Journal: bioRxiv

Article Title: Single-cell profiling uncovers regulatory programs of pathogenic Th2 cells in allergic asthma

doi: 10.1101/2023.08.10.552772

Figure Lengend Snippet: a, Schematic of in vitro differentiation of Th2 cells (left) and pTh2 cells (right). Naïve CD4 + T cells (TCRβ + CD4 + CD62L + CD44 - ) from WT and HDAC1-cKO mice were activated with anti-CD3 and anti-CD28 in the presence of Th2-promoting conditions (IL-4, IL-2, anti-IFN-ψ, and anti-TGF-β; collectively referred as IL-4), or Th2-promoting conditions plus TSLP alone, anti-GITR antibody (DTA-1) alone, or both TSLP and DTA-1, then cultured for 5 days. On day 5, cells were restimulated with PMA and ionomycin in the presence of GolgiStop and GolgiPlug for 4 hours, and cytokine analyses were performed by flow cytometry. b-g, Flow cytometric analysis of cells differentiated under conditions indicated in a . b, Representative plots showing the expression of IL-4 and IL-13 in WT cells (top) and HDAC1-cKO cells (bottom). c, Graph shows the frequency of IL-4 and IL-13 co-expressing cells in b . d, Representative plots showing the expression of IL-5 and IL-13 in WT cells (top) and HDAC1-cKO cells (bottom). e, Graph shows the frequency of IL-5 and IL-13 co-expressing cells in d . f, Histograms depicting the expression of GATA3 in WT cells (blue) and HDAC1-cKO cells (red). Th17 cells were used as controls for gating. g, Graph shows the frequency of GATA3 expressing cells in f. Data are pooled from five independent experiments and presented as the mean ± SEM. Each symbol represents one mouse. Statistical analysis was performed using a Two-way ANOVA with Tukey’s multiple comparisons test. * P <0.05, ** P <0.01, **** P <0.0001. The schematics in a were created using BioRender. TSLP, thymic stromal lymphopoietin; GITR, glucocorticoid-induced TNFR-related protein.

Article Snippet: To investigate the induction of a pathogenic program in Th2 cells, naïve CD4 + T cells were cultured under Th2-promoting conditions together with TSLP (50 ng/ml, R&D, 555-TS-010) and agonistic antibodies for GITR (5 μg/ml, clone DTA-1, BioXCell, BE0063), OX40 (5 μg/ml, clone OX-86, BioXCell, BE0031), or 4-1BB (5 μg/ml, clone 3H3, BE0239) all from BioXCell.

Techniques: In Vitro, Cell Culture, Flow Cytometry, Expressing

Journal: bioRxiv

Article Title: Single-cell profiling uncovers regulatory programs of pathogenic Th2 cells in allergic asthma

doi: 10.1101/2023.08.10.552772

Figure Lengend Snippet: a-c, Analyses of pathogenic mediators in in vitro generated pTh2 cells. Naïve CD4 + T cells (TCRβ + CD4 + CD62L + CD44 - ) from WT and HDAC1-cKO mice were activated with anti-CD3 and anti-CD28 in the presence of Th2-promoting conditions (IL-4, IL-2, anti-IFN-ψ, and anti-TGF-β) or pTh2-promoting conditions (IL-4, IL-2, TSLP, DTA-1, anti-IFN-ψ, and anti-TGF-β) and cultured for 5 days. On day 5, we restimulated the cells with PMA and ionomycin in the presence of GolgiStop and GolgiPlug for 4 hours and performed cytokine analyses by flow cytometry. a, Histogram shows the expression of RANKL (top) and the frequency of Th cells expressing RANKL (bottom). b, Histogram shows the expression of GM-CSF (top) and the frequency of Th cells expressing GM-CSF (bottom). c, Histogram shows the expression of IL-9 (top) and the frequency of Th cells expressing IL-9 (bottom). d,e, Comparing the impact of TNFRSF members in inducing a pathogenic program in Th2 cells. We isolated naïve CD4 + T cells (TCRβ + CD4 + CD62L + CD44 - ) from WT and HDAC1-cKO mice and activated them with anti-CD3 and anti-CD28 in the presence of Th2-promoting conditions (IL-4, IL-2, anti-IFN-ψ, and anti-TGF-β collectively termed IL-4), or IL-4+TSLP alone, or IL-4+TSLP+DTA-1, or IL-4+TSLP+OX86, or IL-4+TSLP+3H3. We cultured the cells for 5 days and restimulated them with PMA and ionomycin in the presence of GolgiStop and GolgiPlug for 4 hours before cytokine analyses by flow cytometry. d, Graphs showing the frequencies of pathogenic Th2 mediators and GATA3 in WT cells (blue). e, Graphs showing the frequencies of pathogenic Th2 mediators and GATA3 in HDAC1-cKO cells (red). Data are pooled from three independent experiments and presented as the mean ± SEM. Each symbol represents one mouse. Statistical analysis was performed using a one-way ANOVA with Tukey’s multiple comparisons test. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001. TSLP, thymic stromal lymphopoietin; DTA-1, agonistic antibody against GITR (TNFRSF18); OX86, agonistic antibody against OX40 (TNFRSF4); 3H3, agonistic antibody against 4-1BB (TNFRSF9).

Article Snippet: To investigate the induction of a pathogenic program in Th2 cells, naïve CD4 + T cells were cultured under Th2-promoting conditions together with TSLP (50 ng/ml, R&D, 555-TS-010) and agonistic antibodies for GITR (5 μg/ml, clone DTA-1, BioXCell, BE0063), OX40 (5 μg/ml, clone OX-86, BioXCell, BE0031), or 4-1BB (5 μg/ml, clone 3H3, BE0239) all from BioXCell.

Techniques: In Vitro, Generated, Cell Culture, Flow Cytometry, Expressing, Isolation

Journal: bioRxiv

Article Title: Single-cell profiling uncovers regulatory programs of pathogenic Th2 cells in allergic asthma

doi: 10.1101/2023.08.10.552772

Figure Lengend Snippet: a-d, Targeting p38 MAPK signalling and AP-1 activity in in vitro generated pTh2 cells. Naïve CD4 + T cells from WT and HDAC1-cKO mice were differentiated under Th2-promoting conditions (IL-4, IL-2, anti-IFN-ψ, and anti-TGF-β) alone, or pTh2-promoting conditions (IL-4, IL-2, TSLP, DTA-1, anti-IFN-ψ, and anti-TGF-β) alone, or pTh2 cells treated with an AP-1 inhibitor (T-5224; 10 µM), or pTh2 cells treated with a p38 MAPK inhibitor (SB 203580; 10 µM), and cultured for 5 days. On day 5, cells were restimulated with PMA and ionomycin in the presence of GolgiStop and GolgiPlug for 4 hours and cytokine analyses were performed by flow cytometry. a, Histograms show expression (top) and frequencies (bottom) of RANKL, GM-CSF, and IL-9, respectively, in WT cells (blue). b, Histograms show expression (top) and frequencies (bottom) of RANKL, GM-CSF, and IL-9, respectively, in HDAC1-cKO cells (red). c,d, Inhibition of ERK1/2 and JNK signalling in in vitro generated pTh2 cells. Naïve CD4 + T cells from WT and HDAC1-cKO mice were differentiated under Th2-promoting conditions (IL-4, IL-2, anti-IFN-ψ, and anti-TGF-β) alone, or pTh2-promoting conditions (IL-4, IL-2, TSLP, DTA-1, anti-IFN-ψ, and anti-TGF-β) alone, or pTh2 cells treated with a MEK1/2 inhibitor (U0126-EtOH; 1 µM) which targets ERK1/2 signalling, or pTh2 cells treated with a JNK inhibitor (SP600125; 1 µM), and cultured for 5 days. On day 5, we restimulated the cells with PMA and ionomycin in the presence of GolgiStop and GolgiPlug for 4 hours and performed cytokine analyses by flow cytometry. a, Histograms show expression (top) and frequencies (bottom) of IL-4, IL-5, and IL-13, respectively, in WT cells (blue). b, Histograms show expression (top) and frequencies (bottom) of IL-4, IL-5, and IL-13, respectively, in HDAC1-cKO cells (red). Data are pooled from three independent experiments and presented as the mean ± SEM. Each symbol represents one mouse. Statistical analysis was performed using a one-way ANOVA with Tukey’s multiple comparisons test. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001. TSLP, thymic stromal lymphopoietin; DTA-1, an agonistic antibody against GITR (TNFRSF18). AP-1, activator protein-1; MAPKs, mitogen-activated protein kinases; p38 MAPK, p38 mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal Kinase.

Article Snippet: To investigate the induction of a pathogenic program in Th2 cells, naïve CD4 + T cells were cultured under Th2-promoting conditions together with TSLP (50 ng/ml, R&D, 555-TS-010) and agonistic antibodies for GITR (5 μg/ml, clone DTA-1, BioXCell, BE0063), OX40 (5 μg/ml, clone OX-86, BioXCell, BE0031), or 4-1BB (5 μg/ml, clone 3H3, BE0239) all from BioXCell.

Techniques: Activity Assay, In Vitro, Generated, Cell Culture, Flow Cytometry, Expressing, Inhibition

Journal: Frontiers in Immunology

Article Title: Targeting Xcr1 on Dendritic Cells Rapidly Induce Th1-Associated Immune Responses That Contribute to Protection Against Influenza Infection

doi: 10.3389/fimmu.2022.752714

Figure Lengend Snippet: Xcl1-OVA induces Th1 polarization of DO11.10 cells in vitro and in vivo . (A) CD4 + cells were purified from spleen of DO11.10 TCR transgenic mice and incubated with BMDCs as APC in the presence of indicated amounts of Xcl1, fliC, or NIP-OVA protein for 72 h. Incorporation of radioactive thymidine was analyzed after 48 h. (B–D) CD4 + from DO11.10 transgenic mice were incubated with BMDC in the presence of Xcl1-, fliC-, or NIP-OVA proteins (0.5 μg/ml) for 72 h. (B) CD4 + DO11.10 + cells were evaluated for expression of T-bet by flow cytometry. MFI for T-bet expression is summarized in the right graph. (C) Ratio of T-bet/GATA3 MFI for CD4 + DO11.10 + cells in mice from (B) and Supplementary Figure 3A . (D) Concentrations of IFNγ, IL-12, TNFα, and IL-13 in supernatants. (E, F) 1 × 10 6 naïve DO11.10 cells were transferred to BALB/c mice that were subsequently injected i.v. with purified Xcl1-OVA, fliC-OVA, or αNIP-OVA proteins (5 μg). Spleens were harvested 72 h later and the percentage of (E) CD4 + DO11.10 + and (F) T-bet + DO11.10 + cells determined by flow cytometry. Data are representative of one (A) or three (B–D) independent experiments with n = 3 samples per group. (E, F) Data from one experiment with n = 3 mice per group. Statistical analysis was performed by one-way ANOVA with Tukey’s multiple-comparison corrections. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: For the early time-point blockade, 500 μg of anti-IL12 antibody (clone R2-9A5) (Bio X Cell, Lebanon, NH, USA) was injected i.p 1 and 2 days after vaccination, while for the later time inhibition the anti-IL12 antibody was injected on days 6 and 7.

Techniques: In Vitro, In Vivo, Purification, Transgenic Assay, Incubation, Expressing, Flow Cytometry, Injection, Comparison

Journal: Frontiers in Immunology

Article Title: Targeting Xcr1 on Dendritic Cells Rapidly Induce Th1-Associated Immune Responses That Contribute to Protection Against Influenza Infection

doi: 10.3389/fimmu.2022.752714

Figure Lengend Snippet: Xcl1-OVA induced Th1 polarization is IL12 dependent. (A, B) CD4 + cells from DO11.10 mice were incubated with BMDCs and Xcl1-OVA, fliC-OVA, or αNIP-OVA proteins (0.5 μg/ml), and either anti-IL-12 or isotype-matched mAbs (10 μg/ml) for 72 h. (A) DO11.10 cells were evaluated for expression of T-bet by flow cytometry, and (B) supernatants tested for secretion of IFNγ by ELISA. (C–E) 1 × 10 6 naïve DO11.10 cells were transferred i.v. to BALB/c mice that were subsequently immunized i.d. with 25 μg DNA encoding Xcl1-OVA. On days 1 and 2 after immunization, mice were injected i.p. with anti-IL12 or isotype-matched mAb (0.5 mg). Skin draining LNs and spleens were harvested after 1 week, and LN analyzed for (C) percentage of T-bet + DO11.10 + cells and (D) MFI of T-bet expression in T-bet + DO11.10 + cells. (E) Secretion of IFNγ from splenocytes stimulated for 24 h with the DO11.110 peptide. Data representative of two (A–D) or pooled from two (E) independent experiments with n = 3 samples per group (A, B) , n = 3–4 mice per group (C, D) or n = 7–8 mice per group (E) . Statistical analysis performed using the parametric t-test. *p < 0.05, ***p < 0.001.

Article Snippet: For the early time-point blockade, 500 μg of anti-IL12 antibody (clone R2-9A5) (Bio X Cell, Lebanon, NH, USA) was injected i.p 1 and 2 days after vaccination, while for the later time inhibition the anti-IL12 antibody was injected on days 6 and 7.

Techniques: Incubation, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Injection

Journal: Frontiers in Immunology

Article Title: Targeting Xcr1 on Dendritic Cells Rapidly Induce Th1-Associated Immune Responses That Contribute to Protection Against Influenza Infection

doi: 10.3389/fimmu.2022.752714

Figure Lengend Snippet: Rapid Th1 induction is essential for induction of an IgG2a dominated response and improves protection induced by a mixed fliC-/Xcl1-HA vaccine. (A, B) DNA immunization of BALB/c mice with 25 μg DNA encoding Xcl1-HA, fliC-HA, a mixture of Xcl1-HA and fliC-HA, or Xcl1-HA and fliC-HA delivered on opposite flanks. (A) Serum titers of IgG1, IgG2a, and IgG2b were determined 2 weeks after vaccination. (B) IgG2a/IgG1 ratio of the serum samples presented in (A) . (C, D) Injection of either anti-IL-12 or isotype-matched mAbs on days 1 and 2 or 6 and 7 after DNA immunization with a mix of Xcl1-HA and fliC-HA plasmids. (C) Serum samples were harvested at the indicated time points and evaluated for the presence of HA-specific IgG1 or IgG2a. (D) Mice in (C) were challenged with 5xLD50 PR8 virus 12 weeks after immunization and weight loss monitored. Data representative of one experiment with n = 6–8 mice per group (A, B) , representative of two independent experiments with n = 4 mice per group (C) or pooled from two independent experiments with n = 8 mice per group (D) . Statistical analysis performed by non-parametric one-way ANOVA with Dunn’s multiple-comparison corrections (A, B) , or two-way ANOVA with Tukey’s multiple-comparison test (C, D) . *p < 0.05, ***p < 0.001.

Article Snippet: For the early time-point blockade, 500 μg of anti-IL12 antibody (clone R2-9A5) (Bio X Cell, Lebanon, NH, USA) was injected i.p 1 and 2 days after vaccination, while for the later time inhibition the anti-IL12 antibody was injected on days 6 and 7.

Techniques: Injection, Virus, Comparison