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: 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 Assay, Functional Assay

Journal: Signal Transduction and Targeted Therapy

Article Title: CD147 contributes to SARS-CoV-2-induced pulmonary fibrosis

doi: 10.1038/s41392-022-01230-5

Figure Lengend Snippet: hCD147 transgenic mice infected with SARS-CoV-2 and delta variant mimicked pulmonary fibrosis pathology. a hCD147 mice or C57BL/6J mice were inoculated via the intranasal with 3 × 10 5 TCID 50 of SARS-CoV-2 or delta variant, and samples were collected at 2, 6, 13, 20 and 27 d.p.i. b RT-qPCR for viral RNA levels in lung tissues at 2 d.p.i. n = 3 for C57BL/6J group, n = 6 for hCD147 groups. c H&E staining of lung tissue sections from 2 to 27 d.p.i. Scale bars, 50 μm. d TEM analysis of lung samples from 2 to 27 d.p.i. The stars indicate collagen fibrils; the arrows indicate elastic fibers. Black scale bars, 20 μm. Yellow scale bars, 2 μm. e Statistics of the percentage of cells positive for α-SMA (fibroblasts), F4/80 (macrophages), CD3 (T cells), CD4, CD8, CD19 (B cells), Ly6G (neutrophils), and NCR1 (NK cells). n = 12 images for each group, one-way ANOVA followed by multiple comparisons

Article Snippet: After blocked with 5% goat serum, the slide was sequentially incubated with primary antibodies recognizing α-SMA (Abcam, ab7817), CD3 (Abcam, ab5690), CD4 (Abcam, ab183685), CD8 (Boster Bio, a02236-1), CD19 (Abcam, ab245235), Ly6G (Abcam, ab238132), NCR1 (Abcam, ab233558), F4/80 (Cell Signaling Technology, 70076 S), TGF-β (Abcam, ab215715), Sftpc (Abcam, ab90716), rabbit anti-mouse CD147 (Abcam, ab188190), mouse anti-human CD147 (prepared in our own lab), FSP1 (Proteintech, 20886-1-AP), rabbit IgG Isotype Control (Invitrogen, 31235), mouse IgG Isotype Control (Invitrogen, 14-4714-82).

Techniques: Transgenic Assay, Infection, Variant Assay, Quantitative RT-PCR, Staining

Journal: Molecular cancer therapeutics

Article Title: CSF-1/CSF-1R Signaling Inhibitor Pexidartinib (PLX3397) Reprograms Tumor-Associated Macrophages and Stimulates T-cell Infiltration in Sarcoma Microenvironment

doi: 10.1158/1535-7163.MCT-20-0591

Figure Lengend Snippet: Systemic treatment of PLX3397 depletes TAMs and increases lymphocyte infiltration into LM8 osteosarcoma. A. Composition of tumor cells and TAMs evaluated by flow-cytometric analysis using the dissociated LM8 tumor cells. Data were represented as mean ± SEM; n = 3; Mann–Whitney test: *p < 0.05. B. Flow cytometry analysis of TAMs (CD45+CD11b+CD206+; left) and CD8 T cells (CD45+CD3+CD8+; right) within the dissociated LM8 tumor cells and representative flow data. C. Composition of infiltrating immune cells (CD3+, CD4+, and CD8+ cells) evaluated by flow-cytometric analysis using the dissociated LM8 tumor cells. Data were represented as mean ± SEM; n = 3; Mann – Whitney test: *p < 0.05. D. Composition of infiltrating FOXP3+ regulatory T cells evaluated by immunohistochemistry using the dissociated LM8 tumor cells. Data were represented as mean ± SEM; n = 3; Mann–Whitney test: *p < 0.05. E. Distribution of infiltrating CD68+ macrophages, CD8+ T cells, and FOXP3+ regulatory T cells in PLX3397- or PBS-treated tumors (green). Nuclei were stained with DAPI (blue). Left, H&E staining images. Scale bars, 50 μm (lower) and 10 μm (upper).

Article Snippet: A rabbit polyclonal anti-CD68 antibody (Boster; Pleasanton, CA; cat#PA1518), a rabbit monoclonal anti-CD8 antibody (Cell Signaling Technology, MA; cat#98941), and a rat monoclonal anti-FOXP3 (eBioscience; cat#14-5773-82) were used in concentrations of 5 μg/ml, 4.8 μg/ml, and 5 μg/ml, respectively.

Techniques: MANN-WHITNEY, Flow Cytometry, Immunohistochemistry, Staining

Journal: Cell Transplantation

Article Title: Leflunomide Inhibits rat-to-Mouse Cardiac Xenograft Rejection by Suppressing Adaptive Immune Cell Response and NF-κB Signaling Activation

doi: 10.1177/09636897211054503

Figure Lengend Snippet: The effect of LEF treatment on CMR- and AVR- mediated immune responses. (A) MLR responses. Recipient splenocytes were isolated at POD 6 (responders) and irradiated naïve SD or BALB/c splenocytes (stimulators) were co-cultured for three days. Data are representative of three independent experiments. (B) Absolute numbers of splenocytes in LEF -treated and normal-saline treated mice recipients. Naïve C57BL/6 mice are shown for comparison ( n = 3 mice/group). (C) Representative proportion of CD4 + and CD8 + T cells in recipient splenocytes. A total of 1 × 10 6 splenocytes were isolated at POD 6, and the percentage of CD4 + and CD8 + T cells was determined by flow cytometry ( n = 3 mice/group). (D) Representative proportion of CD19 + B cells in recipient splenocytes. A total of 1 × 10 6 splenocytes were isolated at POD 6, and the percentage of CD19 + B cells was determined by flow cytometry ( n = 3 mice/group). (E) The number of CD3 + T cells in recipient spleen were determined by flow cytometry ( n = 3 mice/group). (F) The number of CD19 + B cells in recipient spleen were determined by flow cytometry ( n = 3 mice/group). (G) Representative proportion of donor-specific antibodies in recipient serum. Serum was collected from xenograft and allograft recipients at POD 6, and the percent of IgG1, IgG2a, and IgM was determined by flow cytometry ( n = 3 mice/group). (H) Serum levels of proinflammatory cytokines. Peripheral blood was collected at POD 6 and IFN-γ serum levels were measured by ELISA ( n = 3 mice/group). (I) CD3 + T cells were isolated from naïve C57BL/6 mice, and co-cultured with anti-CD3 and anti-CD28 monoclonal antibody in the absence and presence of LEF for 3 days, the supernatant was collected and measured by ELISA ( n = 3 separate experiments). Data are presented as the mean ± SEM of three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 compared to the normal saline-treated group.

Article Snippet: Immunohistochemical staining was performed as described previously , the sections were then stained with primary antibody anti-CD4 Ab (Boster, Wuhan, China), anti-CD8 Ab (Boster), anti-CD20 Ab (Boster), anti-IgM (Boster), and secondary antibody goat anti-rabbit IgG (GB23303; Boster, Wuhan, China).

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

Journal: Cell Transplantation

Article Title: Leflunomide Inhibits rat-to-Mouse Cardiac Xenograft Rejection by Suppressing Adaptive Immune Cell Response and NF-κB Signaling Activation

doi: 10.1177/09636897211054503

Figure Lengend Snippet: Phenotypic characteristics of grafts. Xenografts and allografts were recovered at POD 6. Relative mRNA expression of IL-2, IFN-γ, IL-4, and TNF-α in (A) xenograft and (B) allografts measured by qRT-PCR ( n = 3 mice/group). (C) Immunohistochemistry staining of CD20 + (brown) in xenograft and allograft; bar indicates 20 µm and quantified. (D) IgM in grafts were examined by immunohistochemistry staining; bar indicates 20 µm and quantified. Immunohistochemistry staining of CD4 + (brown), and CD8 + (brown) in (E) xenograft and (H) allografts. (Original magnification: ×400). Data are presented as mean ± SEM from three independent experiments. * P < 0.05, ** P < 0.01 compared to the normal saline-treated group.

Article Snippet: Immunohistochemical staining was performed as described previously , the sections were then stained with primary antibody anti-CD4 Ab (Boster, Wuhan, China), anti-CD8 Ab (Boster), anti-CD20 Ab (Boster), anti-IgM (Boster), and secondary antibody goat anti-rabbit IgG (GB23303; Boster, Wuhan, China).

Techniques: Expressing, Quantitative RT-PCR, Immunohistochemistry, Staining