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: bioRxiv

Article Title: Discovery of CD28-Targeted Small Molecule Inhibitors of T Cell Co-stimulation Using Affinity Selection-Mass Spectrometry (AS-MS) and Ex Vivo Validation

doi: 10.1101/2025.07.31.667814

Figure Lengend Snippet: (A) Schematic of the SEC-coupled AS-MS workflow. A total of 6,336 compounds were screened in pooled format using CD28 protein, followed by SEC and LC-MS to detect protein-bound ligands. (B) Representative SEC/UV chromatograms from the primary screen using 2.5 μM CD28 and 1 μM compound concentration. A distinct protein peak is observed in CD28 + compound samples (P1+, P2+) but absent in the compound-only control (P−), confirming successful protein separation. (C) Hit selection was based on mass error ≤5 ppm, absence in P− control, P+/P− intensity ratio ≥3, consistent retention time, clear isotopic pattern, and signal >1000 counts. A total of 56 compounds were initially selected, and 25 were confirmed as CD28 binders. (D) Fraction bound (%) of selected compounds from the retest screen using 5 μM CD28. Compounds 5MS, 7MS, 12MS, 13MS, 17MS, 19MS , and 20MS showed the highest Fb% values (>30%), indicating strong binding to CD28.

Article Snippet: His-tagged recombinant human CD28 protein (Acro Biosystems) was fluorescently labeled with RED-tris-NTA 2nd Generation dye (NanoTemper, Cat. #MO-L018) following the manufacturer’s protocol.

Techniques: Liquid Chromatography with Mass Spectroscopy, Concentration Assay, Control, Selection, Binding Assay

Journal: bioRxiv

Article Title: Discovery of CD28-Targeted Small Molecule Inhibitors of T Cell Co-stimulation Using Affinity Selection-Mass Spectrometry (AS-MS) and Ex Vivo Validation

doi: 10.1101/2025.07.31.667814

Figure Lengend Snippet: (A) Single-dose binding responses of 25 hits at 100 μM using the Dianthus platform in PBST buffer (PBS + 0.05% Tween-20) with 2% DMSO. Compounds higher or lower three times the SD of CD28 only (Blank) were selected for dose dependent analysis. (B–D) Dose–response MST binding curves for compounds 5MS, 19MS , and 20MS , respectively. Each compound was prepared at an initial concentration of 500 μM and subjected to a 14-point 1:1 serial dilution (final range: 500 to 0.06 μM). Data represent mean ± standard error mean (SEM) from n = 5 independent measurements.

Article Snippet: His-tagged recombinant human CD28 protein (Acro Biosystems) was fluorescently labeled with RED-tris-NTA 2nd Generation dye (NanoTemper, Cat. #MO-L018) following the manufacturer’s protocol.

Techniques: Binding Assay, Concentration Assay, Serial Dilution

Journal: bioRxiv

Article Title: Discovery of CD28-Targeted Small Molecule Inhibitors of T Cell Co-stimulation Using Affinity Selection-Mass Spectrometry (AS-MS) and Ex Vivo Validation

doi: 10.1101/2025.07.31.667814

Figure Lengend Snippet: Each compound was serially diluted from 500 to 0.06 μM in PBST with 2% DMSO, while CD28 protein concentration was held constant. KD values were determined by nonlinear regression using GraphPad Prism, fitted to a 1:1 binding model. Data are presented as mean ± SEM from n = 5 independent experiments.

Article Snippet: His-tagged recombinant human CD28 protein (Acro Biosystems) was fluorescently labeled with RED-tris-NTA 2nd Generation dye (NanoTemper, Cat. #MO-L018) following the manufacturer’s protocol.

Techniques: Protein Concentration, Binding Assay

Journal: bioRxiv

Article Title: Discovery of CD28-Targeted Small Molecule Inhibitors of T Cell Co-stimulation Using Affinity Selection-Mass Spectrometry (AS-MS) and Ex Vivo Validation

doi: 10.1101/2025.07.31.667814

Figure Lengend Snippet: The CD28 Blockade Bioassay (Promega, Cat. #JA6101) was employed to assess the ability of 19MS-5 to inhibit CD28–B7 interactions in a co-culture system of CD28 Effector Cells (Jurkat) and aAPC/Raji Cells. The compound was evaluated in a 10-point dose–response format, and luminescence was quantified using the Bio-Glo™ Luciferase Assay System. Dose–response curve was fitted using nonlinear regression (fourparameter logistic model) in GraphPad Prism. Data are presented as mean ± SEM from n = 5 independent experiments.

Article Snippet: His-tagged recombinant human CD28 protein (Acro Biosystems) was fluorescently labeled with RED-tris-NTA 2nd Generation dye (NanoTemper, Cat. #MO-L018) following the manufacturer’s protocol.

Techniques: Bioassay, Co-Culture Assay, Luciferase

Journal: bioRxiv

Article Title: Discovery of CD28-Targeted Small Molecule Inhibitors of T Cell Co-stimulation Using Affinity Selection-Mass Spectrometry (AS-MS) and Ex Vivo Validation

doi: 10.1101/2025.07.31.667814

Figure Lengend Snippet: (A-B) Compound 5MS docked into CD28, showing surface view (A) and detailed interaction diagram (B). (C-D) Compound 19MS-5 binding mode, displaying surface representation (C) and interaction network (D). (E-F) Compound 5MS-5 docked configuration, illustrated through surface view (E) and binding interactions (F). (G-H) Compound 19MS binding analysis, showing surface representation (G) and detailed interactions (H) . In all panels, the protein surface is colored according to electrostatic potential (blue: positive, red: negative, white: neutral), and green dashed lines indicate hydrogen bonds or favorable electrostatic interactions. The ligands are represented as gray stick models, and interacting residues are labeled with their three-let-ter amino acid codes and residue numbers. Favorable interactions are color-coded as follows: green, hydrogen bonds; light green, carbon–hydrogen interactions; dark pink, π–π stacking interactions; purple, π–sigma interactions; light pink, hydrophobic interactions. All docking studies were performed using Maestro Schrödinger (version 2023.2) with induced fit protocols to account for protein flexibility during ligand binding.

Article Snippet: His-tagged recombinant human CD28 protein (Acro Biosystems) was fluorescently labeled with RED-tris-NTA 2nd Generation dye (NanoTemper, Cat. #MO-L018) following the manufacturer’s protocol.

Techniques: Binding Assay, Labeling, Residue, Ligand Binding Assay

Journal: bioRxiv

Article Title: Discovery of CD28-Targeted Small Molecule Inhibitors of T Cell Co-stimulation Using Affinity Selection-Mass Spectrometry (AS-MS) and Ex Vivo Validation

doi: 10.1101/2025.07.31.667814

Figure Lengend Snippet: RMSD analysis of CD28 protein-ligand complexes during 50 ns molecular dynamics simulations.

Article Snippet: His-tagged recombinant human CD28 protein (Acro Biosystems) was fluorescently labeled with RED-tris-NTA 2nd Generation dye (NanoTemper, Cat. #MO-L018) following the manufacturer’s protocol.

Techniques:

Journal: bioRxiv

Article Title: Discovery of CD28-Targeted Small Molecule Inhibitors of T Cell Co-stimulation Using Affinity Selection-Mass Spectrometry (AS-MS) and Ex Vivo Validation

doi: 10.1101/2025.07.31.667814

Figure Lengend Snippet: (A) Schematic overview of the experimental setup. Human PBMCs were overlaid onto fully differentiated Mu-cilAir™ tissue (Epithelix) and stimulated with plate-bound anti-CD3 and soluble anti-CD28 for 48 h in the presence of vehicle (0.1% DMSO), 19MS-5 (10, 25, or 50 μM), or FR104 (10 μg/mL). (B–D) Quantification of IFN-γ, IL-2, and TNF-α secretion (pg/mL) in apical supernatants by ELISA. 19MS-5 suppressed CD28-induced cytokine production in a dose-dependent manner, with levels at 50 μM comparable to FR104. Statistical comparisons to the “CD3/CD28 + Vehicle” group were performed using one-way ANOVA followed by Dunnett’s post-hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 relative to “CD3/CD28 + Vehicle” group.

Article Snippet: His-tagged recombinant human CD28 protein (Acro Biosystems) was fluorescently labeled with RED-tris-NTA 2nd Generation dye (NanoTemper, Cat. #MO-L018) following the manufacturer’s protocol.

Techniques: Enzyme-linked Immunosorbent Assay

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