Journal: bioRxiv

Article Title: Early life tolerance depends on a subset of specialized dendritic cells and is reinforced by the skin microbiota

doi: 10.1101/2022.06.23.497363

Figure Lengend Snippet: (A-C) derive from CITE-seq data introduced in . (A) Volcano plot of top differentially expressed genes in CD301b + versus CD301b neg DC2 in D10 murine skin, complete list in Table 1 (B) Central functions enriched in each subset based on pathway analysis, complete list in Table 2 (C) Heat map of normalized average expression indicated genes in CD301b + versus CD301b neg cells in either CD11b hi or CD11b lo DC2 clusters and percentage of cells (circle size) in which expression was detected. All genes showed have a p-value <0.05 and were found in two independent scRNAseq experiments. (D-E) Spectral flow cytometry of activation markers in CD301b + versus CD301b neg DC2 from D10 skin 16h after colonization with S. epidermidis . (D) Representative histograms and gates used to delineate positive cells with corresponding (E) quantification of the % positive cells and geometrical mean of fluorescence intensity (gMFI) for the indicated markers. Each dot is biological replicate. 1 of 2 independent experiments are shown. Two-way ANOVA with Šídák’s multiple comparisons test. (F) Schematic of the DC-T cell assay used in (G) and (H) to measure DC capacity to promote Tregs. CD301b + versus CD301b neg DC2 were separately sorted from the migDC fraction of D10 SDLN and incubated with OVA protein overnight then with CTV-labelled OT-II T cells in the presence of IL-2 and TGF-β for 72h before flow cytometry to measure T cell proliferation. (G) Representative plots (pre-gated on Live CD4 + TCRβ + ). (H) Quantification of one of three representative experiments with technical replicates shown. One-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001, ****p<0.0001.

Article Snippet: For suboptimal Treg inducing conditions, IL2 (200 U/mL, GoldBio, # 1310-02-100) and TGFβ (2 ng/mL, Peprotech, #100-21C-10UG) were added to the wells.

Techniques: Expressing, Flow Cytometry, Activation Assay, Fluorescence, Incubation

Journal: bioRxiv

Article Title: Early life tolerance depends on a subset of specialized dendritic cells and is reinforced by the skin microbiota

doi: 10.1101/2022.06.23.497363

Figure Lengend Snippet: (A-B) D10 neonatal mice were colonized with S . epi -zsgreen and skin was harvested 16h later, with commensal antigen-loaded (zsgreen + ) and unloaded (zsgreen neg ) DCs separately sorts and submitted for scRNAseq. (A) UMAP of zsgreen + vs zsgreen neg DCs and (B) heatmaps comparing expression of select differentially genes between zsgreen + vs zsgreen neg CD301b + DC2 (CD301b status defined based Mgl2 expression). (C) Spectral flow cytometry comparing protein-level expression of key markers zsgreen + vs zsgreen neg CD301b + DC2 in neonatal skin 16h after colonization with S. epi- zsgreen colonization. Quantification of the % positive cells and gMFI for the indicated markers in loaded vs non loaded CD301b + DC2. Each dot is a biological replicate. 1 of 2 independent experiments are shown. Two-way ANOVA with Šídák’s multiple comparisons test. (D-F) Human neonatal foreskin was incubated 4 h with S. epi -zsgreen as detailed in Fig. S3B and loaded (zsgreen + ) versus non-loaded (zsgreen neg ) live CD45 + CD16 neg HLADR + cells were separately sorted and submitted for scRNAseq. (D) UMAP of all cells combined, (E) UMAP of DC clusters split by zsgreen status, and (F) heat maps comparing expression of select genes between zsgreen + and zsgreen neg DC2 in human foreskin. (G) Schematic of the DC-T cell assay used in (H) and (I). Indicated DC subsets were sorted from the migDC fraction of D10 SDLN, incubated with S. epi -OVA overnight and then with CTV-labeled OT-II T cells for 72h in the presence of IL-2 and TGF-β. (H) Representative plots (pre-gated on Live CD4 + TCRβ + ) of showing percentage of OT-II Tregs and (I) and quantification thereof. Technical replicates are shown from one of 3 replicate experiments with similar results. One-Way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001, ****p<0.0001, ns = not significant.

Article Snippet: For suboptimal Treg inducing conditions, IL2 (200 U/mL, GoldBio, # 1310-02-100) and TGFβ (2 ng/mL, Peprotech, #100-21C-10UG) were added to the wells.

Techniques: Expressing, Flow Cytometry, Incubation, Labeling

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CD69 regulates type I IFN-induced tolerogenic signals to mucosal CD4 T cells that attenuate their colitogenic potential.

doi: 10.4049/jimmunol.1100765

Figure Lengend Snippet: FIGURE 2. CD69 surface expression is induced by oral Ag challenge in TCR transgenic mice. B6 (A) and OT-II 3 RAG2/2 (B) mice were fed for 2 d with 1 mg OVA protein dissolved in PBS. The expression of the activation Ags CD69 and CD25 by CD4 T cells from spleen (S), MLN, siLP, and cLP was analyzed by multicolor FCM. Data from an individual representative mouse (out of four mice analyzed) are shown. Numbers indicate the percentage of CD4 T cells that express the activation Ags CD69 or CD25. C, OT-II 3 RAG2/2 mice were fed for 2 d with 1 mg OVA protein dissolved in PBS. In- tracellular expression of T-bet, GATA-3, RORgt, and Foxp3 and surface expression of IFN-gR, CD122, CD103, TGF-bRII, IL-21R, IL-10R, ICOS, and LAP/TGF-b1 gated on CD69+ and CD692 CD4 T cells from siLP were analyzed by multicolor FCM. Filled curves represent the respective negative controls. Four mice were analyzed, and the data from a representative individual mouse are presented. Numbers represent the percentage of cells that are positive for the indicated Ag.

Article Snippet: To simulate conditions that favor Foxp3+ Treg cell induction, 2000 U/ml IL-2 (catalog no. 402-ML; R&D Systems) and 5 ng/ml TGF-b1 (catalog no. 240-B; R&D Systems) were added to the cell culture.

Techniques: Expressing, Transgenic Assay, Activation Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CD69 regulates type I IFN-induced tolerogenic signals to mucosal CD4 T cells that attenuate their colitogenic potential.

doi: 10.4049/jimmunol.1100765

Figure Lengend Snippet: FIGURE 4. Reduced Foxp3+ Treg cell number in the absence of CD69. A, Cells were isolated from spleen (S), MLN, siLP, and cLP of nontreated B6 and CD692/2 mice and analyzed for intracellular expression of Foxp3 in CD4 T cell population by multicolor FCM. Numbers indicate the percentage of CD4+

Article Snippet: To simulate conditions that favor Foxp3+ Treg cell induction, 2000 U/ml IL-2 (catalog no. 402-ML; R&D Systems) and 5 ng/ml TGF-b1 (catalog no. 240-B; R&D Systems) were added to the cell culture.

Techniques: Isolation, Expressing

Journal: Investigative Ophthalmology & Visual Science

Article Title: Apolipoprotein A1 Modulates Teff/Treg Balance Through Scavenger Receptor Class B Type I-Dependent Mechanisms in Experimental Autoimmune Uveitis

doi: 10.1167/iovs.63.8.23

Figure Lengend Snippet: APOA1 treatment alters Teffs/Tregs balance in systemic immune. (A-C; a-c) APOA1 inhibited the naive CD4 + T cells (CD44 − CD62L + ) differentiation into memory CD4 + T cells (CD44 + CD62L − ) in DLNs and spleen compared to vehicle. (D – H; d – h) APOA1 decreased the proportion of Th17 cells (CD4 + IL-17A + ), Th1 cells (CD4 + IFN-γ + ) and the secretion of TNF-α in CD4 + T cells (CD4 + TNF-α + ) in DLNs and spleen compared to vehicle. (J – M; j – m) APOA1 increased Treg (CD4 + CD25 + Foxp3 + ) and the secretion of IL-10 in CD4 + T cells (CD4 + IL-10 + ) in DLNs and spleen compared to vehicle (n = 6). The values represent the mean ± SD. * P < 0.05, ** P < 0.01.

Article Snippet: The cultures were also supplemented with Th1 or Treg induction condition (ImmunoCult mouse Th1 or Treg differentiation supplement, Stemcell Technologies) for respective conversion according to the manufacturer's instructions.

Techniques:

Journal: Investigative Ophthalmology & Visual Science

Article Title: Apolipoprotein A1 Modulates Teff/Treg Balance Through Scavenger Receptor Class B Type I-Dependent Mechanisms in Experimental Autoimmune Uveitis

doi: 10.1167/iovs.63.8.23

Figure Lengend Snippet: APOA1 modulates proliferation and differentiation of T cells. (A, B) The viability of T cells was affected by APOA1 inconspicuously at 5 µM and mildly at 10 µM, but significantly at 20 µM and 40 µM (n = 5). (C, D) The proliferation of CD3 + T cells was markedly inhibited by APOA1 at concentrations of 5 µM and 10 µM (n = 5). (E, F) The induction of Th1 differentiation (CD4 + IFN-γ + ) was reduced by APOA1 (n = 5). (G, H) The induction of Treg differentiation (CD4 + CD25 + Foxp3 + ) was expedited by APOA1 (n = 4). The values represent the mean ± SD. nsp>0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: The cultures were also supplemented with Th1 or Treg induction condition (ImmunoCult mouse Th1 or Treg differentiation supplement, Stemcell Technologies) for respective conversion according to the manufacturer's instructions.

Techniques: