RESUMEN

We have developed a widely applicable functional genomics strategy based on alphavirus expression vectors. The technology allows for rapid identification of genes encoding a functional activity such as binding of a defined ligand. Complementary DNA (cDNA) libraries were expressed in mammalian cells following infection with recombinant Sindbis virus (SIN replicon particles), a member of the Alphavirus genus. Virus-infected cells that specifically bound a ligand of choice were isolated using fluorescence-activated cell sorting (FACS). Replication-competent, infective SIN replicon particles harboring the corresponding cDNA were amplified in a next step. Within one round of selection, viral clones encoding proteins recognized by monoclonal antibodies or Fc-fusion molecules could be isolated and sequenced. Moreover, using the same viral libraries, a plaque-lift assay was established that allowed the identification of secreted, intracellular, and membrane proteins.

Asunto(s)

Clonación Molecular/métodos , Virus Sindbis/genética , Animales , Anticuerpos Monoclonales/metabolismo , Línea Celular , Membrana Celular/metabolismo , Separación Celular , Células Cultivadas , Cricetinae , ADN Complementario/metabolismo , Citometría de Flujo , Proteínas Fluorescentes Verdes , Ligandos , Proteínas Luminiscentes/metabolismo , Ratones , Modelos Biológicos , Unión Proteica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo

RESUMEN

Inherited demyelinating neuropathies are chronically disabling human disorders caused by various genetic defects, including deletions, single site mutations, and duplications in the respective myelin genes. We have shown in a mouse model of one distinct hereditary demyelinating neuropathy (heterozygous P0-deficiency, P0+-) that an additional null mutation in the recombination activating gene-1 (RAG-1--) leads to a substantially milder disorder, indicating a disease modifying role of T-lymphocytes. In the present study, we addressed the role of lymphocytes in the mouse model by reconstituting bone marrow of P0+-/RAG-1-- mice with bone marrow from immunocompetent wild-type mice. We compared the pathology and nerve conduction in double mutant mice (P0+-/RAG-1-- on a C57BL/6 background) with that in double mutants after receiving a bone marrow transplant. We found that the milder demyelination seen in the lymphocyte-deficient P0+-/RAG-1-- mutants was reverted to the more severe pathology by reestablishing a competent immune system by bone marrow transfer. These data corroborate the concept that the immune system contributes substantially to the pathologic process in this mouse model and may open new avenues to ameliorate human hereditary neuropathies by exploiting immunosuppressive treatments.

Asunto(s)

Trasplante de Médula Ósea/efectos adversos , Sistema Inmunológico/anomalías , Ratones Mutantes Neurológicos/inmunología , Mutación/fisiología , Vaina de Mielina/inmunología , Polirradiculoneuropatía/inmunología , Animales , Trasplante de Médula Ósea/métodos , Modelos Animales de Enfermedad , Estimulación Eléctrica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Mutantes Neurológicos/genética , Ratones Mutantes Neurológicos/metabolismo , Microscopía Electrónica , Proteína P0 de la Mielina/deficiencia , Proteína P0 de la Mielina/genética , Vaina de Mielina/genética , Vaina de Mielina/metabolismo , Conducción Nerviosa/genética , Conducción Nerviosa/inmunología , Enfermedades del Sistema Nervioso Periférico/genética , Enfermedades del Sistema Nervioso Periférico/inmunología , Enfermedades del Sistema Nervioso Periférico/patología , Polirradiculoneuropatía/genética , Polirradiculoneuropatía/terapia , Raíces Nerviosas Espinales/inmunología , Raíces Nerviosas Espinales/patología , Raíces Nerviosas Espinales/ultraestructura , Linfocitos T/inmunología , Linfocitos T/metabolismo

RESUMEN

Although DNA vaccines have been shown to elicit potent immune responses in animal models, initial clinical trials in humans have been disappointing, highlighting a need to optimize their immunogenicity. Naked DNA vaccines are usually administered either i.m. or intradermally. The current study shows that immunization with naked DNA by direct injection into a peripheral lymph node enhances immunogenicity by 100- to 1,000-fold, inducing strong and biologically relevant CD8(+) cytotoxic T lymphocyte responses. Because injection directly into a lymph node is a rapid and easy procedure in humans, these results have important clinical implications for DNA vaccination.

Asunto(s)

Antígenos Virales/genética , ADN Viral/inmunología , Glicoproteínas/genética , Virus de la Coriomeningitis Linfocítica/genética , Fragmentos de Péptidos/genética , Vacunas de ADN/administración & dosificación , Proteínas Virales/genética , Vacunas Virales/administración & dosificación , Animales , Línea Celular , Epítopos de Linfocito T/genética , Proteínas de Homeodominio/genética , Epítopos Inmunodominantes/genética , Inyecciones Intralinfáticas/métodos , Coriomeningitis Linfocítica/prevención & control , Virus de la Coriomeningitis Linfocítica/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Plásmidos/inmunología , Linfocitos T Citotóxicos/inmunología , Vacunación , Vacunas de ADN/inmunología , Vacunas Virales/inmunología

RESUMEN

Long-term protection against hepatitis B (HB) disease is dependent on persistence of a strong immune memory. This paper presents and discusses new knowledge that allows a better understanding of the mechanisms of long-term protection following hepatitis B vaccination. Studies have revealed links between specific lymphoproliferation, the in vivo humoral response and immune memory. The strength of immune memory and of a subsequent secondary immune response can therefore be predicted by the antibody response following primary vaccination. Vaccine antigen dose and structure have been identified as important influences in the primary antibody response and development of immune memory. The data and considerations presented support the use of highly immunogenic HB vaccines in order to provide long-lasting protection against HB disease.

Asunto(s)

Vacunas contra Hepatitis B/inmunología , Vacunas contra Hepatitis B/farmacología , Hepatitis B/inmunología , Hepatitis B/prevención & control , Memoria Inmunológica , Relación Dosis-Respuesta Inmunológica , Anticuerpos contra la Hepatitis B/biosíntesis , Antígenos de la Hepatitis B/administración & dosificación , Humanos , Inmunidad Celular , Activación de Linfocitos

RESUMEN

Lysis of infected cells by CD8(+) T cells is an important mechanism for the control of virus infections, but remains difficult to quantify in vivo. Here, we study the elimination kinetics of viral antigen-positive lymphocytes by antiviral CD8(+) T cells using flow cytometry and mathematical analysis. In mice acutely infected with lymphocytic choriomeningitis virus, more than 99.99 % of target cells were eliminated each day, corresponding to a half-life of 1.4 h. Even in mice exposed to virus 300 days previously, and with no ex vivo killing activity, 84 % of the target cells were eliminated per day. Unexpectedly, the elimination kinetics of antigen-positive lymphocytes was not significantly impaired in mice deficient in either perforin-, CD95 ligand- or TNF-mediated cytotoxicity. For viruses with a particular tropism for lymphocytes, such as Epstein-Barr virus or HIV, our results illustrate how effectively CD8(+) T cell-mediated elimination of target cells can potentially contribute to virus control and immunosuppression.

Asunto(s)

Linfocitos T CD8-positivos/inmunología , Citotoxicidad Inmunológica , Coriomeningitis Linfocítica/inmunología , Virus de la Coriomeningitis Linfocítica/inmunología , Animales , Presentación de Antígeno , Antígenos Virales/inmunología , Glicoproteínas de Membrana/inmunología , Ratones , Perforina , Proteínas Citotóxicas Formadoras de Poros , Subgrupos de Linfocitos T/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Receptor fas/inmunología

RESUMEN

We studied the impact of the duration of donor cell persistence on CD8+ T cell responsiveness after adoptive transfer of antigen-expressing lymphoid cells. Naive or immunized female mice were treated by adoptive transfer of spleen cells from mice ubiquitously expressing a lymphocytic choriomeningitis virus-derived cytotoxic T lymphocyte (CTL) epitope (gp33-41) either alone or in combination with the male H-Y antigen providing additional antigenic CTL and T helper cell determinants. Low doses of male spleen cells (or sorted B cells) primed CTL, while high doses of the same cells rendered them unresponsive. CTL unresponsiveness induced by high numbers of male spleen cells was dependent upon prolonged persistence of antigen-expressing donor cells. Unresponsive CTL reverted to a state of activation when the duration of donor cell chimerism was limited. Memory CTL could be rendered unresponsive if antigen-expressing donor cells were allowed to persist. These results suggest that, irrespective of the type of antigen-presenting cell and the functional state of the responding T cell, activation and unresponsiveness can represent two different outcomes critically determined by quantitative and kinetic differences of antigen persistence.

Asunto(s)

Antígenos Virales , Linfocitos T CD8-positivos/inmunología , Tolerancia Inmunológica , Activación de Linfocitos , Proteínas Virales , Traslado Adoptivo , Animales , Quimera/inmunología , Femenino , Glicoproteínas/genética , Glicoproteínas/inmunología , Antígeno H-Y/genética , Antígeno H-Y/inmunología , Memoria Inmunológica , Técnicas In Vitro , Virus de la Coriomeningitis Linfocítica/genética , Virus de la Coriomeningitis Linfocítica/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Modelos Biológicos , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Linfocitos T Citotóxicos/inmunología , Linfocitos T Colaboradores-Inductores/inmunología

RESUMEN

In this study, we investigated the potential of a DNA vaccine expressing the minimal cytotoxic T lymphocyte (CTL) epitope gp33 of the lymphocytic choriomeningitis virus glycoprotein to protect against infection of a non-lymphoid organ and compared this to protection against a systemic infection. Furthermore, since immune stimulatory sequences have been shown to augment CTL responses, we examined the capacity of CpG DNA to enhance CTL memory. The data show that DNA vaccination with a gp33-based gene construct induced short-lived gp33-specific CTL which protected against a systemic infection but not against a peripheral infection. Immune stimulatory sequences were incapable of either prolonging CTL memory or promoting protection against infection of a peripheral organ.

Asunto(s)

Antígenos Virales , Islas de CpG/inmunología , Coriomeningitis Linfocítica/prevención & control , Virus de la Coriomeningitis Linfocítica/inmunología , Vacunas de ADN/inmunología , Proteínas Virales , Adyuvantes Inmunológicos , Animales , Citotoxicidad Inmunológica/inmunología , Epítopos de Linfocito T/inmunología , Glicoproteínas/inmunología , Memoria Inmunológica , Coriomeningitis Linfocítica/inmunología , Ratones , Ratones Endogámicos C57BL , Fragmentos de Péptidos/inmunología , Linfocitos T Citotóxicos/inmunología , Factores de Tiempo , Vacunación

RESUMEN

The adhesive cell surface molecule P(0) is the most abundant glycoprotein in peripheral nerve myelin and fulfills pivotal functions during myelin formation and maintenance. Mutations in the corresponding gene cause hereditary demyelinating neuropathies. In mice heterozygously deficient in P(0) (P(0)(+/-) mice), an established animal model for a subtype of hereditary neuropathies, T-lymphocytes are present in the demyelinating nerves. To monitor the possible involvement of the immune system in myelin pathology, we cross-bred P(0)(+/-) mice with null mutants for the recombination activating gene 1 (RAG-1) or with mice deficient in the T-cell receptor alpha-subunit. We found that in P(0)(+/-) mice myelin degeneration and impairment of nerve conduction properties is less severe when the immune system is deficient. Moreover, isolated T-lymphocytes from P(0)(+/-) mice show enhanced reactivity to myelin components of the peripheral nerve, such as P(0), P(2), and myelin basic protein. We hypothesize that autoreactive immune cells can significantly foster the demyelinating phenotype of mice with a primarily genetically based peripheral neuropathy.

Asunto(s)

Enfermedades Desmielinizantes/fisiopatología , Genes RAG-1 , Proteínas de Homeodominio/fisiología , Síndromes de Inmunodeficiencia/fisiopatología , Proteína P0 de la Mielina/fisiología , Enfermedades del Sistema Nervioso Periférico/genética , Enfermedades del Sistema Nervioso Periférico/fisiopatología , Receptores de Antígenos de Linfocitos T alfa-beta/fisiología , Linfocitos T/inmunología , Animales , Cruzamientos Genéticos , Enfermedades Desmielinizantes/genética , Enfermedades Desmielinizantes/patología , Modelos Animales de Enfermedad , Genes Codificadores de la Cadena alfa de los Receptores de Linfocito T , Proteínas de Homeodominio/genética , Síndromes de Inmunodeficiencia/genética , Síndromes de Inmunodeficiencia/patología , Ratones , Ratones Noqueados , Proteína P0 de la Mielina/deficiencia , Proteína P0 de la Mielina/genética , Vaina de Mielina/patología , Vaina de Mielina/fisiología , Degeneración Nerviosa , Conducción Nerviosa , Nervios Periféricos/inmunología , Nervios Periféricos/fisiopatología , Enfermedades del Sistema Nervioso Periférico/patología , Receptores de Antígenos de Linfocitos T alfa-beta/deficiencia , Receptores de Antígenos de Linfocitos T alfa-beta/genética

RESUMEN

Dendritic cells (DC) play a key role in the initiation of T cell-mediated immune responses and may therefore be successfully used in antiviral and antitumor vaccination strategies. Because both strength and duration of an immune response determines the outcome of a vaccination protocol, we evaluated the life span of DC-induced antiviral CTL memory against systemic and peripheral challenge infections with lymphocytic choriomeningitis virus (LCMV). We found that expansion and activation of CTL by DC was transient. Protection against systemic LCMV infection after DC immunization was relatively long-lived (>60 days), whereas complete protection against peripheral infection via intracerebral infection or infection into the footpad with LCMV, where rapid recruitment of effector T cells to the site of infection and elimination of viral pathogen plays a major role, was short-lived (<30 days). Protective immunity was most efficiently restored by administration of antigenic peptides via DC, rather than in combination with IFA or in liposomes. These results suggest that Ag presentation by DC may be crucial for both initiation and maintenance of protective CTL-mediated immunity against viruses infecting solid organs or against peripheral mesenchymal or epithelial tumors.

Asunto(s)

Células Dendríticas/inmunología , Memoria Inmunológica , Activación de Linfocitos , Virus de la Coriomeningitis Linfocítica/inmunología , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/virología , Animales , Línea Celular , Citotoxicidad Inmunológica , Células Dendríticas/trasplante , Inmunoterapia Adoptiva/métodos , Coriomeningitis Linfocítica/inmunología , Coriomeningitis Linfocítica/prevención & control , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Células Tumorales Cultivadas

RESUMEN

Whether memory T cells require persisting antigen for their survival has been a matter of debate. One prominent view that memory T cells do not require persisting antigen is based in part on studies in which T cell populations have been transferred into antigen-free mice. To generate "space" recipients were often irradiated; the functional properties of the transfused T cells were then evaluated after prolonged periods. In this report we show that transferring cytotoxic T lymphocytes (CTL) into irradiated or T and B cell-deficient hosts results in their proliferation and a change of their activation state. Moreover, naïve T cell receptor-transgenic CTL specific for the lymphocytic choriomeningitis virus glycoprotein spontaneously developed cytotoxic effector function under such conditions. Therefore, some of the conclusions based on transfer of T cell populations into irradiated recipients to investigate T cell memory may have to be reevaluated.

Asunto(s)

Memoria Inmunológica , Linfopenia/inmunología , Linfocitos T Citotóxicos/inmunología , Traslado Adoptivo , Animales , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos

RESUMEN

Phenotypically and functionally, the early steps of T cell differentiation are not well characterized. In addition, the effector T cell stage shares several phenotypic characteristics with memory T cells, which has made the analysis of T cell memory difficult. In this study, we have investigated in vitro and in vivo the differentiation of naive CTL into effector and memory CTL as a function of cell division using lymphocytic choriomeningitis virus-specific TCR-transgenic spleen cells labeled with the vital dye carboxyfluorescein diacetate, succinimidyl ester. The following major points emerged. 1) During the first nine cell divisions, the investigated cell surface markers were strongly modulated. 2) The TCR was stepwise down-regulated during viral infection. 3) Cytotoxic effector function was acquired within one cell division and was retained during the next four to five divisions. 4) In vitro, CTL reached a CD44highCD62L+ memory phenotype after 6-10 cell divisions and required restimulation to exert effector function. 5) Lymphocytic choriomeningitis virus memory mice contained two distinct memory populations: a CD44highCD62L- population, predominately located in the spleen and exerting rapid effector function, and a CD44highCD62L+ population found in the spleen and the lymph nodes, which had lost immediate effector function. This finding suggests that two types of memory CTL exist. The correlation between CD62L expression, effector function, and Ag persistence is discussed.

Asunto(s)

Citotoxicidad Inmunológica , Memoria Inmunológica , Inmunofenotipificación , Linfocitos T Citotóxicos/citología , Traslado Adoptivo , Animales , Antígenos CD/biosíntesis , Antígenos de Diferenciación de Linfocitos T/biosíntesis , Diferenciación Celular/inmunología , División Celular/inmunología , Células Cultivadas , Pruebas Inmunológicas de Citotoxicidad , Proteína Ligando Fas , Receptores de Hialuranos/biosíntesis , Selectina L/biosíntesis , Lectinas Tipo C , Ligandos , Glicoproteínas de Membrana/biosíntesis , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptores de Antígenos de Linfocitos T/biosíntesis , Receptores de Interleucina-2/biosíntesis , Bazo/citología , Bazo/trasplante , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/trasplante , Receptor fas/biosíntesis

RESUMEN

To study B cell tolerance to the mitochondrial protein cytochrome c (CYT), the B cell response to pigeon CYT (PCC) was examined in mice transgenic for PCC. PCC was coupled to OVA to provide T cell help, since PCC-specific T cells in PCC-transgenic mice are deleted in the thymus. The frequency of secondary B cells responding to the minor antigenic surface around residue 44 on PCC was decreased about 10-fold in native PCC-transgenic mice compared with that in control mice or in transgenic mice expressing an altered form of PCC that lacked the heme and had a different amino acid sequence at the N-terminus. A similar decrease has been observed in the frequency of B cells in normal mice recognizing the site around residue 44 on mouse CYT compared with the frequency of B cells recognizing the corresponding site on foreign CYT. There were no major decreases but apparently were compensatory increases in the frequencies of B cells recognizing other sites on PCC in the native PCC-transgenic mice compared with those in other mice. These results indicate that B cells in mice are only partially tolerant to self CYT. A possible basis for this partial tolerance relating to the fate of CYT in cell death is discussed. This may be the first example of the use of a transgenic system to study B cell tolerance to a homologous self Ag.

Asunto(s)

Linfocitos B/inmunología , Grupo Citocromo c/inmunología , Epítopos de Linfocito B/inmunología , Autotolerancia/inmunología , Animales , Anticuerpos Monoclonales/metabolismo , Linfocitos B/metabolismo , Sitios de Unión de Anticuerpos , Columbidae , Grupo Citocromo c/genética , Epítopos de Linfocito B/genética , Epítopos de Linfocito B/metabolismo , Variación Genética/inmunología , Sueros Inmunes/biosíntesis , Sueros Inmunes/sangre , Recuento de Linfocitos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Autotolerancia/genética

RESUMEN

In this report we describe a simple method using the vital dye 5- (and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE) to follow splenic graft rejection by flow cytometry. CFSE-labelled spleen cell suspensions were injected intravenously into various recipients and blood samples were taken at different time points to follow the transferred cells. We found that the labelled cells could be readily detected by flow cytometry for up to eleven weeks. The loss of these labelled cells in various transfusion experiments with different major and minor histocompatibility differences followed the rejection kinetics previously described for skin transplants. Thus, this method offers a simple tool to test the histocompatibility of donor cells/grafts with the host in adoptive/transplantation experiments in which donor and host are not completely syngeneic. Furthermore, we developed a method to trace adoptively transferred fluorescent CFSE-labelled cells by light microscopy by converting in situ immunofluorescence staining into immunoenzyme staining.

Asunto(s)

Trasplante de Células , Citometría de Flujo/métodos , Rechazo de Injerto/diagnóstico , Bazo/citología , Traslado Adoptivo , Secuencia de Aminoácidos , Animales , Células Cultivadas , Fluoresceínas , Rechazo de Injerto/inmunología , Inmunohistoquímica/métodos , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Succinimidas , Linfocitos T/inmunología

RESUMEN

Peptide-specific down-regulation of T cell responses may represent a powerful tool to intervene in autoimmune diseases or graft rejections. It is therefore important to know whether peptide treatment tolerizes both naive and antigen-experienced memory T lymphocytes. Here we show that a major histocompatibility complex class I binding peptide, derived from the glycoprotein (GP33 peptide) of lymphocytic choriomeningitis virus (LCMV), specifically tolerized naive cytotoxic T lymphocytes (CTL) when administered three times intraperitoneally in incomplete Freund's adjuvants. However, in the presence of GP33-specific memory CTL in LCMV-primed mice, the same treatment had a general immunosuppressive effect on unrelated third-party antigen-specific T cell responses and caused severe immunopathological damage to the spleen.

Asunto(s)

Antígenos Virales , Epítopos/inmunología , Glicoproteínas/inmunología , Tolerancia Inmunológica , Virus de la Coriomeningitis Linfocítica/inmunología , Fragmentos de Péptidos/inmunología , Bazo/patología , Proteínas Virales , Traslado Adoptivo , Animales , Línea Celular , Glicoproteínas/farmacología , Tolerancia Inmunológica/efectos de los fármacos , Memoria Inmunológica/efectos de los fármacos , Inyecciones Intraperitoneales , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fragmentos de Péptidos/farmacología , Bazo/inmunología , Bazo/trasplante , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T Citotóxicos/inmunología

RESUMEN

This review summarises experimental evidence to illustrate that induction of immune reactivity depends upon antigen reaching and being available in lymphoid organs in a dose- and time-dependent manner. If antigen reaches lymph organs in a localised staggered manner and with a concentration gradient, a response is induced in the draining lymph node. Antigen-presenting cells are of critical importance to transport antigen from the periphery to local organised lymphoid tissue. If antigen is all over the lymphoid system, then it deletes all specific cells in the thymus or induces them within a few days; because of their limited life-span they then die off, leaving the repertoire depleted of this specificity. If antigen does not reach lymphoid organs it is ignored by immune cells. Once a response is induced, activated but not resting T cells will reach antigen outside lymphoid organs, whereas activated B cells differentiate into plasma cells in an inducing environment, mostly in lymphoid tissue including bone marrow, but also in chronic lymphoid-like infiltrations in peripheral organs. In immunopathology (when the infectious agent is known) or in autoimmunity (when the triggering infectious agent is not known or not recognised) lymphoid tissue may become organised close to the antigen (e.g. in organ-specific autoimmune diseases) and may thereby maintain an autoantigen-driven disease-causing immune response for a long time. The notion that native T cells get induced or silenced in the periphery may be questioned because induction can only occur in lymphoid organs providing anatomical structures where critical cell-cell interactions are properly guided and where, therefore, cells are likely to meet sufficiently frequently and in a critical milieu. Since overall immune reactivity critically depends upon the localisation of antigens in a dose- and time-dependent manner, it seems more likely-but this remains to be shown-that activated T cells may get exhausted in non-lymphoid peripheral tissues, whereas they are usually maintained in lymphoid organs. The critical role of antigen in regulating immune responses also has relevance for our understanding of immunological defence against epithelial and mesenchymal tumours, against many infectious diseases and for understanding autoimmunity and immunological memory. Collectively the data indicate that antigen, dependent upon localisation, dose and time, seems to be the simplest regulator of immune responses.

Asunto(s)

Antígenos/inmunología , Sistema Inmunológico/inmunología , Adyuvantes Inmunológicos , Animales , Relación Dosis-Respuesta Inmunológica , Humanos , Tejido Linfoide/inmunología

RESUMEN

This study evaluated whether T-cell memory reflects increased precursor frequencies of specific long-lived T cells and/or a low-level immune response against some form of persistent antigen. Antivirally protective CD8+ T-cell memory was analyzed mostly in the original vaccinated host to assess the role of antigen in its maintenance. T-cell mediated resistance against reinfection was measured in the spleen and in peripheral solid organs with protocols that excluded protection by antibodies. In vivo protection was compared with detectable cytotoxic T-lymphocyte precursor frequencies determined in vitro. In the spleen, in vitro detectable cytotoxic T-lymphocyte precursor frequencies remained stable independently of antigen, conferring resistance against viral replication in the spleen during reinfection. In contrast, T-cell mediated resistance against reinfection of peripheral solid organs faded away in an antigen-dependent fashion within a few days or weeks. We show that only memory T cells persistently or freshly activated with antigen efficiently extravasate into peripheral organs, where cytotoxic T lymphocytes must be able to exert effector function immediately; both the capacity to extravasate and to rapidly exert effector function critically depend on restimulation by antigen. Our experiments document that the duration of T-cell memory protective against peripheral reinfection depended on the antigen dose used for immunization, was prolonged when additional antigen was provided, and was abrogated after removal of antigen. We conclude that T-cell mediated protective immunity against the usual peripheral routes of reinfection is antigen-dependent.

Asunto(s)

Antígenos/inmunología , Antígenos CD8/inmunología , Memoria Inmunológica , Linfocitos T Citotóxicos/inmunología , Animales , Antígenos Virales/inmunología , Activación de Linfocitos , Virus de la Coriomeningitis Linfocítica/inmunología , Ratones , Ratones Endogámicos C57BL , Virus Vaccinia/inmunología , Interferencia Viral

RESUMEN

The process of antigen recognition depends in part on the amount of peptide antigen available and the affinity of the T cell receptor for a particular peptide-major histocompatibility complex (MHC) molecule complex. The availability of self antigen is limited by antigen processing, which is compartmentalized such that peptide antigens presented by MHC class I molecules originate in the cytoplasm, whereas peptide antigens presented by MHC class II molecules are acquired from the endocytic pathway. This segregation of the antigen-processing pathways may limit the diversity of antigens that influence the development and selection of, e.g., CD4-positive, MHC class II-specific T cells. Selection in this case might involve only a subset of self-encoded proteins, specifically those that are plasma membrane bound or secreted. To study these aspects of immune development, we engineered pigeon cytochrome for expression in transgenic mice in two forms: one in which it was expressed as a type II plasma membrane protein, and a second in which it was targeted to the mitochondria after cytoplasmic synthesis. Experiments with these mice clearly show that tolerance is induced in the thymus, irrespective of antigen compartmentation. Using radiation bone marrow chimeras, we further show that cytoplasmic/mitochondrial antigen gains access to the MHC class II pathway by direct presentation. As a result of studying the anatomy of the thymus, we show that the amount of antigen and the affinity of the TCR affect the location and time point of thymocytes under-going apoptosis.

Asunto(s)

Grupo Citocromo c/biosíntesis , Grupo Citocromo c/inmunología , Tolerancia Inmunológica , Linfocitos T Colaboradores-Inductores/inmunología , Transcripción Genética , Animales , Antígenos/biosíntesis , Antígenos/inmunología , Chlorocebus aethiops , Columbidae , Grupo Citocromo c/genética , Citometría de Flujo , Células HeLa , Antígenos de Histocompatibilidad Clase II/inmunología , Humanos , Ratones , Ratones Transgénicos , ARN Mensajero/biosíntesis , Receptores de Antígenos de Linfocitos T/biosíntesis , Receptores de Antígenos de Linfocitos T/inmunología , Mapeo Restrictivo , Timo/inmunología , Transfección

RESUMEN

Immunological memory is a hallmark of the immune system. Evolution can teach us which effector arms of immunological memory are biologically relevant against which virus. Antibodies appear to be the critical protective mechanism against cytopathic viruses. Since these viruses cause cell damage and disease directly, particularly in the absence of an immune response, mothers protect their offspring during a critical immunoincompetent period (a consequence of MHC- restricted T cell recognition) by passive transfer of neutralizing antibodies. In contrast, CTL appear to be the crucial effector mechanism against noncytopathic viruses. Since MHC polymorphism has made vertical transmission of T cell memory impossible, immunoincompetent offspring are not, and need not be, protected against such noncytopathic viruses. During the primary response and again during secondary infection, the most important function of CTL is to eliminate noncytopathic viruses, which may otherwise cause lethal immunopathology. Increased precursor frequencies of B and T cells appear to remain in the host independent of antigen persistence. However, in order to protect against cytopathic viruses, memory B cells have to produce antibody to maintain protective elevated levels of antibody; B cell differentiation into plasma cells is driven by persisting antigen. Similarly, to protect against infection with a noncytopathic virus, CTL have to recirculate through peripheral organs. Activation and capacity to emigrate into solid tissues as well as cytolytic effector function are also dependent upon, and driven by, persisting antigen. Because no convincing evidence is available yet of the existence of identifiable B or T cells with specialized memory characteristics, the phenotype of immunological memory correlates best with antigen-driven activation of low frequency effector T cells and plasma cells.

Asunto(s)

Memoria Inmunológica , Animales , Humanos

RESUMEN

T lymphocytes have been implicated in a variety of autoimmune diseases, and therefore one potential therapeutic approach would be to tolerize the pathogenic self-reactive T cells. In this study, we examined whether retroviral gene therapy could be used to induce tolerance and prevent autoimmunity using a transgenic mouse model for experimentally induced diabetes. In this model, the lymphocytic choriomeningitis virus (LCMV) glycoprotein (gp) is expressed on the beta-islet cells of the pancreas under the control of the rat insulin promoter (RIP). Previous work showed that the T cells specific for the gp remain unaware of the transgenic gp Ag expressed by the iselt cells, and infection with LCMV leads to immune-mediated diabetes. To tolerize the gp-specific pathogenic T cells, a retroviral vector (RV) expressing the LCMV gp was constructed, RV-gp. Replication-defective recombinant retroviruses were used to transduce bone marrow cells, which were subsequently infused into host RIP-gp transgenic animals. Unlike control animals, RV-gp chimeric animals did not possess T cells specific for the gp Ag as measured by proliferation and cytotoxic function, and further analysis suggested that tolerance of the gp-specific self-reactive T cells occurred by clonal deletion. Further experiments demonstrated that chimeric RIP-gp transgenic animals generated using bone marrow transduced with RV-gp did not develop experimentally induced diabetes. Our animal model demonstrates that retroviral gene therapy may cure immune-mediated diabetes by providing long lasting Ag-specific tolerance.

Asunto(s)

Enfermedades Autoinmunes/prevención & control , Diabetes Mellitus Experimental/prevención & control , Terapia Genética/métodos , Virus de la Coriomeningitis Linfocítica/inmunología , Retroviridae/genética , Animales , Secuencia de Bases , Diabetes Mellitus Experimental/etiología , Vectores Genéticos , Tolerancia Inmunológica , Glicoproteínas de Membrana/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Datos de Secuencia Molecular , Quimera por Radiación , Ratas , Linfocitos T/inmunología , Proteínas del Envoltorio Viral/inmunología

RESUMEN

The interactions between CD40 on B cells and its ligand gp39 on activated T helper cells are known to be essential for the development of thymus-dependent humoral immunity. However, CD40 is also functionally expressed on thymic epithelial cells and dendritic cells, suggesting that gp39-CD40 interactions may also play a role in thymic education, the process by which self-reactive cells are deleted from the T cell repertoire. Six systems of negative selection were studied for their reliance on gp39-CD40 interactions to mediate negative selection. In all cases, when the antigen/superantigen was endogenously expressed (in contrast to exogenously administered), negative selection was blocked by loss of gp39 function. Specifically, blockade of gp39-CD40 interactions prevented the deletion of thymocytes expressing V beta 3, V beta 11, and V beta 12, specificities normally deleted in BALB/c mice because of the endogenous expression of minor lymphocyte-stimulating determinants. Independent verification of a role of gp39 in negative selection was provided by studies in gp39-deficient mice where alterations in T cell receptor (TCR) V beta expression were also observed. Studies were also performed in the AND TCR transgenic (Tg) mice, which bear the V alpha 11, V beta 3 TCR and recognize both pigeon cytochrome c (PCC)/IEk and H-2As. Neonatal administration of anti-gp39 to AND TCR Tg mice that endogenously express H-2As or endogenously produce PCC prevented the deletion of TCR Tg T cells. In contrast, deletion mediated by high-dose PCC peptide antigen (administered exogenously) in AND TCR mice was unaltered by administration of anti-gp39. In addition, deletion by Staphylococcus enterotoxin B in conventional mice was also unaffected by anti-gp39 administration. gp39 expression was induced on thymocytes by mitogens or by antigen on TCR Tg thymocytes. Immunohistochemical analysis of B7-2 expression in the thymus indicated that, in the absence of gp39, B7-2 expression was substantially reduced. Taken together, these data suggest that gp39 may influence negative selection through the regulation of costimulatory molecule expression. Moreover, the data support the hypothesis that, for negative selection to some endogenously produced antigens, negative selection may be dependent on TCR engagement and costimulation.

Asunto(s)

Supresión Clonal , Glicoproteínas de Membrana/fisiología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Subgrupos de Linfocitos T/citología , Timo/citología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Antígenos/inmunología , Antígenos CD/biosíntesis , Antígenos CD/inmunología , Apoptosis , Antígeno B7-2 , Ligando de CD40 , Columbidae/genética , Grupo Citocromo c/biosíntesis , Grupo Citocromo c/genética , Grupo Citocromo c/inmunología , Células Dendríticas/inmunología , Epitelio/inmunología , Femenino , Reordenamiento Génico de la Cadena beta de los Receptores de Antígenos de los Linfocitos T , Activación de Linfocitos , Masculino , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/inmunología , Ratones , Ratones Endogámicos BALB C , Antígenos Estimulantes de Linfocito Menor/inmunología , Mitógenos/farmacología , Datos de Secuencia Molecular , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Autotolerancia , Especificidad de la Especie , Subgrupos de Linfocitos T/inmunología , Timo/inmunología