Hierarchical immunogenicity of donor MHC class I peptides in allotransplantation

The recognition of major histocompatibility complex (MHC) allopeptides by recipient MHC class II-restricted CD4(+) T cells via indirect pathway is a prerequisite for the generation of an immune response to the allograft. We tested 13-mer to 24-mer peptides from the MHC class I molecule for their possible immunogenicity in a fully MHC-mismatched rat strain combination. Our results confirm the hierarchical distribution of the immunogenicity of donor MHC class I peptides in the T cell alloactivation via indirect pathway. In addition, we show that allopeptide-induced immune response is critical for acute rejection of heart allografts. Among the seven allopeptides tested, peptide P1 was identified as immunodominant; it induced the greatest T cell proliferation and cytokine production in vitro as well as a significant reduction in allograft survival time. The TCR repertoire of T cells involved in the in vitro and in vivo responses induced by the dominant allopeptide P1 was found to be limited to the Vbeta10 and Vbeta 19 gene families. The identification of dominant allopeptides should greatly facilitate characterization of the specific T cell population responsible for allograft rejection and may be used to modulate the alloimmune response through antigen-specific therapy.     

Intramolecular and intermolecular spreading during the course of organ allograft rejection

Summary: There are two distinct pathways by which T cells may MHC alloantigens. The direct pathway involves T-cell recognition of intact MHC molecules expressed by donor antigen-presenting cells (APCs). The second, or indirect, pathway describes T-cell recognition of peptides derived from the processing and presentation of allogeneic MHC molecules on self APCs. Recent data demonstrates that indirect recognition plays a central role in both acute and chronic rejection of human organ allografts. Our studies have shown that, at the onset of primary acute rejection, recipient T-cell responses lo donor HLA-DR alloantigens are limited to a single dominant determinant present on erne of the disparate alloantigens and restricted by one of the responder's HLA-DR molecules. In allograft recipients with recurring episodes of rejection, and/or at the onset of chronic rejection, recipient T-cell reactivity may spread lo other epitopes within the allogeneic MHC molecule as well as to other alloantigens expressed by graft tissue. Both quantitative and qualitative alterations in T-cell allopeptide reactivity are associated with increased risk of cellular and/or humoral rejection. These studies provide a basis for the design of new therapeutic strategies and for immunologic monitoring of transplant recipients.     

New Approaches to Specific Immunomodulation in Transplantation

T cells can recognize foreign MHC antigens by two distinct routes, either directly as intact molecules, or indirectly as processed peptides. Recent evidence strongly suggests that the indirect pathway of allorecognition plays a key role in initiating and sustaining graft rejection. Theoretically, all mismatched HLA alloantigens could generate immunogenic peptides which may be recognized in the context of any of the two self HLA-DR molecules. However, indirect recognition appears to be limited to a single peptide determinant of an allogeneic HLA-DR molecule and restricted by one self HLA-DR molecule. Furthermore, T cells involved in the self-restricted allopeptide recognition express a limited array of T cell receptor variable genes. These findings suggest that selective immune interventions, such as peptide blockade of the self HLA-DR molecule involved in the presentation of the dominant allopeptide, induction of high-zone tolerance or TCR antagonism, may be devised to prevent graft rejection.     

Indirect allorecognition in solid organ transplantation

Recipient T cell recognition of donor major histocompatibility complex (MHC) alloantigens plays a central role in both acute and chronic rejection of human organ allografts. Two different pathways of T cell recognition of donor MHC alloantigens have been described. The direct pathway involves T cell recognition of intact MHC molecules expressed by donor antigen-presenting cells (APCs). The second, or indirect pathway, operates via T helper cell recognition of peptides derived from the processing and presentation of allogeneic MHC molecules on self-APCs. At the onset of primary acute rejection, recipient CD4+ T cell responses to donor HLA-DR alloantigens are limited to a single dominant determinant present on one of the disparate alloantigens and restricted by one of the responder's HLA-DR molecules. In allograft recipients with recurring episodes of rejection, and/or at the onset of chronic rejection, recipient T cell reactivity may spread to other epitopes within the allogeneic MHC molecule, as well as to other alloantigens expressed by graft tissue. Both quantitative and qualitative alterations in T cell allopeptide reactivity are associated with increased risk of cellular and/or humoral rejection. These studies provide a basis for the design of new therapeutic strategies and for immunologic monitoring of transplant recipients.     

T-cells behaving badly: structural insights into alloreactivity and autoimmunity

T-cells play a critical role in protective immunity, with their broad receptor repertoire capable of engaging diverse foreign pMHC landscapes. While the versatility and specificity of this MHC-restricted response is the hallmark of adaptive immunity, unwanted TCR interactions can profoundly effect the health of the host leading for instance to allograft rejection or autoimmunity. In allogeneic transplantation, such adverse reactions can occur by an indirect pathway when the TCR interacts with self-MHC molecules presenting allogeneic MHC derived peptides. Direct T-cell alloreactivity involves recognition of the allogeneic molecule itself either through molecular mimicry or by novel pMHC binding modes. By contrast, auto-reactive TCRs are considered to interact in a manner distinct from cognate pMHC interactions. Here we review recent advances in the field, focusing on structural data pertaining to alloreactivity and auto-reactivity and discuss implications for T-cell mediated transplant rejection and autoimmune disorders.     

Characterization of MHC- and TCR-binding residues of the myelin oligodendrocyte glycoprotein 38-51 peptide

Myelin oligodendrocyte glycoprotein (MOG) is a major experimental autoimmune encephalomyelitis (EAE) antigen in H-2b mice and a potential autoantigen in multiple sclerosis. How well MOG peptides bind to MHC and how TCR recognize the peptide/MHC complex have important implications for thymic selection as well as T cell activation in the periphery. In this study, we have characterized amino acids in the MOG(38-51) peptide important for peptide binding to I-Ab, and for TCR recognition of the peptide/MHC complex. We found that the amino acids R41, F44, R46 and V47 constituted the major TCR contact residues, as alanine substitution at these positions abrogated T cell responses without decreasing their binding affinity to I-Ab. In addition, G38 and W39 were found to be minor TCR contact residues. Finally, substituting tyrosine for alanine at position 40 decreased binding to I-Ab by approximately 50% and prevented induction of T cell responses in C57BL/6J mice upon immunization. Thus, Y40 is the dominant MHC-binding residue of the MOG(38-51) peptide and most likely occupies the p1 pocket of I-Ab. Our results could be useful to design peptides with altered agretopes and epitopes of the MOG(38-51) peptide to study their therapeutic potential in the EAE model.     

The structural basis of T-cell allorecognition

Foreign allogeneic major histocompatibility complex (MHC) class I and class II molecules elicit an exceptionally vigorous T-cell response. A small component of the alloresponse comprises CD4+ T cells that recognize allogeneic MHC indirectly after processing into peptide fragments that are bound and presented by self-MHC class II. The majority of alloreactive T cells directly recognize intact allogeneic MHC molecules expressed on foreign cells. Some alloreactive T-cell interactions with allogeneic MHC molecules are indifferent to the bound peptide, but evidence suggests that most show specificity to peptide. The vigor and diversity of the direct alloreactive T-cell response can therefore be explained by summation of numerous responses to each of the peptides in the novel set bound by allogeneic MHC molecules. Structural studies definitively show that the overall mechanism of T-cell receptor (TCR) recognition of self-MHC and allogeneic MHC molecules is similar. Many alloreactive T cells recognize several different combinations of MHC and bound peptide that do not necessarily possess structural homology. Flexibility within the TCR structure allows adaptation to the different contact surfaces. Crossreactivity seems to be an intrinsic property of the TCR required, because a single TCR must possess the ability to interact with both self-MHC during positive selection and at least one combination of foreign antigenic peptide presented by self-MHC. Recognition of allogeneic MHC molecules is an inadvertent consequence of the need for TCR crossreactivity.     

Instillation of allogeneic lung antigen-presenting cells deficient in expression of major histocompatibility complex class I or II antigens have differential effects on local cellular and humoral immunity and on pathology in recipient murine lungs

Recognition of allogeneic major histocompatibility complex (MHC) molecules expressed on donor lung antigen-presenting cells (APCs) by host T lymphocytes is believed to stimulate lung allograft rejection. However, the specific roles of donor MHC molecules in the rejection response is unknown. We report a murine model in which instilling allogeneic lung APCs into recipient lungs induces pathology analogous to acute rejection, and the production of interferon (IFN)-gamma, immunoglobulin (Ig) G2a, and alloantibodies in recipient lungs. Using allogeneic lung APCs (C57BL/6, I-a(b), H-2(b)) deficient in MHC class I, II, or both for instillation into lungs of BALB/c mice (I-a(d), H-2(d)), the purpose of the current study was to determine the specific roles of donor MHC molecules in stimulating local alloimmune responses. The data show that MHC class I or II on donor APCs induced IFN-gamma and IgG2a synthesis locally, though less than that induced by wild-type cells. Both MHC class I and II were required to induce alloantibody production. Instillation of wild-type or class I- or class II-deficient APCs induced comparable pathologic lesions in recipient lungs, and more severe than that induced by MHC-deficient cells. These data show that donor MHC class I and II molecules have differential effects in the stimulation of local alloimmune responses.     

T Cell Receptor (TCR) Repertoire in Alloimmune Responses

A large number of alloantigenic determinants could be generated by both the direct and indirect alloantigen presentation pathways. Hence, a heterogeneous population of T cells expressing a wide variety of receptors would be expected to respond to this diverse array of alloantigenic determinants. However, T cells expressing highly restricted T cell receptor (TCR) variable genes have been reported in a variety of alloimmune responses. A similar phenomenon has been observed in a wide variety of other immune responses, from those induced by superantigens, to very specific responses induced by a single peptide presented by a single MHC molecule. Given this scenario, the limited number of T cell clones which dominate an allograft rejection response, or for that matter an autoimmune response or a tumor specific response, could be therapeutically targeted by virtue of the selected TCR expression.     

Prolongation of small bowel allograft survival with a sequential therapy consisting of a synthetic MHC class II peptide and temporarily low-dose cyclosporine A

It has been extensively documented the role of the indirect pathway of allorecognition in allograft rejection. However, recent data demonstrate that the manipulation of this pathway could be also sufficient to promote prolongation of allograft survival. In the present study we evaluated the effect of preoperative immunization with the WF-specific MHC class II peptides RT1.D2 and RT1.B2 in combination with low-dose CsA from days 0 to 7 (5 mg/kg/day) and from days 8 to 30 (1 mg/kg/day) after WF small bowel transplantation. Seven days before and on the day of transplantation, LEW recipients were immunized with the two WF MHC class II peptides RT1.B2 and RT1.D2. The CsA monotherapy induced an allograft survival of 49.3 +/- 6.1 days. MHC class II peptide immunization had a limited effect on allograft survival for RT1.D2 (47.1 +/- 3.8 days) and induced prolongation of allograft survival for RT1.B2 (73.6 +/- 34.6 days). This effect seems to be based on the absence or silence of RT1.B2-reactive T cells and rejection seems to be correlated with the presence of RT1.B2-specific T cells in the late phase. Therefore, the combination of RT1.B2 with low-dose CsA shifts the immunological response and protects small bowel allograft rejection.     

Distinct effects of altered allopeptide analogs on a human self-restricted T cell clone

Alloreactive T cells involved in indirect recognition play a key role in initiating and sustaining graft rejection. One of the most promising approaches to achieve specific immunosuppression of indirect allorecognition resides in the use of chemically modified allopeptides. In order to design and test such peptide analogs, we have defined the dominant immunogenic peptide of the HLA-DRB1^*0101 antigen recognized by DRB1^*1101 responders. Next we engineered structural variants of this peptide (DRB1^*0101/residues 22-35), carrying single amino acid substitutions at postulated MHC and TCR contact residues. These analogs were tested for: (i) binding affinity to recombinant HLA-DRB1^*1101 protein (rDR11), and (ii) stimulatory activity exerted on a human anti-DR1/22-35 self-restricted T cell clone. The binding affinity of the analogs carrying non-homologous substitutions at putative anchor positions (24V/E and 29R/A) was significantly decreased, while little or no effect was observed in either peptide-binding or T cell proliferation assays for conserved substitution (24V/Y and 29R/K). This indicates that positions 24 and 29 are primarily involved in contacting the HLA-DR11 molecule. In contrast, single amino acid substitutions at positions 25 through 28 strongly affected the proliferative response of the clone, even when binding affinity to rDR11 was not altered. This finding suggests that positions 25 through 28 are TCR contact residues. Two peptide analogs (26L/I and 27L/V) displayed a higher stimulatory activity than the wild-type peptide and induced high-zone tolerance. Two other peptides (25R/A and 28E/Q), while binding to rDR11, did not exhibit any stimulatory activity and blocked the presentation and recognition of the wild-type peptide. Our data underscore the therapeutic potential of allopeptide analogs, as well as their value in dissecting the fine antigenic structure of a peptide determinant.     

Structural analysis of two HLA-DR-presented autoantigenic epitopes: crucial role of peripheral but not central peptide residues for T-cell receptor recognition

Specific and major histocompatibility complex (MHC)-restricted T-cell recognition of antigenic peptides is based on interactions of the T-cell receptor (TCR) with the MHC alpha helices and solvent exposed peptide residues termed TCR contacts. In the case of MHC class II-presented peptides, the latter are located in the positions p2/3, p5 and p7/8 between MHC anchor residues. For numerous epitopes, peptide substitution studies have identified the central residue p5 as primary TCR contact characterized by very low permissiveness for peptide substitution, while the more peripheral positions generally represent auxiliary TCR contacts. In structural studies of TCR/peptide/MHC complexes, this has been shown to be due to intimate contact between the TCR complementarity determining region (CDR) three loops and the central peptide residue. We asked whether this model also applied to two HLA-DR presented epitopes derived from an antigen targeted in type 1 diabetes. Large panels of epitope variants with mainly conservative single substitutions were tested for human leukocyte antigen (HLA) class II binding affinity and T cell stimulation. Both epitopes bind with high affinity to the presenting HLA-DR molecules. However, in striking contrast to the standard distribution of TCR contacts, recognition of the central p5 residue displayed high permissiveness even for non-conservative substitutions, while the more peripheral p2 and p8 TCR contacts showed very low permissiveness for substitution. This suggests that intimate TCR interaction with the central peptide residue is not always required for specific antigen recognition and can be compensated by interactions with positions normally acting as auxiliary contacts.     

Mechanism of liver allograft rejection: The indirect recognition pathway

Transplant rejection is mediated by the direct and indirect pathways. To explore the role of the indirect recognition pathway in the rejection of liver allografts, T cells obtained from peripheral blood were expanded in medium containing IL-2 and tested in LDA for reactivity to synthetic peptides corresponding to the hypervariable regions of the mismatched HLA-DR antigen(s) of the donor. Serial investigations of 17 recipients showed that T-cell reactivity to donor HLA-DR peptides was strongly associated with acute rejection episodes. In recipients carrying a graft that was mismatched by two HLA-DR alleles, a single donor antigen was targeted during primary rejection, although allopeptide reactivity against the second HLA-DR antigen was observed during subsequent episodes of acute rejection. The finding that allopeptide reactivity occurs early following transplantation and is predictive of rejection is consistent with the notion that processing of donor alloantigens by recipient APCs activates the indirect T-cell recognition pathway that plays a major role in initiating and amplifying allograft rejection.     

TCR usage in naive and committed alloreactive cells: implications for the understanding of TCR biases in transplantation.

The direct pathway of allorecognition is involved in acute allograft rejection and is characterised by TCR-mediated recognition of the MHC framework; this is thought to occur in a peptide-dependent but not peptide-specific manner. In contrast, the indirect pathway is restricted to the recipient's own MHC molecules and prevails in chronic rejection. In this pathway, the peptide has a major influence on the TCR recognition and selects alloreactive T cells with altered TCR Vbeta usage. However, qualitative analysis of Vbeta usage alone might limit our understanding of alloreactivity. The advantages of a combined quantitative assessment of Vbeta mRNA usage are discussed.     

Diverse usage of human T-cell receptor gene segments in HLA-DR1 allospecific T-cell clones

T-cell recognition of alloantigen involves both the MHC molecule and its associated peptide ligand. To understand the relationship between the specificity of alloantigen recognition and the structure of TCR molecules, we have investigated TCR gene utilization by sequencing TCR genes from well-defined allospecific Tlymphocyte clones. Alloreactive TLC consisted of a panel of clones primed to recognize DR l-related alloantigens. Our sequencing results revealed extensively diverse, but nonrandom, usage of TCR AV and BV gene segments and essentially no conservation in CDR3 or junctional sequences. Such observations are consistent with allospecific TCR that interact with MHC molecules on a generic level while recognizing specific peptides. They also reduce potential enthusiasm for anti-TCR therapy in allograft rejection.     

Induction of T-cell response to cryptic MHC determinants during allograft rejection

The presentation of MHC peptides by recipient and donor antigen presenting cells is an essential element in allorecognition and allograft rejection. MHC proteins contains two sets of determinants: the dominant determinants that are efficiently processed and presented to T cells, and the cryptic determinants that are not presented sufficiently enough to induce T-cell responses in vivo. In transplanted mice, initial T-cell response to MHC peptides is consistently limited to a single or a few immunodominant determinants on donor MHC molecule. However, in this article we show that under appropriate circumstances the hierarchy of determinants on MHC molecules can be disrupted. First, we observed that γIFN can trigger de novo presentation of cryptic self-MHC peptides by spleen cells. Moreover, we showed that allotransplantation is associated with induction of T-cell responses to formerly cryptic determinants on both syngeneic and allogeneic MHC molecules. Our results suggest that cross-reactivity and inflammation are responsible for the initiation of these auto- and alloimmune responses after transplantation.     

So many ways of getting in the way: diversity in the molecular architecture of superantigen-dependent T-cell signaling complexes.

Superantigens (SAGs) elicit massive T-cell proliferation through simultaneous interaction with MHC and TCR molecules. SAGs have been implicated in toxic shock syndrome and food poisoning, and they may also play a pathogenic role in autoimmune diseases. The best-characterized group of SAGs are the pyrogenic bacterial SAGs, which utilize a high degree of genetic variation on a common structural scaffold to achieve a wide range of MHC-binding and T-cell-stimulating effects while assisting pathogen evasion of the adaptive immune response. Several new structures of SAG-MHC and SAG-TCR complexes have significantly increased understanding of the molecular bases for high-affinity peptide/MHC binding by SAGs and for TCR Vbeta domain specificity of SAGs. Using the currently available SAG-MHC and SAG-TCR complex structures, models of various trimolecular MHC-SAG-TCR complexes may be constructed that reveal wide diversity in the architecture of SAG-dependent T-cell signaling complexes, which nevertheless may result in similar signaling outcomes.     

Flexibility in T-cell receptor ligand repertoires depends on MHC and T-cell receptor clonotype.

T-cell receptors (TCR) recognize peptides complexed to self-major histocompatibility complex (MHC) molecules. Recognition of peptide/MHC ligands by the TCR is highly peptide specific. However, certain TCRs can also recognize sequence-related and -unrelated ('mimicry') epitopes presented by homologous MHC molecules. Using two human, human leucocyte antigen-DR1 (HLA-DR1)-restricted T-cell clones specific for HA p307-319, we identified several diverse combinations of peptide-MHC complexes that are functionally equivalent in their ability to trigger T-cell stimulation. These findings demonstrate that a single TCR can productively interact with different peptides complexed to self- as well as non-self-MHC molecules. This extended reactivity is human leucocyte antigen (HLA) allele and TCR clonotype dependent, as the peptide repertoire recognized depends on the presenting HLA-DR molecule and varies among different TCRs that both recognize the HA p307-319/DR1 complex. Importantly, certain peptide analogues can completely change the HLA-restriction pattern of the TCR: T-cell recognition of the wild-type peptide that was absent in the context of a non-self HLA-DR molecule, was restored by complementing substitutions in altered peptide ligands, that could not be presented by the original restriction element. This mechanism may play an important role in allorecognition.     

Peptide competition for antigen presentation

Since each major histocompatibility complex (MHC) molecule can bind many different peptides, it might be predicted that competition for the same MHC-binding site takes place between peptides with unrelated sequences. As Luciano Adorini and Zoltan Nagy report here, this does indeed occur, both in vitro and in vivo. In-vivo competition between peptides for antigen presentation to T lymphocytes is an important influence on the immunodominance of T-cell determinants. In addition, it is possible to modulate T-cell activation by interfering with the binding of antigenic peptides to MHC class II molecules. This could represent a suitable approach to a rational treatment of autoimmune diseases and, possibly, of allograft rejection.