Both rejection and tolerance of allografts can occur in the absence of secondary lymphoid tissues.

In this study, we showed that aly/aly mice, which are devoid of lymph nodes and Peyer's patches, acutely rejected fully allogeneic skin and heart grafts. They mounted potent inflammatory direct alloresponses but failed to develop indirect alloreactivity after transplantation. Remarkably, skin allografts also were rejected acutely by splenectomized aly/aly (aly/aly-spl(-)) mice devoid of all secondary lymphoid organs. In these recipients, the rejection was mediated by alloreactive CD8(+) T cells presumably primed in the bone marrow. In contrast, cardiac transplants were not rejected by aly/aly-spl(-) mice. Actually, aly/aly-spl(-) mice that spontaneously accepted a heart allotransplant and displayed donor-specific tolerance also accepted skin grafts from the same, but not a third-party, donor via a mechanism involving CD4(+) regulatory T cells producing IL-10 cytokine. Therefore, direct priming of alloreactive T cells, as well as rejection and regulatory tolerance of allogeneic transplants, can occur in recipient mice lacking secondary lymphoid organs.

Primary vascularization of allografts governs their immunogenicity and susceptibility to tolerogenesis.

We investigated the influence of allograft primary vascularization on alloimmunity, rejection, and tolerance in mice. First, we showed that fully allogeneic primarily vascularized and conventional skin transplants were rejected at the same pace. Remarkably, however, short-term treatment of mice with anti-CD40L Abs achieved long-term survival of vascularized skin and cardiac transplants but not conventional skin grafts. Nonvascularized skin transplants triggered vigorous direct and indirect proinflammatory type 1 T cell responses (IL-2 and IFN-γ), whereas primarily vascularized skin allografts failed to trigger a significant indirect alloresponse. A similar lack of indirect alloreactivity was also observed after placement of different vascularized organ transplants, including hearts and kidneys, whereas hearts placed under the skin (nonvascularized) triggered potent indirect alloresponses. Altogether, these results suggest that primary vascularization of allografts is associated with a lack of indirect T cell alloreactivity. Finally, we show that long-term survival of vascularized skin allografts induced by anti-CD40L Abs was associated with a combined lack of indirect alloresponse and a shift of the direct alloresponse toward a type 2 cytokine (IL-4, IL-10)-secretion pattern but no activation/expansion of Foxp3(+) regulatory T cells. Therefore, primary vascularization of allografts governs their immunogenicity and tolerogenicity.

The road to transplant tolerance is paved with good dendritic cells.

After transplantation, recipient T cells can recognize donor antigens either by interacting with MHC class II on donor bone marrow-derived cells (direct allorecognition), or by recognizing allogeneic peptides bound to self-MHC class II molecules on recipient antigen presenting cells (indirect allorecognition). The activation of pro-inflammatory T cells via either of these pathways leads to allograft rejection, so the suppression of both of these pathways is needed to achieve transplantation tolerance. A study in this issue of the European Journal of Immunology [Eur. J. Immunol. 2013. 43: 734-746] shows that allogeneic dendritic cells (DCs) modified to either lack expression of CD80/86 or over-express indoleamine 2,3-dioxygenase (IDO) are able to inhibit direct and/or indirect alloresponses in vitro and in vivo in mice. Notably, both allorecognition pathways were suppressed by the coexpression of self- and allo-MHC molecules on semi-allogeneic DCs. This Commentary discusses the challenges and potential of using genetically-modified DCs to suppress alloreactivity in the context of transplant tolerance.

Differential effects of denileukin diftitox IL-2 immunotoxin on NK and regulatory T cells in nonhuman primates.

Denileukin diftitox (DD), a fusion protein comprising IL-2 and diphtheria toxin, was initially expected to enhance antitumor immunity by selectively eliminating regulatory T cells (Tregs) displaying the high-affinity IL-2R (α-ß-γ trimers). Although DD was shown to deplete some Tregs in primates, its effects on NK cells (CD16(+)CD8(+)NKG2A(+)CD3(-)), which constitutively express the intermediate-affinity IL-2R (ß-γ dimers) and play a critical role in antitumor immunity, are still unknown. To address this question, cynomolgus monkeys were injected i.v. with two doses of DD (8 or 18 µg/kg). This treatment resulted in a rapid, but short-term, reduction in detectable peripheral blood resting Tregs (CD4(+)CD45RA(+)Foxp3(+)) and a transient increase in the number of activated Tregs (CD4(+)CD45RA(-)Foxp3(high)), followed by their partial depletion (50-60%). In contrast, all NK cells were deleted immediately and durably after DD administration. This difference was not due to a higher binding or internalization of DD by NK cells compared with Tregs. Coadministration of DD with IL-15, which binds to IL-2Rß-γ, abrogated DD-induced NK cell deletion in vitro and in vivo, whereas it did not affect Treg elimination. Taken together, these results show that DD exerts a potent cytotoxic effect on NK cells, a phenomenon that might impair its antitumoral properties. However, coadministration of IL-15 with DD could alleviate this problem by selectively protecting potentially oncolytic NK cells, while allowing the depletion of immunosuppressive Tregs in cancer patients.

Contributions of direct and indirect alloresponses to chronic rejection of kidney allografts in nonhuman primates.

The relative contribution of direct and indirect allorecognition pathways to chronic rejection of allogeneic organ transplants in primates remains unclear. In this study, we evaluated T and B cell alloresponses in cynomolgus monkeys that had received combined kidney/bone marrow allografts and myeloablative immunosuppressive treatments. We measured donor-specific direct and indirect T cell responses and alloantibody production in monkeys (n = 5) that did not reject their transplant acutely but developed chronic humoral rejection (CHR) and in tolerant recipients (n = 4) that never displayed signs of CHR. All CHR recipients exhibited high levels of anti-donor Abs and mounted potent direct T cell alloresponses in vitro. Such direct alloreactivity could be detected for more than 1 y after transplantation. In contrast, only two of five monkeys with CHR had a detectable indirect alloresponse. No indirect alloresponse by T cells and no alloantibody responses were found in any of the tolerant monkeys. Only one of four tolerant monkeys displayed a direct T cell alloresponse. These observations indicate that direct T cell alloresponses can be sustained for prolonged periods posttransplantation and result in alloantibody production and chronic rejection of kidney transplants, even in the absence of detectable indirect alloreactivity.

Phenotype, distribution and alloreactive properties of memory T cells from cynomolgus monkeys.

The high frequency of memory T cells present in primates is thought to represent a major barrier to tolerance induction in transplantation. Therefore, it is crucial to characterize these memory T cells and determine their functional properties. High numbers of memory T cells were detected in peripheral blood and all lymphoid tissues except lymph nodes, which were essentially the site of naïve T cells. The majority of CD8(+) memory T cells were effector memory cells located in the blood and bone marrow while most CD4(+) memory T cells were central memory cells present in the spleen. Next, memory T cells from over 100 monkeys were tested for their response to alloantigens by ELISPOT. Memory alloreactivity mediated via direct but not indirect allorecognition was detected in all animals. The frequency of allospecific memory T cells varied dramatically depending upon the nature of the responder/stimulator monkey combination tested. MHC gene matching was generally associated with a low-memory alloreactivity. Nevertheless, low anamnestic alloresponses were also found in a significant number of fully MHC-mismatched monkey combinations. These results show that selected donor/recipient combinations displaying a low memory alloresponsiveness can be found. These combinations may be more favorable for transplant tolerance induction.

Modulation of alloreactivity to MHC-derived peptides and transplantation tolerance.

The recognition by T cells of intact foreign MHC molecules at the surface of transplanted cells (direct pathway) was originally thought to represent the driving force behind acute rejection of allogeneic transplants. Over the past decade, however, a body of evidence has been provided demonstrating that T cell recognition of donor peptides presented by recipient APCs (indirect pathway) is sufficient on its own to ensure both acute and chronic rejection of allografts. While the direct allorecognition leads to an exceptionally vigorous inflammatory T cell response, it is thought to be short lived due to the rapid depletion of donor professional APCs and it can be controlled with a short course of immunosuppressive drugs including calcineurin inhibitors. In contrast, while the indirect alloresponse is oligoclonal and much weaker, it is long-lived and tends to spread to formerly cryptic determinants on donor and self-tissue specific antigens. This feature of indirect alloreactivity is presumably associated with the sustained presence of recipient professional APCs that can maintain a chronic inflammatory response similar to that observed in autoimmune diseases. Consequently, the indirect alloresponse may be more difficult to suppress than its direct counterpart. On the other hand, there is accumulating evidence showing that administration of alloantigen in a "tolerogenic fashion" mediates allograft acceptance via the activation of regulatory T cells recognizing donor antigens via the indirect allorecognition pathway. Apparently, these regulatory T cells can suppress both direct and indirect inflammatory T cell responses to donor antigens. This suggests that modulation of indirect alloreactivity may represent the best strategy to achieve long-term donor-specific tolerance to allotransplants.

T-cell response to p53 tumor-associated antigen in patients with colorectal carcinoma.

Despite the radical surgical resection performed in patients with colorectal carcinoma, there is a high rate of tumor recurrence. Over an observation period of 3 years, 18% of the patients in our collective suffered a tumor relapse with local or distinct metastases after initial R0-resection. Some evidence suggests that this may be due to suppression of anti-tumor responses, a phenomenon that might be attributed to regulatory T cells. The aim of our study was to investigate the tumor-specific immune response depending on the UICC stage of patients with colorectal cancer. The cellular immune responses against defined antigens that are overexpressed in most of the patients with colorectal cancer were characterized. For this purpose, the tumor suppressor gene, p53, was chosen as the tumor-associated antigen that exhibits mutations and overexpression in up to 60% of colorectal carcinoma. We observed that p53 induced both IFN-gamma and IL-10 secretion. The predominance of IL-10 production indicated that regulatory T cells directly participate in modulating the anti-tumor immune response. IL-10 levels in the blood as well as the expression of regulatory T-cell specific genes at the tumor site correlate with the UICC stage of the disease. These results may provide an explanation for the poor prognosis and increased recurrence rate in patients with advanced carcinoma.

Donor exosomes rather than passenger leukocytes initiate alloreactive T cell responses after transplantation.

Transplantation of allogeneic organs and tissues represents a lifesaving procedure for a variety of patients affected with end-stage diseases. Although current immunosuppressive therapy prevents early acute rejection, it is associated with nephrotoxicity and increased risks for infection and neoplasia. This stresses the need for selective immune-based therapies relying on manipulation of lymphocyte recognition of donor antigens. The passenger leukocyte theory states that allograft rejection is initiated by recipient T cells recognizing donor major histocompatibility complex (MHC) molecules displayed on graft leukocytes migrating to the host's lymphoid organs. We revisited this concept in mice transplanted with allogeneic skin, heart, or islet grafts using imaging flow cytometry. We observed no donor cells in the lymph nodes and spleen of skin-grafted mice, but we found high numbers of recipient cells displaying allogeneic MHC molecules (cross-dressed) acquired from donor microvesicles (exosomes). After heart or islet transplantation, we observed few donor leukocytes (100 per million) but large numbers of recipient cells cross-dressed with donor MHC (>90,000 per million). Last, we showed that purified allogeneic exosomes induced proinflammatory alloimmune responses by T cells in vitro and in vivo. Collectively, these results suggest that recipient antigen-presenting cells cross-dressed with donor MHC rather than passenger leukocytes trigger T cell responses after allotransplantation.

A salen-manganese catalytic free radical scavenger inhibits type 1 diabetes and islet allograft rejection.

Reactive oxygen species, such as superoxide, and nitrogen oxides, such as peroxynitrite, are thought to contribute to beta-cell destruction during the disease process that leads to type 1 diabetes. EUK-8 is a member of a new class of synthetic salen-manganese compounds with low toxicity that possess catalytic superoxide dismutase, peroxidase, and catalase activity that can inactivate superoxide and nitrogen oxides (e.g., peroxynitrite and nitrogen dioxide). We observed that EUK-8 administration inhibited the adoptive transfer of type 1 diabetes to NOD mice. In addition, administration of EUK-8 to NOD mice with established autoimmunity completely prevented the development of type 1 diabetes for up to 1 year in age, even though the treatment was discontinued after 35 weeks of age. EUK-8 treatment also prolonged the survival of islet allografts in newly diabetic NOD mice. Thus, reactive oxygen and nitrogen species contribute to the pathoetiology of both spontaneous type 1 diabetes and allograft rejection. In cultures of NIT-1 cells, EUK-8 inhibited cytotoxicity caused by superoxide as well as nitric oxide. Collectively, our findings implicate a greater role for nitrogen oxides (other than peroxynitrite) in beta-cell damage. Antioxidants designed to prevent the formation of both cytotoxic reactive oxygen and nitrogen species may effectively protect beta-cells from spontaneous autoimmunity and alloresponses.

Salen-manganese complexes as catalytic scavengers of hydrogen peroxide and cytoprotective agents: structure-activity relationship studies.

Synthetic catalytic scavengers of reactive oxygen species (ROS) may have broad clinical applicability. In previous papers, two salen-manganese complexes, EUK-8 and EUK-134, had superoxide dismutase (SOD) and catalase activities and prevented ROS-associated tissue injury. This study describes two series of salen-manganese complexes, comparing catalytic ROS scavenging properties and cytoprotective activities. The compounds vary widely in ability to scavenge hydrogen peroxide, with this activity most influenced by salen ring alkoxy substitution and aromatic bridge modifications. In contrast, all compounds show comparable SOD activities. The most active alkoxy-substituted catalase mimetics protected cultured cells from hydrogen peroxide, and a subset of these were also neuroprotective in a rodent stroke model. Thus, structural modification of the prototype EUK-8 yields compounds with enhanced catalase activity and, in turn, biological effectiveness. This supports the concept that salen-manganese complexes represent a class of SOD and, in particular, catalase mimetics potentially useful against ROS-associated diseases.

Innate immunity and resistance to tolerogenesis in allotransplantation.

The development of immunosuppressive drugs to control adaptive immune responses has led to the success of transplantation as a therapy for end-stage organ failure. However, these agents are largely ineffective in suppressing components of the innate immune system. This distinction has gained in clinical significance as mounting evidence now indicates that innate immune responses play important roles in the acute and chronic rejection of whole organ allografts. For instance, whereas clinical interest in natural killer (NK) cells was once largely confined to the field of bone marrow transplantation, recent findings suggest that these cells can also participate in the acute rejection of cardiac allografts and prevent tolerance induction. Stimulation of Toll-like receptors (TLRs), another important component of innate immunity, by endogenous ligands released in response to ischemia/reperfusion is now known to cause an inflammatory milieu favorable to graft rejection and abrogation of tolerance. Emerging data suggest that activation of complement is linked to acute rejection and interferes with tolerance. In summary, the conventional wisdom that the innate immune system is of little importance in whole organ transplantation is no longer tenable. The addition of strategies that target TLRs, NK cells, complement, and other components of the innate immune system will be necessary to eventually achieve long-term tolerance to human allograft recipients.

Immune recognition and rejection of allogeneic skin grafts.

The transplantation of allogeneic skin grafts is associated with a potent inflammatory immune response leading to the destruction of donor cells and the rejection of the graft. Shortly after transplantation, skin dendritic cells (DCs) migrate out of the graft through lymphatic vessels and infiltrate the recipient's draining lymph nodes where they present donor antigens via two mechanisms: the direct pathway, in which T cells recognize intact donor MHC antigens on donor DCs; and the indirect pathway, involving T-cell recognition of donor peptides bound to self-MHC molecules on recipient DCs. Some recent studies have suggested that T cells can become activated via recognition of donor MHC molecules transferred on recipient antigen-presenting cells (semidirect pathway). Activation of T cells via direct or indirect allorecognition is sufficient to trigger acute rejection of allogeneic skin grafts. In addition, allospecific antibodies contribute to the rejection process either by killing allogeneic targets in a complement-dependent fashion or by opsonizing donor cells and forming immune complexes. Finally, several studies demonstrate that NK cells, activated due to missing self-MHC class I molecules on allogeneic cells, are involved in allogeneic skin graft rejection via direct killing of donor cells and through the production of proinflammatory cytokines including IFN-γ and TNF-α.

Host alloreactive memory T cells influence tolerance to kidney allografts in nonhuman primates.

Transplant tolerance, defined as indefinite allograft survival without immunosuppression, has been regularly achieved in laboratory mice but not in nonhuman primates or humans. In contrast to laboratory mice, primates regularly have high frequencies of alloreactive memory T cells (TMEMs) before transplantation. These TMEMs are poorly sensitive to conventional immunosuppression and costimulation blockade, and the presence of donor-reactive TMEMs in primates may account for their resistance to transplant tolerance protocols that have proven consistently effective in mice. We measured the frequencies of anti-donor TMEMs before and after transplantation in a series of rejecting and tolerant monkeys that underwent nonmyeloablative conditioning, short-term immunosuppression, and combined allogeneic kidney/cell transplantation. Transplants were acutely rejected in all the monkeys with high numbers of donor-specific TMEMs before transplantation. In contrast, long-term survival was observed in the recipients harboring lower frequencies of anti-donor TMEMs before transplantation. Similar amounts of TMEM homeostatic expansion were recorded in all transplanted monkeys upon hematopoietic reconstitution; however, only the tolerant monkeys had no expansion or activation of donor-reactive TMEMs after transplantation. These results indicate that the presence of high frequencies of host donor-reactive TMEMs before transplantation impairs tolerance induction to kidney allografts in this nonhuman primate model. Indeed, recipients harboring a low anamnestic reactivity to their donor before transplantation were successfully rendered tolerant via infusion of donor cells and short-term immunosuppression. This suggests that selection of allogeneic donors with low memory responses in recipients may be essential to successful transplant tolerance induction in patients.

Prolongation of alloskin graft survival by catalytic scavengers of reactive oxygen species.

We tested the effects of salen manganese (Salen-Mn) complexes, which are scavengers of reactive oxygen species exhibiting superoxide dismutase and catalase activities on the rejection of and alloresponse to fully allogeneic skin grafts in mice. We showed that pre-transplant treatment of C57Bl/6 donor skin or of BALB/c recipients with Salen-Mn complexes significantly delayed allograft rejection. ELISPOT analysis of alloimmune response of treated mice revealed a significant reduction of the frequency of type 1 cytokine (pro-inflammatory) producing T-cells, while the number of activated T-cells producing type 2 cytokines was elevated. In addition, anti-oxidative treatment of graft recipients resulted in a profound inhibition of their donor-specific cytotoxic T-cell response. Our results indicate that salen manganese complexes mediate their effect on graft rejection both by reducing the susceptibility of graft tissue to ROS-mediated injury and by exerting an anti-inflammatory effect in recipients.

The dual response of protein kinase Fyn to neural trauma: early induction in neurons and delayed induction in reactive astrocytes.

In the developing central nervous system, a src-related protein-tyrosine kinase fyn participates in the myelination process, neuronal growth, and cytoskeletal organization. In adults, fyn has been implicated in learning and memory formation. To test if fyn expression is modulated by neuronal activity, we performed quantitative in situ hybridization (ISH) using brain sections of the adult rats that had undergone either kainic acid (KA)-induced seizures or neuronal deafferentation (entorhinal cortex lesion, ECL). In the KA model, a few hours after seizure activities, fyn mRNA was elevated in the dentate gyrus (DG) (+45%), cerebral cortex layer III (+35%), and piriform cortex (+25%). Conversely, fyn mRNA consistently decreased in the hippocampal neurons after transection of the major axonal inputs from the entorhinal cortex. Although fyn expression in the brain has been allegedly limited to neurons and oligodendrocytes, we provide in this study the first evidence that fyn mRNA is highly expressed in the astrocytes involved in reactive gliosis. In the KA model, the occurrence of fyn-overexpressing astrocytes increased with the progress of neuronal damage in the CA1 and CA3 regions of the hippocampus. In contrast, fyn-overexpressing astrocytes were not observed in the granular cell layer of dentate gyrus (DG), where neurons were not damaged. Likewise, in the ECL model, the most drastic change in fyn mRNA expression took place at the reactive astrocytes near the stab wound sites, where fyn mRNA levels were doubled 4-10 d after the lesion. Collectively, our data suggest that (i) an early induction of fyn mRNA in neurons is linked to neuronal activity, and (ii) the delayed induction of fyn mRNA in reactive astrocytes near the damaged cells may play novel signaling roles during glial response.