Journal: PloS one

Article Title: Cross-protective peptide vaccine against influenza A viruses developed in HLA-A*2402 human immunity model.

doi: 10.1371/journal.pone.0024626

Figure Lengend Snippet: Figure 4. An accumulation of murine CD3+ and CD8+ cells around the bronchioles in intranasally immunized mice. A24Tg mice were immunized three times at 7 to 9 days intervals i.n.(A,C,D,E) or s.c.(B) with PA130–138, PB1430–438 and PB2549–557 peptides in the presence of CpG-ODN (B,C,D), Tyrosinase206–214 plus CpG-ODN (E) or CpG-ODN plus empty-liposome solution (A). Lungs were harvested at day 7 after the final immunization, embedded in O.C.T. compound, frozen in dry ice-2-propanol. Ten mm thick frozen sections were prepared. The sections were post- fixed in acetone:ethanol (1:1) solution and blocked endogenous avidin and biotin activity, then stained with anti-mouse CD3 (A,B,C) or anti-mouse CD8a (D,E). doi:10.1371/journal.pone.0024626.g004

Article Snippet: The sections were stained with biotinylated hamster anti-mouse CD3 (eBioscience, San Diego, CA) or biotinylated rat anti-mouse CD8a (R&D Systems ) antibodies.

Techniques: Avidin-Biotin Assay, Activity Assay, Staining

Journal: JCI insight

Article Title: Collectin-11 promotes cancer cell proliferation and tumor growth.

doi: 10.1172/jci.insight.159452

Figure Lengend Snippet: Figure 3. Colec11–/– mice exhibit less immunosuppressive TME. Tumors excised from Colec11+/+ (WT) or Colec11–/– (KO) mice (d14) were used for analyzing TME. (A–C) Tumor infiltrates analyzed by flow cytometry. (A) CD45+ cells. (B) Subsets of tumor-infiltrating leukocytes analyzed by flow cytometry. Data were analyzed by unpaired t test (n = 18 mice per group, pooled from 4 experiments). Each dot represents an individual mouse. (C) A bar chat representing proportion of subsets in CD45+ cells shown in B. (D) Representative microscopy images of immunochemical staining for CD11b (green)/CD3 (red)/DAPI (blue) and F4/80 (green)/CD3 (red)/DAPI (blue). Scale bar: 50 μm. (E) Representative microscopy images of immunochemical staining for CD8 (red)/DAPI (blue) in tumor edge and core areas. Scale bar: 50 μm. (F). qPCR analysis in tumor tissues. Data were analyzed by unpaired t test (n = 8 mice per group). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: The following antibodies were used in immunochemical staining: monoclonal rat anti–mouse CD45 (103120), CD11b (101202), and F4/80 (123102) (all from BioLegend); rat anti–mouse CD31(557355, BD Biosciences); rabbit anti-mouse CD3 (ab237721), CD8α (ab217344), VWF (ab6994), and rabbit anti– human COLEC11 (ab238585) (all from Abcam); rabbit anti–mouse H2-Ab1 (A18658, ABclonal); rat anti– mouse CD68 (FA11) (Bio-Rad); goat anti–mouse CD206(AF2535) (from R&D systems); rabbit anti–mouse Ki67 (9129, Cell Signaling Technology); Alexa Fluor 488 goat anti–rat IgG (catalog 405418), Alexa Fluor 555 goat anti–rat IgG (catalog 405420), and Alexa Fluor 647 donkey anti–rabbit IgG (catalog 406414) (all from BioLegend); and Alexa Fluor 488 goat anti–rabbit IgG (catalog 4412) and Alexa Flour 594 goat anti– rabbit IgG (catalog 8889) (both from Cell Signaling Technology).

Techniques: Flow Cytometry, Microscopy, Staining

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Apoptotic Vesicles Derived from Mesenchymal Stem Cells Ameliorate Hypersensitivity Responses via Inducing CD8 + T Cells Apoptosis with Calcium Overload and Mitochondrial Dysfunction.

doi: 10.1002/advs.202407446

Figure Lengend Snippet: Scheme 1. The schematic diagram demonstrates the ApoVs interact with CD8+ T cells via membrane fusion, triggering cascade reactions including calcium overload, mitochondrial dysfunction, BAX translocation, and eventual apoptosis in CD8+ T cells.

Article Snippet: The antibodies used were: anti-CD31 antibody (FAB3628G, R&D Systems, USA; diluted 1:100), anti-TGF-β antibody (3711S, Cell Signaling Technology, USA, diluted 1:200), anti-TNF-α antibody (11948s, Cell Signaling Technology, USA, diluted 1:200), mouse CD8 alpha Alexa Fluor 488 MAb (FAB116G-100, R&D system, USA, diluted 1:200), anti-Cav1.2 antibody (ACC-003, Alomone, Israel, diluted 1:200), anti-Cavβ1 antibody (PA5109285, Invitrogen, USA, diluted 1:200), anti-BAX antibody(60267-1-Ig, Proteitech, China, diluted 1:200), Alexa Fluor 488 Donkey anti-Mouse secondary antibody (A21202, Invitrogen, USA, diluted 1:200), Alexa Fluor 594 Donkey anti-Mouse secondary antibody (A21203, Invitrogen, USA, diluted 1:200), Alexa Fluor 488 Donkey anti-Rabbit secondary antibody (A21206, Invitrogen, USA, diluted 1:200) and Alexa Fluor 594 Donkey anti-Rabbit secondary antibody (A21207, Invitrogen, USA, diluted 1:200).

Techniques: Membrane, Translocation Assay

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Apoptotic Vesicles Derived from Mesenchymal Stem Cells Ameliorate Hypersensitivity Responses via Inducing CD8 + T Cells Apoptosis with Calcium Overload and Mitochondrial Dysfunction.

doi: 10.1002/advs.202407446

Figure Lengend Snippet: Figure 1. MSCs-ApoVs Treatment Attenuated CD8+ T Cells-mediated Contact Hypersensitivity. A) Schematic illustration of contact hypersensitivity experimental design. B) Representative phenotype of ears captured by dermoscopy. C) Hematoxylin-eosin (H&E) staining of ear samples collected 24 h after the challenge. The lower panel magnifies the boxed area in the top panel. Scale bar: 150 μm for the upper panel and 50 μm for the lower panel. Black arrowheads indicate dilated capillaries. D) Ear thickness was measured 24 h after the elicitation in three groups (n = 15). E) Volcano plots

Article Snippet: The antibodies used were: anti-CD31 antibody (FAB3628G, R&D Systems, USA; diluted 1:100), anti-TGF-β antibody (3711S, Cell Signaling Technology, USA, diluted 1:200), anti-TNF-α antibody (11948s, Cell Signaling Technology, USA, diluted 1:200), mouse CD8 alpha Alexa Fluor 488 MAb (FAB116G-100, R&D system, USA, diluted 1:200), anti-Cav1.2 antibody (ACC-003, Alomone, Israel, diluted 1:200), anti-Cavβ1 antibody (PA5109285, Invitrogen, USA, diluted 1:200), anti-BAX antibody(60267-1-Ig, Proteitech, China, diluted 1:200), Alexa Fluor 488 Donkey anti-Mouse secondary antibody (A21202, Invitrogen, USA, diluted 1:200), Alexa Fluor 594 Donkey anti-Mouse secondary antibody (A21203, Invitrogen, USA, diluted 1:200), Alexa Fluor 488 Donkey anti-Rabbit secondary antibody (A21206, Invitrogen, USA, diluted 1:200) and Alexa Fluor 594 Donkey anti-Rabbit secondary antibody (A21207, Invitrogen, USA, diluted 1:200).

Techniques: Staining

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Apoptotic Vesicles Derived from Mesenchymal Stem Cells Ameliorate Hypersensitivity Responses via Inducing CD8 + T Cells Apoptosis with Calcium Overload and Mitochondrial Dysfunction.

doi: 10.1002/advs.202407446

Figure Lengend Snippet: Figure 2. ApoVs-afforded Anti-hypersensitivity Effects by Promoting the Apoptosis of CD8+ T Cells. A) Schemes of the adoptive transfer experimental design. CD8+ T cells from the draining lymph nodes of oxazolone-sensitized WT mice were isolated and cultured with or without ApoVs. Naïve WT mice then received adoptive transfer of these CD8+ T cells, followed by treatment on the ears of recipient mice with OXA 2 h post-transfer. B) Representative ear lesions visualized by dermoscopy. C) H&E staining of ear samples collected 24 h after the challenge. The lower panel magnifies the boxed area in the top panel. Scale bar: 150 μm for the upper panel and 50 μm for the lower panel. Black arrowheads indicate dilated capillaries. D) Ear thickness was measured 24 h after the elicitation in groups receiving intravenous infusion of CD8+ T cells treated with PBS or ApoVs (n = 10). E) Bubble diagram displayed the top 10 KEGG pathways enriched terms of upregulated DEGs in ApoVs-treated CD8+ T cells compared to PBS-treated CD8+ T cells (n = 3). F) The bar chart showed the top 12 Reactome enrichment pathways of upregulated DEGs in ApoVs-treated CD8+ T cells compared to PBS-treated CD8+

Article Snippet: The antibodies used were: anti-CD31 antibody (FAB3628G, R&D Systems, USA; diluted 1:100), anti-TGF-β antibody (3711S, Cell Signaling Technology, USA, diluted 1:200), anti-TNF-α antibody (11948s, Cell Signaling Technology, USA, diluted 1:200), mouse CD8 alpha Alexa Fluor 488 MAb (FAB116G-100, R&D system, USA, diluted 1:200), anti-Cav1.2 antibody (ACC-003, Alomone, Israel, diluted 1:200), anti-Cavβ1 antibody (PA5109285, Invitrogen, USA, diluted 1:200), anti-BAX antibody(60267-1-Ig, Proteitech, China, diluted 1:200), Alexa Fluor 488 Donkey anti-Mouse secondary antibody (A21202, Invitrogen, USA, diluted 1:200), Alexa Fluor 594 Donkey anti-Mouse secondary antibody (A21203, Invitrogen, USA, diluted 1:200), Alexa Fluor 488 Donkey anti-Rabbit secondary antibody (A21206, Invitrogen, USA, diluted 1:200) and Alexa Fluor 594 Donkey anti-Rabbit secondary antibody (A21207, Invitrogen, USA, diluted 1:200).

Techniques: Adoptive Transfer Assay, Isolation, Cell Culture, Staining

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Apoptotic Vesicles Derived from Mesenchymal Stem Cells Ameliorate Hypersensitivity Responses via Inducing CD8 + T Cells Apoptosis with Calcium Overload and Mitochondrial Dysfunction.

doi: 10.1002/advs.202407446

Figure Lengend Snippet: Figure 3. ApoVs Induced Apoptosis in CD8+ T cells via Affecting Mitochondrial Morphology and Function. A) Proteomic analysis of ApoVs comparing with EVs showed that GO top 10 enrichment terms of upregulated DEPs, categorized into “Molecular Function” (n = 3). B) The bar chart showed the top 10 Reactome enrichment pathways of upregulated DEPs in ApoVs, compared to EVs (n = 3). C) Doubling the resolution of structured illumination microscopy (SIM2) showed the fragmentation of mitochondrial morphology in CD8+ T cells after ApoVs treatment. Scale bar: 2 μm. D-E) The mean perimeter and mean area of mitochondria in ApoVs treated CD8+ T cells significantly decreased (n = 40). F) Transmission electron microscopy images revealed the mitochondrial morphology. The lower panel magnifies the boxed area in the top panel. Scale bar: 1 μm for the upper panel and 0.5 μm for the lower panel. G) SIM2 exhibited the mitochondrial permeability increased in ApoVs treated CD8+ T group, represented by decreasing of relative fluorescence intensity of Calcein AM (n = 5). Scale bar: 2 μm. H) Flow cytometry analysis exhibited the relative fluorescence intensity of Calcein AM (n = 3). I) Mitochondrial membrane potential (∆Ψm) was analyzed by the relative ration of JC-1 aggregates (OD = 525) and monomer (OD = 490) (n = 5). J) Relative mitochondrial ROS level of two groups (n = 5). ** p < 0.01, ***p < 0.001.

Article Snippet: The antibodies used were: anti-CD31 antibody (FAB3628G, R&D Systems, USA; diluted 1:100), anti-TGF-β antibody (3711S, Cell Signaling Technology, USA, diluted 1:200), anti-TNF-α antibody (11948s, Cell Signaling Technology, USA, diluted 1:200), mouse CD8 alpha Alexa Fluor 488 MAb (FAB116G-100, R&D system, USA, diluted 1:200), anti-Cav1.2 antibody (ACC-003, Alomone, Israel, diluted 1:200), anti-Cavβ1 antibody (PA5109285, Invitrogen, USA, diluted 1:200), anti-BAX antibody(60267-1-Ig, Proteitech, China, diluted 1:200), Alexa Fluor 488 Donkey anti-Mouse secondary antibody (A21202, Invitrogen, USA, diluted 1:200), Alexa Fluor 594 Donkey anti-Mouse secondary antibody (A21203, Invitrogen, USA, diluted 1:200), Alexa Fluor 488 Donkey anti-Rabbit secondary antibody (A21206, Invitrogen, USA, diluted 1:200) and Alexa Fluor 594 Donkey anti-Rabbit secondary antibody (A21207, Invitrogen, USA, diluted 1:200).

Techniques: Microscopy, Transmission Assay, Electron Microscopy, Permeability, Flow Cytometry, Membrane

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Apoptotic Vesicles Derived from Mesenchymal Stem Cells Ameliorate Hypersensitivity Responses via Inducing CD8 + T Cells Apoptosis with Calcium Overload and Mitochondrial Dysfunction.

doi: 10.1002/advs.202407446

Figure Lengend Snippet: Figure 4. ApoVs Evoked Calcium Influx through Membrane Fusion with CD8+ T Cells. A) SIM2 showed the ApoVs (red) fused with the membrane of CD8+ T cells (green) in a time manner. Scale bar: 2 μm. B) Relative fluorescence intensity of PKH26 (ApoVs) enhanced after 6 h in CD8+ T cells treated with ApoVs (n = 3). C) SEM showed a sequential observation of ApoVs contact with CD8+ T cells. D) Cytosolic Ca2+ levels in CD8+ T cells after being treated with ApoVs or PBS in 6 min (n = 5). E) Quantification of the mean maximal [Ca2+] rises (Δ[Ca2+]) in CD8+ T cells when treated with ApoVs or

Article Snippet: The antibodies used were: anti-CD31 antibody (FAB3628G, R&D Systems, USA; diluted 1:100), anti-TGF-β antibody (3711S, Cell Signaling Technology, USA, diluted 1:200), anti-TNF-α antibody (11948s, Cell Signaling Technology, USA, diluted 1:200), mouse CD8 alpha Alexa Fluor 488 MAb (FAB116G-100, R&D system, USA, diluted 1:200), anti-Cav1.2 antibody (ACC-003, Alomone, Israel, diluted 1:200), anti-Cavβ1 antibody (PA5109285, Invitrogen, USA, diluted 1:200), anti-BAX antibody(60267-1-Ig, Proteitech, China, diluted 1:200), Alexa Fluor 488 Donkey anti-Mouse secondary antibody (A21202, Invitrogen, USA, diluted 1:200), Alexa Fluor 594 Donkey anti-Mouse secondary antibody (A21203, Invitrogen, USA, diluted 1:200), Alexa Fluor 488 Donkey anti-Rabbit secondary antibody (A21206, Invitrogen, USA, diluted 1:200) and Alexa Fluor 594 Donkey anti-Rabbit secondary antibody (A21207, Invitrogen, USA, diluted 1:200).

Techniques: Membrane

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Apoptotic Vesicles Derived from Mesenchymal Stem Cells Ameliorate Hypersensitivity Responses via Inducing CD8 + T Cells Apoptosis with Calcium Overload and Mitochondrial Dysfunction.

doi: 10.1002/advs.202407446

Figure Lengend Snippet: Figure 5. Harnessing Calcium Influx in CD8+ T Cells Weaken the Efficacy of ApoVs. A) Cytosolic Ca2+ levels and the mean maximal [Ca2+] rises (Δ[Ca2+]) in CD8+ T cells when stimulated with PBS, ApoVs, and ApoVs pretreatment with 1 μM verapamil groups in 6 min (n = 5). B) Quantification of the mean maximal [Ca2+] rises (Δ[Ca2+]) in CD8+ T cells after being treated with PBS, ApoVs, and ApoVs pretreatment with verapamil groups in 6 min (n = 60). C) SIM2 showed the Calcein AM expression in CD8+ T cells treated with PBS, ApoVs, and ApoVs pretreatment with verapamil respectively. Scale bar: 5 μm. D) Quantification of relative fluorescence intensity of Calcein AM in three groups (n = 5). E) H&E staining of ear samples collected 24 h after the challenge. Scale bar: 150 μm. Black arrowheads indicate dilated capillaries. F) Ear thickness was measured 24 h after the elicitation in groups of intravenous infusion of CD8+ T cells (n = 5). G) SIM2 showed the co-localization of BAX (red) and Mitotracker (green) in three groups respectively. Scale bar: 2 μm. Boxed scale bar: 0.5 μm. H) Quantification of percentage of BAX and Mitotracker co-localized Area (n = 5). I) Western blot displayed the expression level of BAX in isolated mitochondria from CD8+ T cells in three groups. J) Western blot displayed the expression level of classical apoptosis protein cleaved-Caspase 9 and cleaved-Caspase 3 in three groups. * p < 0.05, ** p < 0.01, ***p < 0.001.

Article Snippet: The antibodies used were: anti-CD31 antibody (FAB3628G, R&D Systems, USA; diluted 1:100), anti-TGF-β antibody (3711S, Cell Signaling Technology, USA, diluted 1:200), anti-TNF-α antibody (11948s, Cell Signaling Technology, USA, diluted 1:200), mouse CD8 alpha Alexa Fluor 488 MAb (FAB116G-100, R&D system, USA, diluted 1:200), anti-Cav1.2 antibody (ACC-003, Alomone, Israel, diluted 1:200), anti-Cavβ1 antibody (PA5109285, Invitrogen, USA, diluted 1:200), anti-BAX antibody(60267-1-Ig, Proteitech, China, diluted 1:200), Alexa Fluor 488 Donkey anti-Mouse secondary antibody (A21202, Invitrogen, USA, diluted 1:200), Alexa Fluor 594 Donkey anti-Mouse secondary antibody (A21203, Invitrogen, USA, diluted 1:200), Alexa Fluor 488 Donkey anti-Rabbit secondary antibody (A21206, Invitrogen, USA, diluted 1:200) and Alexa Fluor 594 Donkey anti-Rabbit secondary antibody (A21207, Invitrogen, USA, diluted 1:200).

Techniques: Expressing, Staining, Western Blot, Isolation

Journal: Frontiers in Immunology

Article Title: Unveiling spatial complexity in solid tumor immune microenvironments through multiplexed imaging

doi: 10.3389/fimmu.2024.1383932

Figure Lengend Snippet: Immunophenotyping panel for multiplexed tissue imaging of cancer.

Article Snippet: CD8 , REA734 , 50 , 130-110-677 , FITC , Miltenyi Biotec.

Techniques: Imaging

Journal: Frontiers in Immunology

Article Title: TOB1 modulates neutrophil phenotypes to influence gastric cancer progression and immunotherapy efficacy

doi: 10.3389/fimmu.2024.1369087

Figure Lengend Snippet: Expression profiles of TOB1 and immune biomarkers in gastric cancers. mIF images of representative gastric cancer sections analyzed for panel 1 (A) and panel 2 (B) . Paired boxplots were employed to show the density of nTOB1 + (C) and cTOB1 + (D) cells between cancer and adjacent paracancerous tissues by the paired Wilcoxon test analysis. Moreover, the box-violin plots depicted the densities of CD8 + T cells (E) , CD4 + T cells (F) , FOXP3 + Tregs (G) , CD20 + B cells (H) , CD68 + macrophages (I) , and CD66b + neutrophils (J) between gastric cancer and the corresponding adjacent tissues. * P < 0.05, ** P < 0.01, *** P < 0.001, ns, not significant.

Article Snippet: IHC was performed to verify the efficacy of antibodies and determine initial dilution ratios for each marker: CD8 (ZA-0508; Zsbio, China, 1:200), CD4 (ab133616; Abcam, USA, 1:200), FOXP3 (ab4728; Abcam, 1:100), CD20 (ab9475; Abcam, 1:200), CD68 (ab955; Abcam, 1:200), CD66b (555723; BD Biosciences, USA, 1:1000).

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: TOB1 modulates neutrophil phenotypes to influence gastric cancer progression and immunotherapy efficacy

doi: 10.3389/fimmu.2024.1369087

Figure Lengend Snippet: The expression of TOB1 in various immune cell types between gastric cancer and adjacent paracancerous tissues. TOB1 expression of CD8 + T cells (A, B) , CD66b + neutrophils (C, D) , CD4 + T cells (E, F) , FOXP3 + Tregs (G, H) , CD20 + B cells (I, J) , and CD68 + macrophages (K, L) . The densities of double positive cells of each immune marker with nTOB1 (M) or cTOB1 (N) analyzing by pairwise Wilcoxon rank sum test. Bar graphs were employed to illustrate the expression proportions of nTOB1 (O) and cTOB1 (P) within various immune cells, with an ANOVA analysis conducted for assessment. ** P < 0.01, *** P < 0.001, ns, not significant.

Article Snippet: IHC was performed to verify the efficacy of antibodies and determine initial dilution ratios for each marker: CD8 (ZA-0508; Zsbio, China, 1:200), CD4 (ab133616; Abcam, USA, 1:200), FOXP3 (ab4728; Abcam, 1:100), CD20 (ab9475; Abcam, 1:200), CD68 (ab955; Abcam, 1:200), CD66b (555723; BD Biosciences, USA, 1:1000).

Techniques: Expressing, Marker