Helicobacter Pylori UreB Upregulates the Expression of PD-L1 through Myh9 /mTOR Pathway and Inhibits the Activation of CD8+ T Cells

Objectives: Immune regulation mechanism of how Helicobacter pylori urease disrupting the homeostasis of host cells remains unknown. Methods: We thus detected the effect of Helicobacter pylori UreB on macrophage PD-L1 expression with recombinant protein and defective strains. The inuence of UreB induced PD-L1 on CD8 + T cells’ proliferation and perforin and granzyme expression were assessed through co-culture model. Results: Urease subset B (UreB) signicantly promoted PD-L1 expression in Bone marrow-derived macrophages (BMDMs) and thus blocked the proliferation and activity of H. pylori-primed CD8 + T cells. Myosin heavy chain 9 (Myh9) works as the receptor for UreB. The interaction between UreB and Myh9 promoted amino acid anabolism, activated mTOR pathway and induced PD-L1 expression in BMDMs. mTOR inhibitor Temsirolimus reversed UreB-induced PD-L1 expression and the inhibitory effects on CD8 + T cells. Conclusion: Our study reveals a hitherto-unknown immunosuppressive mechanism of UreB during H. pylori infection, provides clues for the development of H. pylori vaccine. or anti-Myh9 immunoblots was detect Myh9.


Introduction
Gram negative H. pylori, which was identi ed as class-I carcinogen, infected half of the adult population worldwide (Hatakeyama, 2017;Hooi et al., 2017). It's well known that H. pylori contributes to the development of chronic gastritis, peptic ulcers and gastric cancer (Uemura et al., 2001). Triple or quadruple antibiotic therapy is the main treatment for H. pylori infection. However, with the increase in resistant H. pylori strains, the cure rate of antibiotic treatment is decreasing year by year (Savoldi et al., 2018). Even though monovalent or multivalent vaccines based on H. pylori's multiple immune antigens, such as CagA, VacA, NAP, and urease are protective in mammalian model (Zeng et al., 2015;Malfertheiner et al., 2018), but it's regrettable that these vaccines are not effective enough in clinical trials.
Urease, which constitutes around 10% of the total H. pylori protein, not only plays a role in acid neutralization, but also disrupts the tight cell junctions, breaches the cellular integrity, and damages the gastric epithelium through producing ammonia (Lytton et al., 2005;Wroblewski et al., 2009). Besides, urease has been reported to promote tumor growth and metastatic dissemination through inducing angiogenesis and it plays a key role in gastric cancer progression (Olivera-Severo et al., 2017). H. pylori urease consists of two different subunits of 26.5 kDa (UreA) and 61.7 kDa (UreB) (Ha et al., 2001). Highly conserved UreB is the foremost active component of urease as well as the most promising candidate vaccine antigens (Guo et al., 2019). An oral vaccine based on UreB showed 55% e cacy against H. pylori infection in children in a two years' follow-up survey (Zeng et al., 2015). On the other hand, H. pylori urease was reported to promote Treg differentiation and asthma protection in neonatally infected mice . What's importantly, H. pylori urease is required for Treg induction and immune tolerance (Koch and Muller, 2015). Furthermore, UreB also induces apoptosis of gastric epithelial cells The wild-type strain H. pylori ATCC43504 used in this study was obtained from the National Institute for Communicable Disease Control and Prevention, Chinese Centers for Disease Control and Prevention (Beijing, China). UreB-knockout H. pylori (H. pylori: ΔUreB) was constructed by our group according to previous reports (Schmalstig et al., 2018). The above strains were cultured on brain-heart infusion medium (10% rabbit blood) under microaerophilic conditions (5% O 2 , 10% CO 2 , and 85% N 2 ) at 37°C.

Cell culture
The murine macrophage cell line RAW264. 7 and Human gastric cancer cell lines AGS and MNK8 were all purchased from the China Center for Type Culture Collection and were cultured in Dulbecco's modi ed Eagle's medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS). Human THP-1 monocyte cell line was suspended in RPMI 1640 culture medium and distributed to 6 well plates at a density of 106 cells per well and differentiated into macrophages during 24 hours of culture with 20 ng/ml of phorbol 12-myristate 13-acetate (PMA) (37°C; 5% CO2). THP-1 monocyte cell line-derived macrophages were attached to the plastic surface of the plates and con rmed by light microscopy.

Preparation of Bone marrow-derived macrophages (BMDMs)
Bone marrow cells isolated from the femurs and tibias of BALB/c mice were treated with AKT Lysis Buffer (Beyotime, Shanghai, China) and cultured in DMEM (Gibco, UK) supplemented with 10% FBS (Gibco), 1% Penicillin-Streptomycin Solution, and 50 ng/ml macrophage colony stimulating factor (M-CSF, Peprotech) for 6 days to induce differentiation into BMDM (the adherent cells). The purity of BMDM (F4/80 + ) was determined by owcytometry (FCM). Amino acid-free medium used for amino acid metabolism test was purchased from Thermo Fisher.

UreB-Ni-NTA bead pull-down assay and MS analysis
The BMDM cell lysates were incubated with 10 μg of the puri ed recombinant UreB protein with 6×Histags at 4°C overnight. The reaction mixtures were then incubated with 20 μl of Ni-NTA agarose at 4°C for 4 hours. After intensive washing with washing buffer A [50 mM Hepes (pH 7.5), 300 mM NaCl, 20 mM imidazole, and 0.1 mM PMSF], the proteins that were bound to the agarose were eluted with elution buffer (elution buffer containing 250 mM imidazole). The eluted proteins were analyzed by SDS-PAGE or WB. For SDS-PAGE, the speci c bands were excised and subjected to LC-MS (TripleTOF 5600+, SCIEX, USA).
Reverse transcription-quantitative real time PCR (RT-qPCR) The total RNA of treated cells was extracted with Trizol (Invitrogen Corp, Carlsbad, CA, USA) and reversetranscribed with a cDNA reverse kit (Toyobo, Osaka, Japan) with Oligo dT primers. The reverse transcribed cDNA was used as a template for qPCR reactions along with SYBR Green Real time PCR Master Mix (Toyobo, Osaka, Japan) and 0.4 μM forward and reverse primers of PD-L1 (Forward 5'-CCTCCGTAACTACTGATACAA-3', Reverse 5'-TGCTATACCTGACACCTATAAG-3'), (TSINGKE, Wuhan). The qPCR reactions were run on an ABI Step One Plus (Applied Biosystems) under standard cycling conditions. Relative RNA levels were calculated by the comparative cycle threshold (CT) method (2 −ΔΔCT method), where CT represents the ampli cation cycle number at which the uorescence generated within a reaction rises above a de ned threshold uorescence and ΔΔCT = experimental groups (Ct Target gene − Ct GAPDH ) − control groups (Ct Target gene − Ct GAPDH ). The mRNA levels of each gene in the experimental groups were then expressed as the fold levels relative to the blank control groups and were calculated by the following formula: 2 −ΔΔCT . Each experimental sample contained three replicate wells.

Magnetic Activated Cell Sorting (MACS) of CD8 + T cells
The EasySep TM Mouse CD8 + T Cell Isolation Kit (STEMCELL Technologies, Canada) was used to isolate CD8 + T cells from splenocytes of BALB/c mice by negative selection. The labeled cells were separated using an EasySep TM magnet, and the desired CD8 + T cells were poured into a new tube. The purity of CD8 + T cells was >95% as determined by FCM.
Co-culture of macrophages with CD8 + T cells Anti-CD3 and anti-CD28 antibodies are used for the activation of CD8 + T cells before assessing the inhibition of treated BMDMs. BMDMs (5×10 5 cells/well) were stimulated with puri ed UreB protein (1 μg/mL) or infected with WT or UreB defective H. pylori strains at MOI=10 for 24 h at 37°C. Then, BMDMs were washed with PBS and co-cultured with CD8 + T cells (1×10 6 /well) at the presence of PD-L1 blocking antibody anti-PD-L1 (10 μg/mL) or mTOR inhibitor Temsirolimus (5 μM) for 24 h. The proliferation of CD8 + T cells was analyzed by CFSE staining. The expression of perforin and granzyme B in CD8 + T cells were detected by FCM with PE anti-mouse Perforin (154306, Biolegend, USA) and FITC antihuman/mouse Granzyme B (515403, Biolegend, USA) respectively.

Amino acid metabolism analysis by LC-MS/MS
BMDMs were stimulated with UreB (1 μg/mL) or solvent for 24 h, cells were washed with cold PBS and collected into 1.5 mL EP tube, add 1 mL cold extraction reagent (70% methanol containing 1 μg/mL 2chlorophenylalanine as an internal standard), Vortex 1 min. Quickly freeze it in liquid nitrogen for 3 minutes and thaw it on ice for 3 minutes, Vortex 2 minutes, and repeat the above 3 steps for 3 cycles.

Graphs and Statistical Analysis
Results were plotted using GraphPad Prism v8 and analyzed using an unpaired, two-tailed t-test or twoway ANOVA as relevant, with p-values represented as P > 0.05 ns, *p < 0.05, **p < 0.01, and ***p < 0.001.

Results
UreB upregulates the PD-L1 expression of host cells H. pyloricolonization results in a local in ltration of macrophages, which play a critical role in the gastric complication (Menaker et al., 2004). Here we infected BMDMs with WT or UreB KO H. pylori which was validated by western blot and urease activity assay ( Figure S1A, S1B) at MOI=10 for 24 h to nd out differently expressed genes related to the immune regulating functions of macrophages through mRNAchip sequencing. As shown in Figure 1A, compared with UreB KO H. pylori infected BMDMs, PD-L1 as well as other immune or in ammatory factors of H. pylori group were upregulated. Then we veri ed this phenomenon as H. pylori infection induced PD-L1 expression was restrained by defecting UreB ( Figure  1B-D). Then we expressed and puri ed the recombinant UreB protein with His tag which was con rmed by Coomassie brilliant blue staining ( Figure S1C) and western blot with anti-His antibody ( Figure S1D). The optimal concentration for UreB protein to induce the expression of PD-L1 in BMDMs was con rmed as 1 μg/mL ( Figure S1E). The PD-L1 expression of UreB protein (1 μg/mL) treated BMDMs were also upregulated with a time gradient dependence ( Figure 1E-G). Besides, UreB induced PD-L1 expression is not limited to Human or murine macrophages as we found UreB also upregulated the PD-L1 expression of gastric cancer cell lines such as AGS and MNK28 ( Figure S1F). These results indicated that UreB contributes to H. pylori induced PD-L1 expression in host cells.

UreB dampens the activation of CD8 + T cells through the induction of macrophage PD-L1 expression
As it has been reported that UreB could be found inside macrophages (Schwartz and Allen, 2006), so, H. pylori may become a kind of intracellular bacteria (Kronsteiner et al., 2014). Since H. pylori-induced PD-L1 can inhibit the proliferation of activated T-cells (Beswick et al., 2007). We use UreB treated BMDMs to coculture with CFSE stained CD8 + T cells which were isolated from the spleen of wild type BALB/c. Then the proliferation ability of CD8 + T cells was measured with FCM. As shown in (Figure 2A, S2A), UreB treated BMDMs inhibited the proliferation of CD8 + T cells, however, when PD-L1 was blocked by PD-L1 blocking antibody, the inhibition effect was partly reversed, suggesting that UreB induced PD-L1 mediated the inhibition on CD8 + T cells proliferation. Next, we get the consistent results from H. pylori-infection assay.
WT H. pylori infected macrophages inhibited the proliferation of CD8 + T cells, but when UreB was knocked out, the inhibition effect was blocked. In addition, when PD-L1 was neutralized with PD-L1 antibody, the proliferation index of both WT and UreB KO group increased ( Figure 2B UreB interacted with Myh9 to upregulate the expression of PD-L1 To explore the exact mechanism through which UreB upregulated the expression of PD-L1, we use UreBhis-Ni-agarose to pulldown the receptor for UreB in BMDMs. As it has been reported that UreB increases MLC (myosin II regulatory light chain) phosphorylation, occludin internalization and barrier dysfunction in gastric epithelial cells during H. pylori infection (Wroblewski et al., 2009). We selected two potential targets Hspe1 and Myosin-9 (Myh9) from all detected proteins in LC-MS ( Figure 3A, S3A-B). When we knock down these two genes with siRNAs respectively, interfering Myh9 rather than Hspe1 blocked UreBinduced PD-L1 expression ( Figure 3B). And then the interaction between UreB and Myh9 during UreB stimulation was further con rmed by co-immunoprecipitation with antibody against His ( Figure 3C) or Myh9 ( Figure 3D), respectively. When BMDMs were infected with WT or UreB KO H. pylori, Myh9 immunoprecipitated UreB in WT H. pylori-infected BMDMs ( Figure 3E), but not UreB KO group. In addition, the interference of Myh9 expression also downregulated H. pylori induced PD-L1 expression of BMDMs, but this effect disappeared when BMDMs were infected with UreB KO strain ( Figure 3F). Taken together, these results suggested that Myh9 works as a receptor for UreB and mediates the upregulation of PD-L1 in H. pylori-infected BMDMs.

mTOR signal pathway mediated UreB induced PD-L1 expression
To further evaluate the effect of UreB on host cells' signal pathway, we make a gene set enrichment analysis on UreB induced differently expressed genes. As shown in Figure 4A, the most enriched genes are related to amino acids metabolism (about 300 genes). And, there are about 100 genes associated with mTOR signal pathway. It is consistent with the report that c-Met/AKT/mTOR pathway activated by MACC1 (metastasis associated in colon cancer-1) contributes to the PD-L1 expression in gastric cancer cells and induces tumor growth in vivo (Tong et al., 2019). We found that H. pylori contributes to the activation of mTOR as well as the PD-L1 expression during H. pylori infection whereas PD-L1 were downregulated when UreB was depleted ( Figure S3C). Therefore, we further examined if mTOR is associated with UreB induced PD-L1 expression. BMDMs, pretreated with inhibitors for PI3K (LY294002), AKT (Ipatasertib), mTOR (Temsirolimus) and Hedgehog/Gli (GANT61) for 1 h, were then infected with H. pylori at MOI=10 for 3 or 12 h, and the mRNA level of PD-L1 was detected with RT-qPCR. Temsirolimus and GANT61 instead of LY294002 and Ipatasertib arrest H. pylori induced PD-L1 expression ( Figure 4B-E). These results mean that mTOR mediated UreB induced PD-L1 just like CagA induced PD-L1 expression through sonic Hedgehog (Shh) signaling pathway (Holokai et al., 2019). Then, BMDMs were pretreated with inhibitors against PI3K/AKT/mTOR, and then stimulated with puri ed UreB protein (1 μg/mL) for 1 or 3 h, and the activity of PI3K/AKT/mTOR signaling pathway was analyzed by immune blots. Even through UreB activated PI3K/AKT/mTOR axis, but only mTOR inhibitor Temsirolimus blocked UreB induced PD-L1 ( Figure 4F), which indicates that UreB activated mTOR is independent of PI3K/AKT.
Then it was con rmed that UreB induced PD-L1 expression during H. pylori infection is independent of shh signaling pathway which mediated CagA upregulated PD-L1 ( Figure 4G). Besides, when the receptor of UreB was interfered with veri ed effective siRNA-Myh9 2 ( Figure S3D), UreB activated mTOR was also weakened ( Figure 4H). These data reach a conclusion that UreB activated mTOR leads to the upregulation of PD-L1 on macrophages. mTOR response to UreB disrupted amino acid metabolism As UreB activated mTOR is independent of PI3K/AKT, and genes about amino acids metabolism are enriched in UreB stimulated BMDMs ( Figure 4A). We wonder if UreB disrupts the amino acids metabolism of host cells to activate mTOR, so the relative content of all detected L-amino acids by LC-MS/MS analysis were shown in Figure 5A. It is obvious that most detected L-amino acids increased after being stimulated with UreB. And we get a consistent result when the amino acids level of WT or UreB KO H. pylori infected BMDMs were detected with L-Amino Acid Quantitation Kit. It's clear that even though H. pylori infection disrupted the amino acids metabolism of host cells which showed a decrease in amino acids level, UreB reversed the amino acids exhaustion ( Figure 5B). To directly evaluate the in uence of UreB on amino acids pool, the autophosphorylation state at Thr 898 of GCN2 (p-GCN2 (T898)) was examined, which increases as a consumed marker of available pools of amino acids (Dever and Hinnebusch, 2005). Compared to the WT H. pylori infected BMDMs, UreB KO group upregulated the expression of phosphorylated GCN2 (p-GCN2 (T898) ( Figure 5C). However, when UreB receptor was disturbed with siRNA, there was no difference between the WT and UreB KO group ( Figure 5D). It is worth noting that despite Myh9 was disabled, WT and UreB KO H. pylori can still induce the expression of p-GCN2 (T898) (Figure 5D), which means that there are some other potential virulence effectors such as CagA contribute to the depletion of amino acids in host cells (Kim et al., 2018). And then our previous hypothesis was con rmed as when BMDMs were cultured in amino acids free medium, neither WT or UreB KO H. pylori could active mTOR signaling pathway and induce the expression of PD-L1 ( Figure 5E). These results unraveled that UreB activated mTOR pathway triggered amino acid anabolism, leading to the induction of PD-L1 expression. mTOR inhibitor blocked the inhibition of UreB up-regulated PD-L1 on CD8 + T cells Given that the above results have con rmed that mTOR mediated the expression of UreB induced PD-L1.
It's of particular interest that whether mTOR is involved in the inhibitory effect of PD-L1 on CD8 + T cells.
UreB treated BMDMs were cocultured with CD8 + T cells at the presence of Temsirolimus. As inhibited mTOR limited the expression of PD-L1, the overall level of proliferation ( Figure 6A, S2C) as well as the secretion of cytotoxic factors perforin and granzyme B ( Figure 6B) are higher than the control groups (DMSO treated). And the inhibition of H. pylori infected macrophages on CD8 + T cells was also blocked by Temsirolimus (Figure 6CD, S2D). However, the blocking effect of Temsirolimus vanished when UreB was KO ( Figure 6C). It's interesting that Temsirolimus could not completely reverse the in uence of H. pylori infected macrophages on the production of cytotoxic factors in CD8 + T cells, which may be caused by other virulence factors (except for UreB) that could inhibit CD8 + T cells' function independent of mTOR signaling pathway ( Figure 6D). The above results suggest that UreB activated mTOR signaling pathway is critical for the inhibition of macrophages on CD8 + T cells during H. pylori infection.

Conclusion
Overall, we found out that UreB upregulates the PD-L1 expression of macrophages through Myh9 /mTOR pathway and inhibits the activity of CD8 + T cells during H. pylori infection. UreB was found to induce PD-L1 expression through interaction with a new receptor Myh9 in macrophages. mTOR, which responds to UreB upregulated amino acid level of macrophages, specially mediated this process. UreB induced PD-L1 arrests the proliferation of CD8 + T cells and inhibits the expression of perforin and granzyme B. In light of these results, we demonstrated the immunosuppressive function of UreB on host cells.

Discussion
To explore the relationship between UreB induced immunosuppressive molecule PD-L1 and CD8 + T cells, we focused on the mechanism research which clari ed how UreB upregulated PD-L1. However, there are so many potential genes that were upregulated by UreB in Fig. 1A. For example, UreB contributes to the expression of many kinds of chemokines and TNF, suggesting that UreB participated in the induction of in ammatory response against H. pylori during infection. A direct interaction between Hsp60 and the N terminal of UreA has been reported in H. pylori (Zhao et al., 2019). Even though Hspe1 was identi ed as a potential receptor for UreB by LC-MS, but no difference in UreB induced PD-L1 was found when Hspe1 was interfered with siRNA, which indicated that UreB may in uence other signaling pathway except for PD-L1 through Hspe1. At least, we found a direct interaction between UreB and the heat shock protein family of host cells, so a further research focused on HSP mediated pathogenic mechanism during H. pylori infection will be valuable. . It's a novel discovery that mTOR response to the amino acids level of host cells which connect the immunosuppressive phenomenon with host amino acids metabolism. But we only detected the mTOR activation in response to amino acids, and it will also be important to examine if UreB in uences the glucose metabolism, as it has been reported that H. pylori CagA upregulates glucose metabolism of gastric cancer cells (Gao et al., 2020). And over expressed PD-L1 accelerates glycolysis by AKT/mTOR/HIF-1α axis in acute myeloid leukemia (Ma et al., 2020). As UreB promoted amino acid anabolism indicates a rich nutrients and energy state which is conducive to proliferation and tumorigenesis. Except for regulating PD-L1, UreB activated PI3K/AKT may in uence the In conclusion, our ndings rstly demonstrated that H. pylori UreB upregulated PD-L1 through activated mTOR, then PD-L1 inhibited the proliferation and activity of CD8 + T cells. Here, we successfully identi ed Myh9 as a new receptor for UreB and mediated the regulation on PD-L1 as well as CD8 + T cells. UreB disrupted amino acids metabolism is involved with the suppression on CD8 + T cells, which revealed that H. pylori inhibits the immune response by disrupting the host metabolism (Fig. 7). In light of our results, it is reasonable that taking the immunosuppressive effect into consideration when developing UreB based H. pylori vaccine.

Ethics statement
All mouse experiments in this study were reviewed and approved by the ethics committee of Tianjin Medical University General Hospital.

Availability of supporting data
All datasets generated for this study are included in the manuscript/Supplementary Files.

Competing interests
The authors declare that they have no conflict of interest. pylori was used in (B). (E-G) BMDMs were treated with UreB (1 μg/mL) for the indicated time, then the expression of PD-L1 was analyzed by RT-qPCR (E), western blotting (F), and FCM (G), respectively. Unpaired t test, versus 0 hours was used for analysis in (E). The data are expressed as the mean ± SEM, n = 3; NS (P > 0.05), *P < 0.05, **P < 0.01, or ***P < 0.001. Unpaired t test, versus 0 hours was used for analysis in (E). The data are expressed as the mean ± SEM, n = 3; NS (P > 0.05), *P < 0.05, **P < 0.01, or ***P < 0.001. pylori was used in (B). (E-G) BMDMs were treated with UreB (1 μg/mL) for the indicated time, then the expression of PD-L1 was analyzed by RT-qPCR (E), western blotting (F), and FCM (G), respectively. Unpaired t test, versus 0 hours was used for analysis in (E). The data are expressed as the mean ± SEM, n = 3; NS (P > 0.05), *P < 0.05, **P < 0.01, or ***P < 0.001.    BMDMs were infected with indicated strains for 30 mins, then co-IP was performed with antibody anti-Myh9 and immunoblots was performed to detect UreB and Myh9. (F) BMDMs were infected with indicated strains for 24 h after being electro-transfected with siRNA-Myh9 or siRNA-control, the mRNA level of PD-L1 was detected by RT-qPCR. Unpaired t test Myh9 KD versus control was used for analysis. The data are expressed as the mean ± SEM, n = 3; NS (P > 0.05), *P < 0.05, **P < 0.01.  BMDMs were infected with indicated strains for 30 mins, then co-IP was performed with antibody anti-Myh9 and immunoblots was performed to detect UreB and Myh9. (F) BMDMs were infected with indicated strains for 24 h after being electro-transfected with siRNA-Myh9 or siRNA-control, the mRNA level of PD-L1 was detected by RT-qPCR. Unpaired t test Myh9 KD versus control was used for analysis. The data are expressed as the mean ± SEM, n = 3; NS (P > 0.05), *P < 0.05, **P < 0.01.   were infected with indicated strains at MOI=10 for 24 h, then the expression of PD-L1 and mTOR were analyzed with immunoblots. The data are expressed as the mean ± SEM, n = 3; NS (P > 0.05), *P < 0.05, ***P < 0.001.   with mTOR inhibitor Temsirolimus were co-cultured with CD8+ T cells for 24 h, then perforin and granzyme B of CD8+ T cells were detected by FCM. The statistical histograms were shown at the right. Unpaired t test versus DMSO (B) or H. pylori (D) were used for analysis. The data are expressed as the mean ± SEM, n = 3; NS (P > 0.05), *P < 0.05, ***P < 0.001. Figure 6 mTOR mediated UreB induced inhibition on CD8+ T cells. (A) BMDMs were treated with puri ed UreB (1 μg/mL) (A) or infected with indicated H. pylori strains (C) at MOI=10 for 24 h, then BMDMs were washed with PBS 3 times and co-cultured with CFSE stained CD8+ T cells at the presences of mTOR inhibitor Temsirolimus for 24 h. The proliferation of CD8+ T cells was analyzed by Flow cytometry. (B, D) UreB (1 μg/mL) stimulated (B) or indicated strains infected (D) (MOI=10) BMDMs which have been pretreated with mTOR inhibitor Temsirolimus were co-cultured with CD8+ T cells for 24 h, then perforin and granzyme B of CD8+ T cells were detected by FCM. The statistical histograms were shown at the right. Unpaired t test versus DMSO (B) or H. pylori (D) were used for analysis. The data are expressed as the mean ± SEM, n = 3; NS (P > 0.05), *P < 0.05, ***P < 0.001. with mTOR inhibitor Temsirolimus were co-cultured with CD8+ T cells for 24 h, then perforin and granzyme B of CD8+ T cells were detected by FCM. The statistical histograms were shown at the right. Unpaired t test versus DMSO (B) or H. pylori (D) were used for analysis. The data are expressed as the mean ± SEM, n = 3; NS (P > 0.05), *P < 0.05, ***P < 0.001.

Figure 7
Illustration of the effects of UreB on macrophages and CD8+ T cells during H. pylori infection.

Figure 7
Illustration of the effects of UreB on macrophages and CD8+ T cells during H. pylori infection. Illustration of the effects of UreB on macrophages and CD8+ T cells during H. pylori infection.

Supplementary Files
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