Ex vivo expansion of regulatory T cells from abdominal aortic aneurysm patients inhibits aneurysm in humanized murine model

Abstract

Objective: Abdominal aortic aneurysm (AAA) is a chronic inflammatory disease. Studies of human aneurysm tissue demonstrate dense inflammatory cell infiltrates with CD4⁺ T cells predominating. Regulatory T cells (Tregs) play an important role in inhibiting pro-inflammatory T cell proliferation, therefore, limiting collateral tissue destruction. The aim of this study was to investigate whether ex vivo augmentation of human Tregs attenuates aneurysm formation in humanized murine model of AAA.

Methods: Circulating Treg population in AAA patients and age- and gender-matched controls were determined by real-time polymerase chain reaction and flow cytometry. To create humanized murine model of AAA, irradiated Rag1-deficient (Rag1^-/-) mice, without mature T lymphocytes, at 7 weeks of age were given 5 × 10⁶ of human CD4⁺ T cells intraperitoneally. Then the mice underwent CaCl₂ aneurysm induction. Aortic diameters were measured before and at 6 weeks after aneurysm induction. Aortic tissue was collected for histology and protein extraction. Verhoeff-Van Gieson stain was used for staining elastic fiber. CD4⁺ T cells in the aortic tissue were detected by immunohistochemical staining.

Results: In human peripheral blood mononuclear cells, the proportion of Tregs are decreased in AAA patients compared with matched control patients with significant vascular disease. We first validated the role of Tregs in the CaCl₂ model of AAA. To determine the role of human T cells in AAA formation, Rag1^-/- mice, resistant to CaCl₂-aneurysm induction, were transplanted with human CD4⁺ T cells. Human CD4⁺ T cells were able to drive aneurysm formation in Rag1^-/- mice. We show that ex vivo augmentation of human Tregs by interleukin-2 resulted in decreased aneurysm progression.

Conclusions: These data suggest that the ex vivo expansion of human Tregs may be a potential therapeutic strategy for inhibiting progression of AAA.

Keywords: Aneurysm; Aorta; Inflammation; Regulatory T cells.

Fig 1.

Treg proportion in the peripheral blood of abdominal aortic aneurysm (AAA) patients was significantly decreased compared with control patients. Human peripheral blood mononuclear cells were isolated from peripheral blood of AAA and control patients. Total RNA was extracted and messenger RNA levels of Foxp3 and CD4 genes were analyzed by reverse transcriptase polymerase chain reaction (RT-PCR). A, Relative messenger RNA expression levels of Foxp3 to CD4 in human Peripheral blood mononuclear cells (PBMCs) from control (n = 52) and AAA (n = 48) patients. The ratio of Foxp3 to CD4 mRNA levels is shown. B, Percentage of Tregs (CD4⁺CD25⁺FoxP3⁺) in the peripheral blood of control (n = 12) and AAA (n = 10) patients determined by flow cytometry; representative dot plot (left). Data represented as mean 6 standard deviation, *P < .05.

Fig 2.

Human CD4⁺ cell infusion promoted aneurysm formation in Rag1^−/− mice. A, The bar graph shows aortic diameters at baseline (Pre) and 6 weeks after (Post) NaCl or CaCl₂ incubation. Verhoeff-Van Gieson (VVG) staining of aortic tissue from (B) NaCl- or (C) CaCl₂-treated Rag1^−/− mice and (D) NaCl- or (E) CaCl₂-treated Rag1^−/− mice infused with CD4⁺ cells from abdominal aortic aneurysm (AAA) patients. F and G, Immunostaining of human CD4⁺ cells in the aortic tissue sections with corresponding VVG staining of (D and E). Three different aortas from each group were stained and representative sections are shown. Positive cells are indicated by arrows. *P < .05 compared CaCl₂-treated Rag1^−/− mice; ǂP < .05 compared NaCl-treated mice.

Fig 3.

Foxp3 deficiency exacerbated aneurysm expansion. A, Aortic diameter changes in Rag1^−/−/WT and Rag1^−/−/Foxp3^−/− mice before and after aneurysm induction. B-E, Histological changes in the mouse aortas as seen with Verhoeff-Van Gieson (VVG) stain. Sections were photographed and shown with the lumen at the top. Five different aortas from each of the four groups were stained and representative sections are shown. F, Immunostaining for mouse CD4⁺ T cells in Rag1^−/−/WT (G) and Rag1^−/−/Foxp3^−/− (H) aortic tissue 6 weeks after aneurysm induction (n = 4/group). CD4⁺ T cell number was quantified by a reviewer blinded to the mouse genotype. The bar graph (F) demonstrates the quantitative differences in CD4⁺ cells. Data are shown as the mean ± standard deviation. *P < .05, compared with NaCl-treated controls, #P < .05, compared with CaCl₂-treated Rag1^−/−/WT mice, ǂP < .05, compared with CaCl₂-treated Rag1^−/−/WT mice. Representative stainings are shown in the left panels. CD4⁺ cells are indicated by arrows.

Fig 4.

Treg augmentation by interleukin-2 (IL-2) treatment inhibited aneurysm formation and elastin degradation. A, Aortic diameter changes in CaCl₂-treated Rag1^−/− mice infused without (baseline Tregs; n = 10) and with IL-2-treated (augmented Tregs; n = 6) human CD4⁺ T cells from abdominal aortic aneurysm (AAA) patients. Histological changes in the mouse aortas as seen with VVG stain. CaCl₂-treated Rag1^−/− infused with untreated (B) and IL-2-treated (C) CD4⁺ T cells from AAA patients. *P < .001 compared with the mice with augmented Tregs. D, Tregs (CD4⁺CD25⁺FoxP3⁺) population with (augmented) and without (baseline) IL-2 stimulation in PBMC of AAA patients (n = 6) determined by flow cytometry.