Fabienne Haspot

Myeloid-Derived Suppressor Cells Accumulate in Kidney Allograft Tolerance and Specifically Suppress Effector T Cell Expansion

The immune tolerance to rat kidney allografts induced by a perioperative treatment with anti-CD28 Abs is associated with a severe unresponsiveness of peripheral blood cells to donor Ags. In this model, we identified an accumulation in the blood of CD3−class II−CD11b+CD80/86+ plastic-adherent cells that additionally expressed CD172a as well as other myeloid markers. These cells were able to inhibit proliferation, but not activation, of effector T cells and to induce apoptosis in a contact-dependent manner. Their suppressive action was found to be under the control of inducible NO synthase, an enzyme also up-regulated in tolerated allografts. Based on these features, these cells can be defined as myeloid-derived suppressor cells (MDSC). Interestingly, CD4+CD25highFoxP3+ regulatory T cells were insensitive in vitro to MDSC-mediated suppression. Although the adoptive transfer of MDSC failed to induce kidney allograft tolerance in recently transplanted recipients, the maintenance of tolerance after administration of anti-CD28 Abs was found to be dependent on the action of inducible NO synthase. These results suggest that increased numbers of MDSC can inhibit alloreactive T cell proliferation in vivo and that these cells may participate in the NO-dependent maintenance phase of tolerance.

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.

Human Cytomegalovirus Entry into Dendritic Cells Occurs via a Macropinocytosis-Like Pathway in a pH-Independent and Cholesterol-Dependent Manner

Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that is able to infect fibroblastic, epithelial, endothelial and hematopoietic cells. Over the past ten years, several groups have provided direct evidence that dendritic cells (DCs) fully support the HCMV lytic cycle. We previously demonstrated that the C-type lectin dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) has a prominent role in the docking of HCMV on monocyte-derived DCs (MDDCs). The DC-SIGN/HCMV interaction was demonstrated to be a crucial and early event that substantially enhanced infection in trans, i.e., from one CMV-bearing cell to another non-infected cell (or trans-infection), and rendered susceptible cells fully permissive to HCMV infection. Nevertheless, nothing is yet known about how HCMV enters MDDCs. In this study, we demonstrated that VHL/E HCMV virions (an endothelio/dendrotropic strain) are first internalized into MDDCs by a macropinocytosis-like process in an actin- and cholesterol-dependent, but pH-independent, manner. We observed the accumulation of virions in large uncoated vesicles with endosomal features, and the virions remained as intact particles that retained infectious potential for several hours. This trans-infection property was specific to MDDCs because monocyte-derived macrophages or monocytes from the same donor were unable to allow the accumulation of and the subsequent transmission of the virus. Together, these data allowed us to delineate the early mechanisms of the internalization and entry of an endothelio/dendrotropic HCMV strain into human MDDCs and to propose that DCs can serve as a Trojan horse to convey CMV from entry sites to other locations that may favor the occurrence of either latency or acute infection.

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.

Anti-CD28 Antibodies Modify Regulatory Mechanisms and Reinforce Tolerance in CD40Ig-Treated Heart Allograft Recipients

Blockade of CD40-CD40 ligand (CD40L) costimulation has been shown to synergize with that of CTLA4/CD28-B7 to promote transplant tolerance. To date, however, CD28-B7 interactions have been prevented using B7-blocking reagents like CTLA4-Ig that inhibit CD28-B7 together with CTLA4-B7 interactions. In this study, we have tested anti-CD28 Abs to prevent selectively CD28-B7 interactions while preserving CTLA4-B7 in addition to CD40-CD40L blockade. In the LEW.1W to LEW.1A rat combination, interfering with CD40-CD40L interactions by CD40Ig administration through gene transfer resulted in indefinite heart allograft survival due to the appearance of clonotypic CD8+CD45RClow regulatory T cells that were capable of transferring the tolerant state to naive animals. However, cardiac transplants in these recipients systematically developed chronic rejection lesions. Whereas anti-CD28 Ab monotherapy only delayed acute rejection and failed to induce tolerance, coadministration of anti-CD28 Abs and CD40Ig resulted in the long-term acceptation of allografts without chronic rejection lesions in 60% of the recipients, reduced the level of intragraft mRNA transcripts for cytokines and immune factors, and fully abrogated alloantibody production. In addition, the nature of regulatory cells was modified: the CD8+CD45RClow clonotypic T cells described in the CD40Ig-treated animals could not be found in cotreated animals, and the other CD8+CD45RClow cells had no regulatory activity and a different cytokine expression profile. Instead, in cotreated recipients we found IDO-dependent non-T cells with regulatory activity in vitro. Thus, the addition of a short-term anti-CD28 treatment with CD40Ig resulted in decreased heart allograft chronic rejection lesions, complete inhibition of Ab production, and modified regulatory mechanisms.

Development of CD25- regulatory T cells following heart transplantation: evidence for transfer of long-term survival.

Donor‐specific heart allograft acceptance can be induced in the MHC‐mismatched LEW.1u2004W to LEW.1A rat by donor‐specific transfusions. Whereas the induction phase of tolerance has been studied in detail, its maintenance remained poorly understood. Here, we performed a side‐by‐side comparison of CD25+ and CD25– splenic T cells of 100‐day tolerant rats. Administration of CD25– T cells from tolerant rats to sublethally irradiated recipients transferred long‐term graft survival. These CD25– T cells displayed a decreased donor‐specific response in the mixed lymphocyte reaction and presented suppressive activity. These CD25– T cells accumulated IFN‐γ, IL‐10 and Foxp3 transcripts. The in vitro suppressive activity of CD25– T cells required both cell contact and soluble factors (IL‐10 and IFN‐γ). The CD25+ T cells from tolerant rats did not show any modification of their regulatory properties. We show that splenic CD25– T cells of tolerant rats contribute to the maintenance of tolerance following the transplantation. Our data show that regulatory T cells are not restricted to the CD4+CD25+ T cell subset and provide new insights on the mechanisms of tolerance to allograft following donor cell priming.

Functional Langerinhigh-Expressing Langerhans-like Cells Can Arise from CD14highCD16− Human Blood Monocytes in Serum-Free Condition

Langerhans cells (LCs) are epithelial APCs that sense danger signals and in turn trigger specific immune responses. In steady-state, they participate in the maintenance of peripheral tolerance to self-antigens whereas under inflammation LCs efficiently trigger immune responses in secondary lymphoid organs. It has been demonstrated in mice that LC-deprived epithelia are rapidly replenished by short half-life langerin-expressing monocyte-derived LCs (MDLCs). These surrogate LCs are thought to be progressively replaced by langerinhigh LCs arising from self-renewing epithelial precursors of hematopoietic origin. How LCs arise from blood monocytes is not fully understood. Hence, we sought to characterize key factors that induce differentiation of langerinhigh-expressing monocyte-derived Langerhans-like cells. We identified GM-CSF and TGF-β1 as key cytokines to generate langerinhigh-expressing cells but only in serum-free conditions. These cells were shown to express the LC-specific TROP-2 and Axl surface markers and contained Birbeck granules. Surprisingly, E-cadherin was not spontaneously expressed by these cells but required a direct contact with keratinocytes to be stably induced. MDLCs induced stronger allogeneic T cell proliferations but released low amounts of inflammatory cytokines upon TLR stimulation compared with donor-paired monocyte-derived dendritic cells. Immature langerinhigh MDLCs were responsive to MIP-3β/CCL20 and CTAC/CCL27 chemokine stimulations. Finally, we demonstrated that those cells behaved as bona fide LCs when inserted in a three-dimensional rebuilt epithelium by becoming activated upon TLR or UV light stimulations. Collectively, these results prompt us to propose these langerinhigh MDLCs as a relevant model to address LC biology–related questions.

Infectious Diseases in Transplantation--Report of the 20th Nantes Actualités Transplantation Meeting.

Abstract The 20th Nantes Actualités Transplantation (NAT) meeting was held on June 11, 2015, and June 12, 2015. This year, the local organizing committee selected an update on infectious diseases in solid organ and hematopoietic stem cell transplantation. With an attendance of close to 170 clinicians, researchers, students, engineers, technicians, invited speakers, and guests from North and South America, Germany, Switzerland, Netherlands, and France, the meeting was well attended. Invited speakers expertise covered basic as well as translational microbiology, immunology, transplantation, and intensive care medicine. This report identifies a number of advances presented during the meeting in the care and management of infectious diseases in transplantation and immunocompromised patients. New antiviral immune responses and their modulation by pathogens in addition to novel antimicrobial therapeutic strategies, cell therapies, and genomic analysis were discussed.