Journal: Health Science Reports

Article Title: CD40LG as a Biomarker in Rheumatoid Arthritis: Links to Bone Destruction and Interstitial Lung Disease ‐ A Bioinformatic Analysis With Clinical Validation

doi: 10.1002/hsr2.71318

Figure Lengend Snippet: Comparison of the expression of genes in RA and RA synovial tissues. (A) There was no difference in CXCL 8 gene expression between RA and OA patients ( p = 0.097). (B) The CD40LG gene expression was higher in RA patients than in OA patients ( p = 0.002). (C) The ITGAX gene expression was higher in RA patients than in OA patients( p = 0.025). (D) There was no difference in IL6 gene expression between RA and OA patients( p = 0.687). (E)The PTPRC gene expression was higher in RA patients than in OA patients( p = 0.003). * p < 0.05; ** p < 0.01.

Article Snippet: The recombinant CD40LG (Novoprotein), ITGAX (Proteintech), and PTPRC (Novoprotein) proteins (0.1 μg/mL) were used to coat 96‐well plates overnight at 4°C, followed by blocking 5% BSA at 37°C for 1 h. After washing three times with PBST, 100 μl of 1:100 dilution serum was added to each well and incubated plates at 37°C for 1 h. After another triple PBST rinse, peroxidase‐conjugated affinipure goat anti‐human IgG (1:10,000; Jackson ImmunoResearch) was added to the wells and incubated at 37°C for 1 h. After triple PBST washes, add the color development solution, once the color appears, immediately apply the stop solution to terminate the reaction.

Techniques: Comparison, Expressing, Gene Expression

Journal: Health Science Reports

Article Title: CD40LG as a Biomarker in Rheumatoid Arthritis: Links to Bone Destruction and Interstitial Lung Disease ‐ A Bioinformatic Analysis With Clinical Validation

doi: 10.1002/hsr2.71318

Figure Lengend Snippet: Receiver operating characteristic (ROC) curve of the 5 specifically expressed hub genes. (A) ROC curves for CXCL8. (B) ROC curves for CD40LG. (C) ROC curves for PTPRC. (D) ROC curves for IL6. (E) ROC curves for ITGAX. (F) 5 genes ROC summary.

Article Snippet: The recombinant CD40LG (Novoprotein), ITGAX (Proteintech), and PTPRC (Novoprotein) proteins (0.1 μg/mL) were used to coat 96‐well plates overnight at 4°C, followed by blocking 5% BSA at 37°C for 1 h. After washing three times with PBST, 100 μl of 1:100 dilution serum was added to each well and incubated plates at 37°C for 1 h. After another triple PBST rinse, peroxidase‐conjugated affinipure goat anti‐human IgG (1:10,000; Jackson ImmunoResearch) was added to the wells and incubated at 37°C for 1 h. After triple PBST washes, add the color development solution, once the color appears, immediately apply the stop solution to terminate the reaction.

Techniques:

Journal: Health Science Reports

Article Title: CD40LG as a Biomarker in Rheumatoid Arthritis: Links to Bone Destruction and Interstitial Lung Disease ‐ A Bioinformatic Analysis With Clinical Validation

doi: 10.1002/hsr2.71318

Figure Lengend Snippet: Comparison of the relevant antibody levels in the serum of different patients. (A) Comparison of OD values of serum CD40LG, ITGAX and PTPRC in RA, OA, and the HC group. (B) ROC curve of CD40LG in RA patients. (C) Higher serum CD40LG antibody levels in RA patients with combined bone destruction and ILD. ns: no significant difference. * p < 0.05; **** p < 0.0001. ROC, receiver operating characteristic.

Article Snippet: The recombinant CD40LG (Novoprotein), ITGAX (Proteintech), and PTPRC (Novoprotein) proteins (0.1 μg/mL) were used to coat 96‐well plates overnight at 4°C, followed by blocking 5% BSA at 37°C for 1 h. After washing three times with PBST, 100 μl of 1:100 dilution serum was added to each well and incubated plates at 37°C for 1 h. After another triple PBST rinse, peroxidase‐conjugated affinipure goat anti‐human IgG (1:10,000; Jackson ImmunoResearch) was added to the wells and incubated at 37°C for 1 h. After triple PBST washes, add the color development solution, once the color appears, immediately apply the stop solution to terminate the reaction.

Techniques: Comparison

Journal: Health Science Reports

Article Title: CD40LG as a Biomarker in Rheumatoid Arthritis: Links to Bone Destruction and Interstitial Lung Disease ‐ A Bioinformatic Analysis With Clinical Validation

doi: 10.1002/hsr2.71318

Figure Lengend Snippet: Molecular docking 3D diagram of key targets and small molecule drugs. (A) CD40LG‐emetine complex, with binding energy of −7.1 kcal/mol. (B) CD40LG‐oligomycin complex, with binding energy of −7.2 kcal/mol. (C) CD40LG‐cromoglicic acid complex, with binding energy of −7.9 kcal/mol. (D) ITGAX‐sulfinpyrazone complex, with binding energy of −7.2 kcal/mol. (E) PTPRC‐oligomycin complex, with binding energy of −8.5 kcal/mol. (F) PTPRC‐buspirone complex, with binding energy of −7.0 kcal/mol. (G) PTPRC‐chlorogenic acid complex, with binding energy of −7.2 kcal/mol.

Article Snippet: The recombinant CD40LG (Novoprotein), ITGAX (Proteintech), and PTPRC (Novoprotein) proteins (0.1 μg/mL) were used to coat 96‐well plates overnight at 4°C, followed by blocking 5% BSA at 37°C for 1 h. After washing three times with PBST, 100 μl of 1:100 dilution serum was added to each well and incubated plates at 37°C for 1 h. After another triple PBST rinse, peroxidase‐conjugated affinipure goat anti‐human IgG (1:10,000; Jackson ImmunoResearch) was added to the wells and incubated at 37°C for 1 h. After triple PBST washes, add the color development solution, once the color appears, immediately apply the stop solution to terminate the reaction.

Techniques: Binding Assay

Journal: Journal of Cancer

Article Title: CD40LG Downregulation in Lung Adenocarcinoma: A Prognostic Biomarker Linked to Immune Cell Infiltration and Survival Outcomes

doi: 10.7150/jca.115525

Figure Lengend Snippet: Figure 1: CD40LG is down-regulated in multiple cancer types, and this down-regulation has significant prognostic implications. (A) Violin plots showed the differential expression of CD40LG in 34 cancer types compared with normal tissues. (B) Body map showing CD40LG expression levels in various normal human tissues. (C) Cox regression analysis of OS demonstrated the prognostic role of CD40LG in a variety of cancers. (D) Cox regression analysis of PFS demonstrated the prognostic role of CD40LG in a variety of cancers. (E) The veen diagram shows those cancers in which CD40LG has the same prognostic predictive efficacy in OS and PFS.

Article Snippet: Subsequently, they were incubated with the primary antibody against GAPDH (1:1,000, 60004-1-lg, Proteintech) and CD40LG (1:1,000, 16668-1-AP, Proteintech) at 4°C overnight.

Techniques: Quantitative Proteomics, Expressing

Journal: Journal of Cancer

Article Title: CD40LG Downregulation in Lung Adenocarcinoma: A Prognostic Biomarker Linked to Immune Cell Infiltration and Survival Outcomes

doi: 10.7150/jca.115525

Figure Lengend Snippet: Figure 2: Correlation Between CD40LG Expression and Immunotherapeutic Efficacy of Various Cancers. (A) Kaplan-Meier plotter analysis showing that patients with high CD40LG expression have better OS after immunotherapy. (B) Kaplan-Meier plotter analysis showing that patients with high CD40LG expression have better PFS after immunotherapy. (C) Comparison of CD40LG expression with traditional biomarkers including PD-L1 expression, TMB, MSI status and CD8+T cell density in predicting immunotherapy outcomes across 25 datasets.

Article Snippet: Subsequently, they were incubated with the primary antibody against GAPDH (1:1,000, 60004-1-lg, Proteintech) and CD40LG (1:1,000, 16668-1-AP, Proteintech) at 4°C overnight.

Techniques: Expressing, Comparison

Journal: Journal of Cancer

Article Title: CD40LG Downregulation in Lung Adenocarcinoma: A Prognostic Biomarker Linked to Immune Cell Infiltration and Survival Outcomes

doi: 10.7150/jca.115525

Figure Lengend Snippet: Figure 3: The important role of CD40LG expression in LUAD. (A-C) Reduced expression of CD40LG in LUAD was analysed in public databases, including TCGA, GSE118370 and GSE43458. (D-F) The association of CD40LG expression with overall survival of LUAD patients was examined in public databases, including GSE30219, GSE31210 and GSE72094. (G) CD40LG expression in LUADs analysed in immunohistochemistry of HPA was lower than in corresponding paraneoplastic tissues. (H) PCR experiments were performed on four patients with LUAD to verify the RNA expression of CD40LG in cancer and corresponding paracancerous tissues. (I) Western blot experiments were performed on four patients with LUAD to verify the protein expression of CD40LG in cancer and corresponding paracancerous tissues.

Article Snippet: Subsequently, they were incubated with the primary antibody against GAPDH (1:1,000, 60004-1-lg, Proteintech) and CD40LG (1:1,000, 16668-1-AP, Proteintech) at 4°C overnight.

Techniques: Expressing, Immunohistochemistry, RNA Expression, Western Blot

Journal: Journal of Cancer

Article Title: CD40LG Downregulation in Lung Adenocarcinoma: A Prognostic Biomarker Linked to Immune Cell Infiltration and Survival Outcomes

doi: 10.7150/jca.115525

Figure Lengend Snippet: Figure 4: CD40LG Expression Correlates with Immune Microenvironment in LUAD. (A) Correlation between CD40LG expression and Immunophenoscore (IPS), including components such as antigen presentation, effector cells, suppressor cells, and immune checkpoints in LUAD. (B-F) Positive correlation of CD40LG expression with infiltration of various immune cells in LUAD tissues, including CD8+ T cells, dendritic cells, macrophages, B cells, and CD4+ T cells, using multiple predictive methods.

Article Snippet: Subsequently, they were incubated with the primary antibody against GAPDH (1:1,000, 60004-1-lg, Proteintech) and CD40LG (1:1,000, 16668-1-AP, Proteintech) at 4°C overnight.

Techniques: Expressing, Immunopeptidomics

Journal: Journal of Cancer

Article Title: CD40LG Downregulation in Lung Adenocarcinoma: A Prognostic Biomarker Linked to Immune Cell Infiltration and Survival Outcomes

doi: 10.7150/jca.115525

Figure Lengend Snippet: Figure 5: CD40LG Expression in LUAD and its Association with Clinical Stages. (A) Representative immunohistochemical staining images of CD40LG in primary LUAD tissues and corresponding paracancerous tissues. (B) Boxplot comparing CD40LG expression levels in tumor tissues versus paracancerous tissues. (C-D) The boxplot analysis illustrates the expression of CD40LG in relation to TNM and T stages.

Article Snippet: Subsequently, they were incubated with the primary antibody against GAPDH (1:1,000, 60004-1-lg, Proteintech) and CD40LG (1:1,000, 16668-1-AP, Proteintech) at 4°C overnight.

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Journal of Cancer

Article Title: CD40LG Downregulation in Lung Adenocarcinoma: A Prognostic Biomarker Linked to Immune Cell Infiltration and Survival Outcomes

doi: 10.7150/jca.115525

Figure Lengend Snippet: Figure 6: CD40LG Expression Correlated with Immune Infiltrate Patterns in LUAD. (A) Representative mIF staining image showing CD40LG expression and immune cell distribution in LUAD tissue microarrays. (B-E) Correlation analysis between CD40LG_IHC_Score and percentages of various immune-related cells in total, tumor, and stromal regions.

Article Snippet: Subsequently, they were incubated with the primary antibody against GAPDH (1:1,000, 60004-1-lg, Proteintech) and CD40LG (1:1,000, 16668-1-AP, Proteintech) at 4°C overnight.

Techniques: Expressing, Staining

Journal: Journal of Cancer

Article Title: CD40LG Downregulation in Lung Adenocarcinoma: A Prognostic Biomarker Linked to Immune Cell Infiltration and Survival Outcomes

doi: 10.7150/jca.115525

Figure Lengend Snippet: Figure 7: CD40LG Expression and Its Effects on LUAD Cell Migration, Invasion, and Proliferation. (A) Western blot analysis of CD40LG expression in five LUAD cell lines. (B) Western blot verification of siRNA-mediated knockdown of CD40LG in H1975 cells and overexpression in A549 cells. (C) EDU assay results showing increased proliferation upon CD40LG knockdown in H1975 cells and decreased proliferation with CD40LG overexpression in A549 cells. (D) Scratch wound healing assay indicating enhanced migration with CD40LG knockdown in H1975 cells and reduced migration with CD40LG overexpression in A549 cells. (E) Transwell assay demonstrating increased migration and invasion capabilities with CD40LG knockdown in H1975 cells and decreased capabilities with CD40LG overexpression in A549 cells.

Article Snippet: Subsequently, they were incubated with the primary antibody against GAPDH (1:1,000, 60004-1-lg, Proteintech) and CD40LG (1:1,000, 16668-1-AP, Proteintech) at 4°C overnight.

Techniques: Expressing, Migration, Western Blot, Knockdown, Over Expression, EdU Assay, Wound Healing Assay, Transwell Assay

Journal: Journal of Cancer

Article Title: CD40LG Downregulation in Lung Adenocarcinoma: A Prognostic Biomarker Linked to Immune Cell Infiltration and Survival Outcomes

doi: 10.7150/jca.115525

Figure Lengend Snippet: Figure 1: CD40LG is down-regulated in multiple cancer types, and this down-regulation has significant prognostic implications. (A) Violin plots showed the differential expression of CD40LG in 34 cancer types compared with normal tissues. (B) Body map showing CD40LG expression levels in various normal human tissues. (C) Cox regression analysis of OS demonstrated the prognostic role of CD40LG in a variety of cancers. (D) Cox regression analysis of PFS demonstrated the prognostic role of CD40LG in a variety of cancers. (E) The veen diagram shows those cancers in which CD40LG has the same prognostic predictive efficacy in OS and PFS.

Article Snippet: Afterwards, they were incubated with the primary antibody against CD40LG (1:150, 16668-1-AP, Proteintech) at 4°C overnight.

Techniques: Quantitative Proteomics, Expressing

Journal: Journal of Cancer

Article Title: CD40LG Downregulation in Lung Adenocarcinoma: A Prognostic Biomarker Linked to Immune Cell Infiltration and Survival Outcomes

doi: 10.7150/jca.115525

Figure Lengend Snippet: Figure 2: Correlation Between CD40LG Expression and Immunotherapeutic Efficacy of Various Cancers. (A) Kaplan-Meier plotter analysis showing that patients with high CD40LG expression have better OS after immunotherapy. (B) Kaplan-Meier plotter analysis showing that patients with high CD40LG expression have better PFS after immunotherapy. (C) Comparison of CD40LG expression with traditional biomarkers including PD-L1 expression, TMB, MSI status and CD8+T cell density in predicting immunotherapy outcomes across 25 datasets.

Article Snippet: Afterwards, they were incubated with the primary antibody against CD40LG (1:150, 16668-1-AP, Proteintech) at 4°C overnight.

Techniques: Expressing, Comparison

Journal: Journal of Cancer

Article Title: CD40LG Downregulation in Lung Adenocarcinoma: A Prognostic Biomarker Linked to Immune Cell Infiltration and Survival Outcomes

doi: 10.7150/jca.115525

Figure Lengend Snippet: Figure 3: The important role of CD40LG expression in LUAD. (A-C) Reduced expression of CD40LG in LUAD was analysed in public databases, including TCGA, GSE118370 and GSE43458. (D-F) The association of CD40LG expression with overall survival of LUAD patients was examined in public databases, including GSE30219, GSE31210 and GSE72094. (G) CD40LG expression in LUADs analysed in immunohistochemistry of HPA was lower than in corresponding paraneoplastic tissues. (H) PCR experiments were performed on four patients with LUAD to verify the RNA expression of CD40LG in cancer and corresponding paracancerous tissues. (I) Western blot experiments were performed on four patients with LUAD to verify the protein expression of CD40LG in cancer and corresponding paracancerous tissues.

Article Snippet: Afterwards, they were incubated with the primary antibody against CD40LG (1:150, 16668-1-AP, Proteintech) at 4°C overnight.

Techniques: Expressing, Immunohistochemistry, RNA Expression, Western Blot

Journal: Journal of Cancer

Article Title: CD40LG Downregulation in Lung Adenocarcinoma: A Prognostic Biomarker Linked to Immune Cell Infiltration and Survival Outcomes

doi: 10.7150/jca.115525

Figure Lengend Snippet: Figure 4: CD40LG Expression Correlates with Immune Microenvironment in LUAD. (A) Correlation between CD40LG expression and Immunophenoscore (IPS), including components such as antigen presentation, effector cells, suppressor cells, and immune checkpoints in LUAD. (B-F) Positive correlation of CD40LG expression with infiltration of various immune cells in LUAD tissues, including CD8+ T cells, dendritic cells, macrophages, B cells, and CD4+ T cells, using multiple predictive methods.

Article Snippet: Afterwards, they were incubated with the primary antibody against CD40LG (1:150, 16668-1-AP, Proteintech) at 4°C overnight.

Techniques: Expressing, Immunopeptidomics

Journal: Journal of Cancer

Article Title: CD40LG Downregulation in Lung Adenocarcinoma: A Prognostic Biomarker Linked to Immune Cell Infiltration and Survival Outcomes

doi: 10.7150/jca.115525

Figure Lengend Snippet: Figure 5: CD40LG Expression in LUAD and its Association with Clinical Stages. (A) Representative immunohistochemical staining images of CD40LG in primary LUAD tissues and corresponding paracancerous tissues. (B) Boxplot comparing CD40LG expression levels in tumor tissues versus paracancerous tissues. (C-D) The boxplot analysis illustrates the expression of CD40LG in relation to TNM and T stages.

Article Snippet: Afterwards, they were incubated with the primary antibody against CD40LG (1:150, 16668-1-AP, Proteintech) at 4°C overnight.

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Journal of Cancer

Article Title: CD40LG Downregulation in Lung Adenocarcinoma: A Prognostic Biomarker Linked to Immune Cell Infiltration and Survival Outcomes

doi: 10.7150/jca.115525

Figure Lengend Snippet: Figure 6: CD40LG Expression Correlated with Immune Infiltrate Patterns in LUAD. (A) Representative mIF staining image showing CD40LG expression and immune cell distribution in LUAD tissue microarrays. (B-E) Correlation analysis between CD40LG_IHC_Score and percentages of various immune-related cells in total, tumor, and stromal regions.

Article Snippet: Afterwards, they were incubated with the primary antibody against CD40LG (1:150, 16668-1-AP, Proteintech) at 4°C overnight.

Techniques: Expressing, Staining

Journal: Journal of Cancer

Article Title: CD40LG Downregulation in Lung Adenocarcinoma: A Prognostic Biomarker Linked to Immune Cell Infiltration and Survival Outcomes

doi: 10.7150/jca.115525

Figure Lengend Snippet: Figure 7: CD40LG Expression and Its Effects on LUAD Cell Migration, Invasion, and Proliferation. (A) Western blot analysis of CD40LG expression in five LUAD cell lines. (B) Western blot verification of siRNA-mediated knockdown of CD40LG in H1975 cells and overexpression in A549 cells. (C) EDU assay results showing increased proliferation upon CD40LG knockdown in H1975 cells and decreased proliferation with CD40LG overexpression in A549 cells. (D) Scratch wound healing assay indicating enhanced migration with CD40LG knockdown in H1975 cells and reduced migration with CD40LG overexpression in A549 cells. (E) Transwell assay demonstrating increased migration and invasion capabilities with CD40LG knockdown in H1975 cells and decreased capabilities with CD40LG overexpression in A549 cells.

Article Snippet: Afterwards, they were incubated with the primary antibody against CD40LG (1:150, 16668-1-AP, Proteintech) at 4°C overnight.

Techniques: Expressing, Migration, Western Blot, Knockdown, Over Expression, EdU Assay, Wound Healing Assay, Transwell Assay

Journal: Science (New York, N.Y.)

Article Title: Identification of antigen-presenting cell-T cell interactions driving immune responses to food

doi: 10.1126/science.ado5088

Figure Lengend Snippet: (A) Schematic representation of LIPSTIC labeling of intercellular contacts in vivo . (B, C, F-J) CD45.2 Cd40 G5/G5 mice were adoptively transferred with 1 × 10 6 naive CD45.1 CD4 + Cd40lg SrtA/Y OT-II T cells prior to one dose of intragastric PBS, OVA or OVA + anti CD40L antibody. Cell-cell interaction was revealed by LIPSTIC protocol 24 h later. (B) Experimental setup for panel c . (C) Representative flow plots showing percentage of labeled DCs in the duodenal gLNs (D-gLNs) (left) and quantification of data (right) (n = 3, 4 mice per group, pool of two independent experiments). (D, E) Mixed bone marrow chimera (BMC) mice reconstituted with Cd40 G5/G5 and Cd40 G5/G5 :H2 −/− cells. Mice were adoptively transferred with 1 × 10 6 naïve CD4 + Cd40lg SrtA/Y OT-II T cells prior to one dose of intragastric OVA. Cell-cell interaction was revealed by LIPSTIC protocol 24 h later. (D) Experimental setup for panel (E). (E) Representative flow plots showing percentage of labeled DCs (n = 4 mice per group, representative of two independent experiments). (F, G) Sorted D-gLNs biotin − or biotin + DCs or DCs derived from OVA-naive mice were co-culture in vitro with naïve OT-II CFSE-labeled T cells for 96 h prior analysis. (F) Representative flow plots showing proliferation of OT-II T cells co-cultured with biotin − or biotin + DCs (left) and quantification of OT-II T cells per well at the end of the culture period with indicated DCs (right). (G) Percentage of Foxp3 + cells among proliferated OT-II T cells co-culture with biotin − or biotin + DCs. Each dot represents one mouse (n = 3 to 5 mice per group, representative of two independent experiments). (H) Representative flow plots showing percentage of Foxp3 + cells among proliferated OT-II T cells co-culture with biotin − or biotin + DCs in the presence of exogenous OT-II peptide (left), and quantification of data (right). Each dot represents one mouse (n = 3, 4 mice per group, pool of three independent experiments). (I, J) CD45.2 Cd40 G5/G5 mice that were adoptively transferred with 1 × 10 6 naive CD45.1 CD4 + Cd40lg SrtA/Y OT-II T cells prior to one dose of intragastric OVA. Analyses were carried out 24 h later. ( I ) Representative flow plots showing gating on resident and migratory DCs in D-gLNs (left), percentage of labeled resident and migratory DCs in D-gLNs (center), and quantification of data (right). Each dot represents one mouse (n = 3 mice per group, pool of three independent experiments). (J) Percentage of cDC1 and cDC2 out of total migratory DCs (left) and percentage of cDC1 and cDC2 out of biotin + DCs (right). Each dot represents one mouse (n = 3 mice per group, pool of three independent experiments). cDC1s were defined as Aqua − CD45.2 + CD45.1 − Lin − CD11c hi MHC-II hi CD103 + CD11b − and cDC2s were defined as Aqua − CD45.2 + CD45.1 − Lin − CD11c hi MHC-II hi CD103 +/− CD11b − . D, duodenum; J, jejunum; I, ileum; C, colon. In graphs, the height of bars indicate mean, and error bars indicate SD. P -values were calculated by one-way ANOVA in (C), two-way ANOVA in (E), (F), and (J) or unpaired t -test in (G) and (I). Statistical significance denoted as not significant (ns), *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Cd40lg SrtAv2 mice (Jackson Laboratories strain 037113) were generated in our laboratory using the Easi-CRISPR method( ).

Techniques: Labeling, In Vivo, Derivative Assay, Co-Culture Assay, In Vitro, Cell Culture

Journal: Science (New York, N.Y.)

Article Title: Identification of antigen-presenting cell-T cell interactions driving immune responses to food

doi: 10.1126/science.ado5088

Figure Lengend Snippet: (A to F) CD45.2 Cd40 G5/G5 mice were adoptively transferred with 1 × 10 6 naive CD45.1 CD4 + Cd40lg SrtA/Y OT-II T cells prior to one dose of intragastric OVA. Cell-cell interaction was revealed by LIPSTIC protocol 24 h later. Biotin − and biotin + D-gLNs DCs were single-cell sorted and subjected to scRNA-seq (263 cells were analyzed). (A) Uniform manifold approximation and projection (UMAP) plot. Cells were pooled from 7 mice from 2 independent experiments. Dotted line indicates the location of resident/migratory DC boundary. (B) Distribution of biotin − (grey) and biotin + (red) DCs. Dotted lines indicate the location of Cluster 0 and 1. (C) Proportion of cells in each transcriptional cluster among biotin − and biotin + DCs. (D) Expression of genes significantly upregulated in both biotin + cDC1 and cDC2 compared to biotin − cDC1 and cDC2. (E, F) OT-II CFSE- or CTV-labeled T cells were co-cultured in vitro with sorted biotin − DCs, biotin + cDC1s or biotin + cDC2s from D-gLNs in the presence of exogenous OT-II peptide for 96 h. Representative flow plots showing percentage of Foxp3 + or Fr4 + Th lin− cells among proliferated OT-II T cells (left), and quantification of data (right). Each dot represents one mouse (n = 3, 4 mice per group, pool of four independent experiments). (G, H) CD45.2 Clec9a +/+ H2-Ab1 fl/fl or Clec9a cre H2-Ab1 fl/fl mice; or bone marrow chimera (BMC) mice reconstituted with C57BL/6 (WT) or Δ1+2+3 cells were adoptively transferred with 1 × 10 6 naive CD45.1 CD4 + OT-II T cells. Mice received two doses of intragastric OVA 48 h and 24 h prior analysis. Percentage of Foxp3 + or Fr4 + Th lin− cells among CD45.1 TCRV 2 + (OT-II) T cells in D-gLNs (n = 3 mice per group, pool of two or three independent experiments). (I to L) CD45.2 Rosa26 uLIPSTIC/uLIPSTIC mice were adoptively transferred with 3 × 10 6 naive CD45.1 Cd4 Cre .Rosa26 uLIPSTIC/uLIPSTIC OT-II T cells prior to one dose of intragastric OVA. Cell-cell interaction was revealed by LIPSTIC protocol at indicated time-points post oral OVA administration. (I) Representative flow plots showing percentage of labeled APCs in the D-gLNs at 4–6 h or 22–24 h after i.g. OVA. (J, K) Percentage of different APCs Biotin + cells among MHC-II + cells. (L) Percentage of Biotin + APCs 4–6h post i.g. OVA with or without MHC-II blocking. (n = 3, 4 mice per group, pool of two or three independent experiments). cDC1s were defined as Aqua − CD45.2 + CD45.1 − Lin − CD11c hi MHC-II hi CD103 + CD11b − and cDC2s were defined as Aqua − CD45.2 + CD45.1 − Lin − CD11c hi MHC-II hi CD103 +/− CD11b − . In all graphs, the height of bars indicate mean, and error bars indicate SD. Wilcoxon signed-rank test was used for (D). P -values were calculated by one-way ANOVA in (E) and (F), or by unpaired t -test in (G) and (H). Statistical significance denoted as not significant (ns), ***P < 0.001. N.d = not detected.

Article Snippet: Cd40lg SrtAv2 mice (Jackson Laboratories strain 037113) were generated in our laboratory using the Easi-CRISPR method( ).

Techniques: Expressing, Labeling, Cell Culture, In Vitro, Blocking Assay

Journal: Science (New York, N.Y.)

Article Title: Identification of antigen-presenting cell-T cell interactions driving immune responses to food

doi: 10.1126/science.ado5088

Figure Lengend Snippet: (A to C) CD45.2 C57BL/6 mice were infected with S. venezuelensis (S.v.) or H. polygyrus (H.p.) 5 days prior adoptively transfer of 1 × 10 6 naive CD45.1 CD4 + OT-II T cells. Mice received two doses of intragastric OVA 48 h and 24 h prior analysis. Non-infected mice (NI) were used as control. (A) Experimental setup for panel (B, C). Percentage of (B) GATA3 + cells among CD45.2 TCRβ + CD4 + T cells and (C) Foxp3 + cells among CD45.1 TCRVα2 + (OT-II) T cells in gLNs. (n = 3 mice per group, pool of two independent experiments). (D) Experimental setup for panel (E-G). (E) Anaphylaxis as measured by survival of mice at the indicated times after intraperitoneal OVA injection (challenge), following four weekly doses of OVA+cholera toxin (CT). (F) OVA-specific IgG1 or (G) OVA-specific IgE levels in serum as measured by ELISA (n = 3, 4 mice per group, pool of three independent experiments). (H to J) CD45.2 Cd40 G5/G5 mice were adoptively transferred with 1 × 10 6 naive CD45.1 CD4 + Cd40lg SrtA/Y OT-II T cells prior to one dose of intragastric OVA. Cell-cell interaction was revealed by LIPSTIC protocol 24 h later. Non-infected mice (NI) were used as control. Percentage of cDC1 and cDC2 out of total migratory DCs (left bar) and percentage of cDC1 and cDC2 out of biotin + DCs (right bar) of NI, H.p - or S.v -infected mice. (K) CD45.2 Rosa26 uLIPSTIC/uLIPSTIC mice were infected with S.v. 5 days prior adoptively transfer of 3 × 10 6 naive CD45.1 Cd4 Cre .Rosa26 uLIPSTIC/uLIPSTIC OT-II T cells. Mice received one dose of intragastric OVA and cell-cell interaction was revealed by LIPSTIC protocol 4 h later. (K) Percentage of Biotin + APCs among MHC-II + cells. (L to O) Sorted biotin − or biotin + DCs from D-gLNs of non-infected mice (NI) or mice infected with H.p (left) or S.v (right) were co-culture with OT-II CFSE-labeled T cells (L, M) or TN CFSE-labeled T cells (N, O) in vitro for 96 h. (L, N) Representative flow plots showing percentage of Foxp3 + and GATA3 + cells among proliferated T cells co-cultured with biotin − or biotin + DCs from NI, H.p (left) or S.v (right) infected mice and (M, O) quantification of data. OT-II or TN peptide were added to the corresponding co-cultures wells. Each dot represents one mouse (n = 3, 4 mice per group, pool of three independent experiments). cDC1s were defined as Aqua − CD45.2 + CD45.1 − Lin − CD11c hi MHC-II hi CD103 + CD11b − and cDC2s were defined as Aqua − CD45.2 + CD45.1 − Lin − CD11c hi MHC-II hi CD103 +/− CD11b − . Height of bars indicate mean, and error bars indicate SD. P -values were calculated by two-way ANOVA in (B), (C), (F), (G), (M) and (O). Statistical significance denoted as not significant (ns), *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Cd40lg SrtAv2 mice (Jackson Laboratories strain 037113) were generated in our laboratory using the Easi-CRISPR method( ).

Techniques: Infection, Control, Injection, Enzyme-linked Immunosorbent Assay, Co-Culture Assay, Labeling, In Vitro, Cell Culture

Journal: Science (New York, N.Y.)

Article Title: Identification of antigen-presenting cell-T cell interactions driving immune responses to food

doi: 10.1126/science.ado5088

Figure Lengend Snippet: CD45.2 Cd40 G5/G5 mice were infected with S. venezuelensis (S.v.) or H. polygyrus (H.p.) 5 days prior adoptive transfer of 1 × 10 6 naive CD45.1 CD4 + Cd40lg SrtA/Y OT-II T cells. Animals received 1 dose of intragastric OVA 18 h after OT-II T cells transfer. Cell-cell interaction was revealed by LIPSTIC protocol 24 h after OVA administration. Non-infected (NI) mice were used as control. (A) Biotin − _and biotin + D-gLNs DCs were single-cell sorted and subjected to scRNA-seq (534 cells were analyzed). UMAP plot showing clustering of sequenced DCs of NI mice or mice infected with H.p or S.v . Cells were pooled from 4–7 mice from 2 independent experiments. Distribution of biotin − (grey) and biotin + (red) DCs in the same plot. Dotted lines indicate the location of Cluster 0. (B) Proportion of cells in each transcriptional cluster among biotin − and biotin + DCs. (C) Dot plot showing expression of genes differentially expressed between Clusters. (D) Ratio of cDC1/cDC2 among biotin + DCs from D-gLNs of NI mice or mice infected with H.p or S.v. (E) Relative frequency (left) and absolute numbers (right) of migratory cDC1 and cDC2 in D-gLNs of naïve C57BL/6 mice or mice infected with S.v. (F) Percentage of Foxp3 + cells among proliferated OT-II CFSE-labeled T cells in vitro after 96 h of co-culture with D-gLN NI or S.v biotin − or biotin + DCs or a combination of NI and S.v DCs (1:1 ratio). (G) Percentage of Foxp3 + cells among proliferated OT-II CFSE-labeled T cells in vitro after 96 h of co-culture with D-gLN NI or S.v . biotin + cDC1 and cDC2 at indicated ratios. OT-II peptide was added to the co-cultures. Each dot represents one mouse (n = 3 to 5 mice per group, representative of two independent experiments). (H and I) Bone marrow chimera (BMC) mice reconstituted with C57BL/6 (WT) or Δ1+2+3 cells. Mice were infected with S.v. 5 days prior adoptively transfer of 1 × 10 6 naive CD45.1 CD4 + OT-II T cells. Mice received two doses of intragastric OVA 48 h and 24 h prior analysis. (H) Experimental set up for panel (I). (I) Percentage of Foxp3 + cells among CD45.1 TCRVα2 + (OT-II) T cells (right) and percentage of GATA3 + cells among CD45.2 TCRβ + CD4 + T cells (left) in D-gLNs (n = 3 mice per group, pool of two independent experiments). (J) Experimental setup for panel (K to M). (K) Anaphylaxis as measured by survival of mice at the indicated times after intraperitoneal OVA injection (challenge), following four weekly doses of OVA+cholera toxin (CT). (L) OVA-specific IgG1 or (M) OVA-specific IgE levels in serum as measured by ELISA (n = 3, 4 mice per group, pool of three independent experiments). cDC1s were defined as Aqua − CD45.2 + CD45.1 − Lin − CD11c hi MHC-II hi CD103 + CD11b − and cDC2s were defined as Aqua − CD45.2 + CD45.1 − Lin − CD11c hi MHC-II hi CD103 +/− CD11b − . In graphs, the height of bars indicate mean, and error bars indicate SD. Wilcoxon signed-rank test was used for (C). P -values were calculated by one-way ANOVA in (D), or by two-way ANOVA in (F), (G), (L) and (M), and by unpaired t -test in (I). Statistical significance denoted as not significant (ns), *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Cd40lg SrtAv2 mice (Jackson Laboratories strain 037113) were generated in our laboratory using the Easi-CRISPR method( ).

Techniques: Infection, Adoptive Transfer Assay, Control, Expressing, Labeling, In Vitro, Co-Culture Assay, Injection, Enzyme-linked Immunosorbent Assay