...survival of CD4 T-cell-derived double-negative T cells |...

来自 : 发布时间：2025-04-07

Critical role of OX40 in the expansion and survival of CD4 T-cell-derived double-negative T cells AbstractCD4+ T-cell-converted CD4鈭?/sup>CD8鈭?/sup> double negative (cDNT) have strong suppressive activity in the maintenance of immune tolerance, whereas IL-2 promotes cDNT proliferation and enhances cDNT resistance to apoptosis. However, the intrinsic mechanisms that regulate the survival of cDNT are still unknown. Here we demonstrate that the OX40 molecule was highly expressed on cDNT. The expression of OX40 was necessary to promote proliferation and inhibit apoptosis of cDNT in vivo and in vitro. OX40 promoted the survival of cDNT by regulating the expression of Bcl-2, Bcl-xL, Survivin, and BCL2L11. Canonical NF-魏B cell signaling played an important role in the transmission of essential division and survival signals through OX40 in cDNT. IL-2 promoted the survival of cDNT in part via elevating the expression of the OX40 molecule. IL-2 promoted OX40 expression via downregulating the PPAR伪 expression. In conclusion, we elucidated that OX40 is a key molecule that regulates cDNT proliferation and survival. IL-2 promoted OX40 expression by downregulating the PPAR伪 binding to the OX40 promoter, leading to the elevated expression of Bcl-2, Bcl-xL, and Survivin in cDNT, which finally resulted in the promoted proliferation and decreased apoptosis of cDNT. IntroductionRegulatory CD4鈭?/sup>CD8鈭?/sup> double-negative T cells (DNT), which express 伪尾 T-cell receptor (TCR) but do not express natural killer (NK) cell markers compose only a small population of T lymphocytes (1鈥?%) in the peripheral blood and lymphoid organs of rodents and humans1,2. DNT cells have strong suppressive activity toward CD4+ T cells and CD8+ T cells3,4,5,6, as well as B cells4,7, dendritic cells (DCs)8, and NK cells9, which are capable of suppressing the immune response and exert significant protection against allograft rejection, graft-versus-host disease, and autoimmune diseases3,6,10,11,12,13.We have identified the differentiation pathway from CD4+ T cells to DNT, which are important for maintaining immune system homeostasis3,14. The DNT can be derived from activated and proliferated CD4+ T cells, which stimulated by bone marrow-derived DCs in vitro4. The over-activated CD4+ T cells can also be converted into DNT in vivo15. The CD4 T-cell-converted DNT (cDNT) are CD25+, CD44+, CD69+, and Foxp3鈭?/sup>. These cDNT potently suppressed vigorous allo- and autoimmune responses, prolonged islet and skin allograft survival, and prevented and cured autoimmune type 1 diabetes with antigen-specificity3,6,16.Interleukin (IL)-2 is a critical regulator of the activation and proliferation of T lymphocytes, also plays an important role in the generation and expansion of cDNT3. cDNT cells were in an anergic state, whereas exogenous IL-2 could restore cDNT responses, promote cDNT proliferation, and enhance cDNT resistance to聽Activation Induced Cell Death (AICD)17. However, the intrinsic mechanism that regulates the survival of cDNT and how IL-2 promotes cDNT proliferation and enhance cDNT resistance to AICD remain unknown.OX40, often recognized as a costimulatory receptor for T cells, is predominantly expressed on activated CD4 T cells. It is essential for regulating the conventional CD4 and CD8 T-cell division, differentiation, and survival. Previous studies reported that the cytokines IL-1, IL-2, IL-4, and tumor necrosis factor stimulation could enhance or prolong OX40 expression15,18. OX40 is also found on Foxp3+ Tregs, but it is dispensable for the genesis and suppressor functions of naturally arising CD4+Foxp3+ Tregs19. However, the expression and function of OX40 in DNT are still unknown. In this study, we have identified OX40 as the key regulator of cDNT survival and the mediator of IL-2 on the promotion of proliferation and resistance to AICD of cDNT.Results OX40 molecule was highly expressed on cDNT and was necessary to promote proliferation and inhibit apoptosis of cDNTAs we reported3, after 7 days鈥?in vitro stimulation with mature DCs, approximately 30% of CD4 T cells lost CD4 expression and became DNT (Fig.聽1a, left). By monitoring the apoptosis of activated CD4+ and cDNT, we found that the percentage of Annexin V+ cells was markedly lower in the cDNT than in activated CD4+ T cells (51.7鈥壜扁€?.7% vs. 8.1鈥壜扁€?.2%, P鈥?lt;鈥?.05, Fig.聽1a, right). Meanwhile, OX40 expression was significantly higher in cDNT than that in activated CD4+ T cells (37.3鈥壜扁€?.91% vs. 18.9鈥壜扁€?.59%, P鈥?lt;鈥?.05; Fig.聽1a, middle). Furthermore, cDNT and CD4+ T cells were sorted from mixed lymphocyte reaction (MLR) and were assessed for OX40 mRNA expression. As shown in Fig.聽1b, the OX40 mRNA expression level of cDNT was also significantly higher than that of CD4+ T cells. No significant differences of CD27, CD28, CD30, CD40, CD95, and ICOS expression between CD4+ T cells and cDNT (supplementary Figure聽1), indicating that OX40, the apoptosis-related gene of activated CD4+ and CD8+ T cells, may also play an important role on the survival of cDNT.Fig. 1: OX40 regulated survival of cDNT.a CD4+CD25鈭?/sup> T cells from C57BL/6 mice were stimulated with mature DBA/2 DCs for 7 days. The converted DNT and activated CD4+ T cells were detected for OX40 expression through flow cytometric analysis. Annexin V staining was used to detect apoptosis of the two cell populations. b The relative mRNA expression of OX40 was determined by real-time PCR in activated CD4+ T cells and cDNT. c Caspase 3/7 activation was determined in B6 cDNT or OX40 KO cDNT after being stimulated with anti-CD3/CD28 antibodies for 24, 48, and 72鈥塰. d The converted C57BL/6 and OX40 KO DNT were incubated with anti-CD3/CD28 antibodies for 24, 48, and 72鈥塰, and apoptosis was assessed via Annexin V staining. A representative flow cytometry image of Annexin V+ cells (% cDNT) from each group is shown (left). Statistical analysis of Annexin V+ cells in OX40 KO cDNT relative to B6 cDNT in each group was determined by flow cytometry (right). e The converted B6 and OX40 KO cDNT were incubated with anti-CD3/CD28 antibodies for 24, 48, and 72鈥塰, and proliferation was assessed via EdU incorporation. Representative flow cytometry image of EdU+ cells (% cDNT) from each group is shown (left). In addition, statistical analysis was determined by flow cytometry (right). f The B6 cDNT and OX40 KO cDNT stimulated with anti-CD3/CD28 antibodies were incubated with Alamar Blue, and the absorbance at 570鈥塶m at different time points was measured. g A total of 5鈥壝椻€?06 converted DNT or OX40 KO DNT were adoptively transferred into B6D2F1 (H-2b/d) recipient mice by tail vein injection. Three days after injection of BrdU (100鈥壜礸/day), splenocytes were isolated, and stained with fluorochrome-conjugated antibodies against mouse H2Dd and CD3, and the cDNT (H2Dd鈭?/sup>CD3+) apoptosis and proliferation were determined by Annexin V and BrdU staining in vivo. Data are representative of three experiments with similar results. The data are depicted as the mean鈥壜扁€塖D, n鈥?鈥? in each group. *p鈥?lt;鈥?.05Full size imageTo further investigate the functional potential of OX40 on the converted DNT survival, OX40 knockout (KO) DNT converted from OX40 KO CD4+ T cells were stimulated with anti-CD3 and CD28 antibodies in vitro, and the apoptotic and proliferation rate was analyzed with Annexin V and EdU incorporation on 24, 48, and 72鈥塰, respectively. Compared with B6 cDNT, caspase 3/7 activity in OX40 KO cDNT was increased (Fig.聽1c), and the apoptotic rates of OX40 KO cDNT were significantly higher (Fig.聽1d). In contrast, proliferation was decreased in OX40 KO cDNT determined by EdU incorporation (Fig.聽1e). We also measured the proliferation of cDNT using an Alamar Blue cell viability reagent, and as shown in Fig.聽1f, C57BL/6 (B6) cDNT had higher fluorescent, which indicated that cDNT had a higher proliferation rate than OX40 KO cDNT.In addition, we also evaluated the regulation of OX40 on the proliferation and apoptosis of converted DNT in vivo. A total of 5鈥壝椻€?06 converted wild-type (WT) or OX40 KO DNT were adoptively transferred into B6D2F1 (H-2b/d) recipient mice by tail vein injection. After 3 days injection of BrdU (100鈥壜礸/day/mouse), splenocytes were isolated and stained with fluorochrome-conjugated antibodies against mouse H2Dd, the proliferation of cDNT (H2Dd-negative cells) was at a lower level when OX40 was deficient; however, the apoptosis of OX40-deficient cDNT was increased (Fig.聽1g).The collective data from in vitro and in vivo studies indicated that the expression of OX40 was necessary to promote proliferation and inhibit apoptosis of cDNT. OX40 promoted the survival of cDNT by regulating the expression of Bcl-2, Bcl-xL, Survivin, and Bcl-2-like protein 11OX40 engagement promotes CD4+ T-cell survival through the induction of the anti-apoptotic molecules, Bcl-2, Bcl-xL20, and cell cycle progression-related protein Survivin21. Meanwhile, it also greatly enhances CD8+ T-cell survival by upregulating Bcl-xL22. Ligation of OX40 on neutrophils results in enhanced survival, which correlates with reduced activation of caspase 3 and with augmented levels of anti-apoptotic markers23. To garner further insight into the molecular mechanism by which OX40 modulated cDNT survival, the apoptosis-related proteins Bcl-2, Bcl-xL, Survivin, and Bcl-2-like protein 11 (BCL2L11) were analyzed on converted WT DNT and OX40 KO DNT. cDNT were cultured up to 72鈥塰 with anti-CD3 and CD28 antibodies in vitro, the apoptosis of OX40 KO cDNT was increased, together with significant reduction of anti-apoptotic molecules, Bcl-2, Bcl-xL, and Survivin (Fig.聽2a) when OX40 was knocked out. In addition, as shown in Fig.聽2b, Bcl-2, Bcl-xL, and Survivin mRNA expression were dramatically reduced in OX40 KO cDNT, whereas BCL2L11 mRNA expression was augmented. These data indicated that OX40 could promote cDNT survival through the induction of apoptosis-related protein Bcl-2, Bcl-xL, and Survivin, and the suppression of pro-apoptotic protein BCL2L11.Fig. 2: OX40 controls cDNT survival by its regulation on Survivin, Bcl-2, Bcl-xL, and BCL2L11.a Flow cytometric analysis of Bcl-2, Bcl-xl, and Survivin protein expression in B6 cDNT and OX40 KO cDNT after anti-CD3/CD28 stimulation for 48鈥塰. b Relative mRNA expression of anti-apoptosis genes (Bcl-2, Bcl-xl, and Survivin) and pro-apoptosis genes (Bcl2l11) in B6 cDNT and OX40 KO cDNT after incubation with CD3/CD28 for 48 and 72鈥塰. c The relative mRNA levels of NF-魏B signaling genes (nfkb1 and rela) in OX40 KO cDNT compared with WT cDNT. d Flow cytometric analysis of RelA, and phosphorylation of I魏B伪 and IKK伪/尾 protein expression in B6 cDNT and OX40 KO cDNT cells after anti-CD3/CD28 stimulation for 48鈥塰. e Nuclear protein extracts were prepared from B6 cDNT cells and OX40 KO cDNT cells after anti-CD3/CD28 stimulation for 48鈥塰. The RelA and p50 protein levels in the cells were analyzed by western blot. The relative density of the RelA and p50 band was normalized to Histone H3. The data are presented as the mean鈥壜扁€塖D, n鈥?鈥? in each group. *p鈥?lt;鈥?.05. NS no significanceFull size imageCanonical nuclear factor (NF)-魏B cell signaling plays an important role in the transmission of essential division and survival signals through OX40 in T cells24,25. Therefore, we examined the NF-魏B1 and RelA mRNA expression of cDNT after 48 and 72鈥塰 cultures in vitro. The mRNA expression of NF-魏B1 and RelA of cDNT were significantly reduced when OX40 was deficient (Fig.聽2c). We also detected NF-魏B signaling pathway through flow cytometry and western blotting at protein level. As shown in Fig.聽2d, OX40 deficiency could downregulate phosphorylation events of IKK伪/尾, I魏B伪, and RelA (p65) expression of cDNT. Meanwhile, western blotting results also showed OX40 KO cDNT decreased p50 and RelA expression in the nucleus (Fig.聽2e), suggesting that canonical NF-魏B signaling was also important for transmission of proliferation and survival signals through OX40 in cDNT.IL-2 promoted the survival of cDNT in part via elevating the expression of OX40 moleculeIL-2 is critical for the activation and proliferation of T cells, plays an important role in the generation and expansion of cDNT. In this study, we found that IL-2 could remarkably promote the expression of OX40 in cDNT (Fig.聽3a, b), however, CD27, CD28, CD30, CD40, CD95, and ICOS were not influenced by IL-2 (supplementary Figure聽2). Therefore, we tested whether the regulation of IL-2 on cDNT proliferation and apoptosis was OX40-dependent. Accordingly, converted WT DNT and OX40 KO DNT were stimulated with anti-CD3 and CD28 antibodies in vitro with or without rIL-2. With exogenous IL-2, the apoptosis rates of cDNT from WT B6 mice and OX40 KO mice were both decreased (Fig.聽3c, d); however, the reduction of apoptosis in converted WT B6 cDNT was markedly greater than that in OX40 KO cDNT (Fig.聽3e). IL-2 could inhibit the activity of caspase 3/7 in cDNT; however, the reduction of caspase 3/7 activity of cDNT with rIL-2 stimulation in vitro was impaired when OX40 was deficient (Fig.聽3f, g). Similarly, the proliferation of cDNT was significantly augmented in the presence of rIL-2 stimulation (Fig.聽3h, i). Nevertheless, the proliferation enhancements of cDNT with rIL-2 stimulation in vitro were reduced when OX40 was knocked out (Fig.聽3j). The Alamar Blue cell proliferation assay further supported that OX40 was involved in the regulation of IL-2 on the survival of cDNT (Fig.聽3k).Fig. 3: IL-2 promoted cDNT survival through OX40 molecule.a Statistical analysis of OX40 expression on cDNT with or without IL-2 for 24, 48, and 72鈥塰 based on flow cytometric analysis. b Relative changes in OX40 expression induced by IL-2 in cDNT were plotted as fold changes of B6 cDNT without IL-2 stimulation. c B6 cDNT and OX40 KO cDNT were stimulated with or without IL-2 for 24, 48, and 72鈥塰, and apoptosis of the cells was detected through Annexin V staining. Representative flow cytometric image of Annexin V+ cDNT from each group. d Statistical analysis of Annexin V+ cells in each group at a different time point. e Relative changes of Annexin V+ cells with or without IL-2 in B6 cDNT were compared with that in OX40 KO cDNT at 24, 48, and 72鈥塰. f Caspase 3/7 activation was determined in B6 cDNT and OX40 KO cDNT after stimulation with or without IL-2. g The relative changes of Caspase 3/7 activation with or without IL-2 in B6 cDNT were compared with the ratio in OX40 KO cDNT at 24, 48, and 72鈥塰. h The percentages of EdU+ cells relative to the total numbers of B6 cDNT or OX40 KO cDNT with or without IL-2 was determined by flow cytometry in the indicated groups. i Statistical analysis of EdU+ cells in each group at different time points. j Relative changes of EdU+ cells with or without IL-2 in B6 cDNT were compared with that in OX40 KO cDNT at 24, 48, and 72鈥塰. k The B6 cDNT and OX40 KO cDNT were stimulated with IL-2 or without IL-2, and Alamar Blue was incubated during the stimulation. The proliferations of the cells were detected at the absorbance of 570鈥塶m at different time points. #P聽 聽0.05, IL-2 + B6 cDNT聽versus B6 cDNT;聽*P 0.05, IL-2 + B6 cDNT聽versus IL-2聽+聽OX40 KO cDNT.聽l A total of 5鈥壝椻€?06 B6 cDNT or OX40 KO cDNT were adoptively transferred into B6D2F1 recipient mice by tail vein injection in the presence or absence of IL-2/Fc protein. After 3 days, cDNT were analyzed by flow cytometry for proliferation and apoptosis with BrdU and Annexin V detection. Data are representative of three experiments with similar results. The data are presented as the mean鈥壜扁€塖D, n鈥?鈥? in each group. *p鈥?lt;鈥?.05, **p鈥?lt;鈥?.01, NS no significanceFull size imageAdditionally, B6D2F1 mice underwent adoptive transfer with 5鈥壝椻€?06 B6 cDNT or OX40 KO cDNT in the presence or absence of IL-2/Fc. Three days after injection of BrdU, cDNT were analyzed by flow cytometry for proliferation and apoptosis with BrdU and Annexin V detection. As shown in Fig.聽3l, the effect of OX40 on IL-2-regulating cDNT survival in vivo was comparable to those in vitro.We also noticed that rIL-2 upregulated anti-apoptotic molecules, Bcl-2 (Fig.聽4a), Bcl-xl (Fig.聽4b), and Survivin (Fig.聽4c) expression and downregulated pro-apoptotic gene Bcl2l11 expression (Fig.聽4d) of cDNT. However, the regulation of IL-2 on these apoptotic-related genes was impaired in OX40-deficient cDNT (Fig.聽4a鈥揹). These data suggested that IL-2 promoted the survival of cDNT at least in part via the elevation of OX40 molecule expression.Fig. 4: Apoptosis-related genes were affected in OX40-deficient cDNT under IL-2 stimulation.a Relative mRNA expression of anti-apoptosis genes Bcl-2 (a), Bcl-xl (b), and Survivin (c), and pro-apoptosis genes Bcl2l11 (d) in B6 cDNT and OX40 KO cDNT stimulated with IL-2 or without IL-2 at different time points. Relative changes of the genes in the presence or absence of IL-2 in B6 cDNT compared with that in OX40 KO cDNT. The data are presented as the mean鈥壜扁€塖D, n鈥?鈥? in each group. *p鈥?lt;鈥?.05, **p鈥?lt;鈥?.01, NS no significanceFull size imageIL-2 promoted OX40 expression by downregulating the PPAR伪 expressionTo investigate the intrinsic mechanism of IL-2 regulation on OX40 molecular expression in cDNT, the upstream factors regulating OX40 were investigated. In the OX40 promoter region (鈭?032/+28 region), potential transcription factor (TF)-binding sites are presented in Fig.聽5a and were based on bioinformatic prediction. To further identify the TFs that bind to the OX40 core promoter region and regulate the activation of the OX40 gene, a competitive promoter-binding TF profiling array for the OX40 core promoter in cDNT was performed. Among the potential TF-binding sites, PPAR has relative higher changes of chemiluminescence activity between the absence (control) of OX40 core promoter vs. presence (control鈥?鈥塸romoter) of the promoter within the OX40 promoter (Fig.聽5b, c). Furthermore, PPAR伪 and PPAR纬 mRNA expression was markedly decreased in the cDNT under IL-2 stimulation (Fig.聽5d).Fig. 5: Identification of the core promoter of mice OX40 gene.a Nucleotide sequence of the OX40 promoter region. Potential regulatory elements identified according to the Transcription Factor Binding Sites database TRANSFAC are shown underlined and identified by the appropriate symbols. b Promoter-binding transcription factor (TF) profiling array assay of mice OX40 core promoter was performed. This is a competitive binding assay performed to identify promoter-bound TFs through comparisons of the results in the presence (with promoter) or absence (without promoter) of the mouse OX40 core promoter. If the OX40 promoter contains a TF-binding sequence, it will display a lower chemiluminescence activity. c Relative change of chemiluminescence activity in the absence (control) of the OX40 core promoter with presence (control鈥?鈥塸romoter) of the promoter. d Relative mRNA levels of the transcription factors in the presence or absence of IL-2. The data are presented as the mean鈥壜扁€塖D, n鈥?鈥? in each group. *p鈥?lt;鈥?.05, **p鈥?lt;鈥?.01, NS no significance. e ChIP analysis of cDNT stimulated with IL-2 (50鈥塶g/ml) for 48鈥塰. Conventional PCR was performed to measure the relative levels of the antibody-bound DNA fragments. Soluble chromatin from the cDNT was immunoprecipitated with the PPAR伪, PPAR纬 antibody, or incubated with normal rabbit serum (IgG) for control purposes. The amplification for soluble chromatin before immunoprecipitation are shown as input. f Real-time PCR was used to determine the levels of PPAR伪 and PPAR纬 bound directly to OX40 promoter sequence (鈭?45/鈭?36鈥塨p and 鈭?017/鈭?50鈥塨p upstream from transcription start site). The data are presented as the mean鈥壜扁€塖D, n鈥?鈥? in each group. *p鈥?lt;鈥?.05, NS no significanceFull size imageThen we performed ChIP assays with the OX40 promoter to test whether PPAR伪 or PPAR纬 was associated with the OX40 gene upregulation of cDNT in the presence of IL-2. As shown in Fig.聽5e, after immunoprecipitation with PPAR伪 antibodies, a positive band of approximately 鈭?45/鈭?36鈥塨p was yielded in the bound DNA of cDNT without IL-2 stimulation, however, lower amplification of bound DNA from cDNT with IL-2 stimulation was shown. Precipitation with PPAR伪 or without antibody did not show significant changes of positive DNA band (approximately 鈭?35/鈭?39鈥塨p). Precipitation with PPAR纬 antibody or without antibody did not show significant changes of positive DNA band between cDNT treated with or without IL-2, which strongly suggested that PPAR伪 is able to bind to the OX40 promoter and is involved in regulation of OX40 expression by IL-2. Real-time PCR further proved that PPAR伪 direct binding to OX40 promoter sequence (鈭?45/鈭?36鈥塨p) was reduced in cDNT treated with IL-2 compared to cDNT without IL-2 stimulation.To ascertain the possible involvement of PPAR伪 activity in regulation of IL-2 on OX40 molecule, the cultures were performed in the presence or absence of the PPAR伪 agonist and rIL-2. As an aforementioned result, OX40 expression and proliferation of cDNT were significantly augmented in the presence of rIL-2 stimulation; however, the upregulation of OX40 expression induced by IL-2 was abrogated by a PPAR伪 agonist dose dependently (Fig.聽6a, b). In addition, the effect of rIL-2 on the upregulation of anti-apoptotic molecules, Bcl-2, Bcl-xl, and Survivin, or the downregulation of the pro-apoptotic gene Bcl2l11 were all impaired in the presence of a PPAR伪 agonist (Fig.聽6c). Unquestionably, PPAR伪 agonist neutralized the anti-apoptotic effect of IL-2 on cDNT, the percent of apoptotic rate was increased in the presence of a PPAR伪 agonist (Fig.聽6d). In contrast, the proliferation rates of cDNT with rIL-2 stimulation in vitro were reduced in the presence of a PPAR伪 agonist, and the changes were dose-dependent (Fig.聽6e).Fig. 6: IL-2 regulated OX40 expression through transcription factor PPAR伪.a Inhibition of OX40 mRNA levels after incubation with or without IL-2 for 48鈥塰 by a PPAR伪 agonist (80鈥壜礛). b The percentage of OX40+ cDNT was detected after stimulation with a PPAR伪 agonist in the presence or absence of IL-2. Statistical analysis of OX40+ cells proportion stimulation with IL-2 relative to the proportion without IL-2 in each group, as determined by flow cytometry. c Relative mRNA levels of anti-apoptosis genes (Bcl-2, Bcl-xl, and Survivin) and pro-apoptosis genes (Bcl2l11) of cDNT after incubation with a PPAR伪 agonist with or without IL-2. Statistical analysis of the genes expression stimulation with IL-2 relative to the genes without IL-2 in each group was determined. d Apoptosis of cDNT (Annexin V+) was detected after stimulation with a PPAR伪 agonist with or without IL-2 by flow cytometry. e The proliferation of cDNT (EdU+) was detected after stimulation with a PPAR伪 agonist with or without IL-2 by flow cytometry. The data are presented as the mean鈥壜扁€塖D, n鈥?鈥? in each group. *p鈥?lt;鈥?.05, **p鈥?lt;鈥?.01, NS no significanceFull size imageTaken together, these results suggested that IL-2 promoted OX40 expression by downregulating the PPAR伪 expression, leading to the elevated expression of Bcl-2, Bcl-xL, and Survivin in cDNT, which finally resulted in promoted proliferation and decreased apoptosis.DiscussionOX40 is induced primarily during T-cell activation and plays an important role in the survival and proliferation of T cells26,27. OX40 is mainly expressed on activated T cells and is preferentially expressed on CD4+ T cells, although activated CD8+ T cells also express OX40, albeit at lower levels24. OX40 is also highly expressed on both natural and induced Foxp3+ Tregs; however, in contrast to its costimulatory role to T effector cells, OX40 is a rather potent negative regulator of Foxp3+ Tregs25,28. OX40 costimulation did not influenced the survival of the Foxp3+ Tregs significantly, but markedly inhibited Foxp3 gene expression25.In this study, we have demonstrated for the first time that OX40 is highly expressed on cDNT; moreover, similar to its costimulatory role to activated T effector cells, unlike Foxp3+ Tregs, OX40 is also a potent regulator on cDNT proliferation and survival. OX40-deficient cDNT had a higher apoptotic rate and lower proliferation rate than WT cDNT. OX40 participated into cDNT survival regulation by inducting the expression of apoptotic-related proteins Bcl-2, Bcl-xL, and Survivin, thus suppressing the expression of pro-apoptotic gene BCL2L11.OX40 signaling can target the canonical NF-魏B (NF-魏B1) pathway in peripheral antigen-responding CD4 T cells24,25. In this study, we also identified significantly lower phosphorylation events of IKK伪/尾, I魏B伪, RelA (p65) expression, and p50, RelA nuclear import in OX40-deficient cDNT accompanied by the changes of Bcl-2 and Bcl-xL indicating that OX40 might promote the survival of cDNT by regulating the expression of Bcl-2 and Bcl-xL via NF-魏B cell signaling. OX40 signals also controls Survivin expression of cDNT. Survivin, a member of the inhibitor of apoptosis family, was found to bind the active forms of the executioner proteases caspase 3 and 7, but not the upstream initiator caspase 8, and this inhibited apoptosis induced by overexpression of procaspase 3 and 7. Moreover, cotransfection of Survivin prevented spontaneous processing of caspase 7 and caspase 3 to their active forms29. In CD4+ T cells, blocking Survivin suppressed S-phase transition and division of T cells and led to apoptosis21. Converted DNT highly that expressed Survivin may also have contributed to cDNT proliferation and anti-apoptosis.Research over the past decade has definitively shown the importance of OX40鈥?i>OX40L interactions in development of immune-mediated disease24, based on that the OX40鈥?i>OX40L checkpoint inhibition may have potential clinical application in the treatment in autoimmune diseases. However, because anti-OX40/OX40L has complicated effects on different cell types, the use of OX40鈥?i>OX40L checkpoint inhibition should be cautious, especially in the combination with cDNT-based cell therapy.IL-2 plays an unequivocal role in priming activated T cells to undergo apoptotic cell death, which serves as a critical feedback regulator of clonal expansion30; however, accumulating evidence suggests that IL-2 is essential for the activation and suppressive function of Foxp3+ Tregs31,32,33 and is sufficient to induce proliferation of Foxp3+ Tregs. Converted DNT were hypo-responsive when stimulated by mature DCs, and IL-2 promoted the expression of OX40 in cDNT. Meanwhile, IL-2 completely restored their responsiveness, and enhanced cDNT proliferation. In addition, IL-2 increased the resistance of cDNT to apoptosis. However, the regulation of IL-2 on cDNT proliferation and apoptosis was impaired when OX40 in cDNT was knocked out. Furthermore, the mRNA expression of Bcl-2, Bcl-xL, and Survivin was reduced in OX40-deficient cDNT, even in the presence of IL-2, whereas mRNA expression of BCL2L11 was augmented. These data suggested that IL-2 promoted the survival of cDNT in part via elevation of the expression of the OX40 molecule.The regulation of IL-2 on OX40 upregulation of cDNT is unknown. In this study, we provide evidence that this regulation is PPAR伪-dependent. Promoter-binding TF profiling arrays revealed that a possible presence of binding site for PPAR伪 and PPAR纬 within the OX40 promoter. The ChIP assay further supported that PPAR伪 but not PPAR纬 is able to bind to the OX40 promoter and involves in the negative regulation of OX40 expression in cDNT. Furthermore, OX40 expression of cDNT were significantly augmented in the presence of rIL-2 stimulation. In contrast, the OX40 expression of cDNT with rIL-2 stimulation in vitro was reduced when a PPAR伪 agonist was presented.The PPARs are members of the nuclear-hormone-receptor superfamily; they transduce a wide variety of signals, including environmental, nutritional, and inflammatory events. Genetic ablation of PPAR伪 or PPAR纬 on CD4 T cells results in higher production of interferon 纬 and IL-2 through PPAR-mediated transrepression activities34. In this study, we found that the administration of IL-2 effectively promoted OX40 expression in cDNT through a PPAR伪-dependent process and consequently enhanced Bcl-2, Bcl-xL, and Survivin expression, and finally enhanced survival of cDNT. However, how IL-2 affects PPAR伪 activities needs to be further studied.In conclusion, we elucidated that OX40 is a key molecule that controls CD4 T-cell-converted DNT proliferation and apoptosis by upregulation of Survivin, Bcl-2, and Bcl-xL, and downregulation of pro-apoptotic gene BCL2L11. IL-2 promotes cDNT proliferation and resistance to apoptosis in part by upregulation of OX40. This regulation of IL-2 on OX40 expression in cDNT is controlled by PPAR伪. These Snew findings may have important implications in cDNT-based cell therapy for the future.Materials and MethodsMiceMale C57BL/6 (H-2b) mice, C57BL/6 OX40 KO (H-2b) mice, DBA/2 (H-2d) mice, and B6D2F1 (H-2b/d) mice were obtained from the Jackson Laboratory (Bar Harbor, ME, USA). The mice were maintained in a pathogen-free, temperature-controlled environment under a 12-h light/dark cycle at the Beijing Friendship Hospital, and all animal protocols were approved by the Institutional Animal Care and Ethics Committee.Antibodies and reagentsRecombinant mouse IL-2 (rmIL-2) and granulocyte-macrophage colony-stimulating factor were purchased from PeproTech (Rocky Hill, NJ, USA). Mouse IL-2/Fc fusion protein (a long-lasting form of IL-2) was obtained from Jiangsu Futai Biotechnology (Jiangsu, China). Fluorochrome-conjugated antibodies against mouse CD3, CD4, CD8, NK1.1, TCR纬未, CD86, OX40, Bcl-2, H2Dd, isotype controls, and BrdU staining kits were obtained from eBioscience (San Diego, CA, USA). Anti-mouse PE-Bcl-xL and Survivin were obtained from CST (Boston, MA, USA). Anti-mouse p105/p50, p65, and IKB alpha (phosphor S36) were obtained from Abcam (Cambridge, MA, USA), while anti-mouse phosphor-IKK伪/尾 (Ser176/180) and Histone H3 were obtained from CST. Purified anti-CD3, CD28 antibodies, and Annexin V-PE antibodies were purchased from BD Pharmingen (San Diego, CA, USA). Paraformaldehyde, saponin, Triton X-100, and PPAR伪 agonist (WY-14643) were obtained from Sigma (St. Louis, MO, USA). A mouse T-cell enrichment column was purchased from R D Systems (Minneapolis, MN). Anti-PE microbeads, and magnetic bead separation columns were obtained from Miltenyi Biotec (Auburn, CA). The ChIP assay kit was from Millipore (Bedford, MA, USA).Conversion of DNT in vitroThe generation of CD4 T cells converted to DNT was described previously3. Briefly, CD4+CD25鈭?/sup> T cells were isolated from spleens and lymph nodes of C57BL/6 mice or C57BL/6 OX40 KO mice. The purified CD4+CD25鈭?/sup> T cells were then cultured with DBA/2 mature DCs and rmIL-2 (50鈥塶g/ml) for 7 days. Converted DNT were sorted from culture through a fluorescence-activated cell sorter (FACS Aria II; BD Biosciences, San Diego, CA, USA).Re-stimulation of cDNT in vitroConverted DNT and OX40 KO DNT (2鈥壝椻€?05/well) were re-stimulated with 5鈥壩糶/ml anti-CD3 and 2鈥壩糶/ml anti-CD28 with or without rmIL-2 (50鈥塶g/ml) in a 96-well flat-bottom culture plates for 3 days. EdU (RiboBio, Guangzhou, China) was added to the plates 12鈥塰 before harvest (final concentrations were 50鈥壩糓). Cell proliferation was measured via EdU incorporation according to the manufacturer鈥檚 instructions (EdU staining kit, RiboBio Corporation, Guangzhou, China).Re-stimulation of cDNT in vivoIn all, 5鈥壝椻€?06 converted DNT or OX40 KO DNT were adoptively transferred into B6D2F1 recipient mice by tail vein injection. IL-2/Fc (1鈥壜礸/day) and BrdU (100鈥壜礸/day) were administered by intraperitoneal injection. Splenocytes were isolated on day 3, cDNT proliferation was measured via BrdU incorporation according to the manufacturer鈥檚 instructions (eBioscience).Alamar Blue assayConverted DNT and OX40 KO DNT (2鈥壝椻€?05/well) were re-stimulated with 5鈥壩糶/ml anti-CD3 and 2鈥壩糶/ml anti-CD28 with or without rmIL-2 (50鈥塶g/ml) in 96-well flat-bottom culture plates for 5 days. A 1/10th volume of Alamar Blue庐 reagent was added directly to cells in culture medium, and they were incubated at 37鈥壜癈 in a cell culture incubator, protected from direct light. The absorbance of Alamar Blue庐 at 570鈥塶m was measured using 600鈥塶m as a reference wavelength (normalized to the 600鈥塶m value) at different time points.Caspase 3/7 activationCaspase 3/7 activities were measured using the Caspase-Glo 3/7 Assay (Promega, USA). Briefly, the converted DNT and OX40 KO DNT (2鈥壝椻€?05/well) were re-stimulated with 5鈥壩糶/ml anti-CD3 and 2鈥壩糶/ml anti-CD28 with or without rmIL-2 (50鈥塶g/ml) in 96-well flat-bottom culture plates for 24, 48, and 72鈥塰. The caspase 3/7 reagent was then added to each well at a different time point, and the plate was incubated on a rotary shaker for 30鈥塵in at room temperature. Luminescence was recorded for each well. The caspase 3/7 activity is presented as the mean of the results from the three experiments.Quantitative real-time PCRTotal RNA was extracted from cells using an RNeasy mini-kit (Qiagen, Valencia, CA, USA) and reverse transcribed into cDNA with the SuperScript III RT-kit (Invitrogen, Carlsbad, CA, USA). Real-time quantitative polymerase chain reaction was performed with the Power SYBR Green master mix (Applied Biosystems, Foster City, CA, USA) and gene amplification was performed on the ABI 7500 Sequence Detection System (Applied Biosystems). Gene-specific primers used for specific genes and GAPDH are shown in Supplementary Table聽1. The relative gene expression was quantitatively analyzed by the comparative Ct method (2鈭捨斘擟T). Data were normalized to the levels of GAPDH mRNA.Western blotNuclear protein extracts from B6 cDNT and OX40 KO cDNT were prepared with NE-PER庐 Nuclear and Cytoplasmic Extraction reagents (Thermo Fisher Scientific, Inc., Pittsburgh, PA, USA). After determination of concentration, the samples containing 20鈥壩糶 protein were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (Millipore). Primary antibodies against p50/p105 (diluted 1:2000), p65 (RelA, diluted 1:2000), and Histone H3 (diluted 1:5000) were used. The membranes were incubated with secondary antibodies conjugated to horseradish peroxidase (HRP), and the proteins were detected by enhanced chemiluminescence (Thermo). The relative density of the protein bands was quantitatively determined using ImageJ software (National Institutes of Health, Bethesda, MD, USA).Promoter-binding TF profiling array assayTo examine the potential TFs that bind to the OX40 core promoter region (鈭?032/+28 region) in cDNT, TF activation profiling plate array kits were used (Signosis, CA, USA). The assay was performed according to the kit鈥檚 instructions. First, 15鈥壩糽 TF-binding buffer, 3鈥壩糽 TF probe, 10鈥壩糶 cDNT nuclear extract, and 5鈥壩糽 OX40 promoter PCR fragment were mixed in a tube, and incubated at 20鈥?3鈥壜癈 for 30鈥塵in to form TF/DNA complex. Separation of TF/DNA complex from free probes. Then eluted the bound probe from the complex, and incubated the probe with hybridization plate overnight at 42鈥壜癈. After washing with hybridization wash buffer, streptavidin-HRP conjugate were added to each well, which contained the bound probe, and incubated for 45鈥塵in gently shaking at room temperature. Finally, incubated with substrate solution for 1鈥塵in and detected the probe relative light units on a microplate luminometer (Wallac 1450, Wallac, MA, USA).Chromatin immunoprecipitation assayThe chromatin immunoprecipitation (ChIP) assay was performed according to the kit鈥檚 instructions (Millipore). After treatment with mouse IL-2 (50鈥塶g/ml) for 48鈥塰, the cDNT were fixed, sonicated, and collected for ChIP assay. DNA fragments were immunoprecipitated with antibodies specific to PPAR伪, PPAR纬, or control rabbit IgG at 4鈥壜癈 overnight. Subsequently, the DNA fragments were de-crosslinked, purified from the complexes, and ethanol-precipitated. The immunoprecipitated chromatin was amplified by primers (Supplementary Table聽1) corresponding to specific regions of the OX40 genomic locus. Meanwhile, the immunoprecipitated DNA fragments were detected by quantitative real-time. To calculate the fold enrichment of the precipitated PPAR element, each sample was normalized to the corresponding input. All ChIP assays were performed in triplicate.Flow cytometric analysisAll samples were acquired on a FACS Aria II flow cytometer (BD Biosciences), and the data were analyzed using FlowJo software (TreeStar, Ashland, OR, USA).Statistical analysisStatistical analysis was performed using the Prism 5.0 software (GraphPad Software, San Diego, CA, USA). The values are expressed as the mean鈥壜扁€塖D. Analyses for significant differences were performed using Student鈥檚 t test and one-way analysis of variance. p Values鈥?lt;鈥?.05 were considered significant. References1.Juvet, S. C. Zhang, L. Double negative regulatory T cells in transplantation and autoimmunity: recent progress and future directions. J. Mol. Cell Biol. 4, 48鈥?8 (2012).Article聽 PubMed聽 PubMed Central聽 CAS聽Google Scholar聽 2.Hillhouse, E. E. Lesage, S. A comprehensive review of the phenotype and function of antigen-specific immunoregulatory double negative T cells. J. Autoimmun. 40, 58鈥?5 (2013).Article聽 PubMed聽 CAS聽Google Scholar聽 3.Zhang, D. et al. New differentiation pathway for double-negative regulatory T cells that regulates the magnitude of immune responses. Blood 109, 4071鈥?079 (2007).Article聽 PubMed聽 PubMed Central聽 CAS聽Google Scholar聽 4.Zhang, Z. X. et al. Double-negative T cells, activated by xenoantigen, lyse autologous B and T cells using a perforin/granzyme-dependent, Fas-Fas ligand-independent pathway. J. Immunol. 177, 6920鈥?929 (2006).Article聽 PubMed聽 CAS聽Google Scholar聽 5.Chen, W., Diao, J., Stepkowski, S. M. Zhang, L. Both infiltrating regulatory T cells and insufficient antigen presentation are involved in long-term cardiac xenograft survival. J. Immunol. 179, 1542鈥?548 (2007).Article聽 PubMed聽 CAS聽Google Scholar聽 6.Zhang, D. et al. Adoptive cell therapy using antigen-specific CD4鈭扖D8鈭?T regulatory cells to prevent autoimmune diabetes and promote islet allograft survival in NOD mice. Diabetologia 54, 2082鈥?092 (2011).Article聽 PubMed聽 CAS聽Google Scholar聽 7.Li, W. et al. Ex vivo converted double negative T cells suppress activated B cells. Int. Immunopharmacol. 20, 164鈥?69 (2014).Article聽 PubMed聽 CAS聽Google Scholar聽 8.Gao, J. F. et al. Regulation of antigen-expressing dendritic cells by double negative regulatory T cells. Eur. J. Immunol. 41, 2699鈥?708 (2011).Article聽 PubMed聽 CAS聽Google Scholar聽 9.He, K. M. et al. Donor double-negative Treg promote allogeneic mixed chimerism and tolerance. Eur. J. Immunol. 37, 3455鈥?466 (2007).Article聽 PubMed聽 CAS聽Google Scholar聽 10.Zhang, Z. X., Yang, L., Young, K. J., DuTemple, B. Zhang, L. Identification of a previously unknown antigen-specific regulatory T cell and its mechanism of suppression. Nat. Med. 6, 782鈥?89 (2000).Article聽 PubMed聽 CAS聽Google Scholar聽 11.Young, K. J., DuTemple, B., Phillips, M. J. Zhang, L. Inhibition of graft-versus-host disease by double-negative regulatory T cells. J. Immunol. 171, 134鈥?41 (2003).Article聽 PubMed聽 CAS聽Google Scholar聽 12.McIver, Z. et al. Double-negative regulatory T cells induce allotolerance when expanded after allogeneic haematopoietic stem cell transplantation. Br. J. Haematol. 141, 170鈥?78 (2008).Article聽 PubMed聽 CAS聽Google Scholar聽 13.Ford, M. S. et al. Peptide-activated double-negative T cells can prevent autoimmune type-1 diabetes development. Eur. J. Immunol. 37, 2234鈥?241 (2007).Article聽 PubMed聽 CAS聽Google Scholar聽 14.Zhao, X. et al. A novel differentiation pathway from CD4+T cells to CD4- T cells for maintaining immune system homeostasis. Cell Death Dis. 7, e2193 (2016).Article聽 PubMed聽 PubMed Central聽 CAS聽Google Scholar聽 15.Toennies, H. M., Green, J. M. Arch, R. H. Expression of CD30 and Ox40 on T lymphocyte subsets is controlled by distinct regulatory mechanisms. J. Leukoc. Biol. 75, 350鈥?57 (2004).Article聽 PubMed聽 CAS聽Google Scholar聽 16.Liu, T. et al. Combination of double negative T cells and anti-thymocyte serum reverses type 1 diabetes in NOD mice. J. Transl. Med. 14, 57 (2016).Article聽 PubMed聽 PubMed Central聽 CAS聽Google Scholar聽 17.Cong, M. et al. Interleukin-2 enhances the regulatory functions of CD4(+)T cell-derived CD4(-)CD8(-) double negative T cells. J. Interferon Cytokine Res. 36, 499鈥?05 (2016).Article聽 PubMed聽 CAS聽Google Scholar聽 18.Redmond, W. L., Triplett, T., Floyd, K. Weinberg, A. D. Dual anti-OX40/IL-2 therapy augments tumor immunotherapy via IL-2R-mediated regulation of OX40 expression. PLoS ONE 7, e34467 (2012).Article聽 PubMed聽 PubMed Central聽 CAS聽Google Scholar聽 19.Vu, M. D. et al. OX40 costimulation turns off Foxp3(+) tregs. Blood 110, 2501鈥?510 (2007).Article聽 PubMed聽 PubMed Central聽 CAS聽Google Scholar聽 20.Rogers, P. R., Song, J., Gramaglia, I., Killeen, N. Croft, M. OX40 promotes Bcl-xL and Bcl-2 expression and is essential for long-term survival of CD4 T cells. Immunity 15, 445鈥?55 (2001).Article聽 PubMed聽 CAS聽Google Scholar聽 21.Song, J., So, T., Cheng, M., Tang, X. Croft, M. Sustained survivin expression from OX40 costimulatory signals drives T cell clonal expansion. Immunity 22, 621鈥?31 (2005).Article聽 PubMed聽 CAS聽Google Scholar聽 22.Bansal-Pakala, P., Halteman, B. S., Cheng, M. H. Croft, M. Costimulation of CD8 T cell responses by OX40. J. Immunol. 172, 4821鈥?825 (2004).Article聽 PubMed聽 CAS聽Google Scholar聽 23.Baumann, R. et al. Functional expression of CD134 by neutrophils. Eur. J. Immunol. 34, 2268鈥?275 (2004).Article聽 PubMed聽 CAS聽Google Scholar聽 24.Croft, M. Control of immunity by the TNFR-related molecule OX40 (CD134). Annu. Rev. Immunol. 28, 57鈥?8 (2010).Article聽 PubMed聽 PubMed Central聽 CAS聽Google Scholar聽 25.Song, J., So, T. Croft, M. Activation of NF-kappaB1 by OX40 contributes to antigen-driven T cell expansion and survival. J. Immunol. 180, 7240鈥?248 (2008).Article聽 PubMed聽 PubMed Central聽 CAS聽Google Scholar聽 26.Calderhead, D. M. et al. Cloning of mouse Ox40: a T cell activation marker that may mediate T-B cell interactions. J. Immunol. 151, 5261鈥?271 (1993).PubMed聽 CAS聽Google Scholar聽 27.Sugamura, K., Ishii, N. Weinberg, A. D. Therapeutic targeting of the effector T-cell co-stimulatory molecule OX40. Nat. Rev. Immunol. 4, 420鈥?31 (2004).Article聽 PubMed聽 CAS聽Google Scholar聽 28.Valzasina, B. et al. Triggering of OX40 (CD134) on CD4(+)CD25+T cells blocks their inhibitory activity: a novel regulatory role for OX40 and its comparison with GITR. Blood 105, 2845鈥?851 (2005).Article聽 PubMed聽 CAS聽Google Scholar聽 29.Tamm, I. et al. IAP-family protein survivin inhibits caspase activity and apoptosis induced by Fas (CD95), Bax, caspases, and anticancer drugs. Cancer Res. 58, 5315鈥?320 (1998).PubMed聽 CAS聽Google Scholar聽 30.Lenardo, K. M. Interleukin-2 programs mouse ab T lymphocytes for apoptosis. Nature 353, 858鈥?61 (1991).Article聽 PubMed聽 CAS聽Google Scholar聽 31.de la Rosa, M., Rutz, S., Dorninger, H. Scheffold, A. Interleukin-2 is essential for CD4鈥?鈥塁D25鈥?鈥塺egulatory T cell function. Eur. J. Immunol. 34, 2480鈥?488 (2004).Article聽 PubMed聽 CAS聽Google Scholar聽 32.Kang, H. G. et al. Effects of cyclosporine on transplant tolerance: the role of IL-2. Am. J. Transplant. 7, 1907鈥?916 (2007).Article聽 PubMed聽 CAS聽Google Scholar聽 33.Mahmud, S. A., Manlove, L. S. Farrar, M. A. Interleukin-2 and STAT5 in regulatory T cell development and function. JAKSTAT 2, e23154 (2013).PubMed聽 PubMed Central聽Google Scholar聽 34.Daynes, R. A. Jones, D. C. Emerging roles of PPARs in inflammation and immunity. Nat. Rev. Immunol. 2, 748鈥?59 (2002).Article聽 PubMed聽 CAS聽Google Scholar聽 Download referencesAcknowledgementsThis work was supported by grants from the National Natural Science Foundation of China (No. 81570510, 81500598, and 81501379), the Natural Science Foundation of Beijing Municipality (No. 7172060), and Open project from Beijing Key Laboratory of Translational Medicine in Liver Cirrhosis.Author informationAuthor notesThese authors contributed equally: Guangyong Sun, Xiaojing Sun, Wei LiAffiliationsExperimental and Translational Research Center, Beijing Friendship Hospital, Capital Medical University, Beijing, 100050, ChinaGuangyong Sun,聽Xiaojing Sun,聽Kai Liu,聽Dan Tian,聽Yiran Dong,聽Hufeng Xu聽 聽Dong ZhangBeijing Clinical Research Institute, Beijing, 100050, ChinaGuangyong Sun,聽Xiaojing Sun,聽Kai Liu,聽Dan Tian,聽Yiran Dong,聽Hufeng Xu聽 聽Dong ZhangBeijing Key Laboratory of Tolerance Induction and Organ Protection in Transplantation, Beijing, 100050, ChinaGuangyong Sun,聽Xiaojing Sun,聽Kai Liu,聽Dan Tian,聽Yiran Dong,聽Hufeng Xu聽 聽Dong ZhangNational Clinical Research Center for Digestive Diseases, Beijing, 100050, ChinaWei Li聽 聽Dong ZhangDepartment of Emergency Medicine, Beijing Friendship Hospital, Capital Medical University, Beijing, 100050, ChinaXuelian SunAuthorsGuangyong SunView author publicationsYou can also search for this author in PubMed聽Google ScholarXiaojing SunView author publicationsYou can also search for this author in PubMed聽Google ScholarWei LiView author publicationsYou can also search for this author in PubMed聽Google ScholarKai LiuView author publicationsYou can also search for this author in PubMed聽Google ScholarDan TianView author publicationsYou can also search for this author in PubMed聽Google ScholarYiran DongView author publicationsYou can also search for this author in PubMed聽Google ScholarXuelian SunView author publicationsYou can also search for this author in PubMed聽Google ScholarHufeng XuView author publicationsYou can also search for this author in PubMed聽Google ScholarDong ZhangView author publicationsYou can also search for this author in PubMed聽Google ScholarContributionsAll listed authors participated meaningfully in the study and that they have seen and approved the submission of this manuscript. G.S., X.S., and W.L. participated in performing the research, analyzing the data, and initiating the original draft of the article. K.L., D.T., Y.D., and X.S. participated in performing the research and collecting the data. D.Z. and H.X. established the hypotheses, supervised the studies, analyzed the data, and co-wrote the manuscript.Corresponding authorsCorrespondence to Hufeng Xu or Dong Zhang.Ethics declarations Conflict of interest The authors declare that they have no conflict of interest. Additional informationPublisher\'s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Edited by M. HeroldElectronic supplementary material