Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Persistent Herpes Simplex Virus Type 1 Infection of Enteric Neurons Triggers CD8 + T Cell Response and Gastrointestinal Neuromuscular Dysfunction

doi: 10.3389/fcimb.2021.615350

Figure Lengend Snippet: Antibodies used in the study.

Article Snippet: CD4 (rabbit) Cy7 conjugate , 50134-R001 , Sino Biological Inc , FC.

Techniques:

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Persistent Herpes Simplex Virus Type 1 Infection of Enteric Neurons Triggers CD8 + T Cell Response and Gastrointestinal Neuromuscular Dysfunction

doi: 10.3389/fcimb.2021.615350

Figure Lengend Snippet: CD3 + cells infiltrate the LMMP following HSV-1 infection. (A) LMMP preparations were obtained from the ileum of sham- and HSV-1-infected mice as described in Methods. The samples were enzymatically digested, and the resulting cell suspensions were labeled with anti-CD11c and anti-F4/80 antibodies and analyzed using flow cytometry. Cells were first selected on a forward scatter and side scatter dot plot and then double positive cells were recorded. The data are reported as the number of CD11c + F4/80 + cells detected in 10 5 events. The experiments were repeated 2 times; n=4 mice per group. (B) Cell suspension obtained as described in (A) were labeled with anti-CD3 antibody and analyzed by flow cytometry. Data are reported as the percentage of CD3 + cells in 10 5 events on side scatter dot plot. The experiments were repeated 2 times; n=4 mice per group. * denotes p < 0.05 vs sham infected mice. (C) Sections of ileum obtained from the sham or HSV-1 infected mice were subjected to immunohistochemistry using anti-CD3 antibody. Scale bars: 50 μm. Representative images of four separate experiments; n=3 mice per group. (D) Cell suspensions obtained from the LMMP, as described in (A) , were labeled with anti-CD3 and anti-CD4 antibodies (E) or with anti-CD3 and anti-CD8 antibodies (F) or with anti-CD3 and anti-CD69 antibodies. The samples were analyzed using flow cytometry. Cells were first selected on a forward scatter and side scatter dot plot and then double positive cells were recorded in 10,000 events. Data are reported as the percentage of double positive cells. Experiments were repeated 3 times n=4 mice per group. * denotes p < 0.05 vs sham infected mice.

Article Snippet: CD4 (rabbit) Cy7 conjugate , 50134-R001 , Sino Biological Inc , FC.

Techniques: Infection, Labeling, Flow Cytometry, Immunohistochemistry

Journal: Nature Communications

Article Title: Molecular and functional heterogeneity of IL-10-producing CD4 + T cells

doi: 10.1038/s41467-018-07581-4

Figure Lengend Snippet: Mouse IL-10-producing CD4 + T cells are heterogeneous. a – c IL-10-producing Foxp3 neg CD4 + T cells (IL-10 pos ) were isolated from small intestine or spleen of aCD3-treated IL-10 eGFP Foxp3 mRFP double reporter mice. Cells were transferred into lymphopenic hosts and colitis development was assessed by weight loss ( c ) and endoscopic colitis score ( b ) 5 weeks upon transfer (IL-10 pos cells small intestine n = 12; IL-10 pos cells Spleen n = 11; lines indicate mean ±SEM). Results are cumulative of three independent experiments. A Mann–Whitney U test was used to calculate significance. d t- SNE analysis of single cell RNA sequencing of IL-10 pos cells (including Foxp3 + cells) isolated from small intestine or spleen of aCD3-treated IL-10 eGFP Foxp3 mRFP double reporter mice. e Bootstrap analysis of HVGs in single cell RNA sequencing data of IL-10 pos cells from small intestine and spleen. f Expression of indicated genes in t- SNE analysis

Article Snippet: Panels for FACS analysis mouse: TCRbeta (BV421, BioLegend, clone H57-597 Dilution 1:200, lot B209221), CD4 (BUV737, BD Bioscience, clone GK1.5, Dilution 1:800, lot 5100736/PE/Cy7, BioLegend, clone RM4-5, Dilution 1:400, lot B19204), CD49b (PE, BioLegend, clone Hma2, Dilution 1:100, lot B148368; FITC, BioLegend, clone Hma2, Dilution 1:100, lot B178538), LAG-3 (APC, BioLegend, clone C9B7W, Dilution 1:100, lot B163789), TIM-3 (Biotin, eBioscience, clone 8B.2C12, Dilution 1:200, lot E02985-1632), Strepdavidin (BUV395, BD Bioscience, clone NA, Dilution 1:200), TIGIT (PerCPCy5.5, eBioscience, clone GIGD7, Dilution 1:200, lot E15928-104), PD-1 (BV605, BioLegend, clone 29 F.1A12, Dilution 1:300, lot B227578), CCR5 (PECy7, BioLegend, clone HM-CCR5, Dilution 1:200, lot B220574), CD45.1 (Pacific Blue, Biolegend, clone A20, Dilution 1:400, lot B191340), CD45.2 (PECy7, BioLegend, clone 104, Dilution 1:400, lot B185406), and IL-10 (PE-TexasRed, BioLegend, clone JES5-16E3, Dilution 1:100, lot B243511).

Techniques: Isolation, MANN-WHITNEY, RNA Sequencing Assay, Expressing

Journal: Nature Communications

Article Title: Molecular and functional heterogeneity of IL-10-producing CD4 + T cells

doi: 10.1038/s41467-018-07581-4

Figure Lengend Snippet: Splenic IL-10-producing CD4 + T cells contain a regulatory cluster. a Spearman correlation coefficient between each cluster from small intestine and spleen. b Fold change analysis of genes enriched in indicated clusters compared to other clusters that were amongst the 100 most enriched genes. c Expression of indicated genes encoding cytokines and chemokines. d Expression of indicated genes encoding transcription factors. e Expression of indicated genes encoding other factors. f Expression of indicated genes encoding surface receptors

Article Snippet: Panels for FACS analysis mouse: TCRbeta (BV421, BioLegend, clone H57-597 Dilution 1:200, lot B209221), CD4 (BUV737, BD Bioscience, clone GK1.5, Dilution 1:800, lot 5100736/PE/Cy7, BioLegend, clone RM4-5, Dilution 1:400, lot B19204), CD49b (PE, BioLegend, clone Hma2, Dilution 1:100, lot B148368; FITC, BioLegend, clone Hma2, Dilution 1:100, lot B178538), LAG-3 (APC, BioLegend, clone C9B7W, Dilution 1:100, lot B163789), TIM-3 (Biotin, eBioscience, clone 8B.2C12, Dilution 1:200, lot E02985-1632), Strepdavidin (BUV395, BD Bioscience, clone NA, Dilution 1:200), TIGIT (PerCPCy5.5, eBioscience, clone GIGD7, Dilution 1:200, lot E15928-104), PD-1 (BV605, BioLegend, clone 29 F.1A12, Dilution 1:300, lot B227578), CCR5 (PECy7, BioLegend, clone HM-CCR5, Dilution 1:200, lot B220574), CD45.1 (Pacific Blue, Biolegend, clone A20, Dilution 1:400, lot B191340), CD45.2 (PECy7, BioLegend, clone 104, Dilution 1:400, lot B185406), and IL-10 (PE-TexasRed, BioLegend, clone JES5-16E3, Dilution 1:100, lot B243511).

Techniques: Expressing

Journal: Nature Communications

Article Title: Molecular and functional heterogeneity of IL-10-producing CD4 + T cells

doi: 10.1038/s41467-018-07581-4

Figure Lengend Snippet: CIR identify regulatory IL-10-producing CD4 + T cells. vi SNE analysis of IL-10 pos Foxp3 neg CD4 + T cells. Clustering is based on MFI of PD-1, LAG-3, TIGIT, TIM-3, CD49b, and CCR5. Blue circle indicates co-inhibitory receptor rich (CIR rich) region. a Analysis of cells from small intestine, spleen, and lung of aCD3-treated IL-10 eGFP Foxp3 mRFP double reporter mice ( n = 4). Data are representative of two independent experiments. b Analysis of untreated IL-10 eGFP Foxp3 mRFP double reporter mice ( n = 3). Data are representative of three independent experiments. c Analysis of P. berghei infected IL-10 eGFP Foxp3 mRFP double reporter mice ( n = 3). Data are representative of two independent experiments

Article Snippet: Panels for FACS analysis mouse: TCRbeta (BV421, BioLegend, clone H57-597 Dilution 1:200, lot B209221), CD4 (BUV737, BD Bioscience, clone GK1.5, Dilution 1:800, lot 5100736/PE/Cy7, BioLegend, clone RM4-5, Dilution 1:400, lot B19204), CD49b (PE, BioLegend, clone Hma2, Dilution 1:100, lot B148368; FITC, BioLegend, clone Hma2, Dilution 1:100, lot B178538), LAG-3 (APC, BioLegend, clone C9B7W, Dilution 1:100, lot B163789), TIM-3 (Biotin, eBioscience, clone 8B.2C12, Dilution 1:200, lot E02985-1632), Strepdavidin (BUV395, BD Bioscience, clone NA, Dilution 1:200), TIGIT (PerCPCy5.5, eBioscience, clone GIGD7, Dilution 1:200, lot E15928-104), PD-1 (BV605, BioLegend, clone 29 F.1A12, Dilution 1:300, lot B227578), CCR5 (PECy7, BioLegend, clone HM-CCR5, Dilution 1:200, lot B220574), CD45.1 (Pacific Blue, Biolegend, clone A20, Dilution 1:400, lot B191340), CD45.2 (PECy7, BioLegend, clone 104, Dilution 1:400, lot B185406), and IL-10 (PE-TexasRed, BioLegend, clone JES5-16E3, Dilution 1:100, lot B243511).

Techniques: Infection

Journal: Nature Communications

Article Title: Molecular and functional heterogeneity of IL-10-producing CD4 + T cells

doi: 10.1038/s41467-018-07581-4

Figure Lengend Snippet: CIR rich CD4 + T cells have a high suppressive capacity. a In vitro suppression of co-inhibitory receptor rich (CIR rich) and co-inhibitory receptor poor (CIR poor) IL-10 pos Foxp3 neg CD4 + T cells isolated from spleen of aCD3-treated IL-10 eGFP Foxp3 mRFP double reporter mice. Representative histograms of five independent experiments, a paired T- test was used to calculate significance. b In vitro suppression of CIR rich and CIR poor IL-10 pos Foxp3 neg CD4 + T cells isolated from spleen of P. berghei infected IL-10 eGFP Foxp3 mRFP double reporter mice. Representative histograms of four independent experiments. c In vitro suppression of CIR rich and CIR poor IL-10 pos Foxp3 neg CD4 + T cells isolated from spleen of aCD3-treated IL-10 eGFP Foxp3 mRFP reporter mice. TIM-3 and LAG-3 were blocked using blocking antibodies. To block IL-10 receptor signaling responder T cells were isolated from IL-10R dominant negative mice (DN IL-10R Responder). Results are cumulative of three independent experiments. d CIR rich and CIR poor IL-10 pos Foxp3 neg CD4 + T cells and IL-10 neg CD4 + T cells were isolated from spleen of aCD3-treated IL-10 eGFP Foxp3 mRFP double reporter mice. Cells were transferred into lymphopenic hosts and colitis development was assessed by weight loss and endoscopic colitis score 5 weeks upon transfer (IL-10 neg n = 6; CIR poor n = 9; CIR rich n = 6; lines indicate mean±SEM). Results are cumulative of three independent experiments. One-way ANOVA (post-test Tukey) was used to calculate significance (* p < 0.05). e CIR rich IL-10-producing Foxp3 neg CD4 + T cells, IL-10 neg CD4 + T cells and Foxp3 + Treg cells were isolated from spleen of aCD3-treated IL-10 eGFP Foxp3 mRFP double reporter mice. IL-10 pos CIR rich or Foxp3 + Treg cells were co-transferred with IL-10 neg CD4 + T cells and colitis development was assessed by weight loss and endoscopic colitis score 5 weeks upon transfer (IL-10 neg n = 7; IL-10 neg + CIR rich n = 6; IL-10 neg + Foxp3 + Treg n = 6; lines indicate mean±SEM). Results are cumulative of three independent experiments. One-way ANOVA (post-test Tukey) was used to calculate significance (* p < 0.05)

Article Snippet: Panels for FACS analysis mouse: TCRbeta (BV421, BioLegend, clone H57-597 Dilution 1:200, lot B209221), CD4 (BUV737, BD Bioscience, clone GK1.5, Dilution 1:800, lot 5100736/PE/Cy7, BioLegend, clone RM4-5, Dilution 1:400, lot B19204), CD49b (PE, BioLegend, clone Hma2, Dilution 1:100, lot B148368; FITC, BioLegend, clone Hma2, Dilution 1:100, lot B178538), LAG-3 (APC, BioLegend, clone C9B7W, Dilution 1:100, lot B163789), TIM-3 (Biotin, eBioscience, clone 8B.2C12, Dilution 1:200, lot E02985-1632), Strepdavidin (BUV395, BD Bioscience, clone NA, Dilution 1:200), TIGIT (PerCPCy5.5, eBioscience, clone GIGD7, Dilution 1:200, lot E15928-104), PD-1 (BV605, BioLegend, clone 29 F.1A12, Dilution 1:300, lot B227578), CCR5 (PECy7, BioLegend, clone HM-CCR5, Dilution 1:200, lot B220574), CD45.1 (Pacific Blue, Biolegend, clone A20, Dilution 1:400, lot B191340), CD45.2 (PECy7, BioLegend, clone 104, Dilution 1:400, lot B185406), and IL-10 (PE-TexasRed, BioLegend, clone JES5-16E3, Dilution 1:100, lot B243511).

Techniques: In Vitro, Isolation, Infection, Blocking Assay, Dominant Negative Mutation

Journal: Nature Communications

Article Title: Molecular and functional heterogeneity of IL-10-producing CD4 + T cells

doi: 10.1038/s41467-018-07581-4

Figure Lengend Snippet: CIR rich CD4 + T cells have a distinct transcriptional program. a Volcano plots of bulk RNA sequencing data of indicated populations (IL-10 pos CIR rich n = 2; IL-10 pos CIR poor n = 2; IL-10 neg n = 2). b Expression of known T R 1 signature genes comparing IL-10 pos CIR rich with IL-10 neg and IL-10 pos CIR rich with IL-10 pos CIR poor. c Differentially expressed transcription factors between IL-10 pos CIR rich and IL-10 pos CIR poor. mRNA expression of indicated genes normalized to Hprt from at least three independent experiments (separated low-high, line represents median). d CD4 + T cells isolated from Lxrα -/- CD45.2 and wildtype CD45.2 mice were co-transferred with wildtype CD4 + T cells (CD45.1/2) into lymphopenic hosts. Animals (WT:KO n = 7; WT:WT n = 6) were treated with aCD3 antibodies 5 weeks upon transfer and cells were isolated from small intestine. Results are cumulative of two independent experiments. A Wilcoxon test was used to calculate significance. e Correlation between genes significantly higher expressed in IL-10 pos CIR rich versus IL-10 pos CIR poor cells and expression pattern of scRNA-seq data

Article Snippet: Panels for FACS analysis mouse: TCRbeta (BV421, BioLegend, clone H57-597 Dilution 1:200, lot B209221), CD4 (BUV737, BD Bioscience, clone GK1.5, Dilution 1:800, lot 5100736/PE/Cy7, BioLegend, clone RM4-5, Dilution 1:400, lot B19204), CD49b (PE, BioLegend, clone Hma2, Dilution 1:100, lot B148368; FITC, BioLegend, clone Hma2, Dilution 1:100, lot B178538), LAG-3 (APC, BioLegend, clone C9B7W, Dilution 1:100, lot B163789), TIM-3 (Biotin, eBioscience, clone 8B.2C12, Dilution 1:200, lot E02985-1632), Strepdavidin (BUV395, BD Bioscience, clone NA, Dilution 1:200), TIGIT (PerCPCy5.5, eBioscience, clone GIGD7, Dilution 1:200, lot E15928-104), PD-1 (BV605, BioLegend, clone 29 F.1A12, Dilution 1:300, lot B227578), CCR5 (PECy7, BioLegend, clone HM-CCR5, Dilution 1:200, lot B220574), CD45.1 (Pacific Blue, Biolegend, clone A20, Dilution 1:400, lot B191340), CD45.2 (PECy7, BioLegend, clone 104, Dilution 1:400, lot B185406), and IL-10 (PE-TexasRed, BioLegend, clone JES5-16E3, Dilution 1:100, lot B243511).

Techniques: RNA Sequencing Assay, Expressing, Isolation

Journal: Nature Communications

Article Title: Molecular and functional heterogeneity of IL-10-producing CD4 + T cells

doi: 10.1038/s41467-018-07581-4

Figure Lengend Snippet: Human IL-10-producing CD4 + T cells are heterogeneous. a RNA single cell sequencing data of IL-10 pos cells (including Foxp3 + T cells) isolated from PBMCs of healthy donors ( n = 3), stimulated with SEB overnight. b vi SNE analysis of IL-10 pos CD25 low CD4 + T cells from PBMCs of healthy donors ( n = 8) and healthy colon biopsies ( n = 4), stimulated with SEB overnight. Clustering is based on MFI of PD-1, LAG-3, TIGIT, TIM-3, CD49b, and CCR5. Blue circle indicates co-inhibitory receptor rich (CIR rich) region. c In vitro suppression of CIR rich and CIR poor IL-10 pos CD25 low CD4 + T cells isolated from PBMCs of healthy donors, stimulated with SEB overnight. Representative histograms of five independent experiments, a paired T- test was used to calculate significance. d , e Analysis of IL-10 pos CD25 low CD4 + T cells ( d ) and expression of CD49b/LAG-3 within those ( e ) of colon biopsies from IBD patients: UC active n = 22 (inflamed n = 20, blue dots; non-inflamed n = 17, black dots); UC Remission n = 2, violet dots; CD active n = 9 (inflamed n = 5, blue dots; non-inflamed n = 6, black dots); CD remission n = 5, violet dots and healthy controls ( n = 18). Cells were stimulated with SEB overnight, One-way ANOVA (multiple comparisons) was used to calculate significance (** p < 0.005)

Article Snippet: Panels for FACS analysis mouse: TCRbeta (BV421, BioLegend, clone H57-597 Dilution 1:200, lot B209221), CD4 (BUV737, BD Bioscience, clone GK1.5, Dilution 1:800, lot 5100736/PE/Cy7, BioLegend, clone RM4-5, Dilution 1:400, lot B19204), CD49b (PE, BioLegend, clone Hma2, Dilution 1:100, lot B148368; FITC, BioLegend, clone Hma2, Dilution 1:100, lot B178538), LAG-3 (APC, BioLegend, clone C9B7W, Dilution 1:100, lot B163789), TIM-3 (Biotin, eBioscience, clone 8B.2C12, Dilution 1:200, lot E02985-1632), Strepdavidin (BUV395, BD Bioscience, clone NA, Dilution 1:200), TIGIT (PerCPCy5.5, eBioscience, clone GIGD7, Dilution 1:200, lot E15928-104), PD-1 (BV605, BioLegend, clone 29 F.1A12, Dilution 1:300, lot B227578), CCR5 (PECy7, BioLegend, clone HM-CCR5, Dilution 1:200, lot B220574), CD45.1 (Pacific Blue, Biolegend, clone A20, Dilution 1:400, lot B191340), CD45.2 (PECy7, BioLegend, clone 104, Dilution 1:400, lot B185406), and IL-10 (PE-TexasRed, BioLegend, clone JES5-16E3, Dilution 1:100, lot B243511).

Techniques: Sequencing, Isolation, In Vitro, Expressing