RESUMEN
The Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder characterized by thrombocytopenia, eczema and various degrees of immune deficiency caused by mutations in the WAS gene, which encodes the WASP protein, the expression of which is restricted to haematopoietic cells. Mild allelic variants are associated with X-linked thrombocytopenia (XLT). Female carriers tend in general to be asymptomatic as a consequence of a positive selection of cells with an active normal X chromosome, which results in a non-random inactivation of the mutated gene in affected cell lineages. We report on six female members of the same family carrying the mutated WAS allele p.V332A, which is known to be associated with XLT. One of them had presented severe thrombocytopenia from birth. Western blotting showed the WASP protein in peripheral blood cells to be normal in size and expression, and scanning electron microscopy revealed a normal distribution of microvilli on T cells. X-chromosome inactivation-pattern analysis showed total inactivation of the non-mutated paternal X chromosome in the patient's peripheral blood cells. All the other female family members were healthy and presented varying X-chromosome inactivation patterns, ranging from random X chromosome inactivation to total X-chromosome inactivation of the mutated chromosome. Our results in these female carriers of p.V332A show that manifestation of the disease requires a total inactivation of the non-mutated X chromosome and allow us to confirm that clinical manifestations in female carriers are highly dependent not only on the mutation characteristics but also on the X-chromosome inactivation pattern of affected line.
Asunto(s)
Enfermedades Genéticas Ligadas al Cromosoma X/genética , Trombocitopenia/genética , Inactivación del Cromosoma X , Alelos , Preescolar , Femenino , Expresión Génica , Enfermedades Genéticas Ligadas al Cromosoma X/diagnóstico , Haplotipos , Humanos , Lactante , Recién Nacido , Masculino , Mutación , Linaje , Linfocitos T/metabolismo , Linfocitos T/ultraestructura , Trombocitopenia/diagnóstico , Proteína del Síndrome de Wiskott-Aldrich/genética , Proteína del Síndrome de Wiskott-Aldrich/metabolismoRESUMEN
The application of new protocols for gene therapy against monogenic diseases requires the development of safer therapeutic vectors, particularly in the case of diseases in which expression of the mutated gene is subject to fine regulation, as it is with CD40L (CD154). CD40L, the gene mutated in the X-linked hyper-immunoglobulin M syndrome (HIGM1), is tightly regulated to allow surface expression of its product only on T cells stimulated by antigen encounter. Previous studies in an HIGM1 animal model showed that transduction of progenitor cells corrected the syndrome but caused a thymic lymphoproliferative disease because of the unregulated expression of the transgene by constitutive vectors. To develop a tissue-specific, activation-inducible, lentiviral vector (LV) for gene therapy to counter HIGM1, we have constructed two self-inactivating LVs, pCD40L-eGFP and pCD40L-CD40L, regulated by a 1.3 kb fragment of the human CD40L proximal promoter. The expression of pCD40L-eGFP LV is restricted to cells in which mRNA transcripts of the endogenous CD40L gene can be detected. Moreover, the expression of the reporter gene in primary T lymphocytes depends on the activation state of the cells. Remarkably, primary HIGM1 lymphocytes transduced with pCD40L-CD40L LV expressed CD40L only after T-cell stimulation. Therefore, the CD40L-promoter-driven vectors are able to achieve a near-physiological expression pattern that follows very closely that of the endogenous CD40L gene.
Asunto(s)
Ligando de CD40/genética , Vectores Genéticos , Lentivirus/genética , Línea Celular , Terapia Genética , Humanos , Síndrome de Inmunodeficiencia con Hiper-IgM Tipo 1/genética , Síndrome de Inmunodeficiencia con Hiper-IgM Tipo 1/terapia , Activación de Linfocitos , Especificidad de Órganos , Regiones Promotoras Genéticas , Linfocitos T/metabolismo , Transducción GenéticaRESUMEN
The development of vectors that express a therapeutic transgene efficiently and specifically in hematopoietic cells (HCs) is an important goal for gene therapy of hematological disorders. In order to achieve this, we used a 500 bp fragment from the proximal WASP gene promoter to drive the expression of the WASP cDNA in the context of a self-inactivating lentiviral vector. Single-round transduction of WASp-deficient herpesvirus saimiri (HVS)-immortalized cells as well as primary allospecific T cells from Wiskott-Aldrich syndrome (WAS) patients with this vector (WW) resulted in expression levels similar to those of control cells. Non-HCs were transduced with similar efficiency, but the levels of WASp were 135-350 times lower than those achieved in HCs. Additionally, transduction of WASp-deficient cells with WW conferred a selective growth advantage in vitro. Therefore, lentiviral vectors incorporating proximal promoter sequences from the WASP gene confer hematopoietic-specific, and physiological protein expression.
Asunto(s)
Terapia Genética/métodos , Regiones Promotoras Genéticas , Proteínas/genética , Linfocitos T/metabolismo , Síndrome de Wiskott-Aldrich/terapia , Animales , Western Blotting/métodos , Línea Celular Transformada , Proliferación Celular , Ensayo de Unidades Formadoras de Colonias , Expresión Génica , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Herpesvirus Saimiriino 2/genética , Humanos , Ratones , Ratones Endogámicos BALB C , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Madre/metabolismo , Transducción Genética/métodos , Transgenes , Síndrome de Wiskott-Aldrich/inmunología , Proteína del Síndrome de Wiskott-AldrichRESUMEN
El síndrome de Wiskott-Aldrich (WAS) es una inmunodeficiencia primaria humana ligada al cromosoma X, caracterizada por inmunodeficiencia, microtrombocitopenia y eccema. El gen causante de WAS (WASP) codifica una proteína de 502 residuos llamada WASp. WASp da nombre a una familia de proteínas recientemente definida e involucrada en la transducción de señales desde la membrana celular al citoesqueleto de actina, así como en los procesos de desarrollo y activación celular. Además de su papel como organizador del citoesqueleto, WASp también es una proteína clave en la regulación de las señales procedentes de TCR/CD3 y que conducen a la activación transcripcional y proliferación de las células T. Además, WASp ha sido involucrada en la maduración de los timocitos. Así mismo, se ha mostrado que los miembros de la familia de WASp son importantes en el desarrollo de organismos multicelulares. Trabajos más recientes muestran que la familia WASp está involucrada también en diferentes enfermedades degenerativas. Esta revisión está centrada en la fisiología de la familia WASp, teniendo especial interés en la estructura de WASp, en la regulación intramolecular y en nuevos avances en terapia génica (AU)
Asunto(s)
Humanos , Síndrome de Wiskott-Aldrich/inmunología , Síndromes de Inmunodeficiencia/inmunología , Citoesqueleto , Actinas/inmunología , Transducción de Señal/inmunología , Fenotipo , Genotipo , Linfocitos T/inmunología , Interleucina-2/inmunologíaRESUMEN
Infection of human T lymphocytes with the Herpesvirus saimiri (HVS) yields immortalized T-cell lines (HVS-T) which retain all the phenotypical and functional characteristics of their parental cells. This represents a new experimental model for studying genetic disorders of T lymphocytes. In spite of the efforts of many laboratories, no satisfactory way has been found so far to modify HVS-T cells genetically. We have analyzed the capacity of oncoretroviral (MLV)- and lentiviral (HIV-1)-based vectors pseudotyped with vesicular stomatitis virus glycoprotein (VSVg) to transduce HVS-T cells. HIV-1-derived vectors efficiently transduced HVS-T cell lines, reaching up to 85% of cells expressing the transgene in a single round of infection. MLV-based vectors, on the other hand, were unable to transduce more than 1% of any of the HVS-T cell lines analyzed. Lentiviral-driven gene expression was maintained constant and stable in HVS-T cells for a minimum of 48 days. We also observed that although the lentiviral transduction efficiency achieved on HVS-T cells is lower than that obtained with tumor or primary endothelial cells, it is nevertheless similar to that found with activated primary T cells.
Asunto(s)
Traslado Adoptivo/métodos , Terapia Genética/métodos , Vectores Genéticos/administración & dosificación , Lentivirus/genética , Inmunodeficiencia Combinada Grave/terapia , Linfocitos T/inmunología , Adulto , Línea Celular , Femenino , VIH-1/genética , Herpesvirus Saimiriino 2 , Humanos , Virus de la Leucemia Murina/genética , Masculino , Inmunodeficiencia Combinada Grave/inmunología , Linfocitos T/virología , Transducción Genética/métodosRESUMEN
We investigated the ability of human peripheral CD4(+) cells to express CD94 and NKG2 molecules as a consequence of CD3-mediated activation. Using highly purified peripheral CD4(+) T cells, we found expression of both CD94 and NKG2A 15 days after CD3-mediated stimulation of cells. We also determined by reverse transcriptase-PCR that all gene members of NKG2 family-namely, NKG2A, -C, -D, and -E-are sequentially expressed on CD4(+) cells. We found that this expression is tightly regulated by cytokines, and we identified transforming growth factor-beta1 and interleukin-10 as the main factors that, on CD3-dependent stimulation, positively contribute to the expression of CD94 and NKG2A on CD4(+) cells. We also investigated the functional role of NKG2A and found that coligation of CD3 and NKG2A by specific monoclonal antibodies results in significant inhibition of interferon gamma and tumor necrosis factor alpha production by stimulated CD4(+) cells. The presence and function of these receptors on CD4(+) lymphocytes support a more general role for NKG2 molecules, whose functions were originally thought to be confined to cytotoxic cells, in the immune system.
Asunto(s)
Antígenos CD/biosíntesis , Complejo CD3/farmacología , Linfocitos T CD4-Positivos/metabolismo , Lectinas Tipo C , Glicoproteínas de Membrana/biosíntesis , Receptores Inmunológicos/biosíntesis , Antígenos CD/efectos de los fármacos , Complejo CD3/fisiología , Linfocitos T CD4-Positivos/fisiología , Células Cultivadas , Citocinas/metabolismo , Citocinas/farmacología , Humanos , Interferón gamma/metabolismo , Interleucina-10/farmacología , Activación de Linfocitos/efectos de los fármacos , Glicoproteínas de Membrana/efectos de los fármacos , Subfamília C de Receptores Similares a Lectina de Células NK , Subfamília D de Receptores Similares a Lectina de las Células NK , Receptores Inmunológicos/efectos de los fármacos , Receptores Inmunológicos/fisiología , Receptores de Células Asesinas Naturales , Transducción de Señal , Factor de Crecimiento Transformador beta/farmacología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
OBJECTIVE: To examine if peripheral T lymphocytes from HIV-infected individuals show abnormalities in the surface expression of CD43, the major sialoglycoprotein of leukocytes. DESIGN: A series of 86 HIV-positive individuals was studied. The subjects, grouped by their peripheral CD4 cell count, were in different stages of the disease as defined by the Centers for Disease Control and Prevention (CDC). METHODS: Peripheral leukocytes and isolated lymphocytes were examined by double and triple immunofluorescence flow cytometric and Western blot analyses with monoclonal antibodies, which discriminate between CD43 isoforms. RESULTS: We found elevated percentages of the surface expression of CD43-hexasaccharide isoform on T lymphocytes from 82 out of 86 individuals tested. Increasing percentages are progressively found in CDC groups 1, 2 and 3 patients. The expression of the molecule is remarkably biased towards the CD8 cell subpopulation. The percentage of cells bearing human leukocyte antigen-DR locus molecules (HLA-DR) is also augmented. Two subsets expressing T305 have been identified: a minor subset that co-expresses HLA-DR and T305; and a second population formed by the majority of T305-positive cells, which lack surface HLA-DR. Finally, we found CD43 bands with altered electrophoretic mobility in lysates from peripheral lymphocytes from all HIV-positive individuals tested. CONCLUSION: The augmented expression of CD43-hexasaccharides and the observed cellular distribution suggest an important regulatory role for this molecule in HIV-specific responses.
Asunto(s)
Antígenos CD , Infecciones por VIH/sangre , Infecciones por VIH/metabolismo , Isoformas de Proteínas/metabolismo , Sialoglicoproteínas/metabolismo , Linfocitos T/metabolismo , Complejo CD3 , Antígenos CD4 , Recuento de Linfocito CD4 , Linfocitos T CD4-Positivos , Linfocitos T CD8-positivos , Progresión de la Enfermedad , Humanos , Leucosialina , Recuento de Linfocitos , Subgrupos de Linfocitos TRESUMEN
CD43 is the major leukocyte sialoglyco-protein that plays important functional roles in neutrophils and lymphocytes. However, the expression of CD43 on human natural killer (NK) cells and its participation in the regulation of NK activity has not been studied. We have therefore investigated the expression of CD43 isoforms on human NK cell subpopulations as well as the role of this molecule in NK cell activation and cytotoxicity. We found that CD56bright and CD56dim NK cells express different sialylated forms of CD43, observing that activation of the CD56bright NK cells induces the change of tetrasaccharide O-glycans to hexasaccharide O-glycans on CD43. Cross-linking of the molecule with mAbs results in a metalloprotease-dependent loss of CD43 from the NK cell surface, whereas soluble anti-CD43 mAbs induce a vigorous NK cell proliferation. This property is distinct from T cells, which proliferate after CD43 cross-linking only in the presence of monocytes. Occupancy of the CD43 receptor on NK cells transduces specific signals, leading to enhanced killing activity and tyrosine phosphorylation and de-phosphorylation of several substrates. We therefore propose that CD43 significantly contributes to the regulation of the NK cell function by participating in the control of effector/target interactions and, if pertinent, by transducing activation signals.
Asunto(s)
Antígenos CD/biosíntesis , Antígenos CD/fisiología , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Sialoglicoproteínas/biosíntesis , Sialoglicoproteínas/inmunología , Anticuerpos Monoclonales/metabolismo , Especificidad de Anticuerpos , Antígeno CD56/biosíntesis , Células Cultivadas , Citotoxicidad Inmunológica , Regulación hacia Abajo/inmunología , Humanos , Interleucina-2/farmacología , Leucosialina , Activación de Linfocitos , Subgrupos Linfocitarios/inmunología , Metaloendopeptidasas/fisiología , Isoformas de Proteínas/biosíntesisRESUMEN
The Wiskott-Aldrich syndrome (WAS) is a severe immunodeficiency and platelet deficiency disease arising from mutation(s) in the WASP gene, which in normal cells encodes an intracellular protein able to interact with other proteins relevant to the control of cytoskeleton organization. Immunodeficiency is mainly due to T-cell progressive malfunction. Salient defects of WAS T cells are a CD3-restricted impairment in proliferative responses and cytoskeletal abnormalities, including the frequent appearance of T cells with atypical morphology. We have investigated the possibility that the CD3-restricted defect and some of the cytoskeletal defects of WAS T cells are linked. For this purpose, we immortalized by means of infection with Herpesvirus Saimiri a number of previously described allospecific WAS T-cell lines. The resulting cells preserve the surface, molecular, and functional phenotypes of their parental lines, including a negligible WASP mRNA expression as well as the CD3-restricted defect and cytoskeleton abnormalities. Results show that, in CD3-stimulated WAS T cells, the pattern of temporal changes in cell shape and F-actin distribution is substantially different from that of control cells. Furthermore, polymerization of actin, a critical step in the CD3-mediated cytoskeleton reorganization, does not occur in WAS T-cell lines in response to OKT3 stimulation. In conclusion, our data link both CD3 and cytoskeletal defects in WAS T cells, strongly suggesting that cytoskeleton abnormalities are an underlying cause for WAS immunodeficiency.
Asunto(s)
Actinas/metabolismo , Complejo CD3/metabolismo , Linfocitos T/metabolismo , Síndrome de Wiskott-Aldrich/inmunología , Actinas/química , Complejo CD3/inmunología , División Celular , Células Cultivadas , Citoesqueleto/ultraestructura , Regulación de la Expresión Génica , Herpesvirus Saimiriino 2 , Humanos , Activación de Linfocitos , Microscopía Electrónica de Rastreo , Fenotipo , Polímeros , Conformación Proteica , Propiedades de Superficie , Linfocitos T/inmunología , Linfocitos T/ultraestructura , Síndrome de Wiskott-Aldrich/sangreRESUMEN
CD43 is the major sialoglycoprotein found on the surface of cells of hemopoietic origin. Because CD43 is a typical member of the surface mucins, molecules that play a relevant role in tumor progression, we have addressed the expression of CD43 in nonhematopoietic tumor cells. For this purpose, we have tested a broad panel of anti-CD43 antibodies on tumors of different embryonic origins. Of the 17 anti-CD43 mAbs used, 9 reacted with leukocytes in paraffin-fixed formalin-embedded lymph node tissues. Two mAbs, BS1 and L10, stained 24 out of 31 (78%) nonhematopoietic tumors analyzed; ie., some tumors were positive for both antibodies, and others were positive for either L10 or BSL We have also found reactivity of these two anti-CD43 mAbs in 13 out of 20 metastases studied.
Asunto(s)
Anticuerpos Monoclonales , Especificidad de Anticuerpos , Antígenos CD , Neoplasias/química , Sialoglicoproteínas/inmunología , Formaldehído , Humanos , Leucosialina , Ganglios Linfáticos/química , Neuraminidasa/farmacología , Adhesión en Parafina , Fijación del TejidoRESUMEN
Defective T cell functions, including IL-2 production and proliferation, have been shown in SLE patients. After T cell stimulation (first signal), a costimulatory signal (second signal) is required to achieve complete T cell activation. Main costimulatory signals are provided to T cells by B7 antigens (CD80 and CD86, expressed on antigen-presenting cells (APC)) upon interaction with its receptor, the CD28 molecule expressed on T cells. The aim of this study was to investigate the role of CD28/B7 interactions in the impaired T cell responses of SLE patients. We show that stimulation of T cells with phytohaemagglutinin (PHA) in the presence, but not in the absence, of anti-CD28 MoAb or B7+ cells results in tyrosine phosphorylation of specific substrates, transcription of mRNA and production of IL-2 that is indistinguishable in SLE patients and healthy controls. Moreover, proliferation of costimulated T cells from SLE and controls was specifically abrogated by blocking the CD28/B7 interactions by means of addition to the culture of the CTLA4-Ig fusion protein. However, in most patients activated APC failed to up-regulate B7 molecules, giving rise to ineffective costimulatory signalling to T cells. These results indicate that the CD28/B7 costimulatory pathway is defective in SLE patients.
Asunto(s)
Antígeno B7-1/inmunología , Antígenos CD28/fisiología , Inmunoconjugados , Lupus Eritematoso Sistémico/inmunología , Linfocitos T/inmunología , Abatacept , Adulto , Células Presentadoras de Antígenos/inmunología , Antígenos CD , Antígenos de Diferenciación/fisiología , Antígeno CTLA-4 , Femenino , Expresión Génica , Humanos , Interleucina-2/genética , Activación de Linfocitos , ARN Mensajero/genética , Transducción de SeñalRESUMEN
The Wiskott-Aldrich syndrome (WAS) is a disease of profound thrombocytopenia and severe immune defects caused by an unidentified defective X chromosome gene. In this study, T lymphocyte function is examined using a panel of allospecific WAS patient T cell lines, previously found to express the abnormal disease gene and the cytoarchitectural defect characteristic of the disease. Although T cell lines from normal individuals proliferate vigorously in response to immobilized anti-CD3 mAb OKT3 and SPV-T3b, five of seven WAS patient T cell lines failed to proliferate and two lines showed significantly decreased proliferation when challenged with the immobilized anti-CD3 mAb. The deficient responsiveness of the WAS T cell lines to immobilized anti-CD3 mAb is a restricted defect, because the cells proliferate normally when challenged with allospecific Ag, PHA, or PMA plus ionomycin. Addition of anti-CD28 mAb did not correct the deficient proliferation of the WAS cells challenged with immobilized anti-CD3. Deficient response of the WAS T cell lines to immobilized anti-CD3 was detected also when earlier events of the proliferation process, IL-2 production and up-regulation of activation Ag CD69 and CD28, were measured. On the other hand, WAS cell lines did not differ from normal cell lines in binding of anti-CD3 mAb, mobilization of Ca2+ in response to soluble OKT3, and tyrosine phosphorylation and GTP binding of the CD3 zeta-chain in response to OKT3. Cumulatively, these findings demonstrate a striking restricted defect in the proliferative response of WAS T cells, which because it is found in cell lines free of secondary changes that occur in the patient circulation must be a reflection of the inherited defective disease gene product.
Asunto(s)
Activación de Linfocitos , Linfocitos T/inmunología , Síndrome de Wiskott-Aldrich/inmunología , Secuencia de Aminoácidos , Anticuerpos Monoclonales/inmunología , Antígenos CD28/fisiología , Complejo CD3/inmunología , Calcio/metabolismo , Línea Celular , Guanosina Trifosfato/metabolismo , Humanos , Interleucina-2/biosíntesis , Datos de Secuencia Molecular , Tirosina/metabolismoRESUMEN
The Wiskott-Aldrich syndrome (WAS) is a severe immunodeficiency and platelet deficiency disease arising from an X-linked defect. The disease is correctable by transplantation of hematopoietic stem cells, but the product of the defective gene is unidentified and the number of defects in patient blood cells is large. The current hurdle is the need to identify the early pathogenic event(s) that are the cause of other defects. As a step toward this goal, we have generated and examined a panel of interleukin 2-dependent allospecific T cell lines from peripheral lymphocytes of seven WAS patients and five normal individuals. WAS cell lines, like normal lines, undergo vigorous proliferation when challenged with specific allostimulant or with phorbol myristate acetate and ionomycin. Both normal and WAS T cell lines express cell surface molecules CD2, CD3, T cell receptor-alpha/beta, human histocompatibility leukocyte antigen class I, CD45 and CD11a, and varying ratios of CD4 and CD8, and are negative for natural killer cell and monocyte surface molecules. WAS T cell lines express CD43 (sialophorin/leukosialin) with molecular weight and in an amount comparable with normal T cell lines. WAS T cell lines thus do not express defects in CD43 (decreased amount, abnormal molecular weight), previously documented in WAS circulating lymphocytes. On the other hand, as detected by scanning electron microscopy, WAS cell lines exhibit severe morphological abnormalities, including decreased size and density of the microvillus surface projections. The morphological abnormalities of WAS T cell lines are similar to, or more extensive than, those previously reported for WAS peripheral lymphocytes, indicating that the generation of morphological (cytoarchitectural) defects is an early pathogenic event in this disease. The findings suggest that the gene that is defective in the WAS encodes a protein that normally functions to maintain or regulate the cytoskeletal structure of blood cells.
Asunto(s)
Linfocitos T/inmunología , Síndrome de Wiskott-Aldrich/etiología , Adolescente , Adulto , Western Blotting , Niño , Citometría de Flujo , Humanos , Lactante , Activación de Linfocitos , Microscopía Electrónica de Rastreo , Fenotipo , Linfocitos T/ultraestructura , Regulación hacia Arriba , Síndrome de Wiskott-Aldrich/inmunología , Síndrome de Wiskott-Aldrich/patologíaRESUMEN
We have addressed the differential regulatory properties that IFN-gamma and IL-4 exert on macrophage (M phi) subpopulations. For this purpose, Thyoglicolate-, Peptone-, and Con A-elicited M phi, as well as bone marrow-derived M phi and P388D1 cells, were cultured in the presence of either IFN-gamma or IL-4. The expression of LFA-1, Mac-1, and Mac-2 after this treatment was studied by FACS analysis. We have found that these surface molecules are differentially modulated by the two lymphokines, depending on the M phi subpopulation studied. Mac-1 is upregulated only in Thyoglicolate-elicited cells after treatment with IFN-gamma, while no change in the expression of Mac-2 was observed in any of the groups. LFA-1 is upregulated by IFN-gamma in Thyoglicolate- and bone marrow-derived M phi and P388D1 cells, while IL-4 does not induce LFA-1 on these cells. Interestingly, however, we have observed the reverse situation on Con A-elicited M phi, where a strong induction of LFA-1 is achieved by treatment of the cells with IL-4, while IFN-gamma does not modify the expression of this antigen. Our results obtained with the lymphokine-stimulated M phi are interpreted in the context of functionally induced M phi subpopulations, which might be regulated by either Th1 or Th2 CD4+ T cells. Thyoglicolate-elicited M phi may represent the in vitro equivalent of a M phi subpopulation regulated in vivo by Th1 cells while Con A-elicited M phi could be the equivalent of a subpopulation regulated by Th2 cells.
Asunto(s)
Interferón gamma/farmacología , Interleucina-4/farmacología , Activación de Macrófagos , Macrófagos/inmunología , Animales , Anticuerpos Monoclonales , Antígenos de Diferenciación/análisis , Citometría de Flujo , Galectina 3 , Antígeno-1 Asociado a Función de Linfocito/análisis , Antígeno de Macrófago-1/análisis , Ratones , Regulación hacia ArribaRESUMEN
We have compared the functional properties of I-Ad expressed on different cell types. Specifically, we have transfected I-A alpha d and I-A beta d cDNA into a panel of T cell thymomas of various phenotypes. Excellent class II surface expression was achieved in all T cell tumors, equivalent in level to that found on the B cell lymphoma A20. Interestingly, however, two allo-I-Ad-specific Thy differed in their recognition of the transfected tumor cells: whereas the 42H11 T cell hybridoma (THy) was stimulated very efficiently by all transfectants, the RK38.2.2 Thy did not react to any of them. Both THy responded equally well to I-Ad on A20 B lymphoma cells. Purified macrophages isolated from various sources were also differentially recognized by the two THy, although there was only a quantitative difference in stimulation. Taken together, these results are best interpreted to show that the TCR of the RK38.2.2 THy is specific for I-Ad in the context of a B cell-specific determinant, possibly a self-peptide that is naturally associated with Ia. A cross-reactive molecule could be expressed by macrophages and COS-1 cells, but not by T cells.
Asunto(s)
Células Presentadoras de Antígenos/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Linfocitos T/inmunología , Animales , Antígenos de Diferenciación/análisis , Linfocitos B/inmunología , Linfocitos T CD4-Positivos/inmunología , Antígenos H-2/inmunología , Antígenos de Histocompatibilidad Clase II/genética , Interleucina-2/biosíntesis , Activación de Linfocitos , Prueba de Cultivo Mixto de Linfocitos , Antígeno-1 Asociado a Función de Linfocito , Macrófagos/fisiología , Complejo Mayor de Histocompatibilidad , Ratones , Ratones Endogámicos , Receptores de Adhesión de Leucocito/análisis , Transfección , Células Tumorales CultivadasRESUMEN
In order to test the tissue distribution of Mlsa determinants, we have generated highly purified stimulator cell populations. First, Mlsa expression in bone marrow derived macrophages (M phi) of Mlsa genotype was tested in primary MLR and on Mlsa-specific T cell hybridomas (THy). Second, a similar experimental approach was used to analyze thioglycolate, peptone or Con A elicited peritoneal M phi. In all cases, these M phi cell populations were able to generate an excellent alloresponse, whereas no functional Mlsa determinants could be detected. Third, to further investigate whether the expression of Mlsa is lymphocyte specific, but dependent on expression of class II molecules, we have transfected I-Ek alpha and beta cDNA into a panel of thymomas of Mlsa genotype. Although we achieved a high level of surface I-Ek expression in all of these T cell tumors, none of them was able to trigger the Mlsa-specific THy. These results strongly suggest that Mlsa expression is limited to B cells. It is likely that Mlsa is a tissue-specific self-peptide that associates with class II molecules.
Asunto(s)
Antígenos de Superficie/análisis , Epítopos/análisis , Activación de Linfocitos , Macrófagos/análisis , Linfocitos T/análisis , Animales , Antígenos de Superficie/genética , Genotipo , Antígenos de Histocompatibilidad Clase II , Hibridomas/inmunología , Prueba de Cultivo Mixto de Linfocitos , Macrófagos/clasificación , Macrófagos/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Antígenos Estimulantes de Linfocito Menor , Linfocitos T/clasificación , Linfocitos T/inmunologíaRESUMEN
We have analysed the genetic relationship between Mlsa and Fc gamma R in mice. Using the Fc gamma R-specific DNA probes, we were unable to detect a restriction fragment length polymorphism (RFLP) which is consistent in DNA derived from Mlsa strains and which differed from that of Mlsb strains, while we could see a polymorphism that distinguishes Ly17.1 from Ly17.2, alleles of the Fc gamma R. These results strongly suggest that Mlsa is neither a product of the alpha Fc gamma R nor of the beta Fc gamma R gene. Furthermore, we have re-examined the tissue distribution of Mlsa determinants using a major histocompatibility complex (MHC) class II antigen-positive T-cell tumour as well as a pure population of bone marrow derived macrophages of Mlsa genotype. Both these cell types were recognized to varying degrees by alloreactive cells; however, none of them expressed functionally detectable Mlsa determinants. We conclude from our studies that Mlsa is a highly stimulatory self peptide that is exclusively expressed in B cells.