Michèle Heslan

Presentation of donor major histocompatibility complex antigens by bone marrow dendritic cell-derived exosomes modulates allograft rejection1

Background. Dendritic cells secrete a population of “antigen-presenting vesicles,” called exosomes, expressing functional class I and II major histocompatibility complex (MHC) and co-stimulatory molecules. The subcutaneous administration of syngeneic exosomes expressing tumor antigens has been shown to induce specific antitumor immune responses in vivo. The authors hypothesized that antigen presentation by exosomes, depending on the context of their administration, may induce tolerance rather than immunity. Methods. The authors therefore tested the capacity of exosomes derived from donor bone marrow dendritic cells, given before transplantation, to modulate heart allograft rejection. Results. The authors show here that donor type but not syngeneic exosomes induced a significant prolongation of allograft survival, with a few recipients having long-term graft survival. During the first week after transplantation, allografts from exosome-treated rats displayed a significant decrease in graft-infiltrating leukocytes and in the expression of interferon-&ggr; mRNA compared with allografts from untreated animals. Moreover, when tested in vitro, spleen CD4+ T cells from exosome-treated recipients displayed a significant decrease in anti-donor responses, suggesting a decrease in anti-donor T-cell responses. However, the authors also found that allogeneic donor-derived exosomes increased anti-donor MHC class II alloantibody production. Conclusions. The authors demonstrate an effect of allogeneic exosomes on the modulation of immune responses in vivo, suggesting that, like donor cells, exosomes can stimulate or regulate antigen-specific immune responses.

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.

Variation in numbers of CD4+CD25highFOXP3+ T cells with normal immuno-regulatory properties in long-term graft outcome

Chronic rejection (CR) is a major cause of long‐term graft loss that would be avoided by the induction of tolerance. We previously showed that renal transplant patients with CR have lower numbers of peripheral CD4+CD25high T cells than operationally tolerant patients, patients with stable graft function and healthy volunteers (HV). We explored here the profile of CD4+CD25high blood T cells in these patients focusing on their expression of the regulatory T cells (Treg) gene Forkhead Box P3 (FOXP3) and their suppressive function. We show that CR is associated with a decreased number of CD4+CD25highFOXP3+T cells with normal regulatory profile, whereas graft acceptance is associated with CD4+CD25highFOXP3+T cell numbers similar to HVs. These data suggest that Treg numbers, rather than their intrinsic suppressive capacity, may contribute to determining the long‐term fate of renal transplants.

Role of IFNγ in Allograft Tolerance Mediated by CD4+CD25+ Regulatory T Cells by Induction of IDO in Endothelial Cells

Regulatory T cells have been described to specifically accumulate at the site of regulation together with effector T cells and antigen‐presenting cells, establishing a state of local immune privilege. However the mechanisms of this interplay remain to be defined. We previously demonstrated, in a fully MHC mismatched rat cardiac allograft combination, that a short‐term treatment with a deoxyspergualine analogue, LF15‐0195, induces long‐term allograft tolerance with a specific expansion of regulatory CD4+CD25+T cells that accumulate within the graft. In this study, we show that following transfer of regulatory CD4+T cells to a secondary irradiated recipient, regulatory CD25+Foxp3+ and CD25+Foxp3− CD4+T cells accumulate at the graft site and induce graft endothelial cell expression of Indoleamine 2, 3‐dioxygenase (IDO) by an IFNγ‐dependent mechanism. Moreover, in vivo transfer of tolerance can be abrogated by blocking IFNγ or IDO, and anti‐IFNγ reduces the survival/expansion of alloantigen‐induced regulatory Foxp3+CD4+T cells. Together, our results demonstrate interrelated mechanisms between regulatory CD4+CD25+T cells and the graft endothelial cells in this local immune privilege, and a key role for IFNγ and IDO in this process.

An Immunomodulatory Role for Follistatin-Like 1 in Heart Allograft Transplantation

Donor‐specific tolerance to heart allografts in the rat can be achieved by donor‐specific blood transfusions (DST) before transplantation. We have previously reported that this tolerance is associated with strong leukocyte infiltration, and that host CD8+ T cells and TGFβ are required. In order to identify new molecules involved in the induction phase of tolerance, we compared tolerated and rejected heart allografts (suppressive subtractive hybridization) 5 days after transplantation. We identified overexpression of Follistatin‐like 1 (FSTL1) transcript in tolerated allografts compared to rejected allografts or syngeneic grafts. We show that FSTL1 is overexpressed during both the induction and maintenance phase of tolerance, and appears to be specific to the tolerance model induced by DST. Analysis of graft‐infiltrating cells revealed predominant expression of FSTL1 in CD8+ T cells from tolerated grafts, and depletion of these cells prior to transplantation abrogated FSTL1 expression and heart allograft survival. Moreover, overexpression of FSTL1 by adenovirus gene transfer in vivo significantly prolonged allograft survival in association with inhibition of the proinflammatory cytokines, IL6, IL17 A and IFNγ. Taken together, these results suggest that FSTL1 could be an active component of the mechanisms mediating heart allograft tolerance.

The C-Type Lectin-Like Receptor CLEC-1, Expressed by Myeloid Cells and Endothelial Cells, Is Up-Regulated by Immunoregulatory Mediators and Moderates T Cell Activation

C-type lectin receptors have recently been described as playing crucial roles in immunity and homeostasis since these proteins are able to recognize pathogens as well as self-Ags. We identified the C-type lectin-like receptor-1, CLEC-1, as being overexpressed in a model of rat allograft tolerance. We previously described in this model the expression of numerous cytoprotective molecules by graft endothelial cells and their interplay with regulatory CD4+CD25+ T cells. In this study, we demonstrate that CLEC-1 is expressed by myeloid cells and specifically by endothelial cells in tolerated allografts and that CLEC-1 expression can be induced in endothelial cells by alloantigen-specific regulatory CD4+CD25+ T cells. Analysis of CLEC-1 expression in naive rats demonstrates that CLEC-1 is highly expressed by myeloid cells and at a lower level by endothelial cells, and that its expression is down-regulated by inflammatory stimuli but increased by the immunoregulators IL-10 or TGFβ. Interestingly, we demonstrate in vitro that inhibition of CLEC-1 expression in rat dendritic cells increases the subsequent differentiation of allogeneic Th17 T cells and decreases the regulatory Foxp3+ T cell pool. Additionally, in chronically rejected allograft, the decreased expression of CLEC-1 is associated with a higher production of IL-17. Taken together, our data suggest that CLEC-1, expressed by myeloid cells and endothelial cells, is enhanced by regulatory mediators and moderates Th17 differentiation. Therefore, CLEC-1 may represent a new therapeutic agent to modulate the immune response in transplantation, autoimmunity, or cancer settings.

IL-22BP is produced by eosinophils in human gut and blocks IL-22 protective actions during colitis

Crohn’s disease and ulcerative colitis, the two major forms of inflammatory bowel diseases (IBDs), are characterized by high levels of IL-22 production. Rodent studies revealed that this cytokine is protective during colitis but whether this is true in IBDs is unclear. We show here that levels of the soluble inhibitor of IL-22, interleukin 22-binding protein (IL-22BP), are significantly enhanced during IBDs owing to increased numbers of IL-22BP-producing eosinophils, that we unexpectedly identify as the most abundant source of IL-22BP protein in human gut. In addition, using IL-22BP-deficient rats, we confirm that endogenous IL-22BP is effective at blocking protective actions of IL-22 during acute colitis. In conclusion, our study provides new important insights regarding the biology of IL-22 and IL-22BP in the gut and indicates that protective actions of IL-22 are likely to be suboptimal in IBDs thus making IL-22BP a new relevant therapeutic target.

Superiority of bone marrow-derived dendritic cells over monocyte-derived ones for the expansion of regulatory T cells in the macaque.

Regulatory T cells (Treg) have been identified as playing a pivotal role in the control of tolerance and in the suppression of pathologic immune responses in autoimmune diseases, transplantation, and graft-versus-host disease. Treg expanded ex vivo by dendritic cells could be potential reagents to promote antigen-specific tolerance in vivo. However, in vivo studies have been carried out mostly in rodents and will need validation in primates before clinical application. We characterized macaque dendritic cell derived either from bone marrow with and without prior CD34+ cell selection (BMDC), or from CD14+ peripheral blood mononuclear cells (Mo-DC). We demonstrate that with a semi-mature phenotype, BMDC are superior to Mo-DC in their capacity to expand freshly isolated allogeneic macaque CD4+ CD25+ CD127- Foxp3+ Treg in vitro in the presence of interleukin-2. Moreover, the expanded Treg maintain their phenotype and suppressive activity. These data provide a step toward the use of macaque dendritic cell to expand Treg for future preclinical testing.

Characterization of Schlafen-3 expression in effector and regulatory T cells

Members of the Slfn protein family have been implicated in the regulation of cell growth, hematopoietic cell differentiation, and T cell development/differentiation in the thymus. Ten members of this family have been described in the mouse, and they have been divided into three subgroups based on the overall sequence homology and the size of the encoded proteins. We have identified Slfn3, a member of Subgroup II, as an overexpressed gene in CD4+ CD25+ T cells in the periphery. Interestingly, we demonstrate that upon activation and proliferation, Slfn3 mRNA is down‐regulated in CD4+ CD25+ Tregs and up‐regulated in CD4+ CD25– Teffs. Moreover, TGF‐β inhibits the expression of Slfn3 in anti‐CD3/CD28‐activated CD4+ T cells, and the same conditions induce FoxP3 mRNA. Our results suggest that Slfn3 could have a role in T cell differentiation and activation.

Regulatory B Cells with a Partial Defect in CD40 Signaling and Overexpressing Granzyme B Transfer Allograft Tolerance in Rodents

Emerging knowledge regarding B cells in organ transplantation has demonstrated that these cells can no longer be taken as mere generators of deleterious Abs but can also act as beneficial players. We previously demonstrated in a rat model of cardiac allograft tolerance induced by short-term immunosuppression an accumulation in the blood of B cells overexpressing inhibitory molecules, a phenotype also observed in the blood of patients that spontaneously develop graft tolerance. In this study, we demonstrated the presence in the spleen of regulatory B cells enriched in the CD24intCD38+CD27+IgD−IgM+/low subpopulation, which are able to transfer donor-specific tolerance via IL-10 and TGF-β1–dependent mechanisms and to suppress in vitro TNF-α secretion. Following anti-CD40 stimulation, IgD−IgM+/low B cells were blocked in their plasma cell differentiation pathway, maintained high expression of the inhibitory molecules CD23 and Bank1, and upregulated Granzyme B and Irf4, two molecules described as highly expressed by regulatory B cells. Interestingly, these B cells recognized specifically a dominant donor Ag, suggesting restricted specificity that could lead to a particular B cell response. Regulatory B cells were not required for induction of tolerance and appeared following Foxp3+CD4+CD25+ regulatory T cells, suggesting cooperation with regulatory T cells for their expansion. Nevertheless, following transfer to new recipients, these B cells migrated to the allograft, kept their regulatory profile, and promoted local accumulation of Foxp3+CD4+CD25+ regulatory T cells. Mechanisms of regulatory B cells and their cell therapy potential are important to decipher in experimental models to pave the way for future developments in the clinic.