Introduction
Natural killer (NK) cells are a vital component of
the innate immune system and serve as the body's first line of
defense against tumors and infections (1). Unlike T and B lymphocytes, NK cells
do not require prior antigen sensitization to mount a rapid
response, enabling them to recognize and eliminate target cells
within hours of encountering a threat. They participate in immune
regulation through multiple mechanisms, including the release of
cytotoxic granules (perforin and granzymes) that induce apoptosis
in malignant or infected cells, secretion of pro-inflammatory
cytokines (such as IFN-γ and tumor necrosis factor-α) and
chemokines that recruit and activate other immune cells, and
dynamic cross-talk with dendritic cells, macrophages and T cells to
orchestrate both innate and adaptive immunity (2). The effector functions of NK cells are
tightly regulated by a complex network of activating and inhibitory
receptors, which integrate extracellular cues to determine the
threshold of NK cell activation. In addition to receptor signaling,
cellular metabolism has emerged as a critical rheostat for NK cell
activity. Key metabolic processes, including glycolysis,
mitochondrial oxidative phosphorylation and fatty acid oxidation,
can be dynamically modulated to meet the bioenergetic and
biosynthetic demands of resting, activated or memory-like NK cells
(3). Dysregulation of these
metabolic pathways has been associated with impaired NK cell
function in various disease settings, including cancer and chronic
viral infections.
The development of NK cells from hematopoietic stem
cells in the bone marrow proceeds through a series of well-defined
stages, including the common lymphoid progenitor, the NK progenitor
and the immature NK cell, followed by their subsequent maturation
in peripheral tissues such as the spleen, liver and lymph nodes
(4). This entire process is
orchestrated by a delicate balance of transcription factors,
including eomesdermin, T-box expressed in T cells, inhibitor of DNA
binding 2 and GATA binding protein 3, which act in a stage-specific
manner to control lineage commitment, survival and functional
maturation (5,6). In parallel, cytokine signals,
primarily IL-15 but also IL-12, IL-18 and type I interferons,
provide essential survival and proliferation cues through receptor
complexes that activate downstream JAK-STAT and PI3K-Akt pathways
(7). Moreover, intrinsic metabolic
checkpoints, such as the balance between glycolysis and oxidative
phosphorylation, as well as mitochondrial fitness, have recently
been recognized as key determinants that shape NK cell development
and homeostasis (8).
Simultaneously, the effector functions of mature NK
cells, including direct cytotoxicity via perforin/granzyme release
and cytokine production are tightly controlled by a highly
integrated network of activating receptors and inhibitory receptors
that recognize self-major histocompatibility class (MHC) class I
molecules (9). The integration of
opposing signals from these receptors determines the threshold for
NK cell activation. In addition, intracellular signaling cascades,
including the PI3K-Akt-mTOR axis, the MAPK/ERK pathway and JAK-STAT
pathways, coordinate cytoskeletal reorganization, granule
polarization and transcriptional reprogramming required for
effective target cell elimination. Furthermore, epigenetic
modifications, such as DNA methylation, histone acetylation and
methylation, as well as non-coding RNAs, have emerged as critical
regulators that fine-tune the expression of receptors and effector
molecules during NK cell education, licensing and memory-like
differentiation (10).
Glucose-regulated protein 75 [GRP75; also known as
Heat shock protein family A (Hsp70) member 9 (Hspa9)] functions as
a critical tether at mitochondria-associated membranes. It
physically interacts with the endoplasmic reticulum calcium release
channel inositol 1,4,5-trisphosphate receptor type 1 (IP3R1) and
the mitochondrial outer membrane protein voltage-dependent
anion-selective channel 1 (VDAC1), facilitating ER-mitochondria
contact and efficient calcium transfer into the mitochondria
(11). However, dysregulation of
this IP3R1/GRP75/VDAC1 axis, resulting in excessive mitochondrial
calcium influx, can trigger calcium overload, mitochondrial
dysfunction and subsequent cellular apoptosis, as observed in
conditions such as skeletal muscle atrophy (12). This highlights the delicate balance
maintained by Hspa9 in cellular physiology.
While the role of Hspa9 in mitochondrial homeostasis
is established, its specific function in NK cells remains largely
unexplored. The present study aimed to investigate the role of
Hspa9 in the development and functional regulation of NK cells, as
well as its modulatory effect on the PI3K-Akt signaling pathway, a
key pathway in NK cells. The aim was to elucidate the regulatory
function and molecular mechanism of Hspa9 in NK cells. The present
findings will broaden the theoretical understanding of NK cell
functional regulation.
Materials and methods
Mice
Hspa9flox/flox mice (stock no.
S-CKO-02995) and β2m-/- mice (stock no.
KOCMP-12010-B2m-B6J-VA) were imported from Cyagen Biosciences, Inc.
Hspa9flox/flox mice were crossed with Vav1-cre mice
(B6/Vav1-cre; stock no. C001019; Cyagen Biosciences, Inc.) to
obtain hematopoietic cell-specific Hspa9-knockout mice. The
experiments were conducted using paired 8-week-old male or female
C57BL/6 mice (18-22 g; n=80). Mice were divided into two groups (40
mice/group): i) Wild-type group (Hspa9fl/fl mice); and
ii) Hspa9 conditional knockout group (Hspa9fl/fl Vav-cre
mice). Animals were housed in an SPF facility at 22±2˚C, 50-60%
humidity, with a 12-h light/dark cycle. They were housed
group-housed, ≤5 per cage with ample bedding, nesting material and
environmental enrichment (tunnels and wooden blocks). Standard chow
and autoclaved water were available ad libitum. Soft diet or
gel was provided for debilitated animals to facilitate feeding and
prevent excessive weight loss. During the experiment, all animals
were clinically observed at least once daily. Body weight was
measured every 3 days and recorded in detail. The humane endpoints
for mice in the present study were established as a body weight
loss of >20% of the initial weight, or the manifestation of
severe clinical signs such as hunched posture, ruffled fur,
lethargy, reduced response to external stimuli, respiratory
distress or persistent diarrhea. For all mice except those with B16
melanoma lung metastasis, animals were anesthetized with
tribromoethanol (125 mg/kg) by intraperitoneal injection.
Subsequently, they were euthanized by cervical dislocation and
dissected to collect the spleen, liver, lungs, and other organs or
tissues for further experiments (13). At the end of the experiment in mice
with B16 melanoma lung metastasis, the mice were euthanized in a
10-liter chamber with a gradual CO2 inflow at a rate of
3 l/min, which corresponds to a volume displacement rate of 30% per
min. The animal experiment protocols were reviewed and approved by
the Animal Ethics Committee of the Sixth Affiliated Hospital of
South China University of Technology (Nanhai District People's
Hospital of Foshan; Foshan, China; approval no.
ZXSY-JY-ZXK-250918-002). The animal experiments were implemented in
strict accordance with the approved protocols. We followed the
methods of Zeng et al (14).
Analysis of Hspa9 expression across
immune cell types in mice using public databases
Data on the RNA expression of Hspa9 in different
mouse cell types were obtained from the Expression Atlas database
(https://www.ebi.ac.uk/gxa/home) and the
expression levels were compared among the cell types.
Antibodies and flow cytometry
The methods were performed as previously described
by Zeng et al (14). The
surface and functional markers of the immune cells were detected
using a flow cytometer from BD Biosciences (CANTO II).
Fluorescein-labeled monoclonal antibodies against mouse CD3e,
NK1.1, CD11b, CD27, CD98, CD71, CD69, CD25, killer cell lectin-like
receptor subfamily G member 1 (KLRG1), IFN-γ, CD107a, CD127, Ly49A,
Ly49G2, NKp46, Ly49C/I, CD117, NKG2A, 2B4, NKG2D and an isotype
control were purchased from Thermo Fisher Scientific, Inc.
(Table SI). To analyze protein
expression on cell surfaces, the mononuclear cells of spleen from
the mice were obtained. A 70-µm cell strainer (Wuxi NEST
Biotechnology Co., Ltd.) was placed in a 35-mm cell culture dish
(Corning, Inc.) containing 2 ml RPMI 1640 medium (Gibco; Thermo
Fisher Scientific, Inc.). The freshly isolated mouse spleen was
placed onto the strainer and gently mashed with the plunger of a
2.5-ml syringe until the tissue passed through the mesh to generate
a single-cell suspension. The suspension was transferred to a 15-ml
centrifuge tube (Corning, Inc.) and centrifuged at 400 x g for 5
min at 4˚C. The supernatant was discarded, and the cell pellet was
resuspended in 1 ml of red blood cell lysis buffer (MedChemExpress)
by gentle pipetting. After thorough mixing, the tube was incubated
on ice for 5 min. Next, 5 ml phosphate-buffered saline (PBS)
(Sangon Biotech Co., Ltd.) was added, and the cells were
centrifuged again at 400 x g for 5 min at 4˚C. The supernatant was
removed, and the pellet was resuspended in 1 ml PBS containing 1%
fetal bovine serum (FBS) (Gibco; Thermo Fisher Scientific, Inc.).
Finally, the cell suspension was filtered through a new 70-µm cell
strainer to obtain the mononuclear cell suspension. The mononuclear
cells (1x106) were stained with the specific antibodies
dissolved in 100 µl PBS containing 2% FBS. Cells were washed once
with PBS and analyzed using the flow cytometer. For the detection
of intracellular proteins, such as Hspa9, lymphocytes were fixed
with Phosflow Lyse/Fix buffer and permeabilized with Phosflow Perm
buffer III (Invitrogen; Thermo Fisher Scientific, Inc.; cat. no.
88-8824-00) prior to staining with antibodies. For Hspa9, Akt,
phosphorylated (p-)Akt and mTOR staining, after the primary
antibody staining was completed, samples were washed with PBS and
secondary antibody (goat anti-Rabbit IgG-PE) was added for
staining. The fluorescence signal was detected using flow
cytometry. The data were analyzed using FlowJo™ software (v10.4;
FlowJo, LLC; BD Biosciences). The mean fluorescence intensity (MFI)
was defined as the MFI of the gated cell population, which is less
sensitive to outliers and therefore suitable for skewed
fluorescence distributions.
Detection of CD107a expression and
intracellular staining of NK cells
The methods of Zeng et al (14) and Liang et al (15) were followed. CD107a is a lysosomal
membrane protein. During the degranulation process, the cell
membrane combines with the lysosome. Therefore, the expression
level of CD107a is related to cell degranulation. First, to
activate NK cells in the mice, the mice were intraperitoneally
injected with 200 µg Poly I:C (cat. no. P9582; MilliporeSigma) for
NK cell activation. After 18 h, the spleen was removed and the
splenocytes were processed to obtain splenic mononuclear cells, as
aforementioned. The splenocytes (2x106 cells) were
stained with CD107a antibody and co-cultured with Yac-1 cells (cat.
no. TIB-160; ATCC) (2x106 cells) for 1 h in DMEM (cat.
no. 11885084; Gibco; Thermo Fisher Scientific, Inc.) with 10% FBS
(cat. no. 10099141; Gibco; Thermo Fisher Scientific, Inc.) in a
culture incubator (37˚C, 5% CO2). Next, a Golgi
inhibitor (GolgiStop™; BD Biosciences) was added to prevent the
release of intracellular proteins. Cells were cultured for a
further 4 h. The cells were collected for extracellular staining
(Nkp46). A total of 250 µl Fixation/Permeabilization solution (cat.
no. 00-5523-00; Thermo Fisher Scientific, Inc.) was added, and the
cells were permeabilized at 4˚C for 20 min protected from light.
The samples were washed twice with 1 ml cytoperm/wash buffer (cat.
no. 00-5523-00; Thermo Fisher Scientific, Inc.). Specific
intracellular staining antibodies for IFN-γ were added and the
samples incubated at room temperature for 30 min protected from
light. The cells were washed twice with 1 ml cytoperm/wash buffer,
then the resuspended cells were analyzed using the flow cytometer.
The data were analyzed using FlowJo™ software (v10.4; FlowJo, LLC;
BD Biosciences).
In vivo β2M-/- cell
clearance assay
β2m-/- mice (stock no.
KOCMP-12010-B2m-B6J-VA) were imported from Cyagen Biosciences, Inc.
The experiments were conducted using 8-week-old male C57BL/6 mice
(18-22 g; n=3). Animals were housed in an SPF facility at 22±2˚C
and 50-60% humidity, with a 12-h light/dark cycle. The mice were
group-housed, with ≤5 animals per cage, with ample bedding, nesting
material and environmental enrichment (tunnels and wooden blocks).
Standard chow and autoclaved water were available ad
libitum. Splenocytes from wild-type and β2M-/- mice
were labeled with 5 µM Carboxyfluorescein Diacetate Succinimidyl
Ester (cat. no. 21888; MilliporeSigma) and 5 µg/ml Cell Tracker™
Violet (CTV) (cat. no. C34557; Thermo Fisher Scientific, Inc.),
respectively. The splenocytes from wild-type and β2M-/-
mice were then intravenously injected at 1:1 ratio
(1x106) into the Hspa9fl/fl and
Hspa9fl/fl Vav-cre mice which were pretreated with poly
I:C 18 h before. After 6 h, the proportion of CTV-positive
β2M-/- cells in the spleens and lymph nodes of the
recipient mice was quantified using flow cytometry (CANTO II; BD
Biosciences). The data were analyzed using FlowJo™ software (v10.4;
FlowJo, LLC; BD Biosciences).
Detection of mitochondrial mass
The splenocytes from Hspa9fl/fl and
Hspa9fl/fl Vav-cre mice were cultured in RPMI 1640
medium containing recombinant human IL-2 (1,000 IU/ml) (cat. no.
200-02-50UG; Thermo Fisher Scientific, Inc.) for 4 h. For the
detection the mitochondrial mass, NK cells were stained with
surface markers CD3 and NK1.1 before being stained with the dye
MitoTracker Green FM (Sigma-Aldrich; Merck KGaA; cat. no. M7514)
for 30 min at room temperature. MitoTracker Green FM in
CD3- NK1.1+ cells was detected using flow
cytometry (CANTO II; BD Biosciences). MFI of MitoTracker Green FM
was used to measure the mitochondrial mass. The data were analyzed
using FlowJo™ software (v10.4; FlowJo, LLC; BD Biosciences).
Detection of reactive oxygen species
(ROS) generation
Splenocytes were cultured in RPMI 1640 medium
containing recombinant human IL-2 (1,000 IU/ml) for 4 h. Rotenone
(MedChemExpress; cat. no. HY-B1756) was used to induce ROS at a
concentration of 1 µM for 1 h. For the detection of ROS generation,
NK cells were stained with surface markers CD3 and NK1.1 before
being stained with the dye 2',7'-dichlorofuorescin diacetate
(DCFH-DA; Sigma-Aldrich; Merck KGaA; cat. no. D6883) for 30 min at
room temperature. DCFH-DA in CD3- NK1.1+
cells was detected using flow cytometry (CANTO II; BD Biosciences).
The data were analyzed using FlowJo™ software (v10.4; FlowJo, LLC;
BD Biosciences). MFI of DCFH-DA was used to assess the ROS
levels.
B16 melanoma lung metastasis mouse
model
B16-F10 melanoma cells (cat no. CRL-6475; ATCC) were
cultured in DMEM (catalog no. 11885084; Gibco; Thermo Fisher
Scientific, Inc.) with 10% FBS (cat. no. 10099141; Gibco; Thermo
Fisher Scientific, Inc.) in a culture incubator (37˚C, 5%
CO2). Logarithmic-phase cells were suspended in 1X HBSS
and intravenously injected into mice (3x105 cells/mouse)
as previously described by He et al (16). After 14 days, the mice were
sacrificed by CO2 inhalation as aforementioned. The
lungs were weighed, and the number of lung surface nodules was
counted under a dissecting microscope.
Statistical analysis
GraphPad Prism 7 software (Dotmatics) was used to
plot the graphs. Unpaired Student's t-tests (two-tailed) were
performed using GraphPad Prism 7. Dunnett's post hoc tests were
performed based on the ANOVA for multivariate data analysis using
SPSS 22.0 (IBM Corp.). P<0.05 was considered to indicate a
statistically significant difference.
Results
Hspa9 is highly expressed in NK cells,
and its expression increases upon tumor cell stimulation
Analysis of public databases revealed that Hspa9 is
highly expressed in mouse common lymphoid progenitors (CLPs), kit
and SCA1-positive hematopoietic stem cell, lymphoid progenitor,
myeloid progenitor, monocyte progenitor within the immune system,
with high levels also observed in NK, T and B cells (Fig. 1A). Yac-1 cells are target cells for
NK cells. Yac-1 cells continuously secrete IL-10, leading to
extremely low expression of MHC-I molecules on their surface. The
lack of MHC-I molecules prevents effective binding of inhibitory
receptors on NK cells, resulting in sustained activation of NK
cells. To investigate the relationship between Hspa9 expression and
NK cell activation, Hspa9 expression was examined in mouse NK cells
following stimulation with the NK-sensitive target cell line Yac-1
for 4 h. Experimental validation confirmed that Yac-1 stimulation
significantly elevated Hspa9 protein levels in mouse NK cells
(Fig. 1B). Stimulation with Yac-1
cells increases the expression of Hspa9, suggesting that Hspa9 may
be involved in the regulation of NK cell activation.
Conditional knockout of Hspa9 in mouse
hematopoietic cells reduces NK cell numbers in the spleen, bone
marrow and liver
To investigate the role of hematopoietic Hspa9 in NK
cells, Hspa9fl/fl Vav-cre mice were utilized and NK cell
populations were analyzed across various tissues. First, the
knockout efficiency of Hspa9 in splenic NK cells from
Hspa9fl/fl Vav-cre mice was examined and demonstrated
that Hspa9 was barely detectable in these cells, indicating that
Hspa9 was successfully knocked out in NK cells of
Hspa9fl/fl Vav-cre mice (Fig. 2A and B). Next, the phenotype of NK cells in
Hspa9fl/fl Vav-cre mice was assessed. The gating
strategy for flow cytometry is shown in Fig. S1. Results showed a significant
reduction in NK cell numbers in the spleen, bone marrow and liver
of Hspa9fl/fl Vav-cre mice compared with
Hspa9fl/fl controls (Fig.
2C-E), indicating that Hspa9 expression in the hematopoietic
cell is crucial for NK cell homeostasis.
 | Figure 2Conditional knockout of Hspa9 in
mouse hematopoietic cells reduces NK cell numbers in the spleen,
bone marrow, and liver. (A) Representative flow cytometry plot
showing Hspa9 expression in splenic NK cells from
Hspa9fl/fl, Vav-cre, and Hspa9fl/fl Vav-cre
mice. (B) Statistical graph of Hspa9 expression in splenic NK cells
from Hspa9fl/fl, Vav-cre, and Hspa9fl/fl
Vav-cre mice. (C) Representative flow cytometry plots of NK cells
in SP, BM, LN, liver and lung from Hspa9fl/fl, Vav-cre,
and Hspa9fl/fl Vav-cre mice. (D) Statistical graphs
showing the proportion of NK cells in SP, BM, LN, liver and lung
from Hspa9fl/fl, Vav-cre, and Hspa9fl/fl
Vav-cre mice. (E) Statistical graphs showing the absolute number of
NK cells in SP, BM, LN, liver and lung from Hspa9fl/fl,
Vav-cre, and Hspa9fl/fl Vav-cre mice. Data are shown as
mean ± SD. One-way ANOVA with Dunnett's post hoc tests was
performed using SPSS 22.0. *P<0.05,
**P<0.01, ***P<0.001,
****P<0.0001. Hspa9, Heat shock protein family A
(Hsp70) member 9; NK, natural killer; SP, spleen; BM, bone marrow;
LN, lymph nodes; MFI mean fluorescence intensity. |
Hspa9 deficiency in hematopoietic
cells of mice may impair NK cell maturation
To further assess the impact of hematopoietic Hspa9
deletion on NK cell development, maturation stages were analyzed
using flow cytometry. In both the spleen and bone marrow of
Hspa9fl/fl Vav-cre mice, the proportion and absolute
number of terminally mature CD11b+ NK cells was
significantly decreased, while the proportion and absolute number
of early mature CD27+CD11b+ NK cells was
markedly increased compared to Hspa9fl/fl controls
(Fig. 3A-D). Consistent findings
were obtained using an alternative marker set: The frequency of NK
cell precursors (NKp) was significantly elevated, whereas the
proportion and absolute number of mature NK cells was reduced in
Hspa9fl/fl Vav-Cre mice compared to
Hspa9fl/fl controls (Fig.
3E-H). These results demonstrate that Hspa9 deficiency may
disrupt NK cell maturation.
 | Figure 3Conditional knockout of Hspa9 in
mouse hematopoietic cells may impair NK cell maturation. (A)
Representative flow cytometry plots delineating mouse NK cell
developmental stages using CD27 and CD11b markers in SP and BM from
Hspa9fl/fl and Hspa9fl/fl Vav-cre mice. (B)
Statistical graphs showing the proportion of NK cells at different
developmental stages in SP and BM from Hspa9fl/fl and
Hspa9fl/fl Vav-cre mice. Statistical graphs showing the
absolute number of NK cells at different developmental stages in
the (C) SP and (D) BM from Hspa9fl/fl and
Hspa9fl/fl Vav-cre mice. (E) Representative flow
cytometry plots delineating mouse NK cell developmental stages
using NK1.1 and CD11b markers in SP and BM from
Hspa9fl/fl and Hspa9fl/fl Vav-cre mice. (F)
Statistical graphs showing the proportion of NK cells at different
developmental stages in SP and BM from Hspa9fl/fl and
Hspa9fl/fl Vav-cre mice. Statistical graphs showing the
absolute number of NK cells at different developmental stages in
the (G) SP and (H) BM from Hspa9fl/fl and
Hspa9fl/fl Vav-cre mice. Data are shown as mean ± SD.
Unpaired Student's t-tests (two-tailed) were performed using Prism
software. *P<0.05, **P<0.01. Hspa9,
Heat shock protein family A (Hsp70) member 9; NK, natural killer;
SP, spleen; BM, bone marrow; DN, double negative NK cells
(CD27-CD11b-); CD27SP, single-positive NK
cells (CD27+CD11b-); DP, double-positive NK
cells (CD27+CD11b+); CD11bSP, single-positive
NK cells (CD27-CD11b-); NKP, double-negative
NK cells (NK1.1-CD11b-); immature,
single-positive NK cells (NK1.1+CD11b-);
mature, double-positive NK cells
(NK1.1+CD11b+). |
Conditional knockout of Hspa9 in
hematopoietic cells of mice disrupts the expression of NK cell
surface receptors
To explore the mechanism underlying the maturation
blockade, the expression of key surface receptors on NK cells from
Hspa9fl/fl Vav-Cre mice was analyzed. In splenic NK
cells, expression of CD117 and CD127 was significantly upregulated,
while expression of Ly49C/I and Ly49D was downregulated on NK cells
from Hspa9fl/fl Vav-Cre mice (Fig. 4A and B). In bone marrow NK cells, CD117
expression was increased, whereas Ly49C/I and KLRG1 expression was
decreased in Hspa9fl/fl Vav-Cre mice (Fig. 4C and D). These findings suggest that Hspa9
deficiency perturbs NK cell development and altering the expression
of critical surface molecules such as CD117, CD127 and Ly49C/I.
NK cells from hematopoietic
cell-specific Hspa9-knockout mice exhibit impaired effector
functions and antitumor function of Hspa9fl/fl Vav-cre
mice is reduced
The function of NK cells from Hspa9fl/fl
Vav-Cre mice was evaluated. Upon Yac-1 stimulation, Hspa9-deficient
NK cells produced significantly less IFN-γ (Fig. 5A and B) and exhibited reduced CD107a expression
compared with wild-type NK cells (Fig.
5C and D). These data indicate
that Hspa9 deficiency in the hematopoietic cell may compromise NK
cell IFN-γ and degranulation function. Furthermore,
Hspa9fl/fl Vav-cre mice showed a diminished ability to
clear β2M-/- target cells (Fig. 5E and F) and developed significantly more lung
metastases in a B16-F10 melanoma model (Fig. 5G and H). This indicates that the antitumor
function of Hspa9fl/fl Vav-cre mice is reduced.
Conditional knockout of Hspa9 in
hematopoietic cells suppresses Akt/mTOR signaling in NK cells of
mice
To elucidate the molecular mechanism, key signaling
pathways which regulate NK cell function were examined. Compared
with Hspa9fl/fl mice, NK cells from
Hspa9fl/fl Vav-cre mice exhibited significantly
decreased mitochondrial mass and significantly increased ROS
production (Fig. 6A-D). Flow
cytometry analysis revealed that Hspa9-deficient NK cells the
activation of the Akt/mTOR pathway was suppressed, as evidenced by
reduced phosphorylation of Akt and decreased mTOR expression
(Fig. 6E-K). This indicates that
in mice with hematopoietic cell-specific knockout of Hspa9, NK cell
function is impaired, and the Akt/mTOR signaling pathway in NK
cells is also inhibited.
Discussion
Hspa9 is a highly conserved molecular chaperone
primarily localized to the mitochondrial matrix and inner membrane.
It serves a central role in maintaining mitochondrial homeostasis,
participating in processes such as mitochondrial protein import,
assembly of oxidative phosphorylation complexes and stabilization
of mitochondrial DNA. Beyond its mitochondrial functions, cytosolic
Hspa9 can interact with various signaling proteins, influencing
pathways related to cell proliferation, migration and apoptosis
(17,18). Notably, elevated Hspa9 expression
is frequently observed in multiple solid tumors, including breast
cancer, hepatocellular carcinoma and glioma, and is associated with
poor patient prognosis (19,20).
However, its regulatory role within the immune system remains less
explored.
The present study identified a novel function for
Hspa9 in NK cell biology. Hspa9 was found to be highly expressed in
CLPs and mature NK cells. Furthermore, its protein levels in NK
cells increased upon stimulation with tumor target cells (Yac-1),
suggesting a potential link between Hspa9 expression and NK cell
activation. Using a hematopoietic-specific knockout mouse model
(Hspa9fl/fl Vav-cre), Hspa9 was demonstrated to be
essential for NK cell homeostasis, as evidenced by a significant
reduction in NK cell numbers in the spleen, bone marrow and liver
of Hspa9fl/fl Vav-cre mice. This finding is consistent
with a prior report implicating Hspa9 as a critical regulator for
thymocyte development (21).
However, the Hspa9fl/fl Vav-cre mice used mediate gene
knockout in hematopoietic cells and all hematopoietic-derived
cells, including T cells, B cells, myeloid cells and NK cells.
Therefore, although changes were observed in the number, phenotype
or function of NK cells in this mouse model, these alterations may
not reflect the intrinsic role of Hspa9 in NK cells. In future
studies, the use of NK cell-specific knockout mice combined with
adoptive transfer experiments will help further clarify the
cell-intrinsic function of Hspa9 in NK cells.
Further analysis revealed that mice with Hspa9
deficiency in hematopoietic cells exhibit altered NK cell
maturation status. A marked decrease was observed in the proportion
of terminally mature CD11b+ NK cells alongside an
accumulation of earlier developmental intermediates. This
maturation block may be associated with dysregulated expression of
surface receptors, such as CD117, CD127 and Ly49C/I, in
Hspa9-deficient NK cells. The crucial role of Hspa9 in cellular
differentiation and survival is supported by studies in other
models. For instance, Hspa9 deficiency in zebrafish leads to
increased apoptosis and defective hematopoiesis (22), while Hspa9 deficiency disrupting
its function can impair energy metabolism and differentiation in
neurons (23,24).
Functionally, NK cells from Hspa9fl/fl
Vav-cre mice exhibited impaired function. Their ability to secrete
IFN-γ and express CD107a was significantly compromised. Although
the ability of Hspa9fl/fl Vav-cre mice to resist B16-F10
lung metastasis was reduced, the present study lacks NK cell
depletion experiments to confirm that the impaired metastasis
control is NK cell-dependent. Therefore, the reduction in antitumor
function of NK cells with Hspa9 deletion cannot be definitively
concluded. The mechanistic studies will be refined further in
future research. Hspa9 deficiency in NK cells was demonstrated to
suppress Akt/mTOR signaling. As Hspa9 is pivotal for energy
metabolism and anti-apoptotic signaling, cytosolic Hspa9 can
inhibit the transcriptional activity of p53, thereby suppressing
apoptosis (25). Notably, in
cancer contexts, Hspa9 often promotes tumor progression by
activating pathways such as PI3K/Akt/mTOR and Wnt/β-catenin
(26). The present findings
suggest that within NK cells, the loss of Hspa9 negatively impacts
activation of the Akt/mTOR pathway.
Long-term Hspa9 deficiency may induce compensatory
upregulation of other heat shock proteins (such as Hspa1 and Hspa8)
by activating the mitochondrial unfolded protein response and heat
shock factor 1 (HSF1) (27). These
chaperone proteins help maintain cytosolic protein homeostasis and
inhibit apoptotic pathways, thereby alleviating cellular
dysfunction caused by Hspa deficiency and modulating the final
phenotype. However, the compensatory effect is highly dependent on
cell type, stress intensity and duration. Upregulation of Hspa1b
and Hspa8b has been observed in certain models, such as Smyd1b
mutant zebrafish (28), supporting
this compensatory mechanism. Therefore, in future studies, the
expression levels of family members including Hspa1 and Hspa8 will
be simultaneously monitored to more accurately assess their
compensatory contributions.
Targeting Hspa9 in NK cells holds promise but faces
notable challenges. To achieve NK cell-specific modulation, two
strategies are feasible: i) NK-directed nanoparticles, such as
anti-CD56-conjugated lipid nanoparticles delivering small
interfering RNA or CRISPR components; and ii) ex vivo
genetic modification of NK cells followed by adoptive transfer. The
nanoparticle approach enables systemic administration but suffers
from liver sequestration and limited targeting efficiency. Ex
vivo modification offers precise control but is complex and
costly. Future efforts should prioritize developing specific Hspa9
tools and optimizing delivery vectors using unique NK cell surface
markers.
In conclusion, the present study uncovers a critical
and previously uncharacterized role for Hspa9 in regulating NK cell
function. Hspa9 is essential for maintaining their cytotoxic and
cytokine-producing capabilities. Hspa9-deficiency also resulted in
suppression of the mTOR/Akt signaling pathway in NK cells. To the
best of our knowledge, these findings demonstrate for the first
time the role of Hspa9 in regulating NK cell function, broadening
the understanding of its function and the regulatory mechanisms of
NK cell biology.
Supplementary Material
Gating strategy for flow cytometry.
(A) Gating strategies for Figs. 1B
and 2A. (B) Gating strategies for
Fig. 2B. (C) Gating strategies for
Fig. 3A. (D) Gating strategies for
Fig. 3E. (E) Gating strategies for
Fig. 4A and B. (F) Gating strategies for Fig. 5A and C. (G) Gating strategies for Fig. 5E. (H) Gating strategies for
Fig. 6.
Antibody information.
Acknowledgements
Not applicable.
Funding
Funding: The present work was funded by Guangdong Basic and
Applied Basic Research Foundation (grant no. 2022A1515140023, to
XZ) and the National Natural Science Foundation of China (grant no.
82201919, to XZ).
Availability of data and materials
The data generated in the present study may be
requested from the corresponding author.
Authors' contributions
XZ, JZ and ZW conceptualized the study and designed
the experiments and data analysis methods. XZ, JZ, CL and JY
conducted all experiments and performed the data analysis. XZ, JZ,
JY and ZW prepared the figures, analyzed the data and drafted the
manuscript. XZ and ZW confirm the authenticity of all the raw data.
All authors have read and approved the final version of the
manuscript and agree to be accountable for all aspects of the
work.
Ethics approval and consent to
participate
The animal experiment protocol was approved by the
Animal Ethics Committee of the Sixth Affiliated Hospital of South
China University of Technology (Nanhai District People's Hospital
of Foshan; approval no. ZXSY-JY-ZXK-250918-002).
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing
interests.
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