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Results for “Allorecognition”

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PubMed2002

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.

Animals↗

PubMed1992

Rejection of skin allografts by indirect allorecognition of donor class I major histocompatibility complex peptides.

LEW (RT1l) rats were immunized with peptides corresponding to the alpha helical region of the alpha 1 domain (peptide 1), the beta sheet of the alpha 2 domain (peptide 2), and the alpha helical region of the alpha 2 domain (peptide 3) of the RT1-Aav1 classical class I molecule of the DA (RT1av1) strain. The immunizations were without carriers, and the objective was to prime to indirect allorecognition without influencing direct recognition of the RT1-Aav1 molecule. The LEW rats mounted strong primary and secondary antibody responses to peptides 1 and 3, but only weak secondary responses to peptide 2. None of the antipeptide antibodies crossreacted with intact RT1-Aav1 class I molecules. The immunization also resulted in LEW antigen-presenting cell-dependent, CD4+ T cell proliferative responses, which were very strong against peptide 1 and weakest against peptide 2. LEW rats immunized with peptides 1 or 3, but most effectively with both peptides 1 and 3 together, showed accelerated rejection of DA skin allografts. This effect was not observed in LEW rats immunized with peptide 2. In response to the DA skin allograft, the peptide-immunized LEW rats showed markedly accelerated kinetics of antibody production to the intact RT1-Aav1 molecule. These data demonstrate that indirect allorecognition can play an important role in allograft rejection and have important implications for understanding allograft rejection and its regulation.

Amino Acid Sequence↗

PubMed2003

Functional analysis of HLA-DP polymorphism: a crucial role for DPbeta residues 9, 11, 35, 55, 56, 69 and 84-87 in T cell allorecognition and peptide binding.

The information available on the specific function of HLA-DP and the structure-function relationships is very limited. Here, single amino acid substitutions of HLA-DPB1*02012 have been used to analyze the role of polymorphic residues of the DPbeta1 domain on DP-mediated T cell allorecognition and peptide binding. Using a panel of specific anti-HLA-DP mAb, we identified the HLA-DP residues involved in the recognition by these mAb, with a crucial role for DPbeta56 for most of the mAb assayed. Individual substitutions at residues 9, 11, 35, 55, 56 and 69 completely abrogated T cell recognition mediated by two different HLA-DPw2-allospecific T cell clones (8.3 and 8.9). Interestingly single changes at positions 9, 11, 35 and 55 of HLA-DPbeta also altered the binding of peptides AAII(12-27) and IIP(53-65), natural ligands of the HLA-DPB1*02012 molecule. Individual changes at residues located in pocket 1 (84, 85, 86 and 87 from HLA-DPbeta) led to a partial reduction in cytotoxic T lymphocyte-mediated lysis and also partially affected peptide binding. However, the simultaneous substitution of these positions completely abolished both T cell allorecognition and peptide binding, suggesting a major role for polymorphisms at pocket 1 in HLA-DP function. Molecular modeling, used to predict changes induced by amino acid substitutions, supported the functional data. Taken together, these results strongly suggest that polymorphic residues 84, 85, 86 and 87 at pocket 1, residues 9, 35 and 55 at pocket 9, and residues 11 and 69 at pockets 6 and 4 respectively play a key role in HLA-DP function, probably by modifying the way the peptide is bound within the groove of HLA-DP2 and determining changes in the conformation of the MHC-peptide complex recognized by the TCR.

Antibodies, Monoclonal↗

PubMed1993

Selective T-cell-receptor gene usage in allorecognition and graft-versus-host disease.

Immune recognition of foreign HLA molecules is initiated by T cell recognition mediated by alloreactive T cell receptor (TCR) molecules. We analyzed the diversity of TCR expression in the clinical setting of allorecognition in a patient with acute graft-versus-host disease following bone marrow transplantation. Nearly 200 TCR transcripts from peripheral blood lymphocytes were cloned and sequenced at two time points during GVHD. HLA genes in the transplant donor and the recipient were mismatched for a very specific HLA-DR subtype: HLA-DRB1 genes in the donor (DR4/Dw4) and the recipient (DR4/Dw14) encode HLA molecules that differ at only two amino acids, providing a very restricted target for allorecognition. We also studied TCR genes from five T cell clones derived in vitro from mixed lymphocyte cultures between Dw4-positive responder and Dw14-positive stimulator cells. Comparisons of the derived TCR sequences implicate nonrandom patterns of TCR selection both in vivo and in vitro.

Acute Disease↗

PubMed1996

Mechanisms of indirect allorecognition in graft rejection: class II MHC allopeptide-specific T cell clones transfer delayed-type hypersensitivity responses in vivo.

Recent animal studies suggest that indirect T-cell recognition of alloantigen plays an important role in allograft rejection and tolerance. In this study, we generated T cell clones from Lewis (LEW, RT1(l)) rat lymph node cells that had been primed in vivo by immunization with immunogenic class II MHC allopeptide RT1.D(u)beta2, representing residues 20-44 of the polymorphic beta chain of RT1.Dbeta(u) (Wistar Furth [WF]). Using reverse transcriptase polymerase chain reaction transcript analysis with specific rat T cell receptor Vbeta primers, we show that six out of nine T cell clones specifically proliferated to RT1.D(u)beta2 and expressed Vbeta 9. One of these clones, clone 2F4, which specifically proliferated to RT1.D(u)beta2 in a dose-response fashion and produced interferon-gamma in response to restimulation by RT1.D(u)beta2, was selected for further studies. The beta-chains of RT1.D(l) and RT1.D(u) residues 20-44 differ by two amino acids at positions 30 and 38. We synthesized two peptides, each containing a single polymorphic site: RT1.D(u)beta 20-33 and RT1.D(u)beta 31-44. Both these peptides were immunogenic by LEW responders, since lymph node cells primed by immunization proliferated equally to the peptides in vitro. Interestingly, in vitro dose-response studies with clone 2F4 showed better proliferative response to peptide RT1.D(u)beta 20-33 than to peptide RT1.D(u)beta 31-44, indicating that this T cell clone preferentially recognizes a single amino acid difference on residue 30. Finally, it has been suggested that indirect allorecognition by CD4+ T cells mediate graft rejection by delayed-type hypersensitivity responses, although definitive studies are lacking. Systemic injection of the 2F4 clone to naive LEW rats elicited an antigen-specific delayed-type hypersensitivity response against RT1.D(u)beta2 peptide and WF splenocytes, confirming indirect presentation in vivo. These data demonstrate that Th1 cell clones generated by in vivo priming via the indirect pathway utilize specific T cell receptor Vbeta and recognize single amino acid differences in the allopeptide. More importantly,these Th1 clones are capable of mediating a specific immune response in vivo. These studies with MHC allopeptide-specific T cell clones further delineate the cellular mechanisms of indirect allorecognition and provide a potential strategy to study its role in acute and chronic rejection, and tolerance.

Adoptive Transfer↗

PubMed1998

Mechanisms of indirect allorecognition: characterization of MHC class II allopeptide-specific T helper cell clones from animals undergoing acute allograft rejection.

BACKGROUND: Recent evidence indicates that T cells primed via the indirect pathway of allorecognition play an important role in allograft rejection, although the effector mechanisms remain unknown. The purpose of this study was to characterize and study the in vivo function of self-restricted MHC allopeptide-specific T-cell clones generated from animals undergoing allograft rejection. METHODS AND RESULTS: We generated self-restricted class II MHC allopeptide-specific T-cell clones from the spleen and kidney of Lewis (LEW; RT1l) rats undergoing acute rejection of MHC-incompatible Wistar Furth (WF; RT1u) renal allografts. RT1.Du/beta20-44 peptide-specific CD4+ T helper 1 clones from the spleen and kidney of rejecting animals expressed a restricted T cell receptor (TCR) Vbeta repertoire: Vbeta4, 8.2, or 9. In comparison, clones generated from RT1.Dubeta20-44 immunized LEW rats all expressed TCR Vbeta9. The amino acid sequence of RT1.Dl (LEW) and RT1.Du (WF) residues 20-44 differ only at positions 30 and 38. T-cell clones expressing TCR Vbeta9 preferentially proliferated to the peptide fragment RT1.Dubeta20-33. T-cell clones expressing TCR Vbeta4 proliferated weakly to peptide fragments RT1.Dubeta20-33 and 31-44, whereas those expressing TCR Vbeta8.2 proliferated preferentially to the peptide fragment 31-44. Adoptive transfer of T-cell clones expressing TCR Vbeta9 or Vbeta8.2, but not Vbeta4, to naive LEW animals elicited significant delayed-type hypersensitivity responses after challenge with the RT1.Dubeta20-44 peptide or allogeneic WF (RT1u) splenocytes. CONCLUSION: This is the first report on the cellular, molecular, and functional characterization of self-restricted MHC allopeptide-specific T-cell clones from animals undergoing acute rejection. Our data provide support for a biologically significant role of indirect allorecognition in allograft rejection.

Acute Disease↗

PubMed1999

Indirect allorecognition in acquired thymic tolerance: induction of donor-specific tolerance to rat cardiac allografts by allopeptide-pulsed host dendritic cells.

BACKGROUND: Presentation of peptides either by recipient or donor MHC molecules displayed on the surface of antigen-presenting cells is an essential element in the induction of T cell responses to transplant antigens. The finding that intrathymic (IT) injection of an immunodominant peptide induces acquired thymic tolerance suggests an indirect pathway of allorecognition in the thymus. To address this theory, we studied the effects of IT injection of host bone marrow (BM)-derived dendritic cells (DC)-pulsed with the immunodominant Wistar Furth (WF) MHC class I (RT1.Au) peptide 5 (93-109) on cardiac allograft survival in the WF-to-ACI rat combination. METHODS: DC were propagated from cultures of ACI (recipient) bone marrow (BM) maintained in a medium supplemented with granulocyte-macrophage colony-stimulating factor and IL-4. The BM-derived DC after 8 days of culture were pulsed in vitro with a single WF MHC class I peptide (Residue 93-109) with the dominant epitope, washed, and injected into the thymus of ACI rats. The ACI recipients received donor-type (WF) or 3rd party (Lewis) cardiac allografts 7 days after IT immunization with peptide-pulsed DC. RESULTS: BM-derived DC cultured in granulocyte-macrophage colony-stimulating factor and interleukin-4 for 8 days have a strong allostimulatory ability and present peptide 5 to naive syngeneic T cells in mixed lymphocyte reaction. IT inoculation of 300 microg RT1.Au peptide 5 combined with transient antilymphocyte serum immunosuppressive therapy induced donor-specific tolerance to cardiac allografts. Extension of this finding to peptide-pulsed self DC showed that IT injection of peptide 5-pulsed host DC consistently led to permanent acceptance (>150 days) of donor-type (WF) cardiac allografts, whereas third-party (Lewis) grafts were acutely rejected. The long-term unresponsive recipients challenged with second-set grafts accepted permanently (>100 days) donor-type(WF) grafts while rejecting third-party (Lewis) grafts without the rejection of the primary WF grafts. CONCLUSION: This novel finding that allopeptide-pulsed host DC induces tolerance to cardiac allografts suggests that the induction of acquired tolerance is dependent on the indirect allorecognition pathway. The results further suggest that genetically engineered DC expressing donor MHC class I or II molecules or a peptide analogue might have therapeutic potential in the induction of transplant tolerance and in the treatment of autoimmune diseases.

Animals↗

PubMed2001

Involvement of the direct and indirect pathways of allorecognition in tolerance induction.

It is generally accepted that there are two pathways of allorecognition, direct and indirect, that together contribute to allograft rejection. Although it has been suggested that the direct pathway predominates during early acute rejection and that the indirect pathway provides a continuous supply of alloantigen responsible for chronic rejection, the true relative contribution of each pathway to the overall rejection process is still not entirely known. It is clear, however, that any strategies designed to achieve the ultimate goal in transplantation, the induction of tolerance, will need to take into account both pathways. This review seeks to explore the involvement of the direct and indirect pathways of allorecognition on a mechanistic level as it relates to the induction of tolerance. A brief historical perspective is included for each pathway as well as a comprehensive review of the mechanisms felt to be active during tolerance induction.

Animals↗

PubMed2005

Direct allorecognition promotes activation of bystander dendritic cells and licenses them for Th1 priming: a functional link between direct and indirect allosensitization.

T-cell sensitization to indirectly presented alloantigens (indirect pathway of allorecognition) plays a critical role in chronic rejection. The usual very efficient priming of such self-restricted, T helper type 1 (Th1)-deviated CD4+ T cells obviously conflicts with the fact that allogeneic MHC molecules are poorly immunogenic per se. The aim of the present study is to elucidate whether direct allosensitization induces production of inflammatory mediators that may affect recruitment and activation of immature bystander (host) dendritic cells (DC). These potential mechanisms were studied in vitro by conducting primary allogeneic mixed leucocyte reactions (MLR), mimicking the priming phase in secondary lymphoid organs, and secondary MLR, mimicking the effector phase within the graft. Primary, and particularly secondary, MLR supernatants were found to contain high levels of monocyte/immature DC-recruiting CC chemokines and pro-inflammatory cytokines. Exposure of immature DC to primary or secondary MLR supernatants was found to upregulate CD40 expression and further enhanced lipopolysaccharide-induced interleukin-12 (IL-12) p70 production. Secondary MLR supernatants additionally induced upregulation of CD86 and deviated allogeneic T-cell responses towards Th1 (enhanced interferon-gamma production without concomitant induction of detectable IL-4 or IL-10 production). These findings indicate that direct allorecognition may act as a Th1-deviating adjuvant for indirect allosensitization.

Antigen Presentation↗

PubMed2006

The direct pathway of human T-Cell allorecognition is not tolerized by stimulation with allogeneic peripheral blood mononuclear cells irradiated with high-dose ultraviolet B.

Transfusions of high-dose (> or =10,000 Joule/m(2)) ultraviolet-B (UVB)-irradiated allogeneic leukocytes in rodent models have been shown to induce immunologic tolerance that is mediated by allospecific regulatory CD4(+) T cells. Whether these regulatory T cells recognize alloantigens through the direct or indirect pathway of allorecognition is controversial. Here, we demonstrate that the proliferative response obtained in standard primary mixed leukocyte reactions (MLRs) with human peripheral blood mononuclear cells (PBMCs) reflected a CD4(+) T-cell-dependent direct pathway of allorecognition and that high-dose UVB irradiation of PBMCs totally inhibited their capacity to induce a proliferative alloresponse. Re-stimulation with gamma-irradiated PBMCs from the same allogeneic donor (secondary MLR) elicited a proliferative and Th1-deviated response that was similar to the response induced in unprimed PBMCs. Finally, high-dose UVB was found to induce a rapid and massive apoptosis of irradiated PBMCs. Collectively, these data indicate that leukocytes irradiated with high-dose UVB are unable to prime for unresponsiveness or immune deviation in T cells directly recognizing allogeneic major histocompatibility complex molecules. Because it is well-established that antigens within transfused apoptotic cells are captured by resident tolerogenic spleen dendritic cells, we propose that tolerance induced by transfusions of high-dose UVB-irradiated leukocytes primarily involve T cells indirectly recognizing alloantigens.

Apoptosis↗

PubMed2004

MD-1 expression regulates direct and indirect allorecognition.

Expression of the molecule MD-1 was previously described to regulate allogeneic and xenogeneic skin graft survival, as documented by the decrease in rejection seen following functional blockade of MD-1 expression in vivo, using antisense oligodeoxynucleotides (ODNs) or anti-MD-1 antibodies. It was unclear from these data whether blockade of expression of MD-1 on donor or recipient cells was crucial. We have investigated the effect on allorecognition of treating skin graft donors, and/or recipients, of either fully major histocompatibility complex (MHC)-mismatched allogeneic skin grafts (C3H with C57BL/6 grafts and vice versa) or grafts differing at only multiple minor alloantigens (C3H with B10.BR grafts; C57BL/6 with C3H.SW), with antisense ODNs to MD-1, or in some cases, following transplantation of class II-deficient cells into class I-deficient mice. Graft-specific cytotoxic T lymphocytes (CTLs) were measured in spleen cells recovered at sacrifice of recipients and following donor-specific restimulation in vitro. In the latter case, we also measured cell proliferation and (by enzyme-linked immunosorbent assay) production of interleukin-2 (IL-2)/interferon-gamma (IFN-gamma) or IL-4/IL-10 in vitro (nominal type-1 vs type-2 cytokines). CTL responses to minor-incompatible grafts were diminished, only if graft recipients were treated with ODNs. However, treatment of graft donor and/or recipient of MHC-incompatible grafts produced inhibition of CTL production. Optimal inhibition came from treating both. Specific suppression of CTL production coincided with inhibition of proliferation and preferential production of IL-4 and IL-10 at the expense of IL-2 and IFN-gamma. Our data are consistent with the hypothesis that MD-1 expression regulates both the direct and indirect pathways of allorecognition and that regulation of MD-1 expression may thus help regulate clinical graft rejection.

Animals↗

PubMed1993

The molecular basis of allorecognition.

Until recently it was generally assumed that the focus of T cell receptor recognition of allogeneic MHC molecules was the polymorphic regions on the molecule that differed between responder and stimulator. It is now clear that all T cell recognition, including self-tolerance and allorecognition, involves both the MHC molecule and its associated peptide ligand. Polymorphic residues located within the peptide binding groove of the MHC and inaccessible to the T cell receptor can profoundly affect selection and recognition of bound peptides. These peptide differences between histoincompatible individuals greatly amplify the antigenic impact of MHC polymorphism and result in the high frequency of alloreactive cells. Evidence for the role of peptides in allorecognition is reviewed.

Animals↗

PubMedDate not supplied

Effect of the substitution of critical residues on the allorecognition of HLA-B27.

The three-dimensional structure of the HLA class I molecules has highlighted the importance of the "groove" formed by the helices. We used site-directed mutagenesis to construct a series of HLA-B27 mutants with different substitutions at the sites of the conserved amino acid residues of HLA-B27 subtypes, specifically residue 77 which is thought to be critical to the binding site of the molecule, and a residue at the CD8 binding site. We formed an anti-B27 CTL line and derived six anti-B27 clones. Each of the six clones showed a different pattern of reaction, reflecting the diversity of the epitopes recognized. All nine mutants were effective in altering allorecognition by HLA-B27 specific CTL, although positions 45 and 77 caused the most drastic effect. The residue in position 77 is also the last amino acid of the peptide sequence shared with Klebsiella. Our results highlight the importance of certain epitopes in allorecognition that may have important implications for the immunotherapy of autoimmune diseases.

Amino Acid Sequence↗

PubMed1996

TCR-beta transgenic mice fail to mediate a GVHR due to defects of allorecognition and subsequent IL-2 generation.

All T cells of TCR-beta transgenic mice bear a single TCR-beta chain and consequently the diversity of the TCR may be reduced by as much as one million-fold. Despite this limited diversity, many measures of lymphocyte function in these mice are normal. We have previously demonstrated that lymphoid cells from TCR-beta mice are unable to mediate an intense graft-versus-host response (GVHR). In order to investigate the mechanism of this hyporesponsiveness, we studied in vivo allorecognition in diverse strains of TCR-beta mice. All tested strains of TCR-beta mice failed to mediate a substantial GVHR across multiple H-2 barriers. In addition, mixtures of cells from several strains of TCR-beta mice only generated mild GVHRs. Sensitive tests of in vitro allorecognition show that lymphoid cells from TCR-beta mice respond less vigorously to alloantigen as measured both by decreased proliferation and decreased IL-2 production in a MLR. In addition, cells from TCR-beta mice fail to use exogenous IL-2 appropriately in their response to alloantigen. We conclude that the fixed TCR-beta chain causes a defective response to alloantigen, which is measured as decreased IL-2 generation and utilization, and that this abnormality results in a decreased GVHR.

Animals↗

PubMed1998

Indirect T cell allorecognition and alloantibody-mediated rejection of MHC class I-disparate heart grafts.

Recent studies in the rat have identified a role for T cell-dependent alloantibody in rejection of MHC class I-disparate allografts. RT1Aa-disparate PVG.R8 heart grafts are rejected acutely in naive, and hyperacutely in sensitized, PVG.RTIu recipients by CD4 T cell-dependent alloantibody. Here, we examined the T cell Ag recognition pathways responsible and show that direct injection into skeletal muscle of plasmid DNA, encoding a water-soluble form of the RT1Aa MHC class I heavy chain (pcmu-tAa), stimulates IgG2b cytotoxic alloantibody and markedly accelerates rejection of PVG.R8 heart grafts (median survival time 2 days). pcmu-tAa injection did not induce CTL to Aa, arguing against direct allorecognition of soluble Aa. Treatment with mAbs confirmed that the alloimmune response to pcmu-tAa injection depended on CD4, not CD8, T cells. Priming T cells for indirect allorecognition by injection of 15-mer peptides spanning the alpha1 and alpha2 domains of Aa failed to stimulate anti-Aa Ab but caused an accelerated Ab response to a PVG.R8 heart and a modest acceleration in graft rejection (median survival time 4 days). These results suggest that both soluble MHC class I and allopeptides prime CD4 T cells by the indirect pathway, but that soluble class I is a more effective immunogen for humoral alloimmunity because its tertiary protein structure provides B cell epitopes. We propose that priming humoral alloimmunity, like CTL priming, requires recognition of intact MHC on donor cells, but essential T cell help can be provided by CD4 T cells recognizing allogeneic class I exclusively by the indirect pathway.

Animals↗

PubMed1993

Position 71 in the alpha helix of the DR beta domain is predicted to influence peptide binding and plays a central role in allorecognition.

Despite all the structural and functional data that have been accumulated regarding major histocompatibility complex (MHC) class II molecules during recent years, the relative contribution of putative T cell receptor (TcR)-contacting residues and peptide-binding MHC polymorphisms to MHC-restricted and allospecific T cell responses remains a point of contention. Some authors emphasize the importance of direct interaction between the allospecific TcR and polymorphic MHC residues whereas other emphasize the role of naturally processed MHC-bound peptides. We have previously described a new HLA-DRB1 allele: DR BON (DRB1*0103). This gene differs from DRB1*0101 by six base pairs clustered in the third variable region of the second exon leading to three amino acid changes at positions 67, 70 and 71 of the beta chain of the HLA-DR molecule. To define the respective role of these residues in allorecognition, we have performed site-directed mutagenesis on the DRB1*0103 allele to create six mutants which are intermediary between the DR BON and the DR1 alleles. These mutant cDNA were expressed in mouse fibroblasts and the transfectants with the highest expression of class II molecules were used as stimulators for a panel of ten anti-DR BON and five anti-DR1 alloreactive T cell clones. We demonstrate that the residue at the peptide-binding position 71 is of paramount importance in the alloresponse of these clones. In addition some clones were sensitive to amino acid substitution at the TcR-contacting position 70, while substitution at position 67 affects very few clones. The dominance of residue 71 was also observed with an influenza hemagglutinin-specific HLA-DR BON-restricted T cell line.

Amino Acid Sequence↗

PubMed2004

Frontline: Peripheral priming of alloreactive T cells by the direct pathway of allorecognition.

Recent studies, though controversial, have suggested that secondary lymphoid organs may not constitute an essential site for the initiation of immune responses to transplant antigens. However, this issue has never been examined in the context of direct and indirect allorecognition. Here, we characterized immune responses arising in draining lymph nodes and skin allografts, in a murine model based on a single T cell clonotype where these two pathways can be independently studied. In this model, graft rejection by the direct or the indirect pathway occurred with similar kinetics, although initiation of the alloreactive responses was clearly different. During indirect responses, expansion and activation of alloreactive T cells were first observed in draining lymph nodes, at day 7 post-transplant, and graft-infiltrating T cells were observed later, at day 11. In striking contrast, directly activated alloreactive T cells were detected at an early stage inside the graft, and only later in the draining lymph nodes, after skin allograft rejection was almost completed. These results suggest that sensitization of naive T cells through the direct pathway could take place outside secondary lymphoid organs.

Animals↗

PubMed1991

Limited regions of the alpha 2-domain alpha-helix control anti-A2 allorecognition: an analysis using a panel of A2 mutants.

The regions of the HLA-A2 molecule controlling anti-A2 alloreactivity were explored using naturally occurring allelic variants of HLA-A, and a panel of transfectants expressing the products of A2.1 genes that had been mutated at multiple positions encoding residues in the alpha 2 domain alpha-helix. As a means of detecting distant conformational effects, these altered A2.1 molecules were also examined serologically. Amino acid substitutions at the carboxy-terminal end of the alpha 2 domain alpha-helix led to diminished staining with the monoclonal antibody (mAb) MA2.1. The epitope for this antibody has previously been mapped to the alpha 1 domain alpha-helix (residues 62-65). This suggests that interdomain contacts may cause conformational alteration, and that mutants can have distant, as well as local effects. Of the 24 positions where substitutions were made, only six led to loss of the anti-A2 alloresponse by the three clones and three lines that were tested. In addition, the mutations that altered the MA2.1 epitope, located on the alpha 1 domain alpha-helix, did not inhibit allorecognition. This suggests that a limited number of regions on the A2.1 molecule are responsible for allodeterminant expression. The most influential substitutions were those at positions 152, 154, 162, and 166. It is notable that three of these are predicted to be T-cell receptor (Tcr)-contacting residues, and one (152) to contribute to peptide binding. These results suggest that the specificity of alloreactive T cells is determined by exposed polymorphisms, directly contacted by the Tcr, and by concealed polymorphisms which influence peptide binding.

Cell Line↗