IL-15和树突状细胞诱导CD4~+CD25~+调节性T细胞增殖的实验研究
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摘要
目前,临床器官移植取得了显著的进步,器官移植后短期(1年)存活率达到90%以上;但是,长期存活率仍不理想。其原因主要是由于慢性排斥反应以及非特异性的免疫抑制药物的毒副作用引起。如何让器官移植受者在不使用或少使用免疫抑制药物的前提下,对移植器官不产生免疫识别和/或免疫攻击,也就是特异性针对移植器官免疫耐受,是目前移植医学难题之一。在移植免疫耐受机制的研究中,CD4~+CD25~+调节性T细胞(Tregs)的作用越来越受到大家的重视。Tregs是一类高表达CD25和叉头/翼形螺旋状转录因子(Foxp3)的CD4~+T细胞,具有很强的免疫抑制功能。研究表明,输注Tregs可控制移植物抗宿主病和移植排斥反应的发生。然而,体内天然产生的Tregs在正常生理状态下,仅占外周血单个核细胞数量的1%,与效应T细胞不同,它们被T细胞表面受体(TCR)激活后仍保持无能状态,极低增殖;而且目前尚无有效的手段诱导体内Tregs大量扩增。显然,为了达到治疗目的,必须在体外大量扩增Tregs再输回患者体内。如何扩增Tregs以用于诱导器官移植免疫耐受,是目前移植医学研究的热点问题之一。
白介素15(IL-15)是1994年由Burton和Grabstein同时发现的一种细胞因子。由于IL-15与IL-2结构中均含4α螺旋结构, IL-15与IL-2一起共享IL-2受体β和γ链(IL-2Rβγ/γc),有学者将IL-15归类于IL-2细胞因子家族,二者对T细胞作用后的许多效应有相同之处。不同在于IL-15主要以与IL-15Rα链高亲和力结合状态存在,即使缺乏IL-2Rβ和IL-2Rγ/γc受体亚单位,IL-15与其受体α私有链仍有着高度的亲和力(Ka≥1011M-1);而在缺乏IL-2Rβγ时,IL-2与IL-2Rα的亲和力却很低(Ka～108M-1)。体外实验表明,IL-2扩增Tregs的能力优于IL-15,然而IL-2或者IL-15缺陷小鼠体内Tregs数量和功能轻度下降或正常,只有当二者均缺陷时才会出现Tregs数量和功能显著下降。表明IL-15在Tregs的扩增中同样发挥重要作用,但IL-15是如何发挥作用还有待于进一步研究。
Rachael等将IL-15、表皮成纤维细胞各自单独与Tregs培养时不能增殖Tregs,但将二者联合后却能显著增殖Tregs,提示IL-15在其他细胞存在的条件下才能发挥增殖Tregs的作用。既往研究表明IL-15联合表皮成纤维细胞促进活化的T细胞增殖时主要是由表皮成纤维细胞以膜结合的形式向T细胞递呈IL-15。在免疫反应的启动中,树突状细胞(DC)是功能最强的激活初始T细胞的抗原递呈细胞,与表皮成纤维细胞一样,DC也能以膜结合的形式向靶细胞递呈IL-15。DC是否同样可以以膜结合的形式向Tregs细胞递呈IL-15而诱导Tregs增殖尚不清楚。因此,本研究从人外周血中分离出Tregs,随后将IL-15、DC单独或联合干预Tregs的生长,从分子水平探讨IL-15和DC对Tregs的作用及机制,为体外更好地扩增Tregs以得到足够数量的Tregs用于诱导器官移植免疫耐受奠定实验基础。
我们将本研究主要方法、获得的主要结果和结论归纳如下:
一、树突状细胞与CD4~+CD25~+调节性T细胞的分离及鉴定。
1.人外周血贴壁的单核细胞经GM-CSF~+IL-4联合诱导,于第6天负载同种异体抗原,培养第7天收集获得的细胞,光镜、电镜观察细胞的形态;流式细胞仪检测细胞表面标志物;将所得细胞经丝裂霉素灭活后与自体CD4~+CD25-T细胞混合淋巴细胞培养72h,3H-TdR掺入法检测CD4~+CD25-T细胞的增殖。结果显示,所得细胞逐渐由贴壁变成悬浮,细胞形态不规则,表面粗糙,有大量层叠状皱襞和毛刺状突起,表达共刺激分子CD80、CD86,MLR显示能刺激自体CD4~+CD25-T细胞增殖。表明所得细胞具有DC的细胞形态、免疫表型及功能特性,说明实验中建立的分离培养DC的方法是可行的。
2.人外周血单个核细胞经免疫磁珠阴性分选和阳性分选,将获得的细胞经台盼蓝染色检测细胞的活力;流式细胞仪检测细胞的纯度和细胞表型;将所得细胞经丝裂霉素灭活后与自体CD4~+CD25-T细胞混合淋巴细胞培养,包被抗CD3/CD28抗体的Dynal磁珠刺激72h,3H-TdR掺入法检测CD4~+CD25-T细胞的增殖。结果显示所得细胞活力为(95. 7±2.1) %,纯度为95.2%,胞内因子Foxp3在Tregs细胞的表达率为94.2%,在体外能抑制致敏T淋巴细胞增殖。表明所得细胞具有Tregs的细胞表型及功能特性,说明本实验建立的方法可以在体外分离出Tregs,为进一步研究Tregs打下基础。
二、IL-15和树突状细胞诱导增殖后CD4~+CD25~+调节性T细胞的表型与功能
1.Tregs按1×104/孔置于U型底96孔板中,依据每孔加入的DC(1×104/孔,经丝裂霉素灭活)或包被抗CD3/CD28抗体的Dynal磁珠(1×104磁珠/孔)不同分为四大组:负载同种异体抗原的DC~+IL-2组(简称DC~+IL-2组)、负载同种异体抗原的DC~+IL-15组(简称DC~+IL-15组)、包被抗CD3/CD28抗体的Dynal磁珠~+IL-2组(简称CD3/CD28~+IL-2组)和包被抗CD3/CD28抗体的Dynal磁珠~+IL-15组(简称CD3/CD28~+IL-15组),各组内依据细胞因子浓度不同分为0、25U/ml、50U/ml、100U/ml、200U/ml等小组,各小组3复孔。培养第5天,3H-TdR掺入法检测Tregs增殖。结果显示在包被抗CD3/CD28抗体的Dynal磁珠刺激下,IL-15在0---100U/ml的浓度范围内,基本不引起Tregs增殖,至200U/ml浓度时,可引起Tregs轻度增殖;而在包被抗CD3/CD28抗体的Dynal磁珠刺激下,IL-2在25U/ml浓度时即可引起Tregs显著性增殖(较同等浓度的IL-15);在负载同种异体抗原的DC刺激下,IL-15和IL-2一样,在0---200U/ml的浓度范围内,成剂量依赖性诱导Tregs增殖,且在100U/ml和200U/ml浓度时,二者诱导Tregs增殖的幅度无显著性差异。说明在DC存在的情况下,IL-15也能诱导Tregs增殖。
2.从培养体系中免疫磁珠阳性分选出CD4~+T细胞,流式细胞仪检测该细胞的纯度、细胞表型和CD62L;3H-TdR掺入法检测该细胞对CD4~+CD25-T细胞的增殖的抑制作用。结果显示,DC跨细胞递呈IL-15增殖的Tregs纯度高,仍具有典型的CD4~+CD25~+Foxp3~+表型特征,在体外对特异性同种异体抗原激活的Teff增殖的抑制功能强,对第三方同种异体抗原激活的Teff的免疫抑制功能弱,且Tregs高表达CD62L。说明DC跨细胞递呈IL-15增殖的Tregs具有天然Tregs的细胞表型,具有的特异性抑制作用,具备到达特定的作用部位发挥作用的能力。
三、IL-15和树突状细胞诱导CD4~+CD25~+调节性T细胞增殖的作用机制
1.将Tregs和DC分别与浓度为100U/ml的IL-15在37℃细胞培养温箱中孵育15分钟后流式细胞仪检测细胞表面IL-15,而细胞表面IL-15Rα则直接检测。结果显示,Tregs细胞表面IL-15Rα的表达极低,基本上未在Tregs细胞表面检测到结合的IL-15;而DC细胞表面IL-15Rα的表达较高,DC表面结合的IL-15远远高于在Tregs细胞表面结合的IL-15(p<0.05)。表明Tregs直接结合IL-15的能力较低,而DC直接结合IL-15的能力较高,特殊ELISA证实DC细胞表面确实形成了IL-15-IL-15Rα复合物。
2.将负载同种异体抗原的DC与Tregs按1:1比例混合培养,培养液中加入IL-15 100U/ml,24h后收集混合培养的细胞于离心管中,分别用PE-抗CD86和FITC-抗CD4标记DC和Tregs。激光共聚焦显示将DC、Tregs与IL-15共培养后,DC与Tregs间形成稳定的细胞团,这些提示DC和IL-15可能通过由DC跨细胞递呈IL-15给Tregs而诱导Tregs增殖。
3.DC和Tregs各以1×105/孔共同培养于平底96孔板,依据是否加入Anti-IL-15Rα不同将实验分为对照组和阻断组:两组均加入100U/ml的IL-15;不同之处在于阻断组在加入IL-15之前先加入40ug/ml的Anti-IL-15Rα,而对照组加入等体积的PBS。两组均在培养的第5天收集细胞、匀浆,特殊ELISA检测IL-15-IL-15Rα复合物。Tregs和经丝裂霉素灭活的DC各以1×104/孔共同培养于U型底96孔板,实验分组及处理同前。在培养的第5天收集细胞,3H-TdR掺入法检测Tregs增殖。结果显示,用IL-15Rα抗体预处理DC后再与与IL-15孵育, DC细胞表面IL-15-IL-15Rα复合物下降,此时Tregs增殖亦显著减少。从反面证实DC和IL-15主要是通过由DC跨细胞递呈IL-15给Tregs而诱导Tregs增殖。
4.在不同时间点检测DC表面IL-15:将DC置于含100U/ml的IL-15的培养液中培养4天,流式细胞仪检测DC表面IL-15;收集细胞,在甘氨酸缓冲液(PH=3)孵育,10分钟后检测DC表面IL-15;再将DC重新培养于无细胞因子的培养液中继续培养24h,流式细胞仪检测DC表面IL-15。DC和Tregs各以各1×105/孔共同培养于平底96孔板,依据处理方式不同将实验分为三组:对照组、维持组、撤退组。三组中对照组不加IL-15;维持组和撤退组均加100U/ml的IL-15,但撤退组于共培养的第4天撤退IL-15再培养24h,而维持组不撤退IL-15。三组均在培养的第5天收集细胞、匀浆,特殊ELISA检测IL-15-IL-15Rα复合物。经丝裂霉素灭活的DC和Tregs各以各1×104/孔共同培养于U型底96孔板,同时加入100U/ml的IL-2或IL-15依据是否在细胞培养的第4天撤退细胞因子分为维持组(不撤退)和撤退组。将细胞继续24h,3H-TdR掺入法检测Tregs增殖。结果显示,在培养体系中撤退IL-15后24h流式细胞仪仍能在DC表面检测到IL-15,培养体系中仍有IL-15-IL-15Rα复合物形成,Tregs可以继续增殖。说明IL-15还可在DC跨胞内体再循环,从而使IL-15持续长时间存在于DC细胞表面,继续通过DC跨细胞递呈IL-15给Tregs而诱导Tregs增殖。
5.将Tregs和DC各1×104/孔共同培养于U型底96孔板,依据是否加入浓度为100U/ml的IL-15分两组:实验组加入IL-15,而对照组不加入IL-15。于细胞培养的第1、2、3、4、5天取上清液ELISA检测IL-2浓度。结果显示,对照组IL-2浓度无明显变化,而实验组IL-2浓度显著性较对照组高,于细胞培养的第四天达到顶峰。但是其峰值仅约60pg/ml,从实验中我们可以看出,此浓度下Tregs基本上无明显增殖,表明在培养体系中IL-15确实调节DC分泌IL-2,但此作用在DC和IL-15诱导Tregs增殖的过程中不起主导作用。
6.实验中重悬细胞的培养液含100U/ml的IL-15,将含Tregs总数为4×104的细胞悬液600μl置于24孔板,依据是否使用Transwell小室将实验分为对照组和实验组,每组3复孔:实验组加上滤膜孔径为3.0um的Transwell小室,小室内再加入含DC总数为4×104的细胞悬液100μl;而对照组直接将含经丝裂霉素灭活的总数为4×104的DC细胞悬液100μl加入24孔板,与Tregs共培养。第5天收集24孔板内细胞,台胎盘蓝染色后直接在光镜下计数。结果显示,使用Transwell小室的实验组基本不增殖,而不加Transwell的对照组显著性增殖,p<0.05。表明在IL-15和DC诱导Tregs增殖的过程中,IL-15与DC作用形成的IL-2和sIL-15-IL-15Rα不起主要作用。
7.Tregs以5×104/孔培养于U型底96孔板中,依据加入经丝裂霉素灭活的DC(5×104/孔)和IL-15(100U/ml)不同分为五组:对照组、DC组、IL-15组、联合诱导组和联合诱导阻断组。其中,对照组不加IL-15和DC;DC组仅加DC;IL-15组仅加IL-15;联合诱导组同时加DC和IL-15;联合诱导阻断组先加入加入40ug/ml的Anti-IL-15Rα和DC,然后再加入IL-15。培养24h后收集细胞、提取蛋白。Western-Blot检测CD4~+CD25~+调节性T细胞p-ERK、p-AKT、p-STAT5和P27kip1的表达。结果显示:DC介导的活化能成功诱导Tregs内PI3K的靶分子Akt活化但不能诱导MEK1/2的靶分子Erk1/2的活化,也不能诱导STAT5的活化和p27kip1的降解;而单独加入外源性IL-15后,由于Tregs的IL-15Rα低表达,IL-15并不能显著诱导Tregs的Akt、Erk1/2和STAT5的活化以及p27kip1的降解;只有同时加入DC和IL-15,才能显著诱导Tregs的Akt、Erk1/2和STAT5的活化以及p27kip1的降解,引起Tregs增殖。此外,加入Anti-IL-15Rα后再加入DC和IL-15,Tregs的Akt、Erk1/2和STAT5的活化以及p27kip1的降解被抑制,Tregs增殖下降,进一步说明DC和IL-15主要以跨细胞递呈方式诱导Tregs增殖。
结论
一、成功建立了从人外周血分离培养DC和Tregs的方法。
二、在DC存在的情况下,IL-15也能大量诱导Tregs增殖。DC和IL-15诱导增殖的Tregs纯度高,具有天然Tregs的细胞表型,具有特异性抑制作用,具备到达特定的作用部位发挥作用的能力,有用于临床诱导移植免疫耐受的潜力。
三、DC和IL-15诱导Tregs增殖的作用形式主要是通过由DC跨细胞递呈IL-15给Tregs而诱导Tregs增殖。IL-15还可在DC跨胞内体再循环,从而使IL-15持续长时间存在于DC细胞表面,继续通过DC跨细胞递呈IL-15给Tregs而诱导Tregs增殖。此外,IL-15调节DC分泌IL-2可能在DC和IL-15诱导Tregs增殖的过程中起辅助作用。
四、DC和IL-15诱导Tregs增殖,其分子机制可能是通过Tregs的Akt、Erk1/2和STAT5的活化以及p27kip1的降解。
Clinical organ transplantation has remarkably advanced and is currently a well-established treatment for all sorts of organs’end-stage disease. Short-term survival rates are currently excellent. Unfortunately, long-term survival is still comparatively poor, mainly because of the chronic rejection and the toxicities of nonspecific immunosuppressants. To gain transplantation tolerance between donor organs and hosts is the ultimate goal of all sorts of organ transplantations. Intriguingly, CD4~+CD25~+ regulatory T cells (Tregs), which express a high level of Foxp3, display strong immune suppressive effect. Studies have shown that Tregs transfusion can prevent the graft-versus-host disease (GVHD) and transplantation rejection. Thus, Tregs have been suggested as potential reagents for adoptive cell therapy. However, the amount of naturally occurring Tregs only account for 1% of peripheral blood mononuclear cells, they are narurally anergic and poorly proliferative after TCR stimulation, such that cell numbers are severely limiting. Apparently, Tregs need to be massively expanded in vitro before they could be transfused back to patients and achieve the therapeutic objective. How to expand Tregs for the induction of transplantation tolerance is one of the hot areas where intensive studies are being performed.
Interleukin-15(IL-15) is a pleiotropic cytokine of the 4-α-helix bundle cytokine family, which was discovered by Burton and Grabstein in 1994 due to its ability to mimic IL-2-dependent T cell proliferation. A special feature of IL-15 is that it shares with IL-2, another member of the 4-α-helix bundle cytokine family, the IL-2 receptor beta (IL-2Rβ) and IL-2 receptor gamma/gamma common (IL-2Rγ/γc). IL-15 and IL-2 produce similar effects after their interactions with the T cells. IL-15 mainly binds to IL-15Rαchain in high affinity, and it maintains a high affinity (Ka≥1011M-1) to the private chain of itsαreceptor, even in the absence of IL-2Rβ和IL-2Rγ/γc receptor subunits. In contrast, the affinity of IL-2 and IL-2Rαis very low (Ka～108M-1) in the absence of IL-2Rβγ. In vitro study showed that the ability of IL-2 to simulate Tregs expanding is superior to IL-15. However, IL-2 or IL-15 deficient mice display normal or slightly reduced Tregs amount and function, which are significantly decreased in IL-2 and IL-15 double deficient mice. This indicates that IL-15 plays an equally important role in the expanding of Tregs. Nevertheless, how IL-15 performs its function remains to be addressed by further studies.
Rachael et al. showed that Tregs do not proliferate in the presence of either IL-15 or dermal fibroblasts, but do significantly proliferate when both IL-15 and dermal fibroblasts are present. This indicates that IL-15 can promote Tregs proliferation, but only in the presence of other types of cells. The underlying reason is that dermal fibroblasts present IL-15 to T cells in the form of membrane-bound IL-15. In the initiation of immune reaction, dendritic cells (DC) are the most potent antigen-presenting cells that activate initial T cells. Like dermal fibroblasts, DC can also present IL-15 to target cells as membrane-bound IL-15; however, it remains unclear whether DC and IL-15 can induce Tregs proliferation. For this reason, we isolated Tregs from human peripheral blood, and utilized IL-15, DC, and DC combined with IL-15 to intervene the proliferation of Tregs, and explored the related molecular mechanisms. Our work aims to improve the in vitro proliferation of Tregs and obtain a sufficient number of Tregs, and to lay a foundation for the induction of immune tolerance in organ transplantation. The main results and conclusions of our study are summarized bellow:
A. Isolation and characterization of dendritic cells and CD4~+CD25~+ regulatory T cells
1. Human peripheral adherent monocytes were induced with GM-CSF and IL-4 and loaded with the allogeneic antigen on the 6th day. The cells were harvested on the 7th day. The results showed that the cells obtained gradually converted from adherent cells to suspended cells that exhibit irregular morphology, coarse surface, large amount of laminated folds, and spike-like processes. These cells were found to express co-stimulating molecules such as CD80 and CD86, which, as shown in MLR, can stimulated the proliferation of autologous CD4~+CD25- T cells. These results indicate that the cells that we obtained have the morphology, immunophenotype, and functional characteristics of DCs.
2. Human peripheral blood mononuclear cells were positively and negatively selected with immunomagnetic beads. The results showed that the viability of the cells was 95. 7± 2.1 %,the purity of the cells was 95.2%,and the expression rate of intracellular factor Foxp3 was 94.2%. The cells we obtained can inhibit the proliferation of sensitized T cells in vitro, indicating these cells have the cellular phenotype and functional characteristics of Tregs. Therefore, the method that we developed can be used to isolate Tregs in vitro, and this laid a foundation for further study on Tregs.
B. The proliferation of CD4~+CD25~+ regulatory T cells induced by IL-15 and Dendritic cells
1. Tregs were cultured (1×104cells/well) in U-shaped 96-well plates, and were divided into 4 different groups according to the presence of Dynal beads or DCs (1×104 cells/well, inactivated with mitomycin). The results showed that in the presence of the magnetic beads coated with anti-CD3/CD28 antibody, the proliferation of Tregs was not observed with 0-100 U/ml IL-15, but was slightly observed with 200 U/ml IL-15. In contrast, 25 U/ml IL-2 induced significant Tregs proliferation in the presence the magnetic beads coated with anti-CD3/CD28 antibody. Furthermore, in the presence of the DCs loaded with allogeneic antigen, both IL-15 and IL-2 induced similar extents of Tregs proliferation in a dose-dependent manner within 0-200 U/ml cytokine. The magnitudes of the induction of Tregs proliferation by IL-15 and IL-2 were not statistically significant, when the concentration of the cytokine was within the range of 100-200 U/ml. These results indicate that IL-15 can also induce Tregs proliferation when combined with DCs.
2. CD4~+ T cells were positively screened using immunomagnetic beads, analyzed for purity, cellular phenotype, and CD62L using flow cytometry, and assayed for the inhibitory of the proliferation of CD4~+CD25- regulatory T cells using 3H-TdR incorporation. The results showed that proliferated Tregs induced by IL-15 presented by DC in trans are of high purity, and maintain the typical CD4~+CD25~+Foxp3~+ phenotype. Tregs have strong inhibitory effect in vitro on the proliferation of Teff activated by specific allogeneic antigen, but not that of Teff activated by third-party allogeneic antigen. Tregs express high level of CD62L. These results indicate that proliferated Tregs, which were induced by IL-15 presented in trans by DCs, display the same phenotypes of naturally produced Tregs. In addition, proliferated Tregs display specific inhibitory effect, and have the capability to migrate to the target location and perform specific functions.
C. The related mechanism of the proliferation of CD4~+CD25~+ regulatory T cells induced by IL-15 and Dendritic cells
1. Tregs and DCs were incubated separately with 100 U/ml IL-15 at 37℃for 15 min, and were analyzed for IL-15Rαand cell-surface IL-15 using flow cytometry. The results showed that the level of Tregs-surface IL-15Rαwas extremely low and Tregs-surface IL-15 was not detectable. In contrast, we detected high level of IL-15Rαon the surface of DCs, and the level of IL-15 bound to DCs surface was far higher than that bound to Tregs surface (p < 0.05). This indicates that the ability of Tregs to bind IL-15 is much lower than that of DCs. In addition, the presence of IL-15-IL-15Rαcomplex on DCs surface was confirmed with special ELISA.
2. Tregs were cultured with DCs loaded with allogeneic antigens at 1:1 ratio in the presence of 100 U/ml IL-15. The cell mixture was harvested, and DCs and Tregs in the mixture were labeled respectively with PE-anti-CD86 antibody and FITC-anti-CD4 antibody. Subsequent confocal microscopy showed that DCs and Tregs form stable cell aggregates, indicating that DCs may present IL-15 in trans to Tregs and hence induce the Tregs proliferation.
3. DCs and Tregs were cultured together in plate-bottom 96-well plates in the presence of 100 U/ml IL-15 and divided into 2 groups. The block group was added 40ug/ml anti-IL-15Rαantibody,while the control group was added equal volume of PBS. The results showed that if DCs were blocked by anti-IL-15Rαantibody before they were incubated with IL-15, the concentration of IL-15-IL-15Rαcomplexes on DCs surface decreased, and so was the proliferation of Tregs. These result support the notion that IL-15 was presented in trans by DCs to Tregs and induce Tregs proliferation.
4. Analysis of DC surface IL-15 as a function of time: DCs were cultured in medium containing 100 U/ml IL-15 for 4 days, and then were assayed for surface IL-15 using flow cytometry. The cells continued to be cultured in the medium containing Glycine buffer (pH = 3), and analyzed for surface IL-15 10 min later. The cells were again cultured in medium without cytokines for 24 hr, and analyzed for DC-surface IL-15 using flow cytometry. DCs and Tregs were co-cultured in plate-bottom 96-well plates each at a density of 1×105 cells/well. These cells were then divided into 3 groups. The control group contained no IL-15, while the maintenance group contained 100 U/ml IL-15 throughout the course. In the withdrawal group, IL-15 was removed on the 4th day of the co-culture, and continued to be cultured for 24 hr. The cells of each group were harvested on the 5th day of the culture, homogenated and assayed for IL-15-IL-15Rαcomplexes using special ELISA. In a separate experiment, Tregs were cultured with DCs inactivated with mitomycin in U-shaped 96-well plates each at a density of 1×104 cells/well with 100 U/ml IL-15. The samples were divided into 2 groups. In the withdrawal group, the cytokine (IL-15 or IL-2) was removed on the 4th day, and continued to be cultured for additional 24 hr. The cytokine remained in the medium of the maintenance group. The proliferation of Tregs was quantified using 3H-TdR incorporation. The results showed that IL-15 was detectable on the surface of DCs 24 hours after the removal of IL-15 in the culture system. This indicates the formation of IL-15-IL-15Rαcomplex in the culture system, and thus Tregs could continue to proliferate. This also indicates that IL-15 could be recycled via trans-endosomal recycling, and thus could remain on DC surface for a long period of time. IL-15 could be presented by DCs to Tregs, and induce Tregs proliferation.
5. Tregs were co-cultured with DCs each at a density of 1×104 cells/well in U-shaped 96-well plates. The cells were divided into the experimental and control group, with and without the addition of IL-15. The supernatant was sampled and analyzed for IL-2 concentration using ELISA on the 1st, 2nd, 3rd, 4th, and 5th day of the culture. The results showed that the concentration of IL-2 remained unchanged in the control group and was much higher in the experimental group, in which the concentration peaked at 60 pg/ml on the 4th day. We observed no Tregs proliferation in this experiment. The results indicate that IL-15 indeed regulates the secretion of IL-2 in DCs, but this does not play a predominant role in the induction of Tregs proliferation by DCs and IL-15.
6. Tregs were resuspended in medium containing 100 U/m IL-15, and 600μl (containing 4×104 cells) cell suspension was added to each well of 24-well plates. The samples were divided into 2 groups. The experimental group utilized TranswellTM chambers equipped with 3.0μm (pore size) filter, and each chamber was loaded with 4×104 suspended DC (100μL). In the control group, Tregs were cultured with 4×104 (100μl) of suspended DCs inactivated with mitomycin in 24-well plates. The cells were harvested on the 5th day, and the cell number was quantified using Trypan blue under light microscopy. Tregs did not proliferate in the experimental group, but did significantly proliferate in the control group (p < 0.05). This indicates that the IL-2 and sIL-15-IL-15Rα, which were produced by the interaction of IL-15 and DCs, do not play an important role in the induction of Tregs proliferation.
7. Tregs were cultured in U-shaped 96-well plates at a concentration of 5×104 well/plates. The cells were divided into 5 groups. The control group contained no IL-15 and DCs. The DC group contains only DCs inactivated with mitomycin (5×104 cells/well). The IL-15 group contained only IL-15 (100 U/ml). The combined induction group contained DCs and IL-15 at the above concentrations. In the blocked combined induction group, the addition of IL-15 was preceded by the addition of DCs and 40ug/ml anti-IL-15Rαantibody. After 24 hours, the cells were harvested, and the proteins in CD4~+CD25~+ regulatory T cells were extracted and analyzed for the expression of p-ERK, p-AKT, p-STAT5 and P27kip1 using Western-Blot. The results showed that DC-mediated activation could successfully induce the activation of Akt (target molecules of PI3K), but not the activation of Erk1/2 (the target molecule of MEK1/2) or STAT5, or the degradation of p27kip1 in Tregs. In contrast, when external IL-15 was added alone, IL-15 could not significantly induced the activation of Akt, Erk1/2, and STAT5 or the degradation of p27kip1, because of the low expression of IL-15Rαin Tregs. It was only in the presence of both DCs and IL-15 that significant induction of the activation of Akt, Erk1/2 and STAT5 and the degradation of p27kip1 in Tregs, and hence the proliferation of Tregs. In addition, if DCs and IL-15 were added after the addition of anti-IL-15Rαantibody, the activation of Akt, Erk1/2 and STAT5 and the degradation of p27kip1 were all inhibited, and the proliferation of Tregs was reduced. These results indicate that DCs and IL-15 mainly induce the proliferation of Tregs via the in trans presentation of IL-15.
Conclusion: We successfully developed a method to isolate and culture DCs and Tregs from peripheral blood. We demonstrate that IL-15 can induce the proliferation of Tregs in the presence of DCs. Proliferated Tregs induced by DCs and IL-15 are of high purity and have the phenotype of naturally occurring Tregs and perform specific inhibitory effect. They have the capability to migrate to the target location and perform their functions. Tregs have the potential to be applied for the induction of transplantation tolerance in clinics. The induction was mediated by IL-15 presented in trans by DCs. IL-15 can be recycled via trans-endosomal recycling, and remain on the surface of DCs for long-period of time. Therefore, DCs can continue to present IL-15 to Tregs and induce the proliferation. IL-15 may also regulate the secretion of IL-2 derived from DC, which plays a supportive role in the induction of Tregs proliferation by DCs and IL-15. The molecular mechanism of Tregs proliferation induced by DCs and IL-15 may involve the activation of Akt, Erk1/2 and STAT5, and the degradation of p27kip1.
白介素15(IL-15)是1994年由Burton和Grabstein同时发现的一种细胞因子。由于IL-15与IL-2结构中均含4α螺旋结构, IL-15与IL-2一起共享IL-2受体β和γ链(IL-2Rβγ/γc),有学者将IL-15归类于IL-2细胞因子家族,二者对T细胞作用后的许多效应有相同之处。不同在于IL-15主要以与IL-15Rα链高亲和力结合状态存在,即使缺乏IL-2Rβ和IL-2Rγ/γc受体亚单位,IL-15与其受体α私有链仍有着高度的亲和力(Ka≥1011M-1);而在缺乏IL-2Rβγ时,IL-2与IL-2Rα的亲和力却很低(Ka～108M-1)。体外实验表明,IL-2扩增Tregs的能力优于IL-15,然而IL-2或者IL-15缺陷小鼠体内Tregs数量和功能轻度下降或正常,只有当二者均缺陷时才会出现Tregs数量和功能显著下降。表明IL-15在Tregs的扩增中同样发挥重要作用,但IL-15是如何发挥作用还有待于进一步研究。
Rachael等将IL-15、表皮成纤维细胞各自单独与Tregs培养时不能增殖Tregs,但将二者联合后却能显著增殖Tregs,提示IL-15在其他细胞存在的条件下才能发挥增殖Tregs的作用。既往研究表明IL-15联合表皮成纤维细胞促进活化的T细胞增殖时主要是由表皮成纤维细胞以膜结合的形式向T细胞递呈IL-15。在免疫反应的启动中,树突状细胞(DC)是功能最强的激活初始T细胞的抗原递呈细胞,与表皮成纤维细胞一样,DC也能以膜结合的形式向靶细胞递呈IL-15。DC是否同样可以以膜结合的形式向Tregs细胞递呈IL-15而诱导Tregs增殖尚不清楚。因此,本研究从人外周血中分离出Tregs,随后将IL-15、DC单独或联合干预Tregs的生长,从分子水平探讨IL-15和DC对Tregs的作用及机制,为体外更好地扩增Tregs以得到足够数量的Tregs用于诱导器官移植免疫耐受奠定实验基础。
我们将本研究主要方法、获得的主要结果和结论归纳如下:
一、树突状细胞与CD4~+CD25~+调节性T细胞的分离及鉴定。
1.人外周血贴壁的单核细胞经GM-CSF~+IL-4联合诱导,于第6天负载同种异体抗原,培养第7天收集获得的细胞,光镜、电镜观察细胞的形态;流式细胞仪检测细胞表面标志物;将所得细胞经丝裂霉素灭活后与自体CD4~+CD25-T细胞混合淋巴细胞培养72h,3H-TdR掺入法检测CD4~+CD25-T细胞的增殖。结果显示,所得细胞逐渐由贴壁变成悬浮,细胞形态不规则,表面粗糙,有大量层叠状皱襞和毛刺状突起,表达共刺激分子CD80、CD86,MLR显示能刺激自体CD4~+CD25-T细胞增殖。表明所得细胞具有DC的细胞形态、免疫表型及功能特性,说明实验中建立的分离培养DC的方法是可行的。
2.人外周血单个核细胞经免疫磁珠阴性分选和阳性分选,将获得的细胞经台盼蓝染色检测细胞的活力;流式细胞仪检测细胞的纯度和细胞表型;将所得细胞经丝裂霉素灭活后与自体CD4~+CD25-T细胞混合淋巴细胞培养,包被抗CD3/CD28抗体的Dynal磁珠刺激72h,3H-TdR掺入法检测CD4~+CD25-T细胞的增殖。结果显示所得细胞活力为(95. 7±2.1) %,纯度为95.2%,胞内因子Foxp3在Tregs细胞的表达率为94.2%,在体外能抑制致敏T淋巴细胞增殖。表明所得细胞具有Tregs的细胞表型及功能特性,说明本实验建立的方法可以在体外分离出Tregs,为进一步研究Tregs打下基础。
二、IL-15和树突状细胞诱导增殖后CD4~+CD25~+调节性T细胞的表型与功能
1.Tregs按1×104/孔置于U型底96孔板中,依据每孔加入的DC(1×104/孔,经丝裂霉素灭活)或包被抗CD3/CD28抗体的Dynal磁珠(1×104磁珠/孔)不同分为四大组:负载同种异体抗原的DC~+IL-2组(简称DC~+IL-2组)、负载同种异体抗原的DC~+IL-15组(简称DC~+IL-15组)、包被抗CD3/CD28抗体的Dynal磁珠~+IL-2组(简称CD3/CD28~+IL-2组)和包被抗CD3/CD28抗体的Dynal磁珠~+IL-15组(简称CD3/CD28~+IL-15组),各组内依据细胞因子浓度不同分为0、25U/ml、50U/ml、100U/ml、200U/ml等小组,各小组3复孔。培养第5天,3H-TdR掺入法检测Tregs增殖。结果显示在包被抗CD3/CD28抗体的Dynal磁珠刺激下,IL-15在0---100U/ml的浓度范围内,基本不引起Tregs增殖,至200U/ml浓度时,可引起Tregs轻度增殖;而在包被抗CD3/CD28抗体的Dynal磁珠刺激下,IL-2在25U/ml浓度时即可引起Tregs显著性增殖(较同等浓度的IL-15);在负载同种异体抗原的DC刺激下,IL-15和IL-2一样,在0---200U/ml的浓度范围内,成剂量依赖性诱导Tregs增殖,且在100U/ml和200U/ml浓度时,二者诱导Tregs增殖的幅度无显著性差异。说明在DC存在的情况下,IL-15也能诱导Tregs增殖。
2.从培养体系中免疫磁珠阳性分选出CD4~+T细胞,流式细胞仪检测该细胞的纯度、细胞表型和CD62L;3H-TdR掺入法检测该细胞对CD4~+CD25-T细胞的增殖的抑制作用。结果显示,DC跨细胞递呈IL-15增殖的Tregs纯度高,仍具有典型的CD4~+CD25~+Foxp3~+表型特征,在体外对特异性同种异体抗原激活的Teff增殖的抑制功能强,对第三方同种异体抗原激活的Teff的免疫抑制功能弱,且Tregs高表达CD62L。说明DC跨细胞递呈IL-15增殖的Tregs具有天然Tregs的细胞表型,具有的特异性抑制作用,具备到达特定的作用部位发挥作用的能力。
三、IL-15和树突状细胞诱导CD4~+CD25~+调节性T细胞增殖的作用机制
1.将Tregs和DC分别与浓度为100U/ml的IL-15在37℃细胞培养温箱中孵育15分钟后流式细胞仪检测细胞表面IL-15,而细胞表面IL-15Rα则直接检测。结果显示,Tregs细胞表面IL-15Rα的表达极低,基本上未在Tregs细胞表面检测到结合的IL-15;而DC细胞表面IL-15Rα的表达较高,DC表面结合的IL-15远远高于在Tregs细胞表面结合的IL-15(p<0.05)。表明Tregs直接结合IL-15的能力较低,而DC直接结合IL-15的能力较高,特殊ELISA证实DC细胞表面确实形成了IL-15-IL-15Rα复合物。
2.将负载同种异体抗原的DC与Tregs按1:1比例混合培养,培养液中加入IL-15 100U/ml,24h后收集混合培养的细胞于离心管中,分别用PE-抗CD86和FITC-抗CD4标记DC和Tregs。激光共聚焦显示将DC、Tregs与IL-15共培养后,DC与Tregs间形成稳定的细胞团,这些提示DC和IL-15可能通过由DC跨细胞递呈IL-15给Tregs而诱导Tregs增殖。
3.DC和Tregs各以1×105/孔共同培养于平底96孔板,依据是否加入Anti-IL-15Rα不同将实验分为对照组和阻断组:两组均加入100U/ml的IL-15;不同之处在于阻断组在加入IL-15之前先加入40ug/ml的Anti-IL-15Rα,而对照组加入等体积的PBS。两组均在培养的第5天收集细胞、匀浆,特殊ELISA检测IL-15-IL-15Rα复合物。Tregs和经丝裂霉素灭活的DC各以1×104/孔共同培养于U型底96孔板,实验分组及处理同前。在培养的第5天收集细胞,3H-TdR掺入法检测Tregs增殖。结果显示,用IL-15Rα抗体预处理DC后再与与IL-15孵育, DC细胞表面IL-15-IL-15Rα复合物下降,此时Tregs增殖亦显著减少。从反面证实DC和IL-15主要是通过由DC跨细胞递呈IL-15给Tregs而诱导Tregs增殖。
4.在不同时间点检测DC表面IL-15:将DC置于含100U/ml的IL-15的培养液中培养4天,流式细胞仪检测DC表面IL-15;收集细胞,在甘氨酸缓冲液(PH=3)孵育,10分钟后检测DC表面IL-15;再将DC重新培养于无细胞因子的培养液中继续培养24h,流式细胞仪检测DC表面IL-15。DC和Tregs各以各1×105/孔共同培养于平底96孔板,依据处理方式不同将实验分为三组:对照组、维持组、撤退组。三组中对照组不加IL-15;维持组和撤退组均加100U/ml的IL-15,但撤退组于共培养的第4天撤退IL-15再培养24h,而维持组不撤退IL-15。三组均在培养的第5天收集细胞、匀浆,特殊ELISA检测IL-15-IL-15Rα复合物。经丝裂霉素灭活的DC和Tregs各以各1×104/孔共同培养于U型底96孔板,同时加入100U/ml的IL-2或IL-15依据是否在细胞培养的第4天撤退细胞因子分为维持组(不撤退)和撤退组。将细胞继续24h,3H-TdR掺入法检测Tregs增殖。结果显示,在培养体系中撤退IL-15后24h流式细胞仪仍能在DC表面检测到IL-15,培养体系中仍有IL-15-IL-15Rα复合物形成,Tregs可以继续增殖。说明IL-15还可在DC跨胞内体再循环,从而使IL-15持续长时间存在于DC细胞表面,继续通过DC跨细胞递呈IL-15给Tregs而诱导Tregs增殖。
5.将Tregs和DC各1×104/孔共同培养于U型底96孔板,依据是否加入浓度为100U/ml的IL-15分两组:实验组加入IL-15,而对照组不加入IL-15。于细胞培养的第1、2、3、4、5天取上清液ELISA检测IL-2浓度。结果显示,对照组IL-2浓度无明显变化,而实验组IL-2浓度显著性较对照组高,于细胞培养的第四天达到顶峰。但是其峰值仅约60pg/ml,从实验中我们可以看出,此浓度下Tregs基本上无明显增殖,表明在培养体系中IL-15确实调节DC分泌IL-2,但此作用在DC和IL-15诱导Tregs增殖的过程中不起主导作用。
6.实验中重悬细胞的培养液含100U/ml的IL-15,将含Tregs总数为4×104的细胞悬液600μl置于24孔板,依据是否使用Transwell小室将实验分为对照组和实验组,每组3复孔:实验组加上滤膜孔径为3.0um的Transwell小室,小室内再加入含DC总数为4×104的细胞悬液100μl;而对照组直接将含经丝裂霉素灭活的总数为4×104的DC细胞悬液100μl加入24孔板,与Tregs共培养。第5天收集24孔板内细胞,台胎盘蓝染色后直接在光镜下计数。结果显示,使用Transwell小室的实验组基本不增殖,而不加Transwell的对照组显著性增殖,p<0.05。表明在IL-15和DC诱导Tregs增殖的过程中,IL-15与DC作用形成的IL-2和sIL-15-IL-15Rα不起主要作用。
7.Tregs以5×104/孔培养于U型底96孔板中,依据加入经丝裂霉素灭活的DC(5×104/孔)和IL-15(100U/ml)不同分为五组:对照组、DC组、IL-15组、联合诱导组和联合诱导阻断组。其中,对照组不加IL-15和DC;DC组仅加DC;IL-15组仅加IL-15;联合诱导组同时加DC和IL-15;联合诱导阻断组先加入加入40ug/ml的Anti-IL-15Rα和DC,然后再加入IL-15。培养24h后收集细胞、提取蛋白。Western-Blot检测CD4~+CD25~+调节性T细胞p-ERK、p-AKT、p-STAT5和P27kip1的表达。结果显示:DC介导的活化能成功诱导Tregs内PI3K的靶分子Akt活化但不能诱导MEK1/2的靶分子Erk1/2的活化,也不能诱导STAT5的活化和p27kip1的降解;而单独加入外源性IL-15后,由于Tregs的IL-15Rα低表达,IL-15并不能显著诱导Tregs的Akt、Erk1/2和STAT5的活化以及p27kip1的降解;只有同时加入DC和IL-15,才能显著诱导Tregs的Akt、Erk1/2和STAT5的活化以及p27kip1的降解,引起Tregs增殖。此外,加入Anti-IL-15Rα后再加入DC和IL-15,Tregs的Akt、Erk1/2和STAT5的活化以及p27kip1的降解被抑制,Tregs增殖下降,进一步说明DC和IL-15主要以跨细胞递呈方式诱导Tregs增殖。
结论
一、成功建立了从人外周血分离培养DC和Tregs的方法。
二、在DC存在的情况下,IL-15也能大量诱导Tregs增殖。DC和IL-15诱导增殖的Tregs纯度高,具有天然Tregs的细胞表型,具有特异性抑制作用,具备到达特定的作用部位发挥作用的能力,有用于临床诱导移植免疫耐受的潜力。
三、DC和IL-15诱导Tregs增殖的作用形式主要是通过由DC跨细胞递呈IL-15给Tregs而诱导Tregs增殖。IL-15还可在DC跨胞内体再循环,从而使IL-15持续长时间存在于DC细胞表面,继续通过DC跨细胞递呈IL-15给Tregs而诱导Tregs增殖。此外,IL-15调节DC分泌IL-2可能在DC和IL-15诱导Tregs增殖的过程中起辅助作用。
四、DC和IL-15诱导Tregs增殖,其分子机制可能是通过Tregs的Akt、Erk1/2和STAT5的活化以及p27kip1的降解。
Clinical organ transplantation has remarkably advanced and is currently a well-established treatment for all sorts of organs’end-stage disease. Short-term survival rates are currently excellent. Unfortunately, long-term survival is still comparatively poor, mainly because of the chronic rejection and the toxicities of nonspecific immunosuppressants. To gain transplantation tolerance between donor organs and hosts is the ultimate goal of all sorts of organ transplantations. Intriguingly, CD4~+CD25~+ regulatory T cells (Tregs), which express a high level of Foxp3, display strong immune suppressive effect. Studies have shown that Tregs transfusion can prevent the graft-versus-host disease (GVHD) and transplantation rejection. Thus, Tregs have been suggested as potential reagents for adoptive cell therapy. However, the amount of naturally occurring Tregs only account for 1% of peripheral blood mononuclear cells, they are narurally anergic and poorly proliferative after TCR stimulation, such that cell numbers are severely limiting. Apparently, Tregs need to be massively expanded in vitro before they could be transfused back to patients and achieve the therapeutic objective. How to expand Tregs for the induction of transplantation tolerance is one of the hot areas where intensive studies are being performed.
Interleukin-15(IL-15) is a pleiotropic cytokine of the 4-α-helix bundle cytokine family, which was discovered by Burton and Grabstein in 1994 due to its ability to mimic IL-2-dependent T cell proliferation. A special feature of IL-15 is that it shares with IL-2, another member of the 4-α-helix bundle cytokine family, the IL-2 receptor beta (IL-2Rβ) and IL-2 receptor gamma/gamma common (IL-2Rγ/γc). IL-15 and IL-2 produce similar effects after their interactions with the T cells. IL-15 mainly binds to IL-15Rαchain in high affinity, and it maintains a high affinity (Ka≥1011M-1) to the private chain of itsαreceptor, even in the absence of IL-2Rβ和IL-2Rγ/γc receptor subunits. In contrast, the affinity of IL-2 and IL-2Rαis very low (Ka～108M-1) in the absence of IL-2Rβγ. In vitro study showed that the ability of IL-2 to simulate Tregs expanding is superior to IL-15. However, IL-2 or IL-15 deficient mice display normal or slightly reduced Tregs amount and function, which are significantly decreased in IL-2 and IL-15 double deficient mice. This indicates that IL-15 plays an equally important role in the expanding of Tregs. Nevertheless, how IL-15 performs its function remains to be addressed by further studies.
Rachael et al. showed that Tregs do not proliferate in the presence of either IL-15 or dermal fibroblasts, but do significantly proliferate when both IL-15 and dermal fibroblasts are present. This indicates that IL-15 can promote Tregs proliferation, but only in the presence of other types of cells. The underlying reason is that dermal fibroblasts present IL-15 to T cells in the form of membrane-bound IL-15. In the initiation of immune reaction, dendritic cells (DC) are the most potent antigen-presenting cells that activate initial T cells. Like dermal fibroblasts, DC can also present IL-15 to target cells as membrane-bound IL-15; however, it remains unclear whether DC and IL-15 can induce Tregs proliferation. For this reason, we isolated Tregs from human peripheral blood, and utilized IL-15, DC, and DC combined with IL-15 to intervene the proliferation of Tregs, and explored the related molecular mechanisms. Our work aims to improve the in vitro proliferation of Tregs and obtain a sufficient number of Tregs, and to lay a foundation for the induction of immune tolerance in organ transplantation. The main results and conclusions of our study are summarized bellow:
A. Isolation and characterization of dendritic cells and CD4~+CD25~+ regulatory T cells
1. Human peripheral adherent monocytes were induced with GM-CSF and IL-4 and loaded with the allogeneic antigen on the 6th day. The cells were harvested on the 7th day. The results showed that the cells obtained gradually converted from adherent cells to suspended cells that exhibit irregular morphology, coarse surface, large amount of laminated folds, and spike-like processes. These cells were found to express co-stimulating molecules such as CD80 and CD86, which, as shown in MLR, can stimulated the proliferation of autologous CD4~+CD25- T cells. These results indicate that the cells that we obtained have the morphology, immunophenotype, and functional characteristics of DCs.
2. Human peripheral blood mononuclear cells were positively and negatively selected with immunomagnetic beads. The results showed that the viability of the cells was 95. 7± 2.1 %,the purity of the cells was 95.2%,and the expression rate of intracellular factor Foxp3 was 94.2%. The cells we obtained can inhibit the proliferation of sensitized T cells in vitro, indicating these cells have the cellular phenotype and functional characteristics of Tregs. Therefore, the method that we developed can be used to isolate Tregs in vitro, and this laid a foundation for further study on Tregs.
B. The proliferation of CD4~+CD25~+ regulatory T cells induced by IL-15 and Dendritic cells
1. Tregs were cultured (1×104cells/well) in U-shaped 96-well plates, and were divided into 4 different groups according to the presence of Dynal beads or DCs (1×104 cells/well, inactivated with mitomycin). The results showed that in the presence of the magnetic beads coated with anti-CD3/CD28 antibody, the proliferation of Tregs was not observed with 0-100 U/ml IL-15, but was slightly observed with 200 U/ml IL-15. In contrast, 25 U/ml IL-2 induced significant Tregs proliferation in the presence the magnetic beads coated with anti-CD3/CD28 antibody. Furthermore, in the presence of the DCs loaded with allogeneic antigen, both IL-15 and IL-2 induced similar extents of Tregs proliferation in a dose-dependent manner within 0-200 U/ml cytokine. The magnitudes of the induction of Tregs proliferation by IL-15 and IL-2 were not statistically significant, when the concentration of the cytokine was within the range of 100-200 U/ml. These results indicate that IL-15 can also induce Tregs proliferation when combined with DCs.
2. CD4~+ T cells were positively screened using immunomagnetic beads, analyzed for purity, cellular phenotype, and CD62L using flow cytometry, and assayed for the inhibitory of the proliferation of CD4~+CD25- regulatory T cells using 3H-TdR incorporation. The results showed that proliferated Tregs induced by IL-15 presented by DC in trans are of high purity, and maintain the typical CD4~+CD25~+Foxp3~+ phenotype. Tregs have strong inhibitory effect in vitro on the proliferation of Teff activated by specific allogeneic antigen, but not that of Teff activated by third-party allogeneic antigen. Tregs express high level of CD62L. These results indicate that proliferated Tregs, which were induced by IL-15 presented in trans by DCs, display the same phenotypes of naturally produced Tregs. In addition, proliferated Tregs display specific inhibitory effect, and have the capability to migrate to the target location and perform specific functions.
C. The related mechanism of the proliferation of CD4~+CD25~+ regulatory T cells induced by IL-15 and Dendritic cells
1. Tregs and DCs were incubated separately with 100 U/ml IL-15 at 37℃for 15 min, and were analyzed for IL-15Rαand cell-surface IL-15 using flow cytometry. The results showed that the level of Tregs-surface IL-15Rαwas extremely low and Tregs-surface IL-15 was not detectable. In contrast, we detected high level of IL-15Rαon the surface of DCs, and the level of IL-15 bound to DCs surface was far higher than that bound to Tregs surface (p < 0.05). This indicates that the ability of Tregs to bind IL-15 is much lower than that of DCs. In addition, the presence of IL-15-IL-15Rαcomplex on DCs surface was confirmed with special ELISA.
2. Tregs were cultured with DCs loaded with allogeneic antigens at 1:1 ratio in the presence of 100 U/ml IL-15. The cell mixture was harvested, and DCs and Tregs in the mixture were labeled respectively with PE-anti-CD86 antibody and FITC-anti-CD4 antibody. Subsequent confocal microscopy showed that DCs and Tregs form stable cell aggregates, indicating that DCs may present IL-15 in trans to Tregs and hence induce the Tregs proliferation.
3. DCs and Tregs were cultured together in plate-bottom 96-well plates in the presence of 100 U/ml IL-15 and divided into 2 groups. The block group was added 40ug/ml anti-IL-15Rαantibody,while the control group was added equal volume of PBS. The results showed that if DCs were blocked by anti-IL-15Rαantibody before they were incubated with IL-15, the concentration of IL-15-IL-15Rαcomplexes on DCs surface decreased, and so was the proliferation of Tregs. These result support the notion that IL-15 was presented in trans by DCs to Tregs and induce Tregs proliferation.
4. Analysis of DC surface IL-15 as a function of time: DCs were cultured in medium containing 100 U/ml IL-15 for 4 days, and then were assayed for surface IL-15 using flow cytometry. The cells continued to be cultured in the medium containing Glycine buffer (pH = 3), and analyzed for surface IL-15 10 min later. The cells were again cultured in medium without cytokines for 24 hr, and analyzed for DC-surface IL-15 using flow cytometry. DCs and Tregs were co-cultured in plate-bottom 96-well plates each at a density of 1×105 cells/well. These cells were then divided into 3 groups. The control group contained no IL-15, while the maintenance group contained 100 U/ml IL-15 throughout the course. In the withdrawal group, IL-15 was removed on the 4th day of the co-culture, and continued to be cultured for 24 hr. The cells of each group were harvested on the 5th day of the culture, homogenated and assayed for IL-15-IL-15Rαcomplexes using special ELISA. In a separate experiment, Tregs were cultured with DCs inactivated with mitomycin in U-shaped 96-well plates each at a density of 1×104 cells/well with 100 U/ml IL-15. The samples were divided into 2 groups. In the withdrawal group, the cytokine (IL-15 or IL-2) was removed on the 4th day, and continued to be cultured for additional 24 hr. The cytokine remained in the medium of the maintenance group. The proliferation of Tregs was quantified using 3H-TdR incorporation. The results showed that IL-15 was detectable on the surface of DCs 24 hours after the removal of IL-15 in the culture system. This indicates the formation of IL-15-IL-15Rαcomplex in the culture system, and thus Tregs could continue to proliferate. This also indicates that IL-15 could be recycled via trans-endosomal recycling, and thus could remain on DC surface for a long period of time. IL-15 could be presented by DCs to Tregs, and induce Tregs proliferation.
5. Tregs were co-cultured with DCs each at a density of 1×104 cells/well in U-shaped 96-well plates. The cells were divided into the experimental and control group, with and without the addition of IL-15. The supernatant was sampled and analyzed for IL-2 concentration using ELISA on the 1st, 2nd, 3rd, 4th, and 5th day of the culture. The results showed that the concentration of IL-2 remained unchanged in the control group and was much higher in the experimental group, in which the concentration peaked at 60 pg/ml on the 4th day. We observed no Tregs proliferation in this experiment. The results indicate that IL-15 indeed regulates the secretion of IL-2 in DCs, but this does not play a predominant role in the induction of Tregs proliferation by DCs and IL-15.
6. Tregs were resuspended in medium containing 100 U/m IL-15, and 600μl (containing 4×104 cells) cell suspension was added to each well of 24-well plates. The samples were divided into 2 groups. The experimental group utilized TranswellTM chambers equipped with 3.0μm (pore size) filter, and each chamber was loaded with 4×104 suspended DC (100μL). In the control group, Tregs were cultured with 4×104 (100μl) of suspended DCs inactivated with mitomycin in 24-well plates. The cells were harvested on the 5th day, and the cell number was quantified using Trypan blue under light microscopy. Tregs did not proliferate in the experimental group, but did significantly proliferate in the control group (p < 0.05). This indicates that the IL-2 and sIL-15-IL-15Rα, which were produced by the interaction of IL-15 and DCs, do not play an important role in the induction of Tregs proliferation.
7. Tregs were cultured in U-shaped 96-well plates at a concentration of 5×104 well/plates. The cells were divided into 5 groups. The control group contained no IL-15 and DCs. The DC group contains only DCs inactivated with mitomycin (5×104 cells/well). The IL-15 group contained only IL-15 (100 U/ml). The combined induction group contained DCs and IL-15 at the above concentrations. In the blocked combined induction group, the addition of IL-15 was preceded by the addition of DCs and 40ug/ml anti-IL-15Rαantibody. After 24 hours, the cells were harvested, and the proteins in CD4~+CD25~+ regulatory T cells were extracted and analyzed for the expression of p-ERK, p-AKT, p-STAT5 and P27kip1 using Western-Blot. The results showed that DC-mediated activation could successfully induce the activation of Akt (target molecules of PI3K), but not the activation of Erk1/2 (the target molecule of MEK1/2) or STAT5, or the degradation of p27kip1 in Tregs. In contrast, when external IL-15 was added alone, IL-15 could not significantly induced the activation of Akt, Erk1/2, and STAT5 or the degradation of p27kip1, because of the low expression of IL-15Rαin Tregs. It was only in the presence of both DCs and IL-15 that significant induction of the activation of Akt, Erk1/2 and STAT5 and the degradation of p27kip1 in Tregs, and hence the proliferation of Tregs. In addition, if DCs and IL-15 were added after the addition of anti-IL-15Rαantibody, the activation of Akt, Erk1/2 and STAT5 and the degradation of p27kip1 were all inhibited, and the proliferation of Tregs was reduced. These results indicate that DCs and IL-15 mainly induce the proliferation of Tregs via the in trans presentation of IL-15.
Conclusion: We successfully developed a method to isolate and culture DCs and Tregs from peripheral blood. We demonstrate that IL-15 can induce the proliferation of Tregs in the presence of DCs. Proliferated Tregs induced by DCs and IL-15 are of high purity and have the phenotype of naturally occurring Tregs and perform specific inhibitory effect. They have the capability to migrate to the target location and perform their functions. Tregs have the potential to be applied for the induction of transplantation tolerance in clinics. The induction was mediated by IL-15 presented in trans by DCs. IL-15 can be recycled via trans-endosomal recycling, and remain on the surface of DCs for long-period of time. Therefore, DCs can continue to present IL-15 to Tregs and induce the proliferation. IL-15 may also regulate the secretion of IL-2 derived from DC, which plays a supportive role in the induction of Tregs proliferation by DCs and IL-15. The molecular mechanism of Tregs proliferation induced by DCs and IL-15 may involve the activation of Akt, Erk1/2 and STAT5, and the degradation of p27kip1.
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