RESUMEN
The chemokine receptors CCR2 and CCR5 play important roles in the recruitment of monocytes/macrophages and T cells. To better understand the role of both receptors in murine models of inflammatory diseases and to recognize potential problems when correlating these data to humans, we have generated mAbs against murine CCR2 and CCR5. In mice CCR2 is homogeneously expressed on monocytes and on 2--15% of T cells, closely resembling the expression pattern in humans. In contrast to humans, murine NK cells are highly CCR5 positive. In addition, CCR5 is expressed on 3--10% of CD4 and 10--40% of CD8-positive T cells and is weakly detectable on monocytes. Using a model of immune complex nephritis, we examined the effects of inflammation on chemokine receptor expression and found a 10-fold enrichment of CCR5(+) and CCR2(+) T cells in the inflamed kidneys. The activity of various chemokines and the antagonistic properties of the mAbs were measured by ligand-induced internalization of CCR2 and CCR5 on primary leukocytes. The Ab MC-21 (anti-CCR2) reduced the activity of murine monocyte chemotactic protein 1 by 95%, whereas the Ab MC-68 (anti-CCR5) blocked over 99% of the macrophage-inflammatory protein 1alpha and RANTES activity. MC-21 and MC-68 efficiently blocked the ligand binding to CCR2 and CCR5 with an IC(50) of 0.09 and 0.6--1.0 microg/ml, respectively. In good correlation to these in vitro data, MC-21 almost completely prevented the influx of monocytes in thioglycollate-induced peritonitis. Therefore, both Abs appear as useful reagents to further study the role of CCR2 and CCR5 in murine disease models.
Asunto(s)
Receptores CCR5/biosíntesis , Receptores CCR5/inmunología , Receptores de Quimiocina/biosíntesis , Receptores de Quimiocina/inmunología , Animales , Anticuerpos Bloqueadores/metabolismo , Anticuerpos Bloqueadores/farmacología , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/metabolismo , Especificidad de Anticuerpos , Apoferritinas/toxicidad , Unión Competitiva/inmunología , Antagonistas de los Receptores CCR5 , Células CHO , Cricetinae , Regulación hacia Abajo/inmunología , Glomerulonefritis/inducido químicamente , Glomerulonefritis/inmunología , Glomerulonefritis/prevención & control , Inyecciones Intraperitoneales , Leucocitos/metabolismo , Ratones , Ratones Endogámicos BALB C , Peritonitis/inducido químicamente , Peritonitis/inmunología , Peritonitis/prevención & control , Ratas , Ratas Wistar , Receptores CCR2 , Receptores CCR5/metabolismo , Receptores de Quimiocina/antagonistas & inhibidores , Receptores de Quimiocina/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Tioglicolatos/toxicidadRESUMEN
The chemokine receptor CCR5 is expressed on the majority of T cells and monocytes in the inflammatory infiltrate of diseases such as rheumatoid arthritis, renal diseases, and multiple sclerosis. In contrast, little expression of CCR5 is found on peripheral blood leukocytes. A specific depletion of CCR5(+) cells could therefore be a useful strategy to reduce the cellular infiltrate in chronic inflammations. Moreover, CCR5 is the major coreceptor for M-tropic HIV-1 strains. Depletion of CCR5(+) leukocytes may help to eliminate cells latently infected with HIV-1. We designed two constructs that specifically destroy chemokine receptor-positive cells. The first construct, a bispecific Ab, binds simultaneously to CCR5 and CD3. Thereby it redirects CD3(+) T cells against CCR5(+) target cells. The Ab specifically depletes CCR5(+) T cells and monocytes, but is inactive against cells that do not express CCR5. Furthermore, ex vivo the bispecific Ab eliminated >95% of CCR5(+) monocytes and T cells from the synovial fluid of patients with arthritis. Also, we designed a fusion protein of the chemokine RANTES and a truncated version of Pseudomonas. exotoxin A. The fusion protein binds to CCR5 and down-modulates the receptor from the cell surface. The chemokine toxin completely destroyed CCR5(+) Chinese hamster ovary cells at a concentration of 10 nM, whereas no cytotoxic effect was detectable against CCR5(-) Chinese hamster ovary cells. Both constructs efficiently deplete CCR5-positive cells, appear as useful agents in the treatment of chronic inflammatory diseases, and may help to eradicate HIV-1 by increasing the turnover of latently infected cells.
Asunto(s)
ADP Ribosa Transferasas , Artritis Reumatoide/terapia , Toxinas Bacterianas , Quimiocinas/toxicidad , Infecciones por VIH/terapia , Inmunotoxinas/toxicidad , Monocitos/inmunología , Receptores CCR5/biosíntesis , Factores de Virulencia , Animales , Anticuerpos Biespecíficos/biosíntesis , Anticuerpos Biespecíficos/genética , Anticuerpos Biespecíficos/metabolismo , Anticuerpos Biespecíficos/toxicidad , Artritis Reumatoide/inmunología , Artritis Reumatoide/patología , Complejo CD3/inmunología , Células CHO , Separación Celular , Células Cultivadas , Quimiocina CCL5/genética , Quimiocina CCL5/inmunología , Quimiocinas/genética , Quimiocinas/metabolismo , Quimiocinas/uso terapéutico , Enfermedad Crónica , Cricetinae , Citotoxicidad Inmunológica/genética , Exotoxinas/síntesis química , Exotoxinas/genética , Exotoxinas/inmunología , Infecciones por VIH/inmunología , Infecciones por VIH/patología , Humanos , Inmunotoxinas/genética , Inmunotoxinas/metabolismo , Inmunotoxinas/uso terapéutico , Depleción Linfocítica , Monocitos/metabolismo , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/inmunología , Receptores CCR5/inmunología , Receptores CCR5/metabolismo , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/toxicidad , Líquido Sinovial/citología , Líquido Sinovial/inmunología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Linfocitos T/patología , Exotoxina A de Pseudomonas aeruginosaRESUMEN
The release of microparticles from eukaryotic cells is a well-recognized phenomenon. We demonstrate here that the chemokine receptor CCR5, the principal co-receptor for macrophage-tropic human immunodeficiency virus (HIV)-1, can be released through microparticles from the surface of CCR5+ Chinese hamster ovary cells and peripheral blood mononuclear cells. Microparticles containing CCR5 can transfer the receptor to CCR5- cells and render them CCR5+. The CCR5 transfer to CCR5-deficient peripheral blood mononuclear cells homozygous for a 32-base-pair deletion in the CCR5 gene enabled infection of these cells with macrophage-tropic HIV-1. In monocytes, the transfer of CCR5 could be inhibited by cytochalasin D, and transferred CCR5 could be downmodulated by chemokines. A transfer of CCR5 from peripheral blood mononuclear cells to endothelial cells during transendothelial migration could be demonstrated. Thus, the transfer of CCR5 may lead to infection of tissues without endogenous CCR5 expression. Moreover, the intercellular transfer of membrane proteins by microparticles might have broader consequences for intercellular communication beyond the effects seen for HIV-1.
Asunto(s)
Membrana Celular/metabolismo , Endotelio Vascular/virología , VIH-1/crecimiento & desarrollo , Receptores CCR5/metabolismo , Animales , Transporte Biológico , Células CHO , Quimiotaxis de Leucocito , Cricetinae , Endotelio Vascular/citología , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/virología , Macrófagos/citología , Macrófagos/virologíaRESUMEN
OBJECTIVE: To study the role of the chemokine receptors CCR5 and CCR2 in patients with arthritis. METHODS: CCR5 expression on peripheral blood leukocytes was compared with the expression on leukocytes isolated from the synovial fluid of 20 patients with different rheumatic joint diseases. Three additional samples were studied for CCR2 expression. The expression of chemokine receptors on blood and synovial fluid leukocytes was determined by 3-color flow cytometry analysis. To test CCR5 receptor down-modulation from the cell surface, leukocytes were incubated in vitro with a RANTES (regulated on activation, normal T cell expressed and secreted) derivative, aminooxypentane (AOP)-RANTES. Patients were genotyped for the delta32 CCR5 deletion by polymerase chain reaction. RESULTS: A high percentage of CCR5-expressing CD4+ and CD8+ T cells (74% and 81%, respectively), monocytes (51%), and natural killer cells (35%) was found in the synovial fluid of all patients, whereas in the peripheral blood, only a small percentage of these cells expressed CCR5 (13%, 32%, 7.8%, and 4%, respectively). Infiltration of CCR5-positive leukocytes was not reduced in CCR5-heterozygous patients. A similar, but less pronounced, distribution was observed for CCR2-positive T cells. In vitro, CCR5 was completely down-modulated on synovial fluid leukocytes by AOP-RANTES. CONCLUSION: The predominance of CCR5-positive mononuclear cells in the synovial effusions of patients with arthritis suggests an important role for CCR5 in the process of joint inflammation, and identifies CCR5 as a possible new target for therapeutic intervention.
Asunto(s)
Artritis/sangre , Receptores CCR5/sangre , Receptores CCR5/genética , Líquido Sinovial/citología , Adolescente , Adulto , Artritis/genética , Relación CD4-CD8 , Femenino , Citometría de Flujo/métodos , Técnica del Anticuerpo Fluorescente , Expresión Génica , Genotipo , Humanos , Leucocitos/química , Masculino , Persona de Mediana Edad , Espondilitis Anquilosante/sangre , Espondilitis Anquilosante/genéticaRESUMEN
The involvement of CD4+ and CD8+ T cells in pathogenesis of spontaneous autoimmune thyroiditis (SAT) in obese strain (OS) chickens has not been studied in depth until now. We depleted CD4+ or CD8+ T cells in OS chickens by treatment with murine monoclonal anti-CD4 or anti-CD8 antibodies at 3 day intervals beginning at hatching. The birds were killed at 19-25 days of age. Treatment with anti-CD4 antibody completely prevented SAT development, while treatment with anti-CD8 antibody partially inhibited SAT. These results show the critical role of CD4+ T cells in the development of SAT in OS chickens, and indicate that CD8+ T cells are also involved in SAT pathogenesis.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Antígenos CD4/inmunología , Linfocitos T CD4-Positivos/inmunología , Antígenos CD8/inmunología , Linfocitos T CD8-positivos/inmunología , Pollos/genética , Modelos Animales de Enfermedad , Obesidad/veterinaria , Enfermedades de las Aves de Corral/prevención & control , Tiroiditis Autoinmune/veterinaria , Factores de Edad , Animales , Anticuerpos Monoclonales/inmunología , Autoanticuerpos/análisis , Pollos/inmunología , Técnicas para Inmunoenzimas , Endogamia , Ratones , Obesidad/genética , Enfermedades de las Aves de Corral/genética , Enfermedades de las Aves de Corral/inmunología , Enfermedades de las Aves de Corral/patología , Tiroglobulina/inmunología , Glándula Tiroides/inmunología , Glándula Tiroides/patología , Tiroiditis Autoinmune/genética , Tiroiditis Autoinmune/inmunología , Tiroiditis Autoinmune/patología , Tiroiditis Autoinmune/prevención & controlRESUMEN
CCR5, a chemokine receptor expressed on T cells and macrophages, is the principal coreceptor for M-tropic HIV-1 strains. Recently, we described an NH2-terminal modification of the CCR5 ligand regulated on activation, normal T cell expressed and secreted (RANTES), aminooxypentane-RANTES (AOP-RANTES), that showed potent inhibition of macrophage infection by HIV-1 under conditions where RANTES was barely effective. To investigate the mechanism of AOP-RANTES inhibition of HIV infectivity we examined the surface expression of CCR5 using a monoclonal anti-CCR5 antibody, MC-1. We demonstrate that AOP-RANTES rapidly caused >90% decrease in cell surface expression of CCR5 on lymphocytes, monocytes/ macrophages, and CCR5 transfected Chinese hamster ovary (CHO) cells. RANTES also caused a loss of cell surface CCR5, although its effect was less than with AOP-RANTES. Significantly, AOP-RANTES inhibited recycling of internalized CCR5 to the cell surface, whereas RANTES did not. When peripheral blood mononuclear cells are cultured for prolonged periods of time in the presence of RANTES, CCR5 expression is comparable to that seen on cells treated with control medium, whereas there is no CCR5 surface expression on cells cultured in the presence of AOP-RANTES. Immunofluorescence indicated that both AOP-RANTES and RANTES induced downmodulation of cell surface CCR5, and that the receptor was redistributed into endocytic organelles containing the transferrin receptor. When RANTES was removed, the internalized receptor was recycled to the cell surface; however, the receptor internalized in the presence of AOP-RANTES was retained in endosomes. Using human osteosarcoma (GHOST) 34/CCR5 cells, the potency of AOP-RANTES and RANTES to inhibit infection by the M-tropic HIV-1 strain, SF 162, correlated with the degree of downregulation of CCR5 induced by the two chemokines. These differences between AOP-RANTES and RANTES in their effect on receptor downregulation and recycling suggest a mechanism for the potent inhibition of HIV infection by AOP-RANTES. Moreover, these results support the notion that receptor internalization and inhibition of receptor recycling present new targets for therapeutic agents to prevent HIV infection.
Asunto(s)
Fármacos Anti-VIH/farmacología , Quimiocina CCL5/análogos & derivados , VIH-1/efectos de los fármacos , Receptores CCR5/metabolismo , Animales , Transporte Biológico , Células CHO , Quimiocina CCL5/farmacología , Cricetinae , Regulación hacia Abajo , Endocitosis , Endosomas/metabolismo , HumanosRESUMEN
Chickens have only two T cell receptor variable beta gene families: V beta 1 and V beta 2 (1). In our previous work we found that IgA production was almost completely suppressed in chickens depleted of V beta 1+ alpha beta T cells by treatment with a TCR V beta 1-specific monoclonal antibody (2), while IgM and IgG production was not affected. Our present results indicate that, in vitro, both V beta 1+ and V beta 2+ chicken cecal tonsil T cells provide help for the differentiation of cecal tonsil IgA B cells, suggesting that the failure of V beta 1+ T cell-depleted chickens to produce IgA is not caused by the inability of V beta 2+ T cells to provide help for IgA production by B cells, but rather by the scarcity of these T cells in mucosal tissues (3), where most IgA responses are induced (4).
Asunto(s)
Linfocitos B/inmunología , Pollos/inmunología , Reordenamiento Génico de la Cadena beta de los Receptores de Antígenos de los Linfocitos T , Inmunoglobulina A/biosíntesis , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Subgrupos de Linfocitos T/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Anticuerpos Monoclonales/farmacología , Ciego/inmunología , Células Cultivadas , Pollos/genética , Activación de Linfocitos/efectos de los fármacos , Cooperación Linfocítica , Mitógenos de Phytolacca americana/farmacología , Receptores de Antígenos de Linfocitos T alfa-beta/genéticaRESUMEN
Untreated control OS chickens develop spontaneous autoimmune thyroiditis (SAT). In contrast, OS chickens treated with a monoclonal anti-CD4 antibody failed to develop SAT. The preventive effect of anti-CD4 antibody on SAT was associated with the marked depletion of CD4+ T-cells by anti-CD4 treatment. These results indicate that CD4+ T-cells play a crucial role in SAT of OS chickens.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Antígenos CD4/inmunología , Pollos , Obesidad/veterinaria , Enfermedades de las Aves de Corral/prevención & control , Tiroiditis Autoinmune/veterinaria , Animales , Anticuerpos Monoclonales/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/patología , Linfocitos T CD4-Positivos/fisiología , Obesidad/complicaciones , Obesidad/genética , Enfermedades de las Aves de Corral/etiología , Tiroiditis Autoinmune/etiología , Tiroiditis Autoinmune/prevención & controlRESUMEN
Antigen stimulation may lead to expansion or deletion of T-cells expressing T-cell receptors that belong to specific V beta gene families. Since such stimulation at the same time will lead to conversion from naive to memory T-cells, we have asked whether a bias in V beta families can be observed when comparing these two populations. We have studied the expression of V beta 3, 8, 13.3, 19, and 22 in peripheral blood T-cells for 12 apparently healthy male donors. For flow cytometry 100,000 CD4+ or CD8+ cells each were analysed in three-colour immunofluorescence for percentage of V beta families among CD45R0- naive and CD45R0+ memory cell. Greater than twofold excess was found in the CD8+CD45R0+ cells in four cases (1 x V beta 13.3, 2 x V beta 19, 1 x V beta 22) and a greater than twofold decrease in CD8+CD45R0+ cells in two cases (1 x V beta 8, 1 x V beta 22). In contrast, among CD4+ cells no such bias was detected. The excess in CD8+CD45R0+ memory cells showed no substantial fluctuation over time in that it was found to be stable for 19 to 70 days. Finally, in vitro conversion of purified CD8+CD45R0- cells to CD45R0+ cells by polyclonal stimulation with PHA did not result in the excess of V beta usage observed in vivo. These data suggest that specific antigen stimulation during past infection or allergy may be responsible for the excess of certain V beta gene families. Clinical studies looking for disease associations will have to test CD4 and CD8 naive and memory subsets in order to precisely identify a bias in V beta usage and these studies will have to consider the pronounced changes observed in healthy controls.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Regulación de la Expresión Génica/inmunología , Memoria Inmunológica/genética , Familia de Multigenes , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Adulto , Humanos , Inmunidad Celular/genética , Antígenos Comunes de Leucocito/genética , Activación de Linfocitos/genética , Masculino , Factores de TiempoRESUMEN
Plant lectins can be potent modulators of vertebrate immune functions. Biochemical characterization of lectins from animal tissues enables the determination of whether these endogenous activities display a comparable immunological potency. Focusing on chicken beta-galactoside-binding lectins, the monomeric intestinal (CL-14) and the dimeric liver lectin (CL-16) were purified and the lack of cross-contamination was ascertained. In very close agreement with the molecular masses of 14,974 and 14,976 calculated on the basis of the available sequence data (Y. Sakakura et al., J. Biol. Chem. 265, 21573-21579, 1990), electrospray mass spectrometric analysis yielded values of 14,969 (CL-14) and 14,972 (CL-16), the reasons for the deviation in gel electrophoretic behavior being unclear. Solid-phase assays with immobilized lactosylated poly-L-lysine demonstrated a comparatively lower affinity and higher extent of binding at saturation for the monomeric lectin than for the dimeric protein, whose properties were similar to those of an immunomodulatory plant lectin. Flow cytometry revealed homogeneous and strong binding of the dimeric lectin within the chicken peripheral blood lymphocyte population, whereas the monomeric lectin stained two subpopulations at different intensities. Two-color flow cytometry disclosed preferential binding of this lectin to B cells. When a B cell line was employed for determination of affinity constants and extents of binding at saturation, qualitatively comparable parameters to those for the solid-phase assays were obtained. The similar profile of lectin-binding glycoproteins in blots of cellular extracts underscored that accessibility to ligands, not qualitatively different ligand display, may explain the differences for the cell line. At up to a concentration of 10 micrograms/ml of the lectins no stimulation of [3H]thymidine incorporation was seen for blood and spleen cell populations. However, the dimeric lectin reduced stimulation of cells that were responsive to an anti-TcR2 antibody. Thus, this lectin can apparently exhibit inhibitory activity to this kind of T cell activation in vitro.
Asunto(s)
Galactósidos/química , Hemaglutininas/química , Hemaglutininas/farmacología , Lectinas/antagonistas & inhibidores , Subgrupos Linfocitarios/química , Animales , Pollos , Galactósidos/inmunología , Galectinas , Hemaglutininas/inmunología , Intestinos/química , Intestinos/inmunología , Lectinas/química , Lectinas/farmacología , Hígado/química , Hígado/inmunología , Activación de Linfocitos/efectos de los fármacos , Subgrupos Linfocitarios/inmunología , Unión Proteica/inmunologíaRESUMEN
We studied T cell receptor variable beta (TCR V beta) gene usage by autoreactive T cells in spontaneous autoimmune thyroiditis (SAT) of obese strain (OS) chickens. Chicken alpha beta T cells may express either V beta 1 or V beta 2 genes, the products of which can be recognized by TCR2 and TCR3 monoclonal antibodies, respectively. Selective depletion of V beta 1+ or V beta 2+ T cells in OS chickens was accomplished by repeated injections of TCR2 or TCR3 antibodies into embryonic and 1-3-week-old chickens. The birds were killed at 20 days of age and their spleens and thyroid glands evaluated by immunohistochemistry. We found that V beta 1+ T cells preferentially infiltrated OS chicken thyroid glands. Antibody treatments resulted in a 41% reduction in frequency of V beta 1+, and a 87% reduction of the frequency of V beta 2+ cells in the circulation, and in a profound decrease of the respective T cells in spleens and thyroid glands. Selective suppression of V beta 1+ T cells partially inhibited SAT development in that thyroid-infiltrating cells and destruction of thyroid follicles were reduced by more than 50%. Thyroglobulin autoantibody serum levels were also reduced in V beta 1+ T cell-depleted OS chickens, whereas selective depletion of V beta 2+ T cells did not inhibit SAT development. These findings indicate preferential TCR V beta 1 gene usage by autoreactive T cells in SAT of OS chickens.
Asunto(s)
Receptores de Antígenos de Linfocitos T alfa-beta/genética , Linfocitos T/inmunología , Tiroiditis Autoinmune/genética , Tiroiditis Autoinmune/inmunología , Animales , Anticuerpos Monoclonales/farmacología , Autoanticuerpos/biosíntesis , Pollos , Depleción Linfocítica , Obesidad/genética , Obesidad/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/análisis , Tiroglobulina/inmunología , Glándula Tiroides/inmunología , Tiroiditis Autoinmune/etiologíaRESUMEN
Six monoclonal antibodies (mAb) were produced to identify and characterize surface antigens of chicken T cells. Determination of their reactivity with different lymphatic cells using immunofluorescence analysis demonstrates that mAb KH8, NA6, PD4 and TH8 stained 32-43% blood lymphocytes, 72-77% thymocytes and 19-27% spleen cells, mAb OC5 approximately 99% thymocytes and 55% blood and spleen lymphocytes each, and mAb OC2 36% blood lymphocytes, 79% thymocytes and 62% spleen cells. The KH8, NA6, PD4 and TH8 antibodies immunoprecipitated from lysates of surface-labeled chicken thymocytes a polypeptide of M(r) 60,000 under non-reducing conditions and the OC5 antibody a glycoprotein of M(r) 68,000 under reducing conditions. MAb OC2 precipitated a single polypeptide of M(r) 40,000 under both conditions. The mAb KH8, NA6, PD4, TH8 and OC2 inhibited ConA-induced proliferative responses of blood T cells in vitro. However, sepharose-bound or soluble OC5 antibody was able to increase DNA synthesis significantly. These results indicate that (a) the mAb KH8, NA6, PD4 and TH8 identify the avian homologue of the mammalian CD4 molecule, (b) the mAb OC2 detects the avian CD2 antigen, and (c) the mAb OC5 recognizes the putative avian CD5 homologue.
Asunto(s)
Anticuerpos Monoclonales , Antígenos de Diferenciación de Linfocitos T , Pollos/inmunología , Linfocitos T/inmunología , Animales , Anticuerpos Monoclonales/farmacología , Especificidad de Anticuerpos , Antígenos de Diferenciación de Linfocitos T/química , Antígenos de Diferenciación de Linfocitos T/aislamiento & purificación , Suero Antilinfocítico , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Peso Molecular , Bazo/inmunología , Timo/inmunología , Distribución TisularRESUMEN
In the chicken three types of T-cell receptors can be defined by monoclonal antibodies TCR1, TCR2 and TCR3, which recognize gamma delta T cells, and V beta 1- and V beta 2-expressing alpha beta T cells, respectively. In the present report we have analysed means of selectively depleting the gamma delta T cells and the V beta 1+ alpha beta T cells. gamma delta T cells, which represent up to 66% of all T cells in blood of a 6-month-old chicken, can be effectively depleted by neonatal thymectomy (Tx) to levels as low as 1%. Immunohistology demonstrates a similar depletion in lymphoid organs while intestinal epithelium-associated gamma delta T cells are affected by Tx to a lesser extent. V beta 1-bearing alpha beta T cells, which comprise about 80% of the alpha beta T cells, were depleted by embryonic and neonatal injection of the TCR2 antibody. In the thymus such treatment depleted only the V beta 1+ alpha beta T cells with high density expression of T-cell receptor. Therefore, we thymectomized TCR2-treated animals in order to prevent development of mature V beta 1+ alpha beta T cells from the low density immature thymocytes. Treatment of chickens with a total of 22 mg of TCR2 antibody plus Tx reduced V beta 1+ alpha beta T cells from an average of 65% to 10% of all T cells. In these TCR2 antibody-treated animals the V beta 2-expressing alpha beta T cells become the predominant type of T cell (average 85%).
Asunto(s)
Depleción Linfocítica , Subgrupos de Linfocitos T/inmunología , Factores de Edad , Animales , Pollos , Hipersensibilidad Tardía , Ratones , Receptores de Antígenos de Linfocitos T alfa-beta/análisis , Receptores de Antígenos de Linfocitos T gamma-delta/análisis , TimectomíaRESUMEN
We induced a virus-specific cytotoxic T lymphocyte (CTL) response in B2 chickens by i.v. inoculation with 100 TCID50 of the reticuloendotheliosis virus (REV). Chickens were sacrificed 7 days after the infection and cytotoxic activity of the spleen cells against various target cells was assayed in a 4 h 51Cr-release assay at an effector to target ratio of 100:1. In addition, T cell receptor (TCR) alpha beta and TCR gamma delta cells were negatively selected from the REV-immune spleen cells and used as effector cells against REV-infected B2 target cells. (On average 40% of spleen T cells express TCR gamma delta in the chicken.) By inhibition of the cytotoxic activity of the immune spleen cells against REV-infected syngeneic target cells with monoclonal antibodies specific for chicken CD3 and CD8 molecules, the effector cells could be identified as CD8+ T cells. The cytotoxic activity was MHC-restricted, as only syngeneic but not allogeneic REV-infected target cells were lysed by REV-immune spleen cells, and virus-specific, as no cytotoxic activity could be found using uninfected syngeneic target cells. When assaying the activity of the negatively selected, > 98% pure alpha beta and gamma delta T cells, it was found that alpha beta T cells exerted virus-specific CTL activity ranging from 26 to 62% specific 51Cr-release, while gamma delta T cells showed only 2-4% 51Cr-release. These data indicate that REV-specific CTL response is mediated by alpha beta T cells and that gamma delta T cells are not involved in virus-specific CTL activity in the spleen of REV-infected chickens.
Asunto(s)
Enfermedades Linfáticas/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Linfocitos T Citotóxicos/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Pollos , Pruebas Inmunológicas de Citotoxicidad , Citotoxicidad Inmunológica , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente , Inmunoglobulina G/biosíntesis , Inmunoglobulina M/biosíntesis , Formación de Roseta , Bazo/inmunologíaRESUMEN
While alpha beta T cells in mammals may express one of many variable (V) gene families in the beta locus, chickens have only two V beta gene families. The avian V beta 2+ T cells are recognized by the T-cell receptor 3 (TCR3) monoclonal antibody and V beta 1+ T cells are recognized by the TCR2 antibody, which we used to selectively suppress development of V beta 1+ T cells in order to examine their functional role. Suppression was accomplished by multiple injections of anti-TCR2 antibodies beginning in embryonic life and perpetuated by thymectomy 8 days after hatching. Young birds thus depleted of V beta 1+ T cells had greater than normal numbers of V beta 2+ T cells and appeared as healthy as thymectomized and untreated controls. While production of IgM and IgG antibodies was unimpaired, IgA antibody production was severely compromised in the V beta 1-depleted birds. The levels of secretory IgA in bile and lung lavage fluid were reduced 1000- to 10,000-fold and secretory IgA antibodies were not produced in response to mucosal immunization. B-cell production of IgA antibodies thus appears to require T cells expressing the V beta 1 genes, whereas T cells that express the V beta 2 genes lack this capacity.
Asunto(s)
Inmunoglobulina A/biosíntesis , Familia de Multigenes , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Animales , Anticuerpos Monoclonales , Formación de Anticuerpos , Antígenos de Diferenciación de Linfocitos T/análisis , Bilis/inmunología , Líquido del Lavado Bronquioalveolar/inmunología , Complejo CD3 , Pollos , Inmunohistoquímica , Sustancias Macromoleculares , Especificidad de Órganos , Receptores de Antígenos de Linfocitos T/análisis , Bazo/citología , Bazo/inmunología , Lágrimas/inmunología , TimectomíaAsunto(s)
Anestesia de Conducción , Anestesia Obstétrica , Cesárea , Anestesia General , Puntaje de Apgar , Urgencias Médicas , Femenino , Humanos , Recién Nacido , EmbarazoRESUMEN
Treatment of chickens as embryos and in the first six days after hatching with mg amounts of anti-TCR2 (alpha beta T cell antigen receptor) antibody plus thymectomy 6 to 8 days after hatching suppressed by more than 90% the TCR2 and TCR1 (TCR gamma delta) subpopulations in the blood. Thymectomy alone resulted only in a profound reduction (greater than 90%) of the absolute number of TCR1 cells, while the absolute number of TCR2 cells was reduced only by 65%. We were interested in the regulation of the immunoglobulin synthesis in these T cell deficient chickens. We determined serum IgG, IgM and IgA levels and found a profound selective reduction (by about 90% on average) of the serum IgA level in anti-TCR2 antibody treated plus thymectomized chickens. In chickens which had been thymectomized only, the serum IgA level was reduced on average only by about 60%. Serum IgG and IgM levels were not reduced in any group. Thus, there was a positive correlation of the serum IgA level and the absolute number of TCR2 cells in all groups. These results indicate that T cells which play a role in IgA specific isotype switching or in IgA B cell differentiation belong to the TCR2 subpopulation.
Asunto(s)
Pollos/inmunología , Inmunización Pasiva , Inmunoglobulinas/análisis , Receptores de Antígenos de Linfocitos T/inmunología , Timectomía/veterinaria , Animales , Embrión de Pollo/inmunologíaRESUMEN
We examined the in vivo generation of alloantigen-specific cytotoxic T lymphocytes in chicken TCR-alpha beta and TCR-gamma delta T cells. The TCR-alpha beta and TCR-gamma delta subpopulations were separated from spleen of alloantigen-immune chickens using a negative selection method. The separated cells were examined for alloantigen-specific CTL activity in a 51Cr release assay. While negatively selected TCR-alpha beta cells exerted alloantigen-specific CTL activity, no cytotoxic activity could be detected with TCR-gamma delta cells.
Asunto(s)
Receptores de Antígenos de Linfocitos T gamma-delta , Linfocitos T Citotóxicos/inmunología , Animales , Antígenos CD , Pollos , Inmunización , Isoantígenos , Receptores de Antígenos de Linfocitos T alfa-betaAsunto(s)
Amenaza de Aborto/diagnóstico , Cardiotocografía/instrumentación , Femenino , Humanos , EmbarazoRESUMEN
We have performed immunoperoxidase staining on cryostat tissue sections and immunofluorescence analysis on cell suspensions to identify cells expressing the alpha/beta T cell antigen receptor during ontogeny and adult life in chickens. We used the mouse monoclonal antibody, TCR2, which was previously shown to recognize the alpha/beta TCR in chickens. TCR2+ cells were observed in thymic cortex and medulla and in T-dependent areas of spleen, intestine, and cecal tonsils of young adult chickens. Some TCR2+ cells were found in the cortex of bursal follicles and in liver. The first TCR2+ cells appear in thymus on Day 13 of the embryonic life and it is only after hatching that TCR2+ cells begin to migrate to the periphery.