T Cells Are Dominant Population in Human Abdominal Aortic Aneurysms and Their Infiltration in the Perivascular Tissue Correlates With Disease Severity

Abstract

Abdominal Aortic Aneurysm (AAA) is a major cause of cardiovascular mortality. Adverse changes in vascular phenotype act in concert with chronic inflammation to promote AAA progression. Perivascular adipose tissue (PVAT) helps maintain vascular homeostasis but when inflamed and dysfunctional, can also promote vascular pathology. Previous studies suggested that PVAT may be an important site of vascular inflammation in AAA; however, a detailed assessment of leukocyte populations in human AAA, their anatomic location in the vessel wall and correlation to AAA size remain undefined. Accordingly, we performed in depth immunophenotyping of cells infiltrating the pathologically altered perivascular tissue (PVT) and vessel wall in AAA samples at the site of maximal dilatation (n = 51 patients). Flow cytometry revealed that T cells, rather than macrophages, are the major leukocyte subset in AAA and that their greatest accumulations occur in PVT. Both CD4⁺ and CD8⁺ T cell populations are highly activated in both compartments, with CD4⁺ T cells displaying the highest activation status within the AAA wall. Finally, we observed a positive relationship between T cell infiltration in PVT and AAA wall. Interestingly, only PVT T cell infiltration was strongly related to tertiles of AAA size. In summary, this study highlights an important role for PVT as a reservoir of T lymphocytes and potentially as a key site in modulating the underlying inflammation in AAA.

Keywords: T cell; abdominal aortic aneurysm; inflammation; macrophages; perivascular adipose tissue.

Figure 1

Aortic abdominal aneurysm (AAA) leukocyte infiltration: comparison and relationship between aneurysmal wall and PVT. (A) Gating strategy depicting identification of total leukocytes (CD45⁺) and leukocyte sub-populations: granulocytes, dendritic cells, B cells, T cells, NK cells and macrophages in AAA (aortic abdominal aneurysm). Gates were applied based on fluorescence minus one (FMO) analysis. (B) Distribution of the main leukocytes: T cells (CD3⁺), B cells (CD19⁺), NK cells (CD16⁺CD56⁺), macrophages (CD11b⁺CD64⁺), dendritic cells (CD83⁺), granulocytes (CD66b⁺) in wall and PVT of AAA tissue (n = 8–11), T cell percentages compared to other leukocyte subpopulations, t-test for related samples, p values presented on graphs only for statistically significant comparisons. (C) Bottom panel: example of immunofluorescence staining of T cells (CD3⁺) shown in red and macrophages (CD68⁺) shown in green in PVT and wall of an AAA. Nuclear staining (DAPI) is shown in blue. Example of T cell and macrophage co-localization shown by yellow/orange staining; white arrow. Representative of n = 5; Top panel: negative control consisting of secondary antibody staining only with DAPI. Scale bar = 50 μM.

Figure 2

Aortic abdominal aneurysm (AAA) leukocyte infiltrate: comparison and relationship between aneurysmal wall and PVT. (A) Example of flow cytometric identification of leukocytes (CD45⁺), total T cells (CD3⁺) and CD4⁺, CD8⁺ T cell subpopulations in aneurysmal wall and PVT. (B) Leukocyte number per mg of aneurysmal wall vs. PVT, n = 40, **p < 0.01 (Wilcoxon matched paired). (C) T cells number per mg of aneurysmal wall vs. PVT, n = 39, *p < 0.05 (Wilcoxon matched paired). (F) Spearman rho correlation between number of leukocytes in aneurysmal wall and PVT; R = 0.384, n = 40, p = 0.015. (G) Spearman rho correlation between number of T cells in aneurysmal wall and PVT; R = 0.418, n = 39, p = 0.007. (D) CD4⁺ T cell number per mg of wall and PVT of AAA tissue, n = 39, p < 0.05 (Wilcoxon matched paired). (E) CD8⁺ T cell number per mg of wall and PVT in AAA, n = 39, NS (Wilcoxon matched paired).

Figure 3

Effect of patient gender on immune cell numbers in AAA wall and PVT. Male vs. female total leukocyte and T cell content in AAA PVT and wall. (A) Leukocyte number per mg of aneurysmal wall and PVT in male group n = 31 vs. female group n = 9 (B). T cell number per mg of aneurysmal wall and PVT in male group n = 30 vs. female group n = 9. Mann-Whitney test for comparison male vs. female for wall and PVT, Wilcoxon test for comparison wall vs. PVT within male and female group. p values presented on graphs only for statistically significant comparisons.

Figure 4

Relationship between T cell number and AAA size. AAA diameter was determined by preoperative CT. Graphs display relationship between AAA size (tertile 1 ≤ 53 mm; tertile 2 ≤ 60 mm; tertile 3 > 60 mm) and CD3⁺ cell infiltration in wall (A) and PVT (B) (mean/SEM/25–75 CI); (n = 20/10/9 for tertiles); statistical comparisons were performed by ANOVA with Neuman-Keuls post-hoc analysis.