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

Article Title: SZB120 Exhibits Immunomodulatory Effects by Targeting eIF2α to Suppress Th17 Cell Differentiation.

doi: 10.4049/jimmunol.2000036

Figure Lengend Snippet: FIGURE 1. SZB120 selectively inhibits Th17 cell differentiation. (A) The synthesis of SZB120. (B) Mouse primary naive CD4+ T cells from IL-17A– GFP mice were differentiated under Th17 cell polarization conditions for 72 h with DMSO and different concentrations of AbBA (0.5, 1, 2, 5 mM) or SZB120 (0.5, 1, 2, 5 mM) (n = 3). (C) Naive CD4+ T cells were differentiated under Th17 polarization conditions for 72 h with DMSO and SZB120 (0.5, 1, 2, 5 mM). CD4+ annexin V+ 7AAD+ cells were analyzed. The black bar represents the percentage of annexin V+ 7AAD2 cells (early apoptosis). The red bar indicates the percentage of annexin V+ 7AAD+ cells (late apoptosis) (n = 3). (D) The proliferation marker Ki67 was tested under the Th17 cell differ- entiation conditions mentioned above with DMSO and SZB120 (0.5, 1, 2, 5 mM). Cells were gated for CD4+ and stained for Ki67. Black bar indicates the mean fluorescence intensity (MFI) of Ki67+ cells (n = 3). (E) T cells were activated under Th1, Th2, and iTreg cell polarization conditions in the presence of DMSO and 0.5, 1, 2, and 5 mM SZB120 (n = 3–6). Data are the mean 6 SEM by two-way ANOVA (B and C) or one-way ANOVA (D and E) with Bonferroni correction for multiple comparisons. *p , 0.05, ***p , 0.001, compared with DMSO. ns, no significance

Article Snippet: The inducible T regulatory cell (iTreg) differentiation condition included 5 ng/ml rmTGF-b (R&D Systems) and 10 ng/ml rmIL-2 (R&D Systems).

Techniques: Cell Differentiation, Marker, Staining

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:

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