Journal: Nature Communications

Article Title: An in-situ peptide-antibody self-assembly to block CD47 and CD24 signaling enhances macrophage-mediated phagocytosis and anti-tumor immune responses

doi: 10.1038/s41467-024-49825-6

Figure Lengend Snippet: a Schematic illustration of tumor immune quiescent microenvironment, in situ assembly of PAC-SABIs on the cancer cell surface, and blockage of CD47 and CD24 phagocytic checkpoints by PAC-SABIs. Figure was created with BioRender.com and released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 international license. b Time-dependent CLSM images of 4T1 cells treated with NBD-labeled PAC-SABIs. Scale bar: 20 μm. Three independent experiments were performed. c Time-dependent CLSM images of PAN02 cells treated with NBD-labeled PAC-SABIs. Scale bar: 20 μm. Three independent experiments were performed. d Merged bright field and fluorescence images of 4T1 cell treated with NBD-labeled PAC-SABIs for 120 min. Scale bar: 20 μm. Three independent experiments were performed. e CLSM images of 4T1 cell treated with Dil dye (red) and NBD-labeled PAC-SABIs for 120 min. Scale bar: 10 μm. Three independent experiments were performed. f , g Fluorescence distribution of NBD-labeled PAC-SABIs on 4T1 cell. Three independent experiments were performed. h Time-dependent SEM images of 4T1 cells treated with PAC-SABIs. The red arrows point to PAC-SABIs on cell membrane. Scale bar: 2 μm. Three independent experiments were performed. i Time-dependent SEM images of PAN02 cells treated with PAC-SABIs. The red arrows point to the PAC-SABIs on cell membrane. Scale bar: 2 μm. Three independent experiments were performed. j CLSM images of 3D 4T1 spheroids treated with Cy5.5 PAC-SABIs, Calcein-AM, and Hoechst 33342. Scale bar: 50 μm. Three independent experiments were performed. k CLSM images of 3D 4T1 spheroids along the z -axis position. Scale bar: 50 μm. Three independent experiments were performed.

Article Snippet: Treatment: anti-mouse CD24 antibody (BioXcell, #BE0360, clone M1/69), anti-human CD24 antibody (Novus Biologics, #NB100-64861, clone SN3), anti-mouse CD47 antibody (BioXcell, #BE0270, clone MIAP301), anti-mouse PD-1 antibody (BioXcell, #BE0273, clone 29F.1A12TM), anti-CSF1R antibody (BioXcell, #BE0213, clone AFS98).

Techniques: In Situ, Labeling, Fluorescence, Membrane

Journal: Nature Communications

Article Title: An in-situ peptide-antibody self-assembly to block CD47 and CD24 signaling enhances macrophage-mediated phagocytosis and anti-tumor immune responses

doi: 10.1038/s41467-024-49825-6

Figure Lengend Snippet: a Phagocytosis images of pHrodo-red + over time. Scale bar: 50 μm. Three independent experiments were performed. b Phagocytosis of 4T1 cells, in the presence of IgG control or PAC-SABIs. The error bars represent the mean ± SD ( n = 3 independent experiments). c Representative 3D CLSM image reconstruction of in vitro phagocytosis of 4T1 cells (pHrodo-red + , red) by BMDMs and RAW264.7 cells (Calcein-AM, green). Three independent experiments were performed. d Representative flow cytometry plots depicting BMDM phagocytosis of 4T1 cells treated with IgG control, anti-CD24 mAb, SAMIs and PAC-SABIs. e Quantitative analysis of BMDM flow cytometry results. The error bars represent the mean ± SD ( n = 3 independent experiments; ** p < 0.01, *** p < 0.001; the p value was analyzed by a two-tailed unpaired Student’s t test). f Representative flow cytometry plots depicting RAW264.7 cell phagocytosis of 4T1 cells treated with IgG control, anti-CD24 mAb, SAMIs and PAC-SABIs. g Quantitative analysis of RAW264.7 cell flow cytometry results. The error bars represent the mean ± SD ( n = 3 independent experiments; ** p < 0.01; the p value was analyzed by a two-tailed unpaired Student’s t test). Source data are provided as a Source Data file.

Article Snippet: Treatment: anti-mouse CD24 antibody (BioXcell, #BE0360, clone M1/69), anti-human CD24 antibody (Novus Biologics, #NB100-64861, clone SN3), anti-mouse CD47 antibody (BioXcell, #BE0270, clone MIAP301), anti-mouse PD-1 antibody (BioXcell, #BE0273, clone 29F.1A12TM), anti-CSF1R antibody (BioXcell, #BE0213, clone AFS98).

Techniques: Control, In Vitro, Flow Cytometry, Two Tailed Test

Journal: Parasites & vectors

Article Title: Upregulation of CD244 promotes CD8 + T cell exhaustion in patients with alveolar echinococcosis and a murine model.

doi: 10.1186/s13071-024-06573-2

Figure Lengend Snippet: Fig. 1 CD244 contributes to CD8+ T cell exhaustion in patients with hepatic AE. A Visualization of various immune cell subtypes using Uniform Manifold Approximation and Projection (UMAP) based on single-cell RNA sequencing data from the CLT of patients with AE (number of patients = 4, total cell count = 29,369, accession no. HRA000553). B The expression levels of CD244 in various immune cell types. C Representative immunohistochemical staining of CD244 in liver tissue sections from patients with AE is displayed (top panel 100×, bottom panel enlarged to 400×). D The percentage of positive staining area was calculated to evaluate the expression of CD244 (n = 20). E Representative images from immunofluorescence co-staining of DAPI (blue), CD8 (green), CD244 (red) and a merged image on the liver tissue sections from patients with AE (n = 6). Boxed areas show ×400 magnification of histological images. Arrows indicate CD244+CD8+ T cells. Lesions are delimited with a white dashed line. F, G FCM analysis of differentiation phenotypes of CD244−CD8+ T cells and CD244+CD8+ T cells in the CLT of patients with AE. H FCM analysis of the ability of CD244−CD8+ T cells and CD244+CD8+ T cells in the CLT of AE patients to secrete IFN-γ and TNF-α. CLT, “close” liver tissue; DLT, “distant” liver tissue. Data were analyzed using paired Student’s t-tests. All data are presented as mean ± SD

Article Snippet: Sections were subsequently blocked with PBS and 10% donkey serum at room temperature for 1 h. Primary antibodies, anti-human CD244 (R&D Systems, 1:500), were applied at appropriate dilutions and incubated overnight at 4 °C.

Techniques: RNA Sequencing, Cell Counting, Expressing, Immunohistochemical staining, Staining, Immunofluorescence

Journal: Parasites & vectors

Article Title: Upregulation of CD244 promotes CD8 + T cell exhaustion in patients with alveolar echinococcosis and a murine model.

doi: 10.1186/s13071-024-06573-2

Figure Lengend Snippet: Fig. 2 High expression of CD244 promotes the terminal differentiation effector phenotype of CD8+ T cells in liver of Echinococcus multilocularis-infected mice after 24 weeks of infection. A Expression changes of CD244 on the CD8+T cells in the liver of mice infected with E. multilocularis. B Representative flow cytometry plots (left) and the frequency (right) of SLECs and MPECs in hepatic CD244− or CD244+CD8+ T cells from mice after 24 weeks of infection (5 mice per group). C Representative flow cytometry plots (left) and the frequency (right) of CD8+Tn, CD8+Tcm and CD8+Tem by CD244− or CD244+CD8+ T cells in the livers from mice after 24 weeks of infection (5 mice per group). D Mean fluorescence intensity (MFI) of T-bet and Ki67 expression by activated CD244− or CD244+CD8+ T cells in the liver from mice after 24 weeks of infection (4 mice per group). E–G Representative flow cytometry plots (left) and the frequency (right) of GZMB, IFN-γ and TNF-α production by activated CD244− or CD244+CD8+ T cells in the liver from mice after 24 weeks of infection (5 mice per group). Data are one representative of two independent experiments. Con, control; Em, E. multilocularis; KLRG1, killer cell lectin-like receptor G1. Data were analyzed using two independent samples t-tests or paired Student’s t-tests. All data are presented as mean ± SD

Article Snippet: Sections were subsequently blocked with PBS and 10% donkey serum at room temperature for 1 h. Primary antibodies, anti-human CD244 (R&D Systems, 1:500), were applied at appropriate dilutions and incubated overnight at 4 °C.

Techniques: Expressing, Infection, Flow Cytometry, Fluorescence, Control

Journal: Parasites & vectors

Article Title: Upregulation of CD244 promotes CD8 + T cell exhaustion in patients with alveolar echinococcosis and a murine model.

doi: 10.1186/s13071-024-06573-2

Figure Lengend Snippet: Fig. 3 CD244 induces functional exhaustion in hepatic CD8+ T cells from mice exposed to Echinococcus multilocularis protoscoleces antigens in vitro. A FCM detection of CD244 expression on hepatic CD8+T cells from WT mice after in vitro stimulation with E. multilocularis protoscoleces antigen (Emp) for 48 h. B FCM analysis of changes in the secretion capacity of the pro-inflammatory cytokine IFN-γ and the inhibitory cytokine IL-10 by CD244−CD8+ T cells and CD244+CD8+ T cells in mouse liver following Emp stimulation for 48 h. C Representative flow cytometry plots (left) and the frequencies (right) of Granzyme-B, IFN-γ and TNF-α production by hepatic CD8+ T cells from control, WT and CD244-KO mice with Emp stimulation for 48 h in vitro. Data are one representative of two independent experiments. Con, control; Em, E. multilocularis; KO, knockout; WT, wild type; Emp, E. multilocularis protoscoleces. Data were analyzed using paired Student’s t-tests or two independent samples t-tests. All data are presented as mean ± SD. ns, P > 0.05

Article Snippet: Sections were subsequently blocked with PBS and 10% donkey serum at room temperature for 1 h. Primary antibodies, anti-human CD244 (R&D Systems, 1:500), were applied at appropriate dilutions and incubated overnight at 4 °C.

Techniques: Functional Assay, In Vitro, Expressing, Flow Cytometry, Control, Knock-Out

Journal: Parasites & vectors

Article Title: Upregulation of CD244 promotes CD8 + T cell exhaustion in patients with alveolar echinococcosis and a murine model.

doi: 10.1186/s13071-024-06573-2

Figure Lengend Snippet: Fig. 4 CD244 deficiency delays disease progression by inhibiting terminal differentiation and functional exhaustion of CD8+ T cells in the liver of Echinococcus multilocularis-infected mice after 19 weeks of infection. A Representative images of metacestode tissue in the liver from WT and CD244-KO mice after 19 weeks of infection. The metacestode tissues are delineated with a yellow line. Owing to the heterogeneity of lesion weight, KO group mice were divided into two subgroups, named Group 1 and Group 2. B, C Lesion volume and weight in the liver of WT and CD244-KO mice after 19 weeks of infection (4–6 mice per group). D, E Representative flow cytometry plots (left) and frequency (right) of differentiation phenotypes of CD8+ T cells in the liver of mice at 19 weeks post-infection with E. multilocularis (4–6 mice per group). F Representative flow cytometry plot and percentage of IFN-γ and TNF-α production by activated CD8+ T cells in the liver from mice after 19 weeks of infection (4–6 mice per group). G MFI of Ki67 expression by activated CD8+ T cells in the liver from mice after 19 weeks of infection (4–6 mice per group). H Flow cytometry assessment of hepatic ROS+CD8+ T cells from CD244-KO and WT mice after 19 weeks of E. multilocularis infection (10–14 mice per group). I Oxygen consumption rate (OCR) of hepatic CD8+ T cells from E. multilocularis-infected mice was measured following treatment with oligomycin, FCCP and rotenone plus antimycin A (Rot/AA). J Statistical analysis of mitochondrial ATP-linked respiration in hepatic CD8+ T cells from mice infected with E. multilocularis. Data are one representative of two independent experiments. KO, knockout; WT, wild type. Data were analyzed using two independent samples t-tests, Kruskal-Wallis test or one-way ANOVA test. ns, P > 0.05

Article Snippet: Sections were subsequently blocked with PBS and 10% donkey serum at room temperature for 1 h. Primary antibodies, anti-human CD244 (R&D Systems, 1:500), were applied at appropriate dilutions and incubated overnight at 4 °C.

Techniques: Biomarker Discovery, Functional Assay, Infection, Flow Cytometry, Expressing, Knock-Out

Journal: Parasites & vectors

Article Title: Upregulation of CD244 promotes CD8 + T cell exhaustion in patients with alveolar echinococcosis and a murine model.

doi: 10.1186/s13071-024-06573-2

Figure Lengend Snippet: Fig. 5 CD244-deficient CD8+ T cells demonstrate enhanced effector function following adoptive transfer into Echinococcus multilocularis-infected CD8-KO mice. A Establishment of the adoptive transfer mouse model: CD8+ T cells were isolated and purified from the spleens of CD244-KO (CD45.2) and WT mice (CD45.1/2) and transferred into CD8-KO recipient mice 30 days post-E. multilocularis infection (5–7 mice per group). B Lesion volume in the livers of two groups of CD8-KO mice 30 days after adoptive transfer. C, D Representative flow cytometry plots and percentages of TNF-α and IFN-γ production by WT and CD244-KO donor CD8+ T cells in the livers of E. multilocularis-infected CD8-KO recipient mice. E, F Representative flow cytometry plots and mean fluorescence intensity (MFI) of T-bet and Ki67 expression by transferred CD8+ T cells in the livers of E. multilocularis-infected CD8-KO mice. KO, knockout; WT, wild type. Data were analyzed using two independent samples t-test or Mann-Whitney test

Article Snippet: Sections were subsequently blocked with PBS and 10% donkey serum at room temperature for 1 h. Primary antibodies, anti-human CD244 (R&D Systems, 1:500), were applied at appropriate dilutions and incubated overnight at 4 °C.

Techniques: Adoptive Transfer Assay, Infection, Isolation, Purification, Flow Cytometry, Fluorescence, Expressing, Knock-Out, MANN-WHITNEY

Journal: Frontiers in Oncology

Article Title: Chordoma cancer stem cell subpopulation characterization may guide targeted immunotherapy approaches to reduce disease recurrence

doi: 10.3389/fonc.2024.1376622

Figure Lengend Snippet: CSCs are identified within the chordoma tumor microenvironment. Representative photomicrographs from one chordoma of merged and single-color immunofluorescence images assessing the presence of CSCs with a validated panel of five biomarkers, PD-L1 (yellow, Opal 570), CD15 (red, Opal 690), ALDH (orange, Opal 620), CD24 (green, Opal 520), and CK (white, Opal 780). Multispectral immunofluorescence images are counterstained with DAPI. Co-localization of CD15, ALDH, and CD24 identified CSCs. Co-localization of these markers with PD-L1 identified PD-L1 + CSCs. CD15 + CD24 + ALDH + CK + CSCs comprised 1.39% of all tumor cells. CK, cytokeratin; DAPI, 4′,6-diamidino-2- phenylindole; CSC, cancer stem cells.

Article Snippet: The CSC panel was comprised of antibodies against CD24 (Novus Biologicals [ML5], #NB100-77903; 1:1000), ALDH1 (Abcam [EP1933Y], #ab52492; 1:400), PD-L1 (Cell Signaling [E1L3N(R)], #13684S; 1:300), CD15 (BD Biosciences [HI98], #555400; 1:1500), and Cytokeratin (Santa Cruz [AE1/AE3], #sc-81714; 1:400) ( ).

Techniques: Immunofluorescence

Journal: Frontiers in Oncology

Article Title: Chordoma cancer stem cell subpopulation characterization may guide targeted immunotherapy approaches to reduce disease recurrence

doi: 10.3389/fonc.2024.1376622

Figure Lengend Snippet: CSC and PD-L1 percent positivity in chordoma cells.

Article Snippet: The CSC panel was comprised of antibodies against CD24 (Novus Biologicals [ML5], #NB100-77903; 1:1000), ALDH1 (Abcam [EP1933Y], #ab52492; 1:400), PD-L1 (Cell Signaling [E1L3N(R)], #13684S; 1:300), CD15 (BD Biosciences [HI98], #555400; 1:1500), and Cytokeratin (Santa Cruz [AE1/AE3], #sc-81714; 1:400) ( ).

Techniques:

Journal: Frontiers in Oncology

Article Title: Chordoma cancer stem cell subpopulation characterization may guide targeted immunotherapy approaches to reduce disease recurrence

doi: 10.3389/fonc.2024.1376622

Figure Lengend Snippet: (A) HALO® density heat map analyses suggest that CSCs are found within clusters in four representative chordoma tumors. Solid blue patches delineate tumor areas included in the analysis. Grouped yellow and green dots, marked using white arrows, denote CSC clusters. (B) CK + chordoma cells triple positive for cancer stem cell markers CD15, CD24, and ALDH are denoted CSCs. CSCs, cancer stem cells.

Article Snippet: The CSC panel was comprised of antibodies against CD24 (Novus Biologicals [ML5], #NB100-77903; 1:1000), ALDH1 (Abcam [EP1933Y], #ab52492; 1:400), PD-L1 (Cell Signaling [E1L3N(R)], #13684S; 1:300), CD15 (BD Biosciences [HI98], #555400; 1:1500), and Cytokeratin (Santa Cruz [AE1/AE3], #sc-81714; 1:400) ( ).

Techniques: