into F4/80 − (P1a) and + (P1b) subsets with a portion of the F4/80+ cells staining positive for iNOS. Isotype controls for F4/80, iNOS, and other markers are shown in supplemental Figure 1. CD11b-negative populations (including the tumor cells) did not express iNOS (supplemental Figure 1). d Kinetics of iNOS expression in TAM. Percent of CD11bint Gr1lo F4/80+ TAM that were iNOS+ were quantified on days 0 (pre-therapy), 2, and 7. *The differences between day 2 and days 0 or 7 were significant (P ≤ 0.0029). Error bars = SD, n = 4 mice per group. e Quantitative analysis of tumor-infiltrating leukocyte subsets. T-regulatory cells (Treg, CD4+ CD25+ Foxp3+), T-helper cells (TH, CD4+ Foxp3−), CD8+ T-cells (CD3+ CD8+), NKT cells (CD3+ CD1d tetramer+), NK cells (CD45+ CD3− DX5α+), dendritic cells (DC, CD11c+ CD11blo Gr1− MHC Class II+), and P1/P2/P3 populations (as defined above) in untreated tumors were quantified. The differences between myeloid cell subsets (P1, P2, P3) and all other populations were highly significant (P ≤ 0.0000002). Error bars = SD, n = 5–9 mice per subset.f Induction of iNOS in wild-type vs IFNγ-KO mice. Fold-changes in iNOS mRNA levels between day 0 and day 2 tumors were quantified by qRT-PCR in wild-type and IFNγ-KO mice. Error bars = SD, n = 4–5 mice per group

Article Snippet: In vivo CD8 T-cell and macrophage depletions Anti-mouse CD8 mAb 53.6.72 (BioXcell) was used to deplete CD8 T cells via i.p. injection of 200 ug of anti-CD8 on days 1 and 5 [ 19 ].

Techniques: Quantitative RT-PCR, Expressing, Control, Membrane, Staining

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Nitric oxide short-circuits interleukin-12-mediated tumor regression

doi: 10.1007/s00262-011-0998-2

Figure Lengend Snippet: Effect of combined therapy with IL-12 and L-NAME on CD8+ T-cell-mediated tumor kill. a Tumor growth patterns during treatment. Mice-bearing subcutaneous Line-1 tumors (70–80 mm3) were treated either with IL-12, L-NAME, or both. Control mice received blank microspheres. Tumor volume was determined daily using the formula a2 × b/2, where a and b are the shortest and longest perpendicular dimensions of the tumor, respectively. *The differences between the IL-12 + L-NAME-treated group and control were significant (P ≤ 0.0013 on days 1–10). The differences between the IL-12 + L-NAME group and IL-12 alone or L-NAME alone groups were also significant between days 3 and 10 (P ≤ 0.025). #,&The differences between IL-12 alone and control groups were significant between days 4 and 10 (P ≤ 0.044) whereas the differences between L-NAME alone and control groups were significant only on days 9 and 10 (P ≤ 0.039). Error bars = SD, n = 5–8 mice per group. b Fold-change in intratumoral CD8 expression. Quantitative RT-PCR was employed to determine the relative levels of CD8 mRNA in pre- (day 0) and post-treatment (day 2) tumors. The fold-changes shown are relative to the control group. *The differences between the combined treatment group and IL-12 or L-NAME-alone groups were significant (P ≤ 0.035). Error bars = SD, n = 5–9 per group. c Intratumoral CD8+ T-cell numbers. CD8+ T-cell numbers were quantified by flow cytometric analysis of day 2 tumors. *The differences between the combined therapy group and other groups were significant (P ≤ 0.013). Error bars = SD, n = 5–8 mice per group. d Effect of CD8+ T-cell depletion on therapeutic efficacy. Mice were injected with anti-CD8 or a control isotype antibody 1 day prior to microsphere treatment and again on day 5 after treatment. Fold-changes in tumor volume between days 0 and 10 were determined. Error bars = SD, n = 5–8 mice/group

Article Snippet: In vivo CD8 T-cell and macrophage depletions Anti-mouse CD8 mAb 53.6.72 (BioXcell) was used to deplete CD8 T cells via i.p. injection of 200 ug of anti-CD8 on days 1 and 5 [ 19 ].

Techniques: Control, Expressing, Quantitative RT-PCR, Drug discovery, Injection

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Nitric oxide short-circuits interleukin-12-mediated tumor regression

doi: 10.1007/s00262-011-0998-2

Figure Lengend Snippet: Effect of L-NAME on post-therapy CD8+ Tem activity. a CD8+ Tem activation. Expression of CD43 and CD69 on day 2 post-treatment CD8+ Tem is shown. *The differences between IL-12-alone or IL-12 + L-NAME groups and control or L-NAME-alone were significant (P ≤ 0.0025). Error bars = SD, n = 4–5 per group. b CD8+ Tem apoptosis. Day 2 post-treatment CD8+ Tem were stained with Annexin V. IL-12-alone group was significantly different from all other groups (P ≤ 0.021). Error bars = SD, n = 4–5 per group. c Post-therapy CD8+ Tem proliferation. CD8+ Tem were stained for BrdU on day 2. *The differences between the IL-12 + L-NAME group and the other 3 groups were significant (P ≤ 0.025). Error bars = SD, n = 4–5 per group. d Effect of macrophage depletion on CD8+ Tem proliferation. The dot plots demonstrate that clodrolip depleted >80% of the iNOS-producing macrophages found in the tumor (42,280 ± 5,800 cells/g tumor vs. 9,200 ± 3,200 cells/g tumor in control vs. clodrolip groups, respectively, P = 0.0000013). The differences between the clodrolip+ IL-12 group and the controls with respect to percent proliferating CD8+ Tem were significant (P ≤ 0.014). Error bars = SD, n = 4 per group

Article Snippet: In vivo CD8 T-cell and macrophage depletions Anti-mouse CD8 mAb 53.6.72 (BioXcell) was used to deplete CD8 T cells via i.p. injection of 200 ug of anti-CD8 on days 1 and 5 [ 19 ].

Techniques: Activity Assay, Activation Assay, Expressing, Control, Staining

Journal: Molecular Vision

Article Title: Conjunctival HLA-DR and CD8 expression detected by impression cytology in ocular graft versus host disease

doi:

Figure Lengend Snippet: Comparison of the clinical data of patients with and without ocular cGvHD following HSCT.

Article Snippet: Slides were stained with primary antibodies against HLA-DR (mouse monoclonal antihuman; DAKO, Glostrup, Denmark; working dilution 1:50), cytokeratin (CK)19 (mouse monoclonal; Santa Cruz Biotechnology, CA; working dilution 1:100) and CD8 (mouse monoclonal; AbD Serotec, Duesseldorf, Germany; working dilution 1:50).

Techniques: Comparison, Expressing

Journal: Molecular Vision

Article Title: Conjunctival HLA-DR and CD8 expression detected by impression cytology in ocular graft versus host disease

doi:

Figure Lengend Snippet: Comparison of the clinical data of the 5 patients with positive CD8 cells in impression cytology and the other patients.

Article Snippet: Slides were stained with primary antibodies against HLA-DR (mouse monoclonal antihuman; DAKO, Glostrup, Denmark; working dilution 1:50), cytokeratin (CK)19 (mouse monoclonal; Santa Cruz Biotechnology, CA; working dilution 1:100) and CD8 (mouse monoclonal; AbD Serotec, Duesseldorf, Germany; working dilution 1:50).

Techniques: Comparison