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.     

Human CD4+CD25low adaptive T regulatory cells suppress delayed-type hypersensitivity during transplant tolerance

Adaptive T regulatory (T(R)) cells mediate the suppression of donor-specific, delayed-type hypersensitivity (DTH) in tolerant organ transplant recipients. We hypothesized that cells belonging to the CD4(+)CD25(+) T cell subset but distinct from natural T(R) cells may fulfill this role. To test this hypothesis, PBMC and biopsy samples from two tolerant kidney transplant recipients (K1 and K2) were analyzed. When transferred with recipient APC into a SCID mouse footpad, CD4(+) T cells were hyporesponsive in DTH to donor type HLA-B Ags and derivative allopeptides. However, anti-human TGF-beta1 Ab revealed a response to immunodominant allopeptides in both patients, suggesting that CD4(+) T effector (T(E)) cells coexisted with suppressive, TGF-beta1-producing CD4(+) T(R) cells. During in vitro culture, allopeptide stimulation induced both IFN-gamma-producing and surface TGF-beta1(+) T cells. The relative strength of the latter response in patient K1 was inversely correlated with the level of systemic anti-donor DTH, which varied over a 6-year interval. Allopeptide-induced surface TGF-beta1 expression was found primarily in Forkhead box P3 (FoxP3)-negative CD4(+)CD25(low) T cells, which could adoptively transfer suppression of donor-specific DTH. Biopsy samples contained numerous surface TGF-beta1(+) mononuclear cells that costained for CD4 and, less frequently CD25, but were negative for FoxP3. The CD4(+)TGF-beta1(+) T cells were localized primarily to the tubulointerstitium, whereas TGF-beta1(-)FoxP3(+)CD25(+) cells were found mainly in lymphoid aggregates. Thus, adaptive T(R) cells suppressing T(E) cell responses to donor allopeptides in two tolerant patients appear to be functionally and phenotypically distinct from CD4(+)CD25(high)FoxP3(+) T cells.     

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.     

Investigation of peptide involvement in T cell allorecognition using recombinant HLA class I multimers

Alloreactive T cells are involved in injurious graft rejection and graft-vs-host disease. However, they can also evoke beneficial responses to tumor Ags restricted by foreign MHC molecules. Manipulation of these alloreactivities requires information on the basis of T cell allorecognition. The vigorous T cell response to foreign MHC molecules may arise from peptide-independent recognition of polymorphic residues of foreign MHC molecules or peptide-specific recognition of novel peptides presented by foreign MHC molecules. We investigated CD8+ T cell allorecognition using recombinant HLA class I/peptide complexes. Peptide-specific allorecognition was examined using tetramers of HLA-A*0201 representing five peptides derived from ubiquitously expressed self-proteins that are known to bind endogenously to HLA-A*0201. Distinct subsets of CD8+ T cells specific for each HLA-A*0201/peptide combination were detected within four in vitro-stimulated T cell populations specific for foreign HLA-A*0201. Peptide-independent allorecognition was investigated using artificial Ag-presenting constructs (aAPCs) coated with CD54, CD80, and functional densities of a single HLA-A*0201/peptide combination for four different peptides. None of the four T cell populations specific for foreign HLA-A*0201 were stimulated by the aAPCs, whereas they did produce IFN-gamma upon stimulation with cells naturally expressing HLA-A*0201. Thus, aAPCs did not stimulate putative peptide-independent allorestricted T cells. The results show that these alloreactive populations comprise subsets of T cells, each specific for a self-peptide presented by foreign class I molecules, with no evidence of peptide-independent components.     

Mechanisms of T cell receptor antagonism: implications in the treatment of disease

The adaptive immune response is often required for the successful clearing of infectious pathogens. Antigen presenting cells (APC) present peptide antigens derived from pathogens to T cells via major histocompatibility complex (MHC) molecules. T cells then become activated and differentiate into effector cells with the capacity to kill infected cells or to induce an anti-pathogen antibody response. In autoimmunity, this T cell response is directed against self-antigens and often leads to deleterious effects on specific tissues. Likewise, T cell responses to allogeneic MHC molecules in transplants also leads to pathology. By introducing subtle changes in the antigenic peptide amino acid content, T cell activation can be inhibited, thereby preventing T cell effector functions. This strategy of TCR antagonism has been used successfully in vitro and in vivo to inhibit models of autoimmunity and allorecognition. In addition, a variety of pathogens that often result in chronic disease following infection, also have seemingly evolved natural mechanisms to inhibit T cell responses by antagonism. These microorganisms express natural variants of certain proteins, that when presented to T cells have the capacity to specifically inhibit T cell responses by functioning as antagonists or by modulating the nature of the T cell response. The understanding of how pathogens mediate this inhibition in vivo will be beneficial to ongoing studies in both autoimmunity and transplantation aimed at suppressing the harmful immune response, thereby controlling disease. TCR antagonism seems to have the potential to be used therapeutically to prevent or inhibit an undesired T cell response that will ultimately lead to disease.     

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.     

Non-hematopoietic allograft cells directly activate CD8+ T cells and trigger acute rejection: an alternative mechanism of allorecognition

Despite evidence that human non-hematopoietic cells, such as vascular endothelium, can activate allogeneic T lymphocytes in vitro, the prevailing view has been that hematopoietic antigen-presenting cells are required to trigger alloimmune responses in vivo. Here we report that mouse non-hematopoietic cells activate alloreactive CD8+ T lymphocytes in vitro and in vivo. We also show that vascularized cardiac allografts are acutely rejected via CD8+ direct allorecognition even if the alloantigen is not presented by hematopoietic professional antigen-presenting cells. Because activation of alloreactive CD8+ T cells by donor-type non-hematopoietic cells can continue for the life of the allograft, these findings present a new clinically relevant mechanism of allorecognition and should be taken into consideration when developing strategies to prevent allograft vasculopathy or to induce tolerance.     

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.     

Mouse vascular endothelium activates CD8+ T lymphocytes in a B7-dependent fashion

Despite several studies examining the contribution of allorecognition pathways to acute and chronic rejection of vascularized murine allografts, little data describing activation of alloreactive T cells by mouse vascular endothelium exist. We have used primary cultures of resting or IFN-gamma-activated C57BL/6 (H-2(b)) vascular endothelial cells as stimulators and CD8(+) T lymphocytes isolated from CBA/J (H-2(k)) mice as responders. Resting endothelium expressed low levels of MHC class I, which was markedly up-regulated after activation with IFN-gamma. It also expressed moderate levels of CD80 at a resting state and after activation. Both resting and activated endothelium were able to induce proliferation of unprimed CD8(+) T lymphocytes, with proliferation noted at earlier time points after coculture with activated endothelium. Activated endothelium was also able to induce proliferation of CD44(low) naive CD8(+) T lymphocytes. Activated CD8(+) T lymphocytes had the ability to produce IFN-gamma and IL-2, acquired an effector phenotype, and showed up-regulation of the antiapoptotic protein Bcl-x(L). Treatment with CTLA4-Ig led to marked reduction of T cell proliferation and a decrease in expression of Bcl-x(L). Moreover, we demonstrate that nonhemopoietic cells such as vascular endothelium induce proliferation of CD8(+) T lymphocytes in a B7-dependent fashion in vivo. These results suggest that vascular endothelium can act as an APC for CD8(+) direct allorecognition and may, therefore, play an important role in regulating immune processes of allograft rejection.     

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.     

I-Ep-bound self-peptides: identification, characterization, and role in alloreactivity

T cell recognition of peptide/allogeneic MHC complexes is a major cause of transplant rejection. Both the presented self-peptides and the MHC molecules are involved; however, the molecular basis for alloreactivity and the contribution of self-peptides are still poorly defined. The murine 2.102 T cell is specific for hemoglobin(64-76)/I-Ek and is alloreactive to I-Ep. The natural self-peptide/I-Ep complex recognized by 2.102 remains unknown. In this study, we characterized the peptides that are naturally processed and presented by I-Ep and used this information to define the binding motif for the murine I-Ep class II molecule. Interestingly, we found that the P9 anchor residue preferred by I-Ep is quite distinct from the residues preferred by other I-E molecules, although the P1 anchor residue is conserved. A degree of specificity for the alloresponse was shown by the lack of stimulation of 2.102 T cells by 19 different identified self-peptides. The binding motif was used to search the mouse genome for candidate 2.102 reactive allopeptides that contain strong P1 and P9 anchor residues and possess previously identified allowable TCR contact residues. Two potential allopeptides were identified, but only one of these peptides, G protein-coupled receptor 128, was able to stimulate 2.102 T cells. Thus, the G protein-coupled receptor 128 peptide represents a candidate allopeptide that is specifically recognized by 2.102 T cells bound to I-Ep and was identified using bioinformatics. These studies highlight the specific involvement of self-peptides in alloreactivity.     

Colonial allorecognition,hemolytic rejection,and viviparity in botryllid ascidians

Allorecognition is a fundamental system that animals use to maintain individuality. Although embryos are usually semiallogeneic with their mother, viviparous animals are required to allow these embryos to develop inside the maternal body, but must also eliminate an "invasion" by nonself. In colonial ascidians of the family Botryllidae, when two colonies are brought into contact at their growing edges, a hemolytic rejection reaction occurs between allogeneic colonies. Morula cells, a type of hemocyte, are the major effector cells in the hemolytic rejection. Morula cells infiltrate and aggregate where the two colonies make contact, and then discharge their vacuolar contents, which contain phenoloxidase and quinones. In viviparous botryllids, colonial contact at artificially cut surfaces always results in colonial fusion and establishment of a common vascular network even between allogeneic colonies in which the growing-edge contact results in rejection. This colonial fusion between incompatible colonies (surgical fusion) suggests that the allorecognition sites are not distributed in the vascular system in which the embryos are brooded. It is supposed that a common ancestor of the viviparous species lost the capacity for allorecognition in their vascular system to protect its embryos from alloreactivity, when it changed from ovoviviparous to viviparous in the course of evolution. The limited distribution of allorecognition sites would be a solution to the embryo-parent histoincompatibility in viviparity.     

Signal transduction via the T cell antigen receptor in naïve and effector/memory T cells

T cells play an indispensable role in immune defense against infectious agents, but can also be pathogenic. These T cells develop in the thymus, are exported into the periphery as naïve cells and participate in immune responses. Upon recognition of antigen, they are activated and differentiate into effector and memory T cells. While effector T cells carry out the function of the immune response, memory T cells can last up to the life time of the individual, and are activated by subsequent antigenic exposure. Throughout this life cycle, the T cell uses the same receptor for antigen, the T cell Receptor, a complex multi-subunit receptor. Recognition of antigen presented by peptide/MHC complexes on antigen presenting cells unleashes signaling pathways that control T cell activation at each stage. In this review, we discuss the signals regulated by the T cell receptor in naïve and effector/memory T cells.     

Innate immunity together with duration of antigen persistence regulate effector T cell induction

Proliferation of T cells is important for the expansion of specific T cell clones during immune responses. In addition, for the establishment of protective immunity against viruses, bacteria, and tumors, the expanded T cells must differentiate into effector T cells. Here we show that effector T cell generation is driven by activation of APCs and duration of antigenic stimulation. Adoptively transferred TCR-transgenic T cells extensively proliferated upon immunization. However, these T cells failed to differentiate into effector cells and died within 1 wk after immunization unless antigenic peptides persisted for >1 day or were presented by activated APCs. The induction of protective immunity in a nontransgenic system was more stringent, since activation of APCs or prolonged Ag persistence alone was not sufficient to drive immunity. In contrast, Ag had to be presented for several days by activated APCs to trigger protective T cell responses. Thus, activation of APCs and duration of Ag presentation together regulate the induction of protective T cell responses.     

Differential helper and effector responses of Lyt-2+ T cells to H-2Kb mutant (Kbm) determinants and the appearance of thymic influence on anti-Kbm CTL responsiveness

The goal of this study was to assess and compare the allorecognition requirements for eliciting Lyt-2+ helper and effector functions from primary T cell populations. By using interleukin 2 (IL 2) secretion as a measure of T helper (Th) function, and cytolytic T lymphocyte (CTL) generation as a measure of effector function, this study compared the responses of Lyt-2+ T cells from wild-type B6 mice against a series of H-2Kb mutant determinants. Although all Kbm determinants stimulated B6 Lyt-2+ T cells to become cytolytic effector cells, the various Kbm determinants differed dramatically in their ability to stimulate Lyt-2+ T cells to function as IL 2-secreting helper cells. For example, in contrast to Kbm1 determinants that stimulated both helper and effector functions, Kbm6 determinants only stimulated B6 Lyt-2+ T cells to become cytolytic and failed to stimulate them to secrete IL 2. The distinct functional responses of Lyt-2+ T cells to Kbm6 determinants was documented by precursor frequency determinations, and was not due to an inability of the Kbm6 molecule to stimulate Lyt-2+ Th cells to secrete IL 2. Rather, it was the specific recognition and response of Lyt-2+ T cells to novel mutant epitopes on the Kbm6 molecule that was defective, such that anti-Kbm6 Lyt-2+ T cells only functioned as CTL effectors and did not function as IL 2-secreting Th cells. The failure of Lyt-2+ anti-Kbm6 T cells to function as IL 2-secreting Th cells was a characteristic of all Lyt-2+ T cell populations examined in which the response to novel mutant epitopes could be distinguished from the response to other epitopes expressed on the Kbm6 molecule. The absence of significant numbers of anti-Kbm6 Th cells in Lyt-2+ T cell populations was examined for its functional consequences on anti-Kbm6 CTL responsiveness. It was found that primary anti-Kbm6 CTL responses could be readily generated in vitro, but unlike responses to most class I alloantigens that can be mediated by Lyt-2+ Th cells, anti-Kbm6 CTL responses were strictly dependent upon self-Ia-restricted L3T4+ Th cells. Because the restriction specificity of L3T4+ Th cells is determined by the thymus, in which their precursors had differentiated, anti-Kbm6 CTL responsiveness, unlike responsiveness to most class I alloantigens, was significantly influenced by the Ia phenotype of the thymus in which the responder cells had differentiated.(ABSTRACT TRUNCATED AT 400 WORDS)     

Innate immune cells contribute to the IFN-gamma-dependent regulation of antigen-specific CD8+ T cell homeostasis

IFN-gamma has a dual function in the regulation of T cell homeostasis. It promotes the expansion of effector T cells and simultaneously programs their contraction. The cellular mechanisms leading to this functional dichotomy of IFN-gamma have not been identified to date. In this study we show: 1) that expansion of wild-type CD8+ T cells is defective in IFN-gamma-deficient mice but increased in IFN-gammaR-deficient mice; and 2) that contraction of the effector CD8+ T cell pool is impaired in both mouse strains. Furthermore, we show that CD11b+ cells responding to IFN-gamma are sufficient to limit CD8+ T cell expansion and promote contraction. The data presented here reveal that IFN-gamma directly promotes CD8+ T cell expansion and simultaneously induces suppressive functions in CD11b+ cells that counter-regulate CD8+ T cell expansion, promote contraction, and limit memory formation. Thus, innate immune cells contribute to the IFN-gamma-dependent regulation of Ag-specific CD8+ T cell homeostasis.     

Modelling T-cell-mediated suppression dependent on interactions in multicellular conjugates

Tolerance to peripheral body antigens involves multiple mechanisms, namely T-cell-mediated suppression of potentially autoimmune cells. Recent in vivo and in vitro evidence indicates that regulatory T cells suppress the response of effector T cells by a mechanism that requires the simultaneous conjugation of regulatory and effector T cells with the same antigen-presenting cell (APC). Despite this strong requirement, it is not yet clear what happens while both cells are conjugated. Several hypotheses are discussed in the literature. Suppression may result from simple competition of regulatory and effector cells for activation resources on the APC; regulatory T cells may deliver an inhibitory signal to effector T cells in the same conjugate; or effector T cells may acquire the regulatory phenotype during their interaction with regulatory T cells. The present article tries to further our understanding of T-cell-mediated suppression, and to narrow-down the number of candidate mechanisms. We propose the first general formalism describing the formation of multicellular conjugates of T cells and APCs. Using this formalism we derive three particular models, representing alternative mechanisms of T-cell-mediated suppression. For each model, we make phase plane and bifurcation analysis, and identify their pros and cons in terms of the relationship with the large body of experimental observations on T-cell-mediated suppression. We argue that accounting for the quantitative details of adoptive transfers of tolerance requires models with bistable regimes in which either regulatory cells or effectors cells dominate the steady state. From this analysis, we conclude that the most plausible mechanism of T-cell-mediated suppression requires that regulatory T cells actively inhibit the growth of effector T cells, and that the maintenance of the population of regulatory T cells is dependent on the effector T cells. The regulatory T cell population may depend on a growth factor produced by effector T cells and/or on a continuous differentiation of effector cells to the regulatory phenotype.     

Variant class II molecules from H-2 haplotypes in wild mouse populations: functional characteristics of closely related class II gene products

Independently derived haplotypes found in wild populations of mice often express class II molecules antigenically related to specific alleles of the A molecules defined in laboratory mice. Tryptic peptide fingerprint comparisons of these antigenically related molecules indicate that they have similar, or possibly identical, primary structures in the A alpha, A beta, or both subunits. By using the Ak and Ap families of independently derived, antigenically related A molecules, we examined the effect that minor structural variations in the A molecule have on allorecognition by T lymphocytes. Data obtained indicate that a) minor structural variations in the A molecule can effect, although not always, major functional changes in allorecognition, b) changes in allorecognition are always detected when the A beta subunit contains structural variations, but not necessarily when the A alpha subunit contains structural variations, and c) more than one site in the A molecule can be recognized by alloreactive T lymphocytes. These results can be interpreted as indicating that specific sites within the A molecule are critically involved in allorecognition and that structural variations must affect these sites to elicit major changes in allorecognition.     

The allorecognition of H-2Kb-specific CD4-CD8- T cell hybridomas is influenced by the substitution at residue 256 of MHC class I molecules

Our previous studies demonstrated that allorecognition of HTB176.10 and HTB177.2, H-2Kb-reactive CD4-CD8- T cell hybridomas is markedly influenced by the exchange of the alpha 3 domain between H-2Kb and H-2Dp. The recombinant genes of the exon 4 between H-2Kb and H-2Dp were constructed to determine the residues of the alpha 3 domain that influence the allorecognition of these T cell hybridomas. Seven recombinant genes of the exon 4 were generated by in vivo recombination in Escherichia coli. Chimeric genes containing these recombinants were transfected into L cells and the transfectants expressing equivalent amounts of chimeric molecules were selected by flow cytometry. Studies on responses of these T cell hybridomas to the chimeric molecules confirmed our previous observation that the primary structure of the alpha 3 domain influences the allorecognition by the hybridomas. Moreover, it was indicated that residue 256 on the alpha 3 domain markedly affects the allorecognition by the T cell hybridomas, although substitutions at residues 184, 193, 195, 197, 262, and 264 exerted some effects on the T cell recognition. Further studies with the use of a single amino acid mutant of H-2Kb at residue 256 confirmed the effect of substitution at residue 256 on allorecognition of the T cell hybridomas. Taken together, results of this study demonstrated that polymorphism of the alpha 3 domain is indeed involved in the formation of allodeterminants recognized by TCR.