Journal: Frontiers in Immunology

Article Title: Establishment and Characterization of a Functionally Competent Type 2 Conventional Dendritic Cell Line

doi: 10.3389/fimmu.2018.01912

Figure Lengend Snippet: CD4 − MutuDC2s are derived from Batf3 −/− Mushi1 splenic tumors. (A) Schematic representation of CD4 − MutuDC2-derivation procedure. Splenocytes from Batf3 −/− Mushi1 (B −/− M) splenic tumors were isolated and seeded in serial two-fold dilutions in 24-well plates. The adherent cells were maintained in 24-well plates for 5–10 passages until they became accustomed to culture conditions. The cultures were progressively expanded into plates and flasks with larger growth surfaces and frozen or used for experiments. (B) Light microscopy image of CD4 − MutuDC2s. (C) Flow cytometric analysis and gating of CD4 − MutuDC2s. (D) Flow cytometric comparison of expression of the SV40LgT-associated reporter EGFP in CD4 − MutuDC2s and in MutuDC1s.

Article Snippet: Ovalbumin-specific CD8 + and CD4 + T cells were isolated from spleens and lymph nodes (brachial, inguinal and mesenteric) of OT-I and OT-II mice, respectively, and purified using the following MACS or EasySep™ kits: CD4 + T Cell Isolation Kit, mouse (130-104-454, Miltenyi Biotec), CD8a + T Cell Isolation Kit, mouse (130-104-07, Miltenyi Biotec), EasySep™ Mouse CD4+ T Cell Isolation Kit (19852, STEMCELL™ TECHNOLOGIES), EasySep™ Mouse CD8+ T Cell Isolation Kit (19853, STEMCELL™ TECHNOLOGIES).

Techniques: Derivative Assay, Isolation, Light Microscopy, Comparison, Expressing

Journal: Frontiers in Immunology

Article Title: Establishment and Characterization of a Functionally Competent Type 2 Conventional Dendritic Cell Line

doi: 10.3389/fimmu.2018.01912

Figure Lengend Snippet: CD4 − MutuDC2s share the surface and intracellular marker expression profile with CD4 − spl-cDC2s. (A–E) Splenocytes from C57BL/6 mice were isolated by digestion of spleens with collagenase D followed by filtration through a 40 μm cell strainer. CD4 − MutuDC2s, MutuDC1s and splenocytes were stained with different antibody cocktails that always contained anti-MHC-II and anti-CD11c antibodies to distinguish spl-cDCs. Antibodies specific for either CD8α or CD11b were included in every staining cocktail to identify spl-cDC1s and spl-DC2s. The cells were analyzed by flow cytometry. (A) Gating strategy to discriminate spl-cDCs and their subsets. The dash-dotted lines show the fluorescence-minus-one (FMO) controls acquired for each marker. (B) CD4 − MutuDC2s and spl-cDC subsets were compared for the expression of the indicated surface markers. The gating strategies applied to analyze CD11c and MHC-II expression in the different spl-cDC subsets are detailed in Supplementary Figure . The dash-dotted lines show the FMO controls acquired for each marker. (C) CD4 − MutuDC2s and spl-cDCs were compared for the expression of the spl-cDC subset-specific transcription factors IRF4 and IRF8. FMO controls: CD4 − MutuDC2s (dash-dotted line), spl-DC1s (dotted line), spl-DC2s (thin solid line). (D) CD4 − MutuDC2s and spl-cDC subsets were compared for the expression of the indicated spl-cDC subset-specific surface markers. The dash-dotted lines show the FMO controls acquired for each marker. (E) CD4 − MutuDC2s and spl-cDC subsets were compared for the expression of the indicated moDC or spl-cDC characterizing surface markers. The dash-dotted lines show the FMO controls acquired for each marker. (F) CD4 − MutuDC2s and MutuDC1s were compared for the expression of the indicated spl-cDC1-specific surface markers. The dash-dotted lines show the FMO controls acquired for each marker. (G) Intracellular analysis of FLT3 expression in CD4 − MutuDCs. The dash-dotted line shows the background fluorescence of unstained cells. All the results are representative of two to six independent experiments.

Article Snippet: Ovalbumin-specific CD8 + and CD4 + T cells were isolated from spleens and lymph nodes (brachial, inguinal and mesenteric) of OT-I and OT-II mice, respectively, and purified using the following MACS or EasySep™ kits: CD4 + T Cell Isolation Kit, mouse (130-104-454, Miltenyi Biotec), CD8a + T Cell Isolation Kit, mouse (130-104-07, Miltenyi Biotec), EasySep™ Mouse CD4+ T Cell Isolation Kit (19852, STEMCELL™ TECHNOLOGIES), EasySep™ Mouse CD8+ T Cell Isolation Kit (19853, STEMCELL™ TECHNOLOGIES).

Techniques: Marker, Expressing, Isolation, Filtration, Staining, Flow Cytometry, Fluorescence

Journal: Frontiers in Immunology

Article Title: Establishment and Characterization of a Functionally Competent Type 2 Conventional Dendritic Cell Line

doi: 10.3389/fimmu.2018.01912

Figure Lengend Snippet: CD4 − MutuDC2s respond weakly to TLR3 and TLR5 ligands and have low TLR3 expression but high levels of TLR5. (A) CD4 − MutuDC2s were stimulated with specific TLR ligands: TLR1/2 ligand Pam3CSK4, TLR3 ligand poly(I:C), TLR4 ligand LPS, TLR5 ligand flagellin, TLR2/6 ligand FSL-1, TLR7 ligand Gardiquimod, TLR9 ligand CpG ODN. After 24 h, the supernatants were collected and analyzed by ELISA to determine the concentration of IL-6, IL-12/IL-23 p40, and MCP-1(CCL2). The graphs show the results of five independent experiments represented as box-and-whiskers plots: mean (+), median (horizontal line), interquartile range (box), min/max values (whiskers). To assess significance, every tested condition was compared with the untreated control by Kruskal-Wallis testing followed by uncorrected Dunn's testing (* p < 0.05, ** p < 0.01, *** p < 0.001, NS, not significant; ND, not detectable). For the statistical analysis, all the measures below the lower detection limit of the assay were replaced with the value of the detection limit. (B) Expression of TLR3 and TLR5 was measured by RT-qPCR in CD4 − MutuDC2s compared with MutuDC1s. Fold expression in CD4 − MutuDC2s relative to MutuDC1s was calculated with the 2 −ΔΔ Ct method using β-actin expression as a reference for normalization. Data are presented as mean and SD of the log 2 fold expression values from two independent experiments. The results were analyzed by two-tailed unpaired t -testing (* p < 0.05). (C) Flow cytometric analysis of TLR5 expression in CD4 − MutuDC2s compared with spl-cDC1s and spl-cDC2s. The isolation of splenocytes and the staining were carried out as described in Figure . The dash-dotted lines show the FMO controls not stained with anti-TLR5 antibody. The results are representative of two independent experiments.

Article Snippet: Ovalbumin-specific CD8 + and CD4 + T cells were isolated from spleens and lymph nodes (brachial, inguinal and mesenteric) of OT-I and OT-II mice, respectively, and purified using the following MACS or EasySep™ kits: CD4 + T Cell Isolation Kit, mouse (130-104-454, Miltenyi Biotec), CD8a + T Cell Isolation Kit, mouse (130-104-07, Miltenyi Biotec), EasySep™ Mouse CD4+ T Cell Isolation Kit (19852, STEMCELL™ TECHNOLOGIES), EasySep™ Mouse CD8+ T Cell Isolation Kit (19853, STEMCELL™ TECHNOLOGIES).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Concentration Assay, Quantitative RT-PCR, Two Tailed Test, Isolation, Staining

Journal: Frontiers in Immunology

Article Title: Establishment and Characterization of a Functionally Competent Type 2 Conventional Dendritic Cell Line

doi: 10.3389/fimmu.2018.01912

Figure Lengend Snippet: CD4 − MutuDC2s upregulate co-stimulatory molecules upon activation and display MHC-I-mediated and MHC-II-mediated activation of T cells but fail to cross-present antigens through MHC-I. (A) CD4 − MutuDC2s were incubated with medium or with CpG ODN. After 20 h they were stained with fluorescent-labeled antibodies specific for MHC-II, CD80, CD86, or CD40 and analyzed by flow cytometry. The graphs relative to untreated CD4 − MutuDC2s are the same shown in Figure . (B,C) CD4 − MutuDC2s or MutuDC1s were pulsed, in the presence of CpG ODN, with full length ovalbumin (OVA FL ) or with the MHC-I-restricted peptide OVA 257−264 or with the MHC-II-restricted peptide OVA 323−339 at the indicated concentrations. OVA-specific CD8 + or CD4 + T cells were isolated respectively from OT-I and OT-II mice and labeled with a proliferation dye. In the MHC-I T cell activation assay ( C , top left), the CD8 + T cells were co-cultured for 3 days with OVA 257−264 -pulsed CD4 − MutuDC2s. In the MHC-I antigen cross-presentation assay ( C , top right), the CD8 + T cells were co-cultured for 3 days with either OVA FL -pulsed CD4 − MutuDC2s or OVA FL -pulsed MutuDC1s. In the MHC-II T cell activation assay ( C , bottom), the CD4 + T cells were co-cultured for 4 days with either OVA 323−339 -pulsed or OVA FL -pulsed CD4 − MutuDC2s. At the end of the co-cultures, the T cells were restimulated with PMA and ionomycin in the presence of brefeldin A and prepared for flow cytometric analysis by staining them with fluorescent-labeled antibodies specific for IFNγ and either CD4 or CD8α. (B) Gating strategy. The gate P1 contains the activated T cells defined as proliferating IFNγ + cells. (C) The percentage of T cells in the gate P1 measured in each tested condition was plotted against the respective antigen concentration. The results are presented as mean and SD of technical triplicates and are representative of two to three independent experiments.

Article Snippet: Ovalbumin-specific CD8 + and CD4 + T cells were isolated from spleens and lymph nodes (brachial, inguinal and mesenteric) of OT-I and OT-II mice, respectively, and purified using the following MACS or EasySep™ kits: CD4 + T Cell Isolation Kit, mouse (130-104-454, Miltenyi Biotec), CD8a + T Cell Isolation Kit, mouse (130-104-07, Miltenyi Biotec), EasySep™ Mouse CD4+ T Cell Isolation Kit (19852, STEMCELL™ TECHNOLOGIES), EasySep™ Mouse CD8+ T Cell Isolation Kit (19853, STEMCELL™ TECHNOLOGIES).

Techniques: Activation Assay, Incubation, Staining, Labeling, Flow Cytometry, Isolation, Cell Culture, Concentration Assay

Journal: PLoS Neglected Tropical Diseases

Article Title: Leishmania infantum Amastigotes Trigger a Subpopulation of Human B Cells with an Immunoregulatory Phenotype

doi: 10.1371/journal.pntd.0003543

Figure Lengend Snippet: Purified human CD25 - CD4 + T cells were either left untreated (resting) or activated with plate-bound anti-CD3 and anti-CD28 antibodies. For activated cells, samples were either left untreated (no supernatant), incubated with cell-free supernatants from untreated B cells (control), or incubated with cell-free supernatants from B cells exposed to L . infantum amastigotes at a final parasite:host cell ratio of 3:1 (AMA). Seventy-two hours later, cells were analysed for cell surface expression of CD25 and CD69 by flow cytometry. Dead cells were excluded by 7-AAD staining. A) A representative dot plot is shown for each tested condition. B) Percentages of CD25 + CD69 + Tcells for studies performed with CD25 - CD4 + T cells from 3 different donors incubated with cell-free supernatants from 5 to 7 different B-cell donors. Statistical significance was evaluated by two-tailed Student’s t-test.

Article Snippet: Primary human CD4 + T cells were isolated from PBMCs using a magnetic Easysep CD4 + T-cell enrichment kit (Stemcell Technologies) and CD25 + T cells (both activated CD4 + T cells and Tregs) were depleted with a CD25 positive selection kit (Stemcell Technologies).

Techniques: Purification, Incubation, Control, Expressing, Flow Cytometry, Staining, Two Tailed Test

Journal: PLoS Neglected Tropical Diseases

Article Title: Leishmania infantum Amastigotes Trigger a Subpopulation of Human B Cells with an Immunoregulatory Phenotype

doi: 10.1371/journal.pntd.0003543

Figure Lengend Snippet: Purified human CD25 - CD4 + T cells were first labeled with CFSE and treated as described in . After 5 days, cell proliferation was assessed by CFSE dilution and dead cells were excluded by 7-AAD staining. A) A representative histogram is shown for each tested condition. B) Proliferation index for experiments performed with CD25 - CD4 + T cells from 2 different donors incubated with cell-free supernatants from 6 or 7 different B-cell donors. Statistical significance was evaluated by two-tailed Student’s t-test.

Article Snippet: Primary human CD4 + T cells were isolated from PBMCs using a magnetic Easysep CD4 + T-cell enrichment kit (Stemcell Technologies) and CD25 + T cells (both activated CD4 + T cells and Tregs) were depleted with a CD25 positive selection kit (Stemcell Technologies).

Techniques: Purification, Labeling, Staining, Incubation, Two Tailed Test

Journal: PLoS Neglected Tropical Diseases

Article Title: Leishmania infantum Amastigotes Trigger a Subpopulation of Human B Cells with an Immunoregulatory Phenotype

doi: 10.1371/journal.pntd.0003543

Figure Lengend Snippet: Purified human CD25 - CD4 + T cells were activated for 72 h with plate-bound anti-CD3 and anti-CD28 antibodies in the presence of cell-free supernatants from B cells either left untreated (control) or incubated overnight with L . infantum amastigotes at a final parasite:host cell ratio of 3:1 (AMA). In some cases, cell-free supernatants from B cells incubated with parasites were treated with a soluble IL-10 receptor (1 μg/ml) before being used with activated CD25 - CD4 + T cells. During the last 5 h of incubation, cells were further stimulated with PMA (50 ng/ml) and ionomycin (1 μg/ml) and Golgiplug™ was added (1 μl per 1 x 10 6 cells). Finally, cells were fixed, permeabilized and stained for intracellular TNF before being analysed by flow cytometry. Dead cells were excluded by 7-AAD staining. Results are expressed as the percentages of TNF + cells multiply by the mean fluorescence intensities (MFI). Data shown are the results from 4 different CD25 - CD4 + T-cell donors incubated with cell-free supernatants from 4 different B-cell donors. Statistical significance was evaluated by two-tailed Student’s t-test.

Article Snippet: Primary human CD4 + T cells were isolated from PBMCs using a magnetic Easysep CD4 + T-cell enrichment kit (Stemcell Technologies) and CD25 + T cells (both activated CD4 + T cells and Tregs) were depleted with a CD25 positive selection kit (Stemcell Technologies).

Techniques: Purification, Control, Incubation, Staining, Flow Cytometry, Fluorescence, Two Tailed Test

Journal: PLoS Neglected Tropical Diseases

Article Title: Leishmania infantum Amastigotes Trigger a Subpopulation of Human B Cells with an Immunoregulatory Phenotype

doi: 10.1371/journal.pntd.0003543

Figure Lengend Snippet: Purified human CD25 - CD4 + T cells were activated for 72 h with plate-bound anti-CD3 and anti-CD28 antibodies in the presence of cell-free supernatants from B cells either left untreated (control) or incubated overnight with L . infantum amastigotes at a final parasite:host cell ratio of 3:1 (AMA). In some cases, cell-free supernatants from B cells incubated with parasites were treated with a soluble IL-10 receptor (1 μg/ml) before being used with activated CD25 - CD4 + T cells. During the last 5 h of incubation, cells were further stimulated with PMA (50 ng/ml) and ionomycin (1 μg/ml) and Golgiplug™ was added (1 μl per 1 x 10 6 cells). Finally, cells were fixed, permeabilized and stained for intracellular IFNγ before being analysed by flow cytometry. Dead cells were excluded by 7-AAD staining. Results are expressed as the percentages of TNF-positive cells multiply by the mean fluorescence intensities (MFI). Data shown are the results from 3 different CD25 - CD4 + T-cell donors incubated with cell-free supernatants from 3 different B-cell donors. Statistical significance was evaluated by two-tailed Student’s t-test.

Article Snippet: Primary human CD4 + T cells were isolated from PBMCs using a magnetic Easysep CD4 + T-cell enrichment kit (Stemcell Technologies) and CD25 + T cells (both activated CD4 + T cells and Tregs) were depleted with a CD25 positive selection kit (Stemcell Technologies).

Techniques: Purification, Control, Incubation, Staining, Flow Cytometry, Fluorescence, Two Tailed Test