Quantification of Vibrio cholerae cholix exotoxin by sandwich bead-ELISA.

Introduction. Cholix toxin (ChxA) is an ADP-ribosylating exotoxin produced by Vibrio cholerae. However, to date, there is no quantitative assay available for ChxA, which makes it difficult to detect and estimate the level of ChxA produced by V. cholerae.Hypothesis/Gap Statement. It is important to develop a reliable and specific quantitative assay to measure the production level of ChxA, which will help us to understand the role of ChxA in V. cholerae pathogenesis.Aim. The aim of this study was to develop a bead-based sandwich ELISA (bead-ELISA) for the quantification of ChxA and to evaluate the importance of ChxA in the pathogenesis of V. cholerae infection.Methodology. Anti-rChxA was raised in New Zealand white rabbits, and Fab-horse radish peroxidase conjugate was prepared by the maleimide method to use in the bead-ELISA. This anti-ChxA bead-ELISA was applied to quantify the ChxA produced by various V. cholerae strains. The production of ChxA was examined in different growth media such as alkaline peptone water (APW), Luria-Bertani broth and AKI. Finally, the assay was evaluated using a mouse lethality assay with representative V. cholerae strains categorized as low to high ChxA-producers based on anti-ChxA bead-ELISA.Results. A sensitive bead-ELISA assay, which can quantify from 0.6 to 60 ng ml-1 of ChxA, was developed. ChxA was mostly detected in the extracellular cell-free supernatant and its production level varied from 1.2 ng ml-1 to 1.6 µg ml-1. The highest ChxA production was observed when V. cholerae strains were cultured in LB broth, but not in APW or AKI medium. The ChxA-producer V. cholerae strains showed 20-80 % lethality and only the high ChxA II-producer was statistically more lethal than a non-ChxA-producer, in the mice model assay. ChxA I and II production levels were not well correlated with mice lethality, and this could be due to the heterogeneity of the strains tested.Conclusion. ChxA I to III was produced mostly extracellularly at various levels depending on strains and culture conditions. The bead-ELISA developed in this study is useful for the detection and quantification of ChxA in V. cholerae strains.

The role of MHC class I recycling and Arf6 in cross-presentation by murine dendritic cells.

Cross-presentation by MHC class I molecules (MHC-I) is critical for priming of cytotoxic T cells. Peptides derived from cross-presented antigens can be loaded on MHC-I in the endoplasmic reticulum and in endocytic or phagocytic compartments of murine DCs. However, the origin of MHC-I in the latter compartments is poorly understood. Recently, Rab22-dependent MHC-I recycling through a Rab11+ compartment has been suggested to be implicated in cross-presentation. We have examined the existence of MHC-I recycling and the role of Arf6, described to regulate recycling in nonprofessional antigen presenting cells, in murine DCs. We confirm folded MHC-I accumulation in a juxtanuclear Rab11+ compartment and partially localize Arf6 to this compartment. MHC-I undergo fast recycling, however, both folded and unfolded internalized MHC-I fail to recycle to the Rab11+Arf6+ compartment. Therefore, the source of MHC-I molecules in DC endocytic compartments remains to be identified. Functionally, depletion of Arf6 compromises cross-presentation of immune complexes but not of soluble, phagocytosed or mannose receptor-targeted antigen, suggesting a role of Fc receptor-regulated Arf6 trafficking in cross-presentation of immune complexes.

Characterization of Functional Primary Cilia in Human Induced Pluripotent Stem Cell-Derived Neurons.

Recent advances in human induced pluripotent stem cells (hiPSCs) offer new possibilities for biomedical research and clinical applications. Neurons differentiated from hiPSCs may be promising tools to develop novel treatment methods for various neurological diseases. However, the detailed process underlying functional maturation of hiPSC-derived neurons remains poorly understood. Here, we analyze the developmental architecture of hiPSC-derived cortical neurons, iCell GlutaNeurons, focusing on the primary cilium, a single sensory organelle that protrudes from the surface of most growth-arrested vertebrate cells. To characterize the neuronal cilia, cells were cultured for various periods and evaluated immunohistochemically by co-staining with antibodies against ciliary markers Arl13b and MAP2. Primary cilia were detected in neurons within days, and their prevalence and length increased with increasing days in culture. Treatment with the mood stabilizer lithium led to primary cilia length elongation, while treatment with the orexigenic neuropeptide melanin-concentrating hormone caused cilia length shortening in iCell GlutaNeurons. The present findings suggest that iCell GlutaNeurons develop neuronal primary cilia together with the signaling machinery for regulation of cilia length. Our approach to the primary cilium as a cellular antenna can be useful for both assessment of neuronal maturation and validation of pharmaceutical agents in hiPSC-derived neurons.

Regulation of Phospholipase D by Arf6 during FcγR-Mediated Phagocytosis.

Phagocytosis is an essential element of the immune response, assuring the elimination of pathogens, cellular debris, and apoptotic and tumoral cells. Activation of phagocytosis by the FcγR stimulates phospholipase D (PLD) activity and triggers the production of phosphatidic acid (PA) at the plasma membrane of macrophages, but the regulatory mechanisms involved are still not clearly understood. In this study, we examined the role of the small GTPase Arf6 in the activation of the PLD isoforms during FcγR-mediated phagocytosis. In RAW 264.7 macrophage cells, expressed Arf6-GFP partially colocalized with PLD1-hemagglutinin on intracellular membrane-bound vesicles and with PLD2-hemagglutinin at the plasma membrane. Both PLD isoforms were found to interact with Arf6 during FcγR-mediated phagocytosis as seen by immunoprecipitation experiments. In macrophages stimulated for phagocytosis, Arf6 was observed to be associated with nascent phagosomes. RNA interference knockdown of Arf6 reduced the amount of active Arf6 associated with phagosomes, revealed by the MT2-GFP probe that specifically binds to Arf6-GTP. Arf6 silencing concomitantly decreased PLD activity as well as the levels of PA found on phagosomes and phagocytic sites as shown with the PA probe Spo20p-GFP. Altogether, our results indicate that Arf6 is involved in the regulation of PLD activity and PA synthesis required for efficient phagocytosis.

Identification and Characterization of a New Enterotoxin Produced by Clostridium perfringens Isolated from Food Poisoning Outbreaks.

There is a strain of Clostridium perfringens, W5052, which does not produce a known enterotoxin. We herein report that the strain W5052 expressed a homologue of the iota-like toxin components sa and sb of C. spiroforme, named Clostridium perfringens iota-like enterotoxin, CPILE-a and CPILE-b, respectively, based on the results of a genome sequencing analysis and a systematic protein screening. In the nicotinamide glyco-hydrolase (NADase) assay the hydrolysis activity was dose-dependently increased by the concentration of rCPILE-a, as judged by the mass spectrometry analysis. In addition, the actin monomer of the lysates of Vero and L929 cells were radiolabeled in the presence of [32P]NAD and rCPILE-a. These findings indicated that CPILE-a possesses ADP-ribosylation activity. The culture supernatant of W5052 facilitated the rounding and killing of Vero and L929 cells, but the rCPILE-a or a non-proteolyzed rCPILE-b did not. However, a trypsin-treated rCPILE-b did. Moreover, a mixture of rCPILE-a and the trypsin-treated rCPILE-b enhanced the cell rounding and killing activities, compared with that induced by the trypsin-treated rCPILE-b alone. The injection of the mixture of rCPILE-a and the trypsin-treated rCPILE-b into an ileum loop of rabbits evoked the swelling of the loop and accumulation of the fluid dose-dependently, suggesting that CPILE possesses enterotoxic activity. The evidence presented in this communication will facilitate the epidemiological, etiological, and toxicological studies of C. perfringens food poisoning, and also stimulate studies on the transfer of the toxins' gene(s) among the Genus Clostridium.

ADP-Ribosylation Factor 3 Mediates Cytidine-Phosphate-Guanosine Oligodeoxynucleotide-Induced Responses by Regulating Toll-Like Receptor 9 Trafficking.

Toll-like receptor 9 (TLR9) trafficking from the endoplasmic reticulum (ER) into endolysosomes is critical for eliciting cytidine-phosphate-guanosine (CpG) DNA-mediated immune responses. ADP-ribosylation factor 3 (ARF3) is a member of the Ras superfamily, which is crucial for a wide variety of cellular events including protein trafficking. In this study, we found that the inhibition of ARF3 by dominant mutants and siRNA impaired CpG oligodeoxynucleotide (ODN)-mediated responses whereas cells expressing the constitutively active ARF3 mutant enhanced CpG ODN-induced NF-x03BA;B activation and cytokine production. Further experiments with MyD88-overexpressing fibroblast cells transfected with a dominant-negative mutant and a constitutively active mutant of ARF3 demonstrated that ARF3 regulated CpG ODN-mediated signaling upstream of MyD88. Additional studies have shown that ARF3 inhibition impairs TLR9 trafficking from the ER into endolysosomes, thereby inhibiting the functional cleavage of TLR9, although it has no significant effect on CpG ODN uptake. Furthermore, activated ARF3 is associated with Unc93B1 and TLR9, suggesting that ARF3 conducts TLR9 trafficking by forming the TLR9-Unc93B1-ARF3 complex. Overall, our findings demonstrate that a novel ARF3 axis pathway mediates CpG ODN-induced responses by regulating TLR9 trafficking.

MHC class II presentation is controlled by the lysosomal small GTPase, Arl8b.

Dendritic cells (DCs) are specialized APCs with the ability to prime naive T cells. DCs first sample Ags from the environment and then orchestrate their processing and loading onto MHC class II (MHC II) Ag-presenting molecules in lysosomes. Once MHC II molecules have bound a peptide, the MHC II-peptide complex is delivered to the cell surface for presentation to CD4(+) T cells. Regulation of Ag uptake via macropinocytosis and phagocytosis has been extensively studied, as well as trafficking in early endocytic vesicles notably regulated by the small GTPase Rab5 and its effectors. However, little is known about the regulators of Ag delivery from early endosomes to lysosomal compartments where the proper pH, proteases, MHC II, invariant chain, and HLA-DM reside, awaiting exogenous Ags for loading. In this article, we report the crucial role of the small GTPase ADP-ribosylation factor-like 8b (Arl8b) in MHC II presentation in DCs. We show for the first time, to our knowledge, that Arl8b localizes to MHC II compartments in DCs and regulates formation of MHC II-peptide complexes. Arl8b-silenced DCs display a defect in MHC II-Ag complex formation and its delivery to the cell surface during infection resulting in a defect in T cell recognition. Our results highlight the role of Arl8b as a trafficking regulator of the late stage of complex formation and MHC II presentation in DCs.

Negative regulation of melanoma differentiation-associated gene 5 (MDA5)-dependent antiviral innate immune responses by Arf-like protein 5B.

RIG-I-like receptors (RLRs), including retinoic acid-inducible gene-I (RIG-I) and MDA5, constitute a family of cytoplasmic RNA helicases that senses viral RNA and mounts antiviral innate immunity by producing type I interferons and inflammatory cytokines. Despite their essential roles in antiviral host defense, RLR signaling is negatively regulated to protect the host from excessive inflammation and autoimmunity. Here, we identified ADP-ribosylation factor-like protein 5B (Arl5B), an Arl family small GTPase, as a regulator of RLR signaling through MDA5 but not RIG-I. Overexpression of Arl5B repressed interferon ß promoter activation by MDA5 but not RIG-I, and its knockdown enhanced MDA5-mediated responses. Furthermore, Arl5B-deficient mouse embryonic fibroblast cells exhibited increased type I interferon expression in response to MDA5 agonists such as poly(I:C) and encephalomyocarditis virus. Arl5B-mediated negative regulation of MDA5 signaling does not require its GTP binding ability but requires Arl5B binding to the C-terminal domain of MDA5, which prevents interaction between MDA5 and poly(I:C). Our results, therefore, suggest that Arl5B is a negative regulator for MDA5.

ARF6 inhibition stabilizes the vasculature and enhances survival during endotoxic shock.

The vascular endothelium responds to infection by destabilizing endothelial cell-cell junctions to allow fluid and cells to pass into peripheral tissues, facilitating clearance of infection and tissue repair. During sepsis, endotoxin and other proinflammatory molecules induce excessive vascular leak, which can cause organ dysfunction, shock, and death. Current therapies for sepsis are limited to antibiotics and supportive care, which are often insufficient to reduce morbidity and prevent mortality. Previous attempts at blocking inflammatory cytokine responses in humans proved ineffective at reducing the pathologies associated with sepsis, highlighting the need for a new therapeutic strategy. The small GTPase ARF6 is activated by a MyD88-ARNO interaction to induce vascular leak through disruption of endothelial adherens junctions. In this study, we show that the MyD88-ARNO-ARF6-signaling axis is responsible for LPS-induced endothelial permeability and is a destabilizing convergence point used by multiple inflammatory cues. We also show that blocking ARF6 with a peptide construct of its N terminus is sufficient to reduce vascular leak and enhance survival during endotoxic shock, without inhibiting the host cytokine response. Our data highlight the therapeutic potential of blocking ARF6 and reducing vascular leak for the treatment of inflammatory conditions, such as endotoxemia.

Specific plasma autoantibody reactivity in myelodysplastic syndromes.

Increased autoantibody reactivity in plasma from Myelodysplastic Syndromes (MDS) patients may provide novel disease signatures, and possible early detection. In a two-stage study we investigated Immunoglobulin G reactivity in plasma from MDS, Acute Myeloid Leukemia post MDS patients, and a healthy cohort. In exploratory Stage I we utilized high-throughput protein arrays to identify 35 high-interest proteins showing increased reactivity in patient subgroups compared to healthy controls. In validation Stage II we designed new arrays focusing on 25 of the proteins identified in Stage I and expanded the initial cohort. We validated increased antibody reactivity against AKT3, FCGR3A and ARL8B in patients, which enabled sample classification into stable MDS and healthy individuals. We also detected elevated AKT3 protein levels in MDS patient plasma. The discovery of increased specific autoantibody reactivity in MDS patients, provides molecular signatures for classification, supplementing existing risk categorizations, and may enhance diagnostic and prognostic capabilities for MDS.

Pivotal role of ADP-ribosylation factor 6 in Toll-like receptor 9-mediated immune signaling.

CpG oligodeoxynucleotide (CpG ODN) cellular uptake into endosomes, the rate-limiting step of Toll-like receptor 9 (TLR9) signaling, is critical in eliciting innate immune responses. ADP-ribosylation factor 6 (ARF6) is a member of the Ras superfamily, which is critical to a wide variety of cellular events including endocytosis. Here, we found that inhibition of ARF6 by dominant mutants and siRNA impaired CpG ODN-mediated responses, whereas cells expressing the constitutively active ARF6 mutant enhanced CpG ODN-induced cytokine production. Inhibition of ARF6 impaired TLR9 trafficking into endolysosomes, thereby inhibiting proceed functional cleavage of TLR9. Additional studies showed that CpG ODN uptake was increased in ARF6-activated cells but impaired in ARF6-defective cells. Furthermore, cells pretreated with CpG ODN but not GpC ODN had increased CpG ODN uptake due to CpG ODN-induced ARF6 activity. Further studies with ARF6-defective and ARF6-activated cells demonstrated that class III phosphatidylinositol 3-kinases (PI3K) was required for downstream ARF6 regulation of CpG ODN uptake. Together, our findings demonstrate that a novel class III PI3K-ARF6 axis pathway mediates TLR9 signaling by regulating the cellular uptake of CpG ODN.

Lysosomal trafficking, antigen presentation, and microbial killing are controlled by the Arf-like GTPase Arl8b.

Antigen presentation and microbial killing are critical arms of host defense that depend upon cargo trafficking into lysosomes. Yet, the molecular regulators of traffic into lysosomes are only partly understood. Here, using a lysosome-dependent immunological screen of a trafficking shRNA library, we identified the Arf-like GTPase Arl8b as a critical regulator of cargo delivery to lysosomes. Homotypic fusion and vacuole protein sorting (HOPS) complex members were identified as effectors of Arl8b and were dependent on Arl8b for recruitment to lysosomes, suggesting that Arl8b-HOPS plays a general role in directing traffic to lysosomes. Moreover, the formation of CD1 antigen-presenting complexes in lysosomes, their delivery to the plasma membrane, and phagosome-lysosome fusion were all markedly impaired in Arl8b silenced cells resulting in corresponding defects in T cell activation and microbial killing. Together, these results define Arl8b as a key regulator of lysosomal cellular and immunological functions.

ARF-like protein 16 (ARL16) inhibits RIG-I by binding with its C-terminal domain in a GTP-dependent manner.

Retinoic acid-inducible gene I (RIG-I) recognizes RNA virus-derived nucleic acids, which leads to the production of type I interferon (IFN) in most cell types. Tight regulation of RIG-I activity is important to prevent ultra-immune responses. In this study, we identified an ARF-like (ARL) family member, ARL16, as a protein that interacts with RIG-I. Overexpression of ARL16, but not its homologous proteins ARL1 and ARF1, inhibited RIG-I-mediated downstream signaling and antiviral activity. Knockdown of endogenous ARL16 by RNAi potentiated Sendai virus-induced IFN-ß expression and vesicular stomatitis virus replication. ARL16 interacted with the C-terminal domain (CTD) of RIG-I to suppress the association between RIG-I and RNA. ARL16 (T37N) and ARL16Δ45-54, which were restricted to the GTP-disassociated form, did not interact with RIG-I and also lost the inhibitory function. Furthermore, we suggest that endogenous ARL16 changes to GTP binding status upon viral infection and binds with the RIG-I CTD to negatively control its signaling activity. These findings suggested a novel innate immune function for an ARL family member, and a GTP-dependent model in which RIG-I is regulated.

Phagocytosis of Leishmania donovani amastigotes is Rac1 dependent and occurs in the absence of NADPH oxidase activation.

Macrophages produce little superoxide during phagocytosis of Leishmania donovani amastigotes. In this study, we characterized molecular events associated with L. donovani amastigotes uptake by mouse macrophages, to further define the mechanisms by which they are internalized without triggering superoxide production. Using transient transfections, we first showed that internalization of L. donovani amastigotes is mediated by the GTPases Rac1 and Arf6, of which Rac1 is recruited and retained on parasite-containing phagosomes. Next, we showed that, whereas internalization of amastigotes induced no superoxide release, co-internalization of serum-opsonized zymozan and amastigotes resulted in superoxide production. Furthermore, in co-internalization experiments, we detected superoxide production in over 95% of phagosomes containing IgG-opsonized SRBC compared to 5% of amastigote-harboring phagosomes. These results suggest that amastigotes evade the ability of macrophages to produce superoxide during phagocytosis. Consistently, we observed that amastigotes induced barely detectable phosphorylation of the NADPH oxidase component p47phox, leading to a defective phagosomal recruitment of p67phox and p47phox. Finally, we showed that amastigotes disrupt phagosomal lipid raft integrity, potentially interfering with NADPH oxidase assembly. Collectively, our results indicate that internalization of L. donovani amastigotes is a Rac1- and Arf6-dependent process that occurs in the absence of significant NADPH oxidase activation.

Arf6: a new player in FcgammaRIIIA lymphocyte-mediated cytotoxicity.

The activation of phosphoinositide metabolism represents a critical step in the signaling pathways leading to the activation of cytolytic machinery, but its regulation is partially understood. We report here that the stimulation of the low-affinity receptor for immunoglobulin G (IgG) (FcgammaRIIIA, CD16) on primary human natural killer (NK) cells induces a phosphatidylinositol 3-kinase (PI3K)-dependent activation of the small G protein Arf6. We first demonstrate a functional role for Arf6-dependent signals in the activation of the antibody-dependent cellular cytotoxicity (ADCC) attributable to the control of secretion of lytic granule content. We also show that Arf6 couples CD16 to the lipid-modifying enzymes phosphatidylinositol4phosphate 5-kinase type I alpha (PI5KIalpha) and phospholipase D (PLD) that are involved in the control of granule secretion; Arf6, but not Rho family small G proteins RhoA and Rac1, is required for receptor-induced PI5KIalpha membrane targeting as well as for PI5KIalpha and PLD activation. Our findings suggest that Arf6 plays a crucial role in the generation of a phosphatidylinositol4,5-bisphosphate (PIP2) plasma membrane pool required for cytolytic granule-mediated target cell killing.

Stabilin-1 localizes to endosomes and the trans-Golgi network in human macrophages and interacts with GGA adaptors.

Stabilin-1 and stabilin-2 constitute a novel family of fasciclin domain-containing hyaluronan receptor homologues recently described by us. Whereas stabilin-1 is expressed in sinusoidal endothelial cells and in macrophages in vivo, stabilin-2 is absent from the latter. In the present study, we analyzed the subcellular distribution of stabilin-1 in primary human macrophages. Using flow cytometry, expression of stabilin-1 was demonstrated on the surface of interleukin-4/dexamethasone-stimulated macrophages (MPhi2). By immunofluorescence and confocal microscopy, we established that stabilin-1 is preferentially localized in early endosome antigen-1-positive early/sorting endosomes and in recycling endosomes identified by transferrin endocytosis. Association of stabilin-1 was infrequently seen with p62 lck ligand-positive late endosomes and with CD63-positive lysosomes but not in lysosome-associated membrane protein-1-positive lysosomes. Stabilin-1 was also found in the trans-Golgi network (TGN) but not in Golgi stack structures. Glutathione S-transferase pull-down assay revealed that the cytoplasmic tail of stabilin-1 but not stabilin-2 binds to recently discovered Golgi-localized, gamma-ear-containing, adenosine 5'-diphosphate-ribosylation factor-binding (GGA) adaptors GGA1, GGA2, and GGA3 long, mediating traffic between Golgi and endosomal/lysosomal compartments. Stabilin-1 did not bind to GGA3 short, which lacks a part of the Vps27p/Hrs/STAM domain. Deletion of DDSLL and LL amino acid motifs resulted in decreased binding of stabilin-1 with GGAs. A small portion of stabilin-1 colocalized with GGA2 and GGA3 in the TGN in MPhi2. Treatment with brefeldin A resulted in accumulation of stabilin-1 in the TGN. Our results suggest that stabilin-1 is involved in the GGA-mediated sorting processes at the interface of the biosynthetic and endosomal pathways; similarly to other GGA-interacting proteins, stabilin-1 may thus function in endocytic and secretory processes of human macrophages.

ADP-ribosylating bacterial enzymes for the targeted control of mucosal tolerance and immunity.

The questions of whether mucosal tolerance and IgA immunity are mutually exclusive or can coexist and whether they represent priming of the local immune system through the same or different activation pathways are addressed. Two strategies were attempted: the first using cholera toxin (CT) or the enzymatically inactive receptor-binding B subunit of CT (CTB), and the second using CTA1-DD or an enzymatically inactive mutant thereof, CTA1R7K-DD. The CTA1-DD adjuvant is a fusion protein composed of the ADP-ribosylating part of CT, CTA1, and DD, which is derived from Staphylococcus areus protein A and targets the molecule to B cells. Here, we provide compelling evidence that delivery of antigen in the absence of ADP ribosylation can promote tolerance, whereas ADP-ribosyltransferase activity induces IgA immunity and prevents tolerance. By linking antigen to the ADP-ribosylating enzymes we could show that CT, although potentially binding to all nucleated cells, in fact, bound preferentially to dendritic cells (DCs) in vivo. On the other hand, DD-bound antigen was distinctly targeted to B cells and probably also to follicular dendritic cells (FDCs) in vivo. Interestingly, the CT and CTA1-DD adjuvants gave equally enhancing effects on mucosal and systemic responses, but appeared to target different APCs in vivo. CT- or CTB-conjugated antigen accumulated in mucosal and systemic DCs. Whereas only CT promoted an active IgA response, CTB induced tolerance to the conjugated antigen. Following intravenous injection of CT-conjugated antigen, DCs in the marginal zone (MZ) of the spleen were selectively targeted. Interestingly, CTB delivered antigen to the same MZ DCs, but failed to induce maturation and upregulation of costimulatory molecules in these cells. Thus, ADP-ribosylation was necessary for a strong enhancing effect of immune responses following CT/CTB-dependent delivery of antigen to the MZ DCs. Moreover, using CTA1-DD, antigen was targeted to the B cell follicle and FDC in the spleen after intravenous injection. Only active CTA1-DD, but not the inactive mutant CTA1R7K-DD, provided enhancing effects on immune responses. By contrast, antigen delivered by the CTA1R7K-DD stimulated specific tolerance in adoptively transferred T cell receptor transgenic CD4(+) T cells. Whether targeting of B cells suffices for tolerance induction or requires participation of DCs remains to be investigated. With CT we found that enzyme-dependent modulation of DCs affects migration, maturation, and differentiation of DCs, which resulted in CD4(+) T cell help for IgA B cell development. On the contrary, antigen presentation in the absence of ADP-ribosylating enzyme, as seen with CTB or CTA1R7K-DD, appears to expand specific T cells to a similar extent as enzymatically active CT or CTA1-DD, but fails to recruit help for germinal center (GC) formation and the necessary expansion of activated B cells. Also, the CD41 T cells that are primed in a suboptimal, tolerogenic, fashion do not migrate to the B cell follicle to provide T cell help. Thus, ADP-ribosylating enzymes may be used to selectively control the induction of an active IgA response or promote the development of tolerance. In particular, on the targeted APC, modulation of the expression of costimulatory molecules, CD80, CD86, CD83, and B7RP-1, plays an important role in the effect of the ADP-ribosylating CTA1-based adjuvants on the development of tolerance or active IgA immunity. For example, the expression of CD86 in vivo was a prominent feature of the enzymatically active CT or CTA1-DD adjuvants. By contrast, CD80 expression appeared not to be important in CTA1-augmented APCs for an adjuvant function.

Recognition of ADP-ribosylation factor 4-like by HLA-A2-restricted and tumor-reactive cytotoxic T lymphocytes from patients with brain tumors.

Although specific immunotherapy is one candidate treatment of brain tumor, the molecular basis of T-cell-mediated recognition of brain tumors has not yet been elucidated. In this study, we tried to identify brain tumor antigens using HLA-A2-restricted and tumor-reactive cytotoxic T lymphocytes (CTLs). As an HLA-A2-restricted OK-CTL line contained CTLs capable of responding to HLA-A2+ malignant glioma cells, this cell line was used for identification of brain tumor antigens. After screening a cDNA library from brain tumor cells, this CTL line was found to produce interferon (IFN)-gamma when cultured with COS-7 cells, which were cotransfected with both a cDNA clone (clone 1) and HLA-A0207 cDNA. Data base searches indicated that the clone 1 was 98% identical to that of the human ADP-ribosylation factor 4-like (ARF4L). Two peptides, ARF4L 15-24 and ARF4L 69-77, possessed the ability to induce HLA-A2-restricted and tumor-reactive CTLs from peripheral blood mononuclear cells of patients with brain tumors. Although ARF4L seemed to be ubiquitously expressed at the mRNA level, ARF4L-reactive CTLs failed to exhibit cytotoxicity against normal lymphoid blasts. These results indicate that these two ARF4L peptides could be targets for immunotherapy of HLA-A2+ patients with brain tumors.

Localization of large ADP-ribosylation factor-guanine nucleotide exchange factors to different Golgi compartments: evidence for distinct functions in protein traffic.

Activation of several ADP-ribosylation factors (ARFs) by guanine nucleotide exchange factors (GEFs) regulates recruitment of coat proteins (COPs) on the Golgi complex and is generally assumed to be the target of brefeldin A (BFA). The large ARF-GEFs Golgi-specific BFA resistance factor 1 (GBF1) and BFA-inhibited GEFs (BIGs) localize to this organelle but catalyze exchange preferentially on class II and class I ARFs, respectively. We now demonstrate using quantitative confocal microscopy that these GEFs show a very limited overlap with each other (15 and 23%). In contrast, GBF1 colocalizes with the cis-marker p115 (86%), whereas BIGs overlap extensively with TGN38 (83%). Consistent with these distributions, GBF1, but not BIG1, partially relocalized to peripheral sites after incubation at 15 degrees C. The new GBF1 structures represent peripheral vesicular tubular clusters (VTCs) because 88% of structures analyzed stained for both GBF1 and p115. Furthermore, as expected of VTCs, they rapidly reclustered to the Golgi complex in a microtubule-dependent manner upon warm-up. These observations suggest that GBF1 and BIGs activate distinct subclasses of ARFs in specific locations to regulate different types of reactions. In agreement with this possibility, COPI overlapped to a greater extent with GBF1 (64%) than BIG1 (31%), whereas clathrin showed limited overlap with BIG1, and virtually none with GBF1.