Human immunodeficiency virus (HIV)-and simian immunodeficiency virus (SIV)-specific CD8؉ T cells are typically largely excluded from lymphoid B cell follicles, where HIV-and SIV-producing cells are most highly concentrated, indicating that B cell follicles are somewhat of an immunoprivileged site. To gain insights into virus-specific follicular CD8؉ T cells, we determined the location and phenotype of follicular SIV-specific CD8 ؉ T cells in situ, the local relationship of these cells to Foxp3 ؉ cells, and the effects of CD8 depletion on levels of follicular SIV-producing cells in chronically SIV-infected rhesus macaques. We found that follicular SIV-specific CD8 ؉ T cells were able to migrate throughout follicular areas, including germinal centers. Many expressed PD-1, indicating that they may have been exhausted. A small subset was in direct contact with and likely inhibited by Foxp3؉ cells, and a few were themselves Foxp3 ؉ . In addition, subsets of follicular SIV-specific CD8 ؉ T cells expressed low to medium levels of perforin, and subsets were activated and proliferating. Importantly, after CD8 depletion, the number of SIVproducing cells increased in B cell follicles and extrafollicular areas, suggesting that follicular and extrafollicular CD8؉ T cells have a suppressive effect on SIV replication. Taken together, these results suggest that during chronic SIV infection, despite high levels of exhaustion and likely inhibition by

Natural killer (NK) cells are lymphocyte effectors that are activated to control certain microbial infections and tumors. Many NK-activating and regulating receptors are involved in regulating NK cell function. In addition, activation of naïve NK cells is fundamentally triggered by cytokines or myeloid dendritic cells (mDC) in various modes. In this study, we synthesized 16 S-[2,3-bis(palmitoyl)propyl]cysteine (Pam2Cys) lipopeptides with sequences designed from lipoproteins of Staphylococcus aureus, and assessed their functional properties using mouse (C57BL/6) bone marrow-derived DC (BMDC) and NK cells. NK cell activation was evaluated by three criteria: IFN-γ production, up-regulation of NK activation markers and cytokines, and NK target (B16D8 cell) cytotoxicity. The diacylated lipopeptides acted as TLR2 ligands, inducing up-regulation of CD25/CD69/CD86, IL-6, and IL-12p40, which represent maturation of BMDC. Strikingly, the Pam2Cys lipopeptides induced mouse NK cell activation based on these criteria. Cell-cell contact by Pam2Cys peptide-stimulated BMDC and NK cells rather than soluble mediators released by stimulated BMDC induced activation of NK cells. For most lipopeptides, the BMDC TLR2/MyD88 pathway was responsible for driving NK activation, while some slightly induced direct activation of NK cells via the TLR2/MyD88 pathway in NK cells. The potential for NK activation was critically regulated by the peptide primary sequence. Hydrophobic or proline-containing sequences proximal to the N-terminal lipid moiety interfered with the ability of lipopeptides to induce BMDC-mediated NK activation. This mode of NK activation is distinctly different from that induced by polyI:C, which is closely associated with type I IFN-inducing pathways of BMDC. These results imply that the MyD88 pathway of BMDC governs an alternative NK-activating pathway in which the peptide sequence of TLR2-agonistic lipopeptides critically affects the potential for NK activation.

Intracellularly expressed antibodies (intrabodies) have been used to inhibit the function of various kinds of protein inside cells. However, problems with stability and functional expression of intrabodies in the cytosol remain unsolved. In this study, we show that single‐chain variable fragment (scFv) intrabodies constructed with a heavy chain variable (VH) leader signal sequence at the N‐terminus were translocated from the endoplasmic reticulum into the cytosol of T lymphocytes and inhibited the function of the target molecule, Wiskott–Aldrich syndrome protein (WASP). WASP resides in the cytosol as a multifunctional adaptor molecule and mediates actin polymerization and interleukin (IL)‐2 synthesis in the T‐cell receptor (TCR) signaling pathway. It has been suggested that an EVH1 domain in the N‐terminal region of WASP may participate in IL‐2 synthesis. In transgenic mice expressing anti‐EVH1 scFvs derived from hybridoma cells producing WASP‐EVH1 mAbs, a large number of scFvs in the cytosol and binding between anti‐EVH1 scFvs and native WASP in T cells were detected by immunoprecipitation analysis. Furthermore, impairment of the proliferative response and IL‐2 production induced by TCR stimulation which did not affect TCR capping was demonstrated in the scFv transgenic T cells. We previously described the same T‐cell defects in WASP transgenic mice overexpressing the EVH1 domain. These results indicate that the EVH1 intrabodies inhibit only the EVH1 domain function that regulates IL‐2 synthesis signaling without affecting the overall domain structure of WASP. The novel procedure presented here is a valuable tool for in vivo functional analysis of cytosolic proteins.

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Wiskott-Aldrich syndrome protein (WASP) plays important roles in TCR signaling. In transgenic (Tg) mice, over-expression of the WASP N-terminal region (exons 1-5) including the enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) homology 1 (EVH1) domain and anti-WASP-EVH1 single-chain variable fragment (scFv) intracellular expressed antibodies (intrabodies) impairs IL-2 production in activated T cells. However, it largely remains unknown that how this domain transduces TCR signaling. Here, we demonstrate for the first time that the WASP N-terminal domain specifically associates with the Fyn SH3 domain; the interaction was uncovered by screening a λgt11 cDNA expression library obtained from the mouse T-cell line KKF. The interaction between Fyn and WASP was inhibited by over-expression of the WASP N-terminal domain and anti-WASP-EVH1 scFv intrabodies in gene-transfected NIH3T3 cells and T cells derived from these Tg mice. WASP-interacting protein binding to the EVH1 domain of WASP was also inhibited in these Tg mice T cells. Furthermore, tyrosine phosphorylation of WASP and nuclear translocation of nuclear factor of activated T cells following TCR stimulation was severely inhibited by over-expression of the WASP N-terminal domain. These observations strongly suggest that the WASP N-terminal domain plays a pivotal role in the TCR signaling cascade by binding to Fyn.