Hspa9^flox/flox mice (stock no. S-CKO-02995) and β2m^-/- mice (stock no. KOCMP-12010-B2m-B6J-VA) were imported from Cyagen Biosciences, Inc. Hspa9^flox/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 (Hspa9^fl/fl mice); and ii) Hspa9 conditional knockout group (Hspa9^fl/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).

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.

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.

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).

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 Hspa9^fl/fl and Hspa9^fl/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).

The splenocytes from Hspa9^fl/fl and Hspa9^fl/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).

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-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.

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.

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.

To investigate the role of hematopoietic Hspa9 in NK cells, Hspa9^fl/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 Hspa9^fl/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 Hspa9^fl/fl Vav-cre mice (Fig. 2A and B). Next, the phenotype of NK cells in Hspa9^fl/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 Hspa9^fl/fl Vav-cre mice compared with Hspa9^fl/fl controls (Fig. 2C-E), indicating that Hspa9 expression in the hematopoietic cell is crucial for NK cell homeostasis.

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 Hspa9^fl/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 Hspa9^fl/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 Hspa9^fl/fl Vav-Cre mice compared to Hspa9^fl/fl controls (Fig. 3E-H). These results demonstrate that Hspa9 deficiency may disrupt NK cell maturation.

To explore the mechanism underlying the maturation blockade, the expression of key surface receptors on NK cells from Hspa9^fl/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 Hspa9^fl/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 Hspa9^fl/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.

The function of NK cells from Hspa9^fl/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, Hspa9^fl/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 Hspa9^fl/fl Vav-cre mice is reduced.

To elucidate the molecular mechanism, key signaling pathways which regulate NK cell function were examined. Compared with Hspa9^fl/fl mice, NK cells from Hspa9^fl/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.