MHC alloantigens elicit secondary,but not primary,indirect in vitro proliferative responses

The relative contributions of direct and indirect pathways of allorecognition to graft rejection remain controversial. Recent reports suggest that the indirect pathway may play a prominent role in both acute and chronic allograft rejection. Such studies suggest that MHC-derived allopeptides are more immunogenic than those derived from minor histocompatibility or other nominal Ags. The aim of this study was to characterize the immunogenicity of MHC alloantigens in MHC-defined miniature swine via primary and secondary MLR culture assays. APCs were selectively depleted from either responder or stimulator cell populations to specifically analyze direct and indirect proliferative responses, respectively. Radio-resistant cytokine secretion and subsequent backstimulation of responder cells was eliminated by using stimulators that were either lysed or unresponsive to the responder MHC haplotypes. When the effect of backstimulation was eliminated from MLR culture assays, indirect proliferative responses were not observed among naive responders. Only after in vivo priming of responder animals could indirect proliferation be detected. These data do not refute the potential importance of indirect allorecognition in graft rejection. However, they suggest that MHC-derived alloantigens behave similarly in vitro to minor histocompatibility Ags, with comparable immunogenicity. These data also suggest that the MLR culture assay does not accurately reflect the importance of indirect mechanisms that have previously been reported in experimental models of graft rejection. A greater understanding of the indirect pathway and the associated immunogenicity of MHC allopeptides has the potential benefit of enabling the development of therapeutic interventions to prevent or halt allograft rejection.     

Clonotype analysis of human alloreactive T cells: a novel approach to studying peripheral tolerance in a transplant recipient

The recognition of allo-MHC and associated peptides on the surface of graft-derived APC by host T cells (direct pathway allorecognition) plays an important role in acute rejection after organ transplantation. However, the status of the direct pathway T cells in stable long term transplants remains unclear. To detect alloreactive T cell clones in PBL and the allograft during the transplant tolerance, we utilized RT-PCR instead of functional assays, which tend to underestimate their in vivo frequencies. We established alloreactive CD4+ and CD8+ T cell clones from peripheral blood sampled during the stable tolerance phase of a patient whose graft maintained good function for 9 years, 7 without immunosuppression. We analyzed the sequence of TCR Vbeta and Valpha genes and made clonotype-specific probes that allowed us to detect each clone in peripheral blood or biopsy specimens obtained during a 1-year period before and after the rapid onset of chronic rejection. We found an unexpectedly high level of donor HLA-specific T cell clonotype mRNA in peripheral blood during the late tolerance phase. Strong signals for two CD4+ clonotypes were detected in association with focal T cell infiltrates in the biopsy. Chronic rejection was associated with a reduction in direct pathway T cell clonotype mRNA in peripheral blood and the graft. Our data are inconsistent with the hypothesis that direct pathway T cells are involved only in early acute rejection events and suggest the possibility that some such T cells may contribute to the maintenance of peripheral tolerance to an allograft.     

Role of CD4+ and CD8+ T cells in allorecognition: lessons from corneal transplantation

Corneal transplantation represents an interesting model to investigate the contribution of direct vs indirect Ag recognition pathways to the alloresponse. Corneal allografts are naturally devoid of MHC class II+ APCs. In addition, minor Ag-mismatched corneal grafts are more readily rejected than their MHC-mismatched counterparts. Accordingly, it has been hypothesized that these transplants do not trigger direct T cell alloresponse, but that donor Ags are presented by host APCs, i.e., in an indirect fashion. Here, we have determined the Ag specificity, frequency, and phenotype of T cells activated through direct and indirect pathways in BALB/c mice transplanted orthotopically with fully allogeneic C57BL/6 corneas. In this combination, only 60% of the corneas are rejected, while the remainder enjoy indefinite graft survival. In rejecting mice the T cell response was mediated by two T cell subsets: 1) CD4+ T cells that recognize alloantigens exclusively through indirect pathway and secrete IL-2, and 2) IFN-gamma-producing CD8+ T cells recognizing donor MHC in a direct fashion. Surprisingly, CD8+ T cells activated directly were not required for graft rejection. In nonrejecting mice, no T cell responses were detected. Strikingly, peripheral sensitization to allogeneic MHC molecules in these mice induced acute rejection of corneal grafts. We conclude that only CD4+ T cells activated via indirect allorecognition have the ability to reject allogeneic corneal grafts. Although alloreactive CD8+ T cells are activated via the direct pathway, they are not fully competent and cannot contribute to the rejection unless they receive an additional signal provided by professional APCs in the periphery.     

TCR cross-reactivity and allorecognition: new insights into the immunogenetics of allorecognition

Alloreactive T cells are core mediators of graft rejection and are a potent barrier to transplantation tolerance. It was previously unclear how T cells educated in the recipient thymus could recognize allogeneic HLA molecules. Recently it was shown that both naïve and memory CD4⁺ and CD8⁺ T cells are frequently cross-reactive against allogeneic HLA molecules and that this allorecognition exhibits exquisite peptide and HLA specificity and is dependent on both public and private specificities of the T cell receptor. In this review we highlight new insights gained into the immunogenetics of allorecognition, with particular emphasis on how viral infection and vaccination may specifically activate allo-HLA reactive T cells. We also briefly discuss the potential for virus-specific T cell infusions to produce GvHD. The progress made in understanding the molecular basis of allograft rejection will hopefully be translated into improved allograft function and/or survival, and eventually tolerance induction.     

CD8 T cells are sufficient to mediate allorecognition and allograft rejection

Different T cell subsets may play different roles in allorecognition and allograft rejection. It has been suggested that CD8 T cells can only initiate rejection with help from CD4 T cells. Since CD8 T cells may have different requirements for allorecognition and for costimulation, it is important to clarify the role of CD8 cells in rejection. We examined the role of CD8 cells in allorecognition using a TCR transgenic mouse transplantation model. In our study, CD8 cells were able to recognize alloantigens and reject allografts in the absence of help from CD4 T cells. Furthermore our study provides a model to study the mechanisms of CD8-mediated allograft rejection. It may be important in the future, to consider that CD8 T cells may need to be targeted independently of CD4 T cells in strategies used to prevent rejection and induce tolerance.     

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.     

Tolerance induced by anti-CD3 immunotoxin plus 15-deoxyspergualin associates with donor-specific indirect pathway unresponsiveness

Peritransplant treatment with anti-CD3 immunotoxin plus deoxyspergualin induces tolerance to kidney allografts in most rhesus macaque recipients. Tolerant recipients maintain normal function for years without evidence of chronic rejection. Indirect alloantigen presentation is implicated in chronic rejection. Accordingly, we determined if anti-CD3 immunotoxin plus deoxyspergualin induced rejection-free tolerance associates with suppression of anti-donor indirect pathway responses. Tolerant recipients exhibited an early decrease in direct anti-donor responses with recovery to baseline levels by 3 years posttransplantation. In contrast, tolerant monkeys were unresponsive to donor antigens presented by the indirect pathway. Recipients that rejected their allografts retained vigorous direct and indirect anti-donor responses. Therefore, following temporary donor-specific hyporesponsiveness, direct responses recover in tolerant recipients >1.5 years after transplantation. However, tolerant recipients tested at 1.9-4 years posttransplant are specifically unresponsive to donor antigens presented by the indirect pathway. Thus, the rejection-free state of tolerant recipients may depend on mechanisms regulating indirect pathway responsiveness.     

Exosomes as a short-range mechanism to spread alloantigen between dendritic cells during T cell allorecognition

Exosomes are nanovesicles released by different cell types including dendritic cells (DCs). The fact that exosomes express surface MHC-peptide complexes suggests that they could function as Ag-presenting vesicles or as vehicles to spread allogeneic Ags for priming of anti-donor T cells during elicitation of graft rejection or induction/maintenance of transplant tolerance. We demonstrate that circulating exosomes transporting alloantigens are captured by splenic DCs of different lineages. Internalization of host-derived exosomes transporting allopeptides by splenic DCs leads to activation of anti-donor CD4 T cells by the indirect pathway of allorecognition, a phenomenon that requires DC-derived, instead of exosome-derived, MHC class II molecules. By contrast, allogeneic exosomes are unable to stimulate direct-pathway T cells in vivo. We demonstrate in mice that although graft-infiltrating leukocytes release exosomes ex vivo, they do not secrete enough concentrations of exosomes into circulation to stimulate donor-reactive T cells in secondary lymphoid organs. Instead, our findings indicate that migrating DCs (generated in vitro or isolated from allografts), once they home in the spleen, they transfer exosomes expressing the reporter marker GFP to spleen-resident DCs. Our results suggest that exchange of exosomes between DCs in lymphoid organs might constitute a potential mechanism by which passenger leukocytes transfer alloantigens to recipient's APCs and amplify generation of donor-reactive T cells following transplantation.     

Loss of direct and maintenance of indirect alloresponses in renal allograft recipients: implications for the pathogenesis of chronic allograft nephropathy.

Chronic allograft nephropathy (CAN) is the principal cause of late renal allograft failure. This complex process is multifactorial in origin, and there is good evidence for immune-mediated effects. The immune contribution to this process is directed by CD4(+) T cells, which can be activated by either direct or indirect pathways of allorecognition. For the first time, these pathways have been simultaneously compared in a cohort of 22 longstanding renal allograft recipients (13 with good function and nine with CAN). CD4(+) T cells from all patients reveal donor-specific hyporesponsiveness by the direct pathway according to proliferation or the secretion of the cytokines IL-2, IL-5, and IFN-gamma. Donor-specific cytotoxic T cell responses were also attenuated. In contrast, the frequencies of indirectly alloreactive cells were maintained, patients with CAN having significantly higher frequencies of CD4(+) T cells indirectly activated by allogeneic peptides when compared with controls with good allograft function. An extensive search for alloantibodies has revealed significant titers in only a minority of patients, both with and without CAN. In summary, this study demonstrates widespread donor-specific hyporesponsiveness in directly activated CD4(+) T cells derived from longstanding recipients of renal allografts, whether they have CAN or not. However, patients with CAN have significantly higher frequencies of CD4(+) T cells activated by donor Ags in an indirect manner, a phenomenon resembling split tolerance. These findings provide an insight into the pathogenesis of CAN and also have implications for the development of a clinical tolerance assay.     

A neural network model of basal ganglia’s decision-making circuitry

The basal ganglia have been increasingly recognized as an important structure involved in decision making. Neurons in the basal ganglia were found to reflect the evidence accumulation process during decision making. However, it is not well understood how the direct and indirect pathways of the basal ganglia work together for decision making. Here, we create a recurrent neural network model that is composed of the direct and indirect pathways and test it with the classic random dot motion discrimination task. The direct pathway drives the outputs, which are modulated through a gating mechanism controlled by the indirect pathway. We train the network to learn the task and find that the network reproduces the accuracy and reaction time patterns of previous animal studies. Units in the model exhibit ramping activities that reflect evidence accumulation. Finally, we simulate manipulations of the direct and indirect pathways and find that the manipulations of the direct pathway mainly affect the choice while the manipulations of the indirect pathway affect the model’s reaction time. These results suggest a potential circuitry mechanism of the basal ganglia’s role in decision making with predictions that can be tested experimentally in the future.     

Induction and comparison of different in vitro morphogenesis pathways using embryo of cumin (<Emphasis Type="Italic">Cuminum cyminum</Emphasis> L.) as a model material

In this study, using cumin embryo as explant and manipulating plant growth regulators (PGRs) in regeneration medium, the main in vitro morphogenesis pathways including direct shoot organogenesis, direct somatic embryogenesis, indirect somatic embryogenesis, and indirect shoot organogenesis were obtained. The effects of PGRs, subculture, and light on the induction and progression of different pathways were studied in detail. Direct shoot organogenesis occurred on the meristematic zone, while direct somatic embryogenesis was observed on hypocotyl part of cumin embryo (more differentiated part). Application of BAP (0.1 mgl⁻¹) was the sole triggering factor for induction of callus and indirect regeneration pathways. Exogenous IAA played the central role in the direct somatic embryogenesis pathway; however, the combined effects of IAA and NAA along with the high endogenous cytokinin level resulted in direct shoot organogenesis. Subculturing revealed accelerating effects on direct somatic embryogenesis pathway and callus formation. Conversely, subculturing had negative effect on direct shoot organogenesis pathway. In certain combinations of PGRs, like 0.4 mgl⁻¹ IAA + 0.4 mgl⁻¹ NAA, co-induction and co-regeneration of different pathways were observed. Investigation of genotype dependencies of different pathways showed that direct pathways are more genotype-dependent, stable, and faster than indirect pathways. This research presents the embryo of cumin as a convenient model material for induction and comparison of different morphogenesis pathways.     

Recipient MHC class II expression is required to achieve long-term survival of murine cardiac allografts after costimulatory blockade.

To study the role of the direct and indirect pathways in achieving tolerance, we used genetically altered mouse strains in two ways: 1) MHC class II-deficient mice were used as donors of skin and cardiac grafts to eliminate the direct CD4(+) T cell response, and 2) B6 II(-)4(+) mice, which are MHC class II-deficient mice expressing an MHC class II transgene only on thymic epithelium, were used as recipients of normal grafts. These mice cannot mount an indirect response. Eliminating the indirect pathway actually made it more difficult to achieve prolonged allograft survival when we used costimulatory blockade than when both pathways were available. Costimulatory blockade was ineffective even when CD4(+) T cells from normal animals were transferred into recipients that lacked MHC class II molecules. These results suggest that an active CD4(+) response through the indirect pathway is necessary for costimulatory blockade to be effective in prolonging allograft survival.     

In vivo helper functions of alloreactive memory CD4+ T cells remain intact despite donor-specific transfusion and anti-CD40 ligand therapy

Memory T cells have specific properties that are beneficial for rapid and efficient protection from pathogens previously encountered by a host. These same features of memory T cells may be deleterious in the context of a transplanted organ. Consistent with this contention is the accumulating evidence in experimental transplantation that previously sensitized animals are resistant to the effects of costimulatory blockade. Using a model of murine cardiac transplantation, we now demonstrate that alloreactive memory CD4(+) T cells prevent long-term allograft survival induced through donor-specific cell transfusion in combination with anti-CD40 ligand Ab (DST/anti-CD40L). We show that memory donor-reactive CD4(+) T cells responding through the direct or indirect pathways of allorecognition provide help for the induction of antidonor CD8(+) T effector cells and for Ab isotype switching, despite DST/anti-CD40L. The induced pathogenic antidonor immunity functions in multiple ways to subsequently mediate graft destruction. Our findings show that the varied functions of alloreactive memory CD4(+) T cells remain intact despite DST/anti-CD40L-based costimulatory blockade, a finding that will likely have important implications for designing approaches to induce tolerance in human transplant recipients.     

Germinal center alloantibody responses are mediated exclusively by indirect-pathway CD4 T follicular helper cells

The durable alloantibody responses that develop in organ transplant patients indicate long-lived plasma cell output from T-dependent germinal centers (GCs), but which of the two pathways of CD4 T cell allorecognition is responsible for generating allospecific T follicular helper cells remains unclear. This was addressed by reconstituting T cell-deficient mice with monoclonal populations of TCR-transgenic CD4 T cells that recognized alloantigen only as conformationally intact protein (direct pathway) or only as self-restricted allopeptide (indirect pathway) and then assessing the alloantibody response to a heart graft. Recipients reconstituted with indirect-pathway CD4 T cells developed long-lasting IgG alloantibody responses, with splenic GCs and allospecific bone marrow plasma cells readily detectable 50 d after heart transplantation. Differentiation of the transferred CD4 T cells into T follicular helper cells was confirmed by follicular localization and by acquisition of signature phenotype. In contrast, IgG alloantibody was not detectable in recipient mice reconstituted with direct-pathway CD4 T cells. Neither prolongation of the response by preventing NK cell killing of donor dendritic cells nor prior immunization to develop CD4 T cell memory altered the inability of the direct pathway to provide allospecific B cell help. CD4 T cell help for GC alloantibody responses is provided exclusively via the indirect-allorecognition pathway.     

Allopeptides and the alloimmune response

The inherent ability of the host immune system to distinguish between self- and non-self forms the basis of allorecognition. T lymphocytes constitute the most important effector arm of allorecognition. Here we describe the fundamentals of direct and indirect pathways by which allopeptides are presented to effector T cells. The nature of allopeptides presented along with tolerogenic strategies like altered peptide ligands and intra- or extra-thymic allopeptide inoculation are discussed. In addition, we speculate on the potential of regulatory T cells to modulate alloimmune responses.     

Differential susceptibility of allogeneic targets to indirect CD4 immunity generates split tolerance

CD4 T cells frequently help to activate CD8 T and B cells that effect transplant rejection. However, CD4 T cells alone can reject transplants, either directly or indirectly. The relative effectiveness of indirect CD4 immunity in rejecting different types of allogeneic grafts is unknown. To address this, we used a TCR transgenic mouse model in which indirect CD4 alloimmunity alone can be studied. We challenged transgenic recipients with hematopoietic cells and shortly thereafter skin transplants that could only be rejected indirectly, and observed Ag-specific indirect donor B cell and skin rejection, but not T cell elimination, reflecting a state of split tolerance. Deficiency of indirect CD4 alloimmunity in donor T cell rejection was also apparent when acute indirect rejection of donor islets occurred despite generation and maintenance of mixed T cell chimerism, due to migration of the few passenger T cells into recipient circulation. Although passenger lymphocytes delayed indirect islet rejection, they enhanced rejection by a full repertoire capable of both direct and indirect reactivity. Interestingly, the persistence of chimerism was associated with the eventual development of tolerance, as demonstrated by acceptance of donor skin grafts given late to hematopoietic cell recipients, and hyporesponsiveness of transgenic T cells from islet recipients in vitro. Mechanistically, tolerance was recessive and associated with progressive down-regulation of CD4. Collectively, our data indicate that indirect CD4 immunity is not equally destructive toward different types of allogeneic grafts, the deficiency of which generates split tolerance. The futility of these responses can convert immunity into tolerance.     

CD4 T cell-mediated alloresistance to fully MHC-mismatched allogeneic bone marrow engraftment is dependent on CD40-CD40 ligand interactions, and lasting T cell tolerance is induced by bone marrow transplantation with initial blockade of this pathway

Costimulatory blockade can be used to promote allogeneic marrow engraftment and tolerance induction, but on its own is not 100% reliable. We sought to determine whether one or the other of the CD4 or CD8 T cell subsets of the recipient was primarily responsible for resistance to allogeneic marrow engraftment in mice receiving costimulatory blockade, and to use this information to develop a more reliable, minimal conditioning regimen for induction of mixed chimerism and transplantation tolerance. We demonstrate that a single anti-CD40 ligand mAb treatment is sufficient to completely overcome CD4 cell-mediated resistance to allogeneic marrow engraftment and rapidly induce CD4 cell tolerance, but does not reliably overcome CD8 CTL-mediated alloresistance. The data suggest that costimulation, which activates alloreactive CTL, is insufficient to activate alloreactive CD4 cells when the CD40 pathway is blocked. The addition of host CD8 T cell depletion to anti-CD40 ligand treatment reliably allows the induction of mixed chimerism and donor-specific skin graft tolerance in 3 Gy-irradiated mice receiving fully MHC-mismatched bone marrow grafts. Thus, despite the existence of multiple costimulatory pathways and pathways of APC activation, our studies demonstrate an absolute dependence on CD40-mediated events for CD4 cell-mediated rejection of allogeneic marrow. Exposure to donor bone marrow allows rapid tolerization of alloreactive CD4 cells when the CD40 pathway is blocked, leading to permanent marrow engraftment and intrathymic tolerization of T cells that develop subsequently.     

Epitope mapping of the indirect T cell response to allogeneic class I MHC: sequences shared by donor and recipient MHC may prime T cells that provide help for alloantibody production

Indirect allorecognition occurs when T cells recognize donor MHC presented as peptide epitopes by recipient APC, but the precise nature of the epitopes involved remains unclear. Rejection of rat MHC class I-disparate PVG.R8 (RT1.A(a)) grafts by PVG.RT1(u) (RT1.A(u)) recipients is mediated by indirectly restricted CD4 T cells that provide help for the generation of alloantibody. In this study, epitope mapping was performed using a functionally relevant readout (alloantibody production) to identify key peptides that prime an indirect alloimmune response, leading to graft rejection. PVG.RT1(u) rats were immunized with a series of overlapping 15-mer peptides (peptides 1-18) that spanned the alpha1 and alpha2 domains of the RT1.A(a) molecule. Several peptides were able to accelerate both the alloantibody response to the intact RT1.A(a) Ag and PVG.R8 heart graft rejection. An immunodominant epitope was identified within the hypervariable region of the alpha1 domain. Fine mapping of this region with a second series of peptides overlapping by single amino acids confirmed the presence of an eight-amino acid core determinant. Additional "subdominant" epitopes were identified, two of which were located within regions of amino acid homology between the RT1.A(a) and RT1.A(u) molecules and not, as had been expected, within other hypervariable regions. The contribution of self-epitopes to indirect allorecognition was emphasized by the demonstration that i.v. administration of a 15-mer peptide encompassing one of the subdominant self-determinants diminished the recipient's ability to mount an alloantibody response on challenge with intact A(a) alloantigen. Our findings suggest that cryptic self-epitopes recognized by autoreactive T cells may contribute to allograft rejection and should be considered when designing novel strategies for inducing tolerance to alloantigen.     

Embryonic thymic epithelium naturally devoid of APCs is acutely rejected in the absence of indirect recognition.

Transplants of tissues depleted of passenger leukocytes are upon in vitro culture usually accepted in allogeneic recipients. Accordingly, fully allogeneic embryonic thymic epithelium was suggested to be poorly immunogenic. However, this tissue is capable of inducing donor-specific tolerance to peripheral tissues, when restoring T cell development in nude mice, through the production of regulatory cells. In the present work, adult immunocompetent allogeneic recipients were grafted with embryonic tissues isolated at stages before hemopoietic colonization or even before the establishment of circulation. Allogeneic thymic epithelium of day 10 embryos and heart primordium of day 8 embryonic donors were always rejected. Acute rejection of the thymic anlagen takes place in less than 12 days, with maximal CD4(+) and CD8(+) T cell infiltrates at 10 days post-transplant. In addition, a significant infiltrate of NK1.1(+) cells is observed, although without any essential role in this process. Furthermore, recipients lacking the indirect pathway of Ag presentation to CD4(+) T cells do not reveal any significant delay in rejection, even when CD8(+) T cells are also eliminated. Thus, our experimental approach reveals acute allograft rejection in the absence of all known pathways of naive T cell activation and therefore unveils a novel graft rejection mechanism that should be mediated by direct recognition of parenchymal cells. Given the importance of dendritic cells in naive T cell activation, it is likely that cross-reactive memory T cells may also drive rejection.