ORIGINAL RESEARCH
published: 31 May 2022
doi: 10.3389/fimmu.2022.866558

Deep Phenotyping of T-Cells
Derived From the Aneurysm
Wall in a Pediatric Case of
Subarachnoid Hemorrhage
Edited by:
Manolo Sambucci,
Santa Lucia Foundation (IRCCS), Italy
Reviewed by:
Gabriela Constantin,
University of Verona, Italy
Claudio Procaccini,
Consiglio Nazionale Delle Ricerche
(CNR), Italy
*Correspondence:
Jens Geginat
geginat@ingm.org
†

These authors have contributed
equally to this work

‡

These authors have contributed
equally to this work and share
the last authorship
Specialty section:
This article was submitted to
Multiple Sclerosis
and Neuroimmunology,
a section of the journal
Frontiers in Immunology
Received: 31 January 2022
Accepted: 28 April 2022
Published: 31 May 2022

Citation:
Moschetti G, Vasco C, Clemente F,
Galeota E, Carbonara M, Pluderi M,
Locatelli M, Stocchetti N, Abrignani S,
Zanier ER, Ortolano F, Zoerle T and
Geginat J (2022) Deep Phenotyping of
T-Cells Derived From the Aneurysm
Wall in a Pediatric Case of
Subarachnoid Hemorrhage.
Front. Immunol. 13:866558.
doi: 10.3389/fimmu.2022.866558

Giorgia Moschetti 1†, Chiara Vasco 1†, Francesca Clemente 1†, Eugenia Galeota 1,
Marco Carbonara 2, Mauro Pluderi 3, Marco Locatelli 3,4, Nino Stocchetti 2,4,
Sergio Abrignani 1,5, Elisa R Zanier 6, Fabrizio Ortolano 2, Tommaso Zoerle 2,4‡
and Jens Geginat 1,5*‡
1 National Institute for Molecular Genetics (INGM), Milan, Italy, 2 Neuroscience Intensive Care Unit, Department of Anesthesia
and Critical Care, Fondazione IRCCS Cà Granda Ospedale Maggiore Policlinico, Milan, Italy, 3 Department of Neurosurgery,
Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy, 4 Department of Pathophysiology and
Transplantation, University of Milan, Milan, Italy, 5 Department of Clinical Sciences and Community Health University Milan,
Milan, Italy, 6 Laboratory of Acute Brain Injury and Therapeutic Strategies, Department of Neuroscience, Mario Negri Institute
for Pharmacological Research IRCCS, Milan, Italy

Intracranial aneurysms (IAs) are very rare in children, and the characteristics of the T-cells
in the IA wall are largely unknown. A comatose 7-years-old child was admitted to our
center because of a subarachnoid hemorrhage due to a ruptured giant aneurysm of the
right middle cerebral artery. Two days after the aneurysm clipping the patient was fully
awake with left hemiparesis. T-cells from the IA wall and from peripheral blood of this
patient were analyzed by multi-dimensional flow cytometry. Unbiased analysis, based on
the use of FlowSOM clustering and dimensionality reduction technique UMAP, indicated
that there was virtually no overlap between circulating and tissue-infiltrating T-cells. Thus,
naïve T-cells and canonical memory T-cells were largely restricted to peripheral blood,
while CD4-CD8-T-cells were strongly enriched in the IA wall. The unique CD4+, CD8+ and
CD4-CD8-T-cell clusters from the IA wall expressed high levels of CCR5, Granzyme B and
CD69, displaying thus characteristics of cytotoxic and tissue-resident effector cells. Low
Ki67 expression indicated that they were nevertheless in a resting state. Among regulatory
T-cell subsets, Eomes+Tr1-like cells were strongly enriched in the IA wall. Finally, analysis
of cytokine producing capacities unveiled that the IA wall contained poly-functional T-cells,
which expressed predominantly IFN-g, TNF and IL-2. CD4+T-cells co-expressed also
CD40L, and produced some IL-17, GM-CSF and IL-10. This report provides to our
knowledge the first detailed characterization of the human T-cell compartment in the
IA wall.
Keywords: intracranial aneurysm, subarachnoid haemorrhage, T-cells, flow cytometry, phenotype

Frontiers in Immunology | www.frontiersin.org

1

May 2022 | Volume 13 | Article 866558

Moschetti et al.

T-Cells in the Aneurysm Wall

INTRODUCTION

Flow Cytometry
T-cell subsets were analysed for the expression of surface
markers, transcription factors and cytokines by staining with
various combinations of fluorochrome-conjugated monoclonal
antibodies (Supplementary Table 1). For intracellular cytokine
detection, T cells were incubated for 4 hours in the presence or
absence of phorbol 12-myristate 13- acetate (PMA), ionomycin
in complete RPMI (10% FBS, 1 mmol/L sodium pyruvate, 10
mmol/L nonessential amino acids, and 1% penicillin/
streptomycin), and with BrefeldinA (Sigma, St Louis, Mo) for
an additional 2 hours. After fixation with Intracellular Fixation &
Permeabilization Buffer Set (Thermo Fisher Scientific, Waltham,
Massachusetts) cells were permeabilized with Permeabilization
Buffer (BD Biosciences). Analysis was performed with a
FACSSymphony™ cytometer (Becton Dickinson, Franklin
Lakes, NJ) and analysed using FlowJo software (BD Biosciences).

An intracranial aneurysm (IA) is a focal dilatation of a cerebral
vessel (1). IAs are very rare in children but they cause more than
10% of hemorrhagic strokes in the pediatric population (2) with
high mortality and morbidity (3, 4). The pathogenesis and the
natural history of these pediatric vascular malformations are
largely unknown with 50% of cases presenting without risk
factors or underlying diseases (e.g. head trauma, infections,
tumors, inflammatory diseases, excessive hemodynamic stress)
(5, 6). Data from adult patients and animal experiments
suggested that endothelial damage and dysfunction, vascular
smooth muscle cell modulation and extracellular matrix
remodeling are the main steps, leading to arterial wall
degeneration and to IA formation (7–10) Different proinflammatory mediators seem to be crucial in these complex
processes, including cytokines like IL-1b, IL-6 and TNF-a,
matrix metalloproteinases (MMP), monocyte chemoattractant
protein 1 (MCP1), reactive oxygen species, complement and
several growth factors. In addition, immune cells were identified
in the aneurysm wall of human subjects, mainly macrophages
and T-cells (11, 12). However, while macrophages are considered
to be fundamental for IA formation, progression and rupture
(13), the role of T-cells is less clear. T-cells play a key role in
orchestrating inflammation, with different sub-populations
involved in the production of pro- or anti-inflammatory
cytokines and in the modulation of the activity of other
immune cells (14). However, the contribution of these subpopulations in the IA pathogenesis and rupture is largely
unexplored, limiting our understanding of these complex
processes that could represent a new therapeutic target for
intracranial bleeding prevention.
The aim of this study was thus to characterize in depth, for the
first time, T-cells that infiltrate the aneurysm wall, derived from a
pediatric idiopathic IA.

Bioinformatic Analysis
Flow cytometry data were imported into FlowJo software
(version 10.8.0) to compensate fluorescence spreading; dead
cells and debris were excluded from the analysis and CD3+ T
cell population was selected for further analysis. A random
down-sample to 5000 events in the CD3+ T-cells compartment
was exported as FCS file for further analysis in R software
(version 4.0.2).
Sample batches were read using read.flowSet (2.6.0) from the
flowCore R package. We applied the Logicle transformation that
allows the use of multiple samples to estimate transformation
parameters. To reduce batch effect due to technical and not to
biological variation we normalized the signal of each marker with
the function gaussNorm from the flowStat package (4.6.0). After
the batch-specific pre-processing, samples were concatenated
into a SingleCellExperiment object in R using the
function prepData from the CATALYST R package (15).
Dimensionality reduction by UMAP was subsequently applied
to visualize relative proximities of cells within reduced
dimensions. Two parallel analyses were applied for each of the
two panels. We performed high-resolution, unsupervised
clustering and meta-clustering using FlowSOM (2.2.0) and
ConsensusClusterPlus (1.58.0) packages following the workflow
in Nowicka et al. (15). The T cell compartment was clustered
based on the expression of 15 markers for Surface panel (CD4,
CD8, CCR7, CD45RA, CD27, CD25, CD127, EOMES,
GRANZYME_K, GRANZYME_B, CCR6, CXCR3, CCR5,
CD69, 41BB) and 15 markers for Cytokines panel (CD4, CD8,
CD127, FOXP3, EOMES, GRANZYME_K, GRANZYME_B,
CCR6, CXCR3, T_BET, IL2, GM_CSF, TNFA, IFNG, CD40L).
We excluded from the analysis markers that identified only very
low numbers of positive cells, i.e. IFN-g, FOXP3, KI67, and
CD40L from the surface panel for unstimulated cells and KI67,
IL10, IL17A and IL17F for the cytokine panel of PMA and
Ionomycin-stimulated cells. Manually annotated clusters were
subsequently visualized on the UMAP. Functional pseudo-time
analysis to infer the differentiation trajectory of cells was carried
out by DiffusionMaps (15) using the function runDiffusionMap
on the SingleCellExperiment object using default parameters.

MATERIAL AND METHODS
Cell Isolation and Purification
A blood sample and a piece of the IA wall were collected during
surgery. As a control, peripheral blood from a sex-matched 4
years-old child was obtained. Written informed consent was
signed by both parents for further analysis and data publication.
The study was approved by the Ethics Committee of the
Fondazione IRCCS Ca’ Granda–Ospedale Maggiore Policlinico
(3.11/2021-680). Peripheral blood mononuclear cells (PBMC)
were isolated by density gradient centrifugation by Ficoll density
gradient (Amersham Pharmacia Biotech, Uppsala, Sweden).
Cells from the aneurysm wall were collected using a Tumor
dissociation kit (Miltenyi Biotec, Bergisch Gladbach; Germany)
according to the manufacturer’s instructions. After dissociation,
the sample was filtered (70µm) to remove any remaining larger
particles from the single-cell suspension and lymphocytes were
isolated by Ficoll density gradient.

Frontiers in Immunology | www.frontiersin.org

2

May 2022 | Volume 13 | Article 866558

Moschetti et al.

T-Cells in the Aneurysm Wall

expression unveiled that the major clusters in peripheral blood
in the patient and in the pediatric control represented naïve T-cells
(Figure 2B). Conversely, T-cells from the IA wall expressed high
levels of CD69 and GzmB (Figure 2B).
We next performed high-resolution, unsupervised clustering
and meta-clustering (Supplementary Figure 1A) (15).
Clustering resolution was chosen based on delta area plots and
visual inspection of the expression of 15 markers
(Supplementary Figure 1B). We first applied an “overclustering” strategy choosing the maximum number of clusters
(K=20) in order to optimally discriminate T cell sub-populations
(Supplementary Figure 1A). Subsequently, clusters with similar
expression profiles (Supplementary Figure 1B) were manually
annotated. In this way we identified 9 distinguishable major
clusters: three CD4+, three CD8+ and three DN T-cell clusters
(Figure 2C and Supplementary Figures 2A–C). Among these
three clusters we identified one naïve (CD45RA+CCR7+) and
one memory (CD45RA-CCR7+/- in the CD4 compartment) or
effector memory subsets (CD45RA+/-CCR7- in the CD4compartments) that were largely restricted to peripheral blood
(Figure 2C and Supplementary Figure 2C). In addition, we
identified in all three T-cell compartments also a third cluster,
which was derived from the IA wall and expressed high levels of
CD69 and GzmB (Figure 2C and Supplementary Figure 2C).
Notably, the UMAP analysis suggested that IA T-cells were
more closely located to circulating memory T-cells then to
naïve T-cells (Figure 2C). Indeed, trajectory analysis by
DiffusionMap unveiled higher proximity of IA-T cells to
circulating memory T-cells as compared to naïve T-cells along
the second diffusion component (Supplementary Figure 3A).
We also observed a clear differentiation pattern from naïve
phenotype towards memory in CD4+, CD8+ and DN T-cells
(Supplementary Figure 3B).

RESULTS
A Pediatric Case of Intracranial Aneurysm
A previously healthy 7-years-old male child had a sudden onset
headache followed by loss of consciousness while he was playing
at home. He was intubated on the scene and transferred to the
emergency department. The head computed tomography scan
(CT) showed a thin subarachnoid hemorrhage (SAH), a large
intra-parenchymal hemorrhage (ICH) and mid-line shift. A
giant aneurysm of the right middle cerebral artery was
identified by angio-CT (Figure 1). The patient underwent
craniotomy for ICH evacuation and intracranial aneurysm (IA)
clipping. He was admitted to the Neuro Intensive Care Unit and
required standard post-operative care with sedation, artificial
ventilation and hemodynamic support. Two days later he was
fully awake with left hemiparesis and was extubated. At 9months follow-up, the parents reported resumption of normal
life without significant disabilities despite a mild left hemiparesis.

Unbiased Analysis of T-Cell Phenotypes
Unveiled Virtually No Overlap Between
T-Cell Subsets From the Blood and the
Aneurysm Wall
T-cells from the IA wall and from peripheral blood were analyzed
by multi-dimensional flow cytometry ex vivo. UMAP analysis
resulted in the clear separation of T-cells derived from the IA wall
and T-cells from peripheral blood (Figure 2A). UMAPs for CD4
and CD8 expression unveiled that CD4+T-cells were separated
from CD4-T-cells, i. e. from both CD8+T-cells and CD4-CD8double-negative (DN) T-cells, in Dimension 1 (Figure 2B).
Notably, Dimension 2 separated instead T-cells from peripheral
blood and from the IA wall, with hardly any overlap between cells
from the two sites (Figures 2A, B). CD45RA and CCR7

A

B

FIGURE 1 | Computed tomography (CT) scan after intracranial aneurysm rupture. (A) The initial head CT showed a thin subarachnoid hemorrhage and a large intraparenchymal hemorrhage (yellow arrow) including an iso-dense region (red arrow). (B) The angio-TC showed a cerebral aneurysm originating from the right middle
cerebral artery (blue arrow).

Frontiers in Immunology | www.frontiersin.org

3

May 2022 | Volume 13 | Article 866558

Moschetti et al.

T-Cells in the Aneurysm Wall

A

B

C

FIGURE 2 | Unsupervised ex vivo flow cytometric analysis reveals unique T-cell clusters in the IA wall. (A) UMAP projections of normalized expression of markers colored
according to the analysed sample. Red: T-cells from the IA wall (IA_WALL), blue: T-cells from peripheral blood of the same patient (PT_PBMCs), green: T-cells from
peripheral blood of a pediatric control (CTR_PBMCs). (B) Two-dimensional illustration of CD4, CD8, CD45RA, CCR7, GRANZYME B and CD69 expression by UMAP.
Blue denotes low, green intermediate and red high expression. (C) Nine different T-cell clusters, i.e. three CD4+, three CD8+ and three DN clusters, were identified by
UMAP and colored according to cell phenotypes as indicated. CCR7, CD45RA, CD69 and GZMB expression identified three naïve (CCR7+CD45RA+CD69-GZMB-), one
memory (CCR7+/-CD45RA-CD69-GZMB-) and two effector memory clusters (“EM”, CCR7-CD45RA+/-CD69-GZMB+/-) in peripheral blood. In addition, three CD69
+GZMB+ clusters were identified in the IA wall (“TRM”). Upper panel: overlay of the 3 samples. Lower panels: UMAP Plots stratified according to sample origin:
“IA_WALL” (left), “PT_PBMCs” (middle) and “CTR_PBMCs” (right).

Frontiers in Immunology | www.frontiersin.org

4

May 2022 | Volume 13 | Article 866558

Moschetti et al.

T-Cells in the Aneurysm Wall

cells from the IA wall expressed instead the chemokine receptor
CCR5 (Figure 4B), which is known to promote migration to
non-lymphoid tissues. This was true both for CD4+, CD8+ and
also DN T-cells. Moreover, a relevant fraction of the
CD4+CCR5+T-cells had down-regulated CD127 expression
(Figure 4B), a phenotype displayed by Eomes+Tr1-like cells
(19). Notably, these cells expressed CD27, but lacked CCR6, as
is characteristic for these Tr1-like cells (19) (Supplementary
Figure 5B). Analysis of intracellular transcription factor
expression in CD4+CD127loFOXP3- cells (gating strategy is
shown in Supplementary Figure 5C) confirmed that
Eomes+Tbet-/lo cells were strongly enriched in the IA wall
(Figure 4C), consistent with the view that these cells represent
Eomes+Tr1-like cells (19–21). In contrast, FOXP3+CD127loTregs
were present at largely similar frequencies in the blood and the
IA wall (Figure 4D).

T-Cells From the IA Wall Lack Canonical
Naïve and Memory Subsets, But Display a
Phenotype of Cytotoxic Effector Cells
We then compared the T-cell compartments of the IA wall and
the blood more in detail. DN T-cells were strongly enriched in
the IA wall as compared to peripheral blood, and conventional
CD4+ and in particular CD8+T-cells were consequently reduced
(Figure 3A, Gating strategy: Supplementary Figure 4, absolute
cell numbers: Supplementary Table 2). We then quantified the
co-expression of differentiation-associated surface markers and
of intracellular expression of cytotoxic mediators (Figures 3B–
E). The large majority of CD4+T-cells from pediatric blood
displayed the canonical CCR7+CD45RA+ phenotype of naïve
T-cells (Figure 3B). Similar results were obtained when CD8+Tcells from peripheral blood were analyzed, while T-cells with a
naive phenotype were virtually absent in the IA wall (Figure 3B).
CD4+T-cells from peripheral blood expressed as expected low
levels of GzmB and CD69 (Figures 3C, D). In marked contrast,
GzmB+, but not GzmK+, T-cells were overall strongly enriched in
the IA wall [Figure 3C and Supplementary Table 3 (reports
MFI)]. Moreover, the majority of T-cells from the IA wall
expressed the activation/tissue residency marker CD69 (16),
but lower levels of the memory-associated costimulatory
receptor CD27 (Figure 3D and Supplementary Table 3). We
next analyzed if T-cells from the IA wall were positive for the
Ki67 proliferation marker, which is expressed in T-cells that have
divided in the last few days. However, both T-cells in peripheral
blood and those from the IA wall expressed only very low levels
of Ki67 (<2%), indicating that the large majority was in a resting
state (Figure 3E). This was true for CD4+T-cells, CD8+T-cells
and also for DN T-cells. Notably, also CD69+T-cells from the IA
wall were largely KI67- (Figure 3E and data not shown),
consistent with the notion that also in the IA wall CD69 is a
marker of tissue-resident rather than of activated cells.

T-Cells From the Aneurism Wall Produce
IFN-g and Contain Poly-Functional Cells
To assess the cytokine producing capacities of T-cells from the
IA wall, cells were poly-clonally stimulated and analyzed for
intracellular cytokines together with selected differentiation and
activation markers (Supplementary Figures 6A, B). UMAP
analysis unveiled again that T-cells from peripheral blood and
the IA wall were largely distinct (Figure 5A). It resulted also
again in the separation of CD4+ from CD4-T-cell subsets
(Figure 5B). We identified 10 different major clusters
(Figure 5C), including 3 clusters that were abundant in the IA
wall, but absent from pediatric blood (Supplementary
Figure 6C). Intriguingly, these 3 clusters represented CD4+,
CD8+ and DN T-cells with polyfunctional capabilities, i.e.
that could produce several different cytokines. Conversely,
CD4+ T-cells that expressed only IL-2 or CD40L were largely
restricted to peripheral blood, whereas T-cells that produced
TNF-a could be identified both in peripheral blood and in the
IA wall (Figure 5C).
A major feature of T-cells in the IA wall was the high
production of IFN-g, TNF and IL-2 from the IA wall
(Figures 6A, B). Thus, approximately 20% of CD4+T-cels from
the IA wall produced IFN-g, while only very few T-cells from
peripheral blood were able to do so. This was true for CD4+ and
CD8+ T-cells (Figure 6A); only double-negative T-cells from the
pediatric control also produced relevant amounts of IFN-g. Tcells from the IA wall produced also IL-2 and TNF, while in
peripheral blood only CD8+T-cells from the IA patient produced
TNF (Figure 6B). Notably, some T-cells from the IA wall were
indeed poly-functional, since they co-produced IFN-g, TNF and
IL-2 (Figure 6B). CD4+T-cells producing IL-17 or GM-CSF were
also enriched in the IA wall (Figure 6C). They were however
rather rare (1-2%), but some of these cells co-produced
nevertheless IFN-g (Figure 6C). Finally, CD4+T-cells from
peripheral blood expressed higher levels of CD40L poststimulation as compared to CD4+T-cells from the IA wall
(Figure 6D). IL-10 production was overall low (<1-2%), but
we detected nevertheless co-expression with IFN-g and a
selective increase of CD4 + T-cells with an IL-10 + CD40L -

Composition of the CD4+ T-Cell
Compartment in the IA Wall
We next focused on well-defined memory/regulatory subsets of
the CD4+T-cell compartment (Figure 4). An unexpected feature
of CD4+T-cells in the IA wall was the low expression of CXCR3
and CCR6, well-established differentiation markers of
respectively Th1 and Th17 memory cells in the blood (15). In
particular, while >15% of cells of the IA patient expressed
CXCR3 in peripheral blood, CXCR3+ cells were rare in the IA
wall (Figure 4A and Supplementary Table 1). This was also true
for CD8+T-cells and DN T-cells. Surprisingly, CXCR3+ and
CCR6 + CD4 + T-cells in pediatric blood were highly
heterogeneous for CD127 and CCR7 expression, indicating
that they contained not only the immature CCR7+central
memory cells, but also the more differentiated CCR7 CD127+effector memory (17) and even some CD127-effectorlike cells (18)(Supplementary Figure 5A). In contrast, the few
CXCR3 + and CCR6 + CD4 + T-cells in the IA wall had
predominantly an effector memory phenotype. T-cells from
pediatric control blood expressed much lower levels of CXCR3
as compared to the IA patient. Importantly, the majority of T-

Frontiers in Immunology | www.frontiersin.org

5

May 2022 | Volume 13 | Article 866558

Moschetti et al.

T-Cells in the Aneurysm Wall

A

B

C

D

E

FIGURE 3 | Differentially expressed T-cell differentiation and activation markers in the IA wall. (A) Donut plots report frequencies of CD4+ (magenta), CD8+ (violet)
and DN cells (blue) among total CD3+T-cells in the IA_WALL and in PBMCs in the three samples. (B) CD45RA and CCR7 surface expression was analysed to
quantify the presence of naïve T-cells (white) and central/effector memory subsets (colored as indicated) in CD4+ (dot plots and left histogram bars) and in CD8+ and
DN T-cells (central and right bar histograms). (C) Intracellular GZMB and GZMK expression in CD4+, CD8+ and DN T-cells. Left dot plots show GZMK and GZMB
expression in CD8+T-cells in the IA wall and in peripheral blood. Right histogram bars report the frequencies of GZMB/K+ cells among CD4+, CD8+ and DN T-cells
in the IA wall (red) and in the blood of the patient (blue) as well as of the pediatric control (green). (D) Surface expression of CD69 versus CD27 in CD4+ (dot plots
and left bar histograms) as well as in CD8+ and DN T-cells (central and right bar histograms). The frequencies of CD27+/-CD69+/- cells are colored as indicated.
(E) Intracellular expression of the proliferation marker KI67 versus CD69 surface expression in CD4+T-cells in the IA wall and in peripheral blood from the patient is
shown in the left dot plots. Percentage of total KI67+ cells among CD4+, CD8+ and DN T-cells in the IA wall (red), in peripheral blood of the patient (blue) and of the
pediatric control (green) is shown in the right panel.

Frontiers in Immunology | www.frontiersin.org

6

May 2022 | Volume 13 | Article 866558

Moschetti et al.

T-Cells in the Aneurysm Wall

regulatory profile (22) in the IA wall. (Figure 6D). In summary,
T-cells from the IA wall had overall higher cytokine producing
capabilities, and produced in particular high amounts of the
Th1-associated cytokines IFN-g, TNF and IL-2 alone or
in combination.

DISCUSSION
The clinical management of ruptured IA is based on early
aneurysm treatment to avoid re-bleeding and ICU admission
to prevent and treat secondary brain injuries. Moreover,

A

B

C

D

FIGURE 4 | Differential distribution of helper and regulatory T-cell subsets in the IA wall and in peripheral blood. (A) Th1 (CXCR3+CCR6-), Th1/17 (CXCR3+ CCR6+) and
Th17 (CXCR3- CCR6+) subsets were analysed on gated CD4+ T-cells. Dot plots show CXCR3 versus CCR6 expression in the IA_WALL, in the peripheral blood of the
patient (PT_PBMCs, middle) and of the pediatric control (CTR_PBMCs, right). (B) CD127 and CCR5 surface expression. Dot plots show CCR5 versus CD127 expression
in the IA wall and in the blood of the patient. Bar Histograms report the frequencies of CD127+/-CCR5+/- cells (colored as indicated) among CD4+, CD8+ and in DN
T-cells in the 3 samples as indicated. (C) EOMES+Tr1-like cells were gated first as CD4+CD127loFOXP3- and then as Eomes+T-bet-/lo. Their frequencies among CD4
+T-cells was reported in the IA wall (red), in peripheral blood of the patient (blue) and of the pediatric control (green). (D) FOXP3+ regulatory T-cells (Treg) cells were gated
as CD127loFOXP3+ and their frequencies among CD4+T-cells in the three samples are reported in the right bar histograms as in C.

Frontiers in Immunology | www.frontiersin.org

7

May 2022 | Volume 13 | Article 866558

Moschetti et al.

T-Cells in the Aneurysm Wall

A

B

C

FIGURE 5 | Unsupervised analysis of cytokine producing capacities identifies polyfunctional T-cell clusters in the IA wall. (A) UMAP obtained using normalized expression
of intracellular cytokines and selected differentiation markers, colored according to the analysed sample (i.e. red: IA_WALL, blue: PT_PBMCs and green: CTR_PBMCs).
(B) UMAP of CD4 and CD8 expression. Blue denotes low, green intermediate and red high expression. (C) UMAP analysis identified ten clusters that were either largely
unique for the IA_WALL or for peripheral blood (PBMCs), but also some that were common to all samples (“ALL”). Clusters were colored according to cytokine
expression as indicated, the sample of origin is indicated in parenthesis. Clusters that were positive for at least 3 different cytokines were named polyfunctional (“POLY”).
The upper UMAP plot shows an overlay of all three samples. The lower UMAP Plots were stratified according to sample of origin: “IA_WALL”, “PT_PBMCs” and
“CTR_PBMCs”.

Frontiers in Immunology | www.frontiersin.org

8

May 2022 | Volume 13 | Article 866558

Moschetti et al.

T-Cells in the Aneurysm Wall

A

B

C

D

FIGURE 6 | Poly-functional T-cell subsets in the IA wall produce mainly IFN-g, TNF-a and IL-2 (A). Left histogram overlay shows IFN-g production by CD4+T-cells in the
IA wall and in the blood of the patient. Right panel: Frequencies of IFN-g producing CD4+, CD8+ and DN T-cells in the IA wall (red) and in peripheral blood of the patient
(blue) and of the pediatric control (green). (B) Analysis of TNF-a and IL2 production (upper left dot plots and upper right bar histograms). Co-expression of TNF-a, IL2
and IFN-g in CD4+, CD8+ and DN subsets in the IA wall is shown in the lower panel. Frequencies were calculated on total CD4+, CD8+ and DN cells. TNF-a and IL2
co-expressing cells among IFN-g+ (right) and IFN-g - (left) cells are in black, cells that produce neither TNF-a nor IL2 in white, and cells that express only TNF-a or IL2 are
colored. (C) Co-expression of GM-CSF, IL17A and IFN-g among CD4+T-cells. Left dot plots show intracellular GM-CSF versus IL17A production in the IA wall and in the
blood of the patient. Co-expression with IFN-g of the two cytokine (IL17 in black and GM-CSF in green) is shown in the right stacked histogram in the IA wall. (D) CD40L
expression versus IL10 production in CD4+T-cells are show in the left dot plots for CD4+T-cells and in the right stacked histograms in the upper panels for all T-cell
subsets. Expression of IL10 and CD40L, alone (coloured as indicated) or in combination (black), in IFN-g + and in IFN-g- cells for all samples is shown in the stacked
histograms of the lower panels.

Frontiers in Immunology | www.frontiersin.org

9

May 2022 | Volume 13 | Article 866558

Moschetti et al.

T-Cells in the Aneurysm Wall

radiological long-term follow-up are necessary to detect
aneurysm recurrence or de-novo formation (1). This approach,
as in our case report, can lead to favorable outcome even in
severe patients. However, the pathogenesis and the natural
history of pediatric IAs is still not fully elucidated and
investigations aimed at identifying the mechanisms underlying
aneurysm rupture could lead to new therapeutic interventions
and primary prevention measures.
The role of T-cells in aneurysms is largely unclear (23). A
recent study of human abdominal aortic aneurysms unveiled an
association of tissue-infiltrating T-cells with disease severity (24).
A study of a mouse model for intracranial aneurysms suggested
that T-cells could be dispensable for the formation of an
aneurysm (10), but their role in aneurysm rupture remained
unclear. It seems likely that the role of tissue-infiltrating T-cells
in IA depends on their pro- or anti-inflammatory properties.
While T-cells were studied in liquor and plasma of patients with
ruptured aneurysm and subarachnoid hemorrhage (25, 26), this
is to our knowledge the first report that analyses the human Tcell compartment directly in the IA wall by multi-dimensional
flow cytometry. We found that the human wall of this pediatric
case of a giant IA was heavily infiltrated by T-cells, comprising
both CD4+ and CD8+ as well as CD4-CD8- DN T-cells. The latter
were enriched, and it seems likely that they contained both a/b
and g/d-T-cells. Comparison with the T-cell compartment of
peripheral blood of the same patient and a pediatric control
unveiled that T-cells in the IA wall were completely different
from the very well-characterized circulating T-cells, and UMAP
analysis confirmed that there was virtually no overlap between
the T-cell clusters in the two tissues. Thus, T-cells from pediatric
blood contained as expected mainly naïve T-cells, which were in
contrast completely absent from the IA wall, excluding also a
contamination of the here analyzed tissue sample. A more
detailed analysis of antigen-experienced CD4+T-cells unveiled
further that also conventional CXCR3+Th1 and CCR6+Th17
memory subsets were large absent in the IA wall.
Unexpectedly, however, in spite of the abundance of
CXCR3 + Th1-cells, which included both CCR7 - effector
memory and CD127- effector-like cells (27), IFN-g production
was hardly detectable in the blood of the SA patient. IFN-g
producing T-cells in the IA wall were in general much more
abundant then in the blood, and co-produced several other
cytokines, in particular TNF and IL-2, indicating that they
were poly-functional. Moreover, they expressed high levels of
the cytotoxic effector molecule Granzyme B. Thus, they had a
cytotoxic effector phenotype, and this was not only true for CD8+
and DN T-cells, but also for CD4+T-cells. Notably, T-cells from
the IA wall expressed also high levels of the chemokine receptor
CCR5, which is considered to be a therapeutic target for recovery
after brain injury (28). In addition, they had up-regulated CD69,
as is characteristic for tissue-infiltrating T-cells (16). CD69 is
rapidly induced upon T-cell activation in vitro, but is associated
with tissue residency in vivo. Indeed, both CD69+ and CD69-Tcells in the IA wall were largely negative for the proliferation
marker Ki67, indicating that they were in a resting state. Finally,
regulatory FOXP3+CD4+T-cells were present at similar low

Frontiers in Immunology | www.frontiersin.org

frequencies in peripheral blood and in the IA wall, where they
might promote brain repair (29, 30). In addition, Eomes+Tr1-like
cells (25) were strongly enriched in the IA wall, indicating the
presence of different regulatory T-cell subsets. IL-10 is
challenging to detect ex vivo intracellularly in particular in
human tissues (21), and it is thus possible that our analysis
underestimated IL-10 production in particular in the IA wall.
Nevertheless, we were able to detect an increase of CD4+T-cells
that expressed IL-10 in the IA wall, consistent with the observed
enrichment of Eomes+Tr1-like cells that produce very high levels
of this anti-inflammatory cytokine. Indeed, IL-10 was produced
by CD4+T-cells that failed to up-regulate the helper molecule
CD40L, as is characteristic for regulatory T-cells (22, 31).
In conclusion, we provided here the first detailed analysis of
the T-cell compartment in the IA wall in a pediatric case. The
dominance of cytotoxic effector T-cells suggests a predominantly
pro-inflammatory, and thus presumably detrimental (32), role of
these T-cells in the IA growth and rupture. The strong
enrichment of Eomes + Tr1-like cells might reflect an
unsuccessful attempt to control the large majority of effector
cells. Future studies including a larger number of patients with
ruptured and unruptured aneurysm could be helpful to
understand the role of immunity in this rare and complex disease.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in
the article/Supplementary Material. Further inquiries can be
directed to the corresponding authors.

ETHICS STATEMENT
The studies involving human participants were reviewed and
approved by Ethics Committee of the Fondazione IRCCS Ca’
Granda–Ospedale Maggiore Policlinico. Written informed
consent to participate in this study was provided by the
participants’ legal guardian/next of kin. Written informed
consent was obtained from the minor(s)’ legal guardian/next of
kin for the publication of any potentially identifiable images or
data included in this article.

AUTHOR CONTRIBUTIONS
GM isolated cells, designed and performed flow cytometry
analysis and participated in manuscript writing, CV designed
and analysed flow cytometry data and participated in Figure's
design and manuscript writing, FC and EG performed
bioinformatic analysis designed Figures and contributed to
manuscript writing, TZ, FO, CM, ERZ and NS provided
human specimens and clinical data and partecipated to the
study design and manuscript writing ML and MP provided
human specimens and clinical data AS pprobided critical

10

May 2022 | Volume 13 | Article 866558

Moschetti et al.

T-Cells in the Aneurysm Wall

Supplementary Figure 2 | (A) Histograms of the marker expression values (surface
and intracellular differentiation markers analysed ex vivo) that were used to distinguish
cell clusters. Clusters were colored according to the tissue of origin: IA wall (red),
PBMCs (green). (B) UMAPs of all analysed markers (as indicated). Blue denotes low,
green intermediate and red high expression. (C) Percentages of the nine cell clusters.

discusiion and contributed to manuscript design JG contributed
to the study design, coordinated the study and wrote the paper.

FUNDING

Supplementary Figure 3 | The temporal order of differentiating cells was inferred
by DiffusionMap. DiffusionMap, colored by sample of origin (A) and clusters (B),
show higher proximity of IA wall T-cells to circulating memory T cells as compared to
naïve cells. A general differentiation pattern from naïve phenotypes to memory and
TRM in CD4+, CD8+ and DN T-cells can be observed.

This study was supported by the Fondazione Cariplo (“Ricerca
biomedica condotta da giovani ricercatori” grant number 2019-1632).

Supplementary Figure 4 | Gating Strategy used to analyse T cell compartment
for CD4+, CD8+ and DN T-cell subsets.

SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online at:
https://www.frontiersin.org/articles/10.3389/fimmu.2022.866558/
full#supplementary-material

Supplementary Figure 5 | (A) CD127 versus CCR7 expression of gated Th1
(CXCR3+ CCR6-), Th1/17 (CXCR3+ CCR6+) and Th17 (CCR6+) were analysed to
identify central memory cells (CCR7+CD127+), effector memory (CCR7- CD127+)
and effector-like (CD127- CCR7-) cells. (B) To assess the presence of CD27
+CCR6- Tr1-like cells the expression of CD27 and CCR6 was analysed on gated
CD4+ CD25- CD127- CCR5+ T-cells. (C) Gating Strategy used to analyse CD4+
effector-like T-cells (CD127loFOXP3-).

Supplementary Table 1 | List of the used antibodies.
Supplementary Table 2 | Absolute cell numbers (CD4+, CD8+ and DN T-cells).
Supplementary Table 3 | MFI values of CD127, CCR5, CCR6, GZMB, GZMK,
CXCR3, CD27 and CD69 expression among CD4+ T cells.

Supplementary Figure 6 | (A) Histograms of the marker (cytokines and
differentiation markers) expression values were used to distinguish cell clusters
following brief polyclonal stimulation. Clusters were colored according to the sample
of origin: IA_WALL (red), PT_PBMCs and IA_WALL (yellow), PBMCs (blue) and ALL
(shared among all tissue, violet). (B) UMAP of all analysed markers, as indicated.
Blue denotes low, green intermediate and red high expression. (C) Frequencies of
all cell clusters among T-cells in each sample as indicated.

Supplementary Figure 1 | (A) UMAP of cells colored according to the clusters as
indicated obtained by FlowSOM using K = 20. (B) Heatmap of median normalized
expression of markers within each cluster. Dendrogram on the left side of the
heatmap shows similarities among clusters.

Smooth Muscle Cells. J Neuroinflamm (2016) 13:185. doi: 10.1186/s12974016-0654-z
12. Frosen J, Piippo A, Paetau A, Kangasniemi M, Niemela M, Hernesniemi J,
et al. Remodeling of Saccular Cerebral Artery Aneurysm Wall is Associated
With Rupture: Histological Analysis of 24 Unruptured and 42 Ruptured
Cases. Stroke (2004) 35:2287–93. doi: 10.1161/01.STR.0000140636.30204.da
13. Muhammad S, Chaudhry SR, Dobreva G, Lawton MT, Niemela M, Hanggi D.
Vascular Macrophages as Therapeutic Targets to Treat Intracranial
Aneurysms. Front Immunol (2021) 12:630381. doi: 10.3389/fimmu.
2021.630381
14. Filiano AJ, Gadani SP, Kipnis J. How and Why do T Cells and Their Derived
Cytokines Affect the Injured and Healthy Brain? Nat Rev Neurosci (2017)
18:375–84. doi: 10.1038/nrn.2017.39
15. Nowicka M, Krieg C, Crowell HL, Weber LM, Hartmann FJ, Guglietta S, et al.
CyTOF Workflow: Differential Discovery in High-Throughput HighDimensional Cytometry Datasets. F1000Research (2017) 6:748. doi:
10.12688/f1000research.11622.1
16. Sathaliyawala T, Kubota M, Yudanin N, Turner D, Camp P, Thome JJ, et al.
Distribution and Compartmentalization of Human Circulating and TissueResident Memory T Cell Subsets. Immunity (2013) 38:187–97. doi: 10.1016/
j.immuni.2012.09.020
17. Sallusto F, Geginat J, Lanzavecchia A. Central Memory and Effector Memory
T Cell Subsets: Function, Generation, and Maintenance. Annu Rev Immunol
(2004) 22:745–63. doi: 10.1146/annurev.immunol.22.012703.104702
18. Haringer B, Lozza L, Steckel B, Geginat J. Identification and Characterization
of IL-10/IFN-Gamma-Producing Effector-Like T Cells With Regulatory
Function in Human Blood. J Exp Med (2009) 206:1009–17. doi: 10.1084/
jem.20082238
19. Gruarin P, Maglie S, De Simone M, Haringer B, Vasco C, Ranzani V, et al.
Eomesodermin Controls a Unique Differentiation Program in Human IL-10
and IFN-Gamma Coproducing Regulatory T Cells. Eur J Immunol (2019)
49:96–111. doi: 10.1002/eji.201847722
20. Bonnal RJP, Rossetti G, Lugli E, De Simone M, Gruarin P, Brummelman J,
et al. Clonally Expanded EOMES(+) Tr1-Like Cells in Primary and Metastatic
Tumors are Associated With Disease Progression. Nat Immunol (2021)
22:735–45. doi: 10.1038/s41590-021-00930-4

REFERENCES
1. Krings T, Mandell DM, Kiehl TR, Geibprasert S, Tymianski M, Alvarez H,
et al. Intracranial Aneurysms: From Vessel Wall Pathology to Therapeutic
Approach. Nat Rev Neurol (2011) 7:547–59. doi: 10.1038/nrneurol.2011.136
2. Beslow LA, Jordan LC. Pediatric Stroke: The Importance of Cerebral
Arteriopathy and Vascular Malformations. Childs Nerv Syst (2010)
26:1263–73. doi: 10.1007/s00381-010-1208-9
3. Xu R, Xie ME, Yang W, Gailloud P, Caplan JM, Jackson CM, et al.
Epidemiology and Outcomes of Pediatric Intracranial Aneurysms:
Comparison With an Adult Population in a 30-Year, Prospective Database.
J Neurosurg Pediatr (2021), 28(6):1–10. doi: 10.3171/2021.6.PEDS21268
4. Proust F, Toussaint P, Garnieri J, Hannequin D, Legars D, Houtteville JP, et al.
Pediatric Cerebral Aneurysms. J Neurosurg (2001) 94:733–9. doi: 10.3171/
jns.2001.94.5.0733
5. Aeron G, Abruzzo TA, Jones BV. Clinical and Imaging Features of
Intracranial Arterial Aneurysms in the Pediatric Population. Radiographics
(2012) 32:667–81. doi: 10.1148/rg.323105224
6. Krings T, Geibprasert S, terBrugge KG. Pathomechanisms and Treatment of
Pediatric Aneurysms. Childs Nerv Syst (2010) 26:1309–18. doi: 10.1007/
s00381-009-1054-9
7. Etminan N, Rinkel GJ. Unruptured Intracranial Aneurysms: Development,
Rupture and Preventive Management. Nat Rev Neurol (2016) 12:699–713.
doi: 10.1038/nrneurol.2016.150
8. Chalouhi N, Ali MS, Jabbour PM, Tjoumakaris SI, Gonzalez LF, Rosenwasser
RH, et al. Biology of Intracranial Aneurysms: Role of Inflammation. J Cereb
Blood Flow Metab (2012) 32:1659–76. doi: 10.1038/jcbfm.2012.84
9. Chyatte D, Bruno G, Desai S, Todor DR. Inflammation and Intracranial
Aneurysms. Neurosurgery (1999) 45:1137–46; discussion 1146-7. doi:
10.1097/00006123-199911000-00024
10. Miyata H, Koseki H, Takizawa K, Kasuya H, Nozaki K, Narumiya S, et al. T
Cell Function is Dispensable for Intracranial Aneurysm Formation and
Progression. PloS One (2017) 12:e0175421. doi: 10.1371/journal.pone.0175421
11. Sawyer DM, Pace LA, Pascale CL, Kutchin AC, O'Neill BE, Starke RM, et al.
Lymphocytes Influence Intracranial Aneurysm Formation and Rupture: Role
of Extracellular Matrix Remodeling and Phenotypic Modulation of Vascular

Frontiers in Immunology | www.frontiersin.org

11

May 2022 | Volume 13 | Article 866558

Moschetti et al.

T-Cells in the Aneurysm Wall

Ischemic Stroke. Immunity (2021) 54:1527–42.e8. doi: 10.1016/j.immuni.
2021.04.022
30. Ito M, Komai K, Mise-Omata S, Iizuka-Koga M, Noguchi Y, Kondo T, et al.
Brain Regulatory T Cells Suppress Astrogliosis and Potentiate Neurological
Recovery. Nature (2019) 565:246–50. doi: 10.1038/s41586-018-0824-5
31. Geginat J, Vasco M, Gerosa M, Tas SW, Pagani M, Grassi F, et al. IL-10
Producing Regulatory and Helper T-Cells in Systemic Lupus Erythematosus.
Semin Immunol (2019) 44:101330. doi: 10.1016/j.smim.2019.101330
32. Iadecola C, Anrather J. The Immunology of Stroke: From Mechanisms to
Translation. Nat Med (2011) 17:796–808. doi: 10.1038/nm.2399

21. Cossarizza A, Chang HD, Radbruch A, Abrignani S, Addo R, Akdis M, et al.
Guidelines for the Use of Flow Cytometry and Cell Sorting in Immunological
Studies (Third Edition). Eur J Immunol (2021) 51:2708–3145.
22. Facciotti F, Gagliani N, Haringer B, Alfen JS, Penatti A, Maglie S, et al. IL-10Producing Forkhead Box Protein 3-Negative Regulatory T Cells Inhibit B-Cell
Responses and are Involved in Systemic Lupus Erythematosus. J Allergy Clin
Immunol (2016) 137:318–21.e5. doi: 10.1016/j.jaci.2015.06.044
23. Lv BJ, Li J, Cheng X. T Lymphocytes and Aortic Aneurysms. Sci China Life Sci
(2014) 57:795–801. doi: 10.1007/s11427-014-4699-x
24. Sagan A, Mikolajczyk TP, Mrowiecki W, MacRitchie N, Daly K, Meldrum A,
et al. T Cells Are Dominant Population in Human Abdominal Aortic Aneurysms
and Their Infiltration in the Perivascular Tissue Correlates With Disease
Severity. Front Immunol (2019) 10:1979. doi: 10.3389/fimmu.2019.01979
25. Mohme M, Sauvigny T, Mader MM, Schweingruber N, Maire CL, Runger A,
et al. Immune Characterization in Aneurysmal Subarachnoid Hemorrhage
Reveals Distinct Monocytic Activation and Chemokine Patterns. Transl
Stroke Res (2020) 11:1348–61. doi: 10.1007/s12975-019-00764-1
26. Roa JA, Sarkar D, Zanaty M, Ishii D, Lu Y, Karandikar NJ, et al. Preliminary
Results in the Analysis of the Immune Response After Aneurysmal Subarachnoid
Hemorrhage. Sci Rep (2020) 10:11809. doi: 10.1038/s41598-020-68861-y
27. Geginat J, Paroni M, Facciotti F, Gruarin P, Kastirr I, Caprioli F, et al. The
CD4-Centered Universe of Human T Cell Subsets. Semin Immunol (2013)
25:252–62. doi: 10.1016/j.smim.2013.10.012
28. Joy MT, Ben Assayag E, Shabashov-Stone D, Liraz-Zaltsman S, Mazzitelli J,
Arenas M, et al. CCR5 Is a Therapeutic Target for Recovery After Stroke and
Traumatic Brain Injury. Cell (2019) 176:1143–57.e13.
29. Shi L, Sun Z, Su W, Xu F, Xie D, Zhang Q, et al. Treg Cell-Derived
Osteopontin Promotes Microglia-Mediated White Matter Repair After

Frontiers in Immunology | www.frontiersin.org

Conflict of Interest: The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be construed as a
potential conflict of interest.
Publisher’s Note: All claims expressed in this article are solely those of the authors
and do not necessarily represent those of their affiliated organizations, or those of
the publisher, the editors and the reviewers. Any product that may be evaluated in
this article, or claim that may be made by its manufacturer, is not guaranteed or
endorsed by the publisher.
Copyright © 2022 Moschetti, Vasco, Clemente, Galeota, Carbonara, Pluderi, Locatelli,
Stocchetti, Abrignani, Zanier, Ortolano, Zoerle and Geginat. This is an open-access
article distributed under the terms of the Creative Commons Attribution License
(CC BY). The use, distribution or reproduction in other forums is permitted, provided
the original author(s) and the copyright owner(s) are credited and that the original
publication in this journal is cited, in accordance with accepted academic practice. No
use, distribution or reproduction is permitted which does not comply with these terms.

12

May 2022 | Volume 13 | Article 866558