Increased-NKG2A-CD8--T-cell-exhaustion-in-patients-with-_2023_Mucosal-Immuno

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ARTICLE
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Increased NKG2A+CD8+ T-cell exhaustion in patients with
adenomyosis
Wei Liu 1,2, Shuman Sheng 2, Chendi Zhu 4,5,6,7,8,9, Changzhong Li 1,3, Yonghui Zou 1,2, Chunrun Yang 1,2, Zi-Jiang Chen 4,5,6,7,8,9,
Fei Wang 1,2,6,✉ and Xue Jiao 4,5,6,7,8,9,10,✉
© 2023 The Author(s). Published by Elsevier Inc. on behalf of Society for Mucosal Immunology.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Immune dysregulation has long been proposed to be associated with adenomyosis, but the underlying mediators and mechanisms
remain largely unexplored. Here, we used flow cytometry to investigate the alterations in immune cell subsets in adenomyotic uteri
and analyze the phenotype and function of abnormal immune cells. We found that an increase in cluster of differentiation (CD)8+ T-
cell number was the predominant alteration in ectopic lesions in patients with adenomyosis and was significantly associated with
the severity of adenomyosis. Importantly, we identified an exhausted natural killer group protein 2A (NKG2A)+CD8+ T-cell subset
that was associated with the severity of adenomyosis and found that the number of these cells was significantly increased in the
eutopic endometrium and ectopic lesions. In addition, the increases in the expression of NKG2A ligand histocompatibility leucocyte
antigen E and interleukin-15 in glandular epithelial cells in the adenomyotic microenvironment might contribute to CD8+ T-cell
exhaustion by promoting NKG2A expression on CD8+ T cells or inhibiting the effector function of these cells. In conclusion, our data
revealed a previously unrecognized role for NKG2A+CD8+ T-cell exhaustion in the pathogenesis of adenomyosis, indicating that
therapeutic interventions designed to target and reinvigorate exhausted CD8+ T cells may be beneficial for patients with
adenomyosis.
Mucosal Immunology (2023) 16:121–134; https://doi.org/10.1016/j.mucimm.2023.02.003
INTRODUCTION
Adenomyosis is a benign uterine disorder characterized by the
presence of endometrial glands and stroma within the myome-
trium surrounded by hypertrophic and hyperplastic smooth
muscle cells1,2. It affects approximately 20% of women of repro-
ductive age worldwide3. Adenomyosis is commonly associated
with a wide variety of symptoms, such as chronic pelvic pain,
dysmenorrhea, dyspareunia, abnormal uterine bleeding, and/or
infertility, and it exerts substantial effects on the health and
quality of life of individuals afflicted by this disease4,5. Currently,
no curative drugs are available for adenomyosis, and hysterec-
tomy remains the primary treatment strategy3. Although differ-
ent disease mechanisms, mainly endometrial invagination,
epithelial metaplasia, and internal invasion of extrauterine
endometriosis lesions, have been proposed, they do not explain
the vast heterogeneity of the phenotypic manifestations of ade-
nomyosis6,7. The etiology and pathophysiology of adenomyosis
remain largely elusive.
The role of immunological dysfunction in adenomyosis
pathogenesis has garnered much attention recently because
evidence for alterations in both systemic and local immunity
has been reported8. An increase in the ratio of T helper 17 cells
to T regulatory cells in the peripheral circulation has been
reported in patients with adenomyosis, and these changes are
positively correlated with the CA125 level and the severity of
dysmenorrhea9. The numbers of macrophages and cluster of dif-
ferentiation (CD)8+ T cells are also increased in both the eutopic
endometrium and ectopic lesions10–12. In addition, an increase in
the levels of cytokines and inflammatory mediators, such as
tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, inter-
feron (IFN)-α, and IFN-γ, has been observed in adenomyotic
uteri. On the other hand, local or circulating levels of
immunoregulatory cytokines, such as IL-10, transforming growth
factor-β1 (TGF-β1), and IL-22, have been reported to be either
increased or decreased in adenomyosis8. However, previous
studies have reported a high heterogeneity of immune cell alter-
ations and contradictory results. Notably, immunohistochemistry
was used to label different immune cell populations in most of
these studies. This method is far from accurate in determining
the numbers of different immune cell types, let alone character-
izing the phenotypic and functional signatures or identifying
specific new immune subpopulations. Moreover, the quantifica-
1Department of Obstetrics and Gynecology, Shandong Provincial Hospital, Shandong University, Jinan, China. 2Department of Obstetrics and Gynecology, Shandong
Provincial Hospital, Affiliated to Shandong First Medical University, Jinan, China. 3Department of Obstetrics and Gynecology, Peking University Shenzhen Hospital, Shenzhen,
China. 4Center for Reproductive Medicine, Shandong University, Jinan, China. 5Key Laboratory of Reproductive Endocrinology of Ministry of Education, Shandong University,
Jinan, China. 6Shandong Key Laboratory of Reproductive Medicine, Jinan, China. 7Shandong Provincial Clinical Research Center for Reproductive Health, Jinan, China.
8Shandong Technology Innovation Center for Reproductive Health, Jinan, China. 9National Research Center for Assisted Reproductive Technology and Reproductive
Genetics, Shandong University, Jinan, China. 10Suzhou Institute of Shandong University, Suzhou, China.
✉
email: feiwangde@163.comthis study, we aimed to comprehensively assess the
changes in immune cell subsets and explore the phenotypes
of CD8+ T-cell exhaustion in individuals with adenomyosis and
the associated microenvironmental alterations. Our data sug-
gested the activation of a negative regulatory mechanism in
exhausted CD8+ T cells from individuals with adenomyosis due
to increased HLA-E/NKG2A engagement, and our results provide
new insights into autoimmune pathogenesis and information for
the development of therapeutic interventions for patients with
adenomyosis.
RESULTS
Alterations in immune cell subsets in adenomyotic uteri
To comprehensively investigate whether immune perturbations
occur in the local uterine microenvironment in adenomyosis, we
first identified the different immune cell subsets in the eutopic
endometrium and ectopic lesions of women with adenomyosis
and compared them with those in the normal endometrium
and myometrium of control female subjects using multicolor
flow cytometry (Table 2; see Supplementary Fig. 1 for the gating
strategies). No differences in the percentages and absolute num-
bers of total infiltrating CD45+ immune cells were observed
between the eutopic endometria (percentage: p = 0.9244; abso-
lute number: p = 0.7643) or ectopic lesions (percentage:
p = 0.4233; absolute number: p = 0.4349) of women with adeno-
myosis and the control endometrium or myometrium (Fig. 1A).
Interestingly, both the proportion and absolute number of
CD3+ T cells (CD45+CD14−CD19−CD56−CD3+) were significantly
increased in the ectopic lesions of women with adenomyosis
compared with the myometrium of control subjects (percent-
age: p = 0.0213; absolute number: p = 0.0377) (Fig. 1B). The per-
centage but not the absolute number of CD19+ B cells
(CD45+CD14−CD19+) showed a marginal decrease in ectopic
lesions compared with the control myometrium (percentage:
p = 0.0396; absolute number: p = 0.1177) (Fig. 1C). Notably,
the proportion and number of CD3+CD56+ NKT cells (CD45+-
CD14−CD19−CD56+CD3+) were significantly increased in the
eutopic endometrium compared with the control endometrium
(percentage: p = 0.0132; absolute number: p = 0.0255) but not in
the ectopic lesions (Fig. 1E). However, no significant differences
in the proportions and numbers of CD3−CD56+ NK (CD45+-
CD14−CD19−CD56+CD3−) cells and CD14+ monocytes and
macrophages (CD45+CD19−CD14+) were observed between
patients with adenomyosis and control subjects (Figs. 1D and
1F).
To further assess changes in T lymphocytes in patients with
adenomyosis, CD4+ T-cell (CD45+CD14−CD19−CD56−CD3+CD4+)
and CD8+ T-cell (CD45+CD14−CD19−CD56−CD3+CD8+) subsets
were detected. Intriguingly, we found that both the percentage
and absolute number of CD8+ T cells (percentage: p = 0.0008;
absolute number: p = 0.0014) but not CD4+ T cells (percentage:
p = 0.0928; absolute number: p = 0.1249) were significantly
increased in ectopic lesions compared with the control myome-
trium (Figs. 1G and 1H). Taken together, these data indicate the
occurrence of immune dysregulation in ectopic lesions in adeno-
myotic uteri characterized by an increase in the number of CD3+
T cells predominantly due to an increase in the number of CD8+
T cells.
Given the important role of mucosal-associated invariant T
(MAIT) cells in mucosal immunity21, we investigated the pres-
ence of MAIT cells in the endometrium and foci of adenomyosis.
We found that the percentage of CD8+ MAIT (CD3+CD8+CD161+-
TCRVα7.2+) cells among the total CD8+ T cells was quite low in
uterine tissues (control endometrium: 1.47% ± 0.69%; eutopic
endometrium: 1.55% ± 0.87%; control myometrium: 1.11% ± 0.
59%; ectopic lesions: 1.52% ± 0.60%), and no differences in
the percentage and absolute number of CD8+ MAIT cells were
observed between patients with adenomyosis and control sub-
jects (Supplementary Fig. 2). Hence, total CD8+ T cells, without
separating the conventional CD8+ T cells or CD8+ MAIT cells,
were further analyzed subsequently.
Increases in CD3+ T-cell and CD8+ T-cell numbers correlate
with the severity of adenomyosis
To confirm that the changes in immune cell subsets are associ-
ated with the severity of adenomyosis, we conducted correlation
analyses between the numbers of different immune cell types in
ectopic lesions and the clinicopathologic features of adeno-
myosis, including dysmenorrhea, phenotypes, and the serum
level of CA125. Interestingly, the number of CD3+ T cells and
CD8+ T cells were significantly increased in patients with diffuse
adenomyosis compared with those with focal adenomyosis
(CD3+ T: p = 0.0142; CD8+ T: p = 0.0070) (Figs. 2C and 2D). Fur-
thermore, both the CD3+ T- and CD8+ T-cell numbers were pos-
itively correlated with diffuse adenomyosis (CD3+ T: r = 0.5521,
p = 0.0142; CD8+ T: r = 0.5966, p = 0.0070; point-biserial correla-
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tion analyses) and the serum level of CA125 (CD3+ T cells:
r = 0.4642, p = 0.0452; CD8+ T cells: r = 0.5255, p = 0.0209; Spear-
man correlation analysis; Figs. 2E and 2F). However, no differ-
ences or associations between the numbers of CD3+ T and
CD8+ T cells and the severity of dysmenorrhea were observed
(both p > 0.05) (Figs. 2A and 2B). In addition, significant differ-
ences or correlations between the number of CD19+ B cells in
ectopic lesions or CD3+CD56+ NKT cells in the eutopic endome-
trium and clinicopathological features were not observed (Sup-
plementary Fig. 3). Overall, the correlation analyses further
suggest a potential causative role for CD8+ T cells in the patho-
genesis of adenomyosis.
CD94/NKG2A expression on tissue-resident CD8+ T cells in
adenomyosis lesions
Generally, NKG2A is recognized as an inhibitory receptor in the
natural killer group 2 (NKG2) family that is mainly expressed on
NK cells22. Emerging evidence suggests that the inhibitory
receptor NKG2A also plays a crucial role in the antitumor
immune response of CD8+ T cells23,24. However, the role of
Fig. 1 Immune cell subsets in uterine tissues from women with adenomyosis and controls. Statistical analyses of the percentages (left panel)
and absolute numbers (right panel) of different immune cell subsets in the EM (n = 26), EU (n = 23), MYO (n = 26), and EC (n = 23). (A) CD45+
immune cells; (B) CD3+ T (CD45+CD14−CD19−CD56−CD3+); (C) CD19+ B (CD45+CD14−CD19+); (D) CD56+ NK (CD45+CD14−CD19−CD56+CD3−);
(E) CD3+CD56+ NKT (CD45+CD14−CD19−CD56+CD3+); (F) CD14+ Monos & MΦs (CD45+CD19−CD14+); (G) CD4+ T (CD45+CD14−CD19−CD56−-
CD3+CD4+); and (G) CD8+ T cells (CD45+CD14−CD19−CD56−CD3+CD8+). Data are shown in scatter plots (means ± standard error of mean).
Statistical significance was assessed using unpaired two-tailed Student’s t test. *pCD8+ T cells mainly localized around the ectopic glands
(Fig. 3C). Because NKG2A was predominantly expressed in
CD8+ T cells in adenomyotic tissue, we further characterized
the tissue residency of these cells by assessing CD103 expres-
sion. Compared with NKG2A-CD8+ T cells, NKG2A+CD8+ T cells
showed a significantly higher proportion of CD103 expression,
suggesting that the majority of NKG2A+CD8+ T cells in ectopic
lesions were tissue-resident cells (64.71% ± 4.24% vs. 31.43% ±
3.14%, p = 0.0003) (Fig. 3D). The data collectively indicate that
the inhibitory receptor CD94/NKG2A is predominantly expressed
on tissue-resident CD8+ T cells in adenomyotic uteri.
Increased NKG2A expression on CD8+ T cells and higher
HLA-E expression in patients with adenomyosis
The presence of tissue-resident NKG2A+CD8+ T cells encouraged
us to explore whether this population is altered in patients with
adenomyosis. We observed a significantly higher percentage
and absolute number of NKG2A+CD8+ T cells in both ectopic
lesions and the eutopic endometrium than in the control myo-
metrium (percentage: p = 0.0004; absolute number: p
 0.05) (Fig. 4C). In addition, no dif-
ferences or associations between the mean fluorescence
intensity of NKG2A on CD8+ T cells and clinicopathological fea-
tures were observed (Supplementary Fig. 4C–E).
The function of NKG2A inhibitory signaling requires the inter-
action of NKG2A with its ligand HLA-E, which suppresses the
immune activity of effector cells. However, the expression level
of HLA-E in adenomyosis tissues remains unclear. Immunohisto-
chemistry revealed that HLA-E was mainly expressed on glandu-
lar epithelial cells rather than stromal cells in the endometrium.
Intriguingly, HLA-E expression was significantly increased in the
eutopic endometrium and ectopic lesions from women with
adenomyosis compared with the normal endometrium
(p = 0.0085, pof
endometrial epithelial cells, with lower expression levels
detected in stromal cells. Here, we found that the expression
of IL-15 and TGF-β was increased in the eutopic endometrium
(p = 0.0190; prevealed by immunohistochemistry, in adenomyosis8.
However, the results were highly heterogeneous, and some
were even contradictory. Here, multicolor flow cytometry was
used to provide a comprehensive picture of dysregulated
immune cells in adenomyotic uteri. Although a significant
increase in the infiltration of total CD45+ immune cells was not
observed, CD3+ T-cell, CD19+ B-cell, and CD3+CD56+ NKT-cell
populations but not CD3−CD56+ NK-cell populations and
CD14+ monocytes or macrophages were dysregulated, suggest-
ing that both innate and adaptive immunity are involved in the
development of adenomyosis8. A previous report documented
increased numbers of both CD4+ and CD8+ T cells in adenomy-
otic uteri11. Furthermore, our data revealed that the increase in
the number of CD3+ T cells in ectopic lesions was predominantly
due to an increase in the number of CD8+ T cells rather than
CD4+ T cells, suggesting that CD8+ T cells are the main underly-
ing population involved in the pathophysiology of adenomyosis.
Notably, the clinical significance of CD8+ T cells has not been
reported. In our study, we first found that the number of CD8+
T cells was positively correlated with adenomyosis phenotypes
and the serum level of CA125, a marker that is used routinely
in the diagnosis of adenomyosis28. Some scholars have pro-
posed that an increase in serum levels of CA125 may be related
to the amount of endometrium that invades the myometrium or
the severity of adenomyosis29. Our data emphasize the associa-
tion between an increase in the CD8+ T-cell number and the clin-
icopathological features of adenomyosis and show that CD8+ T
cells may serve as a predictor of adenomyosis severity and a
therapeutic target.
Exhausted CD8+ T cells have emerged as a clinically relevant
and distinct T-cell type and have been found to play major roles
in immune dysfunction in cancer, autoimmunity, and chronic
infection30. The fact that these infiltrating CD8+ T cells, which
were increased in number, were unable to efficiently exert their
effector functions to eliminate ectopic endometrial cells encour-
aged us to investigate whether functional impairment or
exhaustion of CD8+ T cells occurred in the adenomyotic
microenvironment. Recently, NKG2A was identified as a marker
of CD8+ T-cell exhaustion in cancer and infection16,17. In human
colorectal cancer, the expression of the IC molecules PD-1, TIM-3,
TIGIT, and LAG-3 is significantly higher on NKG2A+ cells than on
NKG2A− cells31. Zheng et al. showed that NKG2A expression on
CD8+ T cells results in the functional exhaustion of CD8+ T cells,
accompanied by decreased production of CD107a, IL-2, and
granzyme B in patients with COVID-1932. In our study, NKG2A
was preferentially expressed on resident CD8+ T cells in the
eutopic endometrium and ectopic lesions but not in the periph-
eral blood, consistent with previous findings from patients with
head and neck squamous cell carcinoma24. NKG2A+CD8+ T cells
exhibited hallmark features of exhaustion, specifically coexpres-
sion of multiple ICs (PD-1, LAG-3, and TIM-3) and a decreased
degranulation capability (indicated by decreased perforin, gran-
zyme B, and CD107a levels). According to previous studies, the
upregulation of NKG2A expression in CD8+ T cells from various
solid tumors facilitates tumor cell escape from immunological
surveillance33–35. Notably, both the percentage and absolute
number of NKG2A+CD8+ T cells were significantly increased in
adenomyotic uteri compared with disease-free tissues in our
study. In contrast, Yang et al. showed that CD94 was expressed
at similar levels on both CD4+ and CD8+ T cells between women
with and without adenomyosis36. CD94 could bind most mem-
bers of the NKG2 family to form heterodimers, such as NKG2A,
NKG2C, and NKG2E37,38. Thus, the level of CD94 expression does
not fully reflect the expression level of NKG2A. Furthermore, the
NKG2A+CD8+ T-cell number was associated with the severity of
adenomyosis. Taken together, our data suggest that NKG2A+-
CD8+ T-cell exhaustion is increased in the adenomyotic microen-
vironment, which might contribute to functional deficits in
targeting invading endometrial cells within the myometrium.
Therefore, targeting and reinvigorating exhausted CD8+ T cells
using checkpoint blockade is likely a major strategy for improv-
ing the clinical response to adenomyosis immunotherapy.
In humans, the binding of the heterodimeric receptor CD94/
NKG2A to its ligand HLA-E induces CD8+ T-cell exhaustion in
cancers, such as gastrointestinal cancer and head and neck
squamous cell carcinoma18,23. In our study, upregulated HLA-E
expression was observed in endometrial glandular epithelial
cells in adenomyotic tissues. Therefore, CD8+ T-cell exhaustion
in adenomyosis might be due to an abnormal increase in HLA-
E/NKG2A signals, but this hypothesis must be verified in subse-
quent in vitro experiments. NKG2A expression was induced in T
cells by T-cell receptor activation, in combination with the secre-
tion of cytokines, such as IL-15 or TGF-β, from tissues26,27,39,40.
Here, we found that the cytokines IL-15 and TGF-β were
expressed at abnormally high levels in endometrial glandular
epithelial cells in adenomyotic tissues, consistent with previous
results41,42. In addition, we confirmed earlier reports that rhIL-
15 but not rhTGF-β increases the expression of NKG2A on acti-
vated CD8+ T cells26,39. This difference might be explained by
the pleiotropic effects of TGF-β signaling on mammals, such as
the inhibition of CD8+ T-cell proliferation43. Our results suggest
that the specific adenomyotic microenvironment can in turn
modulate and promote CD8+ T-cell exhaustion. On the one
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hand, adenomyotic glandular epithelial cells in adenomyotic
uteri secrete IL-15 to promote NKG2A expression on CD8+ T
cells; on the other hand, they express high levels of HLA-E, which
can bind to the CD94/NKG2A receptor, synergistically skewing
CD8+ T-cell responses toward exhaustion and ultimately leading
to continued progression of the disease. Treatments targeting
these molecules represent a potentially attractive strategy for
reversing the effects of the microenvironment on T-cell function
and exhaustion in adenomyosis.
Our study has a few limitations. First, the visual analog scale
scores, which are commonly used to assess the severity of dys-
menorrhea, are somewhat inaccurate due to recall bias and sub-
jectivity. Second, control uterine samples were collected from
patients with uterine myoma. Although these tissues were
pathologically confirmed as normal, the potential effects are
unknown and cannot be excluded. Third, most uterine samples
were collected in the proliferative phase of the menstrual cycle.
Thus, our results only reflect the immune microenvironment
during the proliferative phase. Fourth, due to the difficulty in
sorting immune cells from tissues, in vitro functional assays
showing changes in the behavior of CD8+ T cells were lacking.
Moreover, although no change in the CD8+ MAIT-cell number
was noted, their function and/or phenotype warrant further
exploration. The mutual interactions and regulatory mechanisms
between exhausted CD8+ T cells and other immune cells and
endometrial cells were not studied. Further studies of a larger
independent population and exploration of the functional signif-
icance of CD8+ T-cell exhaustion in adenomyosis pathogenesis
are warranted.
In summary, we observed changes in the innate and adaptive
immunity in adenomyotic uteri, and our findings indicated that
NKG2A expression may be influenced by factors from the adeno-
myotic microenvironment and contributes to CD8+ T-cell
exhaustion. Our study reveals a previously unrecognized role
for CD8+ T-cell exhaustion in the pathogenesis of adenomyosis
and suggests that therapeutic interventions targeting and rein-
vigorating exhausted CD8+ T cells may be beneficial for patients
with adenomyosis.
MATERIALS AND METHODSHuman subjects and specimen collection
All participants were recruited from the Department of Gynecol-
ogy, Shandong Provincial Hospital Affiliated with Shandong First
Medical University from March 2021 to April 2022. A total of 80
patients with adenomyosis without endometriosis or uterine
myoma who underwent hysterectomy were included, and the
ectopic lesions and corresponding eutopic endometrial tissue
were collected. A diagnosis of adenomyosis was based on clini-
cal symptoms, ultrasonography, and a subsequent histological
investigation. Normal endometrium and matched myometrial
tissues were collected from 62 patients with uterine myoma
but not adenomyosis or endometriosis as controls. All samples
were confirmed by a histopathological examination. Post-
menopausal women, pregnant women, women with tumors or
autoimmune diseases, and women who took hormone therapy
or anti-inflammatory drugs within the past 3 months were
excluded. The menstrual cycle status was self-reported and
staged by histopathologists according to endometrial histology.
The severity of dysmenorrhea in patients with adenomyosis was
determined using the visual analog scale: a score of 0 indicated
no pain, 1–3 indicated mild pain, 4–6 indicated moderate pain,
and 7–10 indicated severe pain44. The adenomyosis phenotype
was classified as “diffuse” or “focal” using ultrasound. Women
with a lesion 25% of
the uterine volume or multiple focal lesions were considered
to have diffuse adenomyosis45. The clinical characteristics are
described in Table 1. This study was approved by the ethics
committee of the Shandong Provincial Hospital (No. 2021-776),
and written informed consent was obtained from all
participants.
Cell isolation
The tissue specimens were washed with RPMI 1640 (HyClone,
Thermo Fisher Scientific, Waltham, Massachusetts, USA) and
then cut into small pieces to prepare single-cell suspensions.
The fragmented tissues were digested with collagenase IV (4
mg/ml; Gibco, Rockville, Maryland, USA) and DNase (0.01 mg/
ml; Sigma, St. Louis, Missouri, USA) in RPMI 1640 for 1 hour in
a 37°C shaking incubator and then passed through 70-μm cell
strainers. PBMCs were isolated from peripheral blood using
Ficoll-Hypaque (MP Biomedicals, Santa Ana, California, USA) gra-
dient centrifugation.
Flow cytometry
The dead cells were excluded from the analysis using the
Zombie-NIR Fixable Viability Kit (BioLegend, San Diego, Califor-
nia, USA). For cell surface staining, the samples were incubated
with Fc Receptor Blocking Solution (BioLegend) for 10 minutes
at room temperature in the dark, washed with Dulbecco
phosphate-buffered saline/1% fetal bovine serum, and then
incubated with various antibodies for 15 minutes at room tem-
perature in the dark. For intranuclear staining, cells were fixed/
permeabilized with the FoxP3 Staining Buffer Set (eBioscience,
Thermo Fisher Scientific) at 4°C for 1 hour and then incubated
with antibodies at 4°C for 1 hour. For intracellular cytokine stain-
ing, cells were stimulated with Cell Activation Cocktail (BioLe-
Table 1. Clinical characteristics of women with adenomyosis and controls.
Characteristics Adenomyosis (N =
80)
Control (N =
62)
p-value*
Age (years) 47.08 ± 0.50 46.95 ± 0.59 0.8723
Body mass index (kg/m2) 25.48 ± 0.40 25.04 ± 0.45 0.4708
Smoker 1 (1.25) 1 (1.61) >0.9999
Gravidity 3.04 ± 0.19 2.69 ± 0.16 0.2921
Parity 1.25 ± 0.06 1.26 ± 0.08 0.9345
History of infertility 2 (2.50) 2 (3.23) >0.9999
Menstrual phase 0.1457
Proliferating phase 57 (71.25) 50 (80.65)
Secretory phase 14 (17.50) 4 (6.45)
Unknown 9 (11.25) 8 (12.90)
Abnormal uterine
bleeding
48 (60.00) 35 (56.45) 0.7325
Dysmenorrheafor 10 minutes. Heat-mediated antigen retrieval
with Tris/EDTA buffer (pH 9.0) was performed during each cycle
of staining to remove the Ab TSA complex. After the last TSA
incubation, the sections were counterstained with DAPI at a
1:1000 dilution for 10 minutes. Images of immunofluorescence
were obtained with an Aperio VERSA 8 microscope (Leica, Wet-
zlar, Germany).
Cell sorting and culture
PBMCs were isolated from healthy female donors to obtain CD8+
T cells. The cells were incubated with Zombie-NIR Fixable Viabil-
Table 2. The proportion and number of various immune cell subgroups.
Immune subsets Parameters Control endometrium (N =
26)
Control myometrium (N =
26)
Eutopic endometrium (N =
23)
Ectopic lesions (N =
23)
CD45+ immune cells % 8.73 ± 0.87 6.66 ± 0.70 8.59 ± 1.22 7.53 ± 0.83
# (107/mg) 222.0 ± 21.9 3.6 ± 0.4 231.7 ± 32.9 4.0 ± 0.4
CD3+ T % 45.76 ± 2.69 43.92 ± 2.49 51.89 ± 2.62 52.19 ± 2.35
# (107/mg) 93.4 ± 8.7 1.5 ± 0.2 120.0 ± 17.4 2.1 ± 0.3
CD3+CD4+ T % 45.43 ± 1.71 36.71 ± 2.31 43.81 ± 1.79 32.13 ± 1.30
# (106/mg) 420.0 ± 44.2 4.8 ± 0.6 526.6 ± 73.7 6.4 ± 0.8
CD3+CD8+ T % 50.15 ± 1.63 54.87 ± 2.17 53.41 ± 1.98 64.28 ± 1.40
# (106/mg) 480.6 ± 55.1 7.1 ± 0.8 572.4 ± 76.6 12.8 ± 1.5
CD19+ B % 1.26 ± 0.16 0.80 ± 0.08 1.55 ± 0.18 0.57 ± 0.06
# (105/mg) 232.3 ± 35.0 2.5 ± 0.3 316.9 ± 56.1 1.8 ± 0.3
CD3−CD56+ natural killer % 33.68 ± 2.33 25.82 ± 2.16 30.37 ± 2.77 23.66 ± 2.42
# (107/mg) 72.6 ± 8.7 0.8 ± 0.1 69.5 ± 12.5 0.9 ± 0.2
CD3+CD56+ natural killer T % 3.28 ± 0.51 11.16 ± 1.43 5.55 ± 0.73 11.63 ± 1.20
# (106/mg) 69.2 ± 11.8 3.7 ± 0.7 120.1 ± 18.1 4.7 ± 0.9
CD14+ Monos &
macrophages
% 5.73 ± 0.55 8.03 ± 0.79 6.69 ± 0.68 8.35 ± 0.72
# (106/mg) 116.1 ± 13.1 2.6 ± 0.4 153.8 ± 24.2 3.0 ± 0.4
Values are presented as the means ± standard error of mean.
CD = cluster of differentiation.
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http://
ity Kit reagent (BioLegend) in phosphate-buffered saline (PBS)
for 10 minutes at room temperature and then incubated with
CD3-FITC (clone UCHT1, 300406, BioLegend); CD4-PE (clone
OKT4, 317410, BioLegend); and CD8-APC (clone SK1, 344722,
BioLegend) antibodies. CD3+CD4−CD8+ cells were sorted using
a FACSAria II flow cytometer (BD Biosciences).
For the culture of CD8+ T cells, 96-well plates were coated
with a CD3 mAb (clone OKT3, 16-0037-85, eBioscience) in PBS
at a concentration of 5 µg/ml. After an overnight incubation at
4°C, the unbound antibodies in the wells were removed by
two washes with PBS. Then, CD8+ T cells (105 cells/well) were
seeded in each well in IMEM (Gibco) containing 10% FBS (Biolog-
ical Industries, Beit Haemek, Israel) and 1% penicillin/strepto-
mycin (Thermo Fisher Scientific). rhIL-15 (PeproTech, Rocky Hill,
New Jersey, USA) or TGF-β1 (PeproTech) was added to the cells
at a final concentration of 20 ng/ml. After 8 days of culture, the
cells were harvested for flow cytometry analysis.
Statistical analysis
Statistical analysis was performed using GraphPad Prism 8.0.2
(Version X, La Jolla, CA, USA) software. The details on the statis-
tical tests used are provided in the figure legends. The
d’Agostino-Pearson omnibus normality test was used to test
normality. When continuous data were normally distributed,
they were analyzed using the two-tailed Student’s t test or
one-way analysis of variance; otherwise, the data were com-
pared using the two-tailed Mann-Whitney U test or Kruskal-
Wallis test. Categorical variables were analyzed with the χ2 test.
Point-biserial correlation analysis was used to evaluate the asso-
ciation between the number of immune cells and clinicopatho-
logical features of adenomyosis (dysmenorrhea and phenotype).
Spearman correlation analysis was performed to estimate the
correlation between the number of immune cells and the serum
level of CA125. A paired t test was used to compare the charac-
teristic phenotypes between the NKG2A+ and NKG2A− sub-
groups. When normality was not reached, the statistical
significance of differences between two paired sample popula-
tions was determined using the Wilcoxon paired t test. A p-
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	Increased NKG2A+CD8+ T-cell exhaustion in patients with adenomyosis
	INTRODUCTION
	RESULTS
	Alterations in immune cell subsets in adenomyotic uteri
	Increases in CD3+ T-cell and CD8+ T-cell numbers correlate with the severity of adenomyosis
	CD94/NKG2A expression on tissue-resident CD8+ T cells in adenomyosis lesions
	Increased NKG2A expression on CD8+ T cells and higher HLA-E expression in patients with adenomyosis
	Features of NKG2A+CD8+ T-cell exhaustion in patients with adenomyosis
	High IL-15 expression in the adenomyotic microenvironment is associated with NKG2A expression on CD8+ T cell
	DISCUSSION
	MATERIALS AND METHODS
	Human subjects and specimen collection
	Cell isolation
	Flow cytometry
	Immunohistochemistry
	Immunofluorescence staining
	Cell sorting and culture
	Statistical analysis
	FUNDING
	AUTHOR CONTRIBUTIONS
	DECLARATIONS OF COMPETING INTEREST
	Acknowledgments
	Appendix A Supplementary data
	REFERENCES