RESUMEN
Killer immunoglobulin-like receptors (KIRs) are known to modulate natural killer (NK) and NK T-cell function by interacting with human leucocyte antigen (HLA) class I ligands on target cells. The aim of our study was to investigate the influence of KIR2D genes with their HLA-C ligands in susceptibility to type 1 diabetes. A total of 98 type 1 diabetes patients and 70 healthy subjects from Latvia were typed for KIR genes and HLA-C ligands using polymerase chain reaction-based genotyping. The HLA C1+/C2+ combination was positively, and C1-/C2+ combination was negatively, associated with type 1 diabetes. Stratification analysis of KIR/HLA-C ligand combinations showed 2DL2+/C1+, 2DL3+/C1+, and 2DS2+ /C1+ to be positively, and 2DL2-/C1- and 2DS2-/ C1- to be negatively, associated. The presence of 2DL2-HLA-C1 in the absence of 2DS1, 2DS2 confers maximum susceptibility. Absence of 2DL2 and HLA-C1 along with absence of 2DS1 and 2DS2 confer maximum protection. A hypothetical model of KIR/ligand combinations on immune responses and type 1 diabetes susceptibility is proposed. Our results suggest that a combination KIR2DL2- HLA-C1 plays a critical role in susceptibility or protection in Latvians against type 1 diabetes.
Asunto(s)
Diabetes Mellitus Tipo 1/genética , Predisposición Genética a la Enfermedad , Antígenos HLA-C/genética , Receptores KIR2DL2/genética , Adolescente , Niño , Preescolar , Femenino , Frecuencia de los Genes , Genotipo , Humanos , Lactante , Recién Nacido , Letonia , Masculino , Población Blanca/genéticaRESUMEN
AIM: To identify T cell expansions, i.e. increased frequencies of T cells using a particular T cell receptor (TCR) V alpha or V beta gene segment, in patients with immune thrombocytopenic purpura (ITP). METHODS: The TCR repertoires of CD4+ and CD8+ peripheral blood lymphocytes of 16 patients with chronic ITP were analysed by staining with a panel of anti-TCR V alpha and V beta antibodies followed by flow cytometry. RESULTS: Four of the 16 patients exhibited a total of 6 expansions of CD8+ T cells using a particular V beta segment, but no expansions were detected in the CD4+ subset. For three of the expansions where a follow-up blood sample after treatment with intravenous immunoglobulin was available, only one expansion remained. CONCLUSION: Overall T cell expansion frequency was the same as in healthy individuals. However, the presence of expansions that normalized with treatment suggests the presence of specific T cells implicated in the pathogenesis of ITP.
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Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Genes Codificadores de la Cadena beta de los Receptores de Linfocito T/inmunología , Púrpura Trombocitopénica Idiopática/inmunología , Adolescente , Niño , Preescolar , Femenino , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , MasculinoRESUMEN
CpG motifs originating from bacterial DNA (CpG DNA) can act as danger signals for the mammalian immune system. These CpG DNA motifs like many other pathogen-associated molecular patterns are believed to be recognized by a member of the toll-like receptor family, TLR-9. Here we show results suggesting that heat shock protein 90 (hsp90) is also implicated in the recognition of CpG DNA. Hsp90 was characterized as a binder to oligodeoxynucleotides (ODNs) containing CpG motifs (CpG ODNs) after several purification steps from crude protein extracts of peripheral blood mononuclear cells. This finding was further supported by direct binding of CpG ODNs to commercially available human hsp90. Additionally, immunohistochemistry studies showed redistribution of hsp90 upon CpG ODN uptake. Thus, we propose that hsp90 can act as a ligand transfer molecule and/or play a central role in the signaling cascade induced by CpG DNA.
Asunto(s)
Proteínas HSP90 de Choque Térmico/metabolismo , Oligodesoxirribonucleótidos/metabolismo , Transducción de Señal , Línea Celular , Cromatografía Líquida de Alta Presión , Proteínas de Unión al ADN/metabolismo , Electroforesis en Gel Bidimensional , Ensayo de Cambio de Movilidad Electroforética , Proteínas HSP90 de Choque Térmico/química , Proteínas HSP90 de Choque Térmico/aislamiento & purificación , Humanos , Células Jurkat , Leucocitos Mononucleares/química , Nanotecnología , Receptores de Superficie Celular/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Receptor Toll-Like 9 , Células U937RESUMEN
Immunization with different adjuvants resulted in antithetic outcomes of infection with Chlamydia pneumoniae. Immunization with the outer major protein-2 from C. pneumoniae (OMP-2) emulsified in Freund's complete adjuvant (FCA) thus increased the susceptibility of mice to infection with the bacteria. The detrimental effect was not observed upon inoculation of irrelevant antigens or major outer membrane protein (MOMP) in FCA, but was also observed after immunization with FCA-chlamydial heat shock protein-60 (HSP-60). The harmful effect of FCA-OMP-2 depended on the presence of both CD4+ and CD8+ cells and was mediated by IL-10, as shown using gene-ablated mice. The increased susceptibility to infection caused by FCA-OMP-2 immunization was long-lasting and observed in mice infected 4 months after the last dose of immunogen. In contrast, partial protection against C. pneumoniae was observed when FCA was replaced with oligodeoxynucleotides containing immunostimulatory CpG motifs mixed with Freund's incomplete adjuvant (FIA-IS-CpG). These polar outcomes of infection related to the cytokine pattern: antigen-stimulated spleen cells from FCA-OMP-2-immunized mice showed higher IL-10/IFN-gamma ratios than FIA-IS-CpG-OMP-2-immunized animals. In agreement, sera from FCA-OMP-2 showed higher anti-OMP-2 IgG1/IgG2a ratios than FIA-IS-CpG-OMP-2-immunized animals. Finally, OMP-2 also generated a protective response when delivered by a eukaryotic expression vector in tandem with CTLA4, a procedure that targeted OMP-2 to antigen-presenting cells.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Vacunas Bacterianas/administración & dosificación , Infecciones por Chlamydophila/prevención & control , Chlamydophila pneumoniae/inmunología , Pulmón/inmunología , Neumonía Bacteriana/inmunología , Animales , Anticuerpos/inmunología , Proteínas de la Membrana Bacteriana Externa/genética , Chaperonina 60/administración & dosificación , Infecciones por Chlamydophila/inmunología , Citocinas/inmunología , Ratones , Ratones Endogámicos C57BL , Células TH1/inmunología , Vacunas de ADN/administración & dosificaciónRESUMEN
BACKGROUND: Sarcoidosis is a systemic disease of unknown aetiology frequently affecting the lungs. CD4+ T cells, in particular, accumulate in the lungs, implicating them in the pathogenesis of the disease. METHODS: T cell receptor (TCR) variable (V) gene expression on bronchoalveolar lavage (BAL) fluid T cells and the HLA DR alleles of 121 Scandinavian patients with sarcoidosis was determined. RESULTS: As expected from our previous results, almost every DRB1*0301 (i.e. DR17) positive patient (67/69) had significantly increased numbers of AV2S3+ CD4+ T cells in the BAL fluid but normal levels in peripheral blood (that is, lung restricted expansions) compared with only six of 52 DRB1*0301 negative patients. Detailed genotypic HLA analysis showed that these six DRB1*0301 negative patients with lung restricted AV2S3+ T cell expansions had another HLA allele in common-the HLA-DRB3*0101 allele (also called DR52a)-which was not found in any other DRB1*0301 negative patient. A new group of sarcoidosis patients was therefore identified, characterised by a strict correlation between a distinct HLA allele and lung accumulated T cells expressing a particular TCR V segment. Furthermore, the HLA-DRB1*0301 and HLA-DRB3*0101 encoded molecules showed similarities, with identical amino acid sequences in regions important for antigen binding which may enable them to bind and present the same or similar antigenic peptides. CONCLUSIONS: HLA-DRB3*0101 as well as DRB1*0301 positive sarcoidosis patients may have the capacity to present specific sarcoidosis associated antigens in such a way that AV2S3+ CD4+ T cells are stimulated preferentially, generating lung restricted AV2S3+ T cell expansions.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Antígenos HLA-DR/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Sarcoidosis Pulmonar/inmunología , Adulto , Líquido del Lavado Bronquioalveolar/inmunología , Recuento de Linfocito CD4 , Femenino , Humanos , Masculino , Persona de Mediana Edad , Sarcoidosis Pulmonar/genéticaRESUMEN
Several reports have indicated that cell lineages apart from NK and T cells can also express IFN-gamma. However, the biological relevance of this finding is uncertain. We show in this study that bone marrow-derived macrophages (BMMs) express IFN-gamma at the mRNA and protein level early after infection with Chlamydia pneumoniae. Increased IFN-gamma mRNA accumulation by infected BMMs is early, transient, and requires both bacterial and host protein synthesis. The induction of IFN-gamma mRNA levels is independent of IL-12 and was dramatically enhanced in IL-10(-/-) BMMs. Such IL-10(-/-) BMMs contained less bacteria than the wild-type controls, whereas IFN-gammaR(-/-) BMMs showed increased C. pneumoniae load. Inducible NO synthase (iNOS) also participates in the control of bacterial load, as shown by the enhanced numbers of C. pneumoniae in iNOS(-/-) BMMs. However, the increased accumulation of iNOS mRNA and NO in C. pneumoniae-infected BMMs depended on the presence of IFN-alphabeta, but was independent of IFN-gamma. Interestingly, IFN-alphabeta are also required for increased IFN-gamma mRNA accumulation in C. pneumoniae-infected BMMs. Accordingly, IFN-alphabetaR(-/-) BMMs showed higher levels of C. pneumoniae than wild-type BMMs. Our findings unravel an autocrine/paracrine macrophage activation pathway by showing an IFN-alphabeta-dependent IFN-gamma and iNOS induction in response to infection, which protects macrophages against intracellular bacterial growth.
Asunto(s)
Células de la Médula Ósea/inmunología , Células de la Médula Ósea/microbiología , Chlamydophila pneumoniae/crecimiento & desarrollo , Chlamydophila pneumoniae/inmunología , Interferón Tipo I/fisiología , Interferón gamma/metabolismo , Macrófagos/inmunología , Macrófagos/microbiología , Animales , Antibacterianos/metabolismo , Antibacterianos/farmacología , Células de la Médula Ósea/enzimología , Células de la Médula Ósea/metabolismo , Células Cultivadas , Inhibidores de Crecimiento/metabolismo , Inhibidores de Crecimiento/fisiología , Interferón gamma/biosíntesis , Interferón gamma/genética , Interleucina-10/fisiología , Interleucina-12/fisiología , Líquido Intracelular/enzimología , Líquido Intracelular/inmunología , Líquido Intracelular/microbiología , Macrófagos/enzimología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Óxido Nítrico Sintasa/biosíntesis , Óxido Nítrico Sintasa de Tipo II , ARN Mensajero/biosíntesis , ARN Mensajero/genéticaRESUMEN
Conflicting data have been published concerning the correlation between the length of the second variable region (V2) in the HIV-1 envelope and the biological phenotype of the virus. Here the V2 region length of primary HIV-1 isolates was compared with biological phenotype and coreceptor usage. The V2 region variation was determined by DNA fragment length analysis, virus biological phenotype by the MT-2 cell assay, and coreceptor usage by infection of U87.CD4 cells expressing CCR3, CCR5, or CXCR4. Ninety-three primary virus isolates from 40 patients were analyzed. This panel of viruses included sequential isolates obtained from patients who progressed to AIDS with or without a virus phenotypic switch. We found that NSI MT-2-negative isolates had significantly shorter V2 regions than SI MT-2-positive isolates. However, when V2 region lengths of viruses were analyzed in more detail, we observed that NSI isolates obtained from patients shortly before the phenotypic switch had V2 region lengths similar to those of SI isolates. V2 regions of NSI isolates obtained from patients who progressed to AIDS without a virus phenotypic switch had, in contrast, shorter V2 region than isolates obtained just before virus phenotypic switch. Coreceptor analysis revealed that CCR5-using (R5) isolates generally had shorter V2 regions than virus isolates with the ability to enter CXCR4-expressing cells. Moreover, no significant difference in V2 region length was observed between monotropic SI isolates, that is, X4 isolates, and multitropic SI isolates, that is, R3R5X4 or R5X4 isolates. Thus, we conclude that R5 NSI isolates obtained from patients with stable virus phenotype through the whole disease course display shorter V2 regions than isolates obtained from patients at switch of virus phenotype, suggesting that V2 region length may influence virus coreceptor usage.
Asunto(s)
Antígenos CD4/metabolismo , Proteína gp120 de Envoltorio del VIH/genética , Infecciones por VIH/virología , VIH-1/genética , Receptores CCR5/metabolismo , Receptores CXCR4/metabolismo , Receptores de Quimiocina/metabolismo , Adulto , Secuencia de Aminoácidos , Niño , Proteína gp120 de Envoltorio del VIH/metabolismo , VIH-1/aislamiento & purificación , VIH-1/metabolismo , Humanos , Datos de Secuencia Molecular , Fenotipo , Receptores CCR3RESUMEN
Our data on peripheral blood T cells from Motor neuron disease (MND) patients indicate major immunological disturbances linked to this disease. Both CD4+ and CD8+ T-cell subsets display an increased fraction of cells showing activation markers compared to controls, indicating an unusually high level of activity in both populations. Likewise, an increased number of T-cell expansions were noted in MND patients compared to controls, most dramatically observed in the CD4+ T-cell subset, where 5/144 T-cell V genes analyzed in eight subjects turned out to be expanded in the peripheral blood. In the CD8+ T-cell subset, four out of eight MND patients had peripheral BV gene expansions, 9/144 V genes analyzed. However, the most interesting result was the observation that in three out eight MND patients, expansions concerning the same BV gene were present in both CD4+ and CD8+ subsets (BV8S1 in two and BV12S1 in one patient). Parallel expansions of BV-gene restricted populations in both CD4+ and CD8+ subsets in the same individual, in an major histocompatibility complex (MHC)-unrestricted manner, are normally limited to situations where superantigens are involved. No known superantigen has to date been described with the capacity to simultaneously stimulate both BV8S1 and BV12S1, suggesting that the postulated 'MND-associated' superantigen is a hitherto undefined molecule.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Activación de Linfocitos/inmunología , Enfermedad de la Neurona Motora/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Superantígenos/inmunología , Humanos , Leucocitos Mononucleares/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/genéticaRESUMEN
The objective of this study was to identify disease-associated T cell subsets by characterizing the lung and blood T cell receptor (TCR) repertoires in allergic asthmatics before and after repeated low-dose allergen challenge. Peripheral blood lymphocyte (PBL) and bronchoalveolar lavage (BAL) samples were obtained from eight patients with allergic asthma before and after a period of repeated low-dose allergen inhalations. RT-PCR followed by Southern blot allowed the quantification of relative Vbeta gene segment usage. Thirteen healthy individuals served as controls at PBL level. PBL as well as BAL T cells of asthmatics displayed a higher usage of Vbeta3, Vbeta5.2, and Vbeta6.1-3 and a lower usage of Vbeta16, Vbeta18, and Vbeta19 compared to PBL of healthy controls. Interestingly, TCR Vbeta7 and Vbeta9 usage was significantly higher in BAL than in PBL in asthmatics before as well as after challenge. TCR repertoire alterations after allergen challenge differed between individuals, with relatively mild changes.
Asunto(s)
Alérgenos , Asma/inmunología , Reordenamiento Génico de la Cadena beta de los Receptores de Antígenos de los Linfocitos T , Hipersensibilidad Inmediata/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Administración por Inhalación , Adulto , Alérgenos/administración & dosificación , Asma/patología , Líquido del Lavado Bronquioalveolar/citología , ADN Complementario/genética , Femenino , Humanos , Hipersensibilidad Inmediata/patología , Inmunofenotipificación , Pulmón/patología , Masculino , Polen/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND: Seroepidemiological studies have linked Chlamydia pneumoniae (CP) to coronary heart disease, and recent experimental studies suggest that it may accelerate or even induce atherosclerosis. We therefore evaluated the effect of CP infection on atherosclerosis in atherosclerosis-prone apolipoprotein E-knockout (apoE-KO) and wild-type C57BL/6J mice. METHODS AND RESULTS: Six- to 8-week-old female mice were infected intranasally with live CP and then fed a standard chow diet for 22 weeks. A subgroup of mice was reinfected 18 weeks after primary infection. Polymerase chain reaction analysis of lung tissue confirmed successful infection with CP, and ELISA assays demonstrated development of a humoral immune response. Despite this, no statistically significant differences in aortic atherosclerotic lesions were found between CP-infected and control apoE-KO mice. Furthermore, CP infection did not induce atherosclerosis in C57BL/6J mice. CONCLUSIONS: CP does not induce atherosclerosis in wild-type mice and does not accelerate atherosclerosis in chow-fed apoE-KO mice. Further studies will be necessary to clarify the explanation for the seroepidemiological association between CP and coronary heart disease in humans.
Asunto(s)
Apolipoproteínas E/deficiencia , Arteriosclerosis/etiología , Arteriosclerosis/patología , Infecciones por Chlamydophila/patología , Chlamydophila pneumoniae/patogenicidad , Animales , Anticuerpos Antibacterianos/sangre , Aorta/microbiología , Aorta/patología , Apolipoproteínas E/genética , Arteriosclerosis/sangre , Linfocitos T CD4-Positivos/patología , Infecciones por Chlamydophila/genética , Infecciones por Chlamydophila/inmunología , Infecciones por Chlamydophila/microbiología , Chlamydophila pneumoniae/genética , Chlamydophila pneumoniae/inmunología , Enfermedad Crónica , Modelos Animales de Enfermedad , Femenino , Inmunohistoquímica , Pulmón/microbiología , Pulmón/patología , Macrófagos/microbiología , Macrófagos/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Reacción en Cadena de la Polimerasa , Pruebas SerológicasRESUMEN
OBJECTIVE: To determine whether short peptides corresponding to the RGPGR motif of the V3 loop of gp 120 have anti-human immunodeficiency virus type 1 (anti-HIV-1) activity. DESIGN/METHODS: Short peptides were tested against the HIV-1 laboratory strains and clinical isolates. RESULTS: The tripeptide glycyl-prolyl-glycine amide (GPG-NH2) inhibited the replication of both laboratory strains and 47 clinical isolates, including 19 strains that were resistant to other drugs or that were from patients with failing therapy. The 50% inhibitory concentrations values were 2.7 to 37 microM. Phenotypic change of two isolates from nonsyncytia-inducing to syncytia-inducing did not change their sensitivity to GPG-NH2. The tripeptide added to the antiviral effect of both zidovudine and ritonavir. CONCLUSIONS: The tripeptide GPG-NH2 is a nontoxic compound that inhibits the replication of HIV-1 by an apparently new mode of action. Glycyl-prolyl-glycine-NH2 might prove useful by itself or as a lead compound for the treatment of drug-resistant HIV-1. Glycyl-prolyl-glycine-NH2 is currently undergoing phase I/II human clinical trials in Sweden.
Asunto(s)
Amidas/farmacología , Fármacos Anti-VIH/farmacología , Proteína gp120 de Envoltorio del VIH/química , VIH-1/efectos de los fármacos , Oligopéptidos/farmacología , Fragmentos de Péptidos/química , Replicación Viral/efectos de los fármacos , Fármacos Anti-VIH/química , Farmacorresistencia Microbiana , Sinergismo Farmacológico , Proteína gp120 de Envoltorio del VIH/farmacología , Inhibidores de la Proteasa del VIH/farmacología , VIH-1/aislamiento & purificación , VIH-1/fisiología , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Estructura Molecular , Oligopéptidos/química , Fragmentos de Péptidos/farmacología , Inhibidores de la Transcriptasa Inversa/farmacología , Ritonavir/farmacología , Zidovudina/farmacologíaRESUMEN
In pulmonary sarcoidosis, activated T cells accumulate in the lungs. We hypothesized that the balance between the T-helper type 1 (Th1) cytokines (interferon [IFN]-gamma and interleukin [IL]-2) and Th2 cytokines such as IL-4, IL-5, and IL-10 might explain differences in clinical outcome in pulmonary sarcoidosis, such as why patients of human leukocyte antigen (HLA) type DR17 have a much better prognosis than those of other HLA types. Peripheral blood lymphocytes (PBL) and lymphocytes obtained by bronchoalveolar lavage (BAL) from HLA-typed sarcoidosis patients, as well as PBL from healthy controls, were stimulated in vitro, fixed, and permeabilized with saponin. Thereafter, cells were stained with fluorescence- labeled antibodies specific for intracellular cytokines (IL-2, IL-4, IFN-gamma, and tumor necrosis factor (TNF)-alpha and cell surface markers CD4 and CD8, and were subjected to flow-cytometric analysis. In bronchoalveolar lavage fluid (BALF), there were significantly greater frequencies of T cells positive for IFN-gamma and TNF-alpha than there were among PBL, and significantly fewer cells positive for IL-4, in both the CD4+ and CD8+ subsets. HLA-DR17-positive patients showed a tendency toward a less pronounced Th1 response that may be related to their good prognosis. Sarcoidosis patients had higher frequencies of cells positive for IFN-gamma, IL-4, and IL-2 in their blood than did healthy controls, a finding that may reflect the systemic nature of sarcoidosis. A clear Th1 cytokine profile of CD4+ as well as of CD8+ T cells was demonstrated in BALF from sarcoidosis patients. This was most pronounced for CD8+ cells, which may therefore make an important contribution to the inflammatory process in the lungs in pulmonary sarcoidosis.
Asunto(s)
Linfocitos T CD4-Positivos/química , Linfocitos T CD8-positivos/química , Citocinas/análisis , Sarcoidosis/inmunología , Adulto , Líquido del Lavado Bronquioalveolar/citología , Femenino , Humanos , Líquido Intracelular/química , Pulmón/inmunología , Masculino , Persona de Mediana Edad , Sarcoidosis/sangreRESUMEN
Antibacterial peptides are active defense components of innate immunity. Several studies confirm their importance at epithelial surfaces as immediate barrier effectors in preventing infection. Here we report that early in Shigella spp. infections, expression of the antibacterial peptides LL-37 and human beta-defensin-1 is reduced or turned off. The downregulation is detected in biopsies from patients with bacillary dysenteries and in Shigella- infected cell cultures of epithelial and monocyte origin. This downregulation of immediate defense effectors might promote bacterial adherence and invasion into host epithelium and could be an important virulence parameter. Analyses of bacterial molecules causing the downregulation indicate Shigella plasmid DNA as one mediator.
Asunto(s)
Antibacterianos/metabolismo , Péptidos Catiónicos Antimicrobianos , Proteínas Portadoras/metabolismo , ADN Bacteriano/metabolismo , Regulación hacia Abajo , Disentería Bacilar/metabolismo , Shigella/metabolismo , beta-Defensinas/metabolismo , Adulto , Antibacterianos/biosíntesis , Proteínas Portadoras/biosíntesis , Proteínas Portadoras/genética , Catelicidinas , Niño , Preescolar , Disentería Bacilar/patología , Femenino , Células HT29 , Humanos , Masculino , Persona de Mediana Edad , Shigella/genética , Shigella/fisiología , Shigella boydii/genética , Shigella boydii/metabolismo , Shigella boydii/fisiología , Shigella dysenteriae/genética , Shigella dysenteriae/metabolismo , Shigella dysenteriae/fisiología , Shigella flexneri/genética , Shigella flexneri/metabolismo , Shigella flexneri/fisiología , Células U937 , beta-Defensinas/biosíntesis , beta-Defensinas/genéticaRESUMEN
This study analysed a naturally occurring specific cellular immunity against tumour cells in chronic lymphocytic leukaemia (CLL) patients. Five out of eight patients had blood T lymphocytes able to recognize spontaneously and specifically the autologous tumour B cells (proliferation assay). In these five patients, detection of cytokines by real-time reverse transcription polymerase chain reaction (RT-PCR) revealed that granulocyte-macrophage colony-stimulating factor (GM-CSF) was the most abundant cytokine gene expressed by the T cells that recognized the autologous tumour B cells. Other activated cytokine genes were gamma-interferon (IFN), interleukin (IL)-2 and tumour necrosis factor (TNF)-alpha, but not IL-4. This profile suggests a type 1 anti-B-CLL T-cell response. CD80 and CD54 were relatively downregulated on the native tumour B cells compared with control normal B cells. Upregulation of CD80 on the leukaemic cells was mandatory for the induction of such a specific T-cell response. CD80 and CD54 monoclonal antibodies inhibited the specific T-cell DNA synthesis proliferation. The proliferative T-cell response was either MHC class I or class II restricted (inhibition by monoclonal antibodies). The specific cytokine gene expression could be found in isolated CD4, as well as CD8, T-cell subsets. This study demonstrated the presence of a potential natural specific CD4, as well as a CD8 type 1 T-cell immunity against the leukaemic CLL tumour B cells in CLL. A further detailed analysis of the spontaneous anti-CLL T-cell immunity is warranted that may facilitate the development of effective anti-tumour vaccines in CLL.
Asunto(s)
Linfocitos B/inmunología , Citocinas/genética , Leucemia Linfocítica Crónica de Células B/inmunología , Linfocitos T/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Antígeno B7-1/análisis , Cartilla de ADN , Femenino , Citometría de Flujo , Expresión Génica , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Humanos , Inmunofenotipificación , Molécula 1 de Adhesión Intercelular/análisis , Interferón gamma/genética , Interleucina-2/genética , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
We have previously shown that autologous T cells recognize leukaemic cells from patients with chronic lymphocytic leukaemia (B-CLL) in an MHC class I- and/or II-restricted manner. A candidate recognition structure might be the tumour cell-derived Ig VH complementarity-determining region (CDR)3. Three patients with B-CLL were analysed for the presence of autologous T cells recognizing the tumour-specific VH-CDR3 region. The VH region was shown to be mutated in all three patients. In two patients, a VH-CDR3-specific T-cell response was detected by proliferation assay, as well as by gamma-interferon (IFN) production. The responses could be inhibited by monoclonal antibodies against MHC class II, but not MHC class I. In the third patient, a VH-CDR3 proliferative response was detected, which could be inhibited by an anti-MHC class I monoclonal antibody, but not by anti-MHC class II antibodies. No gamma-IFN response could be detected in this patient. In no patient was an interleukin (IL)-4 response noted. Thus, in patients with B-CLL, naturally occurring T cells recognizing the tumour-unique VH-CDR3 region are present.
Asunto(s)
Genes de Inmunoglobulinas , Leucemia Linfocítica Crónica de Células B/inmunología , Complejo Receptor-CD3 del Antígeno de Linfocito T/inmunología , Linfocitos T/inmunología , Anciano , Secuencia de Aminoácidos , Anticuerpos Monoclonales/farmacología , Secuencia de Bases , Femenino , Antígenos de Histocompatibilidad Clase I/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Humanos , Inmunofenotipificación , Interferón gamma/inmunología , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , MutaciónRESUMEN
We identified antibacterial components in human T and natural killer (NK) cells by using freshly isolated lymphocytes enriched for T and NK cells as starting material. After growing these lymphocytes for 5 days in the presence of interleukin (IL)-2, we isolated and characterized several antibacterial peptides/proteins from the supernatant-alpha-defensins (HNP 1-3), LL-37, lysozyme, and a fragment of histone H2B-although other active components were also present. We then used reverse transcriptase-polymerase chain reaction to search for expression of the gene coding for LL-37 in several B-cell lines, gammadelta T-cell lines, NK clones, and one monocytic cell line, with positive results, but found no expression in several alphabeta T-cell lines. The alpha-defensins (HNP 1-3) were also found to be expressed in several of these cell lines. To confirm the presence of these antibacterial peptides in lymphocytes, we localized them to NK, gammadelta T cells, B cells, and monocytes/macrophages by using double-staining immunohistochemical analysis of freshly isolated lymphocytes. We also found that primary cultures of lymphocytes transcribe and secrete LL-37 and that these processes are affected by IL-6 and interferon-gamma. In addition, we demonstrated that LL-37 has chemotactic activity for polymorphonuclear leukocytes and CD4 T lymphocytes, whereas others have shown chemotactic activity for human alpha-defensins (HNP 1-2). These findings suggest that microbicidal peptides are effector molecules of lymphocytes and that antibacterial activity previously shown to be derived from T and NK cells may be partly mediated by the antibacterial peptides LL-37 and HNP 1-3.
Asunto(s)
Péptidos Catiónicos Antimicrobianos , Linfocitos/fisiología , Monocitos/fisiología , alfa-Defensinas/genética , Antibacterianos/farmacología , Linfocitos B/fisiología , Proteínas Portadoras/farmacología , Catelicidinas , Línea Celular , Quimiotaxis de Leucocito , Clonación Molecular , Histonas/genética , Humanos , Inmunohistoquímica , Técnicas In Vitro , Interferón gamma/farmacología , Interleucina-6/farmacología , Células Asesinas Naturales/fisiología , Activación de Linfocitos , Linfocitos/efectos de los fármacos , Muramidasa/genética , Neutrófilos/efectos de los fármacos , Neutrófilos/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T/fisiología , alfa-Defensinas/farmacología , alfa-Defensinas/fisiologíaRESUMEN
By using mice genomically lacking IFN-gammaR, IL-12, perforin, and recombination-activating gene-1 (RAG-1), we analyzed the regulation and importance of IFN-gamma in the control of infection with Chlamydia pneumoniae. IL-12 participates in resistance of mice to C. pneumoniae, probably by regulating the protective levels of IFN-gamma mRNA. In turn, IFN-gamma is necessary for the increased IL-12p40 mRNA accumulation that occurs in lungs during infection with C. pneumoniae, suggesting a positive feedback regulation between these two cytokines. In experiments including RAG-1-/-/IFN-gammaR-/- mice we showed that IFN-gamma produced by innate cells controls the bacterial load and is necessary for the increased accumulation of transcripts for enzymes controlling high output NO release (inducible NO synthase), superoxide production (gp-91 NADPH oxidase), and catalysis of tryptophan (indoleamine 2, 3-dioxygenase (IDO)), mechanisms probably related to bacterial killing. Adaptive immune responses diminish the levels of IFN-gamma and IL-12 mRNA and thereby the levels of inducible NO synthase, IDO, and gp91 NADPH oxidase transcripts. By using RAG-1-/-/perforin-/- mice, we excluded the overt participation of NK cell cytotoxicity in the control of C. pneumoniae. However, NK cells and probably other innate immune cells release IFN-gamma during the bacterial infection.
Asunto(s)
Infecciones por Chlamydia/genética , Infecciones por Chlamydia/inmunología , Chlamydophila pneumoniae/inmunología , Interferón gamma/biosíntesis , Interferón gamma/fisiología , Animales , Infecciones por Chlamydia/prevención & control , Citotoxicidad Inmunológica/genética , Gangliósido G(M1)/inmunología , Genes RAG-1/inmunología , Predisposición Genética a la Enfermedad , Sueros Inmunes/administración & dosificación , Inmunidad Innata/genética , Inmunosupresores/administración & dosificación , Inyecciones Intravenosas , Interferón gamma/genética , Interferón gamma/metabolismo , Células Asesinas Naturales/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de Interferón/genética , Eliminación de Secuencia , Receptor de Interferón gammaRESUMEN
We have investigated the efficacy of the DNA vaccination using the heat shock protein 60 (HSP-60) gene of C. pneumoniae, for protection of mice against infection with the bacteria. C57Bl/6 mice had a 5-20-fold reduction of C. pneumoniae numbers in lungs when immunized intranasally (i.n.) with plasmids (p) encoding pHSP-60. The reduction of the bacterial load coincided with a decreased severity of disease. No specific antibodies were detected after protective i. n. immunization. In contrast, mice immunized intradermally (i.d.) were not protected against challenge with C. pneumoniae, although specific humoral Immunoglobulin (Ig)G responses were generated. Co-inoculation i.n. of pHSP-60 with pIL-12 but not with pGM-CSF further increased protection of mice against infection with C. pneumoniae. Lungs from pHSP-60 i.n. immunized and infected mice showed higher levels of interferon (IFN)-gamma mRNA, and spleen cells from these mice co-cultured with r-HSP-60 released higher levels of IFN-gamma and displayed higher proliferative responses than nonimmunized and infected controls. pHSP-60 immunized IFN-gamma receptor (R)-/- mice were not protected against infection with C. pneumoniae. Likewise, i.n. administration of pIFN-gamma alone induced significant protection. DNA vaccine-induced protection was CD4+ and CD8+ T-cell dependent, as shown by DNA-vaccination of MHC class II-/-, CD4-/-, CD8-/- and CD4-/-CD8-/-mice. Interestingly, DNA vaccine induced CD4+ T cells, in the absence of CD8+ T cells, were involved in worsening the outcome of infection. This worsening was linked with a shift towards a Th2 cytokine pattern.
Asunto(s)
Chaperonina 60/inmunología , Infecciones por Chlamydia/prevención & control , Chlamydophila pneumoniae/inmunología , Inmunización , Neumonía Bacteriana/prevención & control , Vacunas de ADN/administración & dosificación , Animales , Anticuerpos Antibacterianos/análisis , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Chaperonina 60/genética , Infecciones por Chlamydia/inmunología , Infecciones por Chlamydia/patología , Chlamydophila pneumoniae/genética , Chlamydophila pneumoniae/aislamiento & purificación , Recuento de Colonia Microbiana , Femenino , Inmunoglobulina G/análisis , Interferón gamma/administración & dosificación , Interferón gamma/genética , Interleucina-12/administración & dosificación , Pulmón/microbiología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Mutantes , Mucosa Nasal/inmunología , Plásmidos , Neumonía Bacteriana/inmunología , Neumonía Bacteriana/patología , ARN Mensajero/metabolismoRESUMEN
DNA vaccines that encode encephalitogenic sequences in tandem can protect from subsequent experimental autoimmune encephalomyelitis induced with the corresponding peptide. The mechanism for this protection and, in particular, if it is specific for the amino acid sequence encoding the vaccine are not known. We show here that a single amino acid exchange in position 79 from serine (nonself) to threonine (self) in myelin basic protein peptide MBP68-85, which is a major encephalitogenic determinant for Lewis rats, dramatically alters the protection. Moreover, vaccines encoding the encephalitogenic sequence MBP68-85 do not protect against the second encephalitogenic sequence MBP89-101 in Lewis rats and vice versa. Thus, protective immunity conferred by DNA vaccination exquisitely discriminates between peptide target autoantigens. No bystander suppression was observed. The exact underlying mechanisms remain elusive because no simple correlation between impact on ex vivo responses and protection against disease were noted.