Distinct uptake mechanisms but similar intracellular processing of two different Toll-like receptor ligand-peptide conjugates in dendritic cells
Distinct uptake mechanisms but similar intracellular processing of two different Toll-like receptor ligand-peptide conjugates in dendritic cells
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DOI：
10.1074/jbc.m701705200
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发表时间：
2007-07-20
影响因子：
4.8
通讯作者：
Ossendorp, Ferry
中科院分区：
文献类型：
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作者：
Khan, Selina;Bijker, Martijn S.;Ossendorp, Ferry
Toll样受体配体（TLR-L）与合成抗原肽的共价缀合强烈改善体外抗原呈递和体内T淋巴细胞引发。这些分子上明确定义的TLR-L-肽缀合物构成了一种有吸引力的疫苗接种方式，在一个单一分子中共享肽抗原和确定的佐剂。我们分析了两种TLR-L缀合物在树突状细胞（DC）中的细胞内运输和加工。将含有卵清蛋白细胞毒性T细胞表位的长合成肽化学缀合至两种不同的TLR-L，即TLR 2配体、Pam（3）CysSK（4）（Pam）或TLR 9配体CpG。两种类型的TLR-L结合肽的快速和增强的摄取发生在DC中。此外，TLR-L结合大大增强了抗原呈递，这是一个依赖于内体酸化、蛋白酶体切割和TAP易位的过程。与缀合物类似的CpG的摄取不依赖于如先前报道的内体表达的TLR 9。出乎意料的是，我们发现类似于缀合肽的Pam同样独立于细胞表面表达的TLR 2的表达而内化。对摄取机制的进一步表征显示，TLR 2-L采用与TLR 9-L不同的摄取途径。抑制网格蛋白或小窝蛋白依赖性内吞作用大大降低了帕姆-共轭物的摄取和抗原呈递。相反，类似于缀合物的CpG的内化和抗原呈递不依赖于网格蛋白包被的小凹，但部分依赖于小窝形成。重要的是，与缀合物的TLR非依赖性摄取相反，TLR表达和下游TLR信号传导是树突状细胞成熟和初始CD 8（+）T细胞引发所需的。总之，我们的数据表明，靶向两种不同的TLR需要不同的摄取机制，但遵循类似的运输和细胞内加工途径，从而导致最佳的抗原呈递和T细胞引发。
Covalent conjugation of Toll-like receptor ligands (TLR-L) to synthetic antigenic peptides strongly improves antigen presentation in vitro and T lymphocyte priming in vivo. These molecularly well defined TLR-L-peptide conjugates, constitute an attractive vaccination modality, sharing the peptide antigen and a defined adjuvant in one single molecule. We have analyzed the intracellular trafficking and processing of two TLR-L conjugates in dendritic cells (DCs). Long synthetic peptides containing an ovalbumin cytotoxic T-cell epitope were chemically conjugated to two different TLR-Ls the TLR2 ligand, Pam(3)CysSK(4) (Pam) or the TLR9 ligand CpG. Rapid and enhanced uptake of both types of TLR-L-conjugated peptide occurred in DCs. Moreover, TLR-L conjugation greatly enhanced antigen presentation, a process that was dependent on endosomal acidification, proteasomal cleavage, and TAP translocation. The uptake of the CpG similar to conjugate was independent of endosomally-expressed TLR9 as reported previously. Unexpectedly, we found that Pam similar to conjugated peptides were likewise internalized independently of the expression of cell surface-expressed TLR2. Further characterization of the uptake mechanisms revealed that TLR2-L employed a different uptake route than TLR9-L. Inhibition of clathrin- or caveolin-dependent endocytosis greatly reduced uptake and antigen presentation of the Pam-conjugate. In contrast, internalization and antigen presentation of CpG similar to conjugates was independent of clathrin- coated pits but partly dependent on caveolae formation. Importantly, in contrast to the TLR-independent uptake of the conjugates, TLR expression and downstream TLR signaling was required for dendritic cell maturation and for priming of naive CD8(+) T-cells. Together, our data show that targeting to two distinct TLRs requires distinct uptake mechanism but follows similar trafficking and intracellular processing pathways leading to optimal antigen presentation and T-cell priming.