Fig 1: Disruption of the Dendritic Cell CD40-T cell CD40L axis results in lower interferon-? production.a, b Experimental setup for isolation and confirmation of (a) wild type and CD40-deficient dendritic cells (DCs) and (b) naive T cells for DC-T cell co-culture for in vitro disruption of the CD40-CD40L axis. Specifically, a bone marrow-derived DCs were generated from control ApoE-/- mice and ApoE-/- mice with a global deficiency of CD40 through polarization with Granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) and subsequent maturation with lipopolysaccharide (LPS). CD40 expression was measured by flow cytometry. b Naive T cells (CD62L+CD44-) were isolated from the lymph nodes (LN) and spleens of OT-II transgenic mice with a T cell receptor specific for ovalbumin (OVA) peptide 323–339. Mature DCs were stimulated with OVA peptide 323–339 and cultured with naive OT-II T cells for 72 h for antigen presentation and T cell stimulation. c Mature ApoE-/- DCs were stimulated with the OVA peptide or a vehicle control [n = 5] to confirm the specificity of the T cell receptor on the OT-II T cells. d Flow cytometric analysis of effector T cell (CD62L-CD44+) populations following DC-T cell co-culture [n = 5]. e Supernatant concentration of interferon-? (IFN-?) following DC-T cell co-culture [n = 5]. Data are represented as mean ± s.e.m and n refers to biologically independent animals. Data were analyzed by either a two-tailed unpaired Student’s t test (e) or two-tailed Mann–Whitney test (c, d). Source data are provided as a Source Data file.
Fig 2: T cell CD40L deficiency leads to profound reductions in Th1 and Treg responses.a Flow cytometric analysis of effector memory (EM) T cells in the blood [n = 8], LNs [n = 8], and spleen [n = 8]. b Flow cytometric analysis of effector T cell subpopulations including T helper (Th) 1 (CD62L-CD44+CXCR3+CCR6-), Th2 (CD62L-CD44+CXCR3-CCR6-), and Th17 (CD62L-CD44+CXCR3-CCR6+) in the LNs [n = 8] and spleen [n = 8]. c Representative gating strategy for effector T cell populations. d Flow cytometric analysis of Foxp3+CD25+ T regulatory cells (Tregs) in the blood [n = 8], lymph node (LN) [n = 8], and spleen [n = 8] in Cd40lfl/fl/Cd4Cre mice. Proliferation assay [n: WT = 3, TG = 4 for all data points except 100/50 condition where TG = 2] of Carboxyfluorescein succimidyl ester (CFSE)-stained CD4+ T cells with varying ratios of conventional CD4+ T cells to Tregs (ie. 100/0) analyzed by CFSE-dilution of conventional CD4+ T cells. f Representative gating strategy for Tregs. All data are represented as mean ± s.e.m and n refers to biologically independent animals. Data were analyzed by either a two-tailed unpaired Student’s t test (a, e) or two-tailed Mann–Whitney test (b, d). Source data are provided as a Source Data file.
Fig 3: Estimation of immunoglobulin changes after 3 months of intranasal (IN) treatment with THC or vehicle using ELISA. Blood samples were collected from the NTG and TG mice prior to and post-3-month THC or vehicle intranasal treatment. No significant difference in IgG1, IgG2a, IgG2b, IgG3, IgA, and IgM levels was found before and after intranasal treatment. Data are expressed as mean ± SEM (n = 5 per group). No statistically significant difference in plasma IgG1, IgG2a, or IgG2b levels was observed between any of the study groups. SEM is denoted by error bars. * p < 0.05, ** p < 0.01, and *** p < 0.001 were compared among NTG mice, vehicle-treated APP/PS1 mice, and APP/PS1 mice treated with 0.002 and 0.02 mg/kg THC using a two-way repeated measures ANOVA multiple comparison test.
Fig 4: Lack of T cell CD40L reduces plaque burden with less advanced, more stable plaques.a Atherosclerotic plaque area in cross-sections at indicated positions of the aortic root from Cd40lfl/fl/Cd4Cre mice [n: WT = 17, TG = 16] with representative Oil Red O-stained photomicrographs (scale bar: 200 µm). b, c Immunofluorescent staining assessing plaque phenotypes in cross-sections of the aortic root of mice with quantifications (left) and representative photomicrographs (right). Arrows indicate stained cells and the dashed line separates the atherosclerotic lesion. b CD4+ T cells (green) along with nuclear DAPI staining (blue) [n = 10] and (c) Mac3+ area (green) along with nuclear DAPI staining (blue) [n = 7] (scale bar: 100 µm). d Chi-square distribution analysis of morphological plaque phenotypes describing initial xanthoma (IX), pathological intimal thickening (PIT), and fibrous cap atheroma (FCA) phenotypes [n: WT = 17 animals/51 lesions, TG = 13/39 lesions]. e–g Multi-parameter analysis of plaque stability assessing: e Fibrous cap thickness [n: WT = 20, TG = 14] using a Sirius Red stain (polarized light), (f) Alpha-smooth muscle actin+ (a-SMA+) area [n: WT = 9, TG = 5] (green, scale bar: 75 µm), and (g) Necrotic core content [n = 8] using hematoxylin and Eosin stain all in aortic root sections with quantifications (left) and representative photomicrographs (right). h Gene expression analysis using qPCR to evaluate various mRNA cytokine expression profiles in the descending aorta [n: WT = 6: CD3, TGF-ß, IL-6, CD69; 5: IL-1ß, 4: IFN- ?, IL-10, TNF- a; TG = 7: TGF-ß; 6: IL-6, CD69; 5: IL-1ß, IL-10, IFN- ?, CD3; 4: TNF- a. Data are represented as either mean ± s.e.m. a, h or boxplots (b, c, e–g). In the boxplots, whiskers above and below the boxes indicate the 10th and 90th percentile while the boundaries of the boxes represent the 25th and 75th percentile. The black line within the boxes marks the median. n refers to biologically independent animals. Data were analyzed by either a two-tailed unpaired Student’s t test (a–c, f), two-tailed Mann–Whitney test (e, g, h), or Chi-square test (d). Interleukin-1 beta IL-1ß, Interleukin-6 IL-6, Interferon-gamma IFN-?, Tumor necrosis factor-alpha TNF-a, Transforming growth factor-beta TGB-ß, Interleukin-10 IL-10, Cluster of differentiation 3 CD3. Source data are provided as a Source Data file.
Fig 5: Platelet CD40L deficiency alters thrombosis formation and atherothrombosis.a Atherosclerotic plaque area in cross-sections at indicated positions of the aortic root from Cd40lfl/fl/Pf4Cre mice [n: WT = 21, TG = 18] with representative Oil Red O-stained photomicrographs (scale 200 µm). b–d Immunofluorescent staining assessing plaque phenotypes in cross-sections of the aortic root of mice with quantifications (left) and representative photomicrographs (right). Arrows indicate stained cells and the dashed line separates the atherosclerotic lesion. b CD4+ T cells (green) along with nuclear DAPI staining (blue) [n: WT = 10, TG = 8], (c) Mac3+ area (green) along with nuclear DAPI staining (blue) [n: WT = 6, TG = 5], and (d) Alpha-smooth muscle actin+ (a-SMA+) area [n: WT = 18, TG = 15] (green, scale bar: 75 µm). e–f Multi-parameter assessment of thrombus formation in flowed whole blood with CD40L deficient platelets with quantifications (left) and representative photomicrographs (right, scale bar: 20 µm). Parameters evaluated after 4 min of blood flow were: e 3,3'-Dihexyloxacarbocyanine iodide (DiOC6) labelled platelet adhesion (Surface Area Coverage, SAC%) [n: WT = 19, TG = 12] and (f) Fluorescein isothiocyanate (FITC)-labelled fibrinogen staining of thrombi [n: WT = 20, TG = 12]. g Thrombus build-up [n: WT = 20, KO = 12] was analyzed using micro-spot perfusions comparing Cd40lfl/fl/Pf4Cretg mice and their littermate controls. h Plaque volume [n = 8] was measured in the carotid arteries following wire injury with quantifications (left) and representative photomicrographs (right, scale bar: 200 µm). Data are represented as either mean ± s.e.m. (a, g) or boxplots (b–f, h). In the boxplots, whiskers above and below the boxes indicate the 10th and 90th percentile while the boundaries of the boxes represent the 25th and 75th percentile. The black line within the boxes marks the median. n refers to biologically independent animals. Data were analyzed by either a two-tailed unpaired Student’s t test (a, e, g) or two-tailed Mann–Whitney test (b–d, f, h). Source data are provided as a Source Data file.
Supplier Page from Abcam for Mouse CD40L ELISA Kit