Journal: Cardiovascular research

Article Title: Single-cell profiling reveals age-associated immunity in atherosclerosis.

doi: 10.1093/cvr/cvad099

Figure Lengend Snippet: Figure 1 Aging promotes immunosenescence and atherosclerosis in Ldlr−/− mice. (A) Experimental setup: Ldlr−/− mice aged 3 months (green bars) or 20 months (violet bars) were fed with a standard chow diet (white circles) or a Western diet (grey circles) for 10 weeks. (B) Using flow cytometry, percentages (% from live) of circulating myeloid cells (CD11b+), neutrophils (CD11b+Ly6CintLy6G+), inflammatory monocytes (CD11b+Ly6ChiLy6G−), and (C) circulating CD4+ and CD8+ T-cells were determined (% from live). (D) Splenic naïve (TN: CD44−CD62L+), effector (TEFF: CD44−CD62L−), central memory (TCM: CD44+CD62L+), and effector memory (TEM: CD44+CD62L−) T-cells were quantified as a percentage of CD4+ and CD8+ T-cells and plotted in pie charts. (E) Intracellular cytokine production of interferon-gamma (IFN-γ), interleukin (IL)-4, IL-10, and IL-17 were measured as percentage (mean) of splenic CD4+ and CD8+ T-cells after 4 h of stimulation with PMA and ionomycin. Colour scale is normalized for each cytokine. (F) Circulating CD19+ B-cells were determined with flow cytometry. (G) Total and oxidized LDL (oxLDL)-specific IgM titres were measured in the serum. (H ) Total serum cholesterol levels at sacrifice were measured. (I) Cross sections of the aortic root were stained for lipid and collagen content, and (J ) atherosclerotic lesion volume was quantified. (K) Collagen content and (L) necrotic cores were quantified as percentage of lesion area. (M) Cholesterol crystallization in atherosclerotic lesions was categorized on a scale of 0 (no cholesterol crystallization) to 3, and presence of calcification (purple) or no calcification (grey) was presented as percentage of group. (N) Macrophage content (MOMA-2) was measured as percentage of lesion area. Data are from n = 12–15 mice per group. Statistical significance was tested by one-way ANOVA. Mean ± S.E.M. plotted. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: C D 8 T c el ls Il1 7a + T c el ls T re gs N K T c el ls T c el ls /M C s YC YW OC young chow diet young Western diet old chow diet young chow diet (Cochain, 2018) young Western diet old chow diet Gzmk+ CD8+other CD8+ Nkg7 Ccl5 S100a6 GzmkSatb1 AW112010 S100a4 Id2Lef1 Tcf7 Sell Ccl4Ccr7 Rgs10 Ccr9 0 20 40 60 0.0 2.5 5.0 Log2 fold change NS Log2 FC value p - value and log2 FC chow diet (CD) Western diet (WD) 0 2 4 6 8 10 Natural Killer Cell Signaling Caveolar-mediated Endocytosis Signaling Pathogen Induced Cytokine Storm Signaling Pathway Signaling by Rho Family GTPases Th1 Pathway RHOGDI Signaling Immunogenic Cell Death Signaling Pathway Regulation of Actin-based Motility by Rho Integrin Signaling Th1 and Th2 Activation Pathway -log(p-value) 0 50 100 150 200 # G zm k+ C D 8+ T ce lls * **** * young old Figure 3 Identification of age-associated T-cell populations and gene signatures in atherosclerotic aortas from Ldlr−/− mice. (A) Cd3e+ clusters were extracted from the principal clustering and reclustered, after which the T-cell clusters were identified. (B) UMAP plots and stacked diagrams visualizing the identified T-cell subclusters in young CD, young, WD and old CD aortas, in which Gzmk+CD8+ T-cells are encircled in the dashed red shape. (C ) Dot plot showing the average expression of immune cell cluster-defining markers for each cluster. (D) Heatmap of hierarchically clustered top 25 variable genes across T-cell subclusters. (E) Volcano plot of the differentially expressed genes (DEGs) in the Gzmk+CD8+ T-cell cluster compared to other CD8+ T-cells in clusters 2, 3, 5, and 6. (F ) Top canonical pathways of the Gzmk+CD8+ T-cell cluster compared to CD8+ T-cells in clusters 2, 3, 5, and 6. (G) Heatmap showing average expression of biological process-associated genes in T-cell clusters of young CD, young WD, and old CD Ldlr−/− aortas. (H ) Using flow cytometry, absolute numbers of Ly6C−CD44+Tox+PD-1+ CD8+ T-cells (GzmK+CD8+ T-cells) were measured in aortas of young and aged Ldlr−/− mice (n = 11–15).

Techniques: Western Blot, Flow Cytometry, Staining, Crystallization Assay

Journal: bioRxiv

Article Title: Antithymocyte globulin inhibits CD8 + T cell effector functions via the paracrine induction of PDL-1 on monocytes

doi: 10.1101/2022.07.26.501584

Figure Lengend Snippet: (A) GSEA enrichment plot for regulations of T-cell functions and tolerance induction in monocytes following treatment with ATG. Green lines depict running enrichment score, black vertical lines indicate genes responsible for enrichment and position in ranked list of DEG. (B) Violin plots of core enrichment genes CD274, IDO1, IRF1, HLA-A and HLA-E in monocytes isotype versus monocytes ATG. Expression levels are indicated by violin plot height while width represents proportion of positive cells. Crossbars mark mean expression. **** indicate p-value < 0.0001. (C) Representative histogram of proliferative capacity of activated CD8 + T cells co-cultured with monocytes (black), PDL-1 + monocytes (green) and durvalumab pre-treated PDL1 + monocytes (blue). Non-proliferating (furthest right) and proliferating cell populations are reflected by intensity of cell proliferation dye staining. Dashed red line separates cells with 3 or less divisions from cells with 4 or more divisions. (D) Bar graph depicting mean percentages of CD8 + T cells with 3 or less proliferations and 4 or more proliferations at day 5. N = 4 donors per group. *** p-value = 0.0009; ** p-value = 0.0034. One-way ANOVA with Dennett’s multiple comparison was used to determine statistically significant differences. (E) Concentration of granzyme B in conditioned medium of CD8 + T cells co-cultured with monocytes assessed by ELISA. ** p-value < 0.01.

Article Snippet: In addition, we measured IFN-γ in supernatants of ATG-treated whole blood, purified PBMCs and purified CD8 + T cells as well as human granzyme B in co-cultures via ELISA (Bio-techne, R&D, Minneapolis, Minnesota, USA).

Techniques: Expressing, Cell Culture, Staining, Comparison, Concentration Assay, Enzyme-linked Immunosorbent Assay