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: Journal of Nanobiotechnology

Article Title: Hybrid membrane-coated nanosuspensions for multi-modal anti-glioma therapy via drug and antigen delivery

doi: 10.1186/s12951-021-01110-0

Figure Lengend Snippet: Immune response and cell apoptosis in vitro. A Flow cytometric quantification of the expression of CD80 and CD86 ( B ), the markers for DC maturation ( a PBS; b DCm; c C6m; d [C6&DC]m). C Flow cytometric analyses of the expression of CD8a and CD4, the markers for T cells proliferation ( a PBS; b DCm; c C6m; d [C6&DC]m). D The release of IL-6, TNF-α ( E ), and IFN-γ ( F ) by RAW264.7 cells; n = 6. G Apoptosis of C6 glioma cells after treatment with different DTX-loaded formulations

Article Snippet: After cocultured for 48 h, the T lymphocytes were washed three times with PBS and subsequently stained with anti-CD4-FITC and anti-CD8-PE antibodies (Abcam) for 30 min at 4 °C.

Techniques: In Vitro, Expressing

Journal: Journal of Nanobiotechnology

Article Title: Hybrid membrane-coated nanosuspensions for multi-modal anti-glioma therapy via drug and antigen delivery

doi: 10.1186/s12951-021-01110-0

Figure Lengend Snippet: Immune stimulation efficiency in different tissues and mice serum. A Immunofluorescence staining of CD8 (Red) and CD4 (Green) antibodies in spleen and lymph node ( B ). C Immunofluorescence staining of DiI (Red), CD8 (Green) and CD4 (pink) antibodies in brain of glioma-bearing mice. D Release of cytokines IL-6, TNF-α ( E ), and IFN-γ ( F ) in mice serum. G Immunohistochemical staining of TNF-α of the lymph node and tumors ( H ) (**p < 0.01, ***p < 0.001, ns, not significant; n = 6; ×40 magnification)

Article Snippet: After cocultured for 48 h, the T lymphocytes were washed three times with PBS and subsequently stained with anti-CD4-FITC and anti-CD8-PE antibodies (Abcam) for 30 min at 4 °C.

Techniques: Immunofluorescence, Staining, Immunohistochemical staining

Journal: Nature Communications

Article Title: The chimeric TAC receptor co-opts the T cell receptor yielding robust anti-tumor activity without toxicity

doi: 10.1038/s41467-018-05395-y

Figure Lengend Snippet: TAC design mimics the TCR-CD3:co-receptor complex. a Left: Naturally occurring TCR-CD3 complex interacts directly with the antigen presented by MHC. Meanwhile, the CD8/CD4 co-receptor interacts with MHC I/II in an antigen-independent manner. Together, these interactions comprise the first step in T cell activation. Right: The TAC receptor re-directs the TCR-CD3 complex towards an antigen of choice using an interchangeable antigen binding moiety (here depicted with an scFv, purple). An scFv is used to recruit the TCR-CD3 complex (blue). Co-receptor properties are incorporated by including the CD4 hinge, TM region, and cytosolic tail (green). b The TAC is incorporated into the pCCL DNA backbone containing a truncated NGFR (tNGFR), which lacks cytosolic signaling domains, as a transduction control. The vector features a bi-directional promoter system with tNGFR under control of the mCMV promoter and TAC expression being driven by the EF-1α promoter. TAC is comprised of an antigen binding domain, a CD3-binding domain, and a co-receptor domain. A variety of proteins can be used for each of these three TAC domains allowing the TAC to be modified to best respond to numerous different antigens. The specific domain combinations tested are described below

Article Snippet: IHC antibodies used: CD3 (Abcam Inc.; cat#: ab16669), CD4 (Abcam Inc.; cat#: ab133616), CD8 (Spring Biosciences, Pleasanton, CA; cat#: M3162), HER2 (Cell Signaling Technology, Danvers, MA; cat#: 2242), pan-CK (Sigma Aldrich; cat#: C1801), Ki67 (Spring Biosciences; cat#: M3062), and DAPI, Opal 520, Opal 650, Opal 570, Opal 690, and Opal 620 (Opal 7-ColorfIHC kit; Perkin Elmer; cat# NEL797001KT).

Techniques: Activation Assay, Binding Assay, Transduction, Control, Plasmid Preparation, Expressing, Modification

Journal: Nature Communications

Article Title: The chimeric TAC receptor co-opts the T cell receptor yielding robust anti-tumor activity without toxicity

doi: 10.1038/s41467-018-05395-y

Figure Lengend Snippet: Evaluation of multiple anti-CD3 scFv domains for recruitment of TAC to the TCR-CD3 complex. a , e Schematic representation of evaluated TAC constructs. TAC receptors utilizing the ( a ) anti-HER2 DARPin are paired with either the UCHT1 or OKT3 anti-CD3 scFv. TAC receptors using the ( e ) anti-CD19 scFv are paired with either the huUCHT1, F6A, or L2K anti-CD3 scFv. b , f Relative TAC surface expression is measured by flow cytometry. Cells are stained for CD4, CD8, tNGFR and TAC, and gated on either CD4 + NGFR + or CD8 + NGFR + . Representative data of three independent experiments are presented as histogram analysis of ( b ) HER2-TAC or ( f ) CD19-TAC. Surface expression of OKT3 relative to UCHT1 was significantly higher in CD4 cells ( p = 0.0007) but not in CD8 cells. huUCHT1 expression is significantly higher compared to either L2K ( p = 0.005 (CD4)/0.0002 (CD8)) or F6A ( p < 0.0001 (CD4) p < 0.0001 (CD8)). For the gating strategy see Supplementary Fig. . c , g HER2- and CD19-specific TAC-T cells are stimulated with antigen-positive ( c ) SK-OV-3 and ( g ) Raji tumor cells, respectively. Data are presented as percent of CD4 or CD8 T cells producing cytokine. Cytokine producing cells are compared from ( c ) TAC-T cells bearing UCHT1 (square) or OKT3 (inverted triangle), or ( g ) TAC-T cells bearing huUCHT1 (square), F6A (triangle), or L2K (diamond). Lines represent the mean. Multiple t -test is used to determine significance in all cases. For the gating strategy see Supplementary Fig. . d , h HER2- and CD19-TAC and vector control (vector only carrying tNGFR) T cells are co-cultured with ( d ) SK-OV-3 and ( h ) NALM-6 tumor cells, respectively, to measure TAC-T cell-mediated cytotoxicity. Vector control T cells (circles) are compared against d HER2-specific TAC-T cells bearing UCHT1 (square) or OKT3 (triangle), or ( h ) CD19-specific TAC-T cells bearing huUCHT1 (square), F6A (triangle), or L2K (diamond). Data are from three independent experiments with three different donors, error bars are standard deviation

Article Snippet: IHC antibodies used: CD3 (Abcam Inc.; cat#: ab16669), CD4 (Abcam Inc.; cat#: ab133616), CD8 (Spring Biosciences, Pleasanton, CA; cat#: M3162), HER2 (Cell Signaling Technology, Danvers, MA; cat#: 2242), pan-CK (Sigma Aldrich; cat#: C1801), Ki67 (Spring Biosciences; cat#: M3062), and DAPI, Opal 520, Opal 650, Opal 570, Opal 690, and Opal 620 (Opal 7-ColorfIHC kit; Perkin Elmer; cat# NEL797001KT).

Techniques: Construct, Expressing, Flow Cytometry, Staining, Plasmid Preparation, Control, Cell Culture, Standard Deviation

Journal: Nature Communications

Article Title: The chimeric TAC receptor co-opts the T cell receptor yielding robust anti-tumor activity without toxicity

doi: 10.1038/s41467-018-05395-y

Figure Lengend Snippet: Relative expression of checkpoint receptors and memory T cell subsets in CAR- and TAC-engineered T cells. T cells were transduced with HER2-TAC, or a second-generation anti-HER2 CAR including the CD28 (28ζ CAR) or 4-1BB (BBζ CAR) costimulatory receptor domains, or a vector control (tNGFR). Engineered T cells are stained for surface marker expression and CD4 + NGFR + or CD8 + NGFR + populations are analyzed by flow cytometry for ( a ) expression of checkpoint receptors PD-1, LAG-3, and TIM-3. All data is normalized to TAC-engineered T cells, and lines represent the mean of four donors. Each donor is represented by a unique symbol to highlight donor-to-donor variations. Multiple t -test is used to determine significance. For the gating strategy see Supplementary Fig. . b Memory T cell subsets of TAC-, 28ζ CAR-, and BBζ CAR-T cells, relative to T cells engineered with a vector control (tNGFR). T cell subsets are defined as naïve (CD45RA + , CCR7 + ), central memory (CM) (CD45RA − , CCR7 + ), effector memory (EM) (CD45RA − , CCR7 − ), and terminal effectors (EMRA) (CD45RA + , CCR7 − ). Lines represent the mean of four donors. Multiple t -test is used to determine significance. c CD27 and CD28 expression; representative histograms show data from one donor, median fluorescence intensity is indicated. Data are from two independent experiments with four different donors. For both ( b ) and ( c ), the gating strategy is shown in Supplementary Fig.

Article Snippet: IHC antibodies used: CD3 (Abcam Inc.; cat#: ab16669), CD4 (Abcam Inc.; cat#: ab133616), CD8 (Spring Biosciences, Pleasanton, CA; cat#: M3162), HER2 (Cell Signaling Technology, Danvers, MA; cat#: 2242), pan-CK (Sigma Aldrich; cat#: C1801), Ki67 (Spring Biosciences; cat#: M3062), and DAPI, Opal 520, Opal 650, Opal 570, Opal 690, and Opal 620 (Opal 7-ColorfIHC kit; Perkin Elmer; cat# NEL797001KT).

Techniques: Expressing, Transduction, Plasmid Preparation, Control, Staining, Marker, Flow Cytometry, Fluorescence

Journal: Nature Communications

Article Title: The chimeric TAC receptor co-opts the T cell receptor yielding robust anti-tumor activity without toxicity

doi: 10.1038/s41467-018-05395-y

Figure Lengend Snippet: Engineered T cell distribution and cytokine release in vivo. OVCAR-3 tumor-bearing mice were treated with 6.0 × 10 6 anti-HER2 28ζ CAR- or HER2-TAC-T cells, or a matched total number of vector control cells. Mice are sacrificed at 1, 3, 5, and 7 days post-ACT1 for multiplex serum cytokine analysis or perfusion and fixation of tissues for subsequent histology. a Multicolor IHC is performed on tumor and lung tissue 7 days post-ACT1. Tissues are stained for CD8 (cyan), CD4 (yellow), DNA (blue) and a proliferation marker (Ki-67, purple). Data are representative of 3 mice. Scale bar indicates 100 μm. b Multiplex analysis of human cytokines in mouse serum on day 3 and 7. Measurements that fall below 0.2 pg/mL are below the calibration range and are therefore defined as 0. 0 values are depicted on the graph’s x axis. Statistical analysis is provided in Supplementary Table , analysis was performed using multiple t -test. Individual data points are shown, bars indicate standard deviation and center bars indicate the median

Article Snippet: IHC antibodies used: CD3 (Abcam Inc.; cat#: ab16669), CD4 (Abcam Inc.; cat#: ab133616), CD8 (Spring Biosciences, Pleasanton, CA; cat#: M3162), HER2 (Cell Signaling Technology, Danvers, MA; cat#: 2242), pan-CK (Sigma Aldrich; cat#: C1801), Ki67 (Spring Biosciences; cat#: M3062), and DAPI, Opal 520, Opal 650, Opal 570, Opal 690, and Opal 620 (Opal 7-ColorfIHC kit; Perkin Elmer; cat# NEL797001KT).

Techniques: In Vivo, Plasmid Preparation, Control, Multiplex Assay, Staining, Marker, Standard Deviation

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Fibroblast Common Serum Response Signature-Related Classification Affects the Tumour Microenvironment and Predicts Prognosis in Bladder Cancer

doi: 10.1155/2022/5645944

Figure Lengend Snippet: ANLN to immunity in bladder cancer. (a) Boxplot of the abundance of PD-L1 expression in the CRS groups in the TCGA-BLCA cohort. (b) Correlation analysis between PD-L1 expression and the CRS in TCGA cohort. mRNA levels of (c) ANLN or (d) EML1 in anti-PD-L1 responsiveness in the IMvigor210 cohort. (e, f) Correlation analyses of expression of ANLN and CD8 and PD-L1 in TCGA cohort. (g) Survival probability of patients with differential expression of ANLN of TCGA and GSE13507 cohorts. Levels of ANLN were identified according to the median of ANLN. (h) Correlation analyses of ANLN expression and the CRS. (i) Correlation analyses of expression of ANLN and TNM staging. (j) Correlation analyses of expression of ANLN and grade. Correlation analyses between (k) number of CD8+ T cells or (l) PD-L1 expression and ANLN expression of patients with BLCA in our hospital cohort. (m) Representative IHC or HE images of ANLN, CD8, PD-L1, and Ki67. CR: complete response; PR: partial response; SD: stable disease; PD: progressive disease; CRS: fibroblast common serum response risk score.

Article Snippet: Antibodies against anillin (ANLN) (DF13590, 1 : 200; Affinity Biosciences), CD8 (PB9249, 1 : 200; BOSTER), and PD-L1 (66248-1-lg, 1 : 5000; Proteintech) were used for the immunohistochemical (IHC) staining of these proteins in the tissue samples, which was performed according to previously published methods [ ].

Techniques: Expressing, Quantitative Proteomics

Journal: Phytomedicine : international journal of phytotherapy and phytopharmacology

Article Title: Spatially resolved multi-omics reveals the renal cortex-metabolic reprogramming of Shenhua Tablet for intervention on IgA nephropathy.

doi: 10.1016/j.phymed.2025.156742

Figure Lengend Snippet: Fig. 2. SHT protects from renal dysfunction, mesangial cell proliferation and inflammatory responses in IgAN rats. (A) The 24 h Upro, Scr and BUN levels in the experimental groups (n = 8 in each group). (B) Representative images showing PCNA and PAS staining and semi-quantitative analysis of mesangial cell pro liferation in the experimental groups (n = 6 in each group). Scale bars, 20 μm. (C) Serum IL-6, IL-1β and TNF-α levels in the experimental groups (n = 8 in each group). (D) Immunohistochemical images and semi-quantitative analysis of OX8 and CD68 expression in the experimental groups. (n = 6 in each group). All sta tistical data are presented as mean ± SD and analyzed by ordinary one-way ANOVA followed by Tukey’s multiple comparison test. Comparative analysis, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant.

Article Snippet: The corresponding stain reagent was PAS Stain Kit (Solarbio, G1281), whereas the antibodies used included PCNA rabbit polyclonal antibody (1:200 dilution, Proteintech, 10,205–2-AP), Rabbit Anti-ODC1 antibody (1:200 dilution, Bioss, bs1294R), Col4 Polyclonal antibody (1:300 dilution, Bioss, bsm56208R), CD68 rabbit polyclonal antibody (1:200 dilution, Servicebio, GB113109), OX8 antibodies (1:200 dilution, boster, A02236–1), and HRP-conjugated goat anti-rabbit IgG (H + l) (1:200 dilution, ServiceBio, GB23303).

Techniques: Staining, Immunohistochemical staining, Expressing, Comparison