Andreas Katopodis

Oxysterols direct immune cell migration via EBI2.

Epstein–Barr virus-induced gene 2 (EBI2, also known as GPR183) is a G-protein-coupled receptor that is required for humoral immune responses; polymorphisms in the receptor have been associated with inflammatory autoimmune diseases. The natural ligand for EBI2 has been unknown. Here we describe the identification of 7α,25-dihydroxycholesterol (also called 7α,25-OHC or 5-cholesten-3β,7α,25-triol) as a potent and selective agonist of EBI2. Functional activation of human EBI2 by 7α,25-OHC and closely related oxysterols was verified by monitoring second messenger readouts and saturable, high-affinity radioligand binding. Furthermore, we find that 7α,25-OHC and closely related oxysterols act as chemoattractants for immune cells expressing EBI2 by directing cell migration in vitro and in vivo. A critical enzyme required for the generation of 7α,25-OHC is cholesterol 25-hydroxylase (CH25H). Similar to EBI2 receptor knockout mice, mice deficient in CH25H fail to position activated B cells within the spleen to the outer follicle and mount a reduced plasma cell response after an immune challenge. This demonstrates that CH25H generates EBI2 biological activity in vivo and indicates that the EBI2–oxysterol signalling pathway has an important role in the adaptive immune response.

Removal of anti-Galα1,3Gal xenoantibodies with an injectable polymer

Preformed and elicited Abs against the Galalpha1,3Gal terminating carbohydrate chains (alphaGal Abs) are the primary cause of hyperacute and acute vascular xenograft rejection in pig-to-primate transplantation. alphaGal Abs are produced by long-lived Ab-producing cells that are not susceptible to pharmacological immunosuppression. We reasoned that antigen-specific elimination of alphaGal Abs might be achieved in vivo by systemic administration of nonimmunogenic polyvalent alphaGal structures with high avidity for alphaGal Abs. We devised GAS914, a soluble trisaccharide-polylysine conjugate of approximately 500 kDa that effectively competes for alphaGal binding by alphaGal IgM (IC(50), 43 nM) and IgG (IC(50), 28 nM) in vitro. Injections of GAS914 in cynomolgus monkeys, at the dose of 1 mg/kg, resulted in the immediate decrease of more than 90% of circulating alphaGal Abs and serum anti-pig cytotoxicity. In baboons, repeated injections of GAS914 effectively reduced both circulating alphaGal Abs and cytotoxicity over several months. Studies with [(14)C]GAS914 in rhesus monkeys and Gal(-/-) mice indicate that GAS914 binds to circulating alphaGal Abs and that the complex is quickly metabolized by the liver and excreted by the kidney. Remarkably, posttreatment alphaGal Ab titers never exceeded pretreatment levels and no sensitization to either alphaGal or the polylysine backbone has been observed. Furthermore there was no apparent acute or chronic toxicity associated with GAS914 treatment in primates. We conclude that GAS914 may be used therapeutically for the specific removal of alphaGal Abs.

CLONING OF A HUMAN UDP-GALACTOSE :2-ACETAMIDO-2-DEOXY-D-GLUCOSE 3BETA -GALACTOSYLTRANSFERASE CATALYZING THE FORMATION OF TYPE 1 CHAINS

Biochemical evidence suggests that the galactosyltransferase activity synthesizing type 1 carbohydrate chains is separate from the well characterized enzyme that is responsible for the synthesis of type 2 chains. This was recently confirmed by the cloning, from melanoma cells, of an enzyme capable of synthesizing type 1 chains, which was shown to have no homology to other galactosyltransferases. We report here the molecular cloning and functional expression of a second human β3-galactosyltransferase distinct from the melanoma enzyme. The new β3-galactosyltransferase has homology to the melanoma enzyme in the putative catalytic domain, but has longer cytoplasmic and stem regions and a carboxyl-terminal extension. Northern blots showed that the new gene is present primarily in brain and heart. When transfected into mammalian cells, this gene directs the synthesis of type 1 chains as determined by a monoclonal antibody specific for sialyl Lewisa. A soluble version of the cloned enzyme was expressed in insect cells and purified. The soluble enzyme readily catalyzes the transfer of galactose to GlcNAc to form Gal(β1–3)GlcNAc. It also has a minor but distinct transfer activity toward Gal, LacNAc, and lactose, but is inactive toward GalNAc.

Removal of bowel aerobic gram-negative bacteria is more effective than immunosuppression with cyclophosphamide and steroids to decrease natural α-Galactosyl IgG antibodies

Abstract: Natural α‐Galactosyl (Gal) antibodies play an important role in the rejection of pig xenografts by humans and Old World monkeys. In this study we investigate the efficacy of two different strategies to reduce the serum level of natural anti‐Gal antibodies. On the one hand, removal of aerobic gram‐negative bacteria from the intestinal flora, because anti‐Gal antibodies appear to be produced as a result of the continuous sensitization by these microorganisms. On the other hand, we studied the effect on these antibodies of an immunosuppressive regimen of cyclophosphamide and steroids. Ten baboons were treated for three months with norfloxacin (Nor Group; n = 6) or cyclophosphamide and steroids (CyP Group; n = 4). A further four baboons did not receive any treatment (Control Group). Aerobic gram‐negative bacteria became negative in stools of the Nor Group after two weeks of treatment, and remained undetectable until week 7. Thereafter, a gradual increase on the fecal concentration of aerobic gram‐negative bacteria was observed despite the norfloxacin treatment. The mean anti‐Gal IgG in the Nor Group gradually declined from week 4 to 9 to a mean of 62.7 ± 18% of the baseline level, and during this period were significantly lower than in the CyP (P < 0.02) and the Control (P < 0.05) groups. No differences were observed between the three groups during the 16 weeks of follow‐up in serum levels of anti‐Gal IgM, hemolytic anti‐pig antibodies, total IgG, IgM and IgA. In conclusion, removal of normal aerobic gram‐negative bacteria from the intestinal flora is more effective than immunosuppression with CyP and steroids in reducing the level of natural anti‐Gal antibodies, although there is no discernible effect on IgM antibodies.

Novel glycodendrimers self-assemble to nanoparticles which function as polyvalent ligands in vitro and in vivo

The recognition of oligosaccharides by proteins represents the basis of many biologically important events.[1] Individual protein±carbohydrate interactions are generally weak (KD1⁄4 10 3±10 4m 1).[2] To overcome this, such processes often involve polyvalent binding, which is characterized by the simultaneous contact of multiple ligands (oligosaccharides) on one biological entity to multiple receptors (proteins) on another.[3] Polyvalent carbohydrate±protein interactions occur frequently in recognition events on cellular membranes. Collectively, they can be much stronger than corresponding monovalent interactions rendering it difficult to control them with individual small molecules.[4] Therefore, complex macromolecules have been used as polyvalent antagonists, however, both characterization and preparation of these nonuniform entities is demanding.[4] Here we present an alternative concept for the polyvalent presentation of ligands based on the supramolecular chemistry[5] of small molecules that fulfil single-molecule entity criteria (Figure 1). Novel dendrons capped with carbohydrate ligands (glycodendrimers[6]) were found to self-assemble to noncovalent nanoparticles which function as polyvalent ligands. We demonstrate that these particles–not the individual molecules–efficiently inhibit polyvalent interactions, such as IgM binding (IgM1⁄4 immunoglobulin), to the aGal-epitope[7] (a-d-Gal-(1!3)-b-d-Gal(1!4)-d-GlcNAc), both in vitro and in vivo. As self-assembly is dynamic, optimization of size and shape of the polyvalent ligand could occur utilizing the receptor as a template. Dendrimer cores were prepared by a convergent TMoutsidein∫ approach[8] based on a single building block 1a which was obtained frommethyl 3,5-diaminobenzoate and 4-(tert-butoxycarbonylaminomethyl)benzoic acid (Scheme 1a). Selective deprotection furnished 1b and 1c (first-generation dendrimer core, two end-groups). A one-pot procedure comprising coupling of 1c (1 equiv) and 1b (0.5 equiv) followed by methyl ester cleavage gave 2c (second-generation dendrimer core, four end groups).[9] The third-generation dendrimer 3c (eight end groups) was obtained from 2c (1 equiv) and 1b (0.5 equiv).[9] Applying the same procedure repetitively gave dendrimers with up to 64 end groups (4c, fourth generation, 16 end groups; 5c, fifth generation, 32 end groups; 6c, sixth generation, 64 end groups). Dendrimers 1c±6c were deprotected (!1d±6d) and transformed into their chloroacetamide derivatives (1e±6e) to allow subsequent introduction of thiolated oligosaccharides such as aGal-SH[10] and Lac-SH (Figure 2b) furnishing water-soluble glycodendrimers 1 f±6 f and 3g which were purified by ultrafiltration. Compound 7, which is similar to 2 f but contains butylene chains instead of the disubstituted aromatic rings, was also prepared (Scheme 1a). The integrity of all compounds was established by 1H NMR spectroscopy. Accordingly, the firstto third-generation dendrimers exist as single molecules (purity > 95%). The fourthto sixth-generation dendrimers possibly contain minute quantities of smaller fragments. The 500 MHz 1H NMR spectra of compound 3 f in [D6]DMSO demonstrates the remarkable purity of these compounds (Figure 2). The first indication that our glycodendrimers were aggregating in water came from 1H NMR spectroscopy of 2 f in D2O. At ambient temperature, we observed very broad signals which sharpened at elevated temperatures. The aggregation was quantified using multiangle light scattering (MALS; Table 1). The first-generation dendrimer 1 f forms small aggregates (50 kDa) whereas 2 f forms large particles of 7000 kDa (more than 1500 individual molecules per particle). Interestingly, the particle weight obtained for 3 f±6 f drops (2200 to 200 kDa) with increasing mass of the individual molecule. The root-mean-square radii of the particles formed by 2 f±6 f showed the same trend (for 2 f, 3 f, and 4 f 49, 34, and 12 nm, respectively; for 5 f and 6 f the radii were below the detection limit of 10 nm). Core-modified second-generation glycodendrimer 7 (4 iaGal), which is of comparable size and lipophilicity as 2 f but contains butylene chains instead of the disubstituted aromatic rings, does not form aggregates. The third-generation compounds 3g (8 iLac; 1900 kDa) and 3 f ZUSCHRIFTEN

Deficiency of MALT1 Paracaspase Activity Results in Unbalanced Regulatory and Effector T and B Cell Responses Leading to Multiorgan Inflammation

The paracaspase MALT1 plays an important role in immune receptor-driven signaling pathways leading to NF-κB activation. MALT1 promotes signaling by acting as a scaffold, recruiting downstream signaling proteins, as well as by proteolytic cleavage of multiple substrates. However, the relative contributions of these two different activities to T and B cell function are not well understood. To investigate how MALT1 proteolytic activity contributes to overall immune cell regulation, we generated MALT1 protease-deficient mice (Malt1PD/PD) and compared their phenotype with that of MALT1 knockout animals (Malt1−/−). Malt1PD/PD mice displayed defects in multiple cell types including marginal zone B cells, B1 B cells, IL-10–producing B cells, regulatory T cells, and mature T and B cells. In general, immune defects were more pronounced in Malt1−/− animals. Both mouse lines showed abrogated B cell responses upon immunization with T-dependent and T-independent Ags. In vitro, inactivation of MALT1 protease activity caused reduced stimulation-induced T cell proliferation, impaired IL-2 and TNF-α production, as well as defective Th17 differentiation. Consequently, Malt1PD/PD mice were protected in a Th17-dependent experimental autoimmune encephalomyelitis model. Surprisingly, Malt1PD/PD animals developed a multiorgan inflammatory pathology, characterized by Th1 and Th2/0 responses and enhanced IgG1 and IgE levels, which was delayed by wild-type regulatory T cell reconstitution. We therefore propose that the pathology characterizing Malt1PD/PD animals arises from an immune imbalance featuring pathogenic Th1- and Th2/0-skewed effector responses and reduced immunosuppressive compartments. These data uncover a previously unappreciated key function of MALT1 protease activity in immune homeostasis and underline its relevance in human health and disease.

Combinations of anti-LFA-1, everolimus, anti-CD40 ligand, and allogeneic bone marrow induce central transplantation tolerance through hemopoietic chimerism, including protection from chronic heart allograft rejection.

Central transplantation tolerance through hemopoietic chimerism initially requires inhibition of allogeneic stem cell or bone marrow (BM) rejection, as previously achieved in murine models by combinations of T cell costimulation blockade. We have evaluated LFA-1 blockade as part of regimens to support mixed hemopoietic chimerism development upon fully allogeneic BALB/c BM transfer to nonirradiated busulfan-treated B6 recipient mice. Combining anti-LFA-1 with anti-CD40 ligand (CD40L) induced high incidences and levels of stable multilineage hemopoietic chimerism comparable to chimerism achieved with anti-CD40L and everolimus (40-O-(2-hydroxyethyl)-rapamycin) under conditions where neither Ab alone was effective. The combination of anti-LFA-1 with everolimus also resulted in high levels of chimerism, albeit with a lower incidence of stability. Inhibition of acute allograft rejection critically depended on chimerism stability, even if maintained at very low levels around 1%, as was the case for some recipients without busulfan conditioning. Chimerism stability correlated with a significant donor BM-dependent loss of host-derived Vβ11+ T cells 3 mo after BM transplantation (Tx). Combinations of anti-CD40L with anti-LFA-1 or everolimus also prevented acute rejection of skin allografts transplanted before established chimerism, albeit not independently of allospecific BMTx. All skin and heart allografts transplanted to stable chimeras 3 and 5 mo after BMTx, respectively, were protected from acute rejection. Moreover, this included prevention of heart allograft vascular intimal thickening (“chronic rejection”).

The effect of soluble complement receptor type 1 on acute humoral xenograft rejection in hDAF-transgenic pig-to-primate life-supporting kidney xenografts

Abstract:  Background:  In pig‐to‐nonhuman primate solid organ xenotransplantation using organs from donors transgenic for human decay‐accelerating factor (hDAF), the main type of rejection is antibody‐mediated (acute humoral xenograft rejection, AHXR). This occurs despite the complement‐regulatory function of the transgene, neutralization of natural antibodies to Galα1–3Gal (Gal) using soluble glycoconjugates, and chronic immunosuppression. As complement components play a major role in graft destruction after antibody binding, we evaluated the efficacy of chronic complement inhibition by soluble complement receptor type 1 (TP10).

Hyperacute rejection of hDAF-transgenic pig organ xenografts in cynomolgus monkeys: Influence of pre-existing anti-pig antibodies and prevention by the αGal glycoconjugate GAS914

Abstract:  Background:  Our introductory pig‐to‐cynomolgus monkey heart or kidney transplantation using organs from pigs transgenic for human decay‐accelerating factor (hDAF), showed a high incidence of hyperacute rejection (HAR), which was ascribed to extraordinary high levels of anti‐pig antibodies. We evaluated the efficacy of GAS914, a Galα1–3Gal trisaccharide linked to a poly‐l‐lysine backbone, in inhibition of HAR.

Modulation of T cell homeostasis and alloreactivity under continuous FTY720 exposure

The immunomodulator FTY720 inhibits lymph node (LN) and thymic egress, thereby constraining T cell circulation and reducing peripheral T cell numbers. Here, we analyzed in mouse models the as yet scarcely characterized impact of long-term (up to 6 months) FTY720 exposure on T cell homeostasis and possible consequences for alloreactivity. In green fluorescent protein (GFP) hemopoietic chimeras, the turnover of (initially GFP(-)) peripheral T cell pools was markedly delayed under FTY720, while normal homeostatic differences between CD4 and CD8 T cell sub-populations were retained or amplified further. Homeostatic proliferation was enhanced, and within shrinking T cell pools, the proportions of effector memory phenotype CD4 T cells (CD4T(PEM)) increased in spleens and LNs and of central memory phenotype CD8 T cells (CD8T(PCM)) in LNs. By contrast, the fractions of CD8T(PEM) and CD4T(PCM) remained stably small under FTY720. The enrichment for CD4T(PEM) and CD8T(PCM) correlated with larger proportions of IFNgamma-producing T cells upon nonspecific but not allospecific stimulation. Splenic CD4 T cells from FTY720-treated mice proliferated more strongly upon transfer to semi-allogeneic hosts. However, heart allograft survival was not compromised in FTY720 pre-treated recipients. It correlated with reduced intra-graft CD8 T cells, and the longest surviving transplants contained the highest numbers of CD4 T cells. Thus, continuous FTY720 exposure reveals differential homeostatic responses by memory phenotype CD4 and CD8 T cell sub-populations, and it may enhance alloreactive CD4 T cell proliferation and tissue infiltration without accelerating allograft rejection.