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Hematopoietic cell‑specific knockout Hspa9 impairs natural killer cell function and alters the Akt/mTOR axis in mice

  • Authors:
    • Xiaokang Zeng
    • Jieyu Zhang
    • Caiying Liang
    • Jie Yao
    • Zuqiang Wu
  • View Affiliations / Copyright

    Affiliations: Laboratory Center, School of Medicine, The Sixth Affiliated Hospital of South China University of Technology (Nanhai District People's Hospital of Foshan), Foshan, Guangdong 528200, P.R. China, Clinical Laboratory, School of Medicine, The Sixth Affiliated Hospital of South China University of Technology (Nanhai District People's Hospital of Foshan), Foshan, Guangdong 528200, P.R. China, Laboratory Center, School of Medicine, The Sixth Affiliated Hospital of South China University of Technology (Nanhai District People's Hospital of Foshan), Foshan, Guangdong 528200, P.R. China, Clinical Laboratory, School of Medicine, The Sixth Affiliated Hospital of South China University of Technology (Nanhai District People's Hospital of Foshan), Foshan, Guangdong 528200, P.R. China
    Copyright: © Zeng et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 254
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    Published online on: July 24, 2026
       https://doi.org/10.3892/etm.2026.13249
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Abstract

Natural killer (NK) cells are crucial components of the innate immune system, serving vital roles in tumor killing and antiviral immunity. Heat shock protein family A (Hsp70) member 9 (Hspa9), a molecular chaperone localized to the mitochondrial matrix and inner membrane, is essential for maintaining mitochondrial homeostasis. However, its role in regulating NK cells remains unclear. The present study aimed to investigate the impact of the Hspa9 on NK cell regulation and its underlying molecular mechanism. By knocking out Hspa9 at the hematopoietic cell stage, NK cell numbers were found to be markedly reduced in the spleen, bone marrow and liver of mice. Hspa9‑deficient NK cells exhibited significantly reduced IFN‑γ secretion and CD107a expression. Mechanistically, Hspa9 deficiency led to perturbed expression of surface receptors CD117, CD127 and killer cell lectin‑like receptor subfamily A, member 9, which may underlie the observed alteration in maturation status. Furthermore, the impaired NK cell function may be associated with suppressed activation of the Akt/mTOR signaling pathway. In summary, the present study reveals a novel role for Hspa9 in regulating NK cell function, proposes a potential regulatory mechanism and identifies Hspa9 as a promising molecular target for modulating NK cell biology.
View Figures

Figure 1

Hspa9 is highly expressed in
hematopoietic cells, and its expression in NK cells increases upon
Yac-1 cell stimulation. (A) Analysis of Hspa9 RNA levels across
mouse hematopoietic stem cells and immune cell types using the
Expression Atlas database. (B) Flow cytometry analysis of Hspa9
expression on mouse splenic NK cells with or without Yac-1 cell
stimulation for 4 h. (C) Statistical graph of Hspa9 expression on
mouse splenic NK cells with or without Yac-1 cell stimulation for 4
h. Data are shown as mean ± SD. Unpaired Student's t-tests
(two-tailed) were performed using Prism software.
**P<0.01. Hspa9, Heat shock protein family A (Hsp70)
member 9; NK, natural killer; MFI mean fluorescence intensity;
FPKM, fragments per kilobase million.

Figure 2

Conditional 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.

Figure 3

Conditional 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+).

Figure 4

Hematopoietic cell-specific knockout
of Hspa9 in mice disrupts NK cell surface receptor expression. (A)
Representative flow cytometry plots of surface receptor expression
on splenic NK cells from Hspa9fl/fl and
Hspa9fl/fl Vav-cre mice. (B) Statistical graph of
surface receptor expression on splenic NK cells from
Hspa9fl/fl and Hspa9fl/fl Vav-cre mice. (C)
Representative flow cytometry plots of surface receptor expression
on BM NK cells from Hspa9fl/fl and Hspa9fl/fl
Vav-cre mice. (D) Statistical graph of surface receptor expression
on BM NK cells 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,
***P<0.001. Hspa9, Heat shock protein family A
(Hsp70) member 9; NK, natural killer; SP, spleen; BM, bone
marrow.

Figure 5

Hematopoietic cell-specific knockout
of the mouse Hspa9 gene impairs NK cell function. (A)
Representative flow cytometry plots detecting IFN-γ secretion by
splenic NK cells from Hspa9fl/fl and
Hspa9fl/fl Vav-cre mice. (B) Statistical graph of IFN-γ
secretion by splenic NK cells from Hspa9fl/fl and
Hspa9fl/fl Vav-cre mice. (C) Representative flow
cytometry plots detecting CD107a expression on splenic NK cells
from Hspa9fl/fl and Hspa9fl/fl Vav-cre mice.
(D) Statistical graph of CD107a expression on splenic NK cells from
Hspa9fl/fl and Hspa9fl/fl Vav-cre mice. (E)
Representative flow cytometry plots detecting residual
β2M-/- cells in the SP and LN of Hspa9fl/fl
and Hspa9fl/fl Vav-cre mice at 6 h after cell injection.
CFSE is a live-cell fluorescent labeling dye used to label
wild-type cells. (F) Statistical graph showing the residual amount
of β2M-/- cells in SP and LN from Hspa9fl/fl
and Hspa9fl/fl Vav-cre mice at 6 h after cell injection.
(G) Representative images of the melanoma lung metastasis in
Hspa9fl/fl and Hspa9fl/fl Vav-cre mice. Scale
bar, 1cm. (H) Statistical graph of lung metastatic nodule counts in
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 or one-way ANOVA with Dunnett's
post hoc test were performed using SPSS 22.0.
*P<0.05, **P<0.01,
***P<0.001. Hspa9, Heat shock protein family A
(Hsp70) member 9; NK, natural killer; SP, spleen; BM, bone marrow;
LN, lymph nodes; CFSE, 5(6)-carboxyfluorescein diacetate
N-succinimidyl ester.

Figure 6

Hematopoietic cell-specific knockout
of the mouse Hspa9 gene suppresses Akt/mTOR signaling in NK cells.
(A) Representative flow cytometry plot of MitoTracker staining on
splenic NK cells from Hspa9fl/fl and
Hspa9fl/fl Vav-cre mice. (B) Statistical graph of MFI of
MitoTracker on splenic NK cells from Hspa9fl/fl and
Hspa9fl/fl Vav-cre mice. (C) Representative flow
cytometry plot of ROS staining on splenic NK cells from
Hspa9fl/fl and Hspa9fl/fl Vav-cre mice after
1 µM rotenone treatment for 1 h. (D) Statistical graph of MFI of
ROS on splenic NK cells from Hspa9fl/fl and
Hspa9fl/fl Vav-cre mice after 1 µM rotenone treatment.
(E) Representative flow cytometry plot of total-Akt expression on
splenic NK cells from Hspa9fl/fl and
Hspa9fl/fl Vav-cre mice. (F) Statistical graph of
total-Akt expression on splenic NK cells from Hspa9fl/fl
and Hspa9fl/fl Vav-cre mice. (G) Representative flow
cytometry plot of p-Akt expression on splenic NK cells from
Hspa9fl/fl and Hspa9fl/fl Vav-cre mice. (H)
Statistical graph of p-Akt expression on splenic NK cells from
Hspa9fl/fl and Hspa9fl/fl Vav-cre mice. (I)
Statistical graph of the ratio of p-Akt to total Akt on splenic NK
cells from Hspa9fl/fl and Hspa9fl/fl Vav-cre
mice. (J) Representative flow cytometry plot of mTOR expression on
splenic NK cells from Hspa9fl/fl and
Hspa9fl/fl Vav-cre mice. (K) Statistical graph of mTOR
expression on splenic NK cells 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;
MFI, mean fluorescence intensity; p-, phosphorylated; ROS, reactive
oxygen species.
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Copy and paste a formatted citation
Spandidos Publications style
Zeng X, Zhang J, Liang C, Yao J and Wu Z: Hematopoietic cell‑specific knockout Hspa9 impairs natural killer cell function and alters the Akt/mTOR axis in mice. Exp Ther Med 32: 254, 2026.
APA
Zeng, X., Zhang, J., Liang, C., Yao, J., & Wu, Z. (2026). Hematopoietic cell‑specific knockout Hspa9 impairs natural killer cell function and alters the Akt/mTOR axis in mice. Experimental and Therapeutic Medicine, 32, 254. https://doi.org/10.3892/etm.2026.13249
MLA
Zeng, X., Zhang, J., Liang, C., Yao, J., Wu, Z."Hematopoietic cell‑specific knockout Hspa9 impairs natural killer cell function and alters the Akt/mTOR axis in mice". Experimental and Therapeutic Medicine 32.4 (2026): 254.
Chicago
Zeng, X., Zhang, J., Liang, C., Yao, J., Wu, Z."Hematopoietic cell‑specific knockout Hspa9 impairs natural killer cell function and alters the Akt/mTOR axis in mice". Experimental and Therapeutic Medicine 32, no. 4 (2026): 254. https://doi.org/10.3892/etm.2026.13249
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Spandidos Publications style
Zeng X, Zhang J, Liang C, Yao J and Wu Z: Hematopoietic cell‑specific knockout Hspa9 impairs natural killer cell function and alters the Akt/mTOR axis in mice. Exp Ther Med 32: 254, 2026.
APA
Zeng, X., Zhang, J., Liang, C., Yao, J., & Wu, Z. (2026). Hematopoietic cell‑specific knockout Hspa9 impairs natural killer cell function and alters the Akt/mTOR axis in mice. Experimental and Therapeutic Medicine, 32, 254. https://doi.org/10.3892/etm.2026.13249
MLA
Zeng, X., Zhang, J., Liang, C., Yao, J., Wu, Z."Hematopoietic cell‑specific knockout Hspa9 impairs natural killer cell function and alters the Akt/mTOR axis in mice". Experimental and Therapeutic Medicine 32.4 (2026): 254.
Chicago
Zeng, X., Zhang, J., Liang, C., Yao, J., Wu, Z."Hematopoietic cell‑specific knockout Hspa9 impairs natural killer cell function and alters the Akt/mTOR axis in mice". Experimental and Therapeutic Medicine 32, no. 4 (2026): 254. https://doi.org/10.3892/etm.2026.13249
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