Following transplantation of allogeneic tissues, CD4+ recipient T cells recognize donor antigens via two mechanisms: direct allorecognition, in which T cells interact with intact MHC class II on donor bone marrow-derived passenger leukocytes, and indirect allorecognition, in which T cells recognize allogeneic peptides bound to self-MHC class II molecules on recipient antigen-presenting cells (APCs) . Since the activation of pro-inflammatory T cells through each of these pathways elicits different types of alloresponses leading to the rejection of allografts , the suppression of both of these pathways is needed to achieve transplantation tolerance. A study in this issue of the European Journal of Immunology  shows that allogeneic DCs modified to either lack expression of CD80/86 or overexpress indoleamine 2,3-dioxygenase (IDO) are able to inhibit direct and/or indirect pro-inflammatory T-cell alloresponses in vitro and in vivo. Remarkably, suppression of both pathways was achieved through linked suppression via coexpression of self- and allo-MHC molecules on semi-allogeneic DCs. This approach accomplished long-term survival of allogeneic corneal allografts whose rejection relies on indirect activation of CD4+ T cells specific for minor histocompatibility antigens. This study further demonstrates the tolerogenic potential of genetically modified DCs that could pave the way for the design of DC-based therapy in clinical transplantation.
Pro-inflammatory direct and indirect T-cell alloresponses elicited after transplantation are both capable of causing acute rejection of fully allogeneic skin allografts . Alternatively, early acute rejection of vascularized solid organ allotransplants is essentially mediated through direct allorecognition, while indirect allorecognition is commonly associated with chronic rejection of kidney and heart transplants (a slow process characterized by graft vasculopathy and tissue fibrosis) . Therefore, abrogation of both types of inflammatory alloresponses will be required to achieve tolerance to allogeneic transplants — defined as indefinite graft survival without chronic dysfunction — in the absence of ongoing immunosuppression.
There is ample evidence showing that MHC class II-restricted antigen presentation by APCs incapable of delivering proper costimulatory signals results in CD4+ T-cell unresponsiveness . Actually, peripheral tolerance to autologous antigens is believed to be maintained by continuous presentation of self-determinants by resting DCs in the absence of inflammatory signals, a phenomenon causing T-cell anergy and activation of some regulatory T (Treg) cells [7-9]. Based upon this principle, many transplant tolerance protocols have been designed using donor-cell administration in combination with antibodies blocking key costimulatory pathways like CD28/B7 and CD40/CD40L . For example, infusion of transplant recipients with donor bone marrow or spleen cells, along with anti-CD40L antibodies or CTLA-4-Ig regularly achieves long-term survival of some organ allografts [11-13]. However, in most cases, these treatments do not prevent chronic rejection, suggesting that they fail to tolerize T cells activated in an indirect fashion. This viewpoint is also supported by studies showing that T-cell costimulation blockade using anti-CD40L mAbs prolongs the survival of cardiac, but not skin allografts . This may be explained by the observation that, in contrast to hearts, skin allografts induce potent indirect alloresponses that are somewhat resistant to this antibody treatment (G. Benichou, unpublished data). Consequently, the tolerization of T cells responding to alloantigens through the indirect allorecognition pathway has become a major challenge in the field of transplantation. In this issue of the European Journal of Immunology, Khan et al.  demonstrate that inflammatory CD4+ T cells activated though both direct and indirect allorecognition pathways can be rendered tolerant using either donor DCs lacking CD80/86 co-stimulatory receptor membrane expression, or DCs overexpressing IDO.
In infected tissues, microbial antigen capture by DCs and subsequent antigen presentation in secondary lymphoid organs is clearly the most efficient mechanism to trigger a pro-inflammatory adaptive immune response . This response initiates the destruction of microbes and the generation of memory T cells, conferring long-term protective immunity. This type of antigen presentation occurs during acute infections associated with potent innate immune responses and inflammatory cytokine secretion, causing DC maturation and expression of certain key costimulatory receptors like B71/2 and CD40 . Likewise, alloantigen presentation by donor DCs is known to induce powerful pro-inflammatory T-cell immune responses leading to acute allograft rejection . Based upon these principles, many studies have focused on the elimination of donor DCs in an effort to induce transplant tolerance . However, while elimination of donor passenger leukocytes brought about some prolongation of graft survival by reducing direct alloreactivity, it neither achieved tolerance nor suppressed indirect alloresponses . On the other hand, there is increasing evidence to suggest that inactivation of pro-inflammatory T cells against antigens also relies on antigen presentation by DCs. Indeed, antigens displayed by immature DCs in the absence of innate inflammatory signals may preferentially activate Treg cells and T-cell anergy [7-9]. This mechanism is essential for the maintenance of peripheral tolerance to autoantigens [7-9]. Likewise, there are several lines of data suggesting that donor DCs are necessary for tolerance induction to allografts [18-20]. Therefore, allogeneic DCs are required for both rejection and tolerance of allografts.
Khan et al.  explored two models of DC modification to inhibit allogenenic T-cell proliferation. In the CBK to CBA single MHC class I-disparate model (Model A, Fig. 1), CBK “CTLA-4-DCs” (CBA Kb transgenic) lacking membrane expression of CD80/86 induced both in vivo and in vitro expansion/activation of Treg cells . These Treg cells were capable of suppressing the indirect alloresponse of CD4+ pro-inflammatory CBA T cells recognizing Kb peptides presented by Ak/Ek recipient MHC class II molecules. T-cell suppression was presumably mediated via IL-10 and TGF-β cytokine release, and resulted in anergy confined to donor-specific CD4+ T cells (Fig. 1). In the BALB/c to C3H full MHC-disparate model (Model B), BALB/c “CTLA-4-DCs” (H-2d) were cultured with C3H (H-2k) T cells or administered to C3H mice. In both cases, C3H CD4+ T cells became anergic to BALB/c but not to third party allogeneic cells, and allospecific tolerance could be transferred in vitro and in vivo with C3H CD4+ T cells from cultures or mice exposed to BALB/c CTLA-4-DCs .
The results obtained in model A presumably reflect the mechanism by which Treg cells — activated through the presentation of peptides presented on a nominal antigen — suppress effector T (Teff) cells recognizing the same or other determinants presented by the same MHC class II molecules. The process by which Treg cells mediate suppression in model B is more intriguing. The results suggest that such induced Treg cells recognize alloantigens in the same fashion as pro-inflammatory CD4+ T cells. Indeed, the fact that donor BALB/c DCs and recipient C3H Treg cells do not share any MHC class II molecules hints that Treg cells are activated in a direct fashion, a conclusion that challenges the traditional view that Treg-cell activation occurs exclusively through indirect antigen presentation. Alternatively, one can speculate that Treg cells recognize determinants presented by self-MHC class II molecules present on partially activated Teff cells and not on donor DCs — i.e. in an indirect fashion. This question could be resolved by using Teff cells lacking MHC class II expression.
Next, Khan et al.  showed that Treg cells generated in CBA mice via administration of CBK CTLA-4-DCs inhibit T-cell responses to (B6 × CBA) F1 semi-allogeneic stimulators expressing allogeneic MHC class I (Lb and Db) and class II alleles (Ab) that are not present in CBK mice. These data demonstrate that both indirect alloresponses to Lb/Db and direct alloresponses to Ab of CBA T cells were abrogated though linked suppression, as depicted in Fig. 1. This conclusion was confirmed by lack of suppression to (BALB/c × B6) F1 allogeneic stimulators that express Kb, but not the proper self-MHC class II allele. These observations further illustrate how Treg cells activated indirectly can suppress direct alloresponses by Teff cells [21, 22]. It is not clear whether the effector phase of suppression requires Treg–Teff cell contact. Experiments using transwell plates in which Teff cells, Treg cells, and DCs are cultured in separate compartments could be conducted to answer this question.
Although IDO-DCs suppressed both direct and indirect allo-responses by CD4+ T cells, they failed to induce T-cell anergy and regulatory T-cell immunity. This is in apparent contradiction with the general view that DCs expressing IDO inhibit inflammatory reactions via cell cycle arrest, apoptosis, and anergy of TH1/CT1 cells . The authors speculate that this discrepancy may be explained by the fact that high IDO levels result in nonspecific T-cell hyporesponsiveness and death rather than anergy. Additionally, we surmise that IDO-DC injection does not cause substantial inflammation and subsequent IFN-γ production, a cytokine presumably required for TGF-β production by activated DCs . In this scenario, kynurenines and other metabolites of tryptophan degradation generated through IDO activity may lead to T-cell apoptosis but not anergy and generation of Treg cells, in the absence of TGF-β . This might explain why, apparently, “artificially” increased IDO expression in DCs mitigates some Teff-cell functions in a nonspecific manner but not antigen-dependent induction of the T-cell response.
Last, Khan et al.  investigated the effects of modified DCs on the rejection of corneal allografts in mice. In this model, the alloresponse to, and rejection of, allogeneic transplants are exclusively mediated via indirect alloreactivity by CD4+ T cells directed toward minor histocompatibility antigens [26, 27]. Indeed, lack of corneal APCs expressing allo-MHC class II+ results in the absence of CD4+ T-cell direct alloreactivity . On the other hand, some CD8+ T cells secreting IFN-γ are activated though the direct allorecognition of donor MHC class I antigens, but fail to display cytotoxic functions and do not influence the rejection process . In the C3H to BALB/c model, injection of CTLA-4-DCs resulted in a modest but significant prolongation of graft survival (18 days versus 12 days), while IDO-DCs had no effect. In this setting, it is conceivable that Treg cells recognizing allo-MHC class II directly do not suppress the CD4+ T-cell indirect alloresponse and thereby fail to induce tolerance of corneal allografts. This hypothesis is supported by the observation that administration of CBA recipients with semi-allogeneic (CBA × BALB/c) F1 CTLA-4-DCs, which apparently generates Treg cells blocking both direct and indirect alloresponses by CD4+ T cells, resulted in indefinite survival of corneal allografts (>100d) . It would have been interesting to test the effects of CTLA-4-DCs on the rejection of high-risk corneal transplants, which are performed on an inflamed eye bed, induce direct CD4+ alloresponses, and represent a major problem in clinical settings [29, 30].
In summary, the study by A. George's group  demonstrates that allogeneic DCs can be modified in a fashion that achieves donor-specific (CTLA-4-DCs) or nonspecific (IDO-DCs) tolerance of T cells responding to alloantigens via both direct and indirect allorecognition pathways. It is clear that the injection of transplant recipient with DCs presenting defined alloantigens — while lacking proper costimulatory receptors — is expected to achieve selective suppression of donor-specific inflammatory T cells. In comparison, the administration of donor APCs along with antibodies blocking the same costimulation pathways can potentially suppress other T cells. In the present study, injection of mice with modified allogeneic DCs achieved long-term survival of corneal allografts (that are otherwise rejected by CD4+ T cells activated indirectly). Whether delivery of such tolerogenic DCs would be effective at preventing rejection of more immunogenic transplants such as heart and skin allografts remains to be investigated. In addition, the ability of modified DCs to achieve tolerance in allosensitized mice should be explored, given the documented contribution of donor-reactive memory T cells to transplant tolerance resistance in primates . Finally, since the tolerogenic DCs designed in this study can suppress indirect alloreactivity, it would be interesting to determine whether administration of these cells could prevent or suppress alloantibody production and chronic rejection of vascularized organ transplants in recipients treated with short-term immunosuppressive therapy using calcineurin inhibitors and/or a lymphocyte costimulation blockade.