Immunohistochemical validation of CyTOF single
cell data reveals chronic inflammatory cell
infiltrates comprising T and B cells in the
adventitia of abdominal aortic aneurysms
Bachelor thesis Charid van Stroe Gomez
11704217
Bachelor of Biomedical Sciences University of Amsterdam
Faculty of Science
Daily supervisor LUMC: Laura Bruijn
Principal investigator LUMC: Dr. Jan Lindeman
Assessor UvA: Dr. Maurice van den Hoff
Immunohistochemical validation of CyTOF single cell data reveals
chronic inflammatory cell infiltrates comprising T and B cells in the
adventitia of abdominal aortic aneurysms
Charid van Stroe Gomez, July 15, 2020
Abstract. An abdominal aortic aneurysm (AAA) is a life-threatening disease, characterised by weakening of the aortic wall and chronic inflammation. However, the complex molecular mechanisms of chronic inflammation in AAA are not fully understood and there is no effective drug therapy available. For the development of immunotherapies, it is crucial to do further research on the precise immunological landscape of AAA. This study validates CyTOF single cell data of immune cell subsets in AAA by using IHC, focussing on B, T, and NK cells. This study consists of two aspects. The first aspect of this study has established a consensus classification scheme that covers AAAs, based on their histological characteristics. This has revealed that AAAs comprise a large histological heterogeneity. A minimal group size of n=30 was required as a representative sample set. The second aspect of this study has revealed that the IHC results correspond to the CyTOF data. Regarding the cellular localisation of B, T and NK cells, this study revealed that adventitial inflammatory cell infiltrates present in AAAs contain B and T cells. NK cells were virtually absent. The consensus classification established in this study might help to identify core mechanisms involved with AAA progression. In addition, the IHC validation of the CyTOF data has generated deeper insights for the development of anti-inflammatory drugs for AAA treatment. Yet, further validation of the CyTOF dataset is required to investigate the complete immune cell population in AAAs.
1. Introduction
An abdominal aortic aneurysm (AAA) is a life-threatening disease, affecting 1-7% of the population over the age of 50. When the abdominal aorta is dilated 1.5 times the original vessel diameter to a diameter of least 3 cm, it is classified as an AAA (Kühnl et al., 2017). Risk factors of its development include male gender, older age, smoking, and family history of AAAs (Lederle et al., 2000). Regularly, a patient with AAA does not experience any symptoms and disease progression is slow. Yet, upon aortic rupture, it can cause fatal internal bleeding with a pre-hospitalisation mortality rate between 60 and 80% (LeFevre, 2014; A Rahimi, 2019). Rupture risk of AAA is more than 25% in aneurysms larger than 5 cm in diameter, and only 2% in aneurysms smaller than 4 cm in diameter (Cosford et al., 2007).
AAAs are characterised by weakening of the aortic wall and chronic inflammation (Hellenthal
et al., 2009). Inflammatory cell infiltration from both the adaptive and innate immune system,
have been shown to be crucial for AAA formation and progression (Liu et al., 2015; Sakalihasan et al., 1996). However, the complex molecular mechanisms of chronic inflammation in AAA are not fully understood.
The present focus in aneurysmal care is rupture prevention through surgical repair of the dilated aorta segment. The threshold for surgical repair is an aortic diameter of 5.5 cm. Surgical repair
of smaller aneurysms has shown no clinical benefits, considering that the health risks of this procedure outweighed the possible benefit of reduced chance of AAA rupture (Fleming et al., 2005). Ongoing clinical challenges include the lack of effective drug therapies that can inhibit AAA formation and progression, and thus prevent the need for surgery (Klink et al., 2011). Inhibiting inflammatory responses involved in AAA may bring a halt to its progression and may therefore be a therapeutic approach for its prevention and treatment (Shimizu et al., 2006; Lui et al., 2014). For the development of immunotherapies that can modulate proinflammatory responses, it is crucial to do further research on the precise immunological landscape of AAA.
A study of Cassimjee et al. (2018) uses mass cytometry (CyTOF) to gain deeper understanding of the immunogenicity of AAAs. CyTOF is a method that can simultaneously detect up to 42 metal-conjugated antibodies per cell, making it possible to characterise inflammatory cell subpopulations based on their unique set of markers (Spitzer et al., 2016; Winkels et al., 2018). Nevertheless, challenges encountered with this technology start with the initial isolation of single cells. Furthermore, enzymatic tissue digestion required for the production of a single cell suspension, can destroy potential surface marker epitopes needed for correct cell subset characterisation (Landsdowne & Rehak, 2019). Most importantly, valuable spatial information about the colocalisation of target cells is lost.
This study validates the CyTOF single cell data by Cassimjee et al.(2018), by investigating components of the innate and adaptive immune landscape in human AAAs using immunohistochemistry (IHC). By using IHC, the presence and localisation of target cells in a tissue section can be revealed and potential loss of epitopes avoided. This relatively easy and low-cost method utilises antibodies to identify antigens in cells of tissue sections, without destruction of its histological architecture (Ramos-Vara & Miller, 2014; Schacht & Kern, 2015). In order to correctly choose a representative sample set that portrays the natural histological variation seen in AAAs, first, all wall samples will be analysed according to their histological characteristics. Secondly, based on the histological analysis, an AAA classification scheme will be created. Next, every individual sample will be graded accordingly and will be visually represented in a heatmap. Based on this evaluation, a representative set of samples will be defined for IHC analyses, focussing on T cells, B cells, and NK cells.
2. Materials and methods
2.1. Patients and Tissue Collection
AAAs specimens obtained during elective and emergency open repair surgery, were randomly collected from 73 patients from the Vascular Tissue Bank at the Department of Vascular Surgery, Leiden, the Netherlands. Atherosclerotic infrarenal abdominal aortic wall samples were used as non-aneurysmal controls, due to the presence of atherosclerotic lesions in AAAs. These reference samples were obtained during clinical organ transplantation and were classified according to the Virmani classification, as adaptive intimal thickening (AIT; early stage atherosclerotic lesion), late fibroatheroma (LFA; middle stage atherosclerotic lesion), and fibrocalcific plaque (FCP; late stage atherosclerotic lesion (Yahagi et al., 2016). All samples were acquired in accordance with the guidelines of the Medical and Ethical committee in Leiden and the code of conduct of the Dutch Federation of Biomedical Scientific Societies. After tissue collection, samples were washed briefly in phosphate buffer (0.2 M, pH 7.4),
decalcified in Kristensen’s fluid for 120 hours and immersion fixed in 10% neutral buffered formalin for 24 hours, before being embedded in paraffin for histological and immunohistochemical examination.
2.2. Histological analysis and assessment for sample collection
In order to choose a correct group size of samples that represents the heterogeneity of AAA disease, each sample was histologically analysed. Histological and immunohistochemical analyses were performed on cross-sections (4 µm) of aortic tissue samples. Deparaffinized rehydrated tissue sections were stained with Movat’s pentachrome and haematoxylin and eosin (H&E staining) to asses cellular composition and tissue morphology (see supplemental materials for detailed protocols). Histological assessment was performed on each tissue sample by two independent observers. In case of disagreement a third observed was included in the assessment. Both observers were blinded from patient characteristics. Each histological component was scored according to the highest stage present in the tissue sample. Digitalization of the slides was done using a Philips digital pathology slide scanner (IntelliSite Ultra Fast Scanner).
2.3. Immunohistochemistry for the validation of CyTOF data
To validate the CyTOF single cell data, IHC analyses were performed to localise T cells, B cells, and NK cells. All primary antibodies (table 1) were diluted in PBS-1% albumin and incubated overnight at room temperature. Heat-induced (TRIS/EDTA pH 9.2) antigen retrieval was performed for 10 minutes at 95˚C. Endogenous peroxidase activity was blocked with a 20-minute incubation of 0,3% hydrogen peroxide. Antibody binding was visualised using a polymer-based horseradish peroxidase (HRP) labelled secondary system (mouse or anti-rabbit EnVision+ System, Dako, Amstelveen, the Netherlands) or a polymer based alkaline phosphatase (AP) labelled secondary system (MACH2, Biocare Medical, Concord, CA) along with matching chromogens. Anti-mouse or anti-rabbit Dako EnVision or MACH2 were applied for 30 minutes. Chromogens used for visualisation were diaminobenzidine (DAB) for the HRP label for 10 minutes, and Vulcan Fast Red (product number FR805, Biocare Medical, California, United States) or Ferangi Blue (product number FB813S, Biocare Medical, California, United States) for the AP label, for 5 minutes and 10 minutes, respectively. Vulcan Red and Ferangi Blue chromogens were chosen because they establish easily distinguishable single positive (red or blue in colour) and double positive cells (purple in colour). Afterwards, nuclei were counterstained using Mayer’s haematoxylin (Merck Millipore, The Netherlands) (see supplemental materials for detailed protocols). Human tonsil tissue sections were used as positive controls, and negative controls were AAA tissue samples in which the primary antibody was omitted.
Table 1: Antibodies used in present study
DAB = diaminobenzidine
3. Results
3.1. Patient characteristics
Clinical data of the patients involved in this study were obtained from medical records and are listed in table 2. The patient group consisted of 15 women (21%) and 58 men (79%), with ages ranging between 55 and 89 years (median, 70 years). Regarding the stage of AAA, the diameter of the abdominal aorta ranged between 4,4 and 14 cm (median, 6.7 cm). 78.1% of the patients had a smoking history, 69.9% used statins, and 63.0% used antihypertensive medications.
Antibody, clone Host isotype; subclass Specificity Pre-treatment Dilution Reference/ source Chromogen CD4, H-370 Polyclonal rabbit IgG T-helper cells Tris/EDTA (pH 9.2) 1:800 Santa Cruz Biotechnology, Dallas TX Vulcan red CD7, Catalog # PA5-78998 Polyclonal rabbit IgG T cells, NK cells Tris/EDTA (pH 9.2)
1:100 Invitrogen Vulcan red
CD8, C8/114B Monoclonal mouse IgG1, kappa Cytotoxic T cells, suppressor T cells Tris/EDTA (pH 9.2) 1:200 Dako, Amstelveen, the Netherlands Vulcan red CD20, L26 Monoclonal mouse IgG2a kappa Mature B cells, follicular dendritic cells Tris/EDTA (pH 9.2) 1:4000 Dako, Amstelveen, the Netherlands DAB CD57, TB01 Monoclonal mouse IgM, kappa NK cells, cytotoxic T cells Tris/EDTA (pH 9.2) 1:200 Dako, Amstelveen, the Netherlands Ferangi blue Granzyme B, D6E9W Monoclonal rabbit IgG NK cells, Cytotoxic T cells Tris/EDTA (pH 9) 1:200 Cell signalling technology DAB
Table 2: Clinical characteristics of patients with AAA (n=73) Age in years (mean ± SD) 71 ± 6.5
Gender (male/female) 58/15
Aortic diameter in cm (mean ± SD) 6.9 ± 1.8 Smoking history no. (%)
Current: 32 (45.1%) No: 14 (19.7%) Former: 25 (35.2%) Unknown: 2 (2.7%)
Statin use- no. (%) 51 (69.9%)
Antihypertensive medication use- no. (%) 46 (63.0%)
Continuous data are presented as mean (standard deviation). SD: standard deviation.
3.2. Histological analysis of AAA heterogeneity
3.2.1 AAA histopathology is diverse and can be categorised according to a customised classification scheme
In order to inventory the natural variation in AAAs, first, all 73 individual AAA tissue sections were histologically evaluated. Three AAA samples were too impaired and did not meet the quality requirements for further evaluation and were therefore excluded from histological analysis. After evaluation, prominent histomorphical aspects of AAA disease were inventoried and processed in a consensus classification scheme, according to which all samples were scored. Aspects included were transmural fibrosis, mesenchymal cell loss in the former intima/media zone, diffuse transmural inflammation, lymphoid follicle formation in the adventitia, transmural neovessel formation, atherosclerotic lesions, intraluminal thrombus (ILT) organisation, (micro)calcifications, excessive elastolysis, and adventitial adipogenic degeneration. Detailed results are discussed in the following sections (3.2.2 to 3.2.10). Between AAA tissues, gradations in the extend of these aspects were detected. These are described in table 3. Figures illustrating each histological aspect are shown in figures 2 - 10. Visualisation of the most prominent histological characteristics in AAAs is displayed in the heatmap in figure 1.
Figure 1: Heatmap visualising the broad heterogeneity observed in AAA disease. Heatmap visualising the most prominent histological characteristics in AAAs. Each row represents one AAA sample (n=70). Three original samples did not meet the quality requirements and were excluded from analysis but are included in the patient characteristics. Clustering is based upon intimal-medial fibrosis, mesenchymal cell loss, and inflammation. See table 3 for heatmap index. W a ll th ic k n e s s o f in ti m a /m e d ia z o n e F ib ro s is i n i n ti m a /m e d ia z o n e M e s e n c h y m a l c e ll lo s s i n in ti m a /m e d ia z o n e In fl a m m a ti o n : tr a n s m u ra l ly m p h o id i n fi lt ra ti o n In fl a m m a ti o n : ly m p h o id fo lli c le s i n a d v e n ti ti a E x te n t o f n e o v e s s e l fo rm a ti o n A th e ro s c le ro ti c l e s io n s In tr a lu m in a l th ro m b u s o rg a n is a ti o n C a lc if ic a ti o n E la s ti c f ib e r d e g ra d a ti o n A d v e n ti ti a l a d ip o g e n ic d e g e n e ra ti o n
Table 3: Index to heatmap in figure 1 Fibrosis in intima/media zone 1 10-30% fibrosis
2 30-80% fibrosis in patchy pattern 3 30-80% fibrosis in diffuse pattern 4 >80% fibrosis in patchy pattern 5 >80% fibrosis in diffuse pattern
Mesenchymal cell loss in intima/media zone 1 10-30% loss of mesenchymal cells
2 30-80% loss of mesenchymal cells in patchy pattern 3 30-80% loss of mesenchymal cells in diffuse pattern 4 >80% loss of mesenchymal cells in patchy pattern 5 >80% loss of mesenchymal cells in diffuse pattern
Inflammation: transmural lymphoid infiltration, in vicinity of vasa vasora 0 Low number of perivascular lymphocytes
1 Small (<50 cells) unorganized lymphoid infiltrates 2 Large (≥50 cells) unorganized lymphoid infiltrates Inflammation: lymphoid follicles in adventitia 0 No lymphoid follicles present
1 Early developed tertiary lymphoid-like structure: compaction of lymphocytes, some organization visible (reticular fibres, no germinal centre)
2 Late developed tertiary-like structure: unencapsulated organized lymphoid follicle with a germinal centre and presence of high endothelial venules or lymphatic vessels
Extent of neovessel formation
0 Into part 1 (outer quarter, closest to adventitia) 1 Into part 2 (outer middle, close to adventitia) 2 Into part 3 (inner middle, close to intima) 3 Into part 4 (inner quarter, closest to intima) Atherosclerotic lesions
0 None visible
1 Foam cells/Lipid pool(s) located directly under the intraluminal thrombus (ILT) 2 Foam cells/Lipid pool(s) located not directly under the ILT
3 Necrotic core deeply embedded in the aortic wall, without a classic overlying fibrous cap
4 Superficial necrotic core, resembling a classic atherosclerotic lesion 5 Calcified sheet
Intraluminal thrombus (ILT) organisation 0 No ILT
1 Fibrin and/or cholesterol rich ILT, clearly delineated from intima
2 Reorganizing ILT: incorporation into intima, no immune cell infiltration in ILT 3 Reorganizing ILT: incorporation into intima, immune cell infiltration in ILT
4 Organised: matrix deposition and spindle shaped cell ingrowth in ILT, with(out) calcifications
5 Highly organised: matrix deposition, spindle shaped cell ingrowth and capillary ingrowth, with(out) calcifications
Universal microcalcifications 0 No calcifications
1 Calcifications in intraluminal thrombus 2 Calcifications in inner wall
3 Calcifications in outer wall
4 Calcifications in both inner and outer wall Aorta wall thickness (intima+media) 1 Q1: 0.43-0.8 cm 2 Q2: 0.83-1.095 cm 3 Q3: 1.1-1.49 cm 4 Q4: 1.5-3.05 cm Elastolysis 1 95-98% 2 99-100%
Adventitial adipogenic degeneration 0 Absent
3.2.2. Wall thickness variation in AAAs samples
Wall thickness was measured as the intima-media thickness (IMT) in the abdominal aneurysm: the largest distance from the luminal margin of the intima up to the margin of the vascular plexus or up to lymphoid follicles present in the media-adventitia transition zone (see figure 2). An IMT was found of (mean ± SD) 1.2 mm ± 0.53 mm, ranging between 0.43-3.05 mm.
Figure 2: Measurements of the intima-media thickness (IMT) of the AAA wall. The largest distance between the intima and the vascular plexus/lymphoid follicles was measured. Movat pentachrome stain, colour legend: ochre: collagen, black: nuclei, elastic fibres, blue/green: proteoglycans/ground substance/ECM(extracellular matrix), red: mesenchymal cells.
3.2.3. Intimal/medial fibrosis and elastolysis in AAAs
Evaluation of the elastic and collagen fibres in the AAA tissue samples showed almost complete degradation of the elastic fibres (>95%) in all tissue samples, and highly collagenous ECM in the former intima/media, better known as intimal/medial fibrosis. 51% of the tissue samples showed more than 80% of fibrosis in either a diffuse or patch pattern. Remaining samples demonstrated between 30 to 80% intimal/medial fibrosis in a diffuse or patchy pattern. No samples demonstrated low (10-30%) of intimal/medial fibrosis (see figure 3).
Figure 3: Movat pentachrome stain of complete elastolysis in AAA. (A) Tissue sample displaying 99-100% transmural elastolysis. (B) Higher magnification of image A, showing a lack of elastic fibres in the former media zone of the aneurysmal tissue.
3.2.4. Mesenchymal cell loss in intima/media zone of AAAs
All AAA tissue samples exhibited a loss of more than 30% of mesenchymal cells. Of all samples, 33 (47%) showed between 30 and 80% loss of mesenchymal cells in an either diffuse or patchy pattern. 37 samples (53%) showed more than 80% loss of mesenchymal cells in either a patchy or diffuse pattern (figure 4).
Figure 4: Movat pentachrome stain of intimal/medial fibrosis in AAA and mesenchymal cell loss. (A) Tissue sample displaying 30-80% fibrosis in a patchy pattern. (B) Tissue sample displaying >80% fibrosis in a diffuse pattern. (C) Higher magnification of image A, showing 30-80% loss of mesenchymal cells in a patchy pattern. (D) Higher magnification of image B, showing >80% of mesenchymal cell loss in a diffuse pattern.
3.2.5. Lymphoid cell involvement in AAAs
Two distinct patterns of lymphoid infiltrations were observed in the AAA samples. The first pattern consisted of small (<50 cells) or large (≥50 cells) diffusely distributed and unorganised transmural lymphoid infiltrations. With regard to this pattern, 20 samples (29%) exhibited small unorganized lymphoid infiltrates, while the majority of the samples (71%), exhibited large unorganised lymphoid infiltrates in its vessel wall. The second pattern observed in the AAA samples consisted of organised lymphoid follicles in the adventitia. These compact lymphoid follicles, or tertiary lymphoid structures (TLSs), were identified according to their organisation stage, earlier described by Sato et al (2020). Early TLSs were considered unencapsulated lymphoid follicles with some organisation visible, such as reticular fibres and follicular dendritic cells, but without the presence of a germinal centre (GC). Late TLSs were considered organised lymphoid follicles containing prominent GCs, high endothelial venules (HEVs), and lymphnode-like conduits (Gräbner et al, 2009). Regarding this adventitial follicle pattern, 31 samples (44%) demonstrated early tertiary lymphoid like structures, and 16 samples (23%)
demonstrated late tertiary lymphoid like structures. 23 samples (33%) showed no adventitial lymphoid follicle formation (figure 5).
Figure 5: H&E staining of inflammatory patters seen in AAA. (A) Tissue sample displaying transmural lymphoid infiltration, in vicinity of vasa vasora (indicated with black arrow). (B) Higher magnification of image A, showing lymphocytic infiltrations deep into the vessel wall. (C) Tissue sample with clear lymphoid follicles in adventitial layers. (D) Late developed tertiary lymphoid-like structure. Lymphatic vessel is indicated with a yellow arrow. germinal centre is indicated with a yellow star. (E) Early developed tertiary-lymphoid like structure. Some organisation visible, but germinal centre is absent.
3.2.6. Neovascularisation in AAAs
Another histological feature observed in the AAA samples was transmural neovessel formation, originating from the adventitial vasa vasorum. To score the grade of neovessel ingrowth, the intima-media zone was divided in four zones of equal width (figure 6A). Part one represents the zone in the media closest to the adventitia, and part four represents the zone closest to the lumen. The large majority of the samples displayed neovascularisation up until part four (luminal intimal border) (38 samples, 54%) or part three (29 samples, 41%). Only a total of 3
AAA samples displayed neovascularisation up until part 1. Neovascularisation in the intra luminal thrombus (ILT) was not appreciated here, but in the aspect of ILT organisation (see section 3.2.8).
Figure 6: Movat pentachrome stain of the extent of neovessel formation. (A) To score the grade of neovessel formation, the intima-media zone was divided in four zones of equal width. Part 1 is closest to the adventitia, part 4 is closest to the intimal-luminal border. (B) Tissue sample with neovascularisation up until part 4. Neovessel is indicated with black arrow.
3.2.7. Atherosclerotic lesions in AAAs
In 36 of the 70 AAA samples (51%), atherosclerotic lesions were detected. These lesions included necrotic core(s) deeply embedded in the aortic wall (16 samples, 23%), differing from classic atherosclerotic lesions due to the absence of a classic overlying fibrous cap. In addition, in 19 samples (27%), superficial necrotic cores were found, resembling a classic atherosclerotic lesion. In one sample, a calcified sheet was encountered. The remaining 34 samples (49%) did not display necrotic cores, but solely lipid pools and/or foam cells localised directly under the intraluminal thrombus or deeper in the vessel wall (figure 7).
Figure 7: Movat pentachrome stain of atherosclerotic lesions in AAA. (A) Tissue section showing foam cells and a lipid pool located directly under the ILT. (B) Tissue section displaying lipid pools located deeper in the vessel wall. (C) Tissue sample with a necrotic core located in the former media zone of the vessel wall, without a classic overlying fibrous cap. (D) Tissue sample with a superficial necrotic core, resembling a classic atherosclerotic necrotic core due to its thin cap. (E) Tissue sample with a calcified sheet, localised deep in the vessel wall, in the former media zone. (F) Higher magnification of the calcified sheet in image E. Visible are lymphoid infiltrations in vicinity of the calcified sheet, indicated with a yellow star.
3.2.8. Intraluminal thrombus organisation in AAAs
Organised intra luminal thrombi (ILT) were a common histological feature in most AAA samples. Few cases (3 samples, 4%) demonstrated young, unorganised and erythrocyte rich ILT, clearly delineated from the intima. Often these ILT depicted so called ‘Lines of Zahn’: microscopic alternating layers of platelets mixed with fibrin and layers of erythrocytes (Lee et
al., 2012). Reorganising ILT, with ILT incorporation in the intima and with/without immune
cell infiltration was seen in 17 cases (24%). 12 cases (17%) showed an organised thrombus, with matrix deposition and spindle shaped cell ingrowth, but no neovessel formation. Yet most
cases (26 samples, 37%), demonstrated a highly organised ILT, with visible extracellular matrix deposition, spindle shaped cell ingrowth and neovessel formation (figure 8).
Figure 8: Movat pentachrome and H&E stain of atherosclerotic lesions in AAA. Tissue samples were classified according to table 3 (A) No ILT. (B) Fibrin rich ILT, clearly delineated from intima. (C) Higher magnification of image B, depicting ‘Lines of Zahn’: alternating layers of platelets merged with fibrin (lighter), and layers of red blood cells (darker). (D) Reorganising ILT: incorporation into intima (E) Organized ILT. Matrix deposition and calcification. (F-H) Highly organised ILT: capillary ingrowth (G) and spindle shaped cell ingrowth, indicated with a black arrow (H).
3.2.9. Microcalcification in AAAs
Another aspect observed in all AAA tissue samples was the presence of universal diffuse microcalcifications. These were scored according to their localisation in the vessel wall. From lumen to adventitia, these localisations include calcifications inside the ILT, calcifications in the area that covers 50% of the intima/media zone closest to the lumen (inner aortic wall), calcifications in the area that covers the remaining 50% of the intima/media zone (outer aortic wall), or calcifications in both inner and outer aortic wall. In 100% of the AAA samples, microcalcifications were spotted in both the inner and outer aortic wall (figure 9).
Figure 9: Movat pentachrome stain of microcalcifications in AAA. (A) Tissue samples displaying microcalcifications throughout the vessel wall. (B) Higher magnification of image A. Microcalficication is indicted with black arrow.
3.2.10. Adventitial adipogenic degeneration in AAAs
In 68 samples (97%) the presence of adventitial adipogenic degeneration was identified: infiltration of isolated adipocyte clusters in the former adventitia zone, with intertwining ECM strands and immune cell infiltrations, but without connection to the periaortic adipose tissue (figure 10).
Figure 10: Movat pentachrome stain adventitial adipogenic degeneration in AAA. Tissue section with visible adipogenic degeneration in the former adventitia zone.
500µm
500 µm
3.3 CyTOF data validation
3.3.1 Representative tissue sample sets for IHC staining were formed in consonance with the heatmap
In order to adequately validate the CyTOF data, a representative set of samples had to be chosen that represent the diverse spectrum of AAA disease. In this study, a total of n=26 AAA tissue samples were chosen on which to perform IHC staining. Tissue selection was performed as follows: in consonance with the heatmap, tissues were organised in groups based on their morphology of the histological characteristics: intimal-medial fibrosis, mesenchymal cell loss, and inflammation. Out of each subgroup one representative tissue sample was chosen for IHC staining. IHC staining was performed on AAA and atherosclerotic control wall samples, to characterise the presence and cellular localisations of B and T lymphocytes (see 3.3.4), and natural killer (NK) cells (see 3.3.5).
3.3.2 Single cell characterisation of AAAs by mass cytometry revealed inflammatory cell infiltrates predominantly existing of B cells
Using CyTOF mass cytometry, Cassimjee et al. (2018) discovered that the infiltrating inflammatory cell present in AAA are dominated by B- and T-lymphocytes. The studied CD45+ cells, constituted predominantly of B cells (44%) followed by T cells (41%). In the vessel wall, NK cells were present in low amounts (3%). (figure 11).
Figure 11: Immune cell subset composition of the aneurysmal aortic wall. Aortic wall samples (n=10) were collected intraoperatively from ten patients. Prior to enrichment of CD45+ cells, single cell suspensions were isolated by enzymatic digestion from the AAA using FACS. Dot plots depict the frequencies of immune cell types B Cells, T cells, and NK cells in the aneurysm wall. B cells are the most frequent cell type within the aorta followed by T cells.
Figure 12: Frequencies of T, B, and NK cells in the aorta, ILT, and blood samples. Dot plots depicting the frequencies of T and B cells as a percentage of CD45+ lymphocytes in the aneurysmal aortic wall (n=10), in intra luminal thrombi (n=8), and in peripheral blood (n=9). (A) More T cells are present in the aortic vessel wall, than in the ILT or peripheral blood samples (p <0.0001). (B) There are significantly more B cells present in the aorta than in either ILT or blood (p<0.0001). (C) The presence of NK cells is more frequent in ILT, compared to the aorta and blood. (Dot plot error bars = mean +- SD). (*p<0.01, **p<0.01, ***p<0.001, *p<0.0001, two tailed paired student t-test).
3.3.3. IHC analysis of B and T cells in AAA samples revealed inflammatory cell infiltrates as follicles in the adventitia
Immunohistochemical staining with anti-CD4 (T-helper cells), anti-CD8 (cytotoxic T cells), and anti-CD20 (mature B cells), revealed that mature B cells and T cells were localised as aggregates in the adventitial layers in a band like pattern. Lower amounts of CD20+ B cells and CD4+T cells and CD8+ T cells were also found diffused throughout the vessel wall. In the adventitial follicle-like structures, or tertiary lymphoid structures (TLSs), a greater number of CD20+ cells were found compared to CD4+T cells and CD8+ T cells (figure 13).
Figure 13: IHC analysis of the cellular localisation of B and T lymphocytes in AAAs. Representative photomicrographs of AAA tissue stained with antibodies detecting B-lymphocytes [anti-CD20: diaminobenzidine (DAB; brown)] and T-B-lymphocytes [anti-CD4: Vulcan red (red), anti-CD8: Vulcan red (red)]. (A) Clear positively stained CD4+ and CD8+ T-lymphocytes, localised in the adventitia in a band like pattern. (B) Higher magnification of TLS in adventitia. (C) Diffused CD20+ B lymphocytes, and CD4+ and CD8+ T-lymphocytes in media region.
3.3.4. IHC analysis of NK cells in AAA samples revealed extremely low presence of NK cells in aneurysm wall
Immunohistochemical staining with anti-CD7 (NK cells, T cells), anti-CD57 (NK cells, cytotoxic T cells), and anti-Granzyme B(GB) (granules of NK cells and cytotoxic T cells), revealed that the presence of NK cells in the vessel wall is extremely low; almost no triple-positive cells were detected. Many single triple-positive CD7+ T-cells were detected dispersed throughout the vessel wall and in the adventitia. Single positive CD57+ cytotoxic T-cells were predominalty detected in the adventitial lymphoid follicles. Only a few CD7+ CD57+ cytotoxic T cells were located in lymphoid aggregates in the adventitia and vessel wall. GB+ cells were present in the adventitial lymphoid aggregates and dispersed between loose erythrocytes. In some AAA samples, GB+ cells were absent. Prominent was the presence of CD7+ CD57+ cytotoxic T cells, CD7+ T cells, and GB+ cells between loose erythrocytes and perivascular adipose tissue (figure 14).
Figure 14: IHC analysis of the cellular localisation of NK cells in AAAs. Representative photomicrographs of AAA tissue stained with antibodies detecting NK cells [anti-CD7: Vulcan red (red), anti-CD57: Ferangi blue (blue), anti-Granzyme B: diaminobenzidine (DAB; brown)]. (A) Predominantly CD7+ cells were detected in adventitial lymphoid follicles. (B) Higher magnification of image A. Low amounts of CD57+ and CD7+CD57+ double positive cells were detected in adventitial lymphoid follicles. (C) Between loose erythrocytes and adipose tissue, more granular Granzyme B was detected than in the vessel wall. CD7+CD57+ double positive is indicated by a purple arrow. One triple positive CD7+CD57+GB+ NK cell was detected between loose erythrocytes (indicated by black arrow).
3.3.5. IHC analyses of the non-aneurysmal control samples revealed that the localisation of B- and T-lymphocytes depends on its atherosclerotic stage
Infrarenal control samples included non-aneurysmal atherosclerotic samples of different stadia: adaptive intimal thickening (AIT; early stage atherosclerotic lesion), late fibroatheroma (LFA; middle stage atherosclerotic lesion), and fibrocalcific plaque (FCP; late stage atherosclerotic lesion) (Yahagi et al., 2016). IHC revealed that AIT samples presented minimal amounts of single CD4+ and CD8+ T lymphocytes, localised in the tunica intima or adventitia. AIT samples showed a total absence of CD20+ B lymphocytes. FCP samples displayed the presence of CD4+ and CD8+ T lymphocytes in the intima surrounding lipid pools and necrotic cores. In FCP tissue, low number of CD20+ B lymphocytes were localised in the adventitia. In LFA tissue, CD4+ and CD8+ T lymphocytes were present specifically in the intimal layer of the aorta, in the shoulder and cap regions of necrotic cores, in the adventitia, and dispersed throughout the vessel wall. No CD20+ B lymphocytes were present in LFA tissue samples (figure 15).
Figure 15: IHC analysis of the cellular localisation of B and T lymphocytes in non-aneurysmal control samples. Representative photomicrographs of non-non-aneurysmal control samples AIT, FCP, and LFA, stained with antibodies detecting B-lymphocytes [anti-CD20: diaminobenzidine (DAB; brown)] and T-lymphocytes [anti-CD4: Vulcan red (red), anti-CD8: Vulcan red (red)]. (A-C) IHC of AIT tissue sample showing incident numbers of T-lymphocytes and total absence of B T-lymphocytes. B: T T-lymphocytes localised in the intima of AIT. C: T-lymphocytes in adventitia of AIT. (D-F) IHC of FCP sample. E: B lymphocytes in
the vicinity of adventitial vaso vasora of FCP. F: T-lymphocytes surrounding lipid pools and necrotic cores of FCP. (G-J) IHC of LFA tissue. H: T-lymphocytes in the intima and cap region of a necrotic core in LFA tissue. I: T-lymphocytes dispersed throughout the vessel wall of LFA tissue. J: T-lymphocytes in the shoulder region of a necrotic core in LFA tissue.
4. Discussion
This study has taken the first steps towards validating the full CyTOF dataset by Cassimjee et
al. (2018), by focussing on the cellular localisation of NK, B, and T cells in the AAA wall.
Although no classification scheme exists covering solely AAAs, the first aspect this bipartite study has established a consensus classification scheme that covers AAAs, based on their histological characteristics. Classifying the wall samples according to this consensus classification scheme has revealed that AAAs comprise a large histological heterogeneity. Therefore, in order to correctly choose a representative sample set that portrays the natural histological variation seen in AAAs, a minimal group size of n=30 was required.
The second aspect of this study comprises the validation of the CyTOF single cell data generated by Cassimjee et al. (2018). This validation has revealed that the adventitial inflammatory cell infiltrates present in AAAs comprise B and T cells. NK cells were virtually absent in the aneurysmal samples. These results correspond the the CyTOF single cell data. The presence of lymphoid follicles in the adventitia of the aneurysmal abdominal aorta can be elucidated considering their localisation in the vicinity of microvasculature. Earlier research has shown that endothelial cells are a major participant in the generation of an inflammatory response, because via the microvascular network, inflammatory cells can rapidly migrate towards the site of inflammation. (Pober & Sessa, 2015). Moreover, the excessive presence of B-lymphocytes in the adventitial lymphocyte follicles may be stimulating the progression of AAAs. The GCs of the TLS can produce plasma cells, which produce immunoglobulins and cytokines. These can activate the complement pathway, followed by the activation of macrophages and mast cells, which promote vessel wall destruction and AAA progression (Zhang & Wang, 2015). Furthermore, although NK were present in very low amount in the aortic wall of AAAs, the CyTOF data by Cassimjee et al. (2018), in line with data from other studies, has presented the elevated presence of NK cells in the blood. Due to their increased cytotoxicity per cell, targeted towards aortic smooth muscle cells, NK cells could be a contributing factor in the generation of inflammation in AAAs (Forester et al., 2006).
The consensus classification established in this study might help to identify core mechanisms involved with AAA progression. In addition, IHC validation of the CyTOF data has generated deeper insights for the development of anti-inflammatory drugs for AAA treatment. Yet, further validation of the CyTOF dataset is required to investigate the complete immune cell population in AAAs. Future research could therefore consist of additional multiparameter staining for the detection of smaller subsets of inflammatory cells, such as Th cells and Tregs.
To conclude, this study has established a consensus classification scheme for the evaluation of AAAs and has taken the first steps towards validating the CyTOF dataset by Cassimjee et al. (2018) using IHC. Consequently, these results contributed to filling a crucial gap in knowledge required for the development of possible immunotherapies that can help in the prevention and treatment of this deadly disease.
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6. Supplementary materials
Movat Pentachrome Protocol
Working solutions used in protocol:
(A) 1% Alcian Blue Solution: 1 g Alcian Blue 8 GX (Merck, Burlington, US), 100 ml distilled water, 1 ml Glacial Acetic Acid (Sigma Aldrich, Saint Louis, US)
(B) Alkaline Alcohol solution: 10 ml Ammonium Hydroxide (Merck, Burlington, US), 90 ml Ethanol 100%.
(C) Elastic Hematoxylin Solution: 25 ml 10% Alcoholic Hematoxylin (J), 25 ml Ethanol 100%, 25 ml 10% Ferric Chloride (D), 25 ml Verhoeff’s Iodine Solution (K).
(D) 10% Ferric Chloride Solution: 10 g Ferric Chloride (Sigma Aldrich, Saint Louis, US), 100 ml distilled water
(E) 5% Sodium Thiosulfate Solution: 5g Sodium Thiosulfate (Sigma Aldrich, Saint Louis, US), 100 ml distilled water
(F) Biebrich Scarlet/Acid Fuchsin solution: pre-made from ScyTek Laboratories (Logan, United States).
(G) 1% Acetic Acid Solution: 1 ml Glacial Acetic Acid, 99 ml distilled water
(H) 5% Aqueous Phosphotungstic Acid solution: 5 g Phosphotungstic Acid (Sigma Aldrich, Saint Louis, US), 100 ml distilled water
(I) 4% Alcoholic Saffron Solution: 4 g Saffron (Safranor Safran du Gâtinais, Échilleuses, France), 100 ml Ethanol 100%.
(J) 10% Alcoholic Hematoxylin Solution: 10 g Hematoxylin (Merck, Burlington, US), 100 ml Ethanol 100%
(K) Verhoeff’s Iodine Solution: 2 g Iodine Crystals (Sigma Aldrich, Saint Louis, US), 4 g Potassium Iodide (Sigma Aldrich, Saint Louis, US), 100 ml distilled water
Protocol:
1. Deparaffinization and rehydration of slides. 2. Rinse slides in distilled water.
3. Stain in 2 changes with (A) , both times for 15-25 minutes. 4. Rinse slides in running warm to hot water until clear.
5. Place slides in (B) for 30 minutes, then rinse in running tap water. 6. Stain in (C) for 20 minutes.
7. Rinse in running warm tap water.
8. Differentiate in 2% aqueous (D) for 5 seconds-2 minutes. 9. Place slides in (E) for about 1 minute.
10. Wash in running tap water and rinse in distilled water. 11. Stain in (F) for 1-1.5 minutes.
12. Rinse in distilled water. 13. Rinse in (G) for 7-12 seconds. 14. Place slides in (H) for 7-12 minutes. 15. Rinse in distilled water.
16. Rinse in (G) for 8-10 seconds. 17. Place in 2 changes of Ethanol 100%.
18. Stain in (I) for 1.5 minute and quickly rinse in Ethanol 100%. 19. Dehydration of slides.
H&E Staining protocol:
1. Deparaffinization and rehydration. 2. Rinse in demi water 5 minutes. 3. Haematoxylin 4 minutes.
4. Rinse in tap water and demi water. 5. Eosin 1 minute.
