Dynamic T cell-APC interactions sustain chronic inflammation in atherosclerosis

Abstract

Atherosclerosis is a chronic inflammatory disease of large and medium-sized arteries characterized by leukocyte accumulation in the vessel wall. Both innate and adaptive immune responses contribute to atherogenesis, but the identity of atherosclerosis-relevant antigens and the role of antigen presentation in this disease remain poorly characterized. We developed live-cell imaging of explanted aortas to compare the behavior and role of APCs in normal and atherosclerotic mice. We found that CD4+ T cells were capable of interacting with fluorescently labeled (CD11c-YFP+) APCs in the aortic wall in the presence, but not the absence, of cognate antigen. In atherosclerosis-prone Apoe-/-CD11c-YFP+ mice, APCs extensively interacted with CD4+ T cells in the aorta, leading to cell activation and proliferation as well as secretion of IFN-γ and TNF-α. These cytokines enhanced uptake of oxidized and minimally modified LDL by macrophages. We conclude that antigen presentation by APCs to CD4+ T cells in the arterial wall causes local T cell activation and production of proinflammatory cytokines, which promote atherosclerosis by maintaining chronic inflammation and inducing foam cell formation.

Figure 1. Myeloid cells in aortic plaque and adventitia.

(A) Live CD45⁺ cells from aortas of Apoe^–/– mice fed CD or WD for 12 weeks were stained for CD11b and CD11c. Numbers indicate percentage in each quadrant. (B) Localization of CD11b⁺CD11c^– (green), CD11b⁺CD11c⁺ (yellow), and CD11b^–CD11c⁺ (red) cells in aortic roots of WD-fed Apoe^–/– mice, shown by immunofluorescence. Dotted white lines indicate border of lamina muscularis. L, lumen; P, plaque; A, adventitia. Representative images from at least 3 independent experiments. (C) Examples of CD11b⁺CD11c^–, CD11b⁺CD11c⁺, and CD11b^–CD11c⁺ cells in the adventitia enlarged from boxed regions in B. Localization of CD11c-YFP⁺ cells (green) in normal B6 (D) and in atherosclerotic Apoe^–/– (E) mouse aorta. Blue, second harmonic; brown, autofluorescence. Scale bars: 100 μm (B); 2 μm (C, left); 5 μm (C, middle and right); 150 μm (D and E).

Figure 2. 2-photon imaging of T cell–APC interaction in aortic wall.

(A) Maximum intensity projection along z axis (top view) through image stacks (760 × 760 × 210 μm [x, y, z]). View of CD11c-YFP⁺ (green) and SNARF-labeled OTII T cells (red) from the adventitial side of explanted mouse aorta. (B) Time-lapse images of interacting APCs (green) and T cells (red) in the adventitia (boxed region in A) in the presence of OVA^323–339. Tracks (C) and velocities (D) of individual T cells obtained from 3D projection (1 color per cell) in the presence of OVA^323–339. Long interaction with APCs (>5 minutes) is denoted by a thick line. (E) Velocities of individual noninteracting (free; n = 1,057) and interacting (>5 minutes; n = 99) T cells. Average is indicated by horizontal line. (F) Percent T cells interacting with APCs in the presence and absence of OVA^323–329. Data (mean ± SEM) are representative of at least 3 independent experiments. *P < 0.05. Scale bars: 100 μm (A); 5 μm (B).

Figure 3. 2-photon imaging of antigen-specific T cell–APC interaction in aortic wall.

Interaction of CD11c-YFP⁺ APCs (green) with OTII T cells (red) or SMARTA T cells (blue) in the presence of OVA^323–339 (A and B), OVA (C and D), SMARTA peptide (E and F), or OVA and anti-MHCII antibody (G and H). Aortas were explanted from Apoe^–/– mice fed with WD for 12 weeks. See Supplemental Videos 5 and 6 for time series. (A, C, E, and G) Interacting APCs and T cells (white arrows) were located in the adventitia. Insets in A and E show surface-rendered OTII (A; enlarged ×8) or SMARTA (E; enlarged ×7) T cells interacting with CD11c-YFP⁺ APCs. (B, D, F, and H) Percent interacting OTII and SMARTA T cells. Data are mean ± SEM. Scale bars: 80 μm (A); 70 μm (C and G); 100 μm (E).

Figure 4. 2-photon imaging of Apoe^–/– CD4⁺ T cell–APC interaction in the plaque and adventitia of Apoe^–/– aorta.

(A) Cross-sectional view of innominate artery plaque and media. CD11c-YFP⁺ cells are stained green, and SNARF-labeled Apoe^–/– CD4⁺ T cells are stained red. (B) Velocities of individual freely migrating (n = 126) and interacting (n = 47) T cells. No exogenous antigen was added. Average is indicated by horizontal line. (C) Percent interaction with CD11c-YFP⁺CD4⁺ T cells isolated from spleens and lymph nodes of atherosclerotic WD-fed Apoe^–/– or B6 mice. Data are mean ± SEM of 2 independent experiments. (D) Maximum intensity projection (top view) through image stacks (760 × 760 × 225 μm [x, y, z]). CD11c-YFP⁺ cells are stained green, SNARF-labeled Apoe^–/– CD4⁺ T cells are stained red, and CMRA-labeled OTII CD4⁺ T cells are stained blue. No antigen was added. Interacting cells (arrows) were located in the adventitia. See Supplemental Video 7. (E) Mean velocities of individual prestimulated (CD3/CD28) OTII and freely migrating or interacting Apoe^–/– CD4⁺ T cells, isolated from Apoe^–/– mice fed WD for 20 weeks. Average is indicated by horizontal line. *P < 0.05; **P < 0.01. Scale bars: 100 μm (A); 30 μm (A, inset); 70 μm (D).

Figure 5. 2-photon imaging of interactions of activated B6 or Apoe^–/– CD4⁺ T cells with APCs in the adventitia of atherosclerotic aorta.

(A and B) Maximum intensity projection along z axis (top view) through image stacks (760 × 760 × 225 μm [x, y, z]). CD11c-YFP⁺ cells are stained green, and SNARF-labeled activated CD44^hiCD62L^– B6 (A) or Apoe^–/– (B) CD4⁺ T cells are stained red. (C) Percent interacting CD11c-YFP⁺ APCs and CD44^hiCD62L^–CD4⁺ T cells isolated from atherosclerotic WD-fed Apoe^–/– or B6 mice. (D) Interaction times between CD11c-YFP⁺ APCs and Apoe^–/– or B6 CD44^hiCD62L^–CD4⁺ T cells. Each symbol represents 1 T cell–APC interaction. Data are mean ± SEM. *P < 0.05. Scale bars: 70 μm (A); 100 μm (B).

Figure 6. Cytokine secretion and T cell proliferation in mouse aorta.

(A) IFN-γ and TNF-α protein levels from supernatants of explanted intact Apoe^–/– aortas incubated with sorted CD44^loCD62L⁺ naive or CD44^hiCD62L^– activated (effector-memory) CD4⁺ T cells isolated from Apoe^–/– or B6 mice. No antigen was added. Average of 3 independent experiments. (B and C) Aortic TCRβ⁺ T cell proliferation was analyzed by i.p. BrdU injection 24 and 12 hours prior to collection of aortas. Aortas of B6 (n = 4) and Apoe^–/– (n = 8) mice after 15 weeks of WD feeding were digested and stained for aorta-infiltrating cells. Numbers indicate percentage in each quadrant. Data are mean ± SEM. *P < 0.05.

Figure 7. oxLDL and mmLDL uptake and foam cell formation in mouse CD11b⁺ aortic macrophages.

Uptake of oxLDL (A) or mmLDL (E), CD36, and MHCII expression in CD11b⁺ macrophages in explanted aortas cultured with OTII T cells in the presence or absence of OVA^323–339. Shown are representative experiments. Average ± SEM oxLDL (B) or mmLDL (F) uptake in 3 independent experiments. oxLDL (C) and mmLDL (G) in CD11b⁺ aortic macrophages, measured in the presence of OVA^323–339 with (open histograms) or without (filled filled histograms) MHCII blocking antibody. Shown are representative experiments. CD11b⁺ cells taking up oxLDL (D) or mmLDL (H) in explanted aortas from Apoe^–/–CD11c-YFP⁺ mice cultured with OTII T cells in the presence of OVA^323–339 with or without anti-MHCII antibody. Data (mean ± SEM) are expressed as a percentage of the untreated value. *P < 0.05.

Figure 8. Depletion of CD11b^–CD11c⁺ cells in the mouse aorta decreases oxLDL uptake.

(A). DT was injected into Apoe^–/–Cd11c-DTR⁺ bone marrow chimeric mice fed WD for 12 weeks during the last 2 weeks of WD feeding. Aortas was harvested, digested, and stained for CD11b and CD11c. Percentage (A) and absolute number (B) of live CD45⁺ CD11b⁺CD11c^–, CD11b⁺CD11c⁺, and CD11b^–CD11c⁺ cells in aortas from Apoe^–/–Cd11c-DTR⁺ mice injected with DT (n = 5) or vehicle control (n = 5). (C) Percent CD11b⁺ cells taking up oxLDL in explanted aortas from 24-hour CD11c-depleted and nondepleted Apoe^–/–Cd11c-DTR⁺ mice cultured with OTII T cells in the presence of OVA^323–339. Data (average of 3 independent experiments) are expressed as a percentage of oxLDL uptake in the absence of CD11c depletion. (D) Expression of Il6 and Il12p35, as determined by real-time PCR, in aortas of mice as in A. Data are mean ± SEM. *P < 0.05.