RESUMEN
Alloreactive T lymphocytes are central mediators of graft-versus-host disease and allograft rejection. A public CTL clonotype with specificity for the alloantigens HLA-B*4402 and B*4405 is often expanded to large numbers in healthy HLA-B*0801(+) individuals, driven by cross-reactive stimulation with the common, persistent herpesvirus EBV. Since such alloreactive memory CTL expansions have the potential to influence transplantation outcome, altered peptide ligands (APLs) of the target HLA-B*0801-binding EBV peptide, FLRGRAYGL, were screened as specific antagonists for this immunodominant clonotype. One APL, FLRGRFYGL, exerted powerful antagonism of a prototypic T cell clone expressing this immunodominant TCR when costimulated with target cells presenting HLA-B*0801(FLRGRAYGL). Significantly, this APL also reduced the lysis of allogeneic target cells expressing HLA-B*4402 by up to 99%. The affinities of the agonist and antagonist complexes for the public TCR, measured using solution and solid-phase assays, were 8 and 138 muM, respectively. Surprisingly, the half-life of the agonist and antagonist complexes was similar, yet the association rate for the antagonist complex was significantly slower. These observations were further supported by structural studies that suggested a large conformational hurdle was required to ligate the immunodominant TCR to the HLA-B*0801 antagonist complex. By defining an antagonist APL against an immunodominant alloreactive TCR, these findings raise the prospect of exploiting such peptides to inhibit clinical alloreactivity, particularly against clonal T cell expansions that react with alloantigens.
Asunto(s)
Antígenos Virales/inmunología , Citotoxicidad Inmunológica/inmunología , Rechazo de Injerto/inmunología , Herpesvirus Humano 4/inmunología , Isoantígenos/inmunología , Fragmentos de Péptidos/inmunología , Receptores de Antígenos de Linfocitos T/antagonistas & inhibidores , Linfocitos T Citotóxicos/inmunología , Antígenos Virales/metabolismo , Línea Celular Transformada , Células Clonales , Reactividad Cruzada/inmunología , Pruebas Inmunológicas de Citotoxicidad/métodos , Epítopos de Linfocito T/inmunología , Epítopos de Linfocito T/metabolismo , Rechazo de Injerto/virología , Antígenos HLA-B/inmunología , Antígenos HLA-B/metabolismo , Antígeno HLA-B44 , Antígeno HLA-B8/química , Antígeno HLA-B8/inmunología , Antígeno HLA-B8/metabolismo , Semivida , Humanos , Epítopos Inmunodominantes/inmunología , Epítopos Inmunodominantes/metabolismo , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/metabolismo , Unión Proteica/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T Citotóxicos/virologíaRESUMEN
Epstein-Barr virus (EBV)-encoded nuclear antigen (EBNA)1 is thought to escape cytotoxic T lymphocyte (CTL) recognition through either self-inhibition of synthesis or by blockade of proteasomal degradation by the glycine-alanine repeat (GAr) domain. Here we show that EBNA1 has a remarkably varied cell type-dependent stability. However, these different degradation rates do not correspond to the level of major histocompatibility complex class I-restricted presentation of EBNA1 epitopes. In spite of the highly stable expression of EBNA1 in B cells, CTL epitopes derived from this protein are efficiently processed and presented to CD8+ T cells. Furthermore, we show that EBV-infected B cells can readily activate EBNA1-specific memory T cell responses from healthy virus carriers. Functional assays revealed that processing of these EBNA1 epitopes is proteasome and transporter associated with antigen processing dependent. We also show that the endogenous presentation of these epitopes is dependent on the newly synthesized protein rather than the long-lived stable EBNA1. Based on these observations, we propose that defective ribosomal products, not the full-length antigen, are the primary source of endogenously processed CD8+ T cell epitopes from EBNA1.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Antígenos Nucleares del Virus de Epstein-Barr/inmunología , Herpesvirus Humano 4/inmunología , Linfocitos B/inmunología , Línea Celular , Cicloheximida/farmacología , Epítopos/inmunología , Antígenos Nucleares del Virus de Epstein-Barr/genética , Herpesvirus Humano 4/efectos de los fármacos , Herpesvirus Humano 4/genética , Humanos , Proteínas Recombinantes/genética , Linfocitos T Citotóxicos/inmunología , TransfecciónRESUMEN
Carbohydrate (CHO) ingestion during exercise has been shown to reduce perturbations in immune cell numbers and function, possibly through a reduction in the cortisol response to exercise. We have previously observed that exercise decreases T-lymphocyte responses to mitogen via an increase in cell death of both CD4 and CD8 T lymphocytes (Green KJ and Rowbottom DG. J Appl Physiol. 95: 57-63, 2003). This study tested the hypothesis that CHO ingestion rather than placebo (Pl) would result in an attenuation of the cortisol response to exercise and a reduction of the exercise-associated alterations in cell death. Six well-trained cyclists completed two exercise trials consisting of 2.5 h of cycling at 85% of individual ventilatory threshold. In a random order, trials were completed under either CHO (6% CHO solution, 3.2 g CHO/kg body wt total) or Pl conditions. Blood samples were collected before exercise, midexercise (after 60 min of exercise), immediately after exercise, and after 60 min of recovery. T-lymphocyte responses to mitogen were determined by using carboxyfluorescein diacetate succinimidyl ester fluorescent cell division tracking and expansion rates, and cell death rates were calculated for each sample as well as mitosis rates for each cell generation. Cellular expansion of T lymphocytes was decreased after exercise in Pl only. The reduction in cellular expansion was related to an increase in cell death of both CD4 and CD8 cells in culture rather than a decrease in the ability of cells to undergo mitosis. CHO ingestion compared with Pl was associated with no reductions in cellular expansion or increases in cell death. CHO ingestion during exercise acted to reduce the impairment of T-lymphocyte function by decreasing cell death within mitogen-stimulated cell cultures; however, the mechanism of action appears to be independent of cortisol.
Asunto(s)
Carbohidratos de la Dieta/farmacología , Ejercicio Físico/fisiología , Linfocitos T/fisiología , Adulto , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Ciclismo/fisiología , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/fisiología , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/fisiología , Separación Celular , Células Cultivadas , Prueba de Esfuerzo , Humanos , Hidrocortisona/sangre , Recuento de Leucocitos , Masculino , Mitosis/fisiología , Consumo de Oxígeno/fisiología , Linfocitos T/efectos de los fármacosRESUMEN
Multiple HLA class I alleles can bind peptides with common sequence motifs due to structural similarities in the peptide binding cleft, and these groups of alleles have been classified into supertypes. Nine major HLA supertypes have been proposed, including an A24 supertype that includes A*2301, A*2402, and A*3001. Evidence for this A24 supertype is limited to HLA sequence homology and/or similarity in peptide binding motifs for the alleles. To investigate the immunological relevance of this proposed supertype, we have examined two viral epitopes (from EBV and CMV) initially defined as HLA-A*2301-binding peptides. The data clearly demonstrate that each peptide could be recognized by CTL clones in the context of A*2301 or A*2402; thus validating the inclusion of these three alleles within an A24 supertype. Furthermore, CTL responses to the EBV epitope were detectable in both A*2301(+) and A*2402(+) individuals who had been previously exposed to this virus. These data substantiate the biological relevance of the A24 supertype, and the identification of viral epitopes with the capacity to bind promiscuously across this supertype could aid efforts to develop CTL-based vaccines or immunotherapy. The degeneracy in HLA restriction displayed by some T cells in this study also suggests that the dogma of self-MHC restriction needs some refinement to accommodate foreign peptide recognition in the context of multiple supertype alleles.
Asunto(s)
Pruebas Inmunológicas de Citotoxicidad/métodos , Citotoxicidad Inmunológica , Epítopos de Linfocito T/metabolismo , Antígenos HLA-A/inmunología , Antígenos HLA-A/metabolismo , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/virología , Alelos , Línea Celular , Separación Celular , Células Clonales , Citomegalovirus/inmunología , Citomegalovirus/metabolismo , Relación Dosis-Respuesta Inmunológica , Antígeno HLA-A24 , Herpesvirus Humano 4/inmunología , Herpesvirus Humano 4/metabolismo , Humanos , Oligopéptidos/inmunología , Oligopéptidos/metabolismo , Unión Proteica/inmunología , Linfocitos T Citotóxicos/metabolismo , Proteínas de la Matriz Viral/inmunología , Proteínas de la Matriz Viral/metabolismoRESUMEN
PURPOSE: This study aimed to determine the effect of acute exercise on the proliferation and expression of activation markers on T-lymphocytes. METHODS: Seventeen well-trained male endurance runners completed 60 min of treadmill running at 95% of ventilatory threshold and a resting, no exercise, control session at the same time of day. Five blood samples were collected at each session: before exercise, mid-exercise, immediately after exercise, and 30 min and 60 min after exercise. Isolated peripheral blood mononuclear cells (PBMC) were stimulated with the mitogen PHA. Activation was measured using the expression of CD69 (assessed by three-color flow-cytometry), and cellular proliferation was assessed using 3-(4,5-dimethlthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye uptake. RESULTS: At all sampling points, there was a significant difference (P < 0.05) in the percentage of CD4 and CD8 cells that became activated (CD69+) after mitogen stimulation (68% of CD4 compared with 45% of CD8 cells). Exercise had no effect on the percentage of cells that became activated in response to mitogen. There was a significant exercise-induced decrease in lymphocyte proliferation of PBMC, but when expressed per-T-cell (CD3+), there was no difference between the exercise and control condition. CONCLUSION: This study indicated that on an individual cell basis 1 h of exercise at 95% of ventilatory threshold did not alter the ability of T-lymphocytes (CD3+) or T-lymphocyte subsets (CD3+CD4+ and CD3+CD8+) to become activated and did not alter the ability of T-lymphocytes to proliferate.
Asunto(s)
Antígenos CD/biosíntesis , Antígenos de Diferenciación de Linfocitos T/biosíntesis , Ejercicio Físico/fisiología , Activación de Linfocitos/inmunología , Linfocitos T/inmunología , Adulto , Antígenos CD/análisis , Antígenos de Diferenciación de Linfocitos T/análisis , División Celular , Citometría de Flujo , Humanos , Lectinas Tipo C , Subgrupos Linfocitarios , Masculino , Fitohemaglutininas/administración & dosificación , Fitohemaglutininas/inmunología , Carrera/fisiología , Factores de TiempoRESUMEN
Carboxyfluorescein diacetate succinamidyl ester (CFSE) labeling of lymphocyte populations can provide unique insights into cell function at rest and with exercise, due to its ability to quantify cell division on an individual cell basis. This study aimed to characterize the effect of acute, intense exercise on T-lymphocyte function. Well-trained endurance runners completed 60 min of treadmill running at 95% of individual anaerobic threshold. Blood samples were collected before exercise; after 30 and 60 min of exercise; and after 30, 60, and 90 min of recovery. Isolated peripheral blood mononuclear cells were labeled with CFSE and cultured with or without mitogen (phytohemagglutinin). After culture, cell suspensions were labeled with CD3 (allophycocyanin) and CD8 (phycoerythrin), and expansion rates and cell death rates were calculated for each sample, as well as mitosis rates for each cell generation. Exercise was associated with a 60% decrease in cell expansion in both CD4 and CD8 cell types from before exercise to midexercise (P < 0.05). The significant decrease in expansion rate in the midexercise samples for both cell types was mirrored by a 65% increase in cell death (P < 0.05) in both cell types at that sample point. Exercise had no effect on the mitosis rate of either CD4 or CD8 cells in any cell generation (generations 0-3). This study indicates that 1 h of intense exercise affects in vitro T-lymphocyte function. These data suggest, for the first time, that exercise decreases cell expansion rate via an increase in cell death of both CD4 and CD8 T lymphocytes, rather than a decrease in mitosis.
Asunto(s)
Ejercicio Físico/fisiología , Fluoresceínas , Colorantes Fluorescentes , Mitógenos/farmacología , Succinimidas , Linfocitos T/fisiología , Adulto , Recuento de Linfocito CD4 , Linfocitos T CD4-Positivos/fisiología , Linfocitos T CD8-positivos/fisiología , Muerte Celular/fisiología , Células Cultivadas , Prueba de Esfuerzo , Citometría de Flujo , Humanos , Hidrocortisona/sangre , Recuento de Linfocitos , Masculino , Mitosis/fisiología , Resistencia Física , Fitohemaglutininas/farmacología , Carrera/fisiología , Linfocitos T/efectos de los fármacosRESUMEN
This study utilized recently developed microbead technology to remove natural killer (NK) cells from peripheral blood mononuclear cell (PBMC) preparations to determine the effect of acute exercise on T-lymphocyte function, independent of changes in lymphocyte subpopulations. Twelve well-trained male runners completed a 60-min exercise trial at 95% ventilatory threshold and a no-exercise control trial. Six blood samples were taken at each session: before exercise, midexercise, immediately after exercise, and 30, 60, and 90 min after exercise. Isolated PBMC and NK cell-depleted PBMC were stimulated with the mitogen phytohemagglutinin. Cellular proliferation was assessed by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye uptake. In the PBMC cultures, there was a significantly lower mitogen response to phytohemagglutinin in exercise compared with the control condition immediately postexercise. There were no significant differences between the control and exercise conditions in NK cell-depleted PBMC cultures or in the responses adjusted for the percentage of CD3 cells. The present findings do not support the view that T-lymphocyte function is reduced after exercise.
Asunto(s)
Ejercicio Físico/fisiología , Linfocitos T/fisiología , Adulto , Complejo CD3/análisis , División Celular/efectos de los fármacos , Células Cultivadas , Humanos , Células Asesinas Naturales , Leucaféresis , Recuento de Leucocitos , Masculino , Monocitos/citología , Monocitos/inmunología , Fitohemaglutininas/farmacología , Valores de Referencia , Carrera , Factores de TiempoRESUMEN
It is now widely accepted that athletes undergoing intensive training and competition schedules are at increased risk of developing upper respiratory tract infections (URTI). T-lymphocytes are central to cell-mediated adaptive immune responses and have been the subject of many studies investigating the relationship between T-lymphocyte function, exercise and athlete health. A decrease in T-lymphocyte function following acute intensive exercise has commonly been described, making them a possible factor contributing to increased illness susceptibility in athlete populations. However, there is much controversy regarding the interpretation of traditional methodology (mitogen-induced proliferation assays) used to assess T-lymphocyte function during and after exercise. Carboxyfluorescein diacetate succinimidyl ester (CFSE) labelling of lymphocyte populations can provide unique insights into cell function at rest and with exercise due to its ability to quantify cell division on an individual cell basis. Using this technique, it has been found that the effect of exercise on T-lymphocyte function is mediated via an increase in lymphocyte apoptosis. This paper discusses some recent data in the context of improving understanding of the exercise effects on in vitro T-lymphocyte function.