RESUMO
Mast cells and basophils are innate immune cells with overlapping functions that contribute to anti-helminth immunity. Mast cell function during helminth infection was previously studied using mast cell-deficient Kit-mutant mice that display additional mast cell-unrelated immune deficiencies. Here, we use mice that lack basophils or mucosal and connective tissue mast cells in a Kit-independent manner to re-evaluate the impact of each cell type during helminth infection. Neither mast cells nor basophils participated in the immune response to tissue-migrating Strongyloides ratti third-stage larvae, but both cell types contributed to the early expulsion of parasitic adults from the intestine. The termination of S. ratti infection required the presence of mucosal mast cells: Cpa3Cre mice, which lack mucosal and connective tissue mast cells, remained infected for more than 150 days. Mcpt5Cre R-DTA mice, which lack connective tissue mast cells only, and basophil-deficient Mcpt8Cre mice terminated the infection after 1 month with wild-type kinetics despite their initial increase in intestinal parasite burden. Because Cpa3Cre mice showed intact Th2 polarization and efficiently developed protective immunity after vaccination, we hypothesize that mucosal mast cells are non-redundant terminal effector cells in the intestinal epithelium that execute anti-helminth immunity but do not orchestrate it.
Assuntos
Intestino Delgado/imunologia , Mastócitos/imunologia , Strongyloides ratti/imunologia , Estrongiloidíase/imunologia , Células Th2/imunologia , Animais , Carboxipeptidases A/genética , Quimases/genética , Imunidade nas Mucosas , Intestino Delgado/parasitologia , Larva , Mastócitos/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Carga Parasitária , Ratos , Ratos Wistar , Triptases/genéticaRESUMO
BACKGROUND: Mastocytosis is characterized by the accumulation of mast cells (MCs) associated with activating mutations of KIT. Tumor necrosis factor-related apoptosis-inducing ligand receptors (TRAIL-Rs) are preferentially expressed on neoplastic cells and induce the extrinsic apoptotic pathway. Recent studies reported on the expression of TRAIL-Rs and TRAIL-induced apoptosis in cultured human MCs, which depend on stem cell factor (SCF)-induced or constitutive KIT activation. MATERIAL AND METHODS: We sought to further define the impact of TRAIL-Rs on MCs in vivo and in vitro. Using Cre/loxP recombination, we generated mice with MC-specific and ubiquitous knockout of TRAIL-R. In these mice, anaphylaxis and numbers of MCs were investigated. We also explored the expression and function of TRAIL-Rs in cultured murine and human MCs upon activation of KIT. By conducting immunofluorescence staining, we analyzed the expression of TRAIL-Rs in MCs infiltrating the bone marrow of patients with mastocytosis. RESULTS: MC-specific deletion of TRAIL-R was associated with a slight, but significant increase in anaphylaxis. Numbers of MCs in MC-specific knockouts of TRAIL-R were comparable to controls. Whereas cultured IL-3-dependent murine MCs from wild-type mice were resistant to TRAIL-induced apoptosis, SCF-stimulated MCs underwent apoptosis in response to TRAIL. Interestingly, activating KIT mutations also promoted sensitivity to TRAIL-mediated apoptosis in human MCs. In line with these findings, MCs infiltrating the bone marrow of patients with mastocytosis expressed TRAIL-R1. CONCLUSIONS: Activation of KIT regulates the function of TRAIL-Rs in MCs. TRAIL-R1 may represent an attractive diagnostic and therapeutic target in diseases associated with KIT mutations, such as mastocytosis.
Assuntos
Mastócitos/imunologia , Mastócitos/metabolismo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Medula Óssea/imunologia , Medula Óssea/metabolismo , Medula Óssea/patologia , Contagem de Células , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Regulação da Expressão Gênica , Humanos , Mastocitose/genética , Mastocitose/imunologia , Mastocitose/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Mutação , Proteínas Proto-Oncogênicas c-kit/genética , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Fator de Células-Tronco/metabolismo , Fator de Células-Tronco/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/farmacologiaRESUMO
Aicardi-Goutières syndrome (AGS) is a hereditary autoimmune disease which overlaps clinically and pathogenetically with systemic lupus erythematosus (SLE), and can be regarded as a monogenic variant of SLE. Both conditions are characterized by chronic activation of anti-viral type I interferon (IFN) responses. AGS can be caused by mutations in one of several genes encoding intracellular enzymes all involved in nucleic acid metabolism. Mouse models of AGS-associated defects yielded distinct phenotypes and reproduced important features of the disease. Analysis of these mutant mouse lines stimulated a new concept of autoimmunity caused by intracellular accumulations of nucleic acids, which trigger a chronic cell-intrinsic antiviral type I IFN response and thereby autoimmunity. This model is of major relevance for our understanding of SLE pathogenesis. Findings in gene-targeted mice deficient for AGS associated enzymes are summarized in this review.
Assuntos
Doenças Autoimunes do Sistema Nervoso , Autoimunidade/genética , Interferon Tipo I , Lúpus Eritematoso Sistêmico , Malformações do Sistema Nervoso , Animais , Doenças Autoimunes do Sistema Nervoso/genética , Doenças Autoimunes do Sistema Nervoso/imunologia , Doenças Autoimunes do Sistema Nervoso/patologia , Modelos Animais de Doenças , Marcação de Genes , Humanos , Interferon Tipo I/genética , Interferon Tipo I/imunologia , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/patologia , Camundongos , Camundongos Mutantes , Malformações do Sistema Nervoso/genética , Malformações do Sistema Nervoso/imunologia , Malformações do Sistema Nervoso/patologiaRESUMO
The observation that mast cells accumulate at the periphery of growing tumours is now well documented, and the loss of mast cells correlates with reduced tumour growth. The role of mast cells as innate regulators of both inflammatory and immunosuppressive responses slowly becomes clear as novel tools become available. This review will address the role of mast cells in tumours and how they can interact with the local immune environment to mediate immune suppression contributing to tumour escape.
Assuntos
Mastócitos/imunologia , Neoplasias/imunologia , Animais , Comunicação Celular/imunologia , Células Dendríticas/imunologia , Modelos Animais de Doenças , Humanos , Tolerância Imunológica , Ativação Linfocitária/imunologia , Camundongos , Subpopulações de Linfócitos T/imunologia , Evasão Tumoral/imunologiaRESUMO
For systemic sclerosis, laboratory tests can play a supplementary role to clinical investigations, imaging techniques and functional tests. Typical autoantibodies support early diagnosis and help in assigning patients to subgroups of the disease; negative results for antinuclear antibodies suggest exclusion of the diagnosis. To detect organ involvement and comorbidity, the laboratory contributes by clinical chemistry, in certain cases by histopathological findings and by the cytological assessment of broncho-alveolar lavage fluid. Inflammatory parameters are of minor importance. Multiple autoantibody determinations in the course of the disease are not yet helpful. Numerous additional laboratory parameters are of value for investigating pathogenesis, but have not yet been generally introduced into the routine diagnostics of systemic sclerosis.
Assuntos
Autoanticorpos/sangue , Técnicas de Laboratório Clínico/tendências , Escleroderma Sistêmico/diagnóstico , Escleroderma Sistêmico/imunologia , Alemanha , Humanos , Guias de Prática Clínica como Assunto , Padrões de Prática Médica/tendênciasRESUMO
Angioimmunoblastic lymphadenopathy with dysproteinemia (AILD) is defined in the current lymphoma classifications as a T-cell non-Hodgkin's lymphoma. However, in approximately one third of the cases of this lymphoproliferative disease rearrangements of T-cell receptor (TCR) genes indicating clonal expansion of T cells are not detectable. It is currently believed that these cases may represent early stages of a lymphoma with a minor oligoclonal T-cell population. In the present study, 18 lymph nodes with the characteristic histology of AILD were investigated for clonal T-cell receptor gene rearrangements by analysis of DNA extracted from whole tissue sections. Dominant T-cell clones were detected in 12 of these cases. Single CD4(+) and CD8(+) T cells and proliferating Ki67(+) cells of seven cases were micromanipulated from frozen tissue sections. TCRbeta gene rearrangements were amplified from these cells by polymerase chain reaction and sequenced. In all informative cases, the clonal gene rearrangements were only detected among CD4(+), and not among CD8(+) T cells, indicating that the tumor clones in AILD usually derive from CD4(+) T cells. Minor clonal T-cell populations in those cases in which no clone was found by whole-tissue DNA analysis were not detectable even at single cell resolution. T-cell clones in 4 of 10 cases were found to express similar TCRbeta chains, indicating a potential role of (super) antigen triggering in at least some cases of AILD.
Assuntos
Rearranjo Gênico da Cadeia beta dos Receptores de Antígenos dos Linfócitos T/genética , Genes Codificadores da Cadeia beta de Receptores de Linfócitos T/genética , Linfoma de Células T/genética , Subpopulações de Linfócitos T/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Regiões Determinantes de Complementaridade/genética , DNA de Neoplasias/genética , Amplificação de Genes , Humanos , Dados de Sequência Molecular , Reação em Cadeia da PolimeraseRESUMO
Clonal composition and T cell receptor (TCR) repertoire of CD4(+) and CD8(+) T cells infiltrating actively demyelinating multiple sclerosis (MS) lesions were determined with unprecedented resolution at the level of single cells. Individual CD4(+) or CD8(+) T cells were isolated from frozen sections of lesional tissue by micromanipulation and subjected to single target amplification of TCR-beta gene rearrangements. This strategy allows the assignment of a TCR variable region (V region) sequence to the particular T cell from which it was amplified. Sequence analysis revealed that in both cases investigated, the majority of CD8(+) T cells belonged to few clones. One of these clones accounted for 35% of CD8(+) T cells in case 1. V region sequence comparison revealed signs of selection for common peptide specificities for some of the CD8(+) T cells in case 1. In both cases, the CD4(+) T cell population was more heterogeneous. Most CD4(+) and CD8(+) clones were represented in perivascular infiltrates as well as among parenchymal T cells. In case 2, two of the CD8(+) clones identified in brain tissue were also detected in peripheral blood. Investigation of the antigenic specificities of expanded clones may help to elucidate their functional properties.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Esclerose Múltipla/imunologia , Adulto , Encéfalo/imunologia , Encéfalo/patologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD8-Positivos/citologia , Células Clonais , Feminino , Rearranjo Gênico da Cadeia beta dos Receptores de Antígenos dos Linfócitos T , Humanos , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/patologia , Reação em Cadeia da Polimerase/métodos , Receptores de Antígenos de Linfócitos T alfa-beta/genéticaRESUMO
A minor component (about 25%) of lymphocytes in Hodgkin's disease (HD) are CD8(+) T cells. It is unclear whether the presence of these cells reflects an antitumor cytotoxic response. The goal of the present study was to investigate clonal composition and the T cell receptor (TCR) beta repertoire of the CD8(+) T cell population in HD. Single CD8(+) cells were micromanipulated from frozen tissue sections of lymph nodes affected by primary HD and subjected to single target amplification of TCRbeta gene rearrangements. Sequence analysis of the V region genes revealed the presence of expanded CD8(+) T cell clones in all three cases analyzed. Most of these clonal expansions accounted for less than 10% of the CD8(+) T cell population. In one case, 30% of the CD8(+) T cells belonged to one or two clones. Comparison of V region sequences, however, did not provide evidence that the micromanipulated CD8(+) cells were sampled from a population that was selected for particular antigen specificities. No obvious biases in TCR Vbeta and Jbeta gene segment usage or CDR3 length distribution were found. Similarities of CDR3 amino acid sequences as found in selected CDR3 structures were rare. These results suggest that, like CD4(+) T cells, CD8(+) T cells may also be recruited into the tumor tissue in an antigen-nonspecific manner.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Doença de Hodgkin/imunologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/metabolismo , Células Clonais , DNA/química , DNA/genética , Citometria de Fluxo , Rearranjo Gênico da Cadeia beta dos Receptores de Antígenos dos Linfócitos T/genética , Genes Codificadores da Cadeia beta de Receptores de Linfócitos T/genética , Doença de Hodgkin/genética , Humanos , Região Variável de Imunoglobulina/genética , Imuno-Histoquímica , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Análise de Sequência de DNARESUMO
A subcutaneous, T-phenotypic anaplastic large cell lymphoma (CD30/Ki1-positive, EBV positive) was diagnosed in a HIV-infected bisexual man. Without chemotherapy the patient had a sustained long-term remission of this tumor (more than three years) after the initiation of highly active antiretroviral therapy. By PCR analysis of T-cell receptor beta gene rearrangements the tumor was found to be oligoclonal. Improvement of cellular immune function by antiretroviral therapy is the only recognizable factor which may have led to tumor remission. This hypothesis is supported by parallels to EBV associated polyclonal lymphoproliferation in allogeneic transplantat recipients where regression of lymphoma can be induced by reducing immunosuppressive therapy.
Assuntos
Linfoma Relacionado a AIDS/patologia , Linfoma Anaplásico de Células Grandes/patologia , Regressão Neoplásica Espontânea , Fármacos Anti-HIV/uso terapêutico , Humanos , Linfoma Relacionado a AIDS/tratamento farmacológico , Linfoma Anaplásico de Células Grandes/tratamento farmacológico , Masculino , Pessoa de Meia-IdadeRESUMO
The T helper cell population of human lymph node germinal centers (GCs) was analyzed for clonality and signs of antigen selection. Frozen sections of lymph node biopsies taken from three different individuals were used to micromanipulate single T cells from one particular GC for each of the specimens. T cell receptor (TCR) beta gene rearrangements were amplified from these single cells and directly sequenced. Although only unique rearrangements were amplified from T cells of GC2 and GC3, 11 of 28 potentially functional rearrangements amplified from GC1 originated from four different clones. In all three GCs, TCR gene rearrangements neither showed obvious biases in gene segment usage nor similarities in complementarity determining region 3 amino acid sequence. Thus, it appears that T lymphocytes in human GCs usually represent a diverse population of cells. Sequence analysis of V region genes did not provide evidence that in the human the process of somatic hypermutation acts on the TCRbeta loci. For one of the GCs (GC3), immunoglobulin heavy chain (IgH) gene rearrangements were amplified and sequenced from single micromanipulated GC B cells. The detection of clonal expansions accounting for more than half of the sampled B cells in addition to ongoing somatic hypermutation of Ig V region genes suggested that GC3 was a fully developed GC.
Assuntos
Centro Germinativo , Linfonodos/imunologia , Reação em Cadeia da Polimerase/métodos , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Linfócitos T Auxiliares-Indutores/imunologia , Adolescente , Adulto , Células Clonais , DNA/análise , Feminino , Secções Congeladas , Rearranjo Gênico da Cadeia beta dos Receptores de Antígenos dos Linfócitos T/genética , Centro Germinativo/citologia , Centro Germinativo/imunologia , Humanos , Linfonodos/citologia , Dados de Sequência Molecular , Linfócitos T Auxiliares-Indutores/citologiaRESUMO
Recent work identified Hodgkin and Reed-Sternberg (H/RS) cells in classical Hodgkin's disease (cHD) as clonal progeny of mature B cells. Therefore, it is generally assumed that cHD homogenously represents a B cell lymphoma. In a subset of cHD, however, H/RS cells expressing T cell-associated proteins may be candidates for alternative lineage derivation. Single H/RS cells with cytotoxic T cell phenotype were micromanipulated from three cases of cHD and analyzed by single cell polymerase chain reaction for immunoglobulin heavy (IgH) and light chain (IgL) gene rearrangements, T cell receptor (TCR)-beta gene rearrangements, and germline configuration of the IgH and TCR-beta loci. H/RS cells from two cases of cHD harbored clonal, somatically mutated Ig gene rearrangements, whereas TCR-beta loci were in germline configuration. In contrast, H/RS cells from an additional case harbored clonal TCR-beta variable/diversity/joining (VDJ) and DJ gene rearrangements, whereas the IgH locus was in germline configuration on both alleles. Thus, in two cases of cHD with H/RS cells expressing cytotoxic T cell molecules, the tumor cells are derived from mature B cells that aberrantly express T cell markers. In a third case, however, H/RS cells were derived from a T cell, demonstrating that cHD can also occur as a T cell lymphoma.
Assuntos
Doença de Hodgkin/imunologia , Linfoma de Células T/imunologia , Células de Reed-Sternberg/imunologia , Adulto , Sequência de Bases , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência MolecularRESUMO
Point mutations of the p53 tumor suppressor gene are a frequent finding in human carcinomas and are thought to be an important oncogenic event. In non-Hodgkin lymphomas, p53 mutations occur in a minor fraction of cases. However, conclusive data are still lacking for Hodgkin's disease (HD) where the analysis meets technical problems. The neoplastic tumor cell clone in HD is represented by the large Hodgkin and Reed-Sternberg (HRS) cells, which account for only a minority of all cells in the tumor tissue (often <1%). To identify putative HRS cell-specific mutations, single HRS cells were micromanipulated from frozen tissue sections of HD biopsy specimens. Exons 4 to 8 of the p53 gene (in which more than 90% of p53 mutations associated with human neoplasms occur) were amplified from these single cells and sequenced. Mutations of p53 were not found in HRS cells of any of 8 cases of HD analyzed. We conclude that mutation of the p53 gene is only rarely, if at all, involved in the pathogenesis of HD.
Assuntos
Doença de Hodgkin/genética , Mutação , Células de Reed-Sternberg/patologia , Proteína Supressora de Tumor p53/genética , Adolescente , Adulto , Feminino , Doença de Hodgkin/patologia , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Despite accounting for only a minor fraction of all cells in Hodgkin's lymphoma tissue, the Hodgkin and Reed-Sternberg (HRS) cells represent the malignant tumor cell clone in Hodgkin's disease (HD). By far the most abundant cell type in the tumor tissue are CD4+ T cells. Some of them intimately associate with HRS cells forming rosettes around them. This study addresses the question whether the rosetting phenomenon reflects a specific interaction between T and HRS cells by asking whether the rosettes are composed of T cells expressing a restricted TCR repertoire. Single rosetting T cells were micromanipulated from frozen sections of tumor tissue in two cases of nodular sclerosing HD and one case of lymphocyte predominant HD. TCR Vbeta gene rearrangements were amplified from these single cells by PCR. Of 83 potentially functional Vbeta gene rearrangements obtained altogether from the three cases, 81 were found to be clonally unrelated. Furthermore, they did not show signs of selection of the receptor chains for recognition of common epitopes: The usage of Vbeta and Jbeta gene segments as well as the distribution of complementarity-determining region (CDR) 3 lengths was similar to what was seen in a collection of 60 Vbeta gene rearrangements from blood of healthy donors and no recurrent CDR3 amino acid motifs were found. These data suggest that the HRS cells attract CD4+ T cells nonspecifically.
Assuntos
Rearranjo Gênico da Cadeia beta dos Receptores de Antígenos dos Linfócitos T , Doença de Hodgkin/genética , Doença de Hodgkin/imunologia , Células de Reed-Sternberg/imunologia , Células de Reed-Sternberg/patologia , Linfócitos T/imunologia , Linfócitos T/patologia , Adulto , Sequência de Bases , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Clonagem Molecular , Primers do DNA/genética , Feminino , Amplificação de Genes , Doença de Hodgkin/patologia , Humanos , Masculino , Dados de Sequência Molecular , Formação de RosetaRESUMO
The polymerase chain reaction allows the characterization of RNA and DNA sequences from single cells. Methods were established to analyse single cells isolated from suspension or by multicolour flow cytometry. We established a method to isolate single immunostained cells from frozen tissue sections and to analyse those cells for immunoglobulin gene rearrangements. This method was first used to study B cell differentiation within human germinal centres. In another series of experiments, Hodgkin and Reed-Sternberg (HRS) cells from a total of 14 cases of HD were analysed for B lineage derivation and clonality. In 13 of the 14 cases, clonal V gene rearrangements were identified. This shows that HRS cells generally represent the outgrowth of a clonal population of B cells. The detection of somatic mutations in all VH gene rearrangements amplified from HRS cells and the nature of those mutations identifies a GC B cell as the HRS precursor.
Assuntos
Doença de Hodgkin/genética , Linfoma não Hodgkin/genética , Reação em Cadeia da Polimerase , Rearranjo Gênico , Genes de Imunoglobulinas , Humanos , Células de Reed-Sternberg/ultraestruturaRESUMO
MxA gene expression is known to be regulated tightly and exclusively by type I interferons (IFNs). The kinetics of MxA gene expression was analyzed in peripheral blood mononuclear cells from 11 healthy volunteers vaccinated with the 17-D strain of yellow fever virus. A reliable induction of MxA RNA and MxA protein was found in the absence of easily detectable serum IFN activity. Thus, steady-state MxA RNA levels were elevated 8- to 30-fold above prevaccination levels on day 5 after vaccination. The average increase of MxA protein was approximately 50-fold. In contrast, no induction of MxA RNA or MxA protein was detectable in 3 similarly vaccinated controls who were immune because of previous vaccinations. The IFN marker 2'-5'-oligoadenylate (2-5A) synthetase known to react to both type I and type II IFNs showed a similar response but did not differentiate equally well between nonimmune and immune vaccinees. beta 2-microglobulin and neopterin reacted poorly, remaining at low levels within the normal range. These results demonstrate that MxA gene expression is a good marker for detecting minute quantities of biologically active type I IFN during viral infections.