RESUMEN
CD8(+) T-cell responses against latent viruses can cover considerable portions of the CD8(+) T-cell compartment for many decades, yet their initiation and maintenance remains poorly characterized in humans. A key question is whether the clonal repertoire that is raised during the initial antiviral response can be maintained over these long periods. To investigate this we combined next-generation sequencing of the T-cell receptor repertoire with tetramer-sorting to identify, quantify and longitudinally follow virus-specific clones within the CD8(+) T-cell compartment. Using this approach we studied primary infections of human cytomegalovirus (hCMV) and Epstein Barr virus (EBV) in renal transplant recipients. For both viruses we found that nearly all virus-specific CD8(+) T-cell clones that appeared during the early phase of infection were maintained at high frequencies during the 5-year follow-up and hardly any new anti-viral clones appeared. Both in transplant recipients and in healthy carriers the clones specific for these latent viruses were highly dominant within the CD8(+) T-cell receptor Vß repertoire. These findings suggest that the initial antiviral response in humans is maintained in a stable fashion without signs of contraction or changes of the clonal repertoire.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Infecciones por Citomegalovirus/inmunología , Citomegalovirus/inmunología , Infecciones por Virus de Epstein-Barr/inmunología , Herpesvirus Humano 4/inmunología , Receptores de Antígenos de Linfocitos T/genética , Antígenos Virales/inmunología , Citomegalovirus/genética , Herpesvirus Humano 4/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Trasplante de Riñón/inmunología , Persona de Mediana Edad , Factores de Tiempo , Latencia del Virus , Adulto JovenRESUMEN
Rabbit anti-thymocyte globulin (rATG) induces a long-lasting lymphocytopenia. CD4(+) T cells remain depleted for up to 2 years, whereas the CD8(+) T cell compartment is refilled rapidly by highly differentiated CD27(-) CD45RA(+) CD57(+) effector-type cells. Because the presence of these highly differentiated CD8(+) T cells has been associated with cytomegalovirus (CMV) infection, we questioned to what extent restoration of CMV T cell immunity contributes to the re-emergence of T cells following rATG treatment. We compared T cell repopulation in six CMV-seropositive patients with CMV reactivation (reactivating CMV(+) ) to that in three CMV(+) patients without reactivation (non-reactivating CMV(+) ), and to that in three CMV-seronegative recipients receiving a kidney from a CMV-seronegative donor (CMV(-/-) ). All patients received rATG because of acute allograft rejection. Total CD4 and CD8 counts, frequency and phenotype of virus-specific CD8(+) T cells were determined. In reactivating CMV(+) patients, total CD8(+) T cells reappeared rapidly, whereas in non-reactivating CMV(+) patients they lagged behind. In CMV(-/-) patients, CD8(+) T cell counts had not yet reached pretransplant levels after 2 years. CMV reactivation was indeed followed by a progressive accumulation of CMV-specific CD8(+) T cells. During lymphocytopenia following rATG treatment, serum interleukin (IL)-7 levels were elevated. Although this was most prominent in the CMV-seronegative patients, it did not result in an advantage in T cell repopulation in these patients. Repopulated CD8(+) T cells showed increased skewing in their Vß repertoire in both CMV(-/-) and reactivating CMV-seropositive patients. We conclude that rapid T cell repopulation following rATG treatment is driven mainly by CMV.
Asunto(s)
Suero Antilinfocítico/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Infecciones por Citomegalovirus/inmunología , Citomegalovirus/fisiología , Inmunosupresores/inmunología , Linfopenia/inmunología , Complicaciones Posoperatorias/inmunología , Adulto , Animales , Suero Antilinfocítico/uso terapéutico , Antivirales/uso terapéutico , Infecciones por Citomegalovirus/tratamiento farmacológico , Infecciones por Citomegalovirus/virología , ADN Viral/sangre , Femenino , Ganciclovir/análogos & derivados , Ganciclovir/uso terapéutico , Reordenamiento Génico de la Cadena beta de los Receptores de Antígenos de los Linfocitos T , Rechazo de Injerto/tratamiento farmacológico , Humanos , Inmunosupresores/uso terapéutico , Interleucina-7/sangre , Trasplante de Riñón , Recuento de Linfocitos , Linfopenia/patología , Masculino , Persona de Mediana Edad , Complicaciones Posoperatorias/virología , Conejos , Valganciclovir , Viremia/tratamiento farmacológico , Viremia/inmunología , Viremia/virología , Activación Viral , Adulto JovenRESUMEN
Several assays to measure pre-existing allospecific T cell immunity in renal transplant candidates have been developed in the past years. In 46 patients, we used flow cytometry-based mixed lymphocyte culture to measure the precursor frequency and phenotype of alloreactive T cells before renal transplantation, using donor-specific or third-party cells for allostimulation. Allostimulation induced up-regulation of co-stimulatory molecules, chemokine receptors relevant for migration of T cells into the graft and effector proteins. Recipients prone for acute rejection had a higher precursor frequency of alloreactive CD8(+) T cells and a lower percentage of interleukin (IL)-7Rα expressing alloreactive CD8(+) T cells than non-rejectors. These data point to quantitative and qualitative differences between T cells of patients who will experience acute cellular rejection episodes from those who will not.
Asunto(s)
Trasplante de Riñón/inmunología , Linfocitos T/inmunología , Adulto , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Femenino , Rechazo de Injerto/tratamiento farmacológico , Rechazo de Injerto/inmunología , Humanos , Inmunofenotipificación , Interferón gamma/metabolismo , Subunidad alfa del Receptor de Interleucina-15/metabolismo , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Activación de Linfocitos/inmunología , Recuento de Linfocitos , Prueba de Cultivo Mixto de Linfocitos , Masculino , Persona de Mediana Edad , Receptores de Quimiocina/metabolismo , Receptores de Citocinas/metabolismo , Receptores de Interleucina-7/metabolismo , Donantes de Tejidos , Trasplante Homólogo/inmunologíaRESUMEN
CD97 is an activation-induced antigen on leukocytes with a seven-span transmembrane (7-TM) region homologous to the secretin receptor superfamily. However, in contrast to this group of peptide hormone receptors, CD97 has an extended extracellular region with three EGF domains at the NH2 terminus, two of them with a calcium binding site. By demonstrating that lymphocytes and erythrocytes specifically adhere to CD97-transfected COS cells we here show that CD97 in parallel with its molecular evolution has acquired the ability to bind cellular ligands. A mAb selected on its capacity to block the adhesion between CD97 transfectants and red cells was found to be directed to the NH2-terminal short consensus repeat (SCR) of decay accelerating factor (DAF, CD55), a regulatory protein of the complement cascade. The specificity of the interaction of CD97 with CD55 was established by the observation that erythrocytes that lack CD55, obtained from patients with paroxysmal nocturnal hemoglobinuria (PNH) or the CD55, phenotype Inab, failed to adhere to CD97 transfectants. This is the first demonstration of a cellular ligand for a 7-TM receptor.
Asunto(s)
Antígenos CD55/metabolismo , Glicoproteínas de Membrana/metabolismo , Animales , Antígenos CD , Células COS , Adhesión Celular , Eritrocitos/metabolismo , Humanos , Receptores Acoplados a Proteínas G , TransfecciónRESUMEN
A large number of CD4+ T cell clones, obtained from peripheral blood T lymphocytes by direct limiting dilution, allowed us to address the question whether functional heterogeneity exists within the human CD4+ T cell subset. Cytotoxic capacity of cloned T cells was analyzed with the use of anti-CD3 antibodies and target cells bearing FcR for murine IgG. 6 of 12 CD4+ clones obtained were able to lyse Daudi or P815 cells in the presence of anti-CD3 antibodies. The remaining six CD4+ T cell clones tested did not display anti-CD3-mediated cytotoxic activity and did not acquire this cytotoxic capacity during a culture period of 20 wk. In the absence of anti-CD3 mAb, no lytic activity against Daudi, P815, and K562 target cells was observed under normal culture conditions. Phenotypic analysis of these two distinct types of CD4+ T cells did not reveal differences with regard to reactivity with CDw29 (4B4) and CD45R (2H4) mAbs that have been described to recognize antigens associated with helper suppressor/inducer (respectively) CD4+ cells. The CD4+ clones without anti-CD3-mediated cytotoxic activities (Th2) consistently showed a high expression level of CD28 antigens, whereas the cytotoxic clones (Th1) expressed low amounts of CD28. Th1 CD4+ clones did produce IL-2, IFN-gamma, and TNF-alpha/beta, whereas the Th2 T cell clones produced minimal amounts of IL-2 and only low levels of INF-gamma and TNF-alpha/beta in response to anti-CD3 mAbs and PMA. Although not all CD4+ clones did release IL-4, there was no correlation with cytotoxic activity. Moreover, as compared with the Th1 CD4+ clones, Th2 CD4+ T cell clones proliferated moderately in response to immobilized anti-CD3 mAbs. However, proliferation reached the level of the cytotoxic clones when anti-CD28 mABs were present during culture. Both CD4+ subsets provided help for B cell differentiation upon stimulation with anti-CD3 mAbs. Our data suggest that the human CD4+ subset, in analogy to the murine system, comprises two functionally distinct T cell subpopulations, both of which are able to exert helper activity for polyclonal B cell differentiation, but which differ in cytotoxic capacity, lymphokine production, and requirements for proliferation. A function for these two types of T cells in the immune response is discussed.
Asunto(s)
Linfocitos T CD4-Positivos/clasificación , Antígenos de Diferenciación de Linfocitos T/análisis , Antígenos CD28 , Complejo CD3 , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/fisiología , Células Clonales , Citotoxicidad Inmunológica , Antígenos de Histocompatibilidad Clase II/análisis , Humanos , Inmunidad Celular , Interleucina-2/farmacología , Activación de Linfocitos , Linfocinas/biosíntesis , Receptores de Antígenos de Linfocitos T/análisisRESUMEN
Human CD8+ memory- and effector-type T cells are poorly defined. We show here that, next to a naive compartment, two discrete primed subpopulations can be found within the circulating human CD8+ T cell subset. First, CD45RA-CD45R0(+) cells are reminiscent of memory-type T cells in that they express elevated levels of CD95 (Fas) and the integrin family members CD11a, CD18, CD29, CD49d, and CD49e, compared to naive CD8+ T cells, and are able to secrete not only interleukin (IL) 2 but also interferon gamma, tumor necrosis factor alpha, and IL-4. This subset does not exert cytolytic activity without prior in vitro stimulation but does contain virus-specific cytotoxic T lymphocyte (CTL) precursors. A second primed population is characterized by CD45RA expression with concomitant absence of expression of the costimulatory molecules CD27 and CD28. The CD8+CD45RA+CD27- population contains T cells expressing high levels of CD11a, CD11b, CD18, and CD49d, whereas CD62L (L-selectin) is not expressed. These T cells do not secrete IL-2 or -4 but can produce IFN-gamma and TNF-alpha. In accordance with this finding, cells contained within this subpopulation depend for proliferation on exogenous growth factors such as IL-2 and -15. Interestingly, CD8+CD45RA+CD27- cells parallel effector CTLs, as they abundantly express Fas-ligand mRNA, contain perforin and granzyme B, and have high cytolytic activity without in vitro prestimulation. Based on both phenotypic and functional properties, we conclude that memory- and effector-type T cells can be separated as distinct entities within the human CD8+ T cell subset.
Asunto(s)
Memoria Inmunológica , Linfocitos T Reguladores/clasificación , Linfocitos T Reguladores/inmunología , Adulto , Células Cultivadas , Citocinas/biosíntesis , Citotoxicidad Inmunológica , Epítopos de Linfocito T/análisis , Células Madre Hematopoyéticas/clasificación , Células Madre Hematopoyéticas/inmunología , Humanos , Inmunoglobulinas/biosíntesis , Inmunofenotipificación , Antígenos Comunes de Leucocito/análisis , Activación de Linfocitos , Cooperación Linfocítica , Linfocitos T Citotóxicos/clasificación , Linfocitos T Citotóxicos/inmunología , Linfocitos T Reguladores/metabolismo , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/análisisRESUMEN
We have recently reported that on human B lymphocytes, membrane IgM (mIgM) associates with a heterodimer of 47- and 37-kD polypeptides, the 47-kD subunit being encoded by the mb-1 gene. We show here that expression of mb-1, both at the mRNA and the protein level, is not restricted to IgM+ B cells but can also be found in IgM- pre-B cells and mIgM-IgG+ B cells. Membrane forms of IgD and IgG, isolated from freshly isolated human B cells and B cell lines, are expressed together with heterodimeric protein structures biochemically similar to the mIgM-associated polypeptides, and these were shown to comprise the products of the mb-1 and B29 genes, or homologous genes. Finally, all three classes of antigen receptors are linked to protein kinases, capable of phosphorylating the Ig-associated heterodimers. Our findings provide insight in the structural organization of the different antigen receptors on human B cells and have implications for their function.
Asunto(s)
Linfocitos B/inmunología , Genes de Inmunoglobulinas , Inmunoglobulina D/metabolismo , Inmunoglobulina G/metabolismo , Inmunoglobulina M/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Anticuerpos Monoclonales , Northern Blotting , Línea Celular , Membrana Celular/inmunología , Técnica del Anticuerpo Fluorescente , Humanos , Inmunoglobulina D/análisis , Inmunoglobulina D/genética , Inmunoglobulina G/análisis , Inmunoglobulina G/genética , Inmunoglobulina M/análisis , Inmunoglobulina M/genética , Cadenas lambda de Inmunoglobulina/análisis , Cadenas mu de Inmunoglobulina/análisis , Sustancias Macromoleculares , Peso Molecular , Tonsila Palatina/inmunología , Fosforilación , ARN/genética , ARN/aislamiento & purificación , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Antígenos de Linfocitos B/análisisRESUMEN
Cyclopentenyl cytosine (CPEC) has been shown to induce apoptosis in human T lymphoblastic cell lines and T cells from leukaemia patients. In this study we have addressed the question of whether CPEC is able to decrease proliferation and effector functions of human alloresponsive T lymphocytes and induce T cell anergy. The proliferative capacity of human peripheral blood mononuclear cells in response to allogeneic stimulation was measured by 5,6-carboxy-succinimidyl-diacetate-fluorescein-ester staining. Flow cytometric analysis was performed using surface CD4, CD8, CD25, CD103 and intracellular perforin, granzyme A, granzyme B, caspase-3 and forkhead box P3 (FoxP3) markers. The in vivo immunosuppressive capacity was tested in a murine skin graft model. Addition of CPEC at a concentration of 20 nM strongly decreased the expansion and cytotoxicity of alloreactive T cells. Specific restimulation in the absence of CPEC showed that the cells became anergic. The drug induced caspase-dependent apoptosis of alloreactive T lymphocytes. Finally, CPEC increased the percentage of CD25(high) FoxP3+ CD4+ and CD103+ CD8+ T cells, and potentiated the effect of rapamycin in increasing the numbers of alloreactive regulatory T cells. Treatment with CPEC of CBA/CA mice transplanted with B10/Br skin grafts significantly prolonged graft survival. We conclude that CPEC inhibits proliferation and cytotoxicity of human alloreactive T cells and induces alloantigen non-responsiveness in vitro.
Asunto(s)
Anergia Clonal/efectos de los fármacos , Citidina/análogos & derivados , Inhibidores Enzimáticos/farmacología , Isoantígenos/inmunología , Subgrupos de Linfocitos T/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Apoptosis/inmunología , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Anergia Clonal/inmunología , Citidina/farmacología , Citotoxicidad Inmunológica/efectos de los fármacos , Relación Dosis-Respuesta Inmunológica , Supervivencia de Injerto/efectos de los fármacos , Humanos , Inmunosupresores/farmacología , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos , Trasplante de Piel , Subgrupos de Linfocitos T/inmunologíaRESUMEN
Resident memory T cells (TRM) reside in the lung epithelium and mediate protective immunity against respiratory pathogens. Although lung CD8+ TRM have been extensively characterized, the properties of CD4+ TRM remain unclear. Here we determined the transcriptional signature of CD4+ TRM, identified by the expression of CD103, retrieved from human lung resection material. Various tissue homing molecules were specifically upregulated on CD4+ TRM, whereas expression of tissue egress and lymph node homing molecules were low. CD103+ TRM expressed low levels of T-bet, only a small portion expressed Eomesodermin (Eomes), and although the mRNA levels for Hobit were increased, protein expression was absent. On the other hand, the CD103+ TRM showed a Notch signature. CD4+CD103+ TRM constitutively expressed high transcript levels of numerous cytotoxic mediators that was functionally reflected by a fast recall response, magnitude of cytokine production, and a high degree of polyfunctionality. Interestingly, the superior cytokine production appears to be because of an accessible interferon-γ (IFNγ) locus and was partially because of rapid translation of preformed mRNA. Our studies provide a molecular understanding of the maintenance and potential function of CD4+ TRM in the human lung. Understanding the specific properties of CD4+ TRM is required to rationally improve vaccine design.
Asunto(s)
Linfocitos T CD4-Positivos/fisiología , Pulmón/fisiología , Receptores Notch/metabolismo , Anciano , Animales , Antígenos CD/metabolismo , Citotoxicidad Inmunológica , Femenino , Regulación de la Expresión Génica , Humanos , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/genética , Memoria Inmunológica , Cadenas alfa de Integrinas/metabolismo , Interferón gamma/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Receptores Notch/genética , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/metabolismo , TranscriptomaRESUMEN
In addition to a well-documented depletion of CD4+ T helper cells in later stages of human immunodeficiency virus (HIV) infection, evidence has been provided for a specific unresponsiveness to triggering either by specific antigen in the context of autologous major histocompatibility molecules (self + X) or anti-CD3 monoclonal antibodies (MAb) in both CD4 and CD8 cells from asymptomatic HIV-infected individuals. In the present study we analyzed this unresponsiveness using mitogenic antibodies to distinct T cell membrane receptors. T cells from HIV-infected men who had normal numbers of CD4+ T cells responded poorly to activation signals via the CD3 membrane antigen in both accessory cell-dependent as well as accessory cell-independent culture systems. A similar low response was observed in an anti-CD2-driven system. In contrast, proliferation induced by anti-CD3, anti-CD2, or the phorbol ester Phorbol myristate acetate could be normally enhanced by anti-CD28 MAb. We demonstrated that this unresponsiveness is not due to a failure to induce early events required for activation, such as increased intracellular concentration of free calcium and activation of protein kinase C, but is caused by an imbalance between naive and memory T cells. In HIV-infected asymptomatic men, CD29+ memory T cells are selectively depleted which results in a poor responsiveness to self + X. These findings provide new insights that may have implications for our understanding of the immunopathogenesis of AIDS.
Asunto(s)
Infecciones por VIH/inmunología , Memoria Inmunológica , Linfocitos T/inmunología , Anticuerpos Monoclonales , Antígenos CD/análisis , Antígenos de Diferenciación/análisis , Antígenos de Diferenciación/fisiología , Antígenos de Diferenciación de Linfocitos T/fisiología , Antígenos CD2 , Antígenos CD28 , Complejo CD3 , Calcio/fisiología , Humanos , Integrina beta1 , Antígenos Comunes de Leucocito , Activación de Linfocitos , Masculino , Receptores de Antígenos de Linfocitos T/fisiología , Receptores Inmunológicos/fisiología , Transducción de SeñalRESUMEN
CD4(pos) TH1 T cells are considered to play a central role in a number of human autoimmune diseases such as rheumatoid arthritis (RA) and multiple sclerosis. Experimental treatment protocols aimed at selectively eliminating CD4(pos) T cells thus far have yielded disappointing clinical results. Here we analyzed phenotype and function of circulating T cells in multiple sclerosis patients treated with the chimeric CD4 mAb cM-T412 in a randomized, double-blind, placebo-controlled, magnetic resonance imaging-monitored phase II trial. Treatment resulted in a long-lasting depletion of CD4(pos) T cells but did not affect CD8(pos) T cell numbers. Analysis of CD4(pos) subpopulations showed that unprimed, CD45RA(pos)/R0(neg) lymphocytes were approximately three times more sensitive to the mAb than primed, CD45RA(neg)/R0(pos) T cells. Notably, within the CD45RA(pos) subset, T cells with phenotypic evidence of prior activation, i.e., expressing Fas, were relatively insensitive to cM-T412, compared with Fas(neg) cells. Remarkably, while a decrease in the number of IL-4-producing T helper 2 (TH2)-type cells in the anti-CD4 treated group was observed, numbers of IFN-gamma-producing T helper 1 (TH1)-type cells remained stable, resulting in a significant increase in the TH1/TH2 ratio. Our data show that treatment with depleting CD4 mAb does not eliminate the cells most strongly involved in the disease process, i.e., primed, IFN-gamma-producing TH1-type cells, and may therefore give an explanation for the lack of beneficial clinical effects of depleting CD4 mAb in human chronic autoimmune disease.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Linfocitos T CD4-Positivos/inmunología , Depleción Linfocítica , Esclerosis Múltiple/terapia , Subgrupos de Linfocitos T , Células TH1/inmunología , Adulto , Antígenos CD4/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/inmunología , Método Doble Ciego , Femenino , Humanos , Interferón gamma/biosíntesis , Interferón gamma/inmunología , Interleucina-4/biosíntesis , Interleucina-4/inmunología , Antígenos Comunes de Leucocito/inmunología , Masculino , Persona de Mediana Edad , Esclerosis Múltiple/inmunología , Subgrupos de Linfocitos T/inmunología , Células TH1/metabolismo , Células Th2/inmunología , Células Th2/metabolismo , Receptor fas/inmunologíaRESUMEN
Leukocyte adhesion deficiency (LAD) is characterized by the inability of leukocytes, in particular neutrophilic granulocytes, to emigrate from the bloodstream towards sites of inflammation. Infectious foci are nonpurulent and may eventually become necrotic because of abnormal wound healing. LAD-1 is characterized by the absence of the beta2 integrins (CD11/CD18) on leukocytes. When expression is completely absent, patients often die within the first year. However, low levels of beta2 expression may result in a milder clinical picture of recurrent infection, which offers a better prognosis. In this paper, we describe the in vivo and in vitro findings on a patient with clinical features of a mild LAD-1 disorder, i.e., suffering from bacterial infections without apparent pus formation in the presence of a striking granulocytosis, showing no delayed-type hypersensitivity reaction upon skin testing, no specific antibody generation, but normal in vitro T cell proliferation responses after immunization. Expression levels of CD11/CD18 proteins were completely normal, but leukocyte activation did not result in CD11/ CD18 activation and high-avidity ligand-binding. In vitro chemotaxis and endothelial transmigration of the neutrophils as well as leukocyte aggregation responses were almost absent. On the other hand, beta1 and beta3 integrin-mediated adhesion functions were completely normal. During follow-up, a bleeding tendency related to decreased beta3 activation became clinically apparent, different from previously described cellular adhesion molecule variants. Therefore, this is the first well-documented case of a clinical combined immunodeficiency syndrome that results from nonfunctional CD11/CD18 molecules, and thus designated LAD-1/ variant.
Asunto(s)
Antígenos CD18/inmunología , Integrinas/inmunología , Síndrome de Deficiencia de Adhesión del Leucocito/inmunología , Antígenos CD/inmunología , Antígenos CD11/análisis , Antígenos CD18/análisis , Antígenos CD18/genética , Adhesión Celular , Agregación Celular , Quimiotaxis de Leucocito , Células Clonales , Hemorragia , Humanos , Lactante , Integrina beta1/inmunología , Integrina beta3 , Síndrome de Deficiencia de Adhesión del Leucocito/diagnóstico , Activación de Linfocitos , Masculino , Activación Neutrófila , Agregación Plaquetaria , Glicoproteínas de Membrana Plaquetaria/inmunología , Análisis de Secuencia de ADN , Linfocitos T/inmunologíaRESUMEN
Although virus-specific CD4(+) T cells have been characterized extensively in latently infected individuals, it is unclear how these protective T-cell responses develop during primary virus infection in humans. Here, we analyzed the kinetics and characteristics of cytomegalovirus-specific (CMV-specific) CD4(+) T cells in the course of primary CMV infection in kidney transplant recipients. Our data reveal that, as the first sign of specific immunity, circulating CMV-specific CD4(+) T cells become detectable with a median of 7 days after first appearance of CMV-DNA in peripheral blood. These cells produce the T helper 1 type (Th1) cytokines IFNgamma and TNFalpha, but not the T helper 2 type (Th2) cytokine IL4. In primary CMV infection, the vast majority of these circulating virus-specific T cells have features of recently activated naive T cells in that they coexpress CD45RA and CD45R0 and appear to be in the cell cycle. In contrast, in people who have recovered from CMV infection earlier in life, virus-specific T cells do not cycle and express surface markers characteristic of memory T cells. After the initial rise, circulating virus-specific CD4(+) T cells decline rapidly. During this phase, a strong rise in IgM and IgG anti-CMV antibody titers occurs, concomitant with the reduction of CMV-DNA in the circulation.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Infecciones por Citomegalovirus/inmunología , Estudios Transversales , Citomegalovirus/aislamiento & purificación , ADN Viral/sangre , Humanos , Inmunidad Celular , Trasplante de Riñón/efectos adversos , Estudios Longitudinales , Donantes de TejidosRESUMEN
B-cell chronic lymphocytic leukaemia (B-CLL) is a slowly progressing malignancy of CD5(+) B cells, for which at present no curative treatment is available. In our current study, we apply a novel bridging reagent to redirect cytomegalovirus (CMV)-specific cytotoxic T lymphocytes (CTL) to target B-CLL. A streptavidin-fused anti-CD20 single-chain variable fragment (scFv) is used in combination with biotinylated MHC class I molecules containing CMV pp65 peptide (HLA/CMV). We demonstrate that B-CLL cells coated with this CD20-HLA/CMV complex can be lysed by autologous CMV-specific CTL with similar efficiency as B-CLL cells directly loaded with CMV peptide. Killing is HLA restricted and occurs at scFv CD20 concentrations of >/=100 ng ml(-1) and HLA/CMV concentrations of >/=20 ng ml(-1). Furthermore, complex-coated B-CLL cells induce both proliferation and cytokine production (interferon gamma, tumour necrosis factor alpha and macrophage inflammatory protein-1 beta) in CMV-specific CD8(+) T cells. Hereby, a necessary step towards possible application of CD20-HLA/CMV complexes for immunotherapy of B-cell malignancies is constituted.
Asunto(s)
Antígenos CD20/inmunología , Citomegalovirus/inmunología , Antígenos HLA/inmunología , Leucemia Linfocítica Crónica de Células B/inmunología , Péptidos/inmunología , Linfocitos T Citotóxicos/inmunología , Antígenos CD20/biosíntesis , Proliferación Celular , Citocinas/biosíntesis , Pruebas Inmunológicas de Citotoxicidad , Humanos , Leucemia Linfocítica Crónica de Células B/diagnóstico , Complejos Multiproteicos/inmunología , Células Tumorales CultivadasRESUMEN
We investigated the apoptosis gene expression profile of chronic lymphocytic leukemia (CLL) cells in relation to (1) normal peripheral and tonsillar B-cell subsets, (2) IgV(H) mutation status, and (3) effects of cytotoxic drugs. In accord with their noncycling, antiapoptotic status in vivo, CLL cells displayed high constitutive expression of Bcl-2 and Flip mRNA, while Survivin, Bid and Bik were absent. Paradoxically, along with these antiapoptotic genes CLL cells had high-level expression of proapoptotic BH3-only proteins Bmf and Noxa. Treatment of CLL cells with fludarabine induced only the proapoptotic genes Bax and Puma in a p53-dependent manner. Interestingly, the degree of Puma induction was more pronounced in cells with mutated IgVH genes. Thus, disturbed apoptosis in CLL is the net result of both protective and sensitizing aberrations. This delicate balance can be tipped via induction of Puma in a p53-dependent matter, the level of which may vary between groups of patients with a different tendency for disease progression.
Asunto(s)
Antineoplásicos/farmacología , Leucemia Linfocítica Crónica de Células B/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Fosfato de Vidarabina/análogos & derivados , Fosfato de Vidarabina/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/genética , Apoptosis/fisiología , Proteínas Reguladoras de la Apoptosis , Resistencia a Antineoplásicos , Perfilación de la Expresión Génica/métodos , Regulación Leucémica de la Expresión Génica , Humanos , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteína p53 Supresora de Tumor/efectos de los fármacos , Proteína p53 Supresora de Tumor/genética , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Induction of T-cell activation requires multiple signals provided by cell surface receptor interactions and/or cytokines. T-cell stimulation via the T-cell receptor/CD3 complex provides an important initial activation event which, when combined with the proper costimulatory signals, results in an activated effector T cell. In this report, we have investigated the effectiveness of epithelial glycoprotein-2- (EGP-2) positive tumor target cells to induce specific T-cell stimulation via CD3, CD5, and CD28 using various combinations of bispecific monoclonal antibodies (BsMab) directed against CD3, CD5, or CD28 on the one hand and the pancarcinoma-associated antigen EGP-2 on the other. Induction of T-cell activation was investigated by assessment of CD69 expression, induction of proliferation, and acquirement of cytolytic potential. EGP-2-specific induction of T-cell activation was observed using combinations of BsMab which simultaneous ligated CD3/CD5, CD3/CD28, or CD3/CD5/CD28 with EGP-2. Activation with CD3-, CD5-, or CD28-based BsMab alone did not result in significant induction of T-cell activation in the presence or absence of EGP-2-positive target cells. Simultaneous ligation via CD5/CD28 resulted in partial T-cell activation, including CD69 up-regulation and increased cytolytic activity. Stimulation via CD3 and CD5 or CD28 could be further increased by the addition of exogenously added recombinant Interleukin 2. In contrast, T-cell activation by simultaneous ligation of CD3/CD5/CD28 could not be further augmented by addition of exogenous interleukin 2, indicating that T-cell activation via the combination of CD3, CD5, and CD28 results in complete T-cell activation. Our results show that rapid and target cell-specific induction of T cells is possible using combinations of BsMab directed against different costimulatory molecules. Simultaneous costimulation via CD3/CD5/CD28 results in the most complete activation of T cells.
Asunto(s)
Anticuerpos Biespecíficos/inmunología , Antígenos CD/fisiología , Antígenos CD28/fisiología , Activación de Linfocitos , Linfocitos T/inmunología , Antígenos de Neoplasias/análisis , Antígenos de Neoplasias/inmunología , Complejo CD3/fisiología , Antígenos CD5 , Moléculas de Adhesión Celular/análisis , Moléculas de Adhesión Celular/inmunología , Citotoxicidad Inmunológica , Molécula de Adhesión Celular Epitelial , Humanos , Interleucina-2/farmacología , Células Tumorales CultivadasRESUMEN
The apoptotic pathway activated by chimeric anti-CD20 monoclonal antibodies (rituximab, IDEC.C2B8) was analyzed using the Burkitt lymphoma cell line Ramos. Crosslinking of CD20 (CD20XL) induced apoptosis in Ramos cells, which involved loss of mitochondrial membrane potential (Deltapsi(m)), the release of cytochrome-c (cyt-c), and activation of caspases-9 and -3. Nevertheless, several lines of evidence showed that the apoptotic outcome did not depend on these events. First, under circumstances where Ramos cells display resistance to either CD95- or B cell receptor (BCR)-induced apoptosis, CD20XL-induced apoptosis was not affected, pointing to a distinct pathway. Second, the broad-spectrum caspase inhibitor zVAD-fmk prevented processing of caspase-9, -3 and PARP as well as DNA fragmentation, but did not block apoptosis as measured by annexin V staining, cell size and membrane integrity. Lastly, Bcl-2 overexpression blocked cyt-c release and the decrease in Deltapsi(m), and completely prevented CD95- or BCR-mediated apoptosis; however, it did not affect CD20XL-induced cell death. We conclude that although CD20XL can initiate the mitochondrial apoptosis pathway, CD20-induced apoptosis does not necessarily require active caspases and cannot be blocked by Bcl-2. Since most chemotherapeutic drugs require the activation of caspases to exert their cytotoxicity, these findings provide an important rationale for the use of CD20 mAbs in chemoresistant malignancies.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Antígenos CD20/inmunología , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Linfocitos B/efectos de los fármacos , Caspasas/metabolismo , Mitocondrias/efectos de los fármacos , Clorometilcetonas de Aminoácidos/farmacología , Anticuerpos Monoclonales de Origen Murino , Linfocitos B/patología , Linfoma de Burkitt/tratamiento farmacológico , Linfoma de Burkitt/metabolismo , Linfoma de Burkitt/patología , Inhibidores de Caspasas , Inhibidores de Cisteína Proteinasa/farmacología , Grupo Citocromo c/metabolismo , Activación Enzimática , Humanos , Immunoblotting , Etiquetado Corte-Fin in Situ , Potenciales de la Membrana , Mitocondrias/enzimología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Rituximab , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/metabolismo , Células Tumorales Cultivadas/patologíaRESUMEN
Glycosidase trimming inhibitors may be used to study contribution of N-linked glycan moieties in T cell function. We have studied the effects of castanospermine (Cas), swainsonine (Swain), 1-deoxynojirimycin (dNM), and 1-deoxymannojirimycin (dMM) on T cell activation and differentiation. Our analysis included a new dNM derivative, N-pentyl-1-deoxynojirimycin (pentyldNM). Previous reports showed inhibitory action of trimming inhibitors, such as Swain and Cas, on pokeweed mitogen-driven immunoglobulin (Ig) production. We extend these findings for pentyldNM and observed that glucosidase inhibitors, Cas and pentyldNM were effective in inhibiting CD2 and CD3 monoclonal antibody (mAb) driven Ig production. The pattern of inhibition by mannosidase and glucosidase inhibitors correlated with inhibitory action on T cell activation: only glucosidase trimming inhibitors (Cas and pentyldNM with comparable potency) perturbed mAb-induced T cell activation, in particular if induced by CD2 mAb. Expression of the early activation marker CD69 was not decreased in the presence of these inhibitors, while addition of exogenous recombinant interleukin-2 partially overcame inhibitory effects during proliferation. These findings suggest that glucosidase, but not mannosidase, trimming inhibitors interfere with a late phase of T cell activation. In addition, the enhanced sensitivity of CD2 mAb-induced proliferation for glucosidase trimming inhibitors suggests dependence on N-linked glycans during CD2-mediated adhesion and triggering functions.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Antígenos de Diferenciación de Linfocitos T/inmunología , Glucosidasas/antagonistas & inhibidores , Activación de Linfocitos/efectos de los fármacos , Receptores Inmunológicos/inmunología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , 1-Desoxinojirimicina/análogos & derivados , 1-Desoxinojirimicina/farmacología , Antígenos CD2 , Complejo CD3/inmunología , Concanavalina A/farmacología , Glucosa/metabolismo , Glucosa/farmacocinética , Humanos , Inmunoglobulinas/biosíntesis , Indolizinas/farmacología , Interleucina-2/farmacología , Leucocitos Mononucleares/metabolismo , Manosidasas/antagonistas & inhibidores , Fitohemaglutininas/farmacología , Mitógenos de Phytolacca americana/farmacología , Polisacáridos/metabolismo , Proteínas Recombinantes/farmacología , Estimulación Química , Swainsonina/farmacologíaRESUMEN
Receptor tyrosine kinases (RTK), like the PDGF-receptor, translate information from the extracellular environment into cytoplasmic signals that regulate a spectrum of cellular functions. RTK molecules consist of ligand binding extracellular domains, cytoplasmic kinase domains and tyrosine phosphorylation sites [Ullrich and Schlessinger, 1990 (Cell 61, 203-212); Heldin, 1992 (EMBO J. 11, 4251-4259)]. Upon ligand-induced RTK oligomerization, the kinase domains will become activated and induce auto(trans)phosphorylation of a number of cytoplasmic tyrosine residues. These phosphorylated tyrosine residues are incorporated in distinct sequence motifs and act as specific docking sites for SH2 domain-containing proteins [Songyang et al., 1993 (Cell 72, 767-778)]. In contrast to single- or oligo-chain RTK, immunological receptors such as antigen receptors, FcR and cytokine receptors are multi-chain complexes in which distinct receptor functions appear to be compartmentalized in distinct polypeptides. Here, we summarize current knowledge on the structural and functional characteristics of the B-cell antigen receptor complex (BCR) and address the specific ability of accessory molecules to recruit intracellular signaling intermediates towards the activated receptor complex.
Asunto(s)
Receptores de Antígenos de Linfocitos B/química , Receptores de Antígenos de Linfocitos B/fisiología , Secuencia de Aminoácidos , Humanos , Datos de Secuencia Molecular , Relación Estructura-ActividadRESUMEN
Six men were selected from a large cohort of homosexual men participating in a study on HIV infection that was followed from seroconversion to AIDS. The patients were studied retrospectively for immunological functions of T cells, T-cell subset distribution and biological phenotype of HIV. A severe decrease in anti-CD3 monoclonal antibody (MAb)-induced T-cell proliferation at seroconversion was observed in two out of six men. After this acute phase, CD4+ T-cell numbers were in the normal range in the early asymptomatic period; the proliferative response was subnormal, whereas the capacity to generate cytotoxic T cells (CTL) was normal. From seroconversion on, CD4+CD29+ memory T-cell numbers were decreased to approximately 50% of normal values, which may contribute to loss of T-cell reactivity. In the asymptomatic phase only slow-replicating non-syncytium-inducing HIV variants were observed. The T-cell proliferative response further declined with the depletion of naive CD4+ CD45RA+ T cells and CD4+ T-cell numbers started to decline. This second decrease in T-cell function coincided with the emergence of more rapidly replicating, often (four out of six) syncytium-inducing variants. At diagnosis of AIDS, T-cell proliferation and CD4+ T-cell numbers were extremely low in five out of six patients and CTL function had declined in three out of five individuals tested. Circulating CD8+ cells had gradually shifted to an immature CD38+CD28- phenotype. Our findings support the theory that HIV-induced immune dysfunction allows for the emergence of virulent HIV variants associated with CD4+ cell loss and disease.