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\begin{document}

\title{Impaired Immune Privilege Exclusive to HSCs Mediated by Tregs is
associated with HSCs Exhaustion in children with Aplastic Anemia}



\author[1]{Shayi Jiang}%
\author[1]{Can Huang}%
\author[1]{Shan shan Li}%
\author[1]{Jing Yang}%
\author[1]{Xue lian Liao}%
\author[1]{Yangyang Jiao}%
\author[1]{Ting Zhang}%
\author[2]{Shuanglong Lu}%
\author[1]{Yan hua Li}%
\author[1]{Hui Jiang}%
\author[3]{Fanyi Zeng}%
\affil[1]{Shanghai Jiao Tong University School of Medicine}%
\affil[2]{Shanghai Tongji Hospital}%
\affil[3]{Shanghai Jiao Tong University Institute of Medical Genetics}%


\vspace{-1em}



  
  \date{May 24, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
A series of immune abnormalities presenting in aplastic anemia (AA)
support the immune pathogenesis of AA. However, how abnormal immunity
specifically damages hematopoietic stem cells (HSCs) remains ambiguous.
The discovery of bone marrow immune privilege (IP) sites which are
composed of CD4 \textsuperscript{+}CD25 \textsuperscript{+}FoxP3
\textsuperscript{+} regulatory T cells (Tregs) exclusively to protect
HSCs prompted us boldly assumed that it is a loss of IP protection
leading to HSCs exhausted in AA. A experiment study to clinically
confirm the correlation between HSCs depletion and IP abnormalities in
patients with AA was conducted. The distribution of Tregs in bone marrow
from children with AA, myelodysplastic syndrome (MDS) and control group
was detected by immunohistochemistry. Th1, Th2, Th17 and HSCs as well as
cytokines in bone marrow of these children were examined by flow
cytometry. Tregs near endostea surface of bone marrow of children with
AA was significantly lower than that in control and MDS. Th1 was more
predominant in AA than in the control children. TNF-\selectlanguage{greek}α, \selectlanguage{english}IFN-\selectlanguage{greek}γ \selectlanguage{english}and IL-17
levels were also increased in AA. Compared to the control group, HSCs in
bone marrow of AA, including Long-term HSCs (LT-HSCs) and Short-term
HSCs (ST-HSCs), were at lower level. The results indicate that HSCs
depletion is closely related to bone marrow IP abnormalities in AA, and
the role of IP abnormalities in the pathogenesis of aplastic anemia
deserves further research.%
\end{abstract}%



\sloppy


Impaired Immune Privilege Exclusive to HSCs Mediated by Tregs is
associated with HSCs Exhaustion in children with Aplastic Anemia

Can Huang\textsuperscript{1\#}, Shanshan Li\textsuperscript{1\#},
Jingwei Yang\textsuperscript{1}, Xuelian Liao\textsuperscript{1},
Yangyang Jiao\textsuperscript{1}, Ting Zhang\textsuperscript{1},
Shuanglong Lu\textsuperscript{2}, Yanhua Li\textsuperscript{1}, Hui
Jiang\textsuperscript{1*}, Fanyi Zeng\textsuperscript{3,4*}, Shayi
Jiang\textsuperscript{1,4*}

\textsuperscript{1}Department of Hematology and Oncology, Shanghai
Children's Hospital, School of Medicine, Shanghai Jiao Tong University,
Shanghai, China

\textsuperscript{2}Department of Pediatrics, Tongji Hospital, Tongji
University School of Medicine, Shanghai, China

\textsuperscript{3}Shanghai Institute of Medical Genetics, Shanghai
Children's Hospital, \& Department of Histo-Embryology, Genetics and
Developmental Biology, Shanghai Jiao Tong University School of Medicine,
Shanghai, China

\textsuperscript{4}NHC Key Laboratory of Medical Embryogenesis and
Developmental Molecular Biology \& Shanghai Key Laboratory of Embryo and
Reproduction Engineering, Shanghai, China

\textsuperscript{\#}These authors contribute equally to the work

\textbf{Running head:} HSCs Loss Protection of immune privilege

\textbf{Corresponding authors}

\textsuperscript{\textbf{*}} Shayi Jiang, Department of Hematology and
Oncology, Shanghai children's Hospital, School of medicine, Shanghai
Jiao Tong University. No. 355 Luding Road, Putuo District, Shanghai,
China (e-mail: jiangshayi@163.com).

\textsuperscript{\textbf{*}} Fanyi Zeng, Shanghai Institute of Medical
Genetics, Shanghai Children's Hospital, \& Department of
Histo-Embryology, Genetics and Developmental Biology, Shanghai Jiao Tong
University School of Medicine. No. 24, lane 1400, West Beijing Road,
Jingan District, Shanghai, China (e-mail: fzeng@sjtu.edu.cn).

\textsuperscript{\textbf{*}} Hui Jiang, Department of Hematology and
Oncology, Shanghai children's Hospital, School of medicine, Shanghai
Jiao Tong University. No. 24, lane 1400, West Beijing Road, Jingan
District, Shanghai, China (e-mail: jhui011@126.com).

\textbf{Text word count: 2301}

\textbf{Abstract word count: 224}

\textbf{Total Figures: 6}

\textbf{Number of references: 25}

\textbf{KEYWORDS}

immune privilege, Tregs, hematopoietic stem cells, aplastic anemia

\textbf{Abbreviations}\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
AA & aplastic anemia\tabularnewline
\midrule
\endhead
HSCs & hematopoietic stem cells\tabularnewline
IP & immune privilege\tabularnewline
Tregs &
CD4\textsuperscript{+}CD25\textsuperscript{+}FoxP3\textsuperscript{+}
regulatory T cells\tabularnewline
MDS & myelodysplastic syndrome\tabularnewline
LT-HSCs & Long-term HSCs\tabularnewline
ST-HSCs & Short-term HSCs\tabularnewline
\bottomrule
\end{longtable}

\textbf{Abstract}

A series of immune abnormalities presenting in aplastic anemia (AA)
support the immune pathogenesis of AA. However, how abnormal immunity
specifically damages hematopoietic stem cells (HSCs) remains ambiguous.
The discovery of bone marrow immune privilege (IP) sites which are
composed of
CD4\textsuperscript{+}CD25\textsuperscript{+}FoxP3\textsuperscript{+}regulatory
T cells (Tregs) exclusively to protect HSCs prompted us boldly assumed
that it is a loss of IP protection leading to HSCs exhausted in AA. A
experiment study to clinically confirm the correlation between HSCs
depletion and IP abnormalities in patients with AA was conducted. The
distribution of Tregs in bone marrow from children with AA,
myelodysplastic syndrome (MDS) and control group was detected by
immunohistochemistry. Th1, Th2, Th17 and HSCs as well as cytokines in
bone marrow of these children were examined by flow cytometry. Tregs
near endostea surface of bone marrow of children with AA was
significantly lower than that in control and MDS. Th1 was more
predominant in AA than in the control children. TNF-\selectlanguage{greek}α, \selectlanguage{english}IFN-\selectlanguage{greek}γ \selectlanguage{english}and IL-17
levels were also increased in AA. Compared to the control group, HSCs in
bone marrow of AA, including Long-term HSCs (LT-HSCs) and Short-term
HSCs (ST-HSCs), were at lower level. The results indicate that HSCs
depletion is closely related to bone marrow IP abnormalities in AA, and
the role of IP abnormalities in the pathogenesis of aplastic anemia
deserves further research.

\textbf{1 ? INTRODUCTION}

Acquired aplastic anemia (AA) is characterized by pancytopenia caused by
bone marrow failure without dysplasia or fibrosis. Both clinical and
laboratory evidence support that pathogenesis of bone marrow failure is
due to injury to hematopoietic stem cells (HSCs) by abnormal
immunity\textsuperscript{1,2}. Overall, research on depletion of HSCs in
AA has focused on exploring the mechanism by which abnormal immunity
attacks HSCs. However, no specific antibodies or immune cells targeting
HSCs have been found to date. Therefore, the ''immune-mediated
pathogenesis'' of AA has not yet clarified how immune disorders
specifically damage HSCs rather than other cells in bone marrow.

With in-depth study of bone marrow microenvironment, immune privileged
(IP) sites of bone marrow specific to HSCs are
discovered\textsuperscript{3}. IP sites are mainly comprised of
CD4\textsuperscript{+}CD25\textsuperscript{+}FoxP3\textsuperscript{+}regulatory
T cells (Tregs) which surround HSCs near the endosteal surface,
providing an immune tolerance microenvironment for HSCs to protect HSCs
from infection, radiation and other stress damage. This urged us to
shift the research on depletion mechanism of HSCs in AA from immune
``attack'' to ``protection loss'' of IP. In fact, IP sites also exist in
other tissues, and their abnormal pathogenicity has been a concern in
various diseases\textsuperscript{4}. For example, IP sites in hair
follicle provide protection for epithelial hair follicle stem cells, and
functional impairment leads to destruction of epithelial hair follicle
stem cells, which is the main pathological mechanism of persistent
alopecia in alopecia areata\textsuperscript{5}. The local immune
tolerance status of IP sites can be determined by detecting
FoxP3\textsuperscript{+}/CD4\textsuperscript{+} cells with an
immunohistochemical method\textsuperscript{6}. But the correlation
between the collapse of self-tolerance of bone marrow IP sites and AA
has not been reported. This study aimed to clinically verify that HSCs
failure in AA is closely related to IP abnormalities through the
detection of bone marrow IP sites in AA, MDS, and countrol group,
providing clinical supportive evidence of worth further study for the IP
pathogenesis in AA.

\textbf{2 ? METHODS}

\textbf{2.1 ? Patients}

AA cases and myelodysplastic syndrome (MDS) cases were all from Shanghai
Children's Hospital between. According to the severity, AA patients were
classified into non-severe AA, severe AA and very severe
AA\textsuperscript{7}. MDS was diagnosed based on WHO classification
criteria\textsuperscript{8}. Twenty children with solid tumors without
bone marrow infiltration and any hematological complications were used
as normal controls. Bone marrow biopsy specimens from AA, MDS and normal
controls were used for immunohistochemistry. The bone marrow fluid of
the first suction and peripheral blood from AA and controls were
detected by flow cytometry.

\textbf{2.2 ? Immunohistochemical staining for FoxP3, CD4 and CD3}

Bone marrow specimens from posterior iliac crest were obtained in all
cases. Immunohistochemical staining for FoxP3, CD4 and CD3 was
separately performed using Ventana Benchmark GX Immuno-AutoStainer
(Roche) according to the manufacturer's instructions. The primary
anti-human rabbit monoclonal antibody, incluing anti-FoxP3 (dilution
1:500; Abcam), anti-CD4 (dilution 1:100; Abcam) and anti-CD3 (dilution
1:100; Abcam), were revealed with an indirect biotin streptavidin system
(HRP/DAB Detection ICH kits, Abcam). Cells of
FoxP3\textsuperscript{+}presented brown chromogen on the nucleus, while
CD4\textsuperscript{+}and CD3\textsuperscript{+} cells showed
cytoplasmic brown granules.

All slides were reviewed independently by two skilled pathologists. The
positive cells were counted in 5 visual fields near endosteum of
trabecular bone under high-power magnification (\selectlanguage{ngerman}×600). The mean value of
FoxP3\textsuperscript{+}/CD4\textsuperscript{+} in each case represented
the local immune tolerance status mediated by Tregs.

\selectlanguage{english}\textbf{2.3 ? Flow cytometry analysis of Th1, Th2, Th17 and HSCs}

T-helper 1 (Th1), T-helper 2 (Th2), T-helper 17 (Th17) and HSCs in bone
marrow of children and controls were detected by flow cytometry. The
anti-human antibodies (BD Pharmingen) included APC-H7-CD3, APC-CD4,
FITC-CD183, PE-Cy7-CD196, FITC-CD34,
PE-Cy7-CD38\selectlanguage{english},PE-Cy7-CD45RA\selectlanguage{english},PE-CD90, Alexa Fluor
647-CD49f\selectlanguage{english},PerCP-Cy5.5-CD3\selectlanguage{english},PerCP-Cy5.5-CD56, PerCP-Cy5.5-CD11 and
PerCP-Cy5.5-CD19. Th1, Th2 and Th17 are the lineages of
CD3\textsuperscript{+}CD4\textsuperscript{+}CD183\textsuperscript{+}CD196\textsuperscript{-},
CD3\textsuperscript{+}CD4\textsuperscript{+}CD183\textsuperscript{-}CD196\textsuperscript{-}and
CD3\textsuperscript{+}CD4\textsuperscript{+}CD183\textsuperscript{-}CD196\textsuperscript{+}respectively.
Long-term HSCs (LT-HSCs) are the lineages presenting
lin\textsuperscript{-}CD34\textsuperscript{+}CD38\textsuperscript{-}CD45RA\textsuperscript{-}CD90\textsuperscript{+}CD49f\textsuperscript{+},
while short-term HSCs (ST-HSCs) presenting
lin\textsuperscript{-}CD34\textsuperscript{+}CD38\textsuperscript{-}CD45RA\textsuperscript{-}CD90\textsuperscript{-}CD49f\textsuperscript{-}.
The monocyte was collected from bone marrow aspirates or peripheral
blood with a Ficoll density gradient. Cells were analyzed by a
FACSCalibur (BD Biosciences). Data analysis was performed using FlowJo
software.

\selectlanguage{english}\textbf{2.4 ? Cytokine assays}

Cytokines in bone marrow and peripheral blood were detected by multiplex
bead-based flow cytometric immunoassays. Multiplex assay kits for IL-5,
TNF-\selectlanguage{greek}α, \selectlanguage{english}IL-2, IL-6, IL-1\selectlanguage{greek}β, \selectlanguage{english}IL-10, IFN-\selectlanguage{greek}γ, \selectlanguage{english}IL-8, IL-17 and IL-12 were
purchased from RAISECARE in China. Above cytokines were tested in plasma
samples obtained by centrifugation of heparinized bone marrow or
peripheral blood from AA and control children.

\textbf{2.5 ? Statistical analysis}

All graphs and statistical analysis were generated by using GraphPad
Prism 5 (GraphPad Software). Continuous variables were presented as
mean\selectlanguage{ngerman}±SD. As the data were not distributed normally, nonparametric Mann
Whitney and Kruskal-Wallis tests were used for comparison of
data.\emph{P} \textless{}0.05 was considered as statistically
significant.

\selectlanguage{english}\textbf{2.6 ? Ethical approval}

The Children's Hospital of Shanghai Local Research Ethics Committee
approved this study, and informed written consent was obtained from the
patients' parents.

\selectlanguage{english}\textbf{3 ? Results}

\selectlanguage{english}\textbf{3.1 ? Characteristics of the patients}

Of 30 AA children, 56.70\% (n=17) were boys, and 43.60\% (n=13) were
girls. The age ranged from 2 to 14 years, with a median age of 8 years.
The percentages of non-severe AA, severe AA and very severe AA were
50.00\%, 36.70\% and 13.30\%, respectively, in the 30 AA patients. There
were no statistically significant differences in age or sex among the AA
group, MDS group and control group.

\selectlanguage{english}\textbf{3.2 ? Lower Tregs in IP sites of AA}

HSCs have been mainly detected in close proximity to vasculature and the
endosteum, which is the osteoblast niche\textsuperscript{9}. Tregs
accumulate in bone marrow and provide protection for HSCs via IP
mechanisms. Therefore, FoxP3/CD4 should be able to indicate the immune
tolerance status provided by IP to HSCs, especially in the area near
endosteum, which has been identified as an IP site, overlapping with the
osteoblast niche. We observed FoxP3\textsuperscript{+},
CD4\textsuperscript{+} and CD3\textsuperscript{+} cells near the
endosteum\selectlanguage{english}(Figure 1).
FoxP3\textsuperscript{+}/CD4\textsuperscript{+}were significantly lower
in AA than in normal controls (18.09\%+-5.38\%\emph{vs} . 21.72\%+-4.21\%,
\emph{P} \textless{}0.05) and MDS (18.09\%+-5.38\% \emph{vs} .
22.63\%+-5.98\%, \emph{P} \textless{}0.05) (Figure 2A). Taking the low
number of Tregs in bone marrow into account, we tried to use the
absolute count of FoxP3\textsuperscript{+} cells to represent bone
marrow immune tolerance status. The average number of Tregs in AA was
also significantly lower than that in normal control (4.07+-1.41
\emph{vs} . 5.25+-1.86 cells/HP, \emph{P} \textless{}0.05) and MDS
(4.07+-1.41 \emph{vs} . 5.30+-1.49 cells/HP, \emph{P} \textless{}0.05)
(Figure 2B). No matter FoxP3\textsuperscript{+}/CD4\textsuperscript{+}or
FoxP3\textsuperscript{+} absolute count, there was no statistically
significant difference between MDS patients and normal controls
(\emph{P} \textgreater{}0.05).

\selectlanguage{english}\textbf{3.3 ? Th1/Th2 imbalance in AA}

The normal IP effect also involves homeostasis between Th1 and Th2 with
a predominance action of Th2 cells and a relatively low activity of Th1
and Th17\textsuperscript{10}, so Th1/Th2 can indirectly reflect IP
function\textsuperscript{11}. Th17 cells are also considered to be
similar to Th1 cells in the regulation of immune tolerance. Compared
with the normal control, AA group showed a significantly higher
proportion of Th1 and a lower Th2 in bone marrow (for Th1
39.80\%+-5.20\%\emph{vs} . 22.1\%+-2.9\%, \emph{P} \textless{}0.0001; Th2
59.20\%+-5.30\%\emph{vs} . 77.20\%+-2.60\%, \emph{P} \textless{}0.0001),
with a Th1/Th2 shift. Meanwhile Th17 was also lower in AA group than
that in control group (1.00\%+-0.60\% \emph{vs} . 0.50\%+-0.70\%, \emph{P}
=0.0047) (Figure 3).

For Th1/Th2 in peripheral blood of AA, it was also higher than that in
the control group, but there was no statistically significant difference
(\emph{P} \textgreater{}0.05) (data not shown). The predominance Th1 of
bone marrow also supported the weakening of the local immune tolerance
environment.

\selectlanguage{english}\textbf{3.4 ? Cytokines in bone marrow}

Significant increases of TNF-\selectlanguage{greek}α, \selectlanguage{english}IFN-\selectlanguage{greek}γ \selectlanguage{english}and IL-17 were observed in the
bone marrow of AA patients comparing with that in contral group with
11.20\selectlanguage{ngerman}±35.20 \emph{vs} . 2.80±0.70 pg/mL (\emph{P} \textless{}0.0001),
35.00±7.80 \emph{vs} . 13.00±5.80 pg/mL (\emph{P} \textless{}0.0001) and
28.90±11.20 \emph{vs} 2.60±0.30 pg/mL (\emph{P} \textless{}0.0001),
respectively (Figure 4A). The levels of TNF-\selectlanguage{greek}α, \selectlanguage{english}IFN-\selectlanguage{greek}γ \selectlanguage{english}and IL-17 in
peripheral blood of AA group were also higher than that in the control
group, but there was no statistically significant difference (Figure
4B).

\textbf{3.5 ? Decrease of LT-HSCs}

Hematopoietic stem cell pools include LT-HSCs and ST-HSCs. In order to
understand the status of subpopulations of HSCs in AA, we tested LT-HSCs
and ST-HSCs. The results showed that both of them were significantly
lower in bone marrow of children with AA than that in the control
group(for LT-HSCs 1.13\selectlanguage{ngerman}±0.82/100000 \emph{vs} .
12.09±5.29/100000,\emph{P} \textless{}0.0001; for ST-HSCs
2.79±1.75/100000 \emph{vs} . 22.98±8.76/100000, \emph{P}
\textless{}0.0001\selectlanguage{english})(Figure 5).

\selectlanguage{english}\textbf{4 ? DISCUSSION}

The initial understanding of IP was limited to eyes, testes and brain,
which are organs with blood tissue barriers. Later, research expanded
the concept of IP, which actually exists in many tissues, including hair
follicles, intestinal mucosa, lung and so on. IP actively guides and
controls the immune response through various mechanisms to maintain the
integrity of immune tolerance microenvironment.
Fujisaki\textsuperscript{3} first revealed bone marrow IP sites by using
high-resolution \emph{in vivo} imaging in animal experiments. The main
component cells of IP are Tregs, which are characterized by the
expression of FoxP3 in the nucleus. Through the binding of membrane
CXCR4 receptors with a large amount of chemokine CXCL12 in the HSCs
niche, Tregs aggregate to bone marrow near the endosteum surface of
trabecular bone to surround HSCs\textsuperscript{12, 13}, where the
transplanted allogeneic HSCs could survive for a long time (30 days)
without myeloablative conditioning regimen before transplantation, which
is the gold standard experiment for IP identification. Once the Tregs
were removed from bone marrow by deleting the CXCR4 receptor in mice,
TNF-\selectlanguage{greek}α \selectlanguage{english}and IFN-\selectlanguage{greek}γ \selectlanguage{english}were enhanced, while IL-10 was decreased in the bone
marrow\textsuperscript{3}. Then, the transplanted allogeneic HSCs
described in the above experiment could not survive in the osteoblast
niche. HSCs located in IP immune tolerance microenvironment are
LT-HSCs\textsuperscript{3}. Subsequent studies showed that LT-HSCs were
over mobilized with stress induction after Tregs were
depleted\textsuperscript{12, 14}. Therefore, in bone marrow, IP not only
provides an immune tolerance microenvironment to protect LT-HSCs from
various immune factors but also reduces the overmobilization of LT-HSCs
during stress and maintains the static state of LT-HSCs, which are the
stem cells maintaining lifelong hematopoiesis of bone marrow.

It has been proven in AA patients that the ability of Tregs to migrate
to bone marrow was impaired, and the proportion of Tregs in the bone
marrow detected by flow cytometry was also
decreased\textsuperscript{15,16}. However, there has been no research
linking the immune abnormalities of AA with local immune tolerance
status of IP. Interestingly, a series of immune disorders of AA reported
in the literature and in our previous studies are strikingly similar to
the excessive immune status due to the abnormal IP. The increased
expression of TNF-\selectlanguage{greek}α \selectlanguage{english}and IFN-\selectlanguage{greek}γ \selectlanguage{english}in CD4\textsuperscript{+} and
CD8\textsuperscript{+} T cells was similar to the changes of bone marrow
cytokines in the IP damage assay (Treg deletion)\textsuperscript{3,
17,18}. The Th1/Th2 polarization detected in a previous study of an AA
animal model was highly consistent with the immune disorder caused by
impaired IP function\textsuperscript{19,20}.

Drawing on other experimental methods studying the function of IP in
local tissues \textsuperscript{4,5,21}, we tested the distribution of
Tregs in the bone marrow osteoblast niche by immunohistochemistry. There
were significantly fewer Tregs on the endosteal surface of bone marrow
than in normal controls and MDS patients, accompanied by an increase in
Th1/Th2, TNF-\selectlanguage{greek}α, \selectlanguage{english}IFN-\selectlanguage{greek}γ \selectlanguage{english}and IL-17 in bone marrow, while in another bone
marrow failure disease, MDS, they were not. All these results suggested
that IP structure and function were impaired in bone marrow of children
with AA.

The LT-HSCs protected by IP are usually in a static state to maintain
the hematopoietic stem cell pool\textsuperscript{3}. Generally, LT-HSCs
are appropriately transition into activated ST-HSCs which in turn
differentiate into various blood cells\textsuperscript{22}. This
transition increases during stress. Recent research has shown that Tregs
regulate the level of reactive oxygen species in LT-HSCs to prevent
LT-HSCs from being over mobilized and exhausted during oxidative
stress\textsuperscript{11,13}. Our experiment showed significantly
reduced LT-HSCs in AA, which can be attributed to the loss of the
protective effect of IP on LT-HSCs. Various clinical evidences also
support that the ability to maintain HSCs against stress in AA is
reduced. Patients usually develop AA after infection, exposure to
radioactive substances or certain drugs, and the severity of the disease
is often aggravated during infection\textsuperscript{1, 23}. In clinical
practice, we found that 60\%-70\% of children with NSAA naturally
evolved into SAA during the course of continuous exposure to various
environmental stresses\textsuperscript{24, 25}. After the depletion of
LT-HSCs, there are not enough reserve HSCs to be mobilized, resulting in
the corresponding reduction of ST-HSCs. Therefore, we also detected a
decrease of ST-HSCs in the bone marrow of children with AA.

\textbf{5 ? CONCLUSION}

Our study clinically confirmed the correlation between AA HSCs depletion
and abnormal IP, so the hypothesis of the onset of AA caused by abnormal
IP deserves further research. If this assumption is confirmed, the
specific depletion of HSCs mediated by immune disorder of AA is able
to~be reasonably~explained as the loss of HSCs under stress owing to the
impaired immune tolerance microenvironment provided by IP to HSCs
(Figure 6), being expected to break the bottleneck in the study of the
pathogenesis of AA.

\textbf{AUTHOR CONTRIBUTIONS}

C.H and S.L analyzed data and write the manuscript. J.Y, X.L, Y.J, T.Z,
S.L and Y.L contributed to data collection and sample preparation; S.J,
F.Z and H.J contributed to designed research, performed research,
analyzed and interpreted data, wrote the manuscript as well as to the
approval of the final manuscript.

\textbf{ACKNOWLEDGMENTS}

This study was supported by research funding from the National Natural
Science Foundation of China (No. 81301973) and the Science and
Technology Commission Foundation of Shanghai (No. 14JC1492500).

\textbf{CONFLICT OF INTEREST}

The authors have no relevant financial or non-financial conflicts of
interest to disclose.

\textbf{DATA AVAILABILITY STATEMENT}

The data that support the findings of the study are available on request
from the corresponding author Shayi Jiang.

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\textbf{Figure legends}

\textbf{FIGURE 1. CD3\textsuperscript{+}, CD4\textsuperscript{+} and
FoxP3\textsuperscript{+} cells near endosteum detected by
immunohistochemistry.} The numbers of CD3\textsuperscript{+},
CD4\textsuperscript{+} and FoxP3\textsuperscript{+} cells all decreased
in AA patients.

\textbf{FIGURE 2. Tregs decreased in IP sites} .
FoxP3\textsuperscript{+}/CD4\textsuperscript{+}(A) and
FoxP3\textsuperscript{+}(B) were analyzed in normal control, MDS and AA.
Both FoxP3\textsuperscript{+}/CD4\textsuperscript{+} and
FoxP3\textsuperscript{+} cells in AA were lower than that in normal
control and MDS.

\textbf{FIGURE 3. Proportion of Th1, Th2 and Th17 cells in bone
marrow.}A. The percentages of Th1, Th2 and Th17 in normal control and AA
measured by flow cytometry. B. Compared with the normal control group,
the percentage of Th1 and Th17 in AA group increased significantly.

\selectlanguage{greek}\textbf{FIGURE 4. Expression of TNF-α, IFN-γ and IL-17 among bone marrow
aspirates mononuclear cells.}\selectlanguage{english} A. Compared with the normal control group,
TNF, IFN- \selectlanguage{greek}γ \selectlanguage{english}and IL-17 significantly increased in bone marrow of aplastic
anemia. B. Determination of TNF-\selectlanguage{greek}α, \selectlanguage{english}IFN-\selectlanguage{greek}γ \selectlanguage{english}and IL-17 in peripheral blood
by Flow cytometry, there were no differences between normal control and
AA.

\textbf{FIGURE 5. Bone marrow hematopoietic stem cell subsets: LT-HSCs
and ST-HSCs.} A. The content of LT-HSCs and ST-HSCs in normal and AA
detected by flow cytometry. B. LT-HSCs and ST-HSCs in bone marrow of
aplastic anemia, especially the LT-HSCs, were significantly lower than
those in the control group.

\textbf{FIGURE 6. A schematic diagram illustrating impared IP leading to
HSCs depletion in AA.}

\textbf{FIGURES}\selectlanguage{english}
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FIGURE 1\selectlanguage{english}
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FIGURE 2\selectlanguage{english}
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\end{center}
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FIGURE 3\selectlanguage{english}
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\end{center}
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FIGURE 4\selectlanguage{english}
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\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

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FIGURE 5\selectlanguage{english}
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FIGURE 6

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