Erika Bosio

Tumor-Induced Tolerance and Immune Suppression Depend on the C/EBPβ Transcription Factor

Tumor growth is associated with a profound alteration in myelopoiesis, leading to recruitment of immunosuppressive cells known as myeloid-derived suppressor cells (MDSCs). We showed that among factors produced by various experimental tumors, the cytokines GM-CSF, G-CSF, and IL-6 allowed a rapid generation of MDSCs from precursors present in mouse and human bone marrow (BM). BM-MDSCs induced by GM-CSF+IL-6 possessed the highest tolerogenic activity, as revealed by the ability to impair the priming of CD8(+) T cells and allow long term acceptance of pancreatic islet allografts. Cytokines inducing MDSCs acted on a common molecular pathway and the immunoregulatory activity of both tumor-induced and BM-derived MDSCs was entirely dependent on the C/EBPbeta transcription factor. Adoptive transfer of tumor antigen-specific CD8(+) T lymphocytes resulted in therapy of established tumors only in mice lacking C/EBPbeta in the myeloid compartment, suggesting that C/EBPbeta is a critical regulator of the immunosuppressive environment created by growing cancers.

Innate inflammatory cells are not responsible for early death of donor myoblasts after myoblast transfer therapy.

Background. Myoblast transfer therapy (MTT) is a cell-based gene therapy representing a potential treatment for Duchenne muscular dystrophy. The rapid disappearance of donor myoblasts from transplanted muscles after MTT is one of the most controversial and significant obstacles facing research in this area. Dystrophin-deficient muscles show constitutively high levels of inflammation, thus necessitating an examination of whether inflammatory cells, specifically natural killer (NK) cells, neutrophils, and macrophages, within dystrophic muscle are responsible for poor graft survival. Methods. Female mdx mice were treated with RB6–8C5 monoclonal antibody, PK136 monoclonal antibody, or clodronate liposomes to systemically deplete neutrophils, NK cells, and macrophages, respectively. After each depletion regimen, the mice and age-matched controls received 5.0×105 male myoblasts injected longitudinally into each tibialis anterior muscle. Donor myoblast survival was assessed by Y-chromosome specific quantitative real-time polymerase chain reaction analysis. Results. The systemic depletion of host neutrophils and NK cells resulted in a transient improvement in donor myoblast survival at 72 hr and 7 days post-MTT, respectively. Systemic depletion of macrophages had no significant beneficial effect on myoblast survival. Overall, the number of detectable male donor myoblasts was similar at time 0 and 1 hr post-MTT; however, there was significant loss by 24 hr (∼50%–70%) followed by a continual decline in donor cell numbers. Conclusions. Neutrophils and macrophages do not seem to play a major role in the rapid death of donor myoblasts after transplantation into dystrophic muscle. NK cells similarly seem to have no significant effect, contrary to earlier findings reported by our group.

Efficacy of low-dose oral use of type I interferon in cytomegalovirus infections in vivo

Oral administration of type I interferons (IFNs; murine IFN-alpha and IFN-beta) reduces early replication of murine cytomegalovirus (MCMV) in both the spleen and liver of MCMV-infected BALB/c mice. Examination of a range of doses of IFN (1 to 1000 IU) showed that 10 IU administered daily for 1 week prior to virus infection was optimal for inhibition of MCMV replication. Furthermore, low-dose orally administered IFN (10 IU/day) was effective in mice challenged with lethal and sublethal virus inocula. The antiviral efficacy of low-dose orally administered IFN was not restricted by either the route of virus inoculation or the mouse genotype. Analysis by immunohistochemistry of IFN-alpha receptor-bearing cells of the gastrointestinal tract revealed predominant staining of perivascular smooth muscle and the lamina propria of the anterior tongue, small intestine and rectum. These tissues, dense in IFN-alpha receptor-bearing cells, are likely to be the sites of interaction of the orally administered IFNs with the mucosal immune system. In conclusion, we propose that low-dose oral use of type I IFN therapy may have broad applications in the treatment of CMV infections.

Effects of long-term administration of high-dose recombinant human antithrombin in immunosuppressed primate recipients of porcine xenografts.

Background. Fibrin deposition is central to the acute humoral rejection process occurring in the presence of consumptive coagulopathy when pig organs are transplanted into primates. Methods. To assess whether strategies aimed at preventing fibrin formation may extend xenograft survival, we administered high daily doses of recombinant human antithrombin (rhAT) (500 U/kg twice daily) to obtain both anticoagulant and anti-inflammatory effects in immunosuppressed primate recipients of porcine kidneys. Results. Some degree of consumptive coagulopathy developed in both rhAT-treated (n=3) and untreated (n=3) primates. No major differences in the coagulation parameters analyzed were observed between the 2 groups. Similarly, no difference in survival was seen between rhAT-treated (20.6±4 days; range: 15–23 days) and untreated animals (17.3±11.6 days; range: 7–30 days), although the rhAT-treated primates had a higher bleeding tendency. Despite the high daily dose of rhAT, considerable fibrin deposition was observed in the graft as early as 2 weeks after transplantation. Conclusions. These results suggest that a high daily dose of rhAT fails to influence survival or prevent fibrin formation and deposition in the graft in our pig-to-primate model. However, the potential role of rhAT administered in combination with heparins or other clotting inhibitor concentrates in this model remains to be determined.

De novo Anti-HLA Antibody Responses after Renal Transplantation: Detection and Clinical Impact

Numerous retrospective and prospective studies have been conducted to determine the prevalence and significance on long-term graft survival of de novo post-transplant donor-specific antibodies (DSA), directed against both HLA and non-HLA molecules. Moreover, it has been postulated that the development of anti-HLA antibodies may precede the clinical manifestation of chronic rejection, therefore being considered a predictive marker. In this context, the detection of C4d deposition in the failing kidney in patients presenting de novo DSA supports the hypothesis that antibody production and complement deposition could be involved in the pathogenesis of graft failure. Due to the development of more sensitive meth-ods to detect alloantibodies, the number of transplanted patients which show the appearance of DSA at different times following transplantation has increased. Nevertheless, this increased sensitivity has allowed the identification of circulating donor-specific anti-HLA antibodies in many patients with otherwise good graft function. Such findings are worthy of discussion, as it has yet to be determined whether these circulating antibodies can only be considered an early marker of humoral rejection or whether they could play a protective role. The possible relevance of the post-transplant appearance of non-DSA should also be mentioned. This review will focus primarily on de novo anti-donor HLA antibody responses in kidney transplant patients and will only briefly deal with anti-non HLA and non-DSA that will be discussed elsewhere in this issue.

In vitro and in vivo effects of the carbon monoxide-releasing molecule, CORM-3, in the xenogeneic pig-to-primate context.

Abstract: Background:  Carbon monoxide (CO) interferes with inflammatory and apoptotic processes associated with ischemia–reperfusion injury and graft rejection. Here, the in vitro effects of carbon monoxide releasing molecule‐3 (CORM‐3), a novel water‐soluble carbonyl CO carrier, have been investigated on porcine aortic endothelial cells (PAEC) and primate peripheral blood mononuclear cells (PBMC). Furthermore, the pharmacodynamics and pharmacotolerance of CORM‐3 after administration of single and multiple doses in the primate have been assessed in view of its potential application in pig‐to‐primate xenotransplantation models.

Islet xenotransplantation: current status of preclinical studies in the pig-to-nonhuman primate model.

Purpose of reviewPorcine islets are a major focus of current research in nonhuman primate xenotransplantation models. Major advances have been obtained recently and these are briefly described. Recent findingsReports by three independent centres have described 6-month porcine islet xenograft survival in nonhuman primates. Two of these have obtained such results by interfering with the CD40/CD154 co-stimulatory pathway. While these results are groundbreaking, the immunosuppressive regimens used are not viewed as clinically applicable and will need to be modified before islet xenotransplantation can be considered for clinical trials. In contrast, preliminary results by Gianello and colleagues have demonstrated 6-month survival of diabetic nonhuman primates transplanted subcutaneously with encapsulated porcine islets, in the complete absence of immunosuppression. The confirmation and full assessment of these results are eagerly awaited. Importantly, to date, no evidence of xenozoonoses has been observed following porcine islet xenotransplantation to nonhuman primates. SummaryOf the possible organ candidates for xenotransplantation, porcine islets are the closest to a possible clinical application. Prior to the initiation of ethical and safe clinical trials, however, further efforts to generate additional efficacy and safety data in the nonhuman primate model will be indispensable.

Low-Dose Orally Administered Type I Interferon Reduces Splenic B Cell Numbers in Mice

The beneficial effects of low-dose orally administered type I interferon (LDOA IFN) have been demonstrated in various animal models of disease and in some human clinical trials. The mechanisms by which LDOA IFN therapy has its effects, however, remain to be established. In the present study, groups of mice were administered 10 IU murine IFN-alpha/beta (MuIFN-alpha/beta) orally for 7 days. Spleens were then collected and analyzed. No differences were detected between the spleen weights of treated mice compared with controls, although reductions in total splenic white blood cell (WBC) number ranging from 15.5% to 35% were observed. Further analysis showed this reduction to be largely restricted to the B cell population, with only minor reductions in CD4(+) or CD8(+) populations being detected. Dose-response studies showed the WBC loss from the spleen to be optimal at 1 IU MuIFN-alpha/beta, whereas both higher and lower doses showed less significant effects. Time course studies show these effects had developed after 2 days of treatment. It is hypothesized that this observed WBC movement from the spleen is part of the mechanism of action of LDOA IFN.

Xenotransplantation as a model of integrated, multidisciplinary research.

Xenotransplantation was proposed a long time ago as a possible solution to the world-wide shortage of human organs. For years, researchers in this field have almost exclusively directed their efforts towards combating the immunological barrier that precluded the long-term xenograft survival. Studies have been conducted in both small and large animal models and the most relevant results have been obtained in pre-clincal studies, specifically those utilising the pig-to-nonhuman primate combination. In this context, a better understanding of the immunological mechanisms underlying the rejection of a xenograft have allowed the identification of specific targets of intervention that have resulted in considerable improvements in survival of porcine organs or cells in nonhuman primates. However it has also become apparent that if xenotransplantation has to enter the clinical arena, a multidisciplinary approach will be needed to comprehensively tackle the different issues related to the use of a xenograft to cure human disease. In this regard, the safety, ethics and regulatory aspects of xenotransplantation are currently being aggressively addressed to enable the initiation of xenotransplantation with a favourable risk/benefit ratio.

Antibody mediated rejection in pig-to-nonhuman primate xenotransplantation models.

Antibody-mediated mechanisms are central to the rejection that occurs when pig organs are transplanted into primates. In this article, the histopathological features of the humoral rejection process in these species combinations, namely hyperacute rejection and acute humoral xenograft rejection, will be illustrated. The profile of the natural and elicited antibodies involved will also be discussed. It has now been demonstrated that the natural immune response to a porcine xenograft is primarily directed to Galalpha1-3Gal (alphaGal) specificities, whilst the elicited immune response is directed to both alphaGal and non-alphaGal antigens. The principal characteristics of anti-alphaGal, anti-non-alphaGal and polyreactive antibodies will be described, together with the identification of the molecules recognised by natural and elicited xenoreactive antibodies. The role of the humoral immune response in the rejection of porcine islets in the primate is still uncertain and the current views on the subject will be discussed. Finally, a concise but comprehensive review of the different strategies that have been attempted to prevent the onset of antibody-mediated rejection is presented. These strategies encompass approaches aimed at interfering with the binding of xenoreactive antibodies with their targets, the use of conventional or novel immunosuppressants and splenectomy. It is undeniable that significant progress has been recently achieved in understanding the humoral rejection process of pig organs transplanted into primates. It is expected that a more comprehensive elucidation of the mechanisms underlying accommodation and tolerance may, in the not too distant future, further extend survival of pig organs transplanted into primates.