Anti-OX40L alone or in combination with anti-CD40L and CTLA4Ig does not inhibit the humoral and cellular response to a major grass pollen allergen.

BACKGROUND: IgE-mediated allergy is a common disease characterized by a harmful immune response towards otherwise harmless environmental antigens. Induction of specific immunological non-responsiveness towards allergens would be a desirable goal. Blockade of costimulatory pathways is a promising strategy to modulate the immune response in an antigen-specific manner. Recently, OX40 (CD134) was identified as a costimulatory receptor important in Th2-mediated immune responses. Moreover, synergy between OX40 blockade and 'conventional' costimulation blockade (anti-CD40L, CTLA4Ig) was observed in models of alloimmunity. OBJECTIVE: We investigated the potential of interfering with OX40 alone or in combination with CD40/CD28 signals to influence the allergic immune response. METHODS: The OX40 pathway was investigated in an established murine model of IgE-mediated allergy where BALB/c mice are repeatedly immunized with the clinically relevant grass pollen allergen Phl p 5. Groups were treated with combinations of anti-OX40L, CTLA4Ig and anti-CD40L. In selected mice, Tregs were depleted with anti-CD25. RESULTS: Blockade of OX40L alone at the time of first or second immunization did not modulate the allergic response on the humoral or effector cell levels but slightly on T cell responses. Administration of a combination of anti-CD40L/CTLA4Ig delayed the allergic immune response, but antibody production could not be inhibited after repeated immunization even though the allergen-specific T cell response was suppressed in the long run. Notably, additional blockade of OX40L had no detectable supplementary effect. Immunomodulation partly involved regulatory T cells as depletion of CD25(+) cells led to restored T cell proliferation. CONCLUSIONS AND CLINICAL RELEVANCE: Collectively, our data provide evidence that the allergic immune response towards Phl p 5 is independent of OX40L, although reduction on T cell responses and slightly on the asthmatic phenotype was detectable. Besides, no relevant synergistic effect of OX40L blockade in addition to CD40L/CD28 blockade could be detected. Thus, the therapeutic potential of OX40L blockade for IgE-mediated allergy appears to be ineffective in this setting.

Rapamycin and CTLA4Ig synergize to induce stable mixed chimerism without the need for CD40 blockade.

The mixed chimerism approach achieves donor-specific tolerance in organ transplantation, but clinical use is inhibited by the toxicities of current bone marrow (BM) transplantation (BMT) protocols. Blocking the CD40:CD154 pathway with anti-CD154 monoclonal antibodies (mAbs) is exceptionally potent in inducing mixed chimerism, but these mAbs are clinically not available. Defining the roles of donor and recipient CD40 in a murine allogeneic BMT model, we show that CD4 or CD8 activation through an intact direct or CD4 T cell activation through the indirect pathway is sufficient to trigger BM rejection despite CTLA4Ig treatment. In the absence of CD4 T cells, CD8 T cell activation via the direct pathway, in contrast, leads to a state of split tolerance. Interruption of the CD40 signals in both the direct and indirect pathway of allorecognition or lack of recipient CD154 is required for the induction of chimerism and tolerance. We developed a novel BMT protocol that induces mixed chimerism and donor-specific tolerance to fully mismatched cardiac allografts relying on CD28 costimulation blockade and mTOR inhibition without targeting the CD40 pathway. Notably, MHC-mismatched/minor antigen-matched skin grafts survive indefinitely whereas fully mismatched grafts are rejected, suggesting that non-MHC antigens cause graft rejection and split tolerance.

Donor CD4 T cells trigger costimulation blockade-resistant donor bone marrow rejection through bystander activation requiring IL-6.

Bone marrow (BM) transplantation under costimulation blockade induces chimerism and tolerance. Cotransplantation of donor T cells (contained in substantial numbers in mobilized peripheral blood stem cells and donor lymphocyte infusions) together with donor BM paradoxically triggers rejection of donor BM through undefined mechanisms. Here, nonmyeloablatively irradiated C57BL/6 recipients simultaneously received donor BM (BALB/c) and donor T cells under costimulation blockade (anti-CD154 and CTLA4Ig). Donor CD4, but not CD8 cells, triggered natural killer-independent donor BM rejection which was associated with increased production of IL-6, interferon gamma (IFN-γ) and IL-17A. BM rejection was prevented through neutralization of IL-6, but not of IFN-γ or IL-17A. IL-6 counteracted the antiproliferative effect of anti-CD154 in vitro. Rapamycin and anti-lymphocyte function-associated antigen 1 negated this effect of IL-6 in vitro and prevented BM rejection in vivo. Simultaneous cotransplantation of (BALB/cxB6)F1, recipient or irradiated donor CD4 cells, or late transfer of donor CD4 cells did not lead to BM rejection, whereas cotransplantation of third party CD4 cells did. Transferred donor CD4 cells became activated, rapidly underwent apoptosis and triggered activation and proliferation of recipient T cells. Collectively, these results provide evidence that donor T cells recognizing the recipient as allogeneic lead to the release of IL-6, which abolishes the effect of anti-CD154, triggering donor BM rejection through bystander activation.

Utilization of virus φCh1 elements to establish a shuttle vector system for Halo(alkali)philic Archaea via transformation of Natrialba magadii.

In the study described here, we successfully developed a transformation system for halo(alkali)philic members of the Archaea. This transformation system comprises a series of Natrialba magadii/Escherichia coli shuttle vectors based on a modified method to transform halophilic members of the Archaea and genomic elements of the N. magadii virus Ch1. The shuttle vector pRo-5, based on the repH-containing region of Ch1, stably replicated in E. coli and N. magadii and in several halophilic and haloalkaliphilic members of the Archaea not transformable so far. The Ch1 operon ORF53/ORF54 (repH) was essential for pRo-5 replication and was thus identified as the minimal replication origin. The plasmid allowed homologous and heterologous gene expression, as exemplified by the expression of Ch1 ORF3452, which encodes a structural protein, and the reporter gene bgaH of Haloferax lucentense in N. magadii. The new transformation/vector system will facilitate genetic studies within N. magadii and other haloalkaliphilic archaea and will allow the detailed characterization of the gene functions of N. magadii virus Ch1 in their extreme environments.

Persistent molecular microchimerism induces long-term tolerance towards a clinically relevant respiratory allergen.

BACKGROUND: Development of antigen-specific preventive strategies is a challenging goal in IgE-mediated allergy. We have recently shown in proof-of-concept experiments that allergy can be successfully prevented by induction of durable tolerance via molecular chimerism. Transplantation of syngeneic hematopoietic stem cells genetically modified to express the clinically relevant grass pollen allergen Phl p 5 into myeloablated recipients led to high levels of chimerism (i.e. macrochimerism) and completely abrogated Phl p 5-specific immunity despite repeated immunizations with Phl p 5. OBJECTIVE: It was unclear, however, whether microchimerism (drastically lower levels of chimerism) would be sufficient as well which would allow development of minimally toxic tolerance protocols. METHODS: Bone marrow cells were transduced with recombinant viruses integrating Phl p 5 to be expressed in a membrane-anchored fashion. The syngeneic modified cells were transplanted into non-myeloablated recipients that were subsequently immunized repeatedly with Phl p 5 and Bet v 1 (control). Molecular chimerism was monitored using flow cytometry and PCR. T cell, B-cell and effector-cell tolerance were assessed by allergen-specific proliferation assays, isotype levels in sera and RBL assays. RESULTS: Here we demonstrate that transplantation of Phl p 5-expressing bone marrow cells into recipients having received non-myeloablative irradiation resulted in chimerism persisting for the length of follow-up. Chimerism levels, however, declined from transient macrochimerism levels to persistent levels of microchimerism (followed for 11 months). Notably, these chimerism levels were sufficient to induce B-cell tolerance as no Phl p 5-specific IgE and other high affinity isotypes were detectable in sera of chimeric mice. Furthermore, T-cell and effector-cell tolerance were achieved. CONCLUSIONS AND CLINICAL RELEVANCE: Low levels of persistent molecular chimerism are sufficient to induce long-term tolerance in IgE-mediated allergy. These results suggest that it will be possible to develop minimally toxic conditioning regimens sufficient for low level engraftment of genetically modified bone marrow.

Treg-therapy allows mixed chimerism and transplantation tolerance without cytoreductive conditioning.

Establishment of mixed chimerism through transplantation of allogeneic donor bone marrow (BM) into sufficiently conditioned recipients is an effective experimental approach for the induction of transplantation tolerance. Clinical translation, however, is impeded by the lack of feasible protocols devoid of cytoreductive conditioning (i.e. irradiation and cytotoxic drugs/mAbs). The therapeutic application of regulatory T cells (Tregs) prolongs allograft survival in experimental models, but appears insufficient to induce robust tolerance on its own. We thus investigated whether mixed chimerism and tolerance could be realized without the need for cytoreductive treatment by combining Treg therapy with BM transplantation (BMT). Polyclonal recipient Tregs were cotransplanted with a moderate dose of fully mismatched allogeneic donor BM into recipients conditioned solely with short-course costimulation blockade and rapamycin. This combination treatment led to long-term multilineage chimerism and donor-specific skin graft tolerance. Chimeras also developed humoral and in vitro tolerance. Both deletional and nondeletional mechanisms contributed to maintenance of tolerance. All tested populations of polyclonal Tregs (FoxP3-transduced Tregs, natural Tregs and TGF-beta induced Tregs) were effective in this setting. Thus, Treg therapy achieves mixed chimerism and tolerance without cytoreductive recipient treatment, thereby eliminating a major toxic element impeding clinical translation of this approach.

Highly homologous HERC proteins localize to endosomes and exhibit specific interactions with hPLIC and Nm23B.

Small HERC proteins are defined by the presence of one RCC1-like domain and a HECT domain. Having evolved out of one common ancestor, the four members of the family exhibit a high degree of homology in genomic organization and amino acid sequence, thus it seems possible that they might accomplish similar functions. Here we show that small HERC proteins interact with each other and localize to the same cellular structures, which we identify as late endosomes and lysosomes. We demonstrate interaction of HERC3 with the ubiquitin-like proteins hPLIC-1 and hPLIC-2 and we establish interaction of HERC5 with the metastasis suppressor Nm23B. While hPLIC proteins are not ubiquitinated by HERC3, HERC5 plays an important role in ubiquitination of Nm23B. In summary, although small HERC proteins are highly homologous showing the same subcellular distribution, they undergo different molecular interactions.

The lysogenic region of virus phiCh1: identification of a repressor-operator system and determination of its activity in halophilic Archaea.

phiCh1 is a temperate virus infecting the haloalkaliphilic archaeon Natrialba magadii. As for all temperate viruses, a control of the lysogenic state versus the lytic life cycle is essential. Two open reading frames (ORFs) have been identified as putative repressor encoding genes: ORF48 and ORF49. The protein of ORF48 showed sequence similarities to putative repressor molecules. ORF49 was identified by the analysis of a mutant of phiCh1: the lysogenic strain carrying mutant phiCh1-1 showed a different lysis behavior than wild type virus phiCh1, indicating a dysfunction in the regulation of gene expression. Here, we show that the intergenic region between ORF48 and ORF49 comprises a promoter/operator sequence that is a transcriptionally active region in the model system Haloferax volcanii. Transcription from this region can be repressed by the activity of the ORF48 gene product. Gp43/gp44 has an enhancing effect on this regulatory sequence. Evidence is given for a possible binding site of Rep and gp43/gp44 within the coding region of the rep gene.

Natrialba magadii virus phiCh1: first complete nucleotide sequence and functional organization of a virus infecting a haloalkaliphilic archaeon.

The double-stranded (ds)DNA virus phiCh1 infects the haloalkaliphilic archaeon Natrialba magadii. The complete DNA sequence of 58 498 bp of the temperate virus was established, and the probable functions of 21 of 98 phiCh1-encoded open reading frames (ORFs) have been assigned. This knowledge has been used to propose functional modules each required for specific functions during virus development. The phiCh1 DNA is terminally redundant and circularly permuted and therefore appears to be packaged by the so-called headful mechanism. The presence of ORFs encoding homologues of proteins involved in plasmid replication as well as experimental evidence indicate a plasmid-mediated replication strategy of the virus. Results from nanosequencing of virion components suggest covalent cross-linking of monomers of at least one of the structural proteins during virus maturation. A comparison of the phiCh1 genome with the partly sequenced genome of Halobacterium salinarum virus phiH revealed a close relationship between the two viruses, although their host organisms live in distinct environments with respect to the different pH values required for growth.

The structural protein E of the archaeal virus phiCh1: evidence for processing in Natrialba magadii during virus maturation.

phiCh1 is a lysogenic virus for the haloalkalophilic archaeon Natrialba magadii. The virus morphology resembles other members of Myoviridae infecting Halobacterium species. The gene of the major capsid protein E of virus phiCh1 was cloned and the DNA sequence was determined. Gene E was mapped to a 3.2-kbp ClaI fragment, localized to the 5'-end of the phiCh1 genome. The complete nucleotide sequence of this region was determined and the identity of gene E was confirmed by comparing the experimentally determined N-terminal amino acid sequence of the purified protein to the translated DNA sequence of its open reading frame. We present evidence that the gene E product is proteolytically cleaved between Lys(16) and Asn(17) to yield the 305 residue polypeptides found in the mature viral capsid. Processing of the protein itself during virus development was determined by 2D gel electrophoresis using protein E-specific antibodies. Sequence similarity studies revealed an 80% identity to capsid protein Hp32 of phiH, infecting Halobacterium salinarum. RT-PCR analysis as well as Western blot studies revealed gene E as a late gene. Transcripts and proteins could be detected shortly before onset of lysis of the lysogenic strain N. magadii L11.

The archaeal halophilic virus-encoded Dam-like methyltransferase M. phiCh1-I methylates adenine residues and complements dam mutants in the low salt environment of Escherichia coli.

The genome of the archaeal virus phiCh1, infecting Natrialba magadii (formerly Natronobacterium magadii), is composed of 58.5 kbp linear ds DNA. Virus particles contain several RNA species in sizes of 100-800 nucleotides. A fraction of phiCh1 genomes is modified within 5'-GATC-3' and related sequences, as determined by various restriction enzyme digestion analyses. High performance liquid chromatography revealed a fifth base, in addition to the four nucleosides, which was identified as N6-methyladenosine. Genetic analyses and subsequent sequencing led to the identification of a DNA (N6-adenine) methyltransferase (mtase) gene. The protein product was designated M.phiCh1-I. By the localization of the most conserved motifs (a DPPY motif occurring before FxGxG), the enzyme was placed within the beta-subgroup of the (N6-adenine) methyltransferase class. The mtase gene of phiCh1 was classified as a 'late' gene, as determined by measuring the kinetics of mRNA and protein expression in N. magadii during the lytic cycle of phiCh1. After infection of cells, M.phiCh1-I mRNA and protein could be detected in lower amounts than in the situation of virus induction from lysogenic cells. Consequently, only about 5% of the phiCh1 progeny genomes after infection of N. magadii carry the M.phiCh1-I methylation in contrast to 50% of virus genomes generated by induction of phiCh1-lysogenic N. magadii cells. Heterologous expression of the mtase from a halophile with 3 M cytoplasmic salt concentration showed an unexpected feature: the protein was active in the low environment of Escherichia coli and was able to methylate DNA in vivo. Interestingly, it seemed to exhibit a higher sequence specificity in E. coli that resulted in adenine methylation exclusively in the sequence 5'-GATC-3'. Additionally, expression of M.phiCh1-I in dam- E. coli cells led to a complete substitution of the function of M. Dam in DNA mismatch repair.

Characterization of Natronobacterium magadii phage phi Ch1, a unique archaeal phage containing DNA and RNA.

A novel archaeal bacteriophage, phi Ch1, was isolated from a haloalkalophilic archaeon Natronobacterium magadii upon spontaneous lysis. The phage-cured strain N. magadii(L13) was used to demonstrate infectivity of phage phi Ch1. The turbid-plaque morphology and the fact that N. magadii cells isolated from plaques were able to produce phage indicated that phi Ch1 is a temperate phage. The phage morphology resembles other members of Myoviridae-infecting Halobacterium species. In solution below 2M NaCl, the phage lost its morphological stability and infectivity. One- and two-dimensional SDS-PAGE of phage particles revealed at least four major and five minor proteins with molecular masses ranging from 15 to 80 kDa and acidic isoelectric points. Southern blot analysis of chromosomal DNA of a lysogenic N. magadii strain showed that phi Ch1 exists as a chromosomally integrated prophage. The phage particles contain both double-stranded, linear DNA (approx. 55 kbp) as well as several RNA species (80-700 nucleotides). Hybridization of labelled RNA fragments to total DNA from N. magadii and phi Ch1 showed that the virion-associated RNA is host encoded. Part of the phage DNA population is modified and restriction analysis revealed evidence for adenine methylation. Phage phi Ch1 is the first virus described for the genus natronobacterium, and the first phage containing DNA and RNA in mature phage particles.