Tertiary Lymphoid Organs in Takayasu Arteritis

Abstract

Objective: The role of B cells in the pathogenesis of Takayasu arteritis (TA) is controversial. We aimed to study the presence of tertiary lymphoid organs (TLOs) in the aortic wall of TA patients.

Methods: Hematoxylin and eosin-stained sections from aorta specimens from patients with TA were screened for TLOs. The presence of B cell aggregates (CD20), follicular dendritic cells (FDCs, CD21), and high endothelial venules (HEVs, PNAd) was investigated by immunohistochemistry. Immune cells from the adventitial layer of one patient were characterized by flow cytometry. Demographic, medical history, laboratory, imaging, treatment, and follow-up data were extracted from medical records.

Results: Aorta specimens from Bentall procedures were available from seven patients (5 females, aged 22-57 years) with TA. Surgical treatment was performed at TA diagnosis (n = 4) or at a median of 108 months (84-156) after TA diagnosis. Disease was active at surgery in four patients according to NIH score. B cell aggregates-TLOs containing HEVs were observed in the adventitia of all but one patient. Of note, ectopic follicles containing CD21(+) FDCs were found in all patients (4/4) with increased aortic (18)F-fluoro-deoxyglucose (FDG) uptake before surgery but were absent in all but one patients (2/3) with no FDG uptake. In addition, flow cytometry analysis confirmed the accumulation of memory/germinal center-like B cells in the adventitial layer and showed the presence of antigen-experienced T follicular helper cells.

Conclusion: Ectopic lymphoid neogenesis displaying functional features can be found in the aortic wall of a subset of patients with active TA. The function of these local B cell clusters on the pathogenesis of TA remains to be elucidated.

Keywords: B cells; Takayasu arteritis; immunopathogenesis; tertiary lymphoid organs.

Figure 1

Granulomas versus tertiary lymphoid organs (TLOs) in TA patients. Hematoxylin and eosin staining showing the presence of granuloma [first column (A)] in the aortic wall identified as CD14⁺⁺CD20⁻CD21⁻ cell aggregates by using a combination of CD21 (blue) CD20 (brown) [first column (B)] and CD14 (blue) CD20 (brown) [first column (B)] staining. Other cell aggregates also visible on hematoxylin and eosin-stained sections [second column (A)] are instead TLOs structures displaying few CD14⁺ cells (blue), many CD20⁺ B cells (brown), and CD21⁺ FDCs (blue) (B). Representative picture (C) showing the distribution of granulomas (CD14⁺⁺) in contact with the medial layer and TLOs (CD20⁺⁺) located deeper within the adventitial layer. Orcein staining (D) suggests that granulomas are physically implicated in medial destruction, whereas TLOs might develop in response to sustained inflammation in the adventitia.

Figure 2

Distinct features of granulomas and TLOs. Granulomas were made of clusters of CD14⁺ cells and of CD15⁺ cells (A,B) where very few B cells (inset 1) could be detected. Granulomas were also characterized by an intense angiogenesis [CD31⁺ vessels (C) and inset 2]. These adventitial neovessels were PNAd⁻ (C). Few podoplanin + lymphatic vessels were also associated with blood vessels [(C) and inset 2]. At variance, CD31⁺ blood vessels with endothelial cells displaying a high endothelial venule phenotype and expressing PNAd [(E) and inset 4] were systematically observed within the B cell clusters [(D) and inset 3] that are composed of TLOs. A rich network of CD31⁺ Podoplanin⁺ lymphatics was detected around TLOs [(E) and inset 5].

Figure 3

FDG-PET-CT imaging in TA patient. Coronal (A), sagittal (B), and axial (C) slices of the thoracic aorta with FDG-PET imaging in P05. Note the presence of a linear, high FDG uptake in the ascending thoracic aorta on PET corresponding to the anterior region of the aortic wall from an aortic aneurysm on corresponding CT images. The left and the right imaging are, respectively, ¹⁸FDG PET and fused PET/CT images showing high tracer uptake on the ascending aorta (Red arrows). Hematoxylin and eosin staining (D) of CD21⁺ (blue) CD20⁺ (brown) TLOs in the adventitial layer of the aortic wall of the same P05 patient.

Figure 4

Accumulation of memory B cells, germinal center B cells, and CD4⁺ T follicular helper cells in the adventitia of TA diseased aortas. (A) Accumulation of immune cells (Viability⁻CD45⁺) and B cells (HLA-DR⁺CD19⁺) analyzed by flow cytometry in the adventitial layer of the core of the aortic lesions of a TA patient (P01) as compared to the neck of the same sample. (B) Characterization of B cells in the adventitia of the aortic lesion by flow cytometry shows that most B cells have a memory phenotype (CD27⁺IgD⁻) with some harboring a germinal center phenotype (CD95⁺CD24⁻IgD⁻CD27^high). (C) Flow cytometry analysis of adventitial tissue samples reveals an increase in CD4⁺ T cells in the aortic lesion (top panel), as well as the presence of CD4⁺ T cells harboring a Tfh cell phenotype (PD1⁺CXCR5⁺), as compared to aortic tissues sampled in the aneurysmal neck. (D) CXCR5⁺CD4⁺ T cells from the adventitia of the core aortic lesion are Bcl6^high. (E) CD4⁺ T cells from the adventitia of the neck (gray), non-Tfh cells (CXCR5⁻Bcl6⁻; Red), and Tfh cells (CXCR5⁺Bcl6^high; blue) were analyzed for CD27 and CD45RA expression. As compared to CD4⁺ T cells from the neck, which are a mixture of naive and antigen-experienced (CD27⁻) and memory cells (CD45RA⁻), the adventitia of the core of the aortic lesion contains more antigen-experienced and no naive T cells (CD45RA⁺). Interestingly, Tfh cells in the adventitia are CD45RA^int and display an antigen-experienced phenotype (CD27⁻).