RESUMEN
It is generally considered that myoblasts are unable to prime naive T cell responses without help from professional antigen-presenting cells (APC). However, their ability to present endogenous antigens to previously primed T lymphocytes in the secondary phase of a T cell response has not been well studied. We show here that primary human myoblasts, when stimulated with IFNgamma to express class II MHC, can present an endogenous epitope, probably an acetylcholine receptor (AChR) peptide, to a CD4(+) AChR-specific T helper lymphocyte clone. Presentation leads to secretion of IFNgamma by the T cell clone and, in addition, killing of the myoblast. Our results suggest that, during the effector phase of the immune response, myoblasts could enhance the inflammatory response by presenting endogenous antigen, and thereby become targets for CD4(+) T lymphocyte-induced cytotoxicity; subsequent release of myoblast antigens could then lead to inter- and intra-molecular determinant spreading.
Asunto(s)
Autoinmunidad/inmunología , Epítopos/inmunología , Músculos/inmunología , Miastenia Gravis/inmunología , Receptores Colinérgicos/inmunología , Presentación de Antígeno/inmunología , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , División Celular/inmunología , Células Cultivadas , Células Clonales , Citocinas/biosíntesis , Relación Dosis-Respuesta Inmunológica , Humanos , Músculos/citología , Músculos/metabolismo , Fragmentos de Péptidos/inmunología , Receptores Colinérgicos/biosíntesisRESUMEN
Paraneoplastic cerebellar degeneration (PCD) occurs as a non-metastatic manifestation of cancer in a small proportion of patients with certain breast or gynaecological tumours, and is characterised by widespread Purkinje cell loss. Antibodies against a Purkinje cell cytoplasmic antigen, called Yo, that is expressed by the tumours, are present in the majority of these patients, but the pathogenic role of the antibodies is not clear. To characterise further the immune response in these cases, 13 anti-Yo positive sera were tested for IgG subclasses by immunohistochemistry and western blotting and, in four cases, PHA-stimulated cytokine secretion by peripheral blood lymphocytes was measured. Surprisingly, anti-Yo antibodies were entirely restricted to the IgG1 subclass, whereas antibodies against the small cell cancer-associated antigen, Hu, were found in all four IgG subclasses. There was a trend towards raised IgG1 levels in the total IgG of the anti-Yo positive patients and, in two, PHA-stimulated peripheral blood lymphocytes secreted raised levels of IFN-gamma. By contrast, in the other two cases tested, raised levels of IL-4 were secreted. Patients with PCD associated with anti-Yo antibodies appear to have strong immune responses that are polarised with respect to the IgG subclass and Th cytokine profiles.
Asunto(s)
Proteínas de Unión al ADN/inmunología , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Proteínas de Neoplasias/inmunología , Degeneración Cerebelosa Paraneoplásica/inmunología , Animales , Autoanticuerpos/sangre , Autoanticuerpos/inmunología , Autoantígenos , División Celular/inmunología , Proteínas ELAV , Humanos , Interferón gamma/inmunología , Interleucina-4/inmunología , Proteínas del Tejido Nervioso/inmunología , Proteínas de Unión al ARN/inmunología , Ratas , Ratas Endogámicas Lew , Células TH1/citología , Células TH1/inmunología , Células Th2/citología , Células Th2/inmunologíaRESUMEN
OBJECTIVE: To identify distinctive sequence motifs required for productive peptide presentation by those HLA-DR alleles/DR4 subtypes that predispose to rheumatoid arthritis (RA). METHODS: We tested 10 different HLA-DR4 subtypes for presentation of acetylcholine receptor peptides to 8 different DR4-restricted T cell lines/clones in proliferation assays. RESULTS: Seven of the 8 T cells depended absolutely on either the autologous Lys71 (in Dw4) or Arg71 (e.g., Dw14), despite these alleles' similar charge and RA associations. In contrast, the PM-A T cell was only mildly affected by this interchange. Moreover, after minor modifications, peptides were presented to this unusual T cell preferentially by all the RA-associated subtypes of DR4 as well as by 2 other DR alleles (DR1 and DR1402) that predispose to RA. CONCLUSION: This coincident cross-restriction to all the RA-associated HLA-DR alleles except DR10 shows that there could even be a single arthritogenic peptide; we now suggest a possible consensus motif.
Asunto(s)
Artritis Reumatoide/patología , Células Clonales/inmunología , Antígenos HLA-DR/genética , Linfocitos T/citología , Alelos , Células Presentadoras de Antígenos , Arginina/genética , Reacciones Cruzadas , Glicina/genética , Antígenos HLA-DR/inmunología , Humanos , Lisina/genética , Mutación Puntual , Conformación Proteica , Triptófano/genéticaRESUMEN
Several isolated dimorphisms recur in many HLA class II alleles, but it is not clear whether they merely influence the binding of peptides locally or have more general effects on their recognition by T cells. For example, interchanges in HLA-DRbeta include 86Gly <--> Val and 57Asp <--> Ser at either end of its alpha helix, and 71Arg <--> Lys in the middle. In DR4, the existence of six subtypes differing by single substitutions at these sites enabled us to assess their functional effects--both in isolation and in their natural context--on peptide presentation to a specific T cell clone with unusually broad cross-restrictions. Unexpectedly, the restriction imposed by 86Val was much more severe in the context of 71Arg than 71Lys, but was also more readily overcome by reducing the bulk of the 'p1' peptide 'anchor' residue (149Trp --> Phe). Moreover, when there was also a distant 57Asp-->Ser substitution, compensating similarly for 86Val proved much more difficult. Thus 86Val and 57Ser in combination had far more drastic effects on peptide presentation than they did separately, when peptide binding was also largely unchanged. These and other interactions with position 71 together provide strong evidence that the configuration of the peptide-DR4 complex is critical for T cell recognition, which could be affected by subtle conformational influences on the p1-9 core of the peptide or on the alpha helix of DR4beta (between positions 86 and 57). Ideally, therefore, the effects of individual class II substitutions should be considered in their natural context rather than in isolation.
Asunto(s)
Antígeno HLA-DR4/genética , Fragmentos de Péptidos/inmunología , Linfocitos T/inmunología , Alelos , Secuencia de Aminoácidos , Línea Celular , Epítopos , Antígeno HLA-DR4/química , Humanos , Datos de Secuencia MolecularRESUMEN
A recurring epitope in the human acetylcholine receptor (AChR) alpha subunit (alpha146-160) is presented to specific T cells from myasthenia gravis patients by HLA-DRB3*0101-"DR52a"-or by DR4. Here we first map residues critical for DR52a in this epitope by serial Ala substitution. For two somewhat similar T cells, this confirms the recently deduced importance of hydrophobic "anchor" residues at peptide p1 and p9; also of Asp at p4, which complements this allele's distinctive Arg74 in DRbeta. Surprisingly, despite the 9 sequence differences in DRbeta between DR52a and DR3, merely reducing the bulk of the peptide's p1 anchor residue (Trp149-->Phe) allowed maximal cross-presentation to both T cells by DR3 (which has Val86 instead of Gly). The shared K71G73R74N77 motif in the alpha helices of DR52a and DR3 thus outweighs the five differences in the floor of the peptide-binding groove. A second issue is that T cells selected in vitro with synthetic AChR peptides rarely respond to longer Ag preparations, whereas those raised with recombinant subunits consistently recognize epitopes processed naturally even from whole AChR. Here we compared one T cell of each kind, which both respond to many overlapping alpha140-160 region peptides (in proliferation assays). Even though both use Vbeta2 to recognize peptides bound to the same HLA-DR52a in the same register, the peptide-selected line nevertheless proved to depend on a recurring synthetic artifact-a widely underestimated problem. Unlike these contaminant-responsive T cells, those that are truly specific for natural AChR epitopes appear less heterogeneous and therefore more suitable targets for selective immunotherapy.
Asunto(s)
Presentación de Antígeno , Epítopos de Linfocito B/metabolismo , Epítopos de Linfocito T/metabolismo , Antígenos HLA-DR/inmunología , Receptores Colinérgicos/inmunología , Alelos , Secuencia de Aminoácidos , Sustitución de Aminoácidos/inmunología , Animales , Línea Celular , Epítopos de Linfocito B/química , Epítopos de Linfocito B/inmunología , Epítopos de Linfocito T/química , Epítopos de Linfocito T/inmunología , Antígenos HLA-DR/química , Antígenos HLA-DR/metabolismo , Humanos , Ratones , Datos de Secuencia Molecular , Miastenia Gravis/inmunología , Péptidos/síntesis química , Péptidos/inmunología , Péptidos/metabolismo , Unión Proteica/genética , Unión Proteica/inmunología , Receptores Colinérgicos/química , Receptores Colinérgicos/genética , TorpedoRESUMEN
No immunodominant T-cell epitopes have yet been reported in the human acetylcholine receptor (AChR), the target of the pathogenic autoantibodies in myasthenia gravis (MG). We have selected and characterized T cells from MG patients by restimulation in culture with recombinant human AChR to alpha, gamma and epsilon subunits; the gamma and epsilon distinguish the fetal and adult AChR isoforms, respectively. We obtained clones specific for the epsilon, rather than the alpha or gamma, subunit in 3 of the first 4 early-onset MG cases tested. They all responded to peptide epsilon201-219 and to low concentrations of adult but not fetal AChR. Moreover, although using different T-cell receptor genes, they were all restricted to HLA-DR52a (DRB3*0101), a member of the strongly predisposing HLA-A1-B8-DR3 haplotype. This apparently immunodominant epsilon201-219 epitope (plus DR52a) was also recognized by clones from an elderly patient whose MG had recently been provoked by the drug D-penicillamine. In all 4 cases, however, the serum antibodies reacted better with fetal than adult AChR and may thus be end products of determinant spreading initiated by adult AChR-specific T cell responses. Furthermore, as these T cells had a pathogenic Th1 phenotype, with the potential to induce complement-activating antibodies, they should be important targets for selective immunotherapy.
Asunto(s)
Epítopos de Linfocito T/inmunología , Miastenia Gravis/genética , Miastenia Gravis/inmunología , Receptores Colinérgicos/inmunología , Secuencia de Aminoácidos , Secuencia de Bases , Humanos , Datos de Secuencia Molecular , Fenotipo , Receptores de Antígenos de Linfocitos T/inmunología , Factores de TiempoAsunto(s)
Miastenia Gravis/inmunología , Miastenia Gravis/patología , Receptores Colinérgicos/inmunología , Linfocitos T/inmunología , Timoma/inmunología , Timo/patología , Neoplasias del Timo/inmunología , Secuencia de Aminoácidos , Animales , Variación Genética , Antígenos HLA-DP/inmunología , Humanos , Datos de Secuencia Molecular , Fragmentos de Péptidos/inmunología , Receptores Colinérgicos/química , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Timectomía , Timoma/cirugía , Timo/inmunología , Neoplasias del Timo/patologíaRESUMEN
Using an acetylcholine receptor-specific T-cell clone (TCC) from a myasthenia gravis patient, we have compared the induction of unresponsiveness by three agents: an anti-V beta monoclonal antibody (mAb), complexes of MHC class II with specific peptide (DR4:peptide) and free peptide. Pretreatment with each agent without antigen-presenting cells specifically rendered the TCC unresponsive to a subsequent optimal stimulus. A substantial proportion of the DR4:peptide pretreated cells underwent apoptosis and the remainder were profoundly unresponsive. Apoptosis was less prominent after pretreatment with free peptide and was not significant with anti-V beta mab; with both, the unresponsiveness was transient in the survivors. These diverse effects of engaging the T-cell receptor in the absence of costimulation suggest that these agents act via different mechanisms, and give insights to the potential for specific immunotherapy.
Asunto(s)
Miastenia Gravis/metabolismo , Miastenia Gravis/patología , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores Colinérgicos/metabolismo , Linfocitos T/fisiología , Anticuerpos Monoclonales/inmunología , Apoptosis/fisiología , Supervivencia Celular/fisiología , Células Clonales , Relación Dosis-Respuesta a Droga , Antígenos de Histocompatibilidad Clase II/inmunología , Humanos , Interleucina-2/farmacología , Cinética , Ligandos , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Linfocitos T/metabolismo , Factores de TiempoAsunto(s)
Autoantígenos/inmunología , Miastenia Gravis/inmunología , Miastenia Gravis/terapia , Receptores Colinérgicos/inmunología , Linfocitos T/inmunología , Animales , Anticuerpos Monoclonales/uso terapéutico , Humanos , Inmunoterapia/métodos , Placa Motora/inmunología , Placa Motora/fisiología , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/terapia , Receptores de Antígenos de Linfocitos T/inmunología , Proteínas Recombinantes/inmunología , Timoma/inmunología , Neoplasias del Timo/inmunologíaRESUMEN
1725 Irish females aged 15-70+ were included in this analysis to establish the group bone mineral density (BMD) profiles. Each subject was screened for 27 risk factors by questionnaire. Subjects were segregated into normal (N) and non-normal (NN) subgroups on the basis of the questionnaire. BMD of lumbar spine and hip were measured by dual energy X ray absorptiometry (DEXA). A substantial difference in BMD at all sites was observed between subgroups even following adjustment for body mass index and years of exposure to oestrogen. In the subgroup designated as non-normal 42%, 54%, 72% and 69% of those studied had spinal BMD levels > 2SD below mean peak at 55-59, 60-64, 65-69 and 70+ respectively. In comparison, similar age cohorts in the subgroup designated normal were 26%, 46%, 44% and 68% respectively. A marked difference was also observed in the younger age groups (15-34) where the percentage of individuals with spinal BMD levels > 2SD below mean peak was < 1.9% for N and 18.8% for NN. The results of this study suggest that the use of a questionnaire may be a valuable low cost screening tool in the younger groups.
Asunto(s)
Densidad Ósea , Osteoporosis/epidemiología , Adolescente , Adulto , Distribución por Edad , Anciano , Femenino , Humanos , Incidencia , Irlanda/epidemiología , Persona de Mediana Edad , Osteoporosis/diagnóstico , Análisis de Regresión , Factores de RiesgoRESUMEN
The resistance by T lymphocytes to activation by antigen (anergy) is well documented for CD4+ T-helper (Th) cells, although less is known about CD8+ cytotoxic T lymphocytes (CTL). One widely used method of inducing anergy of CD4+Th is presentation of antigen by ECDI (1-ethyl-3-(3-dimethylamino-propyl)carbodiimide)-fixed antigen-presenting cells (APCs). We report here that in murine mixed lymphocyte reactions (MLRs), a marked reduction in detected cytotoxicity (which is mediated predominantly by CD8+ CTL) occurs on day 7 if the bulk cultures are restimulated 2 days previously with ECDI-fixed allogeneic splenocytes. No differences were seen between untreated cultures on days 5 and 7, or on day 7 of cultures to which were added unfixed allogeneic splenocytes, fixed or unfixed syngeneic splenocytes, or 'third-party' allogeneic splenocytes, 2 days previously. The effect is not mediated directly on CD8+ cells, since MLRs depleted of CD4+ cells immediately prior to exposure to fixed allogeneic splenocytes fail to show reduced lysis. On the other hand, reduced lysis did occur if CD4+ cells, purified from the MLRs on day 4, were exposed to ECDI-fixed allogeneic splenocytes and then returned to MLRs previously depleted of CD4+ cells. Moreover the effect is overcome using exogenous interleukin-2 (IL-2). We propose that CD4+ cells, restimulated by a regimen shown previously to induce their anergy, can cause a reduction in CD(8+)-mediated cytotoxicity in MLRs.