天然杀伤细胞促进记忆性CD8~+T细胞产生及其相关机制的实验研究
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摘要
抗原特异性免疫记忆是免疫的基本特征,其物质基础是初次免疫应答后产生的抗原特异性的记忆性T淋巴细胞和记忆性B淋巴细胞以及浆细胞。CD8T细胞在机体免疫系统抗御多种病原体感染和肿瘤免疫应答中发挥关键作用,因此,记忆性CD8T细胞(memroy CD8 T cells,CD8Tm)产生和维持的调控机制受到广泛关注。
以往的研究大多集中在对CD8Tm细胞产生和功能起关键调控作用的分子上,但对CD8Tm调控的细胞免疫学基础与相关机制研究较少。现有研究表明,树突状细胞(Dendritic cells,DC)能够通过表达IL-15和IL-15Rα促进CD8Tm细胞自稳性增殖,维持CD8Tm细胞数量稳定。但是其它免疫细胞,特别是与CD8T细胞免疫应答密切相关的天然杀伤细胞(Natural killer cells,NK)在CD8T细胞免疫记忆中是否发挥调控作用目前尚不清楚。
NK细胞能够迅速发挥细胞毒性作用,因此在感染早期的机体免疫防御中发挥重要作用。近年来的研究表明,NK细胞的免疫调节作用也不容忽视,在初次应答的天然免疫阶段,NK细胞能够通过分泌穿孔素、颗粒酶以及IFN-γ等可溶性分子直接影响后来的T细胞免疫应答,同时NK细胞还能够通过细胞因子分泌和膜接触依赖的方式促进DC成熟,从而间接调控T细胞免疫应答。这提示NK细胞可能对记忆性CD8T细胞的产生有一定的调控作用,但目前为止尚未见此方面的研究报道。
因此,我们利用小鼠李斯特菌感染模型诱导抗原特异性CD8T细胞免疫应答和免疫记忆,研究了NK细胞在CD8Tm形成过程中可能发挥的调节作用,并对其中的分子机制进行了探索。
1.NK细胞通过调节初次应答收缩期而促进CD8Tm细胞产生
为诱导产生CD8Tm细胞,我们将Thy-1.1~+ OT-I TCR转基因小鼠的CD8 T细胞通过尾静脉注射过继回输到野生型C57小鼠(Thy-1.2)体内,24h后通过尾静脉注射表达OVA的李斯特菌毒力减弱株(△actA LM-OVA)进行感染。感染后7天内,小鼠体内Thy-1.1~+ CD8 T细胞发生强烈的应答并大量增殖。感染40天后,可以在包括外周血、脾脏、淋巴结和肝脏在内的脏器中检测到一定比例的Thy-1.1~+ CD8 T细胞,这群细胞具有记忆性CD8T细胞的表型特征,即CD44~(hi),CD62L~(hi)/CD62L~(lo),更重要的是,这群细胞在体外OVA_(257-264)肽再刺激数小时后即可检测到细胞内IFN-γ分泌。此外,体内存在Thy-1.1~+ CD8 Tm的小鼠再次感染表达OVA的李斯特菌毒力株(LM-OVA)后,能够更快清除细菌。感染100天后,Thy-1.1~+ CD8 T细胞仍然存在并具有同样的表型和功能。这说明,我们诱导出了能够长期存活并具有保护功能的抗原特异性CD8Tm细胞。
接着我们采用抗体体内注射方法清除NK细胞,观察NK细胞清除后CD8T细胞初次应答情况及CD8Tm细胞产生情况。PK136杂交瘤产生特异性针对小鼠NK1.1分子的单克隆抗体,我们制备该杂交瘤腹水并进行抗体纯化定量。day-2和day0(感染当天)时各给予一次PK136抗体或同型对照抗体腹腔注射清除NK1.1~+细胞,通过动态观察发现,在最后一次抗体注射后的3周内,NK细胞的清除率维持在90%以上,NK细胞数在第4周末时基本恢复如常,这可能是由于过量抗体在体内维持并持续清除新产生的少量NK细胞,直至抗体代谢完全。清除NK细胞后,对CD8T细胞初次免疫应答的分析发现,在免疫应答高峰期(感染后7天),NK细胞清除组小鼠Thy-1.1~+ CD8T细胞在各脏器中的比例以及在脾脏中的绝对数均与对照组相似,两组小鼠中Thy-1.1~+效应性CD8T细胞的表型特征(CD27,CD44,CD62L,CD122,CD127,KLRG-1)及细胞因子分泌(IFN-γ,TNF-α,IL-2)均未见明显差异。这说明在针对李斯特菌感染的初次免疫应答过程中,NK细胞对CD8T细胞初次应答的数量和质量上均没有重要影响。
但在感染后第40天,NK细胞清除组小鼠中Thy-1.1~+ CD8Tm细胞在各脏器CD8~+细胞中所占比例和脾脏中的绝对数均显著低于对照组。我们首先排除了Thy-1.1~+ CD8T细胞被PK136抗体直接清除的可能性:流式分析表明,在感染第0,3,7,14,21,40时,该群CD8T细胞均不表达NK1.1分子;如果在感染第7天注射PK136抗体,24h后NK细胞被有效清除,但Thy1.1~+ CD8T细胞数量和比例不变。感染后不同时间点采集外周血进行动态分析发现,两组小鼠中Thy-1.1~+ CD8T细胞在外周血CD8~+细胞中的比例在第7天时基本相同,但到第21天时,NK细胞清除组该比例仅为对照组的1/5,这一1:5的相对比例一直维持到感染40天以后。通过BrdU摄取实验和胞内Bcl-2流式分析发现,虽然两组小鼠中Thy-1.1~+ CD8T细胞在收缩期时增殖无显著差异,但NK细胞清除后,Thy-1.1~+ CD8T细胞胞内Bcl-2分子平均表达水平明显低于对照组。虽然PK136处理组小鼠CD8Tm细胞数显著降低,但两组小鼠中CD8Tm细胞在单个细胞水平上的“质量”并没有明显差异,表现在该群记忆性CD8T细胞群体内部各表型间比例,以及体外再刺激下胞内细胞因子染色的平均荧光强度在两组间没有明显差异。感染后第40天(此时NK细胞数已恢复)给予表达OVA的李斯特菌毒力株(LM-OVA),感染72h后发现对照组肝脏和脾脏中李斯特菌CFU数均显著低于NK细胞清除组。实验结果说明,NK细胞促进活化CD8T细胞在收缩期的存活,进而使CD8Tm细胞数量和保护力维持在较高的水平。
虽然NK细胞清除后对CD8T细胞初次应答的细胞增殖,表型和细胞因子分泌没有显著影响,但这并不能排除NK细胞通过影响CD8T细胞初次应答来影响CD8Tm细胞的产生,而这种对初次应答的影响可能是我们没有检测到的。为进一步阐明NK细胞促进CD8Tm细胞的产生是作用于收缩期而不是扩增期,我们设计了如下的实验:将Thy-1.1~+ OT-I CD8 T细胞过继回输到经PK136抗体或对照抗体处理的C57小鼠(第一受体)体内并用ΔactA LM-OVA感染,7天后将两组小鼠脾脏中的Thy-1.1~+ CD8T细胞分离纯化后等量分别回输到同步感染的经PK136抗体或对照抗体处理的第二受体小鼠体内。三周后,对Thy-1.1~+记忆性CD8T细胞进行分析发现,无论Thy-1.1~+效应性CD8T细胞来自PK136抗体组还是对照组,回输到对照组第二受体小鼠后,均能产生相似数量和比例的CD8Tm细胞;相反,PK136处理组第二受体小鼠体内产生的记忆性细胞显著低于对照。这一证据充分说明NK细胞促进CD8Tm细胞产生的作用时间是初次应答的收缩期。
2.NK细胞通过穿孔素依赖的方式促进收缩期CD8T细胞存活
前面的实验中我们证明了NK细胞通过促进收缩期CD8T细胞存活促进CD8Tm细胞产生,那么,NK细胞通过哪个(些)重要的分子来发挥这一作用呢?
穿孔素(perforin)组成型表达于NK细胞,是NK细胞发挥杀伤作用的重要效应分子。最新研究表明,穿孔素还具有重要的免疫调节功能。例如,在热休克蛋白Gp96-肽复合体所诱导的抗肿瘤免疫应答中,穿孔素基因敲除(PFR)小鼠中单独回输的野生型CD8T细胞不能产生有效的抗肿瘤免疫应答,而同时回输野生型NK细胞能够恢复CTL应答。这一研究结果不仅证明了穿孔素的免疫调节作用,还提示NK细胞和CD8T细胞来源的穿孔素在免疫应答过程中可能发挥不同的作用。我们首先检测了在PFR小鼠中,过继回输的Thy-1.1~+ OTI CD8Tm细胞的产生情况。我们发现,Thy-1.1~+ CD8T细胞回输到PFR小鼠中后,ΔactA LM-OVA感染后CD8T细胞初次应答的数量和质量均没有显著变化。但与NK细胞清除小鼠中的结果相似,回输到PFR小鼠中的CD8T细胞在感染后21天时数量显著低于对照小鼠,感染40天后PFR小鼠中Thy1.1~+CD8Tm数显著低于对照小鼠。但PFR小鼠中Thy-1.1~+ CD8Tm细胞群体中各表型间的比例和单个细胞水平的细胞因子分泌能力与对照小鼠中的CD8Tm细胞相似。
那么,PFR小鼠中的CD8Tm细胞减少是由于其NK细胞不能表达穿孔素所引起的吗?我们对此进行了进一步的研究。我们发现,如果在上述模型中感染后第7天时向PFR小鼠中回输来自同步感染小鼠的野生型NK细胞,那么其体内Thy1.1~+CD8Tm细胞的比例和数量可以恢复到与野生型小鼠相似的水平,而回输穿孔素缺陷的NK细胞或者回输野生型的活化CD8T细胞(表达穿孔素)则没有这种作用。我们还采用了另外一种方式来确认上述结果:在野生型小鼠中回输Thy-1.1~+OTI CD8T细胞并感染,7天后纯化脾脏Thy-1.1~+CD8T细胞,按相同细胞数分别回输到同步感染的野生型C57小鼠或PFR小鼠中,在PFR小鼠中同时回输经活化的野生型或者穿孔素缺陷的NK细胞。我们发现,回输野生型NK细胞后,PFR小鼠中能够产生与野生型小鼠相似数量的Thy1.1~+CD8Tm细胞,而回输穿孔素缺陷的NK细胞则不具有这一作用。以上结果充分证明,NK细胞通过穿孔素依赖的方式促进了CD8Tm细胞产生。
我们从细胞存活和增殖两个方面对NK细胞的收缩期调节作用进行了分析。通过BrdU摄取实验发现,回输到NK细胞清除和对照组野生型C57小鼠中,以及回输到PFR小鼠并回输NK细胞的各组中,Thy-1.1~+CD8T细胞在收缩期的增殖没有显著差异;但是通过胞内Bcl-2分子标记后发现,PFR小鼠体内Thy-1.1~+CD8T细胞在收缩期时的Bcl-2分子表达水平低于对照组,而在PFR小鼠中回输野生型NK细胞后能够提高Thy-1.1~+CD8T细胞在收缩期时Bcl-2分子表达水平。这说明NK细胞通过穿孔素依赖的方式促进CD8T细胞在收缩期的存活,进而促进CD8Tm细胞的产生。
3.NK细胞通过穿孔素依赖的方式促进了树突状细胞表达IL-15和IL-15Rα可能与NK细胞促进了记忆性CD8T细胞产生有关
我们通过前两部分的实验发现,NK细胞能够通过穿孔素依赖的方式促进CD8T细胞在收缩期的存活,进而促进记忆性CD8T细胞产生。现有研究表明,1)NK细胞能够与DC形成免疫突触,促进DC成熟并上调IL-15表达;2)IL-15,特别是DC表达的膜结合型IL-15能够促进CD8T细胞在收缩期的存活。根据上述实验结果和文献报道我们推测,NK细胞可能通过与DC形成免疫突触,以穿孔素依赖的方式促进DC表达IL-15Rα和膜结合型IL-15,进而通过DC间接促进CD8Tm细胞的产生。
我们首先检测了各组小鼠在初次应答的收缩期时DC表达IL-15Rα和膜结合型IL-15的情况。感染后第10天时,取小鼠脾脏进行流式分析发现,清除NK细胞的野生型小鼠和PFR小鼠中CD11c~(hi)la~(b+)的DC表面IL-15Rα和膜结合型IL-15的表达水平低于对照组野生型小鼠。回输野生型NK细胞的PFR小鼠脾脏CD11c~(hi)la~(b+)的DC表面IL-15Rα和膜结合型IL-15的表达恢复到与野生型对照小鼠相似的水平,而回输穿孔素缺陷的NK细胞与不回输细胞组水平相当。感染后第14天时也有相同结果。通过荧光免疫组织化学方法我们发现,在免疫应答的收缩期(day10),NK细胞和DC大多定位于淋巴组织中T细胞区和B细胞区之间的狭小区域中,并且可以观察到NK与DC成紧密的并排排列,这提示在免疫应答的收缩期,NK仍然能够与DC发生直接的细胞接触和相互作用。
形态学观察发现,野生型小鼠的NK细胞经IL-2和IL-15活化后,其胞浆内可检测到均匀分布的穿孔素蛋白,而穿孔素缺陷小鼠的NK细胞中不能检测到穿孔素。经过体外活化的NK细胞与成熟的骨髓来源的DC共同培养1h后,NK细胞与DC能够形成免疫突触,可以观察到NK细胞将穿孔素集中运输到免疫突触部位。这一结果充分说明在NK与DC形成的免疫突触中,NK细胞能够通过分泌穿孔素作用于DC。
将体外活化的NK细胞与骨髓来源的成熟DC共培养后发现,野生型NK细胞能够促进DC上调膜表面IL-15Rα和IL-15的表达水平,而穿孔素缺陷的NK细胞几乎没有促进DC表达IL-15Rα和膜结合型IL-15的能力,而野生型NK细胞和穿孔素缺陷NK细胞等量混合细胞(NK细胞总数保持不变)能够促进DC表达膜结合型IL-15和IL-15Rα。可见,NK细胞通过穿孔素依赖的方式促进了DC表达IL-15和IL-15Rα可能参与了NK细胞促进了CD8Tm细胞产生的过程。
综上,我们的实验结果证明,NK细胞能够在初次免疫应答的收缩期促进活化CD8T细胞存活,进而促进记忆性CD8T细胞产生,而NK细胞表达的穿孔素在这一过程中起到关键作用。我们还通过体内和体外实验证明,在CD8T细胞免疫应答的收缩期,NK细胞能与DC发生相互作用,并且通过穿孔素依赖的方式促进DC表达IL-15Rα和膜结合型IL-15。即NK细胞可能通过DC及其表达的IL-15Rα和膜结合型IL-15间接地促进了记忆性CD8T细胞产生。
在病原体感染所诱导的CD8T细胞免疫记忆中,病原/抗原特异性记忆性CD8T细胞的数量和质量是宿主是否能在再次感染中存活的关键因素。免疫接种的基本原理是利用针对灭活疫苗或者减毒活疫苗的初次应答产生并维持包括记忆性T细胞在内的记忆性细胞群体,接种后免疫记忆的有效产生和维持对于防范此类感染性疾病具有重大意义。因此,对免疫记忆,特别是减毒株病原体接种后保护性免疫记忆的产生和维持的细胞和分子免疫学调控机制的深入研究具有重要的理论和实践价值。我们的研究从一个新的角度揭示了NK细胞对CD8T细胞免疫记忆的调节功能,丰富了人们对NK细胞功能和对CD8T细胞免疫记忆调控的认识,为感染性疾病的免疫治疗和疫苗设计提供了新的思路和一定的理论依据。
Antigen-specific immunological memory is a cardinal feature of immunity, which depends on generation and maintenance of memory T lymphocytes,B lymphocytes and plasma cells with antigen specificity.Mechanisms for generation and maintenance of memory CD8T(CD8Tm) cells are of special interests as they play a crucial role in immune responses against infections and tumors.
Much attention has been paid to key molecules regulating the generation and functions of CD8Tm cells,whereas little is known about the role of populations of cells in regulation of CD8Tm cell generation and functions.Recent data demonstrated that dendritic cells(DCs) promote homeostatic proliferation of memory CD8T cells via IL-15 and IL-15Rα.However,it is unknown whether other immune cells,such as natural killer(NK) cells which are closely related to CD8T cell mediated immune responses,play a role in the regulation of CD8Tm cells.
NK cells mediate fast cytotoxicity against target cells and play crucial roles in immune defense early in an infection.It has been clearly shown in recent years that activated NK cells mediate equally important immuno-modulatory functions. In innate immune response early in an infection,NK cells polarize the ensuing T cell response directly via secretion of soluble factors such as perforin,granzyme and IFN-γ.Meanwhile,NK cells influence T cell response indirectly through promoting maturation of DCs via a both cell-to-cell contact and soluble dependent manner.These data suggest that NK cells may have regulatory functions in CD8Tm generation.
In our current study,we generated antigen specific CD8 T cell immune response and memory using a murine model of Listeria Monocytogenes(LM) infection.We investigated the possible roles of NK cells in the regulation of CD8Tm cell generation and the underlying cellular and molecular mechanisms.
1.Natural killer cells promote CD8 Tm cell generation by regulating the extent of contraction.
To generate CD8Tm cells,we adoptively transferred Thy-1.1 OT-I CD8 T cells in to host C57 mice(Thy-1.2) followed by injection of a virulance attenuated stain of Listeria Monocytogenes(ΔactA LM-OVA)via the tail veil 24h later.On day7 after infection,we detected robust expansion in number of Thy-1.1~+ CD8T cells. On day40 after infection,donor derived Thy-1.1~+ CD8Tm cells were detectable in various organs including peripheral blood,spleen,lymph nodes,and liver.These cells were CD44~(hi),CD62L~(hi)/CD62L~(lo) in phenotype and more importantly,these cells were readily detectable for intracellular IFN-γsecretion several hours after ex vivo restimulation with OVA_(257-264) peptide.Moreover,mice carrying Thy-1.1~+ CD8Tm cells were significantly more resistant to challenge with virulant strain of OVA expressing LM(LM-OVA).Thy-1.1~+ CD8Tm cells were detectable with similar phenotype and function 100 days after infection.These data suggest that we have generated long-lived and functional CD8Tm cells with antigen specificity.
We next investigated whether NK cells are required in primary CD8 T cell response and/or CD8Tm generation by in vivo depletion of NK cells using PK136 monoclonal antibody which is specific to murine NK1.1.We gave one dose intraperitoneally of PK136 Ab or isotype control Ab on day-2 and day0, respectively.We observed a NK cell depletion of over 90%within 3 weeks since the last dose of Ab.Normal NK cell number was observed at the end of week 4. We speculated that excess dose of PK136 Ab existed in mice which depleted newly generated NK cells.We found that after NK cell depletion,the percentage and the absolute number of donor derived Thy-1.1~+ CD8 T cells were comparable in PK136 Ab and isotype Ab treated mice on day 7 after infection,with similar phenotype(CD27,CD44,CD62L,CD122,CD127,KLRG-1)and cytokine secretion profile(IFN-γ,TNF-α,IL-2).Thus,our data demonstrated that NK cells are not required in primary CD8T cell response afterΔactA LM-OVA infection.
Unexpectedly,we observed significantly lowered percentage and number of Thy-1.1~+ CD8 Tm cells 40 days after infection.We first exclude the possibility that Thy-1.1~+CD8T cells were depleted by PK136 Ab by showing that only marginal percentage of Thy-1.1~+ CD8T effector cells were positive for NK1.1 staining on days 0,3,7,14,21 and 40 after infection,and PK136 Ab did not deplete Thy-1.1~+ CD8T effector cells when injected on day 7 after infection and analyzed 24 hours later.By blood sample collection at different time points,we monitored the kinetics of the percentage of Thy-1.1~+ CD8T cells in blood CD8~+ T cells.Though similar percentage of Thy-1.1~+ effector CD8T cells were generated on day 7 after infection,the percentage of Thy-1.1~+ CD8T cells in total blood CD8~+ T cells in NK cell depleted group was about 1/5 that in control group on day 21,and this proportion was essentially fixed in the ensuing days and weeks which resulted in reduced generation of CD8Tm cells.Intracellular detection of Bcl-2 and BrdU incorporation suggested that the survival of Thy-1.1~+ CD8T cells was impaired in NK cell depleted mice,though proliferation of Thy-1.1~+CD8T cells in two groups was indistinguishable.We analyzed surface marker expression as well as cytokine secretion by Thy-1.1~+ CD8Tm cells.Though small in number,Thy-1.1~+ CD8Tm cells generated in NK cell depleted mice were similar in surface marker expression and were equally able in IFN-γ,TNF-α,and IL-2 secretion when restimulated ex vivo with OVA_(257-264)peptide.After challenging withLM-OVA on day 40,we observed reduced protection of CD8Tm cells as a population in NK cell depleted mice as CFUs of LM-OVA in both the spleen and the liver were significantly higher in NK cell depleted mice.Note that NK cells recovered to a normal level on day 40,our data suggested that NK cells promote the survival of activated CD8 T cells in the contraction phase and thus help to generate a protective CD8Tm cell pool.
We did further experiments to confirm that the role of NK cells on activated CD8 T cells was mediated in the contraction phase rather than in the expansion phase,where NK cells may presumably influence the intrinsic quality of CD8 T effector cells which is not detected in our experiments.Thy-1.1~+ OT-I CD8 T cells were adoptively transferred into Thy-1.2~+ host C57 mice treated with either isotype or PK136 Ab as mentioned above followed by injection i.v.withΔactA LM-OVA.On day 7 after infection,equal number of Thy-1.1~+ CD8 T effector cells from either hosts were sorted and adoptively transferred into second host mice(Thy-1.2) which were also on day 7 after infection and treated with isotype or PK136 Ab,respectively.Three weeks after transfer of CD8 effector T cells, absolute number of spleen Thy-1.1~+ CD8 Tm cells and percentages of Thy-1.1~+ cells in CD8 T cells in peripheral blood,spleen and lymph nodes were analyzed. CD8 Tm cell generation was impaired in NK cell depleted second hosts irrespective of whether effector CD8 T cells were generated in NK cell depleted or control mice.In contrast,both effector Thy-1.1~+ CD8T cells isolated from control or NK cell depleted mice generated normal number of CD8Tm cells after transfer into control second hosts.Thus,we provided convincing data showing that the role of NK cells in promoting CD8Tm cell generation were mediated during the contraction phase rather than in the expansion phase after primary infection.
2.NK cells promote the survival of contracting CD8 T cells via a perforin dependent mechanism.
We went on to ask which molecule(s) is required for NK cells in the promotion of the survival of contracting CD8T cells.
Perforin is constitutively expressed by NK cells and is a key molecule in NK mediated cytotoxicity.In heat shock protein gp96-peptide complexes induced tumor rejection model,antigen specific CD8T cells(perforin competent) adoptively transffered into perforin knock out mice failed to proliferate and differentiate into CTLs whereas co-transfer of wide type NK cells rescued the clonal expansion and CTL differentiation of transferred CDST cells.These results suggested important immuno-modulatory roles of perforin and presumably different roles of perforin secreted by NK cells versus by CD8~+ CTLs.Using the same method mentioned above,we adoptively transferred Thy-1.1 OT-I CD8 T cells into wide type B6 mice(W-F) and perforin knock out(PFR)mice on a B6 background followed byΔactA LM-OVA infection.On day 7 after infection, comparable numbers of effector Thy-1.1~+ CD8 T cells with similar phenotype and cytokine secretion profile were generated in WT and PFR mice.However,a more extensive contraction of Thy-1.1~+ CD8 T cells was observed in PFR mice compared with those in WT mice.Consistent with those in NK cell depleted mice, the percentage of effector Thy-1.1~+ CD8 T cells in PFR mice was significantly lower than that in WT control group and number of Thy-1.1~+ CD8Tm cells was significantly decreased compared with that in WT mice 40 days after infection.In accordance with those in NK cell depleted mice,Thy-1.1~+ CD8Tm cells generated in PFR mice were similar in phenotype and cytokine secretion profile on a single cell basis.
To confirm that reduced CD8Tm cell generation in PFR mice was due to perforin incompetence within NK cell population rather than in other cells that could also express perforin after infection(e.g.CTLs),we adoptively transferred WT NK cells or PFR NK cells isolated from simultaneously infected WT or PFR mice on day 7 after infection into PFR mice transferred with Thy-1.1~+ CD8 T cells followed by infection.WT NK cells almost fully rescued generation of CD8Tm cells in PFR mice whereas PFR NK cells failed to do that.Activated CD8 CTLs isolated from infected WT mice could not rescue the generation of Thy-1.1~+ CD8Tm cells in PFR mice,either.Thus,our data clearly showed that perforin is required in NK cell mediated promotion of CD8 Tm cell generation.This conclusion was further confirmed when effector CD8 T cells were generated in WT mice and adoptively transferred into WT or PFR second hosts on day 7 after infection.Co-injection of WT NK cells almost fully rescued CD8Tm cell generation in PFR second host mice whereas PFR NK cells did not.
We analyzed the proliferation and the survival of Thy-1.1~+ effector CD8 T cells in the contraction phase.The proliferation of effector Thy-1.1 ~+CD8 T cells was not different as shown by BrdU incorporation.However,analysis of Bcl-2 expression showed that effector Thy-1.1 ~+CD8 T cells were more resistant to apoptosis in VVT mice than PFR mice.And adoptive transfer of WT NK cells restored Bcl-2 level in effector Thy-1.1 ~+CD8 T cells in PFR mice.Thus,our data demonstrate that perforin is required for NK cells in promotion of CD8Tm generation via promoting survival of contracting CD8T cells.
3.NK cells promote IL-15 and IL-15Rαexpression by dendritic cells in a perforin dependent manner,outlining one of possible mechamisns for the promotion of memory CD8 T cell generation by NK cells
We have shown in the first two parts that NK cells promote CD8Tm cell generation via promoting survival of CD8T cells in a perforin dependent manner.It is reported that a)NK cells form conjugates with DCs and promote IL-15 expression by DCs;b)IL-15,especially membrane bound IL-15 on DCs promotes the survival of CD8T cells in the contraction phase.Based on our experimental data and these reports,we speculate that during the contraction phase of CD8T cell immune response,NK cells may promote IL-15 expression by DCs in a perforin dependent manner and thus indirectly promote the generation of CD8Tm cells via DCs.We first analyzed IL-15Rαand membrane bound IL-15 expression by murine splenic DCs during the contraction phase.Using FACS analysis of splenocytes,we observed reduced IL-15Rαand membrane bound IL-15 expression by CD11c~(hi)la~(b+) splenic DCs in both NK cell depleted WT mice and PFR mice when compared with WT control mice.More importantly,adoptive transferred activated WT NK cells restored IL-15Rαand membrane bound IL-15 expression by CD11c~(hi)la~(b+) splenic DCs in PFR mice,whereas PFR NK cells failed to restore IL-15Rαand IL-15 on DCs.By using fluorenscent immunohistochemical analysis,we observed that during the contraction phase of primary CD8T cell immune response,NK cells and DCs were localized closely in the area between T cell zones and B cell zones.NK-DC juxtaposition could be visualized which indicated direct cell-cell contact between NK cells and DCs in the contraction phase.
We observed that after stimulation with IL-2 and IL-15 in vitro,perforin protein was evenly distributed within cytoplasma of NK cells derived from wild type mice but not PFR mice.When cocultured with bone marrow-derived matured DCs (mDCs)for 1 hour,these in vitro activated NK cells form conjugates with mDCs and more importantly,perforin was centered to the site of NK-DC synapse.These data indicate that NK cells could have certain impact on mDCs via perforin secretion.
Activated wild type NK cells,but not PFR NK cells could promote mDCs to express IL-15Rαand membrane bound IL-15 after coculture in vitro.When wild type NK cells and PFR NK cells were mixed in a 1:1 ratio,they could promote IL-15Rαand membrane bound IL-15 expression by DCs to the same level as wild type NK cells.Considering that IL-15 is crucial in the generation of CD8Tm cells, these data indicate that enhancement of IL-15 and IL-15Rαexpression on DCs by NK cell-drerived perforin might be one of mechanisms by which NK cells promote CD8Tm cell generation.
In conclusion,our data demonstrate that NK cells promote the generation of CD8Tm cells via promoting the survival of activated CD8T cells in the contraction phase after primary immune responses.NK cell-derived perforin is required in the promotion of CD8Tm cell generation by NK cells.We have also shown that NK cells form conjugates with DCs and promote IL-15 and IL-15Rαexpression by DCs in a perforin dependent manner both in vitro and in vivo.Thus,NK cells promote CD8Tm cell generation presumbly via promoting the expression of IL-15Rαand membrane bound IL-15 on DCs.
In CD8Tm cell-mediated immune responses against infections,the quantity and quality of a pathogen/antigen specific CD8 Tm cell pool are key factors that determine whether the host could survive or die of uncontrolled infection.The generation of protective CD8 T cell immunity is also crucial in inoculation with inactivated or virulance attenuated vaccines to fight against possible infections. Thus,exploring the cellular as well as molecular mechanisms that regulate virulance attenuated pathogen-induced CD8Tm cell generation quantitively and qualitively will be valuable both theoretically and practically.In our current study, we provide with a novel perspective in respect to a promoting role of NK cells in the generation of CD8Tm cells.And our data may provide theoretical basis for better immunotherapy as well as vaccination design in the future.
以往的研究大多集中在对CD8Tm细胞产生和功能起关键调控作用的分子上,但对CD8Tm调控的细胞免疫学基础与相关机制研究较少。现有研究表明,树突状细胞(Dendritic cells,DC)能够通过表达IL-15和IL-15Rα促进CD8Tm细胞自稳性增殖,维持CD8Tm细胞数量稳定。但是其它免疫细胞,特别是与CD8T细胞免疫应答密切相关的天然杀伤细胞(Natural killer cells,NK)在CD8T细胞免疫记忆中是否发挥调控作用目前尚不清楚。
NK细胞能够迅速发挥细胞毒性作用,因此在感染早期的机体免疫防御中发挥重要作用。近年来的研究表明,NK细胞的免疫调节作用也不容忽视,在初次应答的天然免疫阶段,NK细胞能够通过分泌穿孔素、颗粒酶以及IFN-γ等可溶性分子直接影响后来的T细胞免疫应答,同时NK细胞还能够通过细胞因子分泌和膜接触依赖的方式促进DC成熟,从而间接调控T细胞免疫应答。这提示NK细胞可能对记忆性CD8T细胞的产生有一定的调控作用,但目前为止尚未见此方面的研究报道。
因此,我们利用小鼠李斯特菌感染模型诱导抗原特异性CD8T细胞免疫应答和免疫记忆,研究了NK细胞在CD8Tm形成过程中可能发挥的调节作用,并对其中的分子机制进行了探索。
1.NK细胞通过调节初次应答收缩期而促进CD8Tm细胞产生
为诱导产生CD8Tm细胞,我们将Thy-1.1~+ OT-I TCR转基因小鼠的CD8 T细胞通过尾静脉注射过继回输到野生型C57小鼠(Thy-1.2)体内,24h后通过尾静脉注射表达OVA的李斯特菌毒力减弱株(△actA LM-OVA)进行感染。感染后7天内,小鼠体内Thy-1.1~+ CD8 T细胞发生强烈的应答并大量增殖。感染40天后,可以在包括外周血、脾脏、淋巴结和肝脏在内的脏器中检测到一定比例的Thy-1.1~+ CD8 T细胞,这群细胞具有记忆性CD8T细胞的表型特征,即CD44~(hi),CD62L~(hi)/CD62L~(lo),更重要的是,这群细胞在体外OVA_(257-264)肽再刺激数小时后即可检测到细胞内IFN-γ分泌。此外,体内存在Thy-1.1~+ CD8 Tm的小鼠再次感染表达OVA的李斯特菌毒力株(LM-OVA)后,能够更快清除细菌。感染100天后,Thy-1.1~+ CD8 T细胞仍然存在并具有同样的表型和功能。这说明,我们诱导出了能够长期存活并具有保护功能的抗原特异性CD8Tm细胞。
接着我们采用抗体体内注射方法清除NK细胞,观察NK细胞清除后CD8T细胞初次应答情况及CD8Tm细胞产生情况。PK136杂交瘤产生特异性针对小鼠NK1.1分子的单克隆抗体,我们制备该杂交瘤腹水并进行抗体纯化定量。day-2和day0(感染当天)时各给予一次PK136抗体或同型对照抗体腹腔注射清除NK1.1~+细胞,通过动态观察发现,在最后一次抗体注射后的3周内,NK细胞的清除率维持在90%以上,NK细胞数在第4周末时基本恢复如常,这可能是由于过量抗体在体内维持并持续清除新产生的少量NK细胞,直至抗体代谢完全。清除NK细胞后,对CD8T细胞初次免疫应答的分析发现,在免疫应答高峰期(感染后7天),NK细胞清除组小鼠Thy-1.1~+ CD8T细胞在各脏器中的比例以及在脾脏中的绝对数均与对照组相似,两组小鼠中Thy-1.1~+效应性CD8T细胞的表型特征(CD27,CD44,CD62L,CD122,CD127,KLRG-1)及细胞因子分泌(IFN-γ,TNF-α,IL-2)均未见明显差异。这说明在针对李斯特菌感染的初次免疫应答过程中,NK细胞对CD8T细胞初次应答的数量和质量上均没有重要影响。
但在感染后第40天,NK细胞清除组小鼠中Thy-1.1~+ CD8Tm细胞在各脏器CD8~+细胞中所占比例和脾脏中的绝对数均显著低于对照组。我们首先排除了Thy-1.1~+ CD8T细胞被PK136抗体直接清除的可能性:流式分析表明,在感染第0,3,7,14,21,40时,该群CD8T细胞均不表达NK1.1分子;如果在感染第7天注射PK136抗体,24h后NK细胞被有效清除,但Thy1.1~+ CD8T细胞数量和比例不变。感染后不同时间点采集外周血进行动态分析发现,两组小鼠中Thy-1.1~+ CD8T细胞在外周血CD8~+细胞中的比例在第7天时基本相同,但到第21天时,NK细胞清除组该比例仅为对照组的1/5,这一1:5的相对比例一直维持到感染40天以后。通过BrdU摄取实验和胞内Bcl-2流式分析发现,虽然两组小鼠中Thy-1.1~+ CD8T细胞在收缩期时增殖无显著差异,但NK细胞清除后,Thy-1.1~+ CD8T细胞胞内Bcl-2分子平均表达水平明显低于对照组。虽然PK136处理组小鼠CD8Tm细胞数显著降低,但两组小鼠中CD8Tm细胞在单个细胞水平上的“质量”并没有明显差异,表现在该群记忆性CD8T细胞群体内部各表型间比例,以及体外再刺激下胞内细胞因子染色的平均荧光强度在两组间没有明显差异。感染后第40天(此时NK细胞数已恢复)给予表达OVA的李斯特菌毒力株(LM-OVA),感染72h后发现对照组肝脏和脾脏中李斯特菌CFU数均显著低于NK细胞清除组。实验结果说明,NK细胞促进活化CD8T细胞在收缩期的存活,进而使CD8Tm细胞数量和保护力维持在较高的水平。
虽然NK细胞清除后对CD8T细胞初次应答的细胞增殖,表型和细胞因子分泌没有显著影响,但这并不能排除NK细胞通过影响CD8T细胞初次应答来影响CD8Tm细胞的产生,而这种对初次应答的影响可能是我们没有检测到的。为进一步阐明NK细胞促进CD8Tm细胞的产生是作用于收缩期而不是扩增期,我们设计了如下的实验:将Thy-1.1~+ OT-I CD8 T细胞过继回输到经PK136抗体或对照抗体处理的C57小鼠(第一受体)体内并用ΔactA LM-OVA感染,7天后将两组小鼠脾脏中的Thy-1.1~+ CD8T细胞分离纯化后等量分别回输到同步感染的经PK136抗体或对照抗体处理的第二受体小鼠体内。三周后,对Thy-1.1~+记忆性CD8T细胞进行分析发现,无论Thy-1.1~+效应性CD8T细胞来自PK136抗体组还是对照组,回输到对照组第二受体小鼠后,均能产生相似数量和比例的CD8Tm细胞;相反,PK136处理组第二受体小鼠体内产生的记忆性细胞显著低于对照。这一证据充分说明NK细胞促进CD8Tm细胞产生的作用时间是初次应答的收缩期。
2.NK细胞通过穿孔素依赖的方式促进收缩期CD8T细胞存活
前面的实验中我们证明了NK细胞通过促进收缩期CD8T细胞存活促进CD8Tm细胞产生,那么,NK细胞通过哪个(些)重要的分子来发挥这一作用呢?
穿孔素(perforin)组成型表达于NK细胞,是NK细胞发挥杀伤作用的重要效应分子。最新研究表明,穿孔素还具有重要的免疫调节功能。例如,在热休克蛋白Gp96-肽复合体所诱导的抗肿瘤免疫应答中,穿孔素基因敲除(PFR)小鼠中单独回输的野生型CD8T细胞不能产生有效的抗肿瘤免疫应答,而同时回输野生型NK细胞能够恢复CTL应答。这一研究结果不仅证明了穿孔素的免疫调节作用,还提示NK细胞和CD8T细胞来源的穿孔素在免疫应答过程中可能发挥不同的作用。我们首先检测了在PFR小鼠中,过继回输的Thy-1.1~+ OTI CD8Tm细胞的产生情况。我们发现,Thy-1.1~+ CD8T细胞回输到PFR小鼠中后,ΔactA LM-OVA感染后CD8T细胞初次应答的数量和质量均没有显著变化。但与NK细胞清除小鼠中的结果相似,回输到PFR小鼠中的CD8T细胞在感染后21天时数量显著低于对照小鼠,感染40天后PFR小鼠中Thy1.1~+CD8Tm数显著低于对照小鼠。但PFR小鼠中Thy-1.1~+ CD8Tm细胞群体中各表型间的比例和单个细胞水平的细胞因子分泌能力与对照小鼠中的CD8Tm细胞相似。
那么,PFR小鼠中的CD8Tm细胞减少是由于其NK细胞不能表达穿孔素所引起的吗?我们对此进行了进一步的研究。我们发现,如果在上述模型中感染后第7天时向PFR小鼠中回输来自同步感染小鼠的野生型NK细胞,那么其体内Thy1.1~+CD8Tm细胞的比例和数量可以恢复到与野生型小鼠相似的水平,而回输穿孔素缺陷的NK细胞或者回输野生型的活化CD8T细胞(表达穿孔素)则没有这种作用。我们还采用了另外一种方式来确认上述结果:在野生型小鼠中回输Thy-1.1~+OTI CD8T细胞并感染,7天后纯化脾脏Thy-1.1~+CD8T细胞,按相同细胞数分别回输到同步感染的野生型C57小鼠或PFR小鼠中,在PFR小鼠中同时回输经活化的野生型或者穿孔素缺陷的NK细胞。我们发现,回输野生型NK细胞后,PFR小鼠中能够产生与野生型小鼠相似数量的Thy1.1~+CD8Tm细胞,而回输穿孔素缺陷的NK细胞则不具有这一作用。以上结果充分证明,NK细胞通过穿孔素依赖的方式促进了CD8Tm细胞产生。
我们从细胞存活和增殖两个方面对NK细胞的收缩期调节作用进行了分析。通过BrdU摄取实验发现,回输到NK细胞清除和对照组野生型C57小鼠中,以及回输到PFR小鼠并回输NK细胞的各组中,Thy-1.1~+CD8T细胞在收缩期的增殖没有显著差异;但是通过胞内Bcl-2分子标记后发现,PFR小鼠体内Thy-1.1~+CD8T细胞在收缩期时的Bcl-2分子表达水平低于对照组,而在PFR小鼠中回输野生型NK细胞后能够提高Thy-1.1~+CD8T细胞在收缩期时Bcl-2分子表达水平。这说明NK细胞通过穿孔素依赖的方式促进CD8T细胞在收缩期的存活,进而促进CD8Tm细胞的产生。
3.NK细胞通过穿孔素依赖的方式促进了树突状细胞表达IL-15和IL-15Rα可能与NK细胞促进了记忆性CD8T细胞产生有关
我们通过前两部分的实验发现,NK细胞能够通过穿孔素依赖的方式促进CD8T细胞在收缩期的存活,进而促进记忆性CD8T细胞产生。现有研究表明,1)NK细胞能够与DC形成免疫突触,促进DC成熟并上调IL-15表达;2)IL-15,特别是DC表达的膜结合型IL-15能够促进CD8T细胞在收缩期的存活。根据上述实验结果和文献报道我们推测,NK细胞可能通过与DC形成免疫突触,以穿孔素依赖的方式促进DC表达IL-15Rα和膜结合型IL-15,进而通过DC间接促进CD8Tm细胞的产生。
我们首先检测了各组小鼠在初次应答的收缩期时DC表达IL-15Rα和膜结合型IL-15的情况。感染后第10天时,取小鼠脾脏进行流式分析发现,清除NK细胞的野生型小鼠和PFR小鼠中CD11c~(hi)la~(b+)的DC表面IL-15Rα和膜结合型IL-15的表达水平低于对照组野生型小鼠。回输野生型NK细胞的PFR小鼠脾脏CD11c~(hi)la~(b+)的DC表面IL-15Rα和膜结合型IL-15的表达恢复到与野生型对照小鼠相似的水平,而回输穿孔素缺陷的NK细胞与不回输细胞组水平相当。感染后第14天时也有相同结果。通过荧光免疫组织化学方法我们发现,在免疫应答的收缩期(day10),NK细胞和DC大多定位于淋巴组织中T细胞区和B细胞区之间的狭小区域中,并且可以观察到NK与DC成紧密的并排排列,这提示在免疫应答的收缩期,NK仍然能够与DC发生直接的细胞接触和相互作用。
形态学观察发现,野生型小鼠的NK细胞经IL-2和IL-15活化后,其胞浆内可检测到均匀分布的穿孔素蛋白,而穿孔素缺陷小鼠的NK细胞中不能检测到穿孔素。经过体外活化的NK细胞与成熟的骨髓来源的DC共同培养1h后,NK细胞与DC能够形成免疫突触,可以观察到NK细胞将穿孔素集中运输到免疫突触部位。这一结果充分说明在NK与DC形成的免疫突触中,NK细胞能够通过分泌穿孔素作用于DC。
将体外活化的NK细胞与骨髓来源的成熟DC共培养后发现,野生型NK细胞能够促进DC上调膜表面IL-15Rα和IL-15的表达水平,而穿孔素缺陷的NK细胞几乎没有促进DC表达IL-15Rα和膜结合型IL-15的能力,而野生型NK细胞和穿孔素缺陷NK细胞等量混合细胞(NK细胞总数保持不变)能够促进DC表达膜结合型IL-15和IL-15Rα。可见,NK细胞通过穿孔素依赖的方式促进了DC表达IL-15和IL-15Rα可能参与了NK细胞促进了CD8Tm细胞产生的过程。
综上,我们的实验结果证明,NK细胞能够在初次免疫应答的收缩期促进活化CD8T细胞存活,进而促进记忆性CD8T细胞产生,而NK细胞表达的穿孔素在这一过程中起到关键作用。我们还通过体内和体外实验证明,在CD8T细胞免疫应答的收缩期,NK细胞能与DC发生相互作用,并且通过穿孔素依赖的方式促进DC表达IL-15Rα和膜结合型IL-15。即NK细胞可能通过DC及其表达的IL-15Rα和膜结合型IL-15间接地促进了记忆性CD8T细胞产生。
在病原体感染所诱导的CD8T细胞免疫记忆中,病原/抗原特异性记忆性CD8T细胞的数量和质量是宿主是否能在再次感染中存活的关键因素。免疫接种的基本原理是利用针对灭活疫苗或者减毒活疫苗的初次应答产生并维持包括记忆性T细胞在内的记忆性细胞群体,接种后免疫记忆的有效产生和维持对于防范此类感染性疾病具有重大意义。因此,对免疫记忆,特别是减毒株病原体接种后保护性免疫记忆的产生和维持的细胞和分子免疫学调控机制的深入研究具有重要的理论和实践价值。我们的研究从一个新的角度揭示了NK细胞对CD8T细胞免疫记忆的调节功能,丰富了人们对NK细胞功能和对CD8T细胞免疫记忆调控的认识,为感染性疾病的免疫治疗和疫苗设计提供了新的思路和一定的理论依据。
Antigen-specific immunological memory is a cardinal feature of immunity, which depends on generation and maintenance of memory T lymphocytes,B lymphocytes and plasma cells with antigen specificity.Mechanisms for generation and maintenance of memory CD8T(CD8Tm) cells are of special interests as they play a crucial role in immune responses against infections and tumors.
Much attention has been paid to key molecules regulating the generation and functions of CD8Tm cells,whereas little is known about the role of populations of cells in regulation of CD8Tm cell generation and functions.Recent data demonstrated that dendritic cells(DCs) promote homeostatic proliferation of memory CD8T cells via IL-15 and IL-15Rα.However,it is unknown whether other immune cells,such as natural killer(NK) cells which are closely related to CD8T cell mediated immune responses,play a role in the regulation of CD8Tm cells.
NK cells mediate fast cytotoxicity against target cells and play crucial roles in immune defense early in an infection.It has been clearly shown in recent years that activated NK cells mediate equally important immuno-modulatory functions. In innate immune response early in an infection,NK cells polarize the ensuing T cell response directly via secretion of soluble factors such as perforin,granzyme and IFN-γ.Meanwhile,NK cells influence T cell response indirectly through promoting maturation of DCs via a both cell-to-cell contact and soluble dependent manner.These data suggest that NK cells may have regulatory functions in CD8Tm generation.
In our current study,we generated antigen specific CD8 T cell immune response and memory using a murine model of Listeria Monocytogenes(LM) infection.We investigated the possible roles of NK cells in the regulation of CD8Tm cell generation and the underlying cellular and molecular mechanisms.
1.Natural killer cells promote CD8 Tm cell generation by regulating the extent of contraction.
To generate CD8Tm cells,we adoptively transferred Thy-1.1 OT-I CD8 T cells in to host C57 mice(Thy-1.2) followed by injection of a virulance attenuated stain of Listeria Monocytogenes(ΔactA LM-OVA)via the tail veil 24h later.On day7 after infection,we detected robust expansion in number of Thy-1.1~+ CD8T cells. On day40 after infection,donor derived Thy-1.1~+ CD8Tm cells were detectable in various organs including peripheral blood,spleen,lymph nodes,and liver.These cells were CD44~(hi),CD62L~(hi)/CD62L~(lo) in phenotype and more importantly,these cells were readily detectable for intracellular IFN-γsecretion several hours after ex vivo restimulation with OVA_(257-264) peptide.Moreover,mice carrying Thy-1.1~+ CD8Tm cells were significantly more resistant to challenge with virulant strain of OVA expressing LM(LM-OVA).Thy-1.1~+ CD8Tm cells were detectable with similar phenotype and function 100 days after infection.These data suggest that we have generated long-lived and functional CD8Tm cells with antigen specificity.
We next investigated whether NK cells are required in primary CD8 T cell response and/or CD8Tm generation by in vivo depletion of NK cells using PK136 monoclonal antibody which is specific to murine NK1.1.We gave one dose intraperitoneally of PK136 Ab or isotype control Ab on day-2 and day0, respectively.We observed a NK cell depletion of over 90%within 3 weeks since the last dose of Ab.Normal NK cell number was observed at the end of week 4. We speculated that excess dose of PK136 Ab existed in mice which depleted newly generated NK cells.We found that after NK cell depletion,the percentage and the absolute number of donor derived Thy-1.1~+ CD8 T cells were comparable in PK136 Ab and isotype Ab treated mice on day 7 after infection,with similar phenotype(CD27,CD44,CD62L,CD122,CD127,KLRG-1)and cytokine secretion profile(IFN-γ,TNF-α,IL-2).Thus,our data demonstrated that NK cells are not required in primary CD8T cell response afterΔactA LM-OVA infection.
Unexpectedly,we observed significantly lowered percentage and number of Thy-1.1~+ CD8 Tm cells 40 days after infection.We first exclude the possibility that Thy-1.1~+CD8T cells were depleted by PK136 Ab by showing that only marginal percentage of Thy-1.1~+ CD8T effector cells were positive for NK1.1 staining on days 0,3,7,14,21 and 40 after infection,and PK136 Ab did not deplete Thy-1.1~+ CD8T effector cells when injected on day 7 after infection and analyzed 24 hours later.By blood sample collection at different time points,we monitored the kinetics of the percentage of Thy-1.1~+ CD8T cells in blood CD8~+ T cells.Though similar percentage of Thy-1.1~+ effector CD8T cells were generated on day 7 after infection,the percentage of Thy-1.1~+ CD8T cells in total blood CD8~+ T cells in NK cell depleted group was about 1/5 that in control group on day 21,and this proportion was essentially fixed in the ensuing days and weeks which resulted in reduced generation of CD8Tm cells.Intracellular detection of Bcl-2 and BrdU incorporation suggested that the survival of Thy-1.1~+ CD8T cells was impaired in NK cell depleted mice,though proliferation of Thy-1.1~+CD8T cells in two groups was indistinguishable.We analyzed surface marker expression as well as cytokine secretion by Thy-1.1~+ CD8Tm cells.Though small in number,Thy-1.1~+ CD8Tm cells generated in NK cell depleted mice were similar in surface marker expression and were equally able in IFN-γ,TNF-α,and IL-2 secretion when restimulated ex vivo with OVA_(257-264)peptide.After challenging withLM-OVA on day 40,we observed reduced protection of CD8Tm cells as a population in NK cell depleted mice as CFUs of LM-OVA in both the spleen and the liver were significantly higher in NK cell depleted mice.Note that NK cells recovered to a normal level on day 40,our data suggested that NK cells promote the survival of activated CD8 T cells in the contraction phase and thus help to generate a protective CD8Tm cell pool.
We did further experiments to confirm that the role of NK cells on activated CD8 T cells was mediated in the contraction phase rather than in the expansion phase,where NK cells may presumably influence the intrinsic quality of CD8 T effector cells which is not detected in our experiments.Thy-1.1~+ OT-I CD8 T cells were adoptively transferred into Thy-1.2~+ host C57 mice treated with either isotype or PK136 Ab as mentioned above followed by injection i.v.withΔactA LM-OVA.On day 7 after infection,equal number of Thy-1.1~+ CD8 T effector cells from either hosts were sorted and adoptively transferred into second host mice(Thy-1.2) which were also on day 7 after infection and treated with isotype or PK136 Ab,respectively.Three weeks after transfer of CD8 effector T cells, absolute number of spleen Thy-1.1~+ CD8 Tm cells and percentages of Thy-1.1~+ cells in CD8 T cells in peripheral blood,spleen and lymph nodes were analyzed. CD8 Tm cell generation was impaired in NK cell depleted second hosts irrespective of whether effector CD8 T cells were generated in NK cell depleted or control mice.In contrast,both effector Thy-1.1~+ CD8T cells isolated from control or NK cell depleted mice generated normal number of CD8Tm cells after transfer into control second hosts.Thus,we provided convincing data showing that the role of NK cells in promoting CD8Tm cell generation were mediated during the contraction phase rather than in the expansion phase after primary infection.
2.NK cells promote the survival of contracting CD8 T cells via a perforin dependent mechanism.
We went on to ask which molecule(s) is required for NK cells in the promotion of the survival of contracting CD8T cells.
Perforin is constitutively expressed by NK cells and is a key molecule in NK mediated cytotoxicity.In heat shock protein gp96-peptide complexes induced tumor rejection model,antigen specific CD8T cells(perforin competent) adoptively transffered into perforin knock out mice failed to proliferate and differentiate into CTLs whereas co-transfer of wide type NK cells rescued the clonal expansion and CTL differentiation of transferred CDST cells.These results suggested important immuno-modulatory roles of perforin and presumably different roles of perforin secreted by NK cells versus by CD8~+ CTLs.Using the same method mentioned above,we adoptively transferred Thy-1.1 OT-I CD8 T cells into wide type B6 mice(W-F) and perforin knock out(PFR)mice on a B6 background followed byΔactA LM-OVA infection.On day 7 after infection, comparable numbers of effector Thy-1.1~+ CD8 T cells with similar phenotype and cytokine secretion profile were generated in WT and PFR mice.However,a more extensive contraction of Thy-1.1~+ CD8 T cells was observed in PFR mice compared with those in WT mice.Consistent with those in NK cell depleted mice, the percentage of effector Thy-1.1~+ CD8 T cells in PFR mice was significantly lower than that in WT control group and number of Thy-1.1~+ CD8Tm cells was significantly decreased compared with that in WT mice 40 days after infection.In accordance with those in NK cell depleted mice,Thy-1.1~+ CD8Tm cells generated in PFR mice were similar in phenotype and cytokine secretion profile on a single cell basis.
To confirm that reduced CD8Tm cell generation in PFR mice was due to perforin incompetence within NK cell population rather than in other cells that could also express perforin after infection(e.g.CTLs),we adoptively transferred WT NK cells or PFR NK cells isolated from simultaneously infected WT or PFR mice on day 7 after infection into PFR mice transferred with Thy-1.1~+ CD8 T cells followed by infection.WT NK cells almost fully rescued generation of CD8Tm cells in PFR mice whereas PFR NK cells failed to do that.Activated CD8 CTLs isolated from infected WT mice could not rescue the generation of Thy-1.1~+ CD8Tm cells in PFR mice,either.Thus,our data clearly showed that perforin is required in NK cell mediated promotion of CD8 Tm cell generation.This conclusion was further confirmed when effector CD8 T cells were generated in WT mice and adoptively transferred into WT or PFR second hosts on day 7 after infection.Co-injection of WT NK cells almost fully rescued CD8Tm cell generation in PFR second host mice whereas PFR NK cells did not.
We analyzed the proliferation and the survival of Thy-1.1~+ effector CD8 T cells in the contraction phase.The proliferation of effector Thy-1.1 ~+CD8 T cells was not different as shown by BrdU incorporation.However,analysis of Bcl-2 expression showed that effector Thy-1.1 ~+CD8 T cells were more resistant to apoptosis in VVT mice than PFR mice.And adoptive transfer of WT NK cells restored Bcl-2 level in effector Thy-1.1 ~+CD8 T cells in PFR mice.Thus,our data demonstrate that perforin is required for NK cells in promotion of CD8Tm generation via promoting survival of contracting CD8T cells.
3.NK cells promote IL-15 and IL-15Rαexpression by dendritic cells in a perforin dependent manner,outlining one of possible mechamisns for the promotion of memory CD8 T cell generation by NK cells
We have shown in the first two parts that NK cells promote CD8Tm cell generation via promoting survival of CD8T cells in a perforin dependent manner.It is reported that a)NK cells form conjugates with DCs and promote IL-15 expression by DCs;b)IL-15,especially membrane bound IL-15 on DCs promotes the survival of CD8T cells in the contraction phase.Based on our experimental data and these reports,we speculate that during the contraction phase of CD8T cell immune response,NK cells may promote IL-15 expression by DCs in a perforin dependent manner and thus indirectly promote the generation of CD8Tm cells via DCs.We first analyzed IL-15Rαand membrane bound IL-15 expression by murine splenic DCs during the contraction phase.Using FACS analysis of splenocytes,we observed reduced IL-15Rαand membrane bound IL-15 expression by CD11c~(hi)la~(b+) splenic DCs in both NK cell depleted WT mice and PFR mice when compared with WT control mice.More importantly,adoptive transferred activated WT NK cells restored IL-15Rαand membrane bound IL-15 expression by CD11c~(hi)la~(b+) splenic DCs in PFR mice,whereas PFR NK cells failed to restore IL-15Rαand IL-15 on DCs.By using fluorenscent immunohistochemical analysis,we observed that during the contraction phase of primary CD8T cell immune response,NK cells and DCs were localized closely in the area between T cell zones and B cell zones.NK-DC juxtaposition could be visualized which indicated direct cell-cell contact between NK cells and DCs in the contraction phase.
We observed that after stimulation with IL-2 and IL-15 in vitro,perforin protein was evenly distributed within cytoplasma of NK cells derived from wild type mice but not PFR mice.When cocultured with bone marrow-derived matured DCs (mDCs)for 1 hour,these in vitro activated NK cells form conjugates with mDCs and more importantly,perforin was centered to the site of NK-DC synapse.These data indicate that NK cells could have certain impact on mDCs via perforin secretion.
Activated wild type NK cells,but not PFR NK cells could promote mDCs to express IL-15Rαand membrane bound IL-15 after coculture in vitro.When wild type NK cells and PFR NK cells were mixed in a 1:1 ratio,they could promote IL-15Rαand membrane bound IL-15 expression by DCs to the same level as wild type NK cells.Considering that IL-15 is crucial in the generation of CD8Tm cells, these data indicate that enhancement of IL-15 and IL-15Rαexpression on DCs by NK cell-drerived perforin might be one of mechanisms by which NK cells promote CD8Tm cell generation.
In conclusion,our data demonstrate that NK cells promote the generation of CD8Tm cells via promoting the survival of activated CD8T cells in the contraction phase after primary immune responses.NK cell-derived perforin is required in the promotion of CD8Tm cell generation by NK cells.We have also shown that NK cells form conjugates with DCs and promote IL-15 and IL-15Rαexpression by DCs in a perforin dependent manner both in vitro and in vivo.Thus,NK cells promote CD8Tm cell generation presumbly via promoting the expression of IL-15Rαand membrane bound IL-15 on DCs.
In CD8Tm cell-mediated immune responses against infections,the quantity and quality of a pathogen/antigen specific CD8 Tm cell pool are key factors that determine whether the host could survive or die of uncontrolled infection.The generation of protective CD8 T cell immunity is also crucial in inoculation with inactivated or virulance attenuated vaccines to fight against possible infections. Thus,exploring the cellular as well as molecular mechanisms that regulate virulance attenuated pathogen-induced CD8Tm cell generation quantitively and qualitively will be valuable both theoretically and practically.In our current study, we provide with a novel perspective in respect to a promoting role of NK cells in the generation of CD8Tm cells.And our data may provide theoretical basis for better immunotherapy as well as vaccination design in the future.
引文
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1. Yokoyama WM, Kim S, French AR. The dynamic life of natural killer cells. Annu.Rev.lmmunol. 2004;22:405-429.
2. Strowig T, Brilot F, Munz C. Noncytotoxic functions of NK cells: direct pathogen restriction and assistance to adaptive immunity. J.Immunol. 2008; 180:7785-7791.
3. Piccioli D, Sbrana S, Melandri E, Valiante NM. Contact-dependent stimulation and inhibition of dendritic cells by natural killer cells. J.Exp.Med. 2002;195:335-341.
4. Adam C, King S, Allgeier T et al. DC-NK cell cross talk as a novel CD4+ T-cell-independent pathway for antitumor CTL induction. Blood 2005; 106:338-344.
5. Gerosa F, Baldani-Guerra B, Nisii C et al. Reciprocal activating interaction between natural killer cells and dendritic cells. J.Exp.Med. 2002; 195:327-333.
6. Flodstrom M, Maday A, Balakrishna D et al. Target cell defense prevents the development of diabetes after viral infection. Nat.lmmunol. 2002;3:373-382.
7. Homann D, Jahreis A, Wolfe T et al. CD40L blockade prevents autoimmune diabetes by induction of bitypic NK/DC regulatory cells. Immunity. 2002; 16:403-415.
8. Moffett-King A. Natural killer cells and pregnancy. Nat.Rev.lmmunol. 2002;2:656-663.
9. Mocikat R, Braumuller H, Gumy A et al. Natural killer cells activated by MHC class l(low) targets prime dendritic cells to induce protective CD8 T cell responses. Immunity. 2003; 19:561-569.
10. Yu YY, Kumar V, Bennett M. Murine natural killer cells and marrow graft rejection. Annu.Rev.lmmunol. 1992; 10:189-213.
11. Fehniger TA, Cooper MA, Nuovo GJ et al. CD56bright natural killer cells are present in human lymph nodes and are activated by T cell-derived IL-2: a potential new link between adaptive and innate immunity. Blood 2003; 101:3052-3057.
12. Jacobs R, Hintzen G, Kemper A et al. CD56bright cells differ in their KIR repertoire and cytotoxic features from CD56dim NK cells. Eur.J.Immunol. 2001 ;31:3121-3127.
13. Moretta A, Bottino C, Vitale M et al. Activating receptors and coreceptors involved in human natural killer cell-mediated cytolysis. Annu.Rev.lmmunol. 2001;19:197-223.
14. Campbell JJ, Qin S, Unutmaz D et al. Unique subpopulations of CD56+ NK and NK-T peripheral blood lymphocytes identified by chemokine receptor expression repertoire. J.Immunol. 2001; 166:6477-6482.
15. Hayakawa Y, Smyth MJ. CD27 dissects mature NK cells into two subsets with distinct responsiveness and migratory capacity. J.Immunol. 2006; 176:1517-1524.
16. Vosshenrich CA, Garcia-Ojeda ME, Samson-Villeger SI et al. A thymic pathway of mouse natural killer cell development characterized by expression of GATA-3 and CD127. Nat.lmmunol. 2006;7:1217-1224.
17. Sun JC, Beilke JN, Lanier LL. Adaptive immune features of natural killer cells. Nature 2009;457:557-561.
18. Street SE, Hayakawa Y, Zhan Y et al. Innate immune surveillance of spontaneous B cell lymphomas by natural killer cells and gammadelta T cells. J.Exp.Med. 2004; 199:879-884.
19. Imai K, Matsuyama S, Miyake S, Suga K, Nakachi K. Natural cytotoxic activity of peripheral-blood lymphocytes and cancer incidence: an 11-year follow-up study of a general population. Lancet 2000;356:1795-1799.
20. Ferlazzo G, Pack M, Thomas D et al. Distinct roles of IL-12 and IL-15 in human natural killer cell activation by dendritic cells from secondary lymphoid organs. Proc.Natl.Acad.Sci.U.S.A 2004;101:16606-16611.
21. Yu Y, Hagihara M, Ando K et al. Enhancement of human cord blood CD34+ cell-derived NK cell cytotoxicity by dendritic cells. J.Immunol. 2001; 166:1590-1600.
22. Orange JS, Biron CA. Characterization of early IL-12, IFN-alphabeta, and TNF effects on antiviral state and NK cell responses during murine cytomegalovirus infection. J.Immunol. 1996;156:4746-4756.
23. Lucas M, Schachterle W, Oberle K, Aichele P, Diefenbach A. Dendritic cells prime natural killer cells by trans-presenting interleukin 15. Immunity. 2007;26:503-517.
24. Orange JS, Biron CA. An absolute and restricted requirement for IL-12 in natural killer cell IFN-gamma production and antiviral defense. Studies of natural killer and T cell responses in contrasting viral infections. J.Immunol. 1996;156:1138-1142.
25. Brilot F, Strowig T, Roberts SM, Arrey F, Munz C. NK cell survival mediated through the regulatory synapse with human DCs requires IL-15Ralpha. J.CIin.Invest 2007;117:3316-3329.
26. Strowig T, Brilot F, Arrey F et al. Tonsilar NK cells restrict B cell transformation by the Epstein-Barr virus via IFN-gamma. PLoS.Pathog. 2008;4:e27.
27. Orange JS, Wang B, Terhorst C, Biron CA. Requirement for natural killer cell-produced interferon gamma in defense against murine cytomegalovirus infection and enhancement of this defense pathway by interleukin 12 administration. J.Exp.Med. 1995; 182:1045-1056.
28. Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature 1998;392:245-252.
29. Morandi B, Bougras G, Muller WA, Ferlazzo G, Munz C. NK cells of human secondary lymphoid tissues enhance T cell polarization via IFN-gamma secretion. Eur.J.Immunol. 2006;36:2394-2400.
30. Strbo N, Oizumi S, Sotosek-Tokmadzic V, Podack ER. Perform is required for innate and adaptive immunity induced by heat shock protein gp96. Immunity. 2003; 18:381-390.
31. Gorski KS, Waller EL, Bjornton-Severson J et al. Distinct indirect pathways govern human NK-cell activation by TLR-7 and TLR-8 agonists. Int.lmmunol. 2006;18:1115-1126.
32. Schmidt KN, Leung B, Kwong M et al. APC-independent activation of NK cells by the Toll-like receptor 3 agonist double-stranded RNA. J.Immunol. 2004;172:138-143.
33. Chan A, Hong DL, Atzberger A et al. CD56bright human NK cells differentiate into CD56dim cells: role of contact with peripheral fibroblasts. J.Immunol. 2007; 179:89-94.
2. Heyzer-Williams LJ, Heyzer-Williams MG. Antigen-specific memory B cell development. Annu.Rev.lmmunol. 2005;23:487-513.
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14. Trapani JA, Smyth MJ. Functional significance of the perforin/granzyme cell death pathway. Nat.Rev.lmmunol. 2002;2:735-747.
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1. Yokoyama WM, Kim S, French AR. The dynamic life of natural killer cells. Annu.Rev.lmmunol. 2004;22:405-429.
2. Strowig T, Brilot F, Munz C. Noncytotoxic functions of NK cells: direct pathogen restriction and assistance to adaptive immunity. J.Immunol. 2008; 180:7785-7791.
3. Piccioli D, Sbrana S, Melandri E, Valiante NM. Contact-dependent stimulation and inhibition of dendritic cells by natural killer cells. J.Exp.Med. 2002;195:335-341.
4. Adam C, King S, Allgeier T et al. DC-NK cell cross talk as a novel CD4+ T-cell-independent pathway for antitumor CTL induction. Blood 2005; 106:338-344.
5. Gerosa F, Baldani-Guerra B, Nisii C et al. Reciprocal activating interaction between natural killer cells and dendritic cells. J.Exp.Med. 2002; 195:327-333.
6. Flodstrom M, Maday A, Balakrishna D et al. Target cell defense prevents the development of diabetes after viral infection. Nat.lmmunol. 2002;3:373-382.
7. Homann D, Jahreis A, Wolfe T et al. CD40L blockade prevents autoimmune diabetes by induction of bitypic NK/DC regulatory cells. Immunity. 2002; 16:403-415.
8. Moffett-King A. Natural killer cells and pregnancy. Nat.Rev.lmmunol. 2002;2:656-663.
9. Mocikat R, Braumuller H, Gumy A et al. Natural killer cells activated by MHC class l(low) targets prime dendritic cells to induce protective CD8 T cell responses. Immunity. 2003; 19:561-569.
10. Yu YY, Kumar V, Bennett M. Murine natural killer cells and marrow graft rejection. Annu.Rev.lmmunol. 1992; 10:189-213.
11. Fehniger TA, Cooper MA, Nuovo GJ et al. CD56bright natural killer cells are present in human lymph nodes and are activated by T cell-derived IL-2: a potential new link between adaptive and innate immunity. Blood 2003; 101:3052-3057.
12. Jacobs R, Hintzen G, Kemper A et al. CD56bright cells differ in their KIR repertoire and cytotoxic features from CD56dim NK cells. Eur.J.Immunol. 2001 ;31:3121-3127.
13. Moretta A, Bottino C, Vitale M et al. Activating receptors and coreceptors involved in human natural killer cell-mediated cytolysis. Annu.Rev.lmmunol. 2001;19:197-223.
14. Campbell JJ, Qin S, Unutmaz D et al. Unique subpopulations of CD56+ NK and NK-T peripheral blood lymphocytes identified by chemokine receptor expression repertoire. J.Immunol. 2001; 166:6477-6482.
15. Hayakawa Y, Smyth MJ. CD27 dissects mature NK cells into two subsets with distinct responsiveness and migratory capacity. J.Immunol. 2006; 176:1517-1524.
16. Vosshenrich CA, Garcia-Ojeda ME, Samson-Villeger SI et al. A thymic pathway of mouse natural killer cell development characterized by expression of GATA-3 and CD127. Nat.lmmunol. 2006;7:1217-1224.
17. Sun JC, Beilke JN, Lanier LL. Adaptive immune features of natural killer cells. Nature 2009;457:557-561.
18. Street SE, Hayakawa Y, Zhan Y et al. Innate immune surveillance of spontaneous B cell lymphomas by natural killer cells and gammadelta T cells. J.Exp.Med. 2004; 199:879-884.
19. Imai K, Matsuyama S, Miyake S, Suga K, Nakachi K. Natural cytotoxic activity of peripheral-blood lymphocytes and cancer incidence: an 11-year follow-up study of a general population. Lancet 2000;356:1795-1799.
20. Ferlazzo G, Pack M, Thomas D et al. Distinct roles of IL-12 and IL-15 in human natural killer cell activation by dendritic cells from secondary lymphoid organs. Proc.Natl.Acad.Sci.U.S.A 2004;101:16606-16611.
21. Yu Y, Hagihara M, Ando K et al. Enhancement of human cord blood CD34+ cell-derived NK cell cytotoxicity by dendritic cells. J.Immunol. 2001; 166:1590-1600.
22. Orange JS, Biron CA. Characterization of early IL-12, IFN-alphabeta, and TNF effects on antiviral state and NK cell responses during murine cytomegalovirus infection. J.Immunol. 1996;156:4746-4756.
23. Lucas M, Schachterle W, Oberle K, Aichele P, Diefenbach A. Dendritic cells prime natural killer cells by trans-presenting interleukin 15. Immunity. 2007;26:503-517.
24. Orange JS, Biron CA. An absolute and restricted requirement for IL-12 in natural killer cell IFN-gamma production and antiviral defense. Studies of natural killer and T cell responses in contrasting viral infections. J.Immunol. 1996;156:1138-1142.
25. Brilot F, Strowig T, Roberts SM, Arrey F, Munz C. NK cell survival mediated through the regulatory synapse with human DCs requires IL-15Ralpha. J.CIin.Invest 2007;117:3316-3329.
26. Strowig T, Brilot F, Arrey F et al. Tonsilar NK cells restrict B cell transformation by the Epstein-Barr virus via IFN-gamma. PLoS.Pathog. 2008;4:e27.
27. Orange JS, Wang B, Terhorst C, Biron CA. Requirement for natural killer cell-produced interferon gamma in defense against murine cytomegalovirus infection and enhancement of this defense pathway by interleukin 12 administration. J.Exp.Med. 1995; 182:1045-1056.
28. Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature 1998;392:245-252.
29. Morandi B, Bougras G, Muller WA, Ferlazzo G, Munz C. NK cells of human secondary lymphoid tissues enhance T cell polarization via IFN-gamma secretion. Eur.J.Immunol. 2006;36:2394-2400.
30. Strbo N, Oizumi S, Sotosek-Tokmadzic V, Podack ER. Perform is required for innate and adaptive immunity induced by heat shock protein gp96. Immunity. 2003; 18:381-390.
31. Gorski KS, Waller EL, Bjornton-Severson J et al. Distinct indirect pathways govern human NK-cell activation by TLR-7 and TLR-8 agonists. Int.lmmunol. 2006;18:1115-1126.
32. Schmidt KN, Leung B, Kwong M et al. APC-independent activation of NK cells by the Toll-like receptor 3 agonist double-stranded RNA. J.Immunol. 2004;172:138-143.
33. Chan A, Hong DL, Atzberger A et al. CD56bright human NK cells differentiate into CD56dim cells: role of contact with peripheral fibroblasts. J.Immunol. 2007; 179:89-94.