RESUMO
MRL/lpr/lpr (MRL/l) mice develop a lupus-like syndrome and a disease histologically and serologically similar to human rheumatoid arthritis. Their sera contain polyclonal IgM rheumatoid factors (RF) reactive with all murine IgG subclasses (frequently strongest with IgG2a) and several heterologous IgG. To examine the repertoire and epitopic specificities of these RF, we fused splenocytes from 3.5-mo-old seropositive MRL/l mice with appropriate myeloma partners and derived 1,723 hybridomas of which 23 secreted IgMRF. These monoclonal IgMRF bound to murine IgG only, not to other murine isotypes. Eight murine IgG subclass-specific clonotypes were identified. Most clones reacted with either multiple IgG subclasses or with IgG2a alone. A few clones reacted solely with IgG2b but none reacted exclusively with IgG1 or IgG3. Monoclonal IgMRF with exclusively anti-IgG2a activity exhibited allotypic specificity, reacting, with few exceptions, with a, c, and e, but not b, d, or j IgG2a allotypes. Four clonotypes could be distinguished by cross-reactivity with IgG from species other than mice. Monoclonals possessing activity against several murine subclasses cross-reacted extensively with heterologous IgG, including all human IgG subclasses without allotypic restrictions. Monoclonal IgMRF specific for murine IgG2a or 2b did not cross-react with heterologous IgG. Based on the absence of cross-reactions by IgG2a-specific monoclonal autoantibodies, certain peptides of the IgG CH2 and CH3 domains appear to generate the antigenic determinants of the anti-IgG2a RF in MRL/l mice. All of the monoclonal RF bound to Fc and, with one exception, not to Fab fragments of murine IgG. Binding of the monoclonal RF to substrate IgG was not inhibited by Clq, thus excluding the Clq-binding site at the CH2 domain as one of the responsible epitopes in the induction of MRL/l RF. mIgMRF could be categorized as strongly, weakly, or noninhibitable by protein A, which interacts with IgG molecules at or near the CH2-CH3 junction. Inhibition appears to be caused by conformational changes and/or steric shielding of certain IgG areas distant from this junction and not by identical binding sites between protein A and RF. Certain of the mIgMRF that were weakly or not at all inhibitable by protein A were found to cross-react equally well with human Fc (CH2-CH3 domains) and pFc' (CH3 domain) fragments, indicating that the binding site for these monoclonals is at the CH3 domain. Monoclonal RF were devoid of anti-double-strand DNA, anticollagen, or antipeptidoglycan pentapeptide cross-reactivity, but one of the monoclonals cross-reacted with histones, four with single-strand DNA, and one with both histones and single-strand DNA.
Assuntos
Anticorpos Monoclonais/genética , Artrite Reumatoide/imunologia , Camundongos Endogâmicos/imunologia , Fator Reumatoide/genética , Animais , Anticorpos Anti-Idiotípicos/análise , Anticorpos Monoclonais/análise , Anticorpos Monoclonais/fisiologia , Sítios de Ligação de Anticorpos , Reações Cruzadas , Modelos Animais de Doenças , Humanos , Hibridomas/imunologia , Alótipos de Imunoglobulina/análise , Alótipos de Imunoglobulina/imunologia , Fragmentos de Imunoglobulinas/imunologia , Imunoglobulina G/análise , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Imunoglobulina M/biossíntese , Imunoglobulina M/imunologia , Imunoglobulina M/metabolismo , Camundongos , Receptores Fc/análise , Receptores de IgG , Fator Reumatoide/análise , Fator Reumatoide/fisiologia , Especificidade da EspécieRESUMO
We have analyzed tolerance-related clonal deletion of Mls-and I-E-reactive thymocytes at the RNA level using a multi-V beta probe RNAse protection assay, and used this phenomenon to identify the maturation stage of the abnormally expanded CD4-8-, TCR-alpha/beta + subset in lpr and gld homozygous mice, and of the phenotypically similar minor thymocyte subset found in normal mice. Essentially complete V beta clonal deletions were detected in lpr and gld cells of all appropriate background strains. Substantial, but not complete, V beta clonal deletions were also detected in the CD4-8- TCR-alpha/beta + subset of normal mice. Since expression of CD4/CD8 is required for V beta clonal deletions to occur, we conclude that lpr and gld cells, and at least a portion of CD4-8- TCR-alpha/beta + thymocytes in normal mice, are derived by secondary loss of CD4/CD8 accessory molecules from more mature CD4+8+ precursors. One possible interpretation of these findings is that such CD4/CD8 loss may affect a class of self-reactive thymocytes that have escaped direct clonal deletion. Exportation and expansion of such cells in the periphery may be an important contributory factor in the induction of systemic autoimmunity.
Assuntos
Antígenos de Diferenciação de Linfócitos T/fisiologia , Doenças Autoimunes/imunologia , Antígenos CD4/fisiologia , Deleção Cromossômica , Tolerância Imunológica , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T/imunologia , Animais , Antígenos CD8 , Transtornos Linfoproliferativos/imunologia , Camundongos , Camundongos Endogâmicos C3H , RNA Mensageiro/análiseRESUMO
B cell hyperactivity, a feature common to all lupus-prone murine strains, may be caused by hyperresponsiveness to, overproduction of, or bypassing of certain signals required for B cell activation, proliferation, and differentiation. In this study, we have compared the responses of B cells from three lupus-prone strains of mice (BXSB males, MRL and NZB/W females) and normal strains in a number of assays for which two or more signals are required to obtain a response. In medium to low density cultures of B cells from BXSB and NZB/W but not MRL/l lupus mice, the cells' proliferation induced by bacterial lipopolysaccharide (LPS) or anti-mu antibody was much higher than that of B cells from normal controls. At low B cell density, polyclonal activation by these substances and subsequent Ig secretion were dependent on accessory signals present in supernatants of concanavalin A-treated normal lymphocytes (CAS) or on the MRL/l proliferating T cell-derived B cell differentiation factor (L-BCDF) in both lupus-prone and immunologically normal mice. However, the responses of B cells from BXSB and NZB/W, but not MRL/l, mice to these accessory signals were higher than those of normal mice. Ig synthesis by fresh B cells of BXSB and NZB/W mice cultured in the absence of mitogens but in the presence of CAS or L-BCDF was higher than by similar cells from other strains, suggesting an increased frequency of B cells activated in vivo in these two autoimmune strains of mice. The patterns of IgG subclass secretion in response to LPS (without added CAS or L-BCDF) were abnormal in all lupus strains, with a predominance of IgG2b and/or IgG2a and low levels of IgG3, contrary to normal B cells for which IgG3 synthesis predominated. However, IgG1 synthesis in vitro by autoimmune and normal B cells alike was highly dependent on T cell-derived soluble mediators. Antigen-specific responses to SRBC in vitro of B cells from all lupus strains, like those of B cells from normal strains, required a minimum of three signals (antigen, LPS, T cell-derived antigen nonspecific helper factors). Yet, once triggered, B cells of BXSB and NZB/W mice gave higher responses than those of the other strains. We conclude that B cells of lupus mice have signal requirements similar to those of normal mice. Nevertheless, B cells of BXSB and NZB/W, but not MRL/l, lupus mice hyperrespond or process some accessory signals abnormally.
Assuntos
Linfócitos B/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Camundongos Endogâmicos/imunologia , Animais , Anticorpos/imunologia , Diferenciação Celular , Divisão Celular , Células Cultivadas , Concanavalina A/farmacologia , Feminino , Imunoglobulina G/biossíntese , Cadeias mu de Imunoglobulina/imunologia , Imunoglobulinas/biossíntese , Lipopolissacarídeos/imunologia , Lúpus Eritematoso Sistêmico/genética , Masculino , Camundongos , Camundongos Endogâmicos NZB/imunologiaRESUMO
In young adulthood, MRL/Mp-lpr/lpr mice develop severe systemic lupus erythematosus (SLE)-like syndrome associated with massive T cell proliferation. The congenic MRL/Mp- mice lack the lpr gene and develop chronic SLE late in life. We have exchanged thymic transplants between these substrains so as to determine the role of the thymus in the development of early, severe SLE and of lymphoproliferation. The median survival times of unmanipulated lpr/lpr and mice were 160 and 510 d, respectively. The lpr/lpr and mice thymectomized when newborn and transplanted at 1 mo with the opposite type of thymus retained the diseases phenotype of their unmanipulated counterparts with 50% mortality at 186 and 498 d, respectively. In contrast, lpr/lpr mice thymectomized when newborn but not transplanted with thymus did not develop lymphoid hyperplasia and glomerulonephritis, and 100% of them were alive at 390 d. Serologically, the thymectomized but untransplanted lpr/lpr mice had significantly reduced levels of autoantibodies, whereas thymectomized and transplanted mice of either substrain were similar to unmanipulated controls. The results indicate that: (a) a thymus is essential for expression of lymphoproliferation and early SLE-like disease in the lpr/lpr phenotype; (b) the lpr/lpr disease is not a result of a unique hormonal or microenvironmental defect(s) of the thymus of this substrain because the genotype of the thymus is irrelevant for the development of T cell proliferation and early SLE; (c) differentiation of stem cells under the hormonal or microenvironmental influences of a thymus that possesses the lpr genotype does not lead to abnormal T cell differentiation or early autoimmunity; and (d) the lpr/lpr disease cannot be caused exclusively by an intrinsic B cell defect or environmental stimuli that cause B cell polyclonal activation.
Assuntos
Lúpus Eritematoso Sistêmico/genética , Linfócitos T/imunologia , Timo/fisiologia , Animais , Formação de Anticorpos , Modelos Animais de Doenças , Genótipo , Glomerulonefrite/genética , Lúpus Eritematoso Sistêmico/imunologia , Ativação Linfocitária , Camundongos , Camundongos Mutantes , Baço/imunologiaRESUMO
We report that approximately 1/4 of monoclonal rheumatoid factors produced by hybridomas derived from fusions of spleen cells from MRL/lpr/lpr mice with systemic lupus erythematosus (SLE) and arthritis exhibited multiple reactivities with other autoantigens, including dDNA , histones, and/or cytoskeletal-cytoplasmic elements. The patterns of reactivities of most of these clones differed, indicating that each had a separate B cell ancestor. Studies with eluted antibodies demonstrated that a single species of antibody molecules was responsible for the observed multiple reactivities. Inhibition experiments suggested that an antibody combining site may be large enough to accommodate dissimilar epitopes. These findings may provide further insights into the generation and extent of antibody diversity as well as the etiopathogenesis of systemic autoimmune diseases.
Assuntos
Anticorpos Monoclonais/fisiologia , Antígenos/imunologia , Autoantígenos/imunologia , Sítios de Ligação de Anticorpos , Fator Reumatoide/fisiologia , Animais , Anticorpos Monoclonais/classificação , Ligação Competitiva , Reações Cruzadas , DNA de Cadeia Simples/imunologia , DNA de Cadeia Simples/metabolismo , Imunofluorescência , Histonas/imunologia , Histonas/metabolismo , Hibridomas/classificação , Hibridomas/imunologia , Hibridomas/metabolismo , Imunoglobulina G/metabolismo , Camundongos , Camundongos Mutantes , Polinucleotídeos/metabolismoRESUMO
Hemopoietic cells have been reciprocally transferred between two lines of mice (MRL lpr/lpr and MRL +/+) that are congenic, differing only at the lpr (lymphoproliferation) and possibly closely linked genes. The lpr strain develops a significantly more severe and fast-paced lupus-like syndrome than +/+ strain, along with a substantially larger lymphoid mass. The results showed that: (a) hemopoietic cells of such mice were sufficient to induce the respective disease phenotypes in lethally irradiated syngeneic recipients; (b) cells of MRL +/+ mice maturing in an MRL lpr/lpr environment essentially retained the disease-producing characteristics of the donor, i.e., they induced late-life lupus without lymphadenopathy; but (c) MRL lpr/lpr cells transferred into irradiated MRL +/+ recipients unexpectedly failed to induce the early-life severe lupus and lymphoid hyperplasia of the donor, instead they caused a severe wasting syndrome resembling, in many respects, graft-vs.-host disease (GVHD). This GVHD-like syndrome developed after transfer of MRL lpr/lpr fetal liver, bone marrow, or spleen cells, and was not abrogated by elimination of T cells from the inocula. Thymectomy of the MRL +/+ recipients retarded, but did not prevent, the wasting disease. The unidirectional nature of this disease suggests that the lpr mutation conferred either a structural or regulatory defect that interfered, blocked, or altered the expression or structure of certain lymphocyte antigen(s). As a result, the MRL +/+ cells that did express this antigen(s) were recognized as foreign, and stimulated a graft-vs.-host reaction. These findings may allow definition of a new kind of rejection phenomenon caused by non-H-2 products, and may extend our understanding of the means by which the lpr gene adversely affects lymphocyte regulation and homeostasis.
Assuntos
Genes , Doença Enxerto-Hospedeiro/genética , Transtornos Linfoproliferativos/genética , Animais , Medula Óssea/imunologia , Feminino , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/patologia , Imunização Passiva , Transtornos Linfoproliferativos/imunologia , Transtornos Linfoproliferativos/patologia , Masculino , Camundongos , Camundongos Endogâmicos , Baço/imunologia , SíndromeRESUMO
Autoantibodies reacting with chromatin and its components, histones and DNA, are characteristic of the human autoimmune disease SLE and drug-induced lupus, but the mechanisms of their induction remain unknown. Serial serum samples collected over short intervals from lupus-prone MRL/MP-lpr/lpr and BXSB mice were tested by ELISA on chromatin and its substructures to characterize the initial autoimmune response to these antigens. Direct binding studies demonstrated that the early autoantibodies recognized discontinuous epitopes on native chromatin and the (H2A-H2B)-DNA subnucleosome. As the immune response progressed, native DNA and other chromatin constituents generally became antigenic. Based on adsorption studies and IgG subclass restriction, antibodies to native DNA were more related to chromatin than to denatured DNA. The kinetics of autoantibody appearance and the Ig class distribution were similar to the kinetics and distribution seen in antibodies induced by immunization with an exogenous T-dependent antigen. These results are most consistent with the view that autoantibodies reacting with chromatin are generated by autoimmunization with chromatin, and antibodies to native DNA are a subset of the wide spectrum of antichromatin autoantibodies.
Assuntos
Autoanticorpos/imunologia , Cromatina/imunologia , Lúpus Vulgar/imunologia , Adsorção , Envelhecimento/fisiologia , Animais , Anticorpos Antinucleares/imunologia , Formação de Anticorpos/fisiologia , DNA/imunologia , Feminino , Histonas/imunologia , Imunização , Masculino , Camundongos , Camundongos Mutantes , Nucleossomos/imunologia , Análise de RegressãoRESUMO
We have investigated the genetic origin of autoantibody production in several strains of mice that spontaneously develop a systemic lupus erythematosus-like disease. Restriction fragment length polymorphism analyses of gene loci encoding kappa light chain variable regions (Igk-V) demonstrated, as shown previously for the Ig heavy chain locus, that autoantibody production and disease occur in different Igk-V haplotypes. Moreover, autoimmune mice with known genetic derivation inherited their Igk-V loci essentially unaltered from their nonautoimmune ancestors. New Zealand black lupus mice, with unknown genetic derivation, had a possibly recombinant Igk-V haplotype, composed of V kappa loci that were primarily indistinguishable from those of nonautoimmune strains from either of the two potential donor haplotypes. The heavy and light chain gene segments (variable, diversity, joining) encoding anti-DNA antibodies were diverse and often closely related, or even identical, to those found in antibodies to foreign antigens in normal mice. Only 1 of 11 sequenced variable region genes could not be assigned to existing variable region gene families; however, corresponding germline genes were present in the genome of normal mice as well. These data argue against abnormalities in the genes and mechanisms generating antibody diversity in lupus mice and suggest a remarkable genetic and structural diversity in the generation of anti-DNA binding sites.
Assuntos
Anticorpos Antinucleares/genética , DNA/imunologia , Genes de Imunoglobulinas , Região Variável de Imunoglobulina/genética , Cadeias kappa de Imunoglobulina/genética , Lúpus Eritematoso Sistêmico/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antinucleares/isolamento & purificação , Sequência de Bases , Feminino , Genes , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/isolamento & purificação , Cadeias kappa de Imunoglobulina/isolamento & purificação , Lúpus Eritematoso Sistêmico/genética , Camundongos , Camundongos Endogâmicos AKR , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NZB , Dados de Sequência MolecularRESUMO
The three-dimensional structure of the complex of a second anti-peptide antibody (Fab 26/9) that recognizes the same six-residue epitope of an immunogenic peptide from influenza virus hemagglutinin (HA1; 75-110) as Fab 17/9 with the peptide has been determined at 2.8 A resolution. The amino acid sequence of the variable region of the 26/9 antibody differs in 24 positions from that of 17/9, the first antibody in this series for which several ligand-bound and free structures have been determined and refined. Comparison of the 26/9-peptide with the 17/9-peptide complex structures shows that the two Fabs are very similar (r.m.s.d. 0.5 to 0.8 A) and that the peptide antigen (101-107) has virtually the same conformation (r.m.s.d. 0.3 to 0.8 A) when bound to both antibodies. A sequence difference in the 26/9 binding pocket (L94; His in 26/9, Asn in 17/9) results in an interaction with a bound water molecule that is not seen in the 17/9 structures. Epitope mapping shows that the relative specificity of 26/9 and 17/9 antibodies for individual positions of the peptide antigen are slightly different. Amino acid substitutions in the peptide, particularly at position SerP107, are tolerated to different extents by 17/9 and 26/9. Structural and sequence analysis suggests that amino acid differences near the peptide-binding site are responsible for altering slightly the specificity of 26/9 for three peptide residues and illustrates how amino acid substitutions can modify antibody-antigen interactions and thereby modulate antibody specificity.
Assuntos
Anticorpos Antivirais/química , Hemaglutininas Virais/imunologia , Fragmentos Fab das Imunoglobulinas/química , Sequência de Aminoácidos , Anticorpos Monoclonais/química , Anticorpos Antivirais/imunologia , Reações Antígeno-Anticorpo , Sequência de Bases , Sítios de Ligação de Anticorpos , Cristalização , Cristalografia , Ensaio de Imunoadsorção Enzimática , Epitopos/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza , Hemaglutininas Virais/química , Fragmentos Fab das Imunoglobulinas/imunologia , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Conformação ProteicaRESUMO
To define age-associated alterations in the immune system at the molecular level, we have analyzed TCR V beta gene expression patterns at the fetal, neonatal, adult, and advanced ages of mice. In contrast to V gamma and VH genes, V beta genes rearranged without any preference related to their chromosomal organization. Endogenous superantigen-mediated clonal deletions were registered for the first time at the neonatal stage, presumably reflecting the late developmental appearance of these molecules. Such deletions, once established, were maintained throughout life with little, if any, leakage in this process. Furthermore, bone marrow transplantation and other studies indicated that an involuted thymus maintained its capacity to perform both its functions, i.e. positive and negative selection. Although overall V beta repertoires showed remarkable stability with advanced age, modifications in expression levels for some V beta, particularly those associated with the CD8 subset and presumably reflecting antigenic stimulation, were recorded. Mice with lupus and early-life thymic involution were fully capable of deleting endogenous superantigen-reactive V beta clones, and even lupus mice with a genetic defect in the apoptosis-promoting Fas gene were normal in this regard. The results indicate that, aside from some anticipated clonal expansions induced by antigenic stimulation, age-associated alterations in immune functions are not caused by any profound changes in the overall TCR repertoire.
Assuntos
Envelhecimento/genética , Envelhecimento/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Linfócitos T/fisiologia , Animais , Diferenciação Celular/genética , Diferenciação Celular/imunologia , CamundongosRESUMO
Diabetes in NOD mice is an autoimmune disease which is characterized by the infiltration of islets of Langerhans by large numbers of T cells. Some of these infiltrating T cells are clearly islet-cells-specific; however, many or most of these T cells could be attracted nonspecificity into these lesions. To study NOD pancreas-infiltrating T cells, we fused these cells with BW5147 to make T cell hybridomas. Ninety-four pancreas-derived T hybrids were analyzed of which 12 responded specifically to islet cells by secreting IL-2. Only CD3+, CD4+ hybrids responded to islet cells in our assay, and a large proportion of these hybrids were islet-cell reactive. T cell receptor (TCR) V beta element usage was heterogeneous in islet-reactive hybridomas.
Assuntos
Hibridomas/imunologia , Pâncreas/imunologia , Linfócitos T/imunologia , Animais , Animais Recém-Nascidos , Antígenos de Diferenciação de Linfócitos T/imunologia , Complexo CD3 , Antígenos CD4/imunologia , Antígenos CD8/imunologia , Diabetes Mellitus Tipo 1/imunologia , Hibridomas/metabolismo , Interleucina-2/metabolismo , Ilhotas Pancreáticas/imunologia , Camundongos , Camundongos Endogâmicos NOD , Receptores de Antígenos de Linfócitos T/imunologiaRESUMO
T cells are primary participants in the pathogenesis of the MHC-dependent autoimmune diseases, and therefore, evidence for association of TCR V-gene repertoires with such disorders has been actively sought. With very few exceptions, no clear-cut evidence for correlation of particular RFLP-defined V-C-region genomic polymorphisms with autoimmune disease predisposition has thus far been demonstrated. With regard to TCR V-gene repertoires engaged in responses to autoantigens, restricted use of certain V beta and V alpha genes in response to myelin basic protein has been documented in animal models. In many spontaneous and experimentally induced animal and human autoimmune diseases, however, the picture is far from clear. Although dominance of certain TCR V genes has been noted, the clonal restrictions are not absolute; they differ from one study to another and from one patient to another. Such variations may be caused by MHC allele-dependent determinant selection mechanisms, secondary T-cell infiltrates in inflammatory sites, different patient populations and stages of disease, or the involvement of different pathogens that, nevertheless, lead to the same clinical entity. Overall, the results indicate that efforts to intervene therapeutically in autoimmune diseases by vaccination with modified T-cell clones, V region-synthetic peptides, or TCR blocking analogues may not be easily applicable. Further studies on the characterization of the specific antigens involved in autoimmune disease pathogenesis is required in order to accurately address the issue of TCR utilization in autoimmune diseases.
Assuntos
Doenças Autoimunes/imunologia , Autoimunidade , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T/imunologia , Animais , Expressão Gênica , Genes , Humanos , Tolerância ImunológicaAssuntos
Doenças Autoimunes/imunologia , Rearranjo Gênico da Cadeia beta dos Receptores de Antígenos dos Linfócitos T , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Linfócitos T/imunologia , Envelhecimento , Animais , Células Clonais , Humanos , Tolerância Imunológica , Camundongos , Camundongos SCID/imunologia , Ratos , Superantígenos , Linfócitos T/citologiaRESUMO
We describe here the use of a sensitive and accurate multiprobe V beta RNase protection assay in characterizing the expression levels of 17 V beta genes in separated CD4+ and CD8+ subsets of selected mouse strains. The IE-reactive V beta genes (V beta s 11, 12, 5.1 and 16) showed various patterns of skewed subset expression in different strains, suggesting additional influences of IA, class I, and non-MHC genes in the selection process. Clonal deletion of V beta 11- and V beta 12-bearing T cells, among others, was skewed strongly towards the CD4+ subset in many IE+ mouse strains, supporting the notion that negative selection can cause incomplete, subset biased, V beta clonal deletions. Broad analysis in separated CD4+ and CD8+ subsets gave improved resolution of V beta repertoire selection, and revealed significant strain and/or subset specific skewing for additional V beta genes; with consistent bias towards higher expression of V beta 7 and V beta 13 in the CD8+ subset, and V beta 15 in the CD4+ subset of most mouse strains. The influence of diverse non-MHC ligands in V beta repertoire selection was further illustrated by the identification of unique V beta repertoires for six different MHC-identical (H2k) strains. Such polymorphisms in TCR repertoire expression may help to define better disease susceptibility phenotypes.
Assuntos
Linfócitos T CD4-Positivos/fisiologia , Receptores de Antígenos de Linfócitos T/genética , Subpopulações de Linfócitos T/fisiologia , Animais , Expressão Gênica , Rearranjo Gênico da Cadeia beta dos Receptores de Antígenos dos Linfócitos T , Antígenos de Histocompatibilidade Classe II/fisiologia , Camundongos , Camundongos Endogâmicos , Sondas RNA , RNA Mensageiro/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta , Baço/citologia , Timo/citologiaRESUMO
To further define the origin, selection, and diversity of hepatic T cells, we have determined V beta gene expression profiles in double negative (DN, CD4-8-) and single positive (SP, CD4+8- or CD8+4-) alpha beta + liver T cells of DBA/2 mice. These I-E+ mice express mouse mammary tumor (Mtv) provirus-encoded endogenous superantigens of the Mlsa,c type, and thus display deletions/depletions of several V beta-bearing SP cells. Total liver alpha beta + T cells of these mice exhibited an overall V beta expression profile similar to splenic T cells, with the notable exception of high V beta 7 and V beta 8.1 expression. As previously reported, DN alpha beta + T cells were enriched highly in the liver. This subset exhibited a V beta expression profile similar to thymic DN alpha beta + cells with deletions/depletions in several V beta s, but high V beta 7 expression in both populations. Surprisingly, hepatic CD4+ cells also displayed high V beta 7 expression compared with splenic T cells, suggesting that hepatic DN alpha beta + and CD4+ T cells are selected via a common pathway. The V beta 7-expressing DN alpha beta + and CD4+ liver T cell populations were polyclonal, as evidenced by cloning and sequencing. High V beta 7 expression in these cells was undiminished with age. On the basis of V beta repertoire and surface phenotype, DN alpha beta + and/or certain CD4+ T cells seem to constitute a distinct population primarily found in the liver, thymus, and bone marrow. These cells may originate from SP T cells that have down-regulated their accessory molecules under certain activation conditions and, because of the accompanying expression of particular adhesion molecules, they accumulate in tissues such as the liver and thymus.
Assuntos
Antígenos CD4/análise , Antígenos CD8/análise , Fígado/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Linfócitos T/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Feminino , Expressão Gênica , Camundongos , Camundongos Endogâmicos DBA , Dados de Sequência Molecular , Receptores de Antígenos de Linfócitos T alfa-beta/análiseRESUMO
Inbred mice infected with Leishmania major promastigotes display two different courses of leishmaniasis: resistant strains develop self-healing local sores, while susceptible strains show progressive systemic disease with lethal outcome. Resistance predominantly correlates with the production of T helper type 1 (TH1) lymphokines and susceptibility with production of TH2-type lymphokines. Here, we analyzed whether this TH phenotype difference correlates with expression of particular T cell receptor V beta chains. Our results show that T cells expand strongly during infection in all groups of mice and invariantly express the same V beta gene families as prior to infection. Our data indicate that TH1 and TH2 cells use similar V beta gene families, and argue against the engagement of a restricted set of V beta by dominant determinants associated with L. major.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Leishmaniose Cutânea/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Animais , Relação CD4-CD8 , Feminino , Expressão Gênica , Rearranjo Gênico da Cadeia beta dos Receptores de Antígenos dos Linfócitos T , Leishmania major/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , RNA Mensageiro/genéticaRESUMO
Activation/proliferation of mouse and human T and B cells is associated with expression and subsequent release of interleukin 2 receptors (IL 2R) into the milieu. The soluble form of IL 2R, at least in part, retains its ability to bind to IL 2 and to anti-receptor antibodies, but its exact structure remains unknown. Because systemic lupus erythematosus (SLE) is associated with T and B cell activation, we have used monoclonal anti-IL 2R antibodies in an ELISA to measure levels of IL 2R in sera of various lupus strains. High levels of the released receptor were found at an active clinical stage in sera of four autoimmune strains of mice homozygous for the lpr (lymphoproliferation) gene that causes T cell expansion, massive lymphoid organ enlargement, and promotes the autoimmune process. High levels were also found in lupus mice characterized primarily by B cell proliferation (BXSB males) and in (NZB X W)F1 mice characterized by T and B cell activation. Similarly high IL 2R serum levels could be induced experimentally in normal mice injected with immunostimulants such as bacterial lipopolysaccharide or Freund's complete adjuvant. The results indicate that IL 2R serum levels may provide a good marker of ongoing lymphoid cell activation/proliferation, and thus might be useful in the follow-up of patients with systemic autoimmune or other lymphoproliferative disorders. The biologic roles, if any, of the soluble form of IL 2R and its effects in normal and abnormal conditions remain to be determined.
Assuntos
Lúpus Eritematoso Sistêmico/sangue , Receptores Imunológicos/sangue , Animais , Anticorpos Monoclonais/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Adjuvante de Freund/farmacologia , Interleucina-2/metabolismo , Lipopolissacarídeos/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Linfócitos/metabolismo , Camundongos , Camundongos Endogâmicos NZB/sangue , Camundongos Endogâmicos/sangue , Camundongos Mutantes/sangue , Receptores de Interleucina-2RESUMO
The effect of thymic selection on the expressed human T-cell antigen receptor beta-chain variable region (V beta) gene repertoire was examined by using a multiprobe RNase protection assay. The relative abundance of transcripts for 22 V beta genes (encompassing 17 of the 20 human V beta gene subfamilies) within a thymus, and among 17 thymuses, was variable. On the basis of the presence of corresponding mRNAs, no genomic deletions were detected, but several coding region polymorphisms were identified. Analysis of mature T-cell subsets revealed the absence of complete "superantigen"-mediated V beta deletions, suggesting that this phenomenon, in contrast to mouse, is uncommon or absent in humans. However, several V beta genes were over- or underexpressed in one or both mature single-positive (CD4+8- or CD8+4-) thymocyte subsets compared to syngeneic total, mostly immature thymocytes. Whether these changes are induced by relatively weak superantigens or conventional antigens and whether the downshifts are caused by negative selection or lack of positive selection remains to be determined.
Assuntos
Variação Genética , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T/imunologia , Transcrição Gênica , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Recém-Nascido , Substâncias Macromoleculares , Masculino , RNA Mensageiro/análise , RNA Mensageiro/genética , Subpopulações de Linfócitos T/imunologia , Timo/imunologiaRESUMO
The effect of neonatal thymectomy on the induction and/or modification of murine SLE disease was examined in several representative groups of mice with early-life SLE (MRL/Mp-lpr/lpr females, BXSB males, (NZB X W)F1 females, (NZW X BXSB)F1 males and females), late-life SLE (MRL/Mp-+/+ and BXSB females), and normal strains (BALB/c and C57BL/6 females). Our results indicated that thymectomy prevented disease only in the MRL/Mp-lpr/lpr SLE mice, and that this effect diminished as thymectomy was delayed beyond 3 wk post-natally. In the other SLE mice studied, neonatal thymectomy did not modify disease symptoms to any significant degree. Moreover, depletion of mature T cells from donor BXSB male bone marrow did not affect the expression of early-life SLE in thymectomized BXSB female recipients. Neonatal thymectomy did not induce SLE in normal mice. Of note, neonatal thymectomy did not completely deplete the Thy-1.2+ cell population, i.e., 10 to 15% remained in the spleens of the thymectomized mice. This incomplete T cell depletion, together with the previously demonstrated dependence on and hyperresponsiveness of BXSB and (NZB X W)F1 B cells to T helper cell-derived accessory signals, cast doubts on earlier conclusions that B cells from some SLE mice can autonomously proliferate and differentiate to autoantibody-secreting cells. It seems more appropriate to conclude that B cells from the various SLE mice vary in their degree of response to, and production of, T cell-derived helper signals, and thus in their expression of B cell hyperactivity and disease.
Assuntos
Lúpus Eritematoso Sistêmico/imunologia , Timectomia , Envelhecimento , Animais , Antígenos de Superfície/genética , Feminino , Glomerulonefrite/mortalidade , Imunização Passiva , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/fisiopatologia , Masculino , Camundongos , Camundongos Mutantes , Fenótipo , Receptores de Antígenos de Linfócitos B/genética , Baço/citologia , Linfócitos T/imunologia , Antígenos Thy-1RESUMO
We used Southern blotting and mRNA analysis to characterize allelic polymorphisms among genes of the T-cell antigen receptor (TCR) alpha-chain variable-region (V alpha) locus in a large panel of normal and autoimmune-susceptible or autoimmune-contributing strains of laboratory mice. Four major V alpha haplotypes were defined on the basis of multiple restriction fragment length polymorphisms for each of nine V alpha subfamily probes used. Southern blotting also revealed haplotype-specific loss of bands within some V alpha subfamilies, consistent with the deletion of particular V alpha genes or sets of genes from haplotype to haplotype. In contrast to the situation in the V beta locus, however, deletion of entire V alpha subfamilies was not observed. The nature of V alpha allelic variability was further explored by using an RNase protection assay to analyze expressed V alpha mRNA sequences in thymocyte RNA. Such analysis revealed both shared and unique patterns of V alpha mRNA expression among the different haplotypes and supported the conclusion that haplotype differences sometimes involve V alpha gene deletions. Interestingly, a disproportionate number of, but not all, autoimmune-susceptible strains, including NZB, SJL, SWR, PL/J, and NOD, share a common V alpha haplotype. The identification of murine TCR V alpha haplotypes should provide a basis for understanding the role of TCR diversity in normal immunoregulatory and immune-response phenomena, as well as autoimmune-disease predisposition.