B. Vokaer

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.

Critical Role of Regulatory T Cells in Th17-Mediated Minor Antigen-Disparate Rejection

Th17-mediated immune responses have been recently identified as novel pathogenic mechanisms in a variety of conditions; however, their importance in allograft rejection processes is still debated. In this paper, we searched for MHC or minor Ag disparate models of skin graft rejection in which Th17 immune responses might be involved. We found that T cell-derived IL-17 is critical for spontaneous rejection of minor but not major Ag-mismatched skin grafts. IL-17 neutralization was associated with a lack of neutrophil infiltration and neutrophil depletion delayed rejection, suggesting neutrophils as an effector mechanism downstream of Th17 cells. Regulatory T cells (Tregs) appeared to be involved in Th17 reactivity. We found that in vivo Treg depletion prevented IL-17 production by recipient T cells. An adoptive cotransfer of Tregs with naive monospecific antidonor T cells in lymphopenic hosts biased the immune response toward Th17. Finally, we observed that IL-6 was central for balancing Tregs and Th17 cells as demonstrated by the prevention of Th17 differentiation, the enhanced Treg/Th17 ratio, and a net impact of rejection blockade in the absence of IL-6. In conclusion, the ability of Tregs to promote the Th17/neutrophil-mediated pathway of rejection that we have described should be considered as a potential drawback of Treg-based cell therapy.

CTLA4-Ig restores rejection of MHC class-II mismatched allografts by disabling IL-2-expanded regulatory T cells

Allograft acceptance and tolerance can be achieved by different approaches including inhibition of effector T cell responses through CD28‐dependent costimulatory blockade and induction of peripheral regulatory T cells (Tregs). The observation that Tregs rely upon CD28‐dependent signals for development and peripheral expansion, raises the intriguing possibility of a counterproductive consequence of CTLA4‐Ig administration on tolerance induction. We have investigated the possible negative effect of CTLA4‐Ig on Treg‐mediated tolerance induction using a mouse model of single MHC class II‐mismatched skin grafts in which long‐term acceptance was achieved by short‐term administration of IL‐2/anti‐IL‐2 complex. CTLA4‐Ig treatment was found to abolish Treg‐dependent acceptance in this model, restoring skin allograft rejection and Th1 alloreactivity. CTLA4‐Ig inhibited IL‐2‐driven Treg expansion, and prevented in particular the occurrence of ICOS+ Tregs endowed with potent suppressive capacities. Restoring CD28 signaling was sufficient to counteract the deleterious effect of CTLA4‐Ig on Treg expansion and functionality, in keeping with the hypothesis that costimulatory blockade inhibits Treg expansion and function by limiting the delivery of essential CD28‐dependent signals. Inhibition of regulatory T cell function should therefore be taken into account when designing tolerance protocols based on costimulatory blockade.

Interleukin 17–producing T helper cells in alloimmunity

Interleukin (IL) 17 is a proinflammatory cytokine already known to play a defense role against microbes and a pathogenic role in a number of autoimmune diseases. Although IL-17 can be produced by a variety of cells including neutrophils, CD8+, NK, and gamma-delta T cells, the concept of IL-17-secreting CD4+ T helper cells (Th17), distinct from Th1 and Th2, recently emerged. Herein, we discuss arguments in favor of a Th17-mediated alternative pathway of allograft rejection based on clinical and experimental observations drawn from the literature. We also discuss the complex interplays among regulatory T cells and Th17 cells in the allogeneic context.

Cyclosporine A Drives a Th17- and Th2-Mediated Posttransplant Obliterative Airway Disease

Calcineurin‐inhibitor refractory bronchiolitis obliterans (BO) represents the leading cause of late graft failure after lung transplantation. T helper (Th)2 and Th17 lymphocytes have been associated with BO development. Taking advantage of a fully allogeneic trachea transplantation model in mice, we addressed the pathogenicity of Th cells in obliterative airway disease (OAD) occurring in cyclosporine A (CsA)‐treated recipients. We found that CsA prevented CD8+ T cell infiltration into the graft and downregulated the Th1 response but affected neither Th2 nor Th17 responses in vivo. In secondary mixed lymphocyte cultures, CsA dramatically decreased donor‐specific IFN‐γ production, enhanced IL‐17 production and did not affect IL‐13. As CD4+ depletion efficiently prevented OAD in CsA‐treated recipients, we further explored the role of Th2 and Th17 immunity in vivo. Although IL‐4 and IL‐17 deficient untreated mice developed an OAD comparable to wild‐type recipients, a single cytokine deficiency afforded significant protection in CsA‐treated recipients. In conclusion, CsA treatment unbalances T helper alloreactivity and favors Th2 and Th17 as coexisting pathways mediating chronic rejection of heterotopic tracheal allografts.

Reviving Function in CD4+ T Cells Adapted to Persistent Systemic Antigen

In bone marrow-transplanted patients, chronic graft-versus-host disease is a complication that results from the persistent stimulation of recipient minor histocompatibility Ag (mHA)-specific T cells contained within the graft. In this study, we developed a mouse model where persistent stimulation of donor T cells by recipient’s mHA led to multiorgan T cell infiltration. Exposure to systemic mHA, however, deeply modified T cell function and chronically stimulated T cells developed a long-lasting state of unresponsiveness, or immune adaptation, characterized by their inability to mediate organ immune damages in vivo. However, analysis of the gene expression profile of adapted CD4+ T cells revealed the specific coexpression of genes known to promote differentiation and function of Th1 effector cells as well as genes coding for proteins that control T cell activity, such as cell surface-negative costimulatory molecules and regulatory cytokines. Strikingly, blockade of negative costimulation abolished T cell adaptation and stimulated strong IFN-γ production and severe multiorgan wasting disease. Negative costimulation was also shown to control lethal LPS-induced toxic shock in mice with adapted T cells, as well as the capacity of adapted T cells to reject skin graft. Our results demonstrate that negative costimulation is the molecular mechanism used by CD4+ T cells to adapt their activity in response to persistent antigenic stimulation. The effector function of CD4+ T cells that have adapted to chronic Ag presentation can be activated by stimuli strong enough to overcome regulatory signals delivered to the T cells by negative costimulation.

IL-17A and IL-2-Expanded Regulatory T Cells Cooperate to Inhibit Th1-Mediated Rejection of MHC II Disparate Skin Grafts

Several evidences suggest that regulatory T cells (Treg) promote Th17 differentiation. Based on this hypothesis, we tested the effect of IL-17A neutralization in a model of skin transplantation in which long-term graft survival depends on a strong in vivo Treg expansion induced by transient exogenous IL-2 administration. As expected, IL-2 supplementation prevented rejection of MHC class II disparate skin allografts but, surprisingly, not in IL-17A-deficient recipients. We attested that IL-17A was not required for IL-2-mediated Treg expansion, intragraft recruitment or suppressive capacities. Instead, IL-17A prevented allograft rejection by inhibiting Th1 alloreactivity independently of Tregs. Indeed, T-bet expression of naive alloreactive CD4+ T cells and the subsequent Th1 immune response was significantly enhanced in IL-17A deficient mice. Our results illustrate for the first time a protective role of IL-17A in CD4+-mediated allograft rejection process.

A brief focus on memory cells in transplantation.

T and B memory cells are critical for host defences against pathogens. However, converging lines of evidence indicate that alloreactive memory T cells can play a detrimental role in the transplantation setting. This emergence of memory cells seems to be facilitated by several induction therapies and immunosuppressive agents, which lead to T cell loss. Herein, we briefly review some clinical and experimental observations from the literature, highlighting the immunological risk associated with enhanced T-cell memory responses.

IL-17A Mediates Early Post-Transplant Lesions after Heterotopic Trachea Allotransplantation in Mice

Primary graft dysfunction (PGD) and bronchiolitis obliterans (BO) are the leading causes of morbidity and mortality after lung transplantation. Reports from clinical and rodent models suggest the implication of IL-17A in either PGD or BO. We took advantage of the heterotopic trachea transplantation model in mice to study the direct role of IL-17A in post-transplant airway lesions. Across full MHC barrier, early lesions were controlled in IL-17A-/- or anti-IL17 treated recipients. In contrast, IL-17A deficiency did not prevent subsequent obliterative airway disease (OAD). Interestingly, this early protection occurred also in syngeneic grafts and was accompanied by a decrease in cellular stress, as attested by lower HSP70 mRNA levels, suggesting the involvement of IL-17A in ischemia-reperfusion injury (IRI). Furthermore, persistence of multipotent CK14+ epithelial stem cells underlined allograft protection afforded by IL-17A deficiency or neutralisation. Recipient-derived γδ+ and CD4+ T cells were the major source of IL-17A. However, lesions still occurred in the absence of each subset, suggesting a high redundancy between the innate and adaptive IL-17A producing cells. Notably, a double depletion significantly diminished lesions. In conclusion, this work implicated IL-17A as mediator of early post-transplant airway lesions and could be considered as a potential therapeutic target in clinical transplantation.

Impact of Interleukin-2–expanded Regulatory T Cells in Various Allogeneic Combinations on Mouse Skin Graft Survival

The impact of in vivo regulatory T cells (Treg) expansion using short-term injections of interleukin-2 (IL-2) coupled to a specific anti-IL-2 antibody was examined in various allogeneic combinations of murine skin transplantations. In a model of a single major histocompatibility complex (MHC) class II disparity, the IL-2-expanded Tregs infiltrated the transplanted skin, inhibited Th1 alloreactivity, and prevented acute graft rejection. However, in the presence of increased load of CD4-recognized alloantigens, exogenous IL-2 only moderately prolonged graft survival as attested by CD8 T cell-depletion in full minor plus major mismatched recipients treated with IL-2. If direct CD8 alloreactivity remained intact, the IL-2/anti-IL-2-mediated Tregs expansion failed to delay allograft rejection. This observation was confirmed by the inability of expanded Tregs to delay rejection of multiple minor disparate (MHC matched) skin allografts. Altogether, these results warn that cross-reactive CD8(+) T cells represent an important hurdle to Treg-based tolerance induction.