Marcelo Hill

CD40Ig treatment results in allograft acceptance mediated by CD8+CD45RClow T cells, IFN-γ, and indoleamine 2,3-dioxygenase

Treatment with CD40Ig results in indefinite allograft survival in a complete MHC-mismatched heart allograft model in the rat. Here we show that serial second, third, and fourth adoptive transfers of total splenocytes from CD40Ig-treated recipients into secondary recipients led to indefinite donor-specific allograft acceptance. Purification of splenocyte subpopulations from CD40Ig-treated recipients demonstrated that only the adoptively transferred CD8(+)CD45RC(low) subset resulted in donor-specific long-term survival, whereas CD8(+)CD45RC(low) T cells from naive animals did not. Accepted grafts displayed increased indoleamine 2,3-dioxygenase (IDO) expression restricted in the graft to ECs. Coculture of donor ECs with CD8(+)CD45RC(low) T cells purified from CD40Ig-treated animals resulted in donor-specific IDO expression dependent on IFN-gamma. Neutralization of IFN-gamma or IDO triggered acute allograft rejection in both CD40Ig-treated and adoptively transferred recipients. This study demonstrates for what we believe to be the first time that interference in CD40-CD40 ligand (CD40-CD40L) interactions induces allospecific CD8(+) Tregs that maintain allograft survival. CD8(+)CD45RC(low) T cells act through IFN-gamma production, which in turn induces IDO expression by graft ECs. Thus, donor alloantigen-specific CD8(+) Tregs may promote local graft immune privilege through IDO expression.

IDO expands human CD4+CD25high regulatory T cells by promoting maturation of LPS-treated dendritic cells

We have previously shown that human monocyte‐derived dendritic cells (DC) express indoleamine 2,3‐dioxygenase (IDO), as well as several other enzymes of the kynurenine pathway at the mRNA level upon maturation. The tolerogenic mechanisms of this pathway remain unclear. Here we show that LPS‐treated DC metabolize tryptophan as far as quinolinate. We found that IDO contributes to LPS and TNF‐α + poly(I:C)‐induced DC maturation since IDO inhibition using two different inhibitors impairs DC maturation. IDO knock‐down using short‐hairpin RNA also led to diminished LPS‐induced maturation. In line with these results, the tryptophan‐derived catabolites 3‐hydroxyanthranilic acid and 3‐hydroxykynurenine increased maturation of LPS‐treated DC. Concerning the molecular mechanisms of this effect, IDO acts as an intermediate pathway in LPS‐induced production of reactive oxygen species and NF‐κB activation, two processes that lead to DC maturation. Finally, we show that mature DC expand CD4+CD25high regulatory T cells in an IDO‐dependent manner. In conclusion, we show that IDO constitutes an intermediate pathway in DC maturation leading to expansion of CD4+CD25high regulatory T cells.

Heme oxygenase-1 inhibits rat and human breast cancer cell proliferation: mutual cross inhibition with indoleamine 2,3-dioxygenase

Heme oxygenase‐1 (HO‐1) is the rate limiting enzyme of heme catabolism whereas indoleamine 2,3 dioxygenase (IDO) catabolizes tryptophan through the kynurenine pathway. We analyzed the expression and biological effects of these enzymes in rat and human breast cancer cell lines. We show that rat (NMU and 13762) but not human cells (MCF‐7 and T47D) express HO‐1. When overexpressed, we found this enzyme to have anti‐proliferative and proapoptotic effects by antioxidant mechanisms in these four cell lines. We show that IDO is expressed by rat and human breast cancer cells. IDO inhibition with 1‐MT and siRNA leads to diminished proliferation in rat cells. In contrast, HO‐1 negative human cell lines increase proliferation upon IDO inhibition. Since we also demonstrate that IDO inhibits the anti‐proliferative HO‐1, we propose that IDO has opposite effects on proliferation depending on the coexpression or not of HO‐1. We also describe that HO‐1 inhibits IDO at the post‐translational level through heme starvation. In vivo, we show that rat normal breast expresses HO‐1 and IDO. In contrast, N‐nitrosomethylurea‐induced breast adenocarcinomas only express IDO. In conclusion, we show that HO‐1/IDO cross‐regulation modulates apoptosis and proliferation in rat and human breast cancer cells.—Hill, M., Pereira, V., Chauveau, C., Zagani, R., Remy, S., Tesson, L., Mazal, D., Ubillos, L., Brion, R., Ashgar, K., Mashreghi, M. F., Kotsch, K., Moffett, J., Doebis, C., Seifert, M., Boczkowski, J., Osinaga, E., Anegon, I. Heme oxygenase‐1 inhibits rat and human breast cancer cells proliferation: mutual cross inhibition with indoleamine 2,3‐dioxygenase. FASEB J. 19, 1957–1968 (2005)

Endotoxin-Induced Myeloid-Derived Suppressor Cells Inhibit Alloimmune Responses via Heme Oxygenase-1

Inflammation and cancer are associated with impairment of T‐cell responses by a heterogeneous population of myeloid‐derived suppressor cells (MDSCs) coexpressing CD11b and GR‐1 antigens. MDSCs have been recently implicated in costimulation blockade‐induced transplantation tolerance in rats, which was under the control of inducible NO synthase (iNOS). Herein, we describe CD11b+GR‐1+MDSC‐compatible cells appearing after repetitive injections of lipopolysaccharide (LPS) using a unique mechanism of suppression. These cells suppressed T‐cell proliferation and Th1 and Th2 cytokine production in both mixed lymphocyte reaction and polyclonal stimulation assays. Transfer of CD11b+ cells from LPS‐treated mice in untreated recipients significantly prolonged skin allograft survival. They produced large amounts of IL‐10 and expressed heme oxygenase‐1 (HO‐1), a stress‐responsive enzyme endowed with immunoregulatory and cytoprotective properties not previously associated with MDSC activity. HO‐1 inhibition by the specific inhibitor, SnPP, completely abolished T‐cell suppression and IL‐10 production. In contrast, neither iNOS nor arginase 1 inhibition did affect suppression. Importantly, HO‐1 inhibition before CD11b+ cell transfer prevented the delay of allograft rejection revealing a new MDSC‐associated suppressor mechanism relevant for transplantation.

Anti‐CD28 Antibody‐Induced Kidney Allograft Tolerance Related to Tryptophan Degradation and TCR− Class II− B7+ Regulatory Cells

B7/CTLA‐4 interactions negatively regulate T‐cell responses and are necessary for transplant tolerance induction. Tolerance induction may therefore be facilitated by selectively inhibiting the B7/CD28 pathway without blocking that of B7/CTLA‐4. In this study, we selectively inhibited CD28/B7 interactions using a monoclonal antibody modulating CD28 in a rat model of acute kidney graft rejection. A short‐term treatment abrogated both acute and chronic rejection. Tolerant recipients presented few alloantibodies against donor MHC class II molecules, whereas untreated rejecting controls developed anti‐MHC class I and II alloantibodies. PBMC from tolerant animals were unable to proliferate against donor cells but could proliferate against third‐party cells. The depletion of B7+, non‐T cells fully restored this reactivity whereas purified T cells were fully reactive. Also, NK cells depletion restored PBMC reactivity in 60% of tolerant recipients. Conversely, NK cells from tolerant recipients dose‐dependently inhibited alloreactivity. PBMC anti‐donor reactivity could be partially restored in vitro by blocking indoleamine‐2,3‐dioxygenase (IDO) and iNOS. In vivo, pharmacologic inhibition of these enzymes led to the rejection of the otherwise tolerated transplants. This study demonstrates that an initial selective blockade of CD28 generates B7+ non‐T regulatory cells and a kidney transplant tolerance sustained by the activity of IDO and iNOS.

Mechanism and Localization of CD8 Regulatory T Cells in a Heart Transplant Model of Tolerance

Despite accumulating evidence for the importance of allospecific CD8+ regulatory T cells (Tregs) in tolerant rodents and free immunosuppression transplant recipients, mechanisms underlying CD8+ Treg-mediated tolerance remain unclear. By using a model of transplantation tolerance mediated by CD8+ Tregs following CD40Ig treatment in rats, in this study, we show that the accumulation of tolerogenic CD8+ Tregs and plasmacytoid dendritic cells (pDCs) in allograft and spleen but not lymph nodes was associated with tolerance induction in vascularized allograft recipients. pDCs preferentially induced tolerogenic CD8+ Tregs to suppress CD4+ effector cells responses to first-donor Ags in vitro. When tolerogenic CD8+ Tregs were not in contact with CD4+ effector cells, suppression was mediated by IDO. Contact with CD4+ effector cells resulted in alternative suppressive mechanisms implicating IFN-γ and fibroleukin-2. In vivo, both IDO and IFN-γ were involved in tolerance induction, suggesting that contact with CD4+ effector cells is crucial to modulate CD8+ Tregs function in vivo. In conclusion, CD8+ Tregs and pDCs interactions were necessary for suppression of CD4+ T cells and involved different mechanisms modulated by the presence of cell contact between CD8+ Tregs, pDCs, and CD4+ effector cells.

Tolerogenic dendritic cells actively inhibit T cells through heme oxygenase-1 in rodents and in nonhuman primates

Clinical translation of dendritic cell (DC)‐based cell therapy requires preclinical studies in nonhuman primates (NHPs). The aim of this work was to establish the in vitro conditions for generation of NHP tolerogenic DCs (Tol‐DCs), as well as to analyze the molecular mechanisms by which these cells could control an immune response. Two populations of NHP bone marrow‐derived DCs (BMDCs) were obtained: adherent and nonadherent. Although both populations displayed a quite similar phenotype, they were very different functionally. We characterized the adherent BMDCs as Tol‐DCs that were poor stimulators of T cells and actively inhibited T‐cell proliferation, whereas the nonadherent population displayed immunogenic properties in vitro. Interestingly, the anti‐inflammatory and immunosuppressive enzyme heme oxygenase‐1 (HO‐1) was up‐regulated in Tol‐DCs, compared to the immunogenic BMDCs. We demonstrated that HO‐1 mediates the immunosuppressive properties of Tol‐DCs in vitro (in NHPs and rats) and that HO‐1 is involved in the in vivo tolerogenic effect of Tol‐DCs in a rat model of allotransplantation. In conclusion, here we characterized the in vitro generation of NHP Tol‐DCs. Furthermore, we showed for the first time that HO‐1 plays a role in the active inhibition of T‐cell responses by rat and NHP Tol‐DCs.—Moreau, A., Hill, M., Thebault, P., Deschamps, J. Y., Chiffoleau, E., Chauveau, C., Moullier, P., Anegon, I., Alliot‐Licht, B., Cuturi M. C. Tolerogenic dendritic cells actively inhibit T cells through heme oxygenase‐1 in rodents and in nonhuman primates. FASEB J. 23, 3070–3077 (2009). www.fasebj.org

Influence of local and systemic CTLA4Ig gene transfer on corneal allograft survival

To analyse the effects of local (ex vivo) or systemic (in vivo) administration of adenovirus type 5 encoding CTLA4Ig (AdCTLA4Ig) on its influence to prolong corneal allograft survival and to study the underlying mechanisms.

Tolerogenic dendritic cells and negative vaccination in transplantation: from rodents to clinical trials

The use of immunosuppressive (IS) drugs to treat transplant recipients has markedly reduced the incidence of acute rejection and early graft loss. However, such treatments have numerous adverse side effects and fail to prevent chronic allograft dysfunction. In this context, therapies based on the adoptive transfer of regulatory cells are promising strategies to induce indefinite transplant survival. The use of tolerogenic dendritic cells (DC) has shown great potential, as preliminary experiments in rodents have demonstrated that administration of tolerogenic DC prolongs graft survival. Recipient DC, Donor DC, or Donor Ag-pulsed recipient DC have been used in preclinical studies and administration of these cells with suboptimal immunosuppression increases their tolerogenic potential. We have demonstrated that autologous unpulsed tolerogenic DC injected in the presence of suboptimal immunosuppression are able to induce Ag-specific allograft tolerance. We derived similar tolerogenic DC in different animal models (mice and non-human primates) and confirmed their protective abilities in vitro and in vivo. The mechanisms involved in the tolerance induced by autologous tolerogenic DC were also investigated. With the aim of using autologous DC in kidney transplant patients, we have developed and characterized tolerogenic monocyte-derived DC in humans. In this review, we will discuss the preclinical studies and describe our recent results from the generation and characterization of tolerogenic monocyte-derived DC in humans for a clinical application. We will also discuss the limits and difficulties in translating preclinical experiments to theclinic.

Dominant Tolerance to Kidney Allografts Induced by Anti-Donor MHC Class II Antibodies: Cooperation between T and Non-T CD103+ Cells

Allograft acceptance can be induced in the rat by pretransplant infusion of donor blood or spleen cells. Although promoting long-term acceptance, this treatment is also associated with chronic rejection. In this study, we show that a single administration of anti-donor MHC class II alloimmune serum on the day of transplantation results in indefinite survival of a MHC-mismatched kidney graft. Long-term recipients accept a donor-type skin graft and display no histological evidence of chronic rejection. The kidney grafts of tolerant animals display an accumulation of TCR Cβ, FoxP3, and IDO transcripts. Moreover, as compared with syngeneic recipients, tolerant recipients harbor a large infiltrate of MHC class II+ cells and CD103+ cells. In vitro, splenocytes from tolerant recipients exhibit decreased donor-specific proliferation, which is restored by depletion of non-T cells and partially restored by the blockade of IDO. Finally, splenocytes from tolerant recipients, but not purified T cell splenocytes, transfer donor-specific infectious tolerance without chronic rejection, after infusion into naive recipients, over two generations. However, splenocytes depleted of T cells or splenocytes depleted of CD103+ cells fail to transfer tolerance. Collectively, these data show that a single administration of anti-donor MHC class II alloimmune serum induces a tolerant state characterized by an infiltration of the kidney graft by regulatory T cells and CD103+ cells. These data also show that the transfer of tolerance requires the presence of both T cells and CD103+ dendritic cells. The precise mechanism of cooperation of these two cell subsets remains to be defined.