The TLRs 7, 8, and 9 stimulate innate immune responses upon recognizing pathogen nucleic acids. U-rich RNA sequences were recently discovered that stimulate human TLR7/8-mediated or murine TLR7-mediated immune effects. In this study we identified single-stranded RNA sequences containing defined sequence motifs that either preferentially activate human TLR8-mediated as opposed to TLR7- or TLR7/8-mediated immune responses. The identified TLR8 RNA motifs signal via TLR8 and fail to induce IFN-α from TLR7-expressing plasmacytoid dendritic cells but induce the secretion of Th1-like and proinflammatory cytokines from TLR8-expressing immune cells such as monocytes or myeloid dendritic cells. In contrast, RNA sequences containing the TLR7/8 motif signal via TLR7 and TLR8 and stimulate cytokine secretion from both TLR7- and TLR8-positive immunocytes. The TLR8-specific RNA sequences are able to trigger cytokine responses from human and bovine but not from mouse, rat, and porcine immune cells, suggesting that these species lack the capability to respond properly to TLR8 RNA ligands. In summary, we describe two classes of single-stranded TLR7/8 and TLR8 RNA agonists with diverse target cell and species specificities and immune response profiles.

p62dok belongs to a newly identified family of adaptor proteins. In T cells, the two members that are predominantly expressed, p56dok and p62dok, are tyrosine phosphorylated upon CD2 or CD28 stimulation, but not upon CD3 ligation. Little is known about the biological role of Dok proteins in T cells. In this study, to evaluate the importance of p62dok in T cell function, we generated Jurkat clones overexpressing p62dok. Our results demonstrate that overexpression of p62dok in Jurkat cells has a dramatic negative effect on CD2-mediated signaling. The p62dok-mediated inhibition affects several biochemical events initiated by CD2 ligation, such as the increase of intracellular Ca2+, phospholipase Cγ1 activation, and extracellular signal-regulated kinase 1/2 activation. Importantly, these cellular events are not affected in the signaling cascade induced by engagement of the CD3/TCR complex. However, both CD3- and CD2-induced NF-AT activation and IL-2 secretion are impaired in p62dok-overexpressing cells. In addition, we show that CD2 but not CD3 stimulation induces p62dok and Ras GTPase-activating protein recruitment to the plasma membrane. These results suggest that p62dok plays a negative role at multiple steps in the CD2 signaling pathway. We propose that p62dok may represent an important negative regulator in the modulation of the response mediated by the TCR.

The Lck tyrosine kinase is involved in signaling by T cell surface receptors such as TCR/CD3, CD2, and CD28. As other downstream protein-tyrosine kinases are activated upon stimulation of these receptors, it is difficult to assign which tyrosine-phosphorylated proteins represent bona fide Lck substrates and which are phosphorylated by other tyrosine kinases. We have developed a system in which Lck can be activated independently of TCR/CD3. We have shown that activation of an epidermal growth factor receptor/Lck chimera leads to the specific phosphorylation of Ras GTPase-activating protein (RasGAP) and two Lck is a member of the Src family PTKs 1 that participates in signal transduction pathways initiated by T cell surface receptors such as TCR/CD3, CD2, CD4, CD8, and CD28. Following cross-linking of these receptors, an increase in its kinase activity has been reported (1-3). Moreover, the absolute requirement of Lck in these pathways has been demonstrated through the use of mutant cell lines that lack Lck expression (4 -8). Two regulatory tyrosine residues, Tyr-505 and Tyr-394, control Lck activity. Dephosphorylation of tyrosine 505, likely by the tyrosine phosphatase CD45, is thought to allow the disruption of an inhibitory intramolecular interaction between the phosphorylated Tyr-505 and the SH2 domain of Lck (9 -11). Moreover, the catalytic and functional activities of Lck are dependent on the phosphorylation of tyrosine 394 (12). Structural domains including the unique SH2 and SH3 domains contribute to regulate Lck kinase activity and specificity (13)(14)(15)(16)(17)(18)(19)(20). In addition, Lck function depends on the ability of the SH3 and SH2 domains to interact with cellular molecules that may represent specific substrates or regulatory proteins (21-25).Although the structural basis for Lck activation is well known, the molecular mechanisms that take place in vivo in Lck activation and function are not well understood. CD4 crosslinking has been shown to enhance Lck activity (3). This might be a consequence of oligomerization of CD4/Lck, a process that takes place upon binding of CD4 to MHC/TCR/Ag (reviewed in Ref. 26). One of the consequences of CD4 activation of Lck is the translocation of Lck to the Nonidet P-40 insoluble cytoskeleton fraction (27). In addition, cross-linking of defined epitopes of the CD4 molecule can lead to the induction of Ca 2ϩ flux, phosphorylation of Shc, and activation of the nuclear factor of activated T cells transcription factor (28). CD4 cross-linking has also been reported to inhibit CD3-mediated signaling by sequestering the CD4-associated Lck from TCR/CD3 (29). Lck can also be activated independently of the CD4 or CD8 coreceptor. Indeed, Lck activity has been reported to increase shortly after CD3 or CD2 cross-linking (2). Stimulation through these receptors results in the phosphorylation of a partially overlapping set of membrane and cytosolic proteins (30, 31). These proteins represent potential in vivo substrates of Lck and several of them such as p95Vav (32), SHP-1 ...

Viruses of the order Mononegavirales encompass life-threatening pathogens with single-stranded segmented or nonsegmented negative-strand RNA genomes. The RNA genomes are characterized by highly conserved sequences at the extreme untranslated 3' and 5' termini that are most important for virus infection and viral RNA synthetic processes. The 3' terminal genome regions of negative-strand viruses such as vesicular stomatitis virus, Sendai virus, or influenza virus contain a high number of conserved U and G nucleotides, and synthetic oligoribonucleotides encoding such sequences stimulate sequence-dependent cytokine responses via TLR7 and TLR8. Immune cells responding to such sequences include NK cells, NK/T cells, plasmacytoid, and myeloid dendritic cells, as well as monocytes and B cells. Strong Th1 and pro-inflammatory cytokine responses are also induced upon in vivo application of oligoribonucleotides. It appears possible that the presence of highly conserved untranslated terminal regions in the viral genome fulfilling fundamental functions for the viral replication may enable the host to induce directed innate immune defense mechanisms, by allowing pathogen detection through essential RNA regions that the virus cannot readily mutate.