Journal: Journal of experimental & clinical cancer research : CR

Article Title: Regnase-1 downregulation promotes pancreatic cancer through myeloid-derived suppressor cell-mediated evasion of anticancer immunity.

doi: 10.1186/s13046-023-02831-w

Figure Lengend Snippet: Fig. 6 Suppression of CTLs by CD11b+ MDSCs is responsible for the acceleration of tumor progression by Regnase-1 downregulation. A-D Evaluation of phenotypes of orthotopic syngeneic tumors of WT or Regnase-1 KO murine pancreatic cancer cells. Representative macro images of pancreatic tumors (A). Relative mRNA levels of Cd8a, Ifng, Fasl, and Gzmb (B) (N = 6 per group). Representative images of HE (C, left panel) and CD8a immunostaining (C, right panel) and the number of CD8-positive cells (C, right) (N = 6 per group). Dot plots of CD3+CD8+ cells evaluated by flow cytometry (D, left) and the proportion of CD8 + cells among CD45+ cells (D, right) (N = 3 per group). E–H Evaluation of phenotypes of orthotopic syngeneic tumors of WT or Regnase-1-KO murine pancreatic cancer cells with or without depletion of CD8+ cells upon anti-CD8a antibody or IgG treatment. Experimental schematic (E). Dot plots of CD3+ and CD8.+ cells in WT or Regnase-1-KO syngeneic tumors upon anti-CD8a antibody or IgG treatment evaluated by flow cytometry (F). Tumor weights (G) (N = 6 per group). The relative mRNA levels of Cd8a, Ifng, Fasl, and Gzmb (H) (N = 6 per group). Student’s t test was used to evaluate differences between 2 groups. One-way ANOVA with Tukey’s post hoc test was used to compare differences among 4 groups. *P < 0.05, scale bars: 100 μm (insets)

Article Snippet: BE0061, a fully neutralizing mAb recognizing CD8a, and control IgG were obtained from Bioxcell.

Techniques: Immunostaining, Flow Cytometry

Journal: BJC reports

Article Title: A detoxified TLR4 agonist inhibits tumour growth and lung metastasis of osteosarcoma by promoting CD8+ cytotoxic lymphocyte infiltration.

doi: 10.1038/s44276-024-00120-3

Figure Lengend Snippet: Fig. 3 Lipo-MP-LPS induces immunological changes in both primary tumours and lung metastases. a, c Representative immunohis- tochemistry images of xenograft tumours and lung metastases stained with mouse specific anti-CD8a antibodies. Scale bar = 50 μm. b, d Quantification of CD8a positive cells in xenograft tumours and lung metastases. Data are represented as means ± SD; *p < 0.05, using Mann–Whitney U test. e, g Representative immunohistochemistry images of xenograft tumours and lung metastases stained with mouse specific anti-F4/80 antibodies. Scale bar = 50 μm. f, h Quantification of F4/80 positive cells in xenograft tumours and lung tissues. Data are represented as means ± SD; n = 6 mice per group, *p < 0.05, using Mann–Whitney U test. (i) iNOS, MHC II, TNF-α, CD206, and IL10 gene expression in xenograft tumours and lung metastases determined by RT-qPCR. Data are presented as means ± SD; n = 6 mice per group, *p < 0.05, **p < 0.01, using two-tailed t-test. NS not significant.

Article Snippet: In vivo depletion of CD8+ T cells Mice were depleted of CD8+ T cells by the intraperitoneal administration of an anti-CD8a antibody (YTS169.4, rat IgG2b; BioXCell, West Lebanon, NH, USA).

Techniques: Staining, MANN-WHITNEY, Immunohistochemistry, Gene Expression, Quantitative RT-PCR, Two Tailed Test

Journal: BJC reports

Article Title: A detoxified TLR4 agonist inhibits tumour growth and lung metastasis of osteosarcoma by promoting CD8+ cytotoxic lymphocyte infiltration.

doi: 10.1038/s44276-024-00120-3

Figure Lengend Snippet: Fig. 4 The tumour suppressive effect of Lipo-MP-LPS depends on CD8+ T cells. a Representative images of IHC staining for CD8a positive cells of xenograft tumours from mice treated with anti-CD8a antibody or IgG2b isotype. Scale bar = 100 μm. b CD8a expression in spleen tissues determined by RT-qPCR. Data are represented as means ± SD; n = 4. ***p < 0.001, using two-tailed t-test. c Tumour growth curve in C3H/HeN mice treated with empty liposome or Lipo-MP-LPS in addition to injection with anti-CD8a or isotype IgG2b antibodies (n = 6 mice per group). Data are shown as mean tumour volume ± SD. *p < 0.05, using Mann–Whitney U test. d Kaplan–Meier survival curves. p < 0.05 was considered significant using the log-rank test. Representative images of H&E staining of xenograft tumours (e) and lungs (g) for each group. Scale bar = 200 μm. f Quantification of necrotic areas in tumour tissues. h Quantification of lung metastasis areas. Data are presented as means ± SD; *p < 0.05, **p < 0.01; NS not significant, using Mann–Whitney U test.

Article Snippet: In vivo depletion of CD8+ T cells Mice were depleted of CD8+ T cells by the intraperitoneal administration of an anti-CD8a antibody (YTS169.4, rat IgG2b; BioXCell, West Lebanon, NH, USA).

Techniques: Immunohistochemistry, Expressing, Quantitative RT-PCR, Two Tailed Test, Injection, MANN-WHITNEY, Staining

Journal: BJC reports

Article Title: A detoxified TLR4 agonist inhibits tumour growth and lung metastasis of osteosarcoma by promoting CD8+ cytotoxic lymphocyte infiltration.

doi: 10.1038/s44276-024-00120-3

Figure Lengend Snippet: Fig. 6 Survival analysis of patients with osteosarcoma according to immune cell infiltration levels estimated by consensus TME. Of the 84 patients with osteosarcoma, the top third (28 patients) and bottom third (28 patients) were classified into high and low score groups, respectively. Kaplan–Meier curves of (a) CD8+ T cells (b) macrophage, (c) M1 macrophage, and (d) M2 macrophage infiltration for OS (left) and PFS (right) are presented. p < 0.05 was considered significant using the log-rank test.

Article Snippet: In vivo depletion of CD8+ T cells Mice were depleted of CD8+ T cells by the intraperitoneal administration of an anti-CD8a antibody (YTS169.4, rat IgG2b; BioXCell, West Lebanon, NH, USA).

Techniques:

Journal: Translational Lung Cancer Research

Article Title: Targeting complement C5a to improve radiotherapy sensitivity in non-small cell lung cancer

doi: 10.21037/tlcr-23-258

Figure Lengend Snippet: Infiltration of recruited CD8 + T cells increased after treatment of RT. (A) Mice were inoculated subcutaneously with LLC tumor cells and treated with 8 Gy of local RT daily for 3 times. Tumor tissues were collected at the indicated time points for subsequent analysis. (B) Tumor tissues in RT and control groups. (C) Immunofluorescence of tumors in RT and control groups. Sections were stained with an antibody recognizing CD8 (green) and DAPI (blue). Scale bars represent 50 µm. Representative images are shown. (D) Flow cytometry assays displayed that infiltration of CD8 + T cells were upregulated after RT. RT, radiotherapy; DAPI, 4',6-diamidino-2-phenylindole; FSC-A, forward scatter-A; FITC-A, fluorescein isothiocyanate-A; LLC, Lewis lung carcinoma; con, control.

Article Snippet: After processes of dewaxing, rehydration, and antigen retrieval with ethylenediaminetetraacetic acid (EDTA) antigen retrieval buffer (pH 8.0) (Servicebio, Wuhan, China; G1206), the slides were then blocked with blocking solution [PBS with 10% fetal calf serum (FCS) and 0.1% TritonX] at room temperature for 1 hour, then washed in PBS and incubated overnight at 4 oC in the appropriate primary anti-C5aR antibody (Abcam, 1:500) and anti-CD8a antibody (Boster, 1:50) diluted in blocking solution.

Techniques: Control, Immunofluorescence, Staining, Flow Cytometry

Journal: Translational Lung Cancer Research

Article Title: Targeting complement C5a to improve radiotherapy sensitivity in non-small cell lung cancer

doi: 10.21037/tlcr-23-258

Figure Lengend Snippet: RT upregulated C5aR1 expression in RT-recruited CD8 + T cells. (A) CD8 + T cells were sorted by magnetic beads for RNA-seq. (B) RNA-seq analysis was displayed and the differential genes are shown in the heat map. (C) DEG counts in RT and control groups. (D) Top 20 of GO enrichment of the up-regulated genes. The red arrows indicate that these functional pathways exist C5aR1 gene enrichment. (E) Volcano map of RNA-seq showing the DEGs. The red dots indicate significantly upregulated genes. RNA-seq, RNA sequencing; RT, radiotherapy; DEGs, differentially expressed genes; GO, Gene Ontology.

Article Snippet: After processes of dewaxing, rehydration, and antigen retrieval with ethylenediaminetetraacetic acid (EDTA) antigen retrieval buffer (pH 8.0) (Servicebio, Wuhan, China; G1206), the slides were then blocked with blocking solution [PBS with 10% fetal calf serum (FCS) and 0.1% TritonX] at room temperature for 1 hour, then washed in PBS and incubated overnight at 4 oC in the appropriate primary anti-C5aR antibody (Abcam, 1:500) and anti-CD8a antibody (Boster, 1:50) diluted in blocking solution.

Techniques: Expressing, Magnetic Beads, RNA Sequencing, Control, Functional Assay

Journal:

Article Title: Suppressor role of Rat CD8 + CD45RC low T cells in Experimental Autoimmune Uveitis (EAU)

doi: 10.1016/j.jneuroim.2006.11.021

Figure Lengend Snippet: A rat encephalitogenic T cell line (S1), uveitogenic T cell line (IRBP22), and EAE suppressor T cell line (anti-S1) were double-stained with antibodies against CD4 or CD8 and CD45RC, then analyzed by FACS. The values represent the purity of each cell population. Our results showed that both pathogenic T cell lines expressed CD4 and high levels of CD45RC and the suppressor anti-S1 cell line expressed CD8 and low level of CD45RC.

Article Snippet: The monoclonal antibodies (mAbs) used for the purification of T cell subpopulations and for flow cytometry were OX35 (mouse anti-rat CD4), OX6 (mouse anti-rat major histocompatibility complex [MHC] class II), OX8 (mouse anti-rat CD8), OX12 (mouse anti-rat kappa light chain), and OX22 (mouse anti-rat CD45RC) [BD Biosciences, CA].

Techniques: Staining

Journal:

Article Title: Suppressor role of Rat CD8 + CD45RC low T cells in Experimental Autoimmune Uveitis (EAU)

doi: 10.1016/j.jneuroim.2006.11.021

Figure Lengend Snippet: (A&B) T cells were separated from pooled spleens and draining lymph nodes of disease-inducing rats. The abundance of CD8+CD45RClow T cells was tested by flow cytometry analysis. Remitting rats were chosen after the acute episode and before the first relapse of the disease. (C) Proliferative response of unfractionated T cells and T cells depleted of CD8+CD45RClow T cells from recovered EAU rats. (D) Proliferative response of unfractionated T cells and T cells depleted of CD8+CD45RClow T cells from recurrent EAU rats, harvested at the peak of the first relapse. T cells (4 x 105, from pooled spleens and lymph nodes of rats) were stimulated for 48 h in the presence of irradiated APCs. [3H]thymidine being added to each cultured well for 8 h before assessing incorporation. The data shown are representative of those obtained in 3 independent experiments.

Article Snippet: The monoclonal antibodies (mAbs) used for the purification of T cell subpopulations and for flow cytometry were OX35 (mouse anti-rat CD4), OX6 (mouse anti-rat major histocompatibility complex [MHC] class II), OX8 (mouse anti-rat CD8), OX12 (mouse anti-rat kappa light chain), and OX22 (mouse anti-rat CD45RC) [BD Biosciences, CA].

Techniques: Flow Cytometry, Irradiation, Cell Culture

Journal:

Article Title: Suppressor role of Rat CD8 + CD45RC low T cells in Experimental Autoimmune Uveitis (EAU)

doi: 10.1016/j.jneuroim.2006.11.021

Figure Lengend Snippet: (A) Unfractionated T cells depleted of CD8+CD45RClow were used as responder cells. Responder T cells were prepared from pooled spleens and draining lymph nodes of recovered rats (18 days p.i.) as described in the Materials and Methods, then 3 x 105 cells were stimulated with a suboptimal dose of immunizing antigen (R16, 0.1 μg/ml) in the presence of irradiated syngeneic spleen (APCs) and graded numbers of CD8CD45RClow T cells from recovered rats and proliferation were assessed. (B) As in (A), but using CD8CD45RClow T cells from rats with progressive, recurrent disease, harvested at the peak of the first relapse. The results shown are representative of those for 3 independent experiments. (C) R16-specific T responder cells from recovered or progressive, recurrent EAU rats were stimulated with R16 and APCs, then the supernatants were collected 48h later for IFN-γ measurement. (D) R16-specific responder cells from recovered rats were co-cultured with CD8+CD45RClow T cells from recovered rats or recurrent rats, harvested at the peak of the first relapse at a ratio of 2:1 and the supernatants collected after 48h culture and analyzed for IFN-γ production by ELISA. The data shown are representative of those obtained in 3 independent experiments (**, p < 0.01). (E) As in A, CD8+CD45RClow T cells were isolated from monophasic or recurrent EAU rats at different time point after the initiation of disease. The results shown are representative of those for 3 independent experiments.

Article Snippet: The monoclonal antibodies (mAbs) used for the purification of T cell subpopulations and for flow cytometry were OX35 (mouse anti-rat CD4), OX6 (mouse anti-rat major histocompatibility complex [MHC] class II), OX8 (mouse anti-rat CD8), OX12 (mouse anti-rat kappa light chain), and OX22 (mouse anti-rat CD45RC) [BD Biosciences, CA].

Techniques: Irradiation, Cell Culture, Enzyme-linked Immunosorbent Assay, Isolation

Journal:

Article Title: Suppressor role of Rat CD8 + CD45RC low T cells in Experimental Autoimmune Uveitis (EAU)

doi: 10.1016/j.jneuroim.2006.11.021

Figure Lengend Snippet: (A) Adoptive transfer of IRBP-specific T cells depleted of CD8+CD45RClow T cells induces more severe uveitis in recipient rats.

Article Snippet: The monoclonal antibodies (mAbs) used for the purification of T cell subpopulations and for flow cytometry were OX35 (mouse anti-rat CD4), OX6 (mouse anti-rat major histocompatibility complex [MHC] class II), OX8 (mouse anti-rat CD8), OX12 (mouse anti-rat kappa light chain), and OX22 (mouse anti-rat CD45RC) [BD Biosciences, CA].

Techniques: Adoptive Transfer Assay

Journal:

Article Title: Suppressor role of Rat CD8 + CD45RC low T cells in Experimental Autoimmune Uveitis (EAU)

doi: 10.1016/j.jneuroim.2006.11.021

Figure Lengend Snippet: (A) CD8+CD45RClow T cells were isolated from naive, recovered, and recurrent rats. After surface staining of FITC-labeled CD8, Foxp3 was detected as described in the Materials and Methods.

Article Snippet: The monoclonal antibodies (mAbs) used for the purification of T cell subpopulations and for flow cytometry were OX35 (mouse anti-rat CD4), OX6 (mouse anti-rat major histocompatibility complex [MHC] class II), OX8 (mouse anti-rat CD8), OX12 (mouse anti-rat kappa light chain), and OX22 (mouse anti-rat CD45RC) [BD Biosciences, CA].

Techniques: Isolation, Staining, Labeling

Journal:

Article Title: Development of organised conjunctival leucocyte aggregates after corneal transplantation in rats

doi:

Figure Lengend Snippet: Antibodies used in histological staining

Article Snippet: Positive control sections of spleen were included in each staining run. table ft1 table-wrap mode="anchored" t5 Table 2 caption a7 Clone (antigen) Specificity Source W3/25 (CD4) T helper cells and monocytes Harlan Seralab, Loughborough, UK OX8 (CD8α) Cytotoxic T lymphocytes and dendritic cells Harlan Seralab OX33 (CD45RA) B cells Harlan Seralab ED2 Tissue macrophage Serotec, Oxford, UK HIS48 Granulocytes Pharmingen, Oxford, UK OX39 (CD25) IL2 receptor Harlan Seralab WT.3 (CD18) LFA1-β Harlan Seralab IA29 (CD54) ICAM-1 Harlan Seralab OX34 (CD2) T cells and dendritic cells Harlan Seralab OX6 MHC class II Serotec 10/78 (CD161) NK cells Serotec SILK6 IL1β Serotec Rabbit Polyclonal TNFα Serotec RP64 IFNγ Harlan Seralab 6B10 Cytokeratin peptide 4 Sigma, St Louis, MI, USA LL002 Cytokeratin peptide 14 Biogenex, San Ramon, CA, USA C-11 Anti-pan cytokeratin Sigma Open in a separate window Antibodies used in histological staining Quantification of cells Sections stained by the immunoperoxide method were examined and stained cells were quantified at ×400 magnification using a Leica Leitz DMRB upright light microscope and an image analysis system (Quantimet; Leica, Cambridge, UK).

Techniques:

Journal:

Article Title: Development of organised conjunctival leucocyte aggregates after corneal transplantation in rats

doi:

Figure Lengend Snippet: (A) Appearance of normal conjunctiva of the temporal half of the upper lid (lid everted with forceps) without clinically evident leucocytic aggregate. Note absence of oedema and normal vasculature (original magnification ×15). (B) ED2+ macrophage (arrowheads) and (C) CD8α+ cells in normal conjunctiva. (D) Clinical appearance of aggregate on day 10 after transplantation showing hyperaemia, overhanging edge (arrow), and swelling, clinical score 4 (original magnification ×10).

Article Snippet: Positive control sections of spleen were included in each staining run. table ft1 table-wrap mode="anchored" t5 Table 2 caption a7 Clone (antigen) Specificity Source W3/25 (CD4) T helper cells and monocytes Harlan Seralab, Loughborough, UK OX8 (CD8α) Cytotoxic T lymphocytes and dendritic cells Harlan Seralab OX33 (CD45RA) B cells Harlan Seralab ED2 Tissue macrophage Serotec, Oxford, UK HIS48 Granulocytes Pharmingen, Oxford, UK OX39 (CD25) IL2 receptor Harlan Seralab WT.3 (CD18) LFA1-β Harlan Seralab IA29 (CD54) ICAM-1 Harlan Seralab OX34 (CD2) T cells and dendritic cells Harlan Seralab OX6 MHC class II Serotec 10/78 (CD161) NK cells Serotec SILK6 IL1β Serotec Rabbit Polyclonal TNFα Serotec RP64 IFNγ Harlan Seralab 6B10 Cytokeratin peptide 4 Sigma, St Louis, MI, USA LL002 Cytokeratin peptide 14 Biogenex, San Ramon, CA, USA C-11 Anti-pan cytokeratin Sigma Open in a separate window Antibodies used in histological staining Quantification of cells Sections stained by the immunoperoxide method were examined and stained cells were quantified at ×400 magnification using a Leica Leitz DMRB upright light microscope and an image analysis system (Quantimet; Leica, Cambridge, UK).

Techniques: Transplantation Assay

Journal:

Article Title: Development of organised conjunctival leucocyte aggregates after corneal transplantation in rats

doi:

Figure Lengend Snippet: (A) MHC class II expressing cells are numerous in superficial leukocytic aggregates. (B) CD4+ T cells colocalise with (C) CD25+ cells. (D) CD8α staining is greater in stromal areas and expressed on round and dendritic cells. (E) Granulocytes in aggregates within both superficial and stromal areas. (F) IFNγ expression and (G) TNFα expression in leucocytic aggregates. Immunofluorescence double staining showing cells (arrows) positive for (H) IFNγ (red) and CD8α (green) (×20); (I) IFNγ (red) and ED2+ macrophages (green) (×40); (J) TNFα (red) and ED2+ macrophages (green) (×40). Unlabelled bars are 50 μm.

Article Snippet: Positive control sections of spleen were included in each staining run. table ft1 table-wrap mode="anchored" t5 Table 2 caption a7 Clone (antigen) Specificity Source W3/25 (CD4) T helper cells and monocytes Harlan Seralab, Loughborough, UK OX8 (CD8α) Cytotoxic T lymphocytes and dendritic cells Harlan Seralab OX33 (CD45RA) B cells Harlan Seralab ED2 Tissue macrophage Serotec, Oxford, UK HIS48 Granulocytes Pharmingen, Oxford, UK OX39 (CD25) IL2 receptor Harlan Seralab WT.3 (CD18) LFA1-β Harlan Seralab IA29 (CD54) ICAM-1 Harlan Seralab OX34 (CD2) T cells and dendritic cells Harlan Seralab OX6 MHC class II Serotec 10/78 (CD161) NK cells Serotec SILK6 IL1β Serotec Rabbit Polyclonal TNFα Serotec RP64 IFNγ Harlan Seralab 6B10 Cytokeratin peptide 4 Sigma, St Louis, MI, USA LL002 Cytokeratin peptide 14 Biogenex, San Ramon, CA, USA C-11 Anti-pan cytokeratin Sigma Open in a separate window Antibodies used in histological staining Quantification of cells Sections stained by the immunoperoxide method were examined and stained cells were quantified at ×400 magnification using a Leica Leitz DMRB upright light microscope and an image analysis system (Quantimet; Leica, Cambridge, UK).

Techniques: Expressing, Staining, Immunofluorescence, Double Staining