Following syngeneic or autologous stem cell transplantation, a syndrome closely resembling allogeneic graft versus host disease (GvHD) occurs in 5% to 20% of recipients,1,2 although there are no genetic differences between the cell therapy product and the host tissues. In a cohort of patients receiving transplants from an identical twin to treat aplastic anemia, GvHD-like features only developed among those recipients treated with pretransplant conditioning.3 Moreover, in allogeneic stem cell transplantation, host resident memory T cells persist in peripheral tissues even after myeloablative conditioning and contribute to the pathogenesis of GvHD.4 These findings imply that the inflammatory response consequent upon pretransplant conditioning can trigger T-cell autoreactivity. Among the factors that have been suggested to contribute to this phenomenon are destruction with inadequate reconstitution of T regulatory cells (Treg) in the peritransplant period, and disruption of the gut microbiome with reduced diversity and impaired production of short-chain fatty acids (reviewed in Rafei and Jenq5). Here, we propose an additional mechanism to account for these findings.
Differences in minor histocompatibility antigens (MiHA) between the donor and recipient of hematopoietic stem cell transplants are well-recognized precipitants of GvHD in the setting of HLA-matched sibling and unrelated donation. MiHA arise through genetically encoded polymorphism in the source proteins of the endogenous peptide antigens that are presented by donor and recipient HLA. The capacity of MiHA to precipitate GvHD reflects the sensitivity of the donor immune cell precursors toward a small proportion of peptide antigens. By analogy, we hypothesize that pretransplant conditioning can trigger donor T-cell reactivity against the host through the generation of both conventional and unconventional peptide epitopes that are not present in the thymus during T-cell development. Autoreactive T cells able to recognize this “induced/altered self” are not removed by negative selection6 and persist within the mature T-cell repertoires of both the donor and recipient. Observations made in the context of autologous or syngeneic transplantation are also relevant to matched and haploidentical stem cell transplantation and solid organ transplantation in which matched and allogeneic HLA are often coexpressed by the donor organ. In these settings, autoreactive T cells could synergize with T cells responding to MiHA and with those directly or indirectly recognizing allogeneic major histocompatibility complex (MHC) to augment damage to target tissues. Both memory T cells and previously naive T cells could contribute to this phenomenon. In addition to self-restricted self-peptides and indirectly presented peptides processed from allogeneic donor MHC, self-peptides directly presented by donor MHC or MiHA could also be subject to induction/modification but, although they may influence the alloresponse, they would not be targets for autoreactivity.
Transplantable organs are subjected to many sources of cellular stress (Figure 1). Donor brain death is accompanied by a catecholamine storm, erratic perfusion of peripheral tissues, and a surge in proinflammatory cytokines and chemokines.7 Circulatory death exposes donor organs to an extended period of warm ischemia,8 whereas some degree of ischemia-reperfusion injury affects all solid organ transplants. Recipient preparation for hematopoietic stem cell transplantation typically involves both irradiation and administration of cytoreductive chemotherapy. The early stages of alloreactive T-cell responses, whether directed toward the graft or the host, are marked by the release of cytokines and other soluble mediators that act upon the target tissues. These factors may alter tissue proteomes and immunopeptidomes in various ways. Among these are the generation of proteasome-spliced, or “hybrid” peptides,9 and immunoproteasome-dependent peptides, modification of the amino acid side chains of peptides or their source proteins,10-13 and the utilization of noncanonical start codons and alternative open reading frames (ORFs)14,15 for protein translation, in parallel with the standard templates. The resulting “dark proteome” yields novel peptide epitopes termed “cryptic” because standard analysis pipelines do not reveal them.16 Beyond directly altering the interacting surfaces of the T-cell receptor-peptide-MHC (pMHC), posttranslational modification (PTM) of proteins can perturb classical antigen processing pathways, exposing epitopes that are normally degraded before MHC loading. An increased density of previously rare epitopes may be sufficient to stimulate weakly reactive T cells past the threshold of activation.17,18
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FIGURE 1.: Factors promoting the generation of induced/altered self-peptides in transplantation. Ischemia-reperfusion injury, pretransplant conditioning with cytoreductive agents, and irradiation and exposure to cytokines all contribute to oxidative stress. Cytokines such as IFN-γ increase the expression of immunoproteasome-dependent peptides, plus spliced or hybrid peptides generated in the proteasome, whereas PTMs of amino acid residues result from specific enzyme activity or exposure to a chemically reactive milieu or can be spontaneous. Oxidative stress increases the usage of noncanonical start codons (CUG, UUG), and proteins translated from these alternative ORFs give rise to cryptic peptides. Both native and altered self-peptides presented by allogeneic MHC are recognized by alloreactive T cells, whereas presentation of altered-self by donor–recipient matched MHC may give rise to de novo autoreactivity. IFN-γ, interferon gamma; MHC, major histocompatibility complex; ORF, open reading frame; PTM, posttranslational modification.
Exposure of cells to interferon gamma (IFN-γ) increases the diversity of the peptide antigens presented by MHC molecules (ie, the immunopeptidome), substantially augmenting presentation of both conventional linear and proteasome-spliced peptides.9 IFN-γ upregulates expression of numerous potential source proteins and induces transcription of the specialized immunoproteasome subunits β1i, β2i, and β5i.19 Immunoproteasomes differ in proteolytic activity from constitutive proteasomes, giving rise to more hydrophobic peptides,19 some of which are exclusively produced by the immunoproteasome. Although medullary thymic epithelial cells express both constitutive proteasomes and immunoproteasomes,20 participation of immunoproteasome-dependent self-peptides in negative selection may be limited. Immunoproteasome-dependent antigens have been implicated in the development of autoimmunity,21 whereas selective inhibition of the b5i subunit blocks development of diabetes in a model in which an immunoproteasome-dependent epitope is expressed in pancreatic islets.22 Consistent with immunoproteasome activity, conventional self-peptides that do not cause negative selection in the thymus have been detected in cells treated with IFN and in rejecting grafts, where they were presented by syngeneic MHC.23 Proteasome-mediated peptide splicing involves the joining of 2 noncontiguous peptide segments, either from the same protein (cis-splicing) or from different proteins (trans-splicing), resulting in novel, nongenomically templated peptide antigens.24
PTM of amino acids can occur spontaneously, through exposure to reactive oxygen species, or via the action of enzymes such as acetyl or methyltransferases, kinases, ubiquitin ligases, glycosyltransferases, and tissue transglutaminase 2.17 Ischemia-reperfusion injury induces the expression of peptidyl-arginine deiminases, which convert arginine residues to citrulline,25 whereas protein cysteinylation (disulfide bond-formation with free cysteine) occurs as a consequence of acute inflammation.26 Irradiation of tumors increases neoantigen presentation and subsequently broadens the repertoire of responding T cells,27,28 suggesting that irradiation of normal tissues may also give rise to neoepitopes. Finally, oxidative stress is associated with dysregulation of protein translation14,15 promoting presentation of cryptic peptides.
The immunogenicity of PTM, spliced, and cryptic peptide neoepitopes is increasingly well documented. Deamidation of asparagine yields both aspartyl and isoaspartyl residues. Isoaspartyl modifications are linked to increased immunogenicity of self, tumor, and viral vector capsid antigens.29-31 Acetylation and citrullination feature prominently in the pathogenesis of autoimmune disorders,18,32,33 whereas cysteinylation and disulfide bond rearrangements have been implicated in both alloreactivity and autoreactivity.34,35 Tumor antigens derived from alternative ORFs elicit CD8+ T-cell responses,36,37 and an H-2Kb-restricted self-peptide derived from an upstream ORF of α-tubulin is recognized by alloreactive T cells.38 Immunogenic spliced peptides have been isolated from human renal cancer,39 melanoma cells,40 acute myeloid leukemia,41 and murine cells infected with Listeria monocytogenes,42 whereas hybrid epitopes formed by the splicing of insulin peptides with fragments of other islet proteins are recognized by autoreactive T cells from human subjects with type 1 diabetes.43 The altered self-peptide concept of de novo autoreactivity in transplantation does not exclude the contributions of dysbiosis or Treg loss to autoimmune components of GvHD or rejection; rather, these factors may act synergistically to promote autoreactivity.
Although it has been well recognized that the graft microenvironment influences transplant outcomes, this has been viewed primarily as a consequence of innate immunity through mechanisms such as the promotion of leukocyte infiltration, upregulation of costimulatory ligand expression on antigen-presenting cell, and interference with Treg functions. Recently, intrarenal B-cell populations from kidneys undergoing chronic rejection were shown to contain cells recognizing tissue-specific autoantigens (rather than alloantigens), some of which were only present in inflamed kidneys.44 Given that these antibodies were isotype-switched and extensively mutated, a role for CD4+ T cells that recognize inflammation-related neoepitopes presented by B cells and promote autoantibody production through the provision of T cell help seems likely.
Based on our hypothesis, we would predict that transplanted organs procured from deceased donors would be more likely to trigger autoreactivity than those obtained through living donation. Localized tissue injury can also result in systemic inflammation,45,46 in turn setting up the conditions under which de novo autoimmunity could occur in organs other than the graft. Similarly, we anticipate that organs exposed to environmental antigens would harbor a tissue immunopeptidome incorporating more altered self-peptides with a greater propensity for recognition by autoreactive T cells. In part, this may relate to augmentation of graft inflammation due to innate signaling by microbial products, although this does not explain all the relevant findings. The half-life of male to female C57BL/6 skin grafts colonized by commensal bacteria in otherwise germ-free recipient mice is shortened compared with that of sterile grafts to germ-free recipients.47 Pattern-recognition receptor signaling alone is not sufficient to drive the acceleration of graft rejection resulting from Staphylococcus epidermidis colonization of donor skin,47 but metabolic or biochemical alterations in colonized skin might change the immunopeptidome with a resultant autoreactive response that hastens graft destruction. Mice primed against S epidermidis or Staphylococcus aureus mounted a brisk immune response against sex-matched syngeneic skin grafts colonized by these commensals, resulting in moderate graft damage in the absence of alloreactivity.48 In parallel to a memory response against the commensal organisms per se, autoreactive memory against induced/altered self-peptides could explain these findings. There may also be a role for molecules like MR1 that present bacterial metabolites.49
Discovery of immunogenic altered self-epitopes expressed by host target tissues and transplantable organs is feasible. Novel immunopeptidomic workflows enable the unbiased capture of the full spectrum of immunogenic peptides. Cryptic epitopes can be revealed using methods such as immunopeptidogenomics, whereby whole transcriptome data are translated in 3 frames and used to create a database of potential proteins resulting from use of alternative ORFs,16,50-53 whereas further recently established pipelines are suitable for the detection of spliced24,54 and other PTM55 peptides. These pipelines can be used in combination with cellular assays for the identification of immunogenic epitopes that are recognized by alloreactive and autoreactive CD8+ T cells,56 thus providing a means of testing the hypothesis proposed herein (Figure S1, SDC, https://links.lww.com/TP/C668).
Dissection of the relative contributions of altered self-peptide antigens, dysbiosis, and Treg loss to GvHD could be attempted using available experimental approaches. If T cells specific for matched MHC complexed with altered self-peptides were depleted (eg, using toxin-coupled57,58 or column-bound tetramers), any resulting amelioration of GvHD severity would suggest a functional role for those pMHC ligands, distinct from the effects of dysbiosis or Treg depletion. This could be compared with the effects of fecal microbiota transfers or regulatory T-cell transfer, although it should be noted that those interventions may also change the peptide landscape by reducing inflammation in the host tissues. Other predictions flowing from the hypothesis are also amenable to empirical testing. Mouse models of donation after brain death or circulatory death versus living donation could be combined with haploidentical (F1 to parent) organ transplantation, with examination of the tissue immunopeptidome and T-cell responses in the different groups. Broad-spectrum antibiotic treatment of donors or recipients or manipulation of extra-intestinal microbiota alone could be accompanied by the assessment of changes to the immunopeptidome and to auto and alloreactive T-cell responses.
New workflows to interrogate the full diversity of the immunopeptidome, along with a robust methodology for assessing pMHC recognition by alloreactive or autoreactive T cells, allow us to ask whether a proinflammatory or metabolically altered microenvironment causes modifications of the pMHC epitopes at the heart of allorecognition and altered self-recognition, thus rendering donor organs or host GvHD target tissues more immunogenic. Recognizing the unknown contributors to rejection and GvHD will allow us to develop strategies to address these.
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