DENDRITIC CELLS: REGULATORS OF ALLOIMMUNITY AND AUTOIMMUNITY

Dendritic cells (DCs) are rare, uniquely well-equipped, heterogeneous antigen-presenting cells derived from CD34+ haematopoietic stem cells. They are regarded both as instigators and critical regulators of immune reactivity.1^–4 Assessment of their in vivo functional significance has been greatly enhanced by studies in the murine system.5^–11 Thus, in addition to their role in intrathymic self-tolerance, evidence has accumulated that DCs play a key role in the maintenance of peripheral (self) tolerance in the normal steady state. Although regarded traditionally as instigators of immune responses, including organ graft rejection and autoimmunity, more recent data have implicated DCs in the induction and even maintenance of tolerance to allo- or autoantigens in experimental models.12 13

This functional dichotomy of DCs is governed by various factors, the most important of which appear to be their stage of differentiation/activation/maturation and their haematopoietic lineage affiliation. We and others have shown that immature or “semimature” conventional myeloid (m)DCs, deficient in costimulatory molecules (CD80, CD86 and inducible costimulatory ligand (ICOSL)), can inhibit alloantigen-specific T-cell responses. These DCs can prolong organ graft survival after their adoptive transfer across major histocompatibility complex (MHC) barriers—in some studies indefinitely—in the absence of immunosuppressive therapy.14^–16 Similarly, immature DCs can prevent or even reverse arthritis and diabetes in murine models. In humans, immature mDCs can drive the generation of T regulatory cells (Treg) in vitro17 and induce antigen-specific T-cell tolerance to model antigens in vivo in healthy volunteers.18 DC subsets, other than conventional DCs, have emerged as candidate “tolerogenic” DCs, with potential for therapeutic application. Specifically, plasmacytoid DCs (pDCs), first generated from DC precursors in human tissue by Grouard et al,19 and which are an important source of type I interferons (IFNs), can regulate T-cell responses, including induction of Treg.20^–22

DC IMMUNOBIOLOGY: MATURATION IN RELATION TO FUNCTION

DCs are ubiquitously distributed migratory leucocytes. They convey antigen from peripheral sites, such as the skin and all commonly transplanted organs, to T cells in secondary lymphoid tissues. When freshly isolated, mDCs express few surface MHC and accessory molecules (eg, CD40, CD54, CD80 (B7–1) CD86 (B7–2) and ICOSL) and are at best, poor stimulators of naïve T cells. These antigen-presenting cells, however, are extremely well equipped to both capture and load foreign antigen onto MHC molecules for export to the cell surface. Mature conventional mDCs, which express high levels of MHC and costimulatory molecules, efficiently present antigen peptides bound to MHC class II to CD4+ T helper (Th) cells and antigen peptides complexed with MHC class I to CD8+ cytotoxic T cells. DC maturation is essential for the initiation of acquired immune reactivity and is stimulated by microbial products (eg, through Toll-like receptors (TLRs): bacterial lipopolysaccharide (LPS), unmethylated cytosine polyguanine motifs, double-stranded RNA), endogenous inflammatory factors (eg, through TLR: high mobility group box (HMGB)1, heat shock protein 70, proinflammatory cytokines (granulocyte/macrophage colony-stimulating factor, interleukin (IL)1β, tumour necrosis factor (TNF)α and IFNα) and cyclo-oxygenase metabolites. Ligation of TLR 2, 3, 4, 7 or 9 triggers DC maturation. Upon maturation, DCs synthesise high levels of the Th1-cell-driving cytokine IL12 and also play a prominent role in the induction of IL17-producing Th cells and Treg. IL10, which is produced by Th2 cells, blocks IL12 synthesis by DCs, downregulates their expression of costimulatory molecules and potentiates their tolerogenicity.23 It is generally considered that resistance of DCs to maturation is a prerequisite for exhibition of their tolerogenic potential as “negative cellular vaccines”.

MAJOR DC SUBSETS: MDCS AND PDCS IN MICE AND HUMANS

Besides conventional mDCs, a second DC subset termed “pre-plasmacytoid” DCs (pre-pDCs) is found in secondary lymphoid tissue. Pre-pDCs, the immediate precursors of pDCs, which develop into DCs after culture in IL3 and CD40 ligand (L), are located in T-dependent areas of secondary lymphoid tissues and in blood.19 While human mDCs induce Th1- and Th17-cell differentiation, pDCs, which make high levels of type-1 IFN after microbial challenge, can selectively induce Th2 cells.24 25 Both DC subsets can regulate innate and adaptive immune responses. Like mDCs, pDCs are well described in mice.9^–11 Our findings26 27 indicate that in a mouse vascularised organ allograft model pre-pDCs of donor origin, like their immature mDC counterparts,15 can markedly prolong fully MHC-mismatched organ graft survival. Moreover, recent data suggest that pDCs that have acquired donor antigen and migrated to lymph nodes of heart-allografted mice given a tolerogenic regimen of donor-specific transfusion and anti-CD40L monoclonal antibody (mAb) play critical roles in tolerance induction.13

MECHANISMS BY WHICH DCS REGULATE IMMUNE REACTIVITY

DCs can regulate immune reactivity and promote/maintain tolerance by a variety of (non-exclusive) mechanisms. For more extensive details, the reader is referred to recent reviews.2 4 12 28^–33

Induction of T-cell anergy or apoptosis in naïve or memory T cells

Many reports show that conventional mDCs, whose T-cell allostimulatory function is impaired, by incomplete maturation, selective blockade of B7 costimulatory molecules, the influence of anti-inflammatory cytokines (eg, IL10 or transforming growth factor β (TGFβ)) or genetic engineering (to express viral IL10, cytotoxic T-lymphocyte antigen (CTLA4)Ig, or FasL (CD95L)), can induce alloantigen-specific T-cell hyporesponsiveness, anergy or apoptosis in vitro and suppress immune reactivity34 35 (reviewed by several authors12 29 36^–38).Our work and that of others has shown that expression of molecules associated with induction of apoptosis—that is, FasL,39 40 nitric oxide (NO)41 42 or indoleamine deoxygenase (IDO),43 44 may confer on DCs the ability to subvert T-cell responses by promoting activation-induced cell death. Blockade of the B7/CD28 pathway by CTLA4Ig increases mDC-induced apoptosis of alloactivated T cells.39 On the other hand, upregulation of IDO production by DCs after ligation of B7 molecules by CTLA4Ig results in decreased clonal expansion and enhanced deletion of T cells.45

Memory T cells exhibit little or no need for the strong costimulatory signals required for full activation of naïve T cells. Thus, it has generally been thought that memory T cells are released from the capacity to be inactivated. However, there is recent evidence46 that steady-state (immature) DCs, which constitutively present an endogenously expressed antigen, can inactivate fully differentiated memory CD8+ T cells in vivo via deletion and inactivation. In addition, “alternatively activated” DCs, which are generated with immunosuppressive agents (dexamethasone and vitamin D3), then activated with LPS, can induce hyporesponsiveness (anergy) in memory CD4+ T cells.47

Selective activation of Th2 cells

Immune deviation (skewing of T cells toward the Th2 type) and the role of Th2 cytokines in immune tolerance has been discussed for many years.48 49 DCs can induce immune deviation in autoimmune disease and transplant models.50 51 Reports of DCs with blocked costimulatory capacity inducing Th2 responses are readily available.

Induction of Tregs

DCs can promote the induction of Tregs, a mechanism that could insure long-term, alloantigen-specific T-cell unresponsiveness. Three populations of CD4+ T cells exhibiting suppressor mechanisms are known: T regulatory type 1 (Tr1) and Th3 cells, which are induced by IL10 and/or TGFβ, and CD4+ CD25+ cells, which are present in the periphery of normal mice. CD8+ TCR+CD4−CD8− and natural killer (NK)T cells with regulatory activities also have been described.52 IL10-secreting, non-proliferating CD4+ Tregs can be induced by repeated stimulation with allogeneic immature human53 or mouse mDCs.54 Moreover, immature, antigen-pulsed mDCs induce antigen-specific IL10-producing CD8+ T cells in normal human volunteers.18 In rats, pretreatment with immature (F1) mDCs expressing donor alloantigen, followed 1 day later by CTLA4Ig, leads to indefinite kidney transplant survival and freedom from transplant vasculopathy, associated with the induction of indirect pathway Treg.55 In a mouse organ transplant model, pDCs in host lymph nodes appear to play a crucial role in tolerance induction and induce alloantigen-specific Treg.13 Although DCs seem to trigger Treg mainly at the immature stage,56 there are also reports that mature human pDCs can induce CD8+ Treg in vitro.20 Furthermore, mature murine pulmonary DCs57 and murine DCs with plasmacytoid morphology, which secrete high levels of IL10 after activation,22 or that have been activated by CD4020 or TLR9 ligation,21 can stimulate the development of Tr1 cells. Collectively, these reports provide strong evidence that DCs can promote the induction of Treg.

CELL THERAPY WITH TOLEROGENIC DCS

Therapeutic strategies with tolerogenic DCs in transplantation have involved the use of donor or recipient-derived DCs, with or without short-term immunosuppression using conventional anti-rejection drugs or biological agents. Donor DC therapies often involve targeting of costimulatory molecule expression or interactions with their T-cell-expressed ligands, through generation of immature DCs,58^–61 which are then administered with or without mAb or CTLA4Ig targeting of key costimulatory molecules expressed by mature/activated DCs.60 62^–64 In rodent models, the addition of costimulation blockade to donor DC therapy results in a striking synergistic effect, with >100 day prolongation of organ graft survival. To examine directly the problem of chronic vascular rejection, we evaluated the influence of immature donor DCs administered in conjunction with anti-CD40L mAb in a murine aortic allograft model.58 Immature donor DCs were administered on days −7, 0, 4 and 10, without or with anti-CD40L mAb. While either treatment alone resulted in diminished intimal smooth muscle cell proliferation compared with untreated animals, the combination resulted in near-complete inhibition of vascular sclerosis. This effect was associated with significant reductions in T-cell (direct pathway) and humoral immunity to donor.

Strategies using recipient DCs have also included generation of immature DCs,65 66 as well as enhanced targeting of the indirect pathway through pulsing of recipient DCs with donor peptide or donor cell lysate,66^–69 with or without additional pharmacological or biological treatment. Theoretically, using recipient DCs to impact the indirect pathway could have significant benefit in counteracting chronic rejection, the major cause of late graft and patient loss in human organ transplantation. In addition, recipient DCs may be more readily available (at least in the setting of deceased donor transplantation) than donor DCs. Recently, Beriou et al have used this approach and demonstrated that donor-specific, indefinite heart graft survival can be achieved in rats given ex vivo generated, recipient-derived, immature DCs, together with only a short course of a perioperative deoxyspergualin derivative (LF 15–0195) to control the direct pathway.70 While this effect implies induction of indirect pathway regulation, the mechanism(s) that underlies graft prolongation has yet to be elucidated.

Human volunteer studies add further support to the use of immature autologous mDCs to induce antigen-specific T-cell tolerance. Thus, Dhodapkar et al18 found that a single subcutaneous injection of influenza matrix peptide (MP)—pulsed immature, autologous mDCs—led to inhibition of MP-specific CD8+ T-cell effector function, as evidenced by diminished IFNγ production and cytolytic function.18 This effect was associated with detection of IL10-producing CD8+ Treg. Silencing of CD8+ T effector cells was specific for MP, as cytomegalovirus-specific CD8+ T effector cells were unaffected. This important study demonstrates that human immature DCs administered in the absence of danger signals promote the development of Treg capable of regulating immunity in an antigen-specific manner and provides further impetus for assessment of this approach in human autoimmune disease or organ transplantation.

In autoimmune disease and transplantation, one of the potential hurdles facing tolerogenic DC therapy is the influence of danger signals/proinflammatory factors present during inflammation and following surgical trauma and ischaemia–reperfusion injury. Under the influence of these factors, the administered immature DCs could potentially undergo maturation and lose their tolerogenic function. This difficulty can, in principle, be overcome if the immature, tolerogenic DCs are administered sufficiently in advance of transplantation, such that their immunological effect can be achieved by the time of transplant surgery (the approach taken in most experimental animal models). Such an approach is possible in live donor transplantation, but not when the donor is deceased. Alternatively, the DCs can be rendered stably maturation-resistant. In this regard, several strategies have been evaluated to develop maturation-resistant DCs, with perhaps the most common being pharmacological treatment in vitro.

MANIPULATION OF DCS TO GENERATE TOLEROGENIC DCS

One solution to the problem of the inflammatory environment and the risk of DC maturation is to manipulate DCs in vitro to produce maturation-resistant, immature DCs with stable tolerogenic properties. Various biological agents (including ultraviolet B radiation, the cytokines IL10 and TGFβ and CTLA4Ig) and pharmacological agents (including corticosteroids, ciclosporin A (CsA), rapamycin, mycophenolate mofetil, vitamin D3 and prostaglandin E2) have been used to confer tolerogenic properties on DCs.71 72 Of these various strategies, pharmacological manipulation stands out as a safe, often predictable and clinically applicable option.

Many conventional immunosuppressant drugs and a wide variety of other pharmacological agents, with known immunoregulatory effects have been investigated for their impact on DC generation and function (reviewed by Hackstein and Thomson71 and Adorini et al72). CsA can inhibit maturation and allostimulatory capacity of mouse mDCs, by inhibiting NF-κB translocation. CsA also impairs IL6 and IL12 production by DCs and DC-triggered production of IFN-γ, IL2 and IL4 by T cells in the bidirectional DC–T-cell system.73 By contrast, human monocyte-derived DCs appear resistant to the inhibitory effects of CsA on DC maturation and allostimulatory capacity. Similarly, another calcineurin inhibitor, tacrolimus (FK506), appears to exert heterogeneous effects on DC maturation, depending on the stimuli used to trigger DC maturation.71 Tacrolimus, however, consistently inhibits T-cell allostimulatory capacity of both mouse and human DCs, irrespective of their maturation status.71 Glucocorticoids inhibit LPS- or CD40L-induced DC maturation and DC production of IL12 and TNFα. DCs exposed to dexamethasone fail to prime Th1 cells efficiently and repeated stimulation of T cells with these DCs generates IL10-producing Treg.

Our studies have shown that rapamycin inhibits mouse bone marrow-derived DC maturation and T-cell stimulatory capacity, both in vitro and in vivo.35 66 74 75 Rapamycin-treated DCs become poor producers of the inflammatory cytokines IL12 and TNFα, and render T cells hyporesponsive to further stimuli when infused into mice. Similar changes in phenotype and function have been reported for rapamycin-treated human monocyte-derived DCs.76 Also, in recent work, we have demonstrated that rapamycin treatment does not block alloantigen uptake by DCs, or impair their in vivo homing to T-cell areas of secondary lymphoid tissue.66 In these studies, a single infusion of rapamycin-treated, donor lysate-pulsed DCs (rapamycin-DCs) 1 week before transplant resulted in significant prolongation of murine organ allograft survival, an effect that was augmented by a short course of immunosuppression (tacrolimus). Significantly, repeated infusion of rapamycin-DCs led to indefinite (>100 day) graft survival in 40% of graft recipients. The effect was associated with donor-specific, T-cell hyporesponsiveness, in both the direct and indirect pathways. Combination of rapamycin-DC infusion with a short postoperative course of rapamycin, led to indefinite graft survival in 80% of transplant recipients.35

In addition to these immunosuppressant drugs, several other pharmacological agents with anti-inflammatory properties (eg, vitamin D, aspirin, N-acetylcysteine) have been reported to target DC function.71 However, the advantage of using the aforementioned classic immunosuppressive agents to “programme” tolerogenic DCs lies in the fact that this approach provides a relatively safe passage into preclinical and, potentially, clinical trials, as these agents currently constitute front-line treatment for graft rejection. Thus, administration of tolerogenic DCs in the setting of conventional immunosuppression would not require a dramatic departure from current clinical strategies and, in addition, those immunosuppressive agents may further promote retention of the tolerogenic DC phenotype. Based on these studies, it is becoming increasingly evident that tolerogenic DC administration, probably in combination with other biological and pharmacological agents, can play a significant role in delaying (and potentially preventing) acute and chronic allograft rejection.

TOLEROGENIC DCS FOR TREATING AUTOIMMUNE DISEASE

We and others have examined the ability of immature/maturation-resistant (as a result of exposure to anti-inflammatory/immunosuppressive agents or cytokines) or genetically modified DCs to confer a therapeutic effect in animal models of autoimmune disease.77^–87 In particular, injection of DCs genetically modified to express either IL4, FasL or IDO into mice with established arthritis at day 32 resulted in significant regression of established diseased.77 Indeed, more than half of the DC-IL4 and DC-IDO-treated mice became disease-free for at least 2 months after treatment. The DC-FasL group also showed significant regression of disease, but disease amelioration was more transient.78 Treatment of mice with DC-IL4 or DC-FasL suppressed IFNγ production from spleen-derived lymphocytes and reduced T-cell proliferation after collagen stimulation, but had no significant effect on anticollagen antibody production. These results demonstrate that systemic administration of DCs, rendered suppressive by gene transfer, can inhibit collagen-reactive T cells, resulting in effective and sustained treatment of established collagen-induced arthritis. It is important to note that the effect of DCs expressing soluble IL4 is similar to the therapeutic effect of DCs expressing a membrane-bound version of IL4.79

In addition to arthritis, suppressive DCs have been used to prevent the onset of hyperglycaemia in non-obese diabetic (NOD) mice. For example, DCs genetically modified to express IL4 can prevent the onset of hyperglycaemia if administered at late stages of insulitis, 12–14 weeks.80 It has been demonstrated that in vitro administration of NF-κB decoys to DCs, as well as direct targeting of CD40, CD80 and CD86 with antisense oligodeoxyribonucleotides, reduce costimulatory molecule levels in bone marrow DCs.81 82 The resulting functionally immature DCs could prevent or reverse new-onset diabetes in the NOD mouse after different routes of injection. The therapeutic effect of the DCs was accomplished while maintaining T-cell responsiveness to alloantigens in animals that received repeated injections of modified DCs. The costimulation-reduced DCs also augmented the number of CD4+ CD25+ Foxp3+ Treg, apparently through an IL7-dependent mechanism.

Interestingly, similar to the studies using DCs to treat rheumatoid arthritis (RA), the therapeutic efficacy in NOD mice was greater if the cells were not pulsed with autoantigens.80^–82 Thus it is likely that suppressive DCs can modulate the function of endogenous DCs, T cells and B cells, preventing or reversing the autoimmune response. Possibly also, tolerogenic DCs can acquire antigens in vivo, resulting in an antigen-specific suppression of autoimmunity. Indeed, it has been observed that DCs can move to arthritic paws in mice with arthritis or to the pancreatic lymph nodes in NOD mice.

ARE TOLEROGENIC DCS READY FOR THE CLINIC?

Support for the clinical translation of tolerogenic DC therapy can be found in DC vaccine trials for cancer. The first report of a clinical study using a DC vaccine was published in Nature Medicine, 12 years ago.88 Subsequently, many patients have received DC vaccines in an effort to promote immunity to tumours. Most of these studies have used mDCs generated from circulating blood monocytes (or alternatively CD34+ cells) as monocytes can readily be recovered and mDCs are easily generated from monocytes.89 Testing of DC immunotherapy has generally proved to be safe, with minimal side effects and has been effective in some patients (even though most patients had late-stage, advanced cancer).89 Some of the early DC vaccine trials used immature rather than mature DCs, without untoward effects (indeed, this was the impetus for the previously discussed landmark paper of Dhodapkar et al,18 in which pre-existing effector T-cell function was silenced in healthy human volunteers in an antigen-specific manner with administration of antigen-pulsed immature DCs, resulting in induction of Treg). Although it has been suggested that more preclinical work is needed before clinical trials, it was also suggested that well-performed, phase I–II studies with quality control measures and appropriate clinical and immunological outcomes should proceed.89

There is still much to learn about optimisation of tolerogenic DC therapy for clinical transplantation or autoimmune disease therapy from a scientific perspective. Variables such as cell dose, single versus multiple doses (and frequency), route of administration (although intravenously seems the most appropriate route for systemic tolerance and has been well tolerated with unmodified bone marrow cell infusion after organ transplantation90 91 as well as DC vaccines)89 and timing relative to transplantation or disease onset remain in question and warrant further preclinical investigation. However, given the relative permissiveness of small animals models to tolerance induction and perhaps species differences in DC biology, many of these questions may not be fully answered until phase I–II trials are initiated in human transplant recipients. We may not be far from that position. Cell therapy (bone marrow infusion, DC vaccines and others) has a good safety record,92 93 and for a patient group with significant need, the potential benefits may justify the risks.

Our own work in this area continues in a preclinical, non-human primate model. Recently, we generated tolerogenic DCs from rhesus monkey blood monocytes using vitamin D3 and IL10. These cells are inferior stimulators of T cells in vitro and are resistant to maturation induced by a potent proinflammatory cytokine cocktail (IL1β, TNFα, IFNγ, IL6, prostaglandin E2). Their potential to induce antigen-specific, T-cell hyporesponsiveness suggests a tolerogenic DC-generating strategy that coupled with conventional immunosuppressant cover could improve outcomes in clinical organ transplantation (ie, reduce dependence on chronic immunosuppressive drug therapy). These DCs are currently being evaluated for their ability to regulate alloimmune responses in vivo and organ transplant outcome.

An obvious patient group to which regulatory DC therapy could apply is live donor organ allograft recipients, with kidney the most prevalent organ transplanted in this manner. Tolerogenic DCs could be generated, modified and administered, well in advance of transplantation during steady-state conditions. As discussed by Mirenda et al,94 prospective infusion of mobilised tolerogenic donor DCs95^–97 or tolerogenic DCs propagated from leukapheresis products into graft recipients, followed by conventional immunosuppression cover, with the goal of inducing immunoregulation, is applicable in the clinic—albeit in the context of live donor transplants. Further exploratory work with tolerogenic DCs given at the time of transplant and/or subsequently is needed to ascertain the efficacy/applicability of these cells in deceased donor transplantation. Well-designed, phase I–II studies for allotransplantation with appropriate safety, as well as immunological monitoring, may not be far off.

CLINICAL TRIALS FOR AUTOIMMUNE DISEASES

As outlined above, in vitro administration of NF-κB decoys to DCs, as well as direct targeting of CD40, CD80 and CD86 with antisense oligodeoxyribonucleotides, reduces the level of costimulatory molecule expression. These functionally immature DCs are effective in preventing or reversing new-onset diabetes in the NOD mouse, while maintaining T-cell responsiveness to alloantigens in animals that received repeated injections of modified DCs. These results have resulted in a phase I clinical trial at the University of Pittsburgh in an adult cohort with insulin-requiring type-1 diabetes of at least 5 years’ duration. In this continuing trial, leucocytes from the patient are obtained by aphaeresis and DCs are generated in vitro and engineered in GMP facilities with the addition of antisense oligodeoxyribonucleotides. These DCs, which express low levels of CD40, CD80 and CD86, are administered by intradermal injection, at a site proximal to the pancreas, from where it is hypothesised that the DCs will migrate to the nearest lymph nodes to reduce islet-specific inflammation. The abrogation of the autoimmune diabetogenic insult should result in the rescue of still-present, insulin-producing β cells and/or neogenesis of other insulin-producing cells in the host endocrine pancreas, even after the onset of the disease. This trial is currently underway and once safety has been demonstrated, a phase II efficacy trial will start, involving patients with new-onset diabetes.

Tolerogenic DCs also are close to entering the clinic for RA. In an approved study at the University of Queensland in Brisbane, Australia, autologous, modified monocyte-derived DCs will be pulsed with a mixture of four citrullinated peptide antigens and administered subcutaneously once to each group of nine patients with RA on the usual disease-modifying drugs in an escalating dose regimen. In this study, safety will be assessed by haematological, biochemical and lipid profile, autoantibodies and measures of inflammation over a 6-month period. Tolerance induction with be measured by peripheral blood T-cell proliferative and cytokine responses to citrullinated peptides, control peptides and tetanus toxoid antigen, for up to 6 months after treatment.

A second clinical trial for treating RA with immunosuppressive DCs will be initiated at the University of Newcastle, England. In this study, DCs rendered suppressive by treatment with dexamethasone and vitamin D3 will be used to treat patients with RA for whom at least one disease-modifying antirheumatic drug has failed. The tolerogenic DCs, not treated with autoantigens, will be administered by intra-articular injection into the affected knee joint. Although the main end point of this trial is safety, secondary end points, such as time to flare and a transcriptional profile of the synovial membrane will be evaluated.

CONCLUSION

In conclusion, there are strong indications that tolerogenic DCs will one day play a role in the treatment of clinical transplantation and chronic inflammatory disease. Indeed, suppressive DCs are in the clinic for type 1 diabetes and soon will be in the clinic for the treatment of RA. As we contemplate the widespread introduction of this treatment in the clinic, the safety of DC vaccines is encouraging and it is reassuring that this treatment can be introduced in the context of current immunosuppressive strategies.