T cell responses to MHC-mismatched transplants can be mediated via direct recognition of allogeneic MHC

Phosphatidylinositol 3'-kinase (PI 3'-kinase) is activated in insulin-stimulated cells by the binding of the SH2 domains in its 85-kDa regulatory subunit to insulin receptor substrate-1 (IRS-1). We have previously shown that both tyrosyl-phosphorylated IRS-1 and mono-phosphopeptides containing a single YXXM motif activate PI 3'-kinase in vitro. However, activation by the monophosphopeptides was significantly less potent than activation by the multiply phosphorylated IRS-1. We now show that the increased potency of PI 3'-kinase activation by IRS-1 relative to phosphopeptide is not due to tertiary structural features IRS-1, as PI 3'-kinase is activated normally by denatured, reduced, and carboxymethylated IRS-1. Furthermore, activation of PI 3'-kinase by bis-phosphorylated peptides containing two YXXM motifs is 100-fold more potent than the corresponding mono-phosphopeptides and similar to activation by IRS-1. These data suggest that tyrosyl-phosphorylated IRS-1 or bis-phosphorylated peptides bind simultaneously to both SH2 domains of p85. However, these data cannot differentiate between an activation mechanism that requires two-site occupancy for maximal activity as opposed to one in which bivalent binding enhances the occupancy of a single activating site. To distinguish between these possibilities, we produced recombinant PI 3'-kinase containing either wild-type p85 or p85 mutated in its N-terminal, C-terminal, or both SH2 domains. We find that mutation of either SH2 domains significantly reduced phosphopeptide binding and decreased PI 3'-kinase activation by 50%, whereas mutation of both SH2 domains completely blocked binding and activation. These data provide the first direct evidence that full activation of PI 3'-kinase by tyrosylphosphorylated proteins requires occupancy of both SH2 domains in p85.

Objectives Intravascular thrombosis remains a major barrier to successful pig-to-primate xenotransplantation. However, the precise factors initiating thrombosis are unknown. In this study, we investigated the contribution of recipient platelets and monocytes. Methods Primary pig aortic endothelial cells (PAEC) were incubated with combinations of fresh or heat-inactivated (HI) human plasma, platelets, or monocytes, following which they were separated and analysed individually by flow cytometry for tissue factor (TF) expression and for their ability to clot recalcified normal or FVII-deficient plasma. Results Procoagulant porcine TF was induced on PAEC only by fresh human plasma, not HI plasma, platelets or monocytes. In contrast, procoagulant human TF was induced on platelets and monocytes after incubation with PAEC, irrespective or whether plasma was present on not. In addition, human platelets caused the shedding of procoagulant TF-expressing aggregates from PAEC. Conclusions This work defines a cell-based in vitro assay system to address complex interactions between PAEC, human platelets and monocytes. The induction of procoagulant TF on PAEC by fresh human plasma was most likely dependent on xenoreactive natural antibody and complement present in fresh human plasma. In contrast, the shedding of procoagulant platelet-PAEC aggregates, induced by human platelets, and the induction of procoagulant TF on human platelets and monocytes by PAEC, occurred independently of these factors. These results suggest that different mechanisms may contribute to the initiation of thrombosis after xenotransplantation, some of which may not be influenced by further manipulation of the immune response against pig xenografts.

Xenotransplantation promises an unlimited supply of organs for clinical transplantation. However, an aggressive humoral immune response continues to limit the survival of pig organs after transplantation into primates. Because intravascular thrombosis and systemic coagulopathy are prominent features of acute humoral xenograft rejection, we hypothesized that expression of anticoagulants on xenogeneic vascular endothelium might inhibit the process. Hearts from novel transgenic mice, expressing membrane-tethered fusion proteins based on human tissue factor pathway inhibitor and hirudin, respectively, were transplanted into rats. In contrast to control non-transgenic mouse hearts, which were all rejected within 3 days, 100% of the organs from both strains of transgenic mice were completely resistant to humoral rejection and survived for more than 100 days when T-cell-mediated rejection was inhibited by administration of ciclosporin A. These results demonstrate the critical role of coagulation in the pathophysiology of acute humoral rejection and the potential for inhibiting rejection by targeting the expression of anticoagulants to graft endothelial cells. This genetic strategy could be applied in a clinically relevant species such as the pig.

We have generated transgenic mice expressing the leech anticoagulant hirudin and human tissue factor pathway inhibitor tethered to the cell surface by fusion with fragments of human CD4 and P-

Thrombin and other coagulation proteases mediate a variety of eff ects independently of thrombosis through specifi c protease-activated receptors (PARs), stimulation of which can amplify infl ammation initiated by several diverse stimuli ( 1 ). Although the proinfl ammatory consequences of PAR stimulation have been implicated in several diseases ( 2 -4 ), it has yet to be established whether thrombin or PAR activation provides a unique stimulus responsible for a nonthrombotic manifestation of infl ammation.Working in a model of acute humoral xenograft rejection (mouse heart into rat), we previously reported that inhibiting thrombin generation or inhibiting thrombin itself inside the graft (using organs from transgenic [Tg] mice expressing endothelial cell [EC]-tethered anticoagulants) completely inhibited humoral rejection so that hearts were rejected by infi ltrating T lymphocytes ( 5 ). These fi ndings were surprising because the rejected hearts had signifi cant Ig and C deposition on graft ECs. Inhibiting thrombosis by depleting fi brinogen from the recipients (using a snake venom protein, ANCROD) failed to achieve the same degree of survival, and under these conditions, infi ltrating NK cells and macrophages (M ⌽ s), rather than T cells, were observed in rejected grafts ( 6 ). From these studies we hypothesized that thrombin was providing a stimulus in the humoral rejection process that was necessary for the infi ltration of NK cells and M ⌽ s. We have tested this hypothesis and confi rmed our conclusions in a second model of thioglycollateinduced M ⌽ recruitment into the peritoneum. RESULTS AND DISCUSSION Donor monocyte chemoattractant protein (MCP)-1 is required for NK cell and M ⌽ recruitmentWe hypothesized that infi ltration of NK cells and M ⌽ s into rejecting mouse hearts was due to the establishment of a chemokine gradient, most likely MCP-1, a CC chemokine known to be essential for NK cell and M ⌽ recruitment. Thrombin, acting through a family of protease-activated receptors (PARs), is known to amplify infl ammatory responses, but the in vivo importance of PARs in infl ammation is not fully appreciated. In a mouse heart-to-rat transplant model, where it is possible to distinguish graft (mouse) from systemic (rat) chemokines, we show that donor PAR-1 is required to generate the local monocyte chemoattractant protein (MCP)-1 needed to recruit rat natural killer cells and macrophages into the hearts. We have confi rmed the importance of this mechanism in a second model of thioglycollate-induced peritonitis and also show that PAR-1 is important for the production of MCP-3 and MCP-5. Despite the presence of multiple other mediators capable of stimulating chemokine production in these models, these data provide the fi rst evidence that thrombin and PAR activation are required in vivo to initiate infl ammatory cell recruitment. CORRESPONDENCE

CD4+CD25+ T regulatory cells (Tregs) can actively suppress immune responses and thus have substantial therapeutical potential. Clinical application is, however, frustrated by their scarcity, anergic status, and lack of defined specificity. We found that a single injection of a small number of expanded but not fresh HY-specific Tregs protected syngeneic male skin grafts from rejection by immune-competent recipients. The expanded Tregs were predominantly located in the grafts and graft-draining lymph nodes. In vitro expanded Tregs displayed a phenotype of CD25highCD4lowFoxp3+CTLA4+, and also up-regulated IL10 and TGFβ while down-regulating IFN-γ, GM-CSF, IL5, and TNF-α production. Furthermore, expanded Tregs appeared to express a reduced level of Foxp3, which could be prevented by adding TGFβ to the culture, and they also tended to lose Foxp3 following the repeated stimulation. Finally, a proportion of expanded HY-specific Tregs secreted IL2 in response to their cognate peptide, and this finding could be confirmed using Tregs from Foxp3GFP reporter mice. We not only demonstrated that expanded Tregs are superior to fresh Tregs in suppressing T cell responses against alloantigens, but also revealed some novel immunobiological properties of expended Tregs which are very instructive for modifying current Treg expansion procedures.

We have developed a polyclonal antibody that activates the heterodimeric p85-p110 phosphatidylinositol (PI) 3-kinase in vitro and in microinjected cells. Affinity purification revealed that the activating antibody recognized the N-terminal SH2 (NSH2) domain of p85, and the antibody increased the catalytic activity of recombinant p85-p110 dimers threefold in vitro. To study the role of endogenous PI 3-kinase in intact cells, the activating anti-NSH2 antibody was microinjected into GRC؉LR73 cells, a CHO cell derivative selected for tight quiescence during serum withdrawal. Microinjection of anti-NSH2 antibodies increased bromodeoxyuridine (BrdU) incorporation fivefold in quiescent cells and enhanced the response to serum. These data reflect a specific activation of PI 3-kinase, as the effect was blocked by coinjection of the appropriate antigen (glutathione S-transferase-NSH2 domains from p85␣), coinjection of inhibitory anti-p110 antibodies, or treatment of cells with wortmannin. We used the activating antibodies to study signals downstream from PI 3-kinase. Although treatment of cells with 50 nM rapamycin only partially decreased anti-NSH2-stimulated BrdU incorporation, coinjection with an anti-p70 S6 kinase antibody effectively blocked anti-NSH2-stimulated DNA synthesis. We also found that coinjection of inhibitory anti-ras antibodies blocked both serum-and anti-NSH2-stimulated BrdU incorporation by approximately 60%, and treatment of cells with a specific inhibitor of MEK abolished antibody-stimulated BrdU incorporation. We conclude that selective activation of physiological levels of PI 3-kinase is sufficient to stimulate DNA synthesis in quiescent cells. PI 3-kinasemediated DNA synthesis requires both p70 S6 kinase and the p21 ras /MEK pathway.Phosphatidylinositol (PI) 3Ј-kinases are a family of enzymes with homologous catalytic subunits and varied regulatory domains or subunits (26). Isoforms of PI 3Ј-kinase include heterodimeric PI 3Ј-kinases that are stimulated by binding of regulatory SH2 domains to tyrosine phosphoproteins, an isoform form that is regulated by ␤␥ subunits from trimeric G proteins, a monomeric form that is homologous to the VPS-34 yeast PI 3Ј-kinase, and isoforms containing C2 regulatory domains (13,20,22,37,43,48,57,58,63,64). Substrate specificities of the PI 3Ј-kinases also vary: the p85-p110 and ␤␥-stimulated PI 3Ј-kinases utilize PI, PI 4-P and PI 4,5-P 2 , whereas the VPS-34-like PI kinases are specific for PI (20,58,64) and the C2-domain-containing isoforms preferentially utilize PI and PI 4-P (37, 63). The catalytic subunit of the p85-p110 PI 3Ј-kinase is also homologous to a yeast PI 4-kinase, the ataxia telangiectasia gene product, the DNA-dependent protein kinase, and the TOR2/FRAP/RAFT proteins, which are upstream regulators of p70 S6 kinase and targets of the immunosuppressant rapamycin (14,18,30,55,56).The heterodimeric PI 3Ј-kinase is composed of an 85-kDa regulatory subunit (p85) and a 110-kDa catalytic subunit (p110) (reviewed in reference 26). The p85 regulatory subunit...