Simian Immunodeficiency Virus-Producing Cells in Follicles Are Partially Suppressed by CD8+ Cells In Vivo.

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 SIV-producing 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 Foxp3+ cells, a subset of follicular SIV-specific CD8+ T cells are functional and suppress viral replication in vivo These findings support HIV cure strategies that augment functional follicular virus-specific CD8+ T cells to enhance viral control. IMPORTANCE: HIV- and SIV-specific CD8+ T cells are typically largely excluded from lymphoid B cell follicles, where virus-producing cells are most highly concentrated, suggesting that B cell follicles are somewhat of an immunoprivileged site where virus-specific CD8+ T cells are not able to clear all follicular HIV- and SIV-producing cells. To gain insights into follicular CD8+ T cell function, we characterized follicular virus-specific CD8+ T cells in situ by using an SIV-infected rhesus macaque model of HIV. We found that subsets of follicular SIV-specific CD8+ T cells are able to migrate throughout the follicle, are likely inhibited by Foxp3+ cells, and are likely exhausted but that, nonetheless, subsets are likely functional, as they express markers consistent with effector function and show signs of suppressing viral replication in vivo These findings support HIV cure strategies that increase the frequency of functional follicular virus-specific CD8+ T cells.

Comparison of Vibratome and Compresstome sectioning of fresh primate lymphoid and genital tissues for in situ MHC-tetramer and immunofluorescence staining.

BACKGROUND: For decades, the Vibratome served as a standard laboratory resource for sectioning fresh and fixed tissues. In skilled hands, high quality and consistent fresh unfixed tissue sections can be produced using a Vibratome but the sectioning procedure is extremely time consuming. In this study, we conducted a systematic comparison between the Vibratome and a new approach to section fresh unfixed tissues using a Compresstome. We used a Vibratome and a Compresstome to cut fresh unfixed lymphoid and genital non-human primate tissues then used in situ tetramer staining to label virus-specific CD8 T cells and immunofluorescent counter-staining to label B and T cells. We compared the Vibratome and Compresstome in five different sectioning parameters: speed of cutting, chilling capability, specimen stabilization, size of section, and section/staining quality. RESULTS: Overall, the Compresstome and Vibratome both produced high quality sections from unfixed spleen, lymph node, vagina, cervix, and uterus, and subsequent immunofluorescent staining was equivalent. The Compresstome however, offered distinct advantages; producing sections approximately 5 times faster than the Vibratome, cutting tissue sections more easily, and allowing production of larger sections. CONCLUSIONS: A Compresstome can be used to generate fresh unfixed primate lymph node, spleen, vagina, cervix and uterus sections, and is superior to a Vibratome in cutting these fresh tissues.

Identification of Fyn as the binding partner for the WASP N-terminal domain in T cells.

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.

Cytoplasmic expression and specific binding of the VH/VL single domain intrabodies in transfected NIH3T3 cells.

Intracellularly expressed antibody fragments (intrabodies) have been utilized as powerful tools not only for clinical applications but also for the functional analysis of proteins inside the cell. Among several types of intrabodies developed so far, single domain types composed of only the variable regions (V(H) or V(L)) of antibodies are the smallest and thus the easiest to design. In this study, four types of single domain intrabodies were evaluated against a cytosolic protein, Wiskott-Aldrich syndrome protein (WASP), in gene-transfected NIH3T3 cells. These single domains were composed of the V(H) and V(L) region with or without their leader sequences. Although these single domains were expressed at similar levels in NIH3T3 cells, the binding activity to the cytosolic target was higher in the single domain constructs with leader sequences. These results suggest the usefulness of the single domain intrabody constructs to analyze the functional domains of cytosolic proteins in cells.

Intrabodies against the EVH1 domain of Wiskott-Aldrich syndrome protein inhibit T cell receptor signaling in transgenic mice T cells.

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 (V(H)) 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.

Single domain intrabodies against WASP inhibit TCR-induced immune responses in transgenic mice T cells.

Intrabody technology provides a novel approach to decipher the molecular mechanisms of protein function in cells. Single domains composed of only the variable regions (V(H) or V(L)) of antibodies are the smallest recombinant antibody fragments to be constructed thus far. In this study, we developed transgenic (Tg) mice expressing the V(H) or V(L) single domains derived from a monoclonal antibody raised against the N-terminal domain of Wiskott-Aldrich syndrome protein (WASP), which is an adaptor molecule in immune cells. In T cells from anti-WASP V(H) and V(L) single domain Tg mice, interleukin-2 production induced by T cell receptor (TCR) stimulation were impaired, and specific interaction between the WASP N-terminal domain and the Fyn SH3 domain was strongly inhibited by masking the binding sites in WASP. These results strongly suggest that the V(H)/VL single domain intrabodies are sufficient to knockdown the domain function of target proteins in the cytosol.

Failure of mycoplasma lipoprotein MALP-2 to induce NK cell activation through dendritic cell TLR2.

Macrophage-activating lipopeptide 2 (MALP-2), a mycoplasmal diacylated lipopeptide with palmitic acid moiety (Pam2), activates Toll-like receptor (TLR) 2 to induce inflammatory cytokines. TLR2 is known to mature myeloid dendritic cells (mDC) to drive mDC contact-mediated natural killer (NK) cell activation. Here we tested if MALP-2 activates NK cells through stimulation of TLR2 on mDC. Although synthetic MALP-2 with 6 or 14 amino acids (a.a.) stretch (designated as s and f) matured mDC to induce IL-6, IL-12p40 and TNF-α to a similar extent, they far less activated NK cells than Pam2CSK4, a positive control of 6 a.a.-containing diacyl lipopeptide. MALP-2s and f were TLR2/6 agonists and activate the MyD88 pathway similar to Pam2CSK4, but MALP-2s having the CGNNDE sequence acted on mDC TLR2 to barely induce external NK activation. Even the s form, with slightly high induction of IL-6 compared to the f form, barely induced in vivo growth retardation of NK-sensitive implant tumor. Pam2CSK4 and MALP-2 have the common lipid moiety but different peptides, which are crucial for NK cell activation. The results infer that MALP-2 is applicable to a cytokine inducer but not to an adjuvant for antitumor NK immunotherapy.

The peptide sequence of diacyl lipopeptides determines dendritic cell TLR2-mediated NK activation.

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-gamma 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.

Impaired LPS-induced signaling in microglia overexpressing the Wiskott-Aldrich syndrome protein N-terminal domain.

Wiskott-Aldrich syndrome protein (WASP) plays important roles in TCR signaling, but its roles in signal transduction in innate immune cells have not been well characterized. As microglia are the primary immune effector cells in the brain, WASP may possibly have important roles in microglial activation, such as production of inflammatory and anti-inflammatory cytokines and neurotoxic factors. Here, we established a microglial cell line from WASP dominant-negative transgenic (Tg) mice overexpressing the N-terminal enabled/vasodilator-stimulated phosphoprotein homology 1 (EVH1) domain. WASP Tg microglia were impaired in production of inflammatory cytokines such as tumor necrosis factor-alpha, IL-6 and IL-1beta upon LPS stimulation, whereas anti-inflammatory IL-10 production was significantly enhanced. Also, LPS-induced phosphorylation of nuclear factor kappaB was reduced in WASP Tg microglia. Furthermore, WASP Tg microglia exhibited less cytotoxicity against co-cultured neurons after stimulation by LPS and IFN-gamma, with a concordant decrease in nitric oxide production. These results strongly suggest that WASP may have pivotal roles through the EVH1 domain in the LPS signaling cascade, either directly or indirectly, and modulates inflammatory immune responses in microglia.

Scarabaecin, a novel cysteine-containing antifungal peptide from the rhinoceros beetle, Oryctes rhinoceros.

A novel antifungal peptide, scarabaecin (4080Da), was isolated from the coconut rhinoceros beetle, Oryctes rhinoceros. Scarabaecin cDNA was cloned by reverse transcriptase-polymerase chain reactions (RT-PCR) using a primer based on the N-terminal amino acid sequence. The amino acid sequence deduced from scarabaecin cDNA showed no significant similarity to those of reported proteins. Chemically synthesized scarabaecin indicated antifungal activity against phytopathogenic fungi such as Pyricularia oryzae, Rhizoctonia solani, and Botrytis cinerea, but not against phytopathogenic bacteria. It showed weak activity against Bauberia bassiana, an insect pathogenic fungus, and Staphylococcus aureus, a pathogenic bacterium. Scarabaecin showed chitin binding property and its K(d) was 1.315 microM. A comparison of putative chitin-binding domains among scarabaecin, invertebrate, and plant chitin-binding proteins suggests that scarabaecin is a new member of chitin-binding antimicrobial proteins.