RESUMEN
Abacavir administration is associated with drug-induced hypersensitivity reactions in HIV+ individuals expressing the HLA-B*57:01 allele. However, the immunological effects of abacavir administration in an HLA-B57 mismatched transplantation setting have not been studied. We hypothesized that abacavir exposure could induce de novo HLA-B57-specific allorecognition. HIV-specific CD8 T cell clones were generated from HIV+ individuals, using single cell sorting based on HIV peptide/HLA tetramer staining. The T cell clones were assayed for alloreactivity against a panel of single HLA-expressing cell lines, in the presence or absence of abacavir. Cytokine assay, CD137 upregulation, and cytotoxicity were used as readout. Abacavir exposure can induce de novo HLA-B57 allorecognition by HIV-specific T cells. A HIV Gag RK9/HLA-A3-specific T cell did exhibit interferon-γ production, CD137 upregulation, and cytolytic effector function against allogeneic HLA-B57, but only in the presence of abacavir. Allorecognition was specific to the virus specificity, HLA restriction, and T cell receptor TRBV use of the T cell. We provide proof-of-principle evidence that administration of a drug could induce specific allorecognition of mismatched HLA molecules in the transplant setting. We suggest that HIV-seropositive recipients of an HLA-B57 mismatched graft should not receive abacavir until further studies are completed.
Asunto(s)
Fármacos Anti-VIH/efectos adversos , Didesoxinucleósidos/efectos adversos , Hipersensibilidad a las Drogas/inmunología , Infecciones por VIH/sangre , Antígenos HLA-B/inmunología , Alelos , Linfocitos T CD8-positivos/inmunología , Citocinas/metabolismo , Infecciones por VIH/complicaciones , Infecciones por VIH/inmunología , Seropositividad para VIH/inmunología , Prueba de Histocompatibilidad , Humanos , Interferón gamma/metabolismo , Leucocitos Mononucleares/citología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismo , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
We hypothesized that HIV-specific CD8 T cell clonotypes can be stimulated by allogeneic HLA molecules. Multiple HIV-specific CD8 T cell clones were derived from 12 individuals with chronic HIV infection, specific for 13 different HIV Gag antigens and restricted to 7 different HLA molecules. The generated T cell clones were assayed for alloreactivity against a panel of single HLA class I expressing cell lines (SALs). HIV-specific T cells recognising at least one allogeneic HLA molecule could be identified from 7 of 12 patients tested. Allorecognition was associated with IFNγ cytokine production, CD137 upregulation and cytotoxicity, suggesting high avidity allo-stimulation. Allo-HLA recognition by HIV-specific T cells was specific to the HIV target peptide/HLA restriction and TCR TRBV usage of the T cells. HIV-specific T cells do crossreact against allogeneic HLA molecules in an epitope and TRBV specific manner. Therefore allo-HLA stimulation could be exploited to induce or augment HIV-specific T cell responses.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Antígenos VIH/inmunología , Infecciones por VIH/inmunología , Antígenos HLA-A/inmunología , Antígenos HLA-B/inmunología , Linfocitos T CD8-positivos/patología , Células Cultivadas , Células Clonales/inmunología , Estudios de Cohortes , Reacciones Cruzadas , Infecciones por VIH/patología , Humanos , Memoria Inmunológica , Receptores de Antígenos de Linfocitos T/inmunología , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/inmunologíaAsunto(s)
Didesoxinucleósidos/efectos adversos , Hipersensibilidad a las Drogas/inmunología , Infecciones por VIH , VIH-1/inmunología , Memoria Inmunológica/efectos de los fármacos , Didesoxinucleósidos/administración & dosificación , Hipersensibilidad a las Drogas/patología , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/inmunología , Infecciones por VIH/patología , HumanosRESUMEN
On 24 January 2023, Eurotransplant has introduced the virtual crossmatch for kidney and pancreas allocation as a better alternative for the physical Complement Dependent Cytotoxicity (CDC) crossmatches at the donor centre, which were associated with a longer cold ischaemia time and false positive reactions. For the time being, the physical CDC crossmatch at the recipient centre will remain in place as the final histocompatibility check. While Eurotransplant is certainly not the first organ allocation organisation to introduce virtual crossmatching, several novel aspects have been introduced, such as calculation of the virtual panel reactive antibody (vPRA) on 11 loci at the second-field level in addition to the serological broad and split level, electronic HLA typing data transmission using Histoimmunogenetics Markup Language (HML) file format, and the actual virtual crossmatch based on ambiguous, second-field HLA typing of the donor on all 11 loci. This short communication will focus on these novel aspects of the virtual crossmatch in Eurotransplant.
Asunto(s)
Antígenos HLA , Prueba de Histocompatibilidad , Trasplante de Riñón , Donantes de Tejidos , Humanos , Prueba de Histocompatibilidad/métodos , Antígenos HLA/inmunología , Antígenos HLA/genética , Trasplante de Riñón/métodos , Obtención de Tejidos y Órganos/métodos , Trasplante de Páncreas/métodos , Europa (Continente) , Isoanticuerpos/sangreRESUMEN
In the absence of treatment, most HIV-1-infected humans develop AIDS. However, a minority are long-term nonprogressors, and resistance is associated with the presence of particular HLA-B*27/B*57 molecules. In contrast, most HIV-1-infected chimpanzees do not contract AIDS. In comparison with humans, chimpanzees experienced an ancient selective sweep affecting the MHC class I repertoire. We have determined the peptide-binding properties of frequent chimpanzee MHC class I molecules, and show that, like HLA-B*27/B*57, they target similar conserved areas of HIV-1/SIV(cpz). In addition, many animals appear to possess multiple molecules targeting various conserved areas of the HIV-1/SIV(cpz) Gag protein, a quantitative aspect of the immune response that may further minimize the chance of viral escape. The functional characteristics of the contemporary chimpanzee MHC repertoire suggest that the selective sweep was caused by a lentiviral pandemic.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/prevención & control , VIH-1/genética , VIH-1/inmunología , Antígenos HLA-B/genética , Antígeno HLA-B27/genética , Antígenos de Histocompatibilidad Clase I/genética , Pan troglodytes/genética , Pan troglodytes/inmunología , Virus de la Inmunodeficiencia de los Simios/genética , Virus de la Inmunodeficiencia de los Simios/inmunología , Síndrome de Inmunodeficiencia Adquirida/genética , Síndrome de Inmunodeficiencia Adquirida/inmunología , Secuencia de Aminoácidos , Animales , Línea Celular , Secuencia Conservada , Productos del Gen gag/genética , Productos del Gen gag/metabolismo , Genes MHC Clase I , Sobrevivientes de VIH a Largo Plazo , Humanos , Datos de Secuencia Molecular , Unión Proteica , Especificidad de la Especie , Linfocitos T Citotóxicos/inmunologíaRESUMEN
The Eurotransplant (The Eurotransplant International Foundation) acceptable mismatch programme has been shown to be a successful tool to enhance transplantation of highly sensitized patients(HSPs). However, patients with rare HLA phenotypes in relation to the Eurotransplant donor population remain on the waiting list. EUROSTAM is an European Union funded project to explore the feasibility of a Europe-wide acceptable mismatch programme enabling transplantation of HSPs with rare HLA phenotypes within their own organ exchange organization. The present study, which forms part of the EUROSTAM project, assesses the differences in the practices of the laboratories in different countries with respect to their HLA antibody profiling and risk adverseness. In the serum exchange exercises of 18 samples, a high level of variability has been shown in both assays and interpretation of results. In the data exchange exercise when all participants were given the same Luminex raw data for analysis, a high degree of consensus was reached where the median fluorescent intensity values of beads were <500 and >2000 for standard single antigen bead assays, or <500 and >5000 for assignment of acceptable mismatches. The risk adverseness analysis has showed distinct patterns of attitudes towards the perceived risks based on HLA antibody assay results, most probably influenced by the local protocols of the clinical transplant programme of each laboratory. In order to ensure fairness and maintain consistencies of organ exchange among partner transplant centres, a centralized facility will be instrumental for a uniform definition of acceptable mismatches.
Asunto(s)
Prueba de Histocompatibilidad , Inmunización , Consenso , Fluorescencia , Humanos , Suero/metabolismoRESUMEN
Non-classical Human Leukocyte Antigen (HLA)-E preferentially presents leader peptides derived from classical HLA-class I molecules. HLA-E can trigger opposed immune responses by interacting with inhibitory NKG2A or by activating NKG2C receptors on NK and T-cells. We studied the impact of HLA-E on renal allograft survival during acute cellular rejection. HLA-E expression was up-regulated in acute cellular rejection (ACR) biopsies (n=12) compared to biopsies from 13 renal allografts with no rejection-signs. HLA-E up-regulation was correlated with numbers of HLA-class I leader peptide mismatches (p=0.04). CD8+ and CD56+ infiltrating cells correlated with HLA-E expression (p<0.0001 and p=0.0009, respectively). Activating NKG2C receptor dominated on effector cells in biopsies and peripheral blood during ACR potentially allowing HLA-E-mediated immune activation. Moreover, HLA-E expression correlated with deterioration in renal allograft function (p<0.008) and reduced allograft survival (p=0.002). Our findings provide evidence that during renal allograft rejection HLA-E along with high numbers of mismatched HLA-class I leader peptides might represent additional targets for immune-activating responses.
Asunto(s)
Rechazo de Injerto/inmunología , Supervivencia de Injerto/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Trasplante de Riñón , Biomarcadores , Biopsia , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Femenino , Expresión Génica , Rechazo de Injerto/genética , Supervivencia de Injerto/genética , Antígenos de Histocompatibilidad Clase I/química , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Inmunohistoquímica , Riñón/inmunología , Riñón/metabolismo , Riñón/patología , Riñón/fisiopatología , Pruebas de Función Renal , Trasplante de Riñón/efectos adversos , Recuento de Linfocitos , Masculino , Persona de Mediana Edad , Subfamilia A de Receptores Similares a Lectina de Células NK/metabolismo , Subfamília C de Receptores Similares a Lectina de Células NK/metabolismo , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Trasplante Homólogo , Antígenos HLA-ERESUMEN
BACKGROUND: Human leukocyte antigen (HLA)-C is expressed on nucleated cells and platelets in lower levels than HLA-A,B, and its antigens are in linkage disequilibrium with HLA-B antigens. Therefore, HLA-C antibody detection is difficult. The authors questioned whether HLA-C could serve as a target in clinical kidney transplantation using a newly developed assay. METHODS: Flow cytometry was performed with sera from patients (n=34) awaiting a kidney retransplant using nine cell lines expressing a single HLA-C antigen (single-antigen lines [SAL]). RESULTS: The SAL were validated with HLA-C-specific alloantisera and human monoclonal antibodies against HLA-A, -B, and -C. The results were in agreement with the specificities previously reported. Exceptions, because of new HLA-C specificities used here, could be explained by epitope sharing between the antigens. With respect to patient sera, 15 of the 34 patients tested (44%) showed serum reactivity toward one or more HLA-C SAL. CONCLUSIONS: In contrast to peripheral blood lymphocytes, SAL are excellent targets for detecting HLA-C-reactive alloantibodies by flow cytometry. This preliminary analysis revealed that HLA-C-reactive antibodies are frequently present in sera of retransplant patients, serving as possible targets in clinical transplantation.
Asunto(s)
Antígenos/inmunología , Antígenos HLA-C/inmunología , Trasplante de Riñón/inmunología , Leucocitos/inmunología , Anticuerpos/sangre , Biomarcadores/análisis , Línea Celular , Epítopos/análisis , Humanos , Selección de Paciente , Reproducibilidad de los ResultadosRESUMEN
Definition of the antibody specificity in the serum of patients waiting for a renal transplant or in need for platelet transfusion is a crucial step for finding adequate donors. Confounding factors are the complexity of the serum antibodies and the expression of several, up to six, different human leukocyte antigens (HLA) on peripheral blood lymphocytes used as target cells in the antibody screening. Single antigen-expressing (SAL) cell lines were generated by transfecting human major histocompatibility complex (MHC) class I sequences into K562, an erythroleukemia-derived cell line lacking MHC class I and II expression. Thirty-seven different SALs have been generated so far. In this study, we present the validation of 16 of those SALs by flow cytometry against a panel of 84 human HLA-specific monoclonal antibodies (30 HLA-A [8 IgG/22 IgM], 45 HLA-B [18 IgG/27 IgM], 6 HLA-A, B [3 IgG/3 IgM], and 3 HLA-C [all IgM]) developed in our laboratory. The SALs proved to be suitable tools to determine acceptable mismatches for highly sensitized patients. This concept of transfecting target sequences in immortalized cell lines opens up new avenues in the definition of serum and cellular reactivity for sensitized patients awaiting a suitable organ or blood component.
Asunto(s)
Anticuerpos/inmunología , Antígenos HLA/inmunología , Anticuerpos/sangre , Anticuerpos Monoclonales/inmunología , Afinidad de Anticuerpos/inmunología , Especificidad de Anticuerpos/inmunología , Línea Celular Tumoral , Proteínas del Sistema Complemento/inmunología , Reacciones Cruzadas/inmunología , Pruebas Inmunológicas de Citotoxicidad , Femenino , Citometría de Flujo , Antígenos HLA/genética , Antígenos HLA/metabolismo , Antígeno HLA-A2/genética , Antígeno HLA-A2/inmunología , Antígeno HLA-A3/genética , Antígeno HLA-A3/inmunología , Antígeno HLA-B7/genética , Antígeno HLA-B7/inmunología , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/inmunología , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Inmunoensayo/métodos , Inmunoglobulina G/inmunología , Inmunoglobulina M/inmunología , Células K562 , Leucocitos Mononucleares/inmunología , TransfecciónRESUMEN
The ability to directly measure virus-specific lymphocytes using fluorochrome-labeled tetrameric complexes has proven a great advancement for the transplantation field. Viral peptide/HLA tetrameric complexes allow the rapid generation of virus-specific clones using single cell sorting apparatus, permitting the determination of alloreactivity from a single TCR with known specificity. When combined with new target "detector" cells called single HLA antigen-transfected K562 cells (SALs), the human alloresponse can for the first time be examined specifically and reliably. Here we describe a method for detection of "heterologous immunity" from virus-specific memory T-cells using single HLA expressing cell lines as allogeneic targets.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Antígenos HLA/genética , Antígenos HLA/inmunología , Memoria Inmunológica/inmunología , Virus/inmunología , Linfocitos T CD8-positivos/citología , Células Clonales , Reacciones Cruzadas/inmunología , Humanos , Células K562 , TransfecciónRESUMEN
BACKGROUND: Allo-human leukocyte antigen (HLA) reactivity by naturally acquired viral-specific memory T cells is common. However, the effect of successful vaccination on the alloreactive memory T-cell repertoire is unclear. We hypothesized that vaccination could specifically induce allo-HLA-reactive memory T cells. METHODS: A varicella-zoster virus (VZV) immediate early 62 (IE62)-specific CD8 memory T-cell clone was single cell sorted from a VZV seronegative renal transplant candidate after response to live attenuated varicella vaccination. To analyze the allo-HLA reactivity, the VZV IE62-specific T-cell clone was tested against HLA-typed target cells and target cells transfected with HLA molecules, in both cytokine production and cytotoxicity assays. RESULTS: The varicella vaccine-induced VZV IE62-specific T-cell clone specifically produced interferon-γ when stimulated with HLA-B*55:01-expressing Epstein-Barr virus-transformed B cells and HLA-B*55:01-transfected K562 cells (single HLA antigen expressing cell line [SALs]) only. The clone also demonstrated specific cytolytic effector function against HLA-B*55:01 SALs and phytohemagglutinin blasts. Cytotoxicity assays using proximal tubular epithelial cell and human umbilical vein endothelial cell targets confirmed the kidney tissue specificity of the allo-HLA-B*55:01 reactivity, and the relevance of the cross-reactivity to clinical kidney transplantation. The results also suggest that molecular mimicry, and not bystander proliferation, is the mechanism underlying vaccine-induced alloreactivity. CONCLUSIONS: Varicella vaccination generated a de novo alloreactive kidney cell-specific cytolytic effector memory T cell in a patient awaiting renal transplantation. Vaccination-induced alloreactivity may have important clinical implications, especially for vaccine timing and recipient monitoring.
Asunto(s)
Vacuna contra la Varicela/inmunología , Antígenos HLA/inmunología , Proteínas Inmediatas-Precoces/inmunología , Memoria Inmunológica , Trasplante de Riñón/inmunología , Linfocitos T/inmunología , Transactivadores/inmunología , Proteínas del Envoltorio Viral/inmunología , Reacciones Cruzadas , Citotoxicidad Inmunológica , Humanos , Masculino , Persona de Mediana Edad , Especificidad de ÓrganosRESUMEN
A recent phase 1 trial has demonstrated that the generation of tumor-reactive T lymphocytes by transfer of specific T-cell receptor (TCR) genes into autologous lymphocytes is feasible. However, compared with results obtained by infusion of tumor-infiltrating lymphocytes, the response rate observed in this first TCR gene therapy trial is low. One strategy that is likely to enhance the success rate of TCR gene therapy is the use of tumor-reactive TCRs with a higher capacity for tumor cell recognition. We therefore sought to develop standardized procedures for the selection of well-expressed, high-affinity, and safe human TCRs. Here we show that TCR surface expression can be improved by modification of TCR alpha and beta sequences and that such improvement has a marked effect on the in vivo function of TCR gene-modified T cells. From a panel of human, melanoma-reactive TCRs we subsequently selected the TCR with the highest affinity. Furthermore, a generally applicable assay was used to assess the lack of alloreactivity of this TCR against a large series of common human leukocyte antigen alleles. The procedures described in this study should be of general value for the selection of well- and stably expressed, high-affinity, and safe human TCRs for subsequent clinical testing.