Khan Selina, Bijker Martijn S, Weterings Jimmy J, Tanke Hans J, Adema Gosse J, van Hall Thorbald, Drijfhout Jan W, Melief Cornelis J M, Overkleeft Hermen S, van der Marel Gijsbert A, Filippov Dmitri V, van der Burg Sjoerd H, Ossendorp Ferry
Department of Immunohematology and Blood Transfusion, Molecular Cell Biology, and Clinical Oncology, Leiden University Medical Centre, P. O. Box 9600, 2300 RC Leiden, The Netherlands.
J Biol Chem. 2007 Jul 20;282(29):21145-59. doi: 10.1074/jbc.M701705200. Epub 2007 Apr 26.
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 approximately conjugate was independent of endosomally-expressed TLR9 as reported previously. Unexpectedly, we found that Pam approximately 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 approximately 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 naïve 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.
将Toll样受体配体（TLR-L）与合成抗原肽共价偶联，可在体外显著增强抗原呈递，并在体内启动T淋巴细胞。这些分子结构明确的TLR-L-肽偶联物构成了一种有吸引力的疫苗接种方式，在单个分子中同时包含肽抗原和特定佐剂。我们分析了两种TLR-L偶联物在树突状细胞（DC）中的细胞内运输和加工过程。含有卵清蛋白细胞毒性T细胞表位的长合成肽与两种不同的TLR-L化学偶联，即TLR2配体Pam(3)CysSK(4)（Pam）或TLR9配体CpG。两种类型的TLR-L偶联肽在DC中均快速且增强摄取。此外，TLR-L偶联显著增强了抗原呈递，这一过程依赖于内体酸化、蛋白酶体裂解和TAP易位。如先前报道，CpG偶联物摄取独立于内体表达的TLR9。出乎意料的是，我们发现Pam偶联肽同样独立于细胞表面表达的TLR2内化。摄取机制的进一步表征显示，TLR2-L采用与TLR9-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可大大降低Pam偶联物的摄取和抗原呈递。相反，CpG偶联物的内化和抗原呈递独立于网格蛋白包被小窝，但部分依赖于小窝形成。重要的是，与偶联物的TLR非依赖性摄取相反，树突状细胞成熟和初始CD8(+) T细胞启动需要TLR表达和下游TLR信号传导。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，但遵循相似的运输和细胞内加工途径，从而实现最佳抗原呈递和T细胞启动。
