Journal: Science advances

Article Title: CD8 + T cell-derived CD40L mediates noncanonical cytotoxicity in CD40-expressing cancer cells.

doi: 10.1126/sciadv.adr9331

Figure Lengend Snippet: Fig. 1. CD40L expression on SV40 TAg–specific CD8+ T cells during a protective immune response. (A) Splenocytes of WT mice challenged with 16.113 TAg+ cancer cells were stimulated with peptide IV at indicated time points. d, day. The dot plots show the intracellular IFN-γ and CD40L staining of CD3+CD8+CD4−-gated lymphocytes from one representative mouse (n = 4 mice). (B) The diagram summarizes the frequencies of CD40L+IFN-γ+ and CD40L−IFN-γ+CD8+ T cells and (C) the frequency of IL-2– producing cells among CD40L+ and CD40L− tumor-specific CD8+ T cells at the different time points (both means ± SD).

Article Snippet: The following mice were obtained from the Jackson Laboratory and bred and housed under specific pathogen–free conditions at the institution’s animal facility (Charité): CD40L−/− (RRID: IMSR_JAX:002428), CD4−/− (RRID: IMSR_JAX:002663), CD8−/− (RRID: IMSR_JAX:002665), CD45.1 (RRID: IMSR_JAX:002014), and CD40−/− (RRID: IMSR_ JAX:002928).

Techniques: Expressing, Staining

Journal: Science advances

Article Title: CD8 + T cell-derived CD40L mediates noncanonical cytotoxicity in CD40-expressing cancer cells.

doi: 10.1126/sciadv.adr9331

Figure Lengend Snippet: Fig. 2. Prevention of tumor outgrowth is dependent on CD40L expression on CD8+ T cells. (A) RAG1−/− mice were injected subcutaneously with 1 × 106 9.27 TAg+ cancer cells and treated in parallel with intravenously injected CD8+ T cells from WT or CD40L−/− mice and/or with WT CD4+ T cells. The means ± SD of the tumor size of four or five mice per group are shown from one representative of five experiments. (B) The tumor sizes of individual mice in different groups are shown at day 26. (C) Sum- mary of the tumor formation data obtained from five independent experiments. Tumor formation was defined as an established tumor when its volume reached >500 mm3. Statistical analysis: Analysis of variance (ANOVA) with Bonferroni multiple comparisons posttest: *P < 0.05, **P < 0.01, and ***P < 0.001.

Article Snippet: The following mice were obtained from the Jackson Laboratory and bred and housed under specific pathogen–free conditions at the institution’s animal facility (Charité): CD40L−/− (RRID: IMSR_JAX:002428), CD4−/− (RRID: IMSR_JAX:002663), CD8−/− (RRID: IMSR_JAX:002665), CD45.1 (RRID: IMSR_JAX:002014), and CD40−/− (RRID: IMSR_ JAX:002928).

Techniques: Expressing, Injection

Journal: Science advances

Article Title: CD8 + T cell-derived CD40L mediates noncanonical cytotoxicity in CD40-expressing cancer cells.

doi: 10.1126/sciadv.adr9331

Figure Lengend Snippet: Fig. 3. Impaired tumor rejection in nonlymphopenic CD8+ T cell–specific CD40L KO mice. (A) Strategy for the generation of CD40Lfl/fl mice. UTR, untranslated region; FRT, flippase recognition target. (B) E8I-Cre × CD40Lfl/fl, E8I-Cre, and CD40L fl/fl mice as WT control were injected subcutaneously with 1 × 106 9.27 TAg+ cancer cells. Sum- mary of the tumor formation data obtained from three individual experiments, each with 5 to 10 mice per group. Tumor was defined as established when its volume reached >500 mm3. (C and D) WT and E8I-Cre × CD40Lfl/fl mice were subcutaneously injected with 1 × 106 9.27 TAg+ cancer cells, and 7 days later, splenocytes and cells from the draining lymph nodes (dLNs) were isolated and stimulated with peptide IV. (C) The dot plots show the intracellular IFN-γ and CD40L staining of CD3+CD8+CD4−- gated splenocytes from one representative mouse of five mice. (D) The diagram summarizes the frequencies of TAg-specific IFN-γ+CD8+ T cells measured among spleno- cytes and lymph node cells. Statistical analysis: Log-rank test: **P < 0.01.

Article Snippet: The following mice were obtained from the Jackson Laboratory and bred and housed under specific pathogen–free conditions at the institution’s animal facility (Charité): CD40L−/− (RRID: IMSR_JAX:002428), CD4−/− (RRID: IMSR_JAX:002663), CD8−/− (RRID: IMSR_JAX:002665), CD45.1 (RRID: IMSR_JAX:002014), and CD40−/− (RRID: IMSR_ JAX:002928).

Techniques: Control, Injection, Isolation, Staining

Journal: Science advances

Article Title: CD8 + T cell-derived CD40L mediates noncanonical cytotoxicity in CD40-expressing cancer cells.

doi: 10.1126/sciadv.adr9331

Figure Lengend Snippet: Fig. 5. CD8+ T cell–mediated CD40 signaling in cancer cells prevents tumor formation. (A) Cell surface expression of CD40 was analyzed after culturing TRAMP-C1 and CD40tg TRAMP-C1 cells with or without TGFβ for 24 hours. (B) Both TRAMP-C1 cancer cell lines were stimulated for 24 hours with multimeric CD40L. Thereafter, cas- pase-8 activity was determined with the fluorescence marker FITC-IETD-FMK. The representative dot plots show the gating of 4′,6-diamidino-2-phenylindole (DAPI) and FITC-IETD-FMK–stained cancer cells after triggering CD40. The diagrams summarize the frequencies of active caspase-8+ cancer cells. (C and D) E8I-Cre × CD40Lfl/fl and E8I-Cre as WT control mice were injected subcutaneously with 5 × 106 TRAMP-C1 (C) or CD40tg TRAMP-C1 (D) cancer cells. In the diagrams [(C) and (D)], the data from one of two representative experiments are shown. Per group, five or six mice were challenged. Tumor was defined as established when its volume reached >500 mm3. Statis- tical analysis: (B) Mann-Whitney U test: **P < 0.01 and [(C) and (D)] log-rank test: *P < 0.05 and **P < 0.01.

Article Snippet: The following mice were obtained from the Jackson Laboratory and bred and housed under specific pathogen–free conditions at the institution’s animal facility (Charité): CD40L−/− (RRID: IMSR_JAX:002428), CD4−/− (RRID: IMSR_JAX:002663), CD8−/− (RRID: IMSR_JAX:002665), CD45.1 (RRID: IMSR_JAX:002014), and CD40−/− (RRID: IMSR_ JAX:002928).

Techniques: Expressing, Activity Assay, Fluorescence, Marker, Staining, Control, Injection, MANN-WHITNEY

Journal: Science advances

Article Title: CD8 + T cell-derived CD40L mediates noncanonical cytotoxicity in CD40-expressing cancer cells.

doi: 10.1126/sciadv.adr9331

Figure Lengend Snippet: Fig. 6. CD40L+CD8+ T cells mediate cell death in human CD40+ carcinoma cell lines by caspase-8 activation. (A) Histograms represent the CD40 expression on EJ138 or A704 transfected with nontargeting single guide RNA (black, sgNon) or cells depleted for CD40 by CRISPR-Cas9 and sgRNA against human CD40 (red). Gray-filled his- tograms show isotype staining. (B) sgNon (black) or caspase-8 sgRNA–treated (red) EJ138 and A704 cells were treated with multimeric CD40L for 24 hours, and caspase-8 activation was monitored by FITC-IETD-FMK staining. Gray-filled histograms represent the basal caspase-8 activation of unstimulated WT cells. (C and D) sgNon, CD40 sgRNA-, or caspase-8 (Casp8) sgRNA–treated variants of EJ138 (C) or A704 (D) were cocultivated with CD40L-enriched CD8+ T cells for 24 hours, and apoptosis was de- tected by annexin V and DAPI staining. Shown are representative dot plots with frequencies of technical triplicates (left) and bar graphs summarizing the data from ex- periments with eight different T cell donors. Statistical analysis: [(C) and (D)] Repeated measures ANOVA, followed by Dunnett’s test: **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Article Snippet: The following mice were obtained from the Jackson Laboratory and bred and housed under specific pathogen–free conditions at the institution’s animal facility (Charité): CD40L−/− (RRID: IMSR_JAX:002428), CD4−/− (RRID: IMSR_JAX:002663), CD8−/− (RRID: IMSR_JAX:002665), CD45.1 (RRID: IMSR_JAX:002014), and CD40−/− (RRID: IMSR_ JAX:002928).

Techniques: Activation Assay, Expressing, Transfection, CRISPR, Staining

Journal: Science advances

Article Title: CD8 + T cell-derived CD40L mediates noncanonical cytotoxicity in CD40-expressing cancer cells.

doi: 10.1126/sciadv.adr9331

Figure Lengend Snippet: Fig. 7. Resistance pattern for CD40-mediated cell death and correlations between CD8 and CD40L in different RCC cohorts. (A) In the histograms, the dark gray areas display the CD40 expression, and the light gray areas display the corresponding isotype control on eight different RCC lines. The included numbers represent the mean fluorescence intensity fold change between CD40 and isotype control staining. (B) Percentages of CD40L-induced lysis per RCC line after stimulation for 48 hours are plotted. Representative lysis values after backgorund substraction of each cell line of at least three individual experiments are depicted. (C) The heatmap represents the top 10 differentially expressed genes between CD40-resistant and CD40-sensitive RCC cell lines. (D) In the Kaplan-Meier survival plot, the resistance score of low and high groups of the TCGA-KIRC patient cohort is compared with each other. (E) Partial correlation networks of different patient groups are displayed, and the numbers are the partial correlation coefficients. (F) In the Kaplan-Meier survival plots, the resistance score of low and high groups of the cohorts is compared with all patients treated within the checkmate-009, checkmate-010, and checkmate-025 studies (left) or divided into two arms of the checkmate-025 study (middle nivolumab arm and right everolimus arm). Statistical analysis: (C) Described in Materials and Methods; [(D) and (F)] log-rank test.

Article Snippet: The following mice were obtained from the Jackson Laboratory and bred and housed under specific pathogen–free conditions at the institution’s animal facility (Charité): CD40L−/− (RRID: IMSR_JAX:002428), CD4−/− (RRID: IMSR_JAX:002663), CD8−/− (RRID: IMSR_JAX:002665), CD45.1 (RRID: IMSR_JAX:002014), and CD40−/− (RRID: IMSR_ JAX:002928).

Techniques: Expressing, Control, Fluorescence, Staining, Lysis

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: iScience

Article Title: Natural killer cells and dendritic epidermal γδ T cells orchestrate type 1 conventional DC spatiotemporal repositioning toward CD8 + T cells

doi: 10.1016/j.isci.2021.103059

Figure Lengend Snippet: NK cells are a main source of XCL1 but not a critical one upon MCMV infection (A and B) Analysis of mTFP1 expression in splenocytes of Wt (dark grey) and Xcl1-mTfp1 fl/fl (white) mice at steady state (A) or 40 h after infection (B). Cells were gated as follow: NK cells (TCRβ - CD19 - NK1.1 + ), ILC1 (NK1.1 + TCRβ - CD19 - CD127 + ), NKT cells (CD19 - CD1d + ), CD8 + T (CD1d - NK1.1 - TCRβ + CD19 - CD8 + ), CD4 + T (CD1d - NK1.1 - TCRβ + CD19 - CD4 + ), γδ T cells (CD1d - NK1.1 - CD3ε + TCRβ - TCRγδ + ) and B cells (NK1.1 - TCRβ - CD19 + ). One representative experiment of 4 independent ones with at least three mice per group is shown. (C and D) Proportion of immune populations within mTFP1-positive cells at steady state (C) and 40 h after MCMV infection (D). Others: sum of all the other cell subsets not detailed in the pie charts. One representative experiment of 4 independent ones with at least three mice per group is shown. (E) IFN-γ production by NK cells in Wt, Xcr1 −/− , Xcl1 fl/fl , and Nkp46 Cre ;Xcl1 fl/fl mice 40 h after infection. One representative experiment of two independent ones with at least four mice per group is shown. A one-way ANOVA statistical analysis with a Tukey's multiple comparison test was applied. ∗, p < 0.05; ∗∗, p < 0.01, n.s., nonsignificant; ANOVA, analysis of variance. See also Figure S3 .

Article Snippet: CD8 + Dendritic Cell Isolation Kit, mouse , Miltenyi Biotec , #130-091-169.

Techniques: Infection, Expressing, Comparison

Journal: iScience

Article Title: Natural killer cells and dendritic epidermal γδ T cells orchestrate type 1 conventional DC spatiotemporal repositioning toward CD8 + T cells

doi: 10.1016/j.isci.2021.103059

Figure Lengend Snippet: CCR7-mediated cDC1 relocalization into the T cell zone promotes MCMV-specific CD8+ T cell responses and host resistance to MCMV infection (A) Analysis of m45-specific CD8 + T cell response in Xcr1 −/− mice and littermate controls 6 days after MCMV infection. One representative experiment of two independent ones with at least 4 mice per group is shown. ∗, p < 0.05. (B) Analysis of m45-specific CD8 + T cell response in Wt : Xcr1 - Dta, Wt : Ccr7 −/− and Xcr1 - Dta : Ccr7 −/− BM chimera mice 6 days after MCMV infection (10 4 PFU). Two independent experiments with at least 4 mice per infected group were pooled. A one-way ANOVA statistical analysis with a Tukey's multiple comparison test was applied. ∗∗, p < 0.01; ∗, p < 0.05, n.s., nonsignificant. Statistical analysis between all other groups is nonsignificant. (C) MCMV titers in spleens and livers of Wt and Xcr1 −/− mice 5 days after MCMV infection (10 4 PFU). Two independent experiments with at least 3 mice per infected group were pooled. NI, noninfected. ∗∗∗, p < 0.001. See also Figure S6 .

Article Snippet: CD8 + Dendritic Cell Isolation Kit, mouse , Miltenyi Biotec , #130-091-169.

Techniques: Infection, Comparison

Journal: iScience

Article Title: Natural killer cells and dendritic epidermal γδ T cells orchestrate type 1 conventional DC spatiotemporal repositioning toward CD8 + T cells

doi: 10.1016/j.isci.2021.103059

Figure Lengend Snippet: Dermal cDC1 make cell/cell contacts with Xcl1 -expressing DETCs (A) Analysis of the lymphocyte heterogeneity within mTFP1 + cells in the skin of Xcl1 mTfp1 fl/fl mice at steady state. One representative experiment of 2 independent ones with at least three mice per group is shown. (B) DETC staining in the skin at steady state. DETCs are CD3ε + TCRγδ + lymphocytes localized in the epidermis. The dotted line delineates the basal layer separating the dermis (D) from the epidermis (EP). C, cartilage. Scale bar: 50 μm (C–E) Analysis of cDC1 localization and contacts with DETCs in the skin, at steady state in Karma Cre ;Rosa26 tdRFP ;Xcr1 +/− mice (C), and at 40 h after MCMV infection in Karma Cre ;Rosa26 tdRFP ;Xcr1 +/− (D) and Karma Cre ;Rosa26 tdRFP ;Xcr1 −/− (E) mice. The dotted line delineates the basal layer separating the dermis from the epidermis. Skin sections (scale bar: 50 μm) were stained for tdRFP (red; cDC1), CD3ε (cyan), TCRγδ (yellow), IE-1 (purple), and Avidin (green; mast cell). EP, epidermis; D, dermis; C, cartilage. (F) 3D visualization of DETC/cDC1 contacts identified in (D). (G) Kinetics of cDC1/DETC contacts occurring in the epidermis and through the epidermis-dermis border per mm of skin length, during MCMV infection. Data are represented as mean (+/− SEM). p.i., postinfection. (H) Quantification of cDC1 nuclear bodies inside the epidermis per mm of skin length 40 h after skin infection. For (G-H), two independent experiments with at least 4 mice per infected group were pooled. n.s., nonsignificant; ∗∗, p < 0.01; ∗∗∗, p < 0.001. (I) Proportion of CD103 + migcDCs in the ear-draining LN of Xcr1 −/− and littermate controls 48 h after skin infection. (J) Analysis of CCR7 expression on migcDCs in ear-draining LN 48 h after skin infection of Xcr1 −/− and Wt mice. Two independent experiments with at least 3 mice per infected group were pooled. ∗∗, p < 0.01. (K and L) Analysis of m45-specific CD8 + T cell response in Xcl1 −/− mice (K), Xcr1 −/− mice (L) and their respective controls 6 days after MCMV infection of the ear. CD8 + T cells from ear-draining LN were stimulated in vitro for 4 h with m45 peptide. Two independent experiments with at least 3 mice per infected group were pooled for (K), and one experiment with at least 4 mice per group is shown for (L). ∗, p < 0.05∗∗, p < 0.01. See also Figure S7 .

Article Snippet: CD8 + Dendritic Cell Isolation Kit, mouse , Miltenyi Biotec , #130-091-169.

Techniques: Expressing, Staining, Infection, Avidin-Biotin Assay, In Vitro

Journal: iScience

Article Title: Natural killer cells and dendritic epidermal γδ T cells orchestrate type 1 conventional DC spatiotemporal repositioning toward CD8 + T cells

doi: 10.1016/j.isci.2021.103059

Figure Lengend Snippet:

Article Snippet: CD8 + Dendritic Cell Isolation Kit, mouse , Miltenyi Biotec , #130-091-169.

Techniques: Purification, Avidin-Biotin Assay, Virus, Recombinant, High Molecular Weight, Cell Isolation, Microarray, Software