Natural killer cell alloreactivity and haplo-identical hematopoietic transplantation

In haplo-identical hematopoietic transplantation, donor vs. recipient natural killer (NK) cell alloreactivity derives from a mismatch between donor NK clones bearing inhibitory killer cell Ig-like receptors (KIR) for self-HLA class I molecules and their HLA class I ligands (KIR ligands) on recipient cells. When faced with mismatched allogeneic targets, these NK clones sense the missing expression of self-HLA class I alleles and mediate alloreactions. KIR ligand mismatches in the GvH direction trigger donor vs. recipient NK cell alloreactions, which improve engraftment, do not cause GvHD and control relapse in AML patients . The mechanism whereby alloreactive NK cells exert their benefits in transplantation has been elucidated in mouse models. The infusion of alloreactive NK cells ablates (i) leukemic cells, (ii) recipient T cells that reject the graft and (iii) recipient DC that trigger GvHD, thus protecting from GvHD.     

Insulin-specific human T cells. Epitope specificity, major histocompatibility complex restriction, and alloreactivity to a diabetes-associated haplotype

T cells from an insulin-treated diabetic (ML, HLA DR1, w6) were stimulated in vitro with insulin, cloned at limiting dilution, and examined for their fine specificity and genetic restriction. T cell lines (TCL) derived from beef insulin stimulation were highly specific for epitopes on beef insulin, whereas pork insulin stimulation generated T cells that recognized determinants shared with beef insulin. Included among TCL reactive with pork insulin is one line (P2/9) that is autoreactive with human insulin. Antigen-presenting cells of known HLA type and monoclonal antibodies directed at class II major histocompatibility complex antigens were used to confirm the role of HLA-DR in restricting the response of insulin immune T cells. No preference or determinant selection within the donor's haplotypes was identified because either DR1 or DRw6 antigen-presenting cells could present the A loop of beef insulin. A TCL that recognized the A loop of beef insulin in association with DR1 was also alloreactive to HLA DR3, or a molecule closely linked to it, in the absence of insulin. A second T cell clone with insulin specificity and alloreactivity was also derived by allo stimulation of the donor's cells with DR3+ cells. When tested with a series of DR3+ stimulator cells, the alloreactivity was directed at diabetes-associated haplotypes. These data show that the T cell repertoire for insulin of a single diabetic donor encompasses that of multiple inbred animal strains and includes fine specificity for one to two amino acids, recognition of autologous insulin, and cross-reactivity with an allogeneic major histocompatibility complex antigen.     

HLA mismatches and hematopoietic cell transplantation: Structural simulations assess the impact of changes in peptide binding specificity on transplant outcome

The success of hematopoietic cell transplantation from an unrelated donor depends in part on the degree of Human Histocompatibility Leukocyte Antigen (HLA) matching between donor and patient. We present a structure-based analysis of HLA mismatching, focusing on individual amino acid mismatches and their effect on peptide binding specificity. Using molecular modeling simulations of HLA-peptide interactions, we find evidence that amino acid mismatches predicted to perturb peptide binding specificity are associated with higher risk of mortality in a large and diverse dataset of patient-donor pairs assembled by the International Histocompatibility Working Group in Hematopoietic Cell Transplantation consortium. This analysis may represent a first step toward sequence-based prediction of relative risk for HLA allele mismatches.     

Structural analysis of an HLA-B7 antigen variant detected by cytotoxic T lymphocytes

It has been demonstrated previously that lymphocytes of donor CF (HLA-A29,w33; B7,14) are not recognized by the HLA-B7-specific CTL clone HG-31. This report presents a structural comparison of the HLA-B7 antigen of donor CF with a "normal" HLA-B7 antigen, derived from the cell line JY. Isoelectric focusing showed that CF HLA-B7 heavy chains were more acidic than JY HLA-B7 heavy chains by the equivalent of a single charge. High pressure liquid chromatography and ion exchange chromatography comparisons of double-labeled tryptic peptides revealed a single detectable difference, which corresponded to the tryptic peptide spanning residues 112 to 121 on the HLA-B7 heavy chain. Although the complete amino acid sequence of this peptide was not obtained, the partial sequence indicates a substitution of an unidentified amino acid for tyrosine at position 116 of the heavy chain. This residue is found to vary among HLA specificities and to be altered in many H-2Kb mutants.     

Alloreactivity between disparate cognate and allogeneic pMHC-I complexes is the result of highly focused, peptide-dependent structural mimicry

Our understanding of the molecular mechanisms of T cell alloreactivity remains limited by the lack of systems for which both the T cell receptor allo- and cognate ligand are known. Here we provide evidence that a single alloreactive T cell receptor interacts with analogous structural regions of its cognate ligand, HLA-B*0801(FLRGRAYGL), as its allogeneic ligand, HLA-B*3501(KPIVVLHGY). The crystal structures of the binary peptide-major histocompatibility complexes show marked differences in the conformation of the heavy chains as well as the bound peptides. Nevertheless, both epitopes possess a prominent solvent-exposed aromatic residue at position 7 flanked by a small glycine at position 8 of the peptide determinant. Moreover, regions of close structural homology between the heavy chains of HLA B8 and HLA B35 coincided with regions that have previously been implicated in "hot spots" of T cell receptor recognition. The avidity of this human T cell receptor was also comparable for the allo- and cognate ligand, consistent with the modes of T cell receptor binding being broadly similar for these complexes. Collectively, it appears that highly focused structural mimicry against a diverse structural background provides a basis for the observed alloreactivity in this system. This cross-reactivity underpins the T cell degeneracy inherent in the limited mature T cell repertoire that must respond to a vast diversity of microbial antigens.     

Human mesenchymal stem cells suppress induction of cytotoxic response to alloantigens

Mesenchymal stem cells (MSC) fail to induce allogeneic responses in mixed lymphocyte reaction assays. Because MSC express HLA class I molecules, here we investigated whether they could be recognized as allogeneic targets by cytolytic T lymphocytes (CTL). With this aim, CTL precursor (CTLp) frequencies were measured following stimulation of T cells with either allogeneic mononuclear cells (MNC) or MSC originated from the same human bone marrow donor. Lysis of MSC was measured at day 10 of culture in standard chromium release assays. In addition, allogeneic PHA blast T cells or B-EBV lymphoblastoid cell lines (LCLs) generated from the same donor were used as positive controls of lysis. Our results showed that when allogeneic MNC were used to stimulate T cells, a high CTLp frequency was detected towards MSC targets. However, when MSC were used as stimulators, CTLp frequencies were markedly altered whatever the targets used, i.e.: MSC, PHA blast T cells or EBV-B LCLs. Moreover, when graded concentrations of MSC were added together with MNC upon stimulation of alloreactive T cells, we observed a dose-dependent decrease in CTLp frequencies towards MSC targets. This inhibition of MSC lysis was partially overcome by adding exogenous rh-IL-2 from the beginning of cultures. In addition, this suppressive effect was totally reproduced when, instead of MSC, supernatant harvested from MSC cultures was added to allogeneic MNC, upon stimulation of alloreactive T cells. In conclusion, our results demonstrate that MSC which can be recognized as targets by pre-activated alloreactive CTLs, may be able to suppress differentiation of CTL precursors into CTL effectors through secretion of suppressive factors.     

Estimation of the size of the alloreactive NK cell repertoire: studies in individuals homozygous for the group A KIR haplotype

Stem cell transplantation across HLA barriers may trigger NK cell-mediated graft-vs-leukemia effects leading to improved survival for patients with hematological malignancies. However, the genetic algorithm based on killer cell Ig-like receptor (KIR) and HLA genes used to predict NK cell alloreactivity have yielded discrepant results. Accordingly, it has been difficult to define transplantation settings that favor NK cell alloreactivity. In this study, we have used multiparameter flow cytometry to simultaneously analyze the cell surface expression of all four major inhibitory KIR and CD94/NKG2A to determine the size of the alloreactive NK cell repertoires in 31 individuals homozygous for the group A KIR haplotype. We observed a vast variability in the frequencies of cells with an alloreactive potential, ranging from 0 to 62% of the total NK cell population depending on which, and how many, KIR ligands were missing in theoretical recipients. This analysis required a functional examination of KIR3DL2-single positive NK cells, showing that this subset was hyporesponsive in individuals harboring the cognate ligands HLA-A3/A11. The results provide new insights into the variability of the functional alloreactive NK cell repertoire and have implications for donor selection in hematopoietic stem cell transplantation and adoptive NK cell-based immunotherapy.     

Identification of two T-cell epitopes on the candidate Epstein-Barr virus vaccine glycoprotein gp340 recognized by CD4+ T-cell clones.

Current efforts to develop an Epstein-Barr virus subunit vaccine are based on the major envelope glycoprotein gp340. Given the central role of CD4+ T cells in regulating immune responses to subunit vaccine antigens, the present study has begun the work of identifying linear epitopes which are recognized by human CD4+ T cells within the 907-amino-acid sequence of gp340. A panel of gp340-specific CD4+ T-cell clones from an Epstein-Barr virus-immune donor were first assayed for their proliferative responses to a series of truncated gp340 molecules expressed from recombinant DNA vectors in rat GH3 cells, by using an autologous B lymphoblastoid cell line as a source of antigen-presenting cells. The first four T-cell clones analyzed all responded to a truncated form of gp340 which contained only the first 260 N-terminal amino acids. These clones were subsequently screened for responses to each of a panel of overlapping synthetic peptides (15-mers) corresponding to the primary amino acid sequence of the first 260 N-terminal amino acids of gp340. One clone (CG2.7) responded specifically to peptides from the region spanning amino acids 61 to 81, while three other clones (CG5.15, CG5.24, and CG5.36) responded specifically to peptides from the region spanning amino acids 163 to 183. Work with individual peptides from these regions allowed finer mapping of the T-cell epitopes and also revealed the highly dose-dependent nature of peptide-induced responses, with inhibitory effects apparent when the most antigenic peptides were present at supraoptimal concentrations. Experiments using homozygous typing B lymphoblastoid cell lines as antigen-presenting cells showed that the T-cell clones with different epitope specificities were restricted through different HLA class II antigens; clone CG2.7 recognized epitope 61-81 in the context of HLA DRw15, whereas clones CG5.15, CG5.24, and CG5.36 recognized epitope 163-183 in the context of HLA DRw11. The present protocol therefore makes a systematic analysis of CD4+ T-cell epitopes within gp340 possible; it will be necessary to screen gp340-specific T-cell clones from a variety of donors to assess the wider influence of HLA class II polymorphism upon epitope choice.     

Imidazole-imidazole pair as a minor groove recognition motif for T:G mismatched base pairs.

The T:G mismatched base pair is associated with many genetic mutations. Understanding its biological consequences may be aided by studying the structural perturbation of DNA caused by a T:G base pair and by specific probing of the mismatch using small molecular ligands. We have shown previously that AR-1-144, a tri-imidazole (Im-Im-Im) minor groove binder, recognizes the sequence CCGG. NMR structural analysis of the symmetric 2:1 complex of AR-1-144 and GAACCGGTTC revealed that each AR-1-144 binds to four base pairs with the guanine N2 amino group forming a bifurcated hydrogen bond to a side-by-side Im/Im pair. We predicted that the free G-N2 amino group in a T:G wobble base pair can form two individual hydrogen bonds to a side-by-side Im/Im pair. Thus an Im/Im pair may be a good recognition motif for a T:G base pair in DNA. Cooperative and tight binding of an AR-1-144 homodimer to GAACTGGTTC permits a detailed structural analysis by 2D NOE NMR refinement and the refined structure confirms our prediction. Surprisingly, AR-1-144 does not bind to GAATCGGTTC. We further show that both the Im-Im-Im/Im-Py-Im heterodimer and the Im-Im-Im/Im-Im-Im homodimer bind strongly to the CACGGGTC + GACTCGTG duplex. These results together suggest that an Im/Im pair can specifically recognize a single T:G mismatch. Our results may be useful in future design of molecules (e.g. linked dimers) that can recognize a single T:G mismatch with specificity.     

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.     

Pretransplant frequency of donor-specific, IFN-gamma-producing lymphocytes is a manifestation of immunologic memory and correlates with the risk of posttransplant rejection episodes.

While matching for MHC Ags improves renal allograft survival, closely matched grafts sometimes fail due to rejection, and poorly matched allografts are often well tolerated by the recipient. The severity of the rejection process may partially depend on the presence of environmentally primed T cells in the recipient that cross-react with donor Ags. To test for the presence of primed, donor-specific T cells in humans before transplantation, we used an enzyme-linked immunospot assay for detection of allospecific cytokines produced by individual human PBLs. We demonstrate that this approach detects cytokine production at single cell resolution and detects production of IFN-gamma only when there is defined immunologic priming, thus representing a measure of primed donor-specific immunity. Because the environmental Ag exposure of the recipient is not a function of the HLA mismatch between donor and potential recipient, the number of HLA mismatches may not correlate with the frequency of pretransplant, donor-specific IFN-gamma-producing PBLs. Studies of donor-specific IFN-gamma-producing lymphocytes in a cohort of patients being evaluated for renal transplantation corroborated this hypothesis. Moreover, for recipients of both living and cadaver renal allografts, the pretransplant frequency of donor-specific memory cells correlated with the posttransplant risk of developing acute rejection episodes. This improved ability to define the strength of the allospecific immune response by enzyme-linked immunospot assay may allow improved pairing of recipients with donors and identification of kidney allograft donor-recipient pairs at high risk for acute rejection, thus permitting targeted interventions aimed at prolonging graft survival.     

Differential susceptibility of cytotoxic and helper T cell precursors to neonatal tolerization to histocompatibility antigens

The ability of various (C57BL/6J X CBA/HT6T6)F1 spleen cell subpopulations to induce tolerance to allogeneic histocompatibility antigens after injection into neonatal CBA/HT6T6 mice was examined. The requirements for tolerization of cytotoxic T lymphocyte precursors (CTLp) and IL 2-producing helper T cell precursors (IL 2Tp) appear to be coordinated but not identical. CTLp frequencies measured in limiting dilution analysis (LDA) were found to be decreased by 90 to 99% in mice injected neonatally with unseparated or a variety of semiallogeneic spleen cell fractions, including T cells, T cell-depleted spleen, the Ig+ and Ig- fractions of nylon-adherent, T-depleted spleen cells, Sephadex-G10 (G10)-nonadherent spleen cells, and T-depleted allogeneic C57BL/6J spleen cells. In contrast, IL 2Tp showed tolerization only after neonatal injection of unseparated or T cell-depleted F1 spleen cells, and not after injection of T or B cells or of G10-nonadherent or T-depleted allogeneic spleen cells. These studies show that the CTLp and IL 2Tp compartments have different requirements for neonatal tolerization, which appear to correlate with the presence of cells expressing class I or class II alloantigens in the inoculum: all spleen cell types tested were capable of tolerizing the CTLp compartment, whereas only whole spleen and T-depleted spleen cells could tolerize IL 2Tp; donor T cells, although capable of inducing CTLp tolerance, are not necessary for either CTLp or IL 2Tp tolerance induction; Ig+ B cells alone are marginally effective in tolerization of IL 2Tp, and G10-nonadherent cells are ineffective, suggesting that macrophages or another type of G10-adherent accessory cell may be required for tolerization of IL 2Tp, although it is not clear whether they are sufficient; and tolerization of CTLp can occur in the presence of a normal IL 2Tp compartment when certain inocula, such as T cells, are used for tolerance induction at birth.     

Persistence of donor-specific IL-2-secreting cells and cytotoxic T lymphocyte precursors in human kidney transplant recipients evidenced by limiting dilution analysis

The low reactivity to donor alloantigens reported in PBL from kidney transplant recipients might be related to clonal deletion and/or suppression of donor-specific alloreactive cells. To discriminate between these two hypotheses, we quantified the number of IL-2 secreting cells (IL-2-SC) and of cytotoxic precursors (CTLp) in the T cells from tolerant recipients when stimulated with either donor specific or nonrelated third-party LCL. To eliminate the irrelevant reactivity, we used as responding cells high-density T cells that had been depleted of such reactivity by 4 days preculture with autologous lymphoblastoid cell line in the presence of bromodeoxyuridine. Thus, frequencies of IL-2-SC and CTLp specifically directed at alloantigens could be measured. In 11 recipients, there was no strong decrease in the frequency of donor-reactive T cells when compared to the frequency of those directed at a third-party lymphoblastoid cell line, either for IL-2-SC (tested in 11 patients) or for CTLp (tested in 6 patients). In three cases of seven, a suppression was observed only when T cells were stimulated by donor cells. These data suggest that donor-reactive cells are still present in PBL of kidney-transplant recipients tested from 6 mo to 4 y posttransplantation. Moreover, suppression of donor-specific cells can be demonstrated in peripheral T cells of some recipients, which may account in part for the absence of rejection.     

The Cholinergic Anti-Inflammatory Pathway Delays TLR-Induced Skin Allograft Rejection in Mice: Cholinergic Pathway Modulates Alloreactivity

Activation of innate immunity through Toll-like receptors (TLR) can abrogate transplantation tolerance by revealing hidden T cell alloreactivity. Separately, the cholinergic anti-inflammatory pathway has the capacity to dampen macrophage activation and cytokine release during endotoxemia and ischemia reperfusion injury. However, the relevance of the α7 nicotinic acetylcholine receptor (α7nAChR)-dependent anti-inflammatory pathway in the process of allograft rejection or maintenance of tolerance remains unknown. The aim of our study is to investigate whether the cholinergic pathway could impact T cell alloreactivity and transplant outcome in mice. For this purpose, we performed minor-mismatched skin allografts using donor/recipient combinations genetically deficient for the α7nAChR. Minor-mismatched skin grafts were not rejected unless the mice were housed in an environment with endogenous pathogen exposure or the graft was treated with direct application of imiquimod (a TLR7 ligand). The α7nAChR-deficient recipient mice showed accelerated rejection compared to wild type recipient mice under these conditions of TLR activation. The accelerated rejection was associated with enhanced IL-17 and IFN-γ production by alloreactive T cells. An α7nAChR-deficiency in the donor tissue facilitated allograft rejection but not in recipient mice. In addition, adoptive T cell transfer experiments in skin-grafted lymphopenic animals revealed a direct regulatory role for the α7nAChR on T cells. Taken together, our data demonstrate that the cholinergic pathway regulates alloreactivity and transplantation tolerance at multiple levels. One implication suggested by our work is that, in an organ transplant setting, deliberate α7nAChR stimulation of brain dead donors might be a valuable approach for preventing donor tissue inflammation prior to transplant.     

Multiplicity of strand incision at G:T base mismatches in DNA by human cell extracts

Cell extract from the HT29 human colon carcinoma cell line (lacking mutator phenotype) was used to study the ATP-dependent G:T mismatch repair. We found that when a 45-bp (model) DNA with a single CpG/TpG mispair was incubated with the cell extract and ATP, it was incised immediately 5' and 3' to the mismatched T, and we noted that the actual 5'- and 3'-labeled fragments were similar to the cleaved products of thymine DNA glycosylase (TDG). This TDG-like cleavage product was enhanced (5-fold) with stimulation of several novel fragments, as inferred from the effect on incision at CpG/TpG site of the addition of G:U competitor DNA and ATP to the HT29 extract. The novel fragments were compatible with a strand incision on both sides of the mismatch (the third phosphodiester bond 5' and the second phosphodiester bond 3' to the mismatched T) and an incision 3' to the mismatched T, respectively. This suggests that while the ATP-dependent (TDG-like) incision activity, contrary to expectation, shows a lack of substrate competition, its catalytic property is likely modified by an interaction with G:U mispair. These multiple ATP-dependent incision events were not detected when extracts of the mismatch repair (MMR) defective HCT15 or HCT116 cell line were augmented with ATP and G:U. We postulate that these multiple ATP-dependent incision events possibly require the same MMR factors, and together they constitute a modified single ATP-dependent G:T incision activity. This activity toward the CpG/TpG was competitively inhibited by a 45-bp DNA with an ApG/TpT mispair; incision at a single site 5' to the latter mismatch compares with one of the multiple sites incised 5' to the former mismatch. These results suggest that one of several mismatch-incision factors is required by the human ATP-dependent G:T incision activity, in addition to MMR factors and ATP.     

Computational characterization of residue couplings and micropolymorphism-induced changes in the dynamics of two differentially disease-associated human MHC class-I alleles

Human major histocompatibility complex class I (MHC I) – or human leukocyte antigen (HLA) – proteins present intracellularly processed peptides to cytotoxic T lymphocytes in the adaptive immune response to pathogens. A high level of polymorphism in human MHC I proteins defines the peptide-binding specificity of thousands of different MHC alleles. However, polymorphism as well as the peptide ligand can also affect the global dynamics of the complex. In this study, we conducted classical molecular dynamics simulations of two HLA alleles, the ankylosing spondylitis (AS) associated/tapasin-dependent HLA-B*27:05 and nondisease-associated/tapasin-independent HLA-B*27:09, both in peptide-free forms as well as complex with four different peptides ligands. Our results indicate that in peptide-free form, the single amino acid substitution distinguishing the two alleles (D116H), leads to a weaker dynamic coupling of residues in the tapasin-dependent HLA-B*27:05. In peptide-bound form, several residues of the binding-groove, mostly in A and B pockets, show hinge-like behavior in the global motion of the MHC. Moreover, allele-dependent changes are shown in residue interactions, affecting the B-pocket as well as the beta-2-microglobulin (β2m)-facing residues of the HLA chain.     

Molecular basis for attenuation of neurovirulence of a yellow fever Virus/Japanese encephalitis virus chimera vaccine (ChimeriVax-JE)

A yellow fever virus (YFV)/Japanese encephalitis virus (JEV) chimera in which the structural proteins prM and E of YFV 17D are replaced with those of the JEV SA14-14-2 vaccine strain is under evaluation as a candidate vaccine against Japanese encephalitis. The chimera (YFV/JEV SA14-14-2, or ChimeriVax-JE) is less neurovirulent than is YFV 17D vaccine in mouse and nonhuman primate models (F. Guirakhoo et al., Virology 257:363-372, 1999; T. P. Monath et al., Vaccine 17:1869-1882, 1999). Attenuation depends on the presence of the JEV SA14-14-2 E protein, as shown by the high neurovirulence of an analogous YFV/JEV Nakayama chimera derived from the wild JEV Nakayama strain (T. J. Chambers, A. Nestorowicz, P. W. Mason, and C. M. Rice, J. Virol. 73:3095-3101, 1999). Ten amino acid differences exist between the E proteins of ChimeriVax-JE and the YFV/JEV Nakayama virus, four of which are predicted to be neurovirulence determinants based on various sequence comparisons. To identify residues that are involved in attenuation, a series of intratypic YFV/JEV chimeras containing either single or multiple amino acid substitutions were engineered and tested for mouse neurovirulence. Reversions in at least three distinct clusters were required to restore the neurovirulence typical of the YFV/JEV Nakayama virus. Different combinations of cluster-specific reversions could confer neurovirulence; however, residue 138 of the E protein (E(138)) exhibited a dominant effect. No single amino acid reversion produced a phenotype significantly different from that of the ChimeriVax-JE parent. Together with the known genetic stability of the virus during prolonged cell culture and mouse brain passage, these findings support the candidacy of this experimental vaccine as a novel live-attenuated viral vaccine against Japanese encephalitis.     

The role of DRB1 amino acid residue differences on donor-specific antibody production and acute rejection after cadaveric renal transplant.

The correlation between DRB1 amino acid residue matching, post-transplant humoral response and acute rejection (ARj) episodes was analysed in 51 renal transplant donor-recipient pairs in order to determine new criteria for organ assignment based on the alloreactivity of the residue within the peptide binding groove. HLA class I and II compatibility was defined using serological and genomic techniques; a sequence-based typing (SBT) was used for a higher resolution of DRB1 alleles. Humoral response was monitored in the first post-transplant year using triple staining flow cytometric analysis of donor-specific antibodies (Abs). Our data showed that DRB1 residue compatibility was always correlated to the absence of ARj while the presence of one or more aminoacid differences was associated with a similar frequency of ARj. Analysis of the mismatched DRB1 amino acid residue localised in the beta-pleated sheet and the alpha-helix of the DRB 1 molecule revealed that the frequency of beta-pleated sheet residue mismatches (MMs) was higher in the ARj-positive than in the ARj-negative group. A significant increase in the alpha-helix residue MMs was observed in patients with anti-class II Ab production (p = 0.034). Furthermore, analysing in detail DRB 1 MMs at the level of single amino acid residue, we found that the frequency of the mismatches localized in codon 9 and codon 28 in the beta-pleated sheet, as well as in codon 57 in the alpha-helix, was higher in patients who experienced ARj; on the other hand, MMs in codon 58 of the alpha-helix were more frequently associated with anti-class II Ab production. The identification of the residues more involved in alloreactivity onset will make it possible to define the existence of "permissive" or immunogenic" allele combinations which could simplify and increase the chances of a successful transplant.     

Analysis of binding of KIR3DS1*014 to HLA suggests distinct evolutionary history of KIR3DS1

NK cell activity is regulated by the integration of positive and negative signals. One important source of these signals for human NK cells is the killer Ig-like receptor (KIR) family, which includes both members that transduce positive and those that generate negative signals. KIR3DL1 inhibits NK cell activity upon engagement by its ligand HLA-Bw4. The highly homologous KIR3DS1 is an activating receptor, which is implicated in the outcome of a variety of pathological situations. However, unlike KIR3DL1, direct binding of KIR3DS1(+) cells to HLA has not been demonstrated. We analyzed four key amino acid differences between KIR3DL1*01502 and KIR3DS1*013 to determine their role in KIR binding to HLA. Single substitutions of these residues dramatically reduced binding by KIR3DL1. In the reciprocal experiment, we found that the rare KIR3DS1 allotype KIR3DS1*014 binds HLA-Bw4 even though it differs from KIR3DS1*013 at only one of these positions (position 138). This reactivity was unexpectedly dependent on residues at other variable positions, as HLA-Bw4 binding was lost in receptors with KIR3DL1-like residues at both positions 199 and 138. These data provide the first evidence, to our knowledge, for the direct binding of KIR3DS1(+) cells to HLA-Bw4 and highlight the key role for position 138 in determining ligand specificity of KIR3DS1. They also reveal that KIR3DS1 reactivity and specificity is dictated by complex interactions between the residues in this region, suggesting a unique functional evolution of KIR3DS1 within the activating KIR family.     

Structural analysis of HLA-A2.4 functional variant KNE. Implications for the mapping of HLA-A2-specific T-cell epitopes

HLA-A2 antigens are divided into four subtypes, designated A2.1 to A2.4, by the use of cytolytic T lymphocytes (CTL). The A2.4 subtype consists of a functionally heterogeneous group of variants that are not recognized by A2.1-, A2.2-, or A2.3-specific CTL lines while it is indistinguishable from A2.1 by isoelectric focusing. The structure of an A2.4 variant expressed on donor KNE has been established by comparative peptide mapping with A2.1 and radiochemical sequencing. It was found to differ from A2.1 by a single amino acid change of Cys to Tyr at position 99. This position is only moderately polymorphic and has not previously been found to vary in any other HLA or H-2 variants. The nature of the change is compatible with its generation by one-point mutation from A2.1. The only other previously characterized A2.4 variant, CLA, differs from A2.1 by a single amino acid replacement at position 9. Both residues 9 and 99 are located in homologous positions within the 1 and 2 domains, respectively. The results shown contribute to the molecular interpretation of the heterogeneity of CTL recognition within the HLA-A2.4 group of antigens.Abbreviations used in this paper CTL cytotoxic T lymphocytes - HPLC high performance liquid chromatography