RESUMEN
The early responses of CD4+ T cells to particle-mediated DNA immunisation were investigated using OVA-specific TCR-transgenic CD4+ T cells. Following adoptive transfer of these cells, mice were immunised by delivery into the skin of a plasmid encoding ovalbumin. Transgenic T cells underwent a rapid and transient antigen-specific activation, followed by clonal expansion (up to approximately 6% of total lymphocytes). Immunisation with ovalbumin in CFA evoked similar responses with slightly faster kinetics. Numerous antigen-specific T cells synthesising IFN-gamma (Th1) and IL-4 (Th2) were detectable using both intracellular staining and ELISPOT assays. This study provides a quantitative analysis of both T cell proliferation and Th1/Th2 balance following particle-mediated DNA immunisation and establishes a robust and sensitive model in which to assess modulation of helper T cell responses in DNA vaccination.
Asunto(s)
Traslado Adoptivo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/trasplante , Vacunas de ADN/administración & dosificación , Vacunas de ADN/inmunología , Animales , Biomarcadores/análisis , División Celular , Células Clonales/citología , Células Clonales/inmunología , Interferón gamma/análisis , Interferón gamma/metabolismo , Interleucina-2/biosíntesis , Interleucina-2/inmunología , Interleucina-4/análisis , Interleucina-5/análisis , Interleucina-5/metabolismo , Cinética , Ganglios Linfáticos/inmunología , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Microesferas , Ovalbúmina/inmunología , Receptores de Interleucina-2/inmunología , Receptores de Interleucina-2/metabolismo , Células TH1/inmunología , Células Th2/inmunología , VacunaciónRESUMEN
The non-obese diabetic (NOD) mouse develops insulin dependent diabetes mellitus (IDDM) spontaneously with a higher incidence in females than in males. There are many similarities to the human disease, making it an ideal model. Our group is examining the role that CD4(+) and CD8(+) T cells play in IDDM in the NOD mouse, as it is known that both T cell subsets are required for onset of disease. Although IDDM has an autoimmune etiology, the initial triggering event is unknown and the autoantigen involved has not been identified. This investigation focussed on one of the potential autoantigens involved, the enzyme glutamic acid decarboxylase (GAD). We raised GAD peptide-specific CD8(+) T cells by immunising NOD mice with the GAD peptide alongside an irrelevant peptide that induced a CD4(+) T cell response. In order to maintain these peptide specific T cells in vitro and generate clones, it was found that antibodies specific to CD4(+) and MHC class II molecules needed to be included in the culture medium. This paper outlines the methods we employed to generate and maintain these CD8(+) T cells in vitro.
Asunto(s)
Autoantígenos , Linfocitos T CD8-positivos/inmunología , Técnicas de Cultivo de Célula/métodos , Secuencia de Aminoácidos , Animales , Autoantígenos/química , Linfocitos T CD4-Positivos/inmunología , Células Clonales , Pruebas Inmunológicas de Citotoxicidad , Diabetes Mellitus Tipo 1/enzimología , Diabetes Mellitus Tipo 1/inmunología , Epítopos/química , Femenino , Glutamato Descarboxilasa/inmunología , Humanos , Inmunización , Masculino , Ratones , Ratones Endogámicos NOD , Oligopéptidos/química , Oligopéptidos/inmunología , Ovalbúmina/inmunologíaRESUMEN
L-selectin has become established as a key molecule in the recirculation of naïve T cells from the blood to peripheral lymph nodes, yet little is known about its role in the migration of effector or memory cells. While differentiating naïve CD4+ T cells into Th1 and Th2 subsets in vitro, it was noted that L-selectin levels were maintained on the Th1 subset of cells. The expression of L-selectin on the Th1 cells appeared to be dependent on the presence of IL-12. Th2 cells, differentiated in the absence of IL-12, failed to maintain L-selectin expression. Coculture with IL-12, IL-18, IL-4, TNF-alpha, or IFN-alpha, -beta, or -gamma demonstrated a dependence on IL-12 alone for L-selectin expression. In addition, the inclusion of heat-killed Listeria monocytogenes in the cultures also maintained L-selectin expression on the Th1 cells. In all cultures, the maintenance of L-selectin on the T cell surface could be blocked by the inclusion of anti-IL-12 Abs. Analysis of the mRNA levels for L-selectin in T cells, differentiated in the presence or absence of IL-12, showed that the cytokine appears to exert its effect on L-selectin at the transcriptional level. Given the key role played by IL-12 in the differentiation of naïve T cells into the Th1 subset, the observation that IL-12 can also regulate L-selectin expression has implications for the migration of Th1 effector cells both through the lymphatic system and to sites of inflammation.
Asunto(s)
Adyuvantes Inmunológicos/fisiología , Interleucina-12/fisiología , Selectina L/biosíntesis , Células TH1/metabolismo , Secuencia de Aminoácidos , Animales , Células Cultivadas , Calor , Selectina L/genética , Listeria monocytogenes/inmunología , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Datos de Secuencia Molecular , ARN Mensajero/biosíntesis , Células TH1/inmunología , Células TH1/microbiología , Células Th2/metabolismo , Transcripción Genética/inmunologíaRESUMEN
The CAMPATH-1H (CD52) antigen is a 21 000-28 000 MW glycopeptide antigen that is highly expressed on T and B lymphocytes and is coupled to the membrane by a glycosylphosphatidylinositol (GPI) anchoring structure. The humanized CAMPATH-1H anti-CD52 antibody is extremely effective at mediating depletion of both normal and tumorigenic lymphocytes in vivo and has been used in clinical trials for lymphoid malignancy and rheumatoid arthritis. Cross-linking GPI-anchored molecules, including CD52, on the surface of T lymphocytes in the presence of phorbol 12-myristate 13-acetate or anti-CD3, results in cellular activation. In the present study we have investigated the functional effects of cross-linking CD52 on T and B tumour cell lines. Cross-linking CD52 on either a B-cell line, Wien 133, which expresses high levels of endogenous CD52 or Jurkat T cells transfected and selected to express high levels of CD52 resulted in growth inhibition. This effect showed slower kinetics and occurred in a lower percentage of cells than growth inhibition stimulated via T- or B-cell receptors. Growth inhibition of the Wien 133 line was followed by the induction of apoptosis, which appeared independent of the Fas/Fas L pathway. Wien 133 cells surviving anti-CD52 treatment were selected and cloned and found to have down-regulated CD52 expression, with a characteristic biphasic pattern of 10% CD52-positive, 90% negative by fluorescence-activated cell sorter analysis. Interestingly, surface expression of other GPI-linked molecules, such as CD59 and CD55, was also down-regulated, but other transmembrane molecules such as surface IgM, CD19, CD20, HLA-DR were unaffected. The present study and previous work show that this is due to a defect in the synthesis of mature GPI precursors. Separation of CD52-positive and negative populations in vitro resulted in a rapid redistribution to the mixed population. Injection of CD52-negative cells into nude mice to form a subcutaneous tumour resulted in a substantial increase in expression of CD52. These results suggest that the defect in the Wien 133 cells is reversible, although the molecular mechanism is not clear. These observations have relevance to the clinical situation as a similar GPI-negative phenotype has been reported to occur in lymphocytes following CAMPATH-1H treatment in vivo.
Asunto(s)
Antígenos CD/metabolismo , Antígenos de Neoplasias/metabolismo , Glicoproteínas/metabolismo , Linfoma de Células B/inmunología , Linfoma de Células T/inmunología , Animales , Apoptosis/inmunología , Western Blotting , Antígeno CD52 , División Celular/inmunología , Humanos , Inmunoglobulina M/inmunología , Linfoma de Células B/patología , Linfoma de Células T/patología , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Receptores de Antígenos de Linfocitos B/inmunología , Células Tumorales CultivadasRESUMEN
A fully humanized immunoglobulin G1 (IgG1) anti-CD4 monoclonal antibody is currently being evaluated in phase I/II clinical trials for rheumatoid arthritis. In order to understand the mode of action of this antibody in vivo, we have carried out a detailed functional analysis in vitro of the effects of this antibody on T-cell activation. The anti-CD4 antibody was found to inhibit both antigen-specific responses involving recognition of human leucocyte antigen (HLA) class II and processed antigenic peptides as well as non-class II dependent responses via anti-CD3 antibodies. The antibody did not cause total blockade of T-cell proliferation, but rather induced a shift in the dose-response curve, decreasing the sensitivity of cells to antigen or anti-CD3-mediated stimulation. The antibody appears to allow at least a partial early signal into the T cell as it does not inhibit the increase in tyrosine phosphorylation induced by anti-CD3 antibodies. A comparison of the intact antibody with that of either the F(ab')2 fragment or an engineered non-Fc receptor (FcR) binding form revealed that the intact antibody was the most effective at inhibiting proliferation of resting peripheral blood CD4+ T cells. However, this difference was only apparent when excess antibody was removed from culture prior to antigen or anti-CD3 mediated stimulation. The intact antibody induced both CD4 down-modulation and increases in CD4-associated tyrosine phosphorylation of resting CD4+ T cells, which were not seen with the non-FcR binding versions, which may account for the enhanced potency of the intact antibody at inhibiting T-cell activation. Interestingly, the anti-CD4 antibody induced a differential effect on activated CD4+ T cell clones compared with resting CD4+ T cells with respect to degree of CD4 cross-linking required to induce functional effects in the T cell. Both intact and non-FcR binding antibodies were equally effective at inhibiting T-cell proliferation of activated T-cell clones. In addition CD4 down-modulation and increased CD4-associated tyrosine phosphorylation were observed with T-cell clones in the absence of secondary cross-linking. Such observations may be of relevance when studying the effects of the antibody at sites of inflammation, where there will be CD4+ T cells of differing activation states as well as varying numbers of FcR positive cells.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Antígenos CD4/inmunología , Activación de Linfocitos/inmunología , Linfocitos T/inmunología , Antígenos/inmunología , Complejo CD3/inmunología , Linfocitos T CD4-Positivos/inmunología , División Celular/inmunología , Células Cultivadas , Regulación hacia Abajo/inmunología , Humanos , Leucocitos Mononucleares/inmunología , Receptores Fc/inmunología , Transducción de Señal/inmunologíaRESUMEN
Cloning of the CD52 from a B-lymphocyte tumour cDNA library revealed two closely related sequences differing only at two amino acids C-terminal to the proposed point of glycosylphosphatidylinositol (GPI)-linkage. When transfected into CHO cells only one of these sequences gave high-level expression of the antigen recognized by the prototypic anti-CD52 antibody CAMPATH-1 whereas in JURKAT cells good expression levels were obtained with both sequences. Fusion of the sequence from the second sequence to DNA encoding the extracellular domain of CD4 indicated that this sequence was capable of directing GPI linkage. The possible implications for the function of CD52 and serotherapy with anti-CD52 antibodies are discussed.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Antineoplásicos/inmunología , Reacciones Antígeno-Anticuerpo , Antígenos CD/genética , Antígenos de Neoplasias , Glicoproteínas , Alemtuzumab , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales Humanizados , Antígenos CD/inmunología , Secuencia de Bases , Antígeno CD52 , Células CHO , Línea Celular , Clonación Molecular , Cricetinae , Cartilla de ADN/genética , Expresión Génica , Técnicas Genéticas , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADN , Linfocitos T/inmunologíaRESUMEN
Patients with severe rheumatoid arthritis who had failed treatment with conventional therapies were treated with a course of five or 10 daily intravenous infusions of CAMPATH-1H, a humanized antibody against the CD52 antigen, resulting in profound depletion of peripheral blood mononuclear cells. During the subsequent 18 months, lymphocytes were analysed for sub-populations by fluorescence-activated cell sorter (FACS) and for proliferation in response to polyclonal T-cell stimulation with anti-CD3 or staphylococcal enterotoxin B (SEB). Treatment resulted in almost complete depletion of lymphocytes from the blood followed by gradual repopulation. CD16+ natural killer (NK) cells and CD14+ monocytes returned to pretreatment levels within 1-2 months. CD19+ B cells returned to within 50% of pre-treatment levels by day 66 and to within normal range by day 150, whereas CD8+ T cells recovered to 50% of pretreatment levels by day 66, but did not show any further increase during the rest of the study period. The most profound effects were on the CD4+ T lymphocyte sub-population, as the mean CD4+ count did not increase above 20% of pre-treatment level at any time during the study period (550 days), at all the doses tested. The T cells which initially repopulated the blood 1-2 months after treatment, nearly all expressed the activation markers human leucocyte antigen (HLA)-DR and CD45RO, although the percentage of T cells expressing these molecules gradually declined to normal levels over time. Proliferative responses to polyclonal T-cell stimulation (anti-CD3 and SEB) were also significantly reduced in the first few months after treatment, but recovered to pre-treatment levels by day 250. The relationship between these observations and the clinical response is discussed.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Antígenos CD/farmacología , Antígenos de Neoplasias , Artritis Reumatoide/terapia , Glicoproteínas , Subgrupos Linfocitarios/fisiología , Antígenos CD/inmunología , Artritis Reumatoide/inmunología , Linfocitos B/inmunología , Antígeno CD52 , Linfocitos T CD8-positivos/inmunología , Femenino , Citometría de Flujo , Antígenos HLA-DR/inmunología , Humanos , Células Asesinas Naturales/inmunología , Antígenos Comunes de Leucocito/inmunología , Recuento de Leucocitos , Activación de Linfocitos , Subgrupos Linfocitarios/efectos de los fármacos , Masculino , Persona de Mediana Edad , Monocitos/inmunología , Factores de TiempoRESUMEN
CD52 is a glycosylphosphatidyl-inositol (GPI)-linked glycoprotein expressed at high levels on normal T and B lymphocytes and at lower levels on monocytes, while being absent on granulocytes and bone marrow stem cell precursors. The emergence of CD52- lymphocytes of both T and B cell lineages was observed in three out of 25 rheumatoid arthritis patients treated with teh humanized antibody Campath-1H in phase II clinical trial. Whereas the majority of CD52- B cells had disappeared from the peripheral blood by 3 months post-treatment, both CD52- CD4+ and CD8+ T cells persisted in the circulation for at least 20 months. In the two patients that were tested, the GPI-anchored surface molecules CD55 and CD59 were also absent on the CD52- cells, although expression of other cell surface transmembrane, proteins (CD3, CD4 and CD2) was unaffected. The CD52- cells maintained a stable phenotype in vitro despite repeated re-stimulation in culture. Both CD52- and C52+ clones, established from one of the patients, responded to a similar extent to several T cell mitogens, as assessed by proliferation, suggesting that a general defect in expression of GPI-linked molecules does not impair T cell activation. These data show that an immune attack against a GPI-anchored surface molecule can result in the selection of a GPI-anchor-deficient cell population. Despite the persistence of CD52- T cells in the peripheral blood, no adverse reactions associated with the presence of these cells were noted in any of the patients; in fact they responded with longer remission times after Campath-1H treatment than the other patients in the trial.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Anticuerpos Antineoplásicos/farmacología , Antígenos CD/biosíntesis , Antígenos de Neoplasias , Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/inmunología , Glicoproteínas , Linfocitos/química , Adulto , Alemtuzumab , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Monoclonales Humanizados , Anticuerpos Antineoplásicos/uso terapéutico , Linfocitos T CD4-Positivos/química , Antígeno CD52 , Linfocitos T CD8-positivos/química , Células Cultivadas , Femenino , Humanos , Activación de Linfocitos , Subgrupos Linfocitarios/inmunología , Linfocitos/inmunología , Masculino , Persona de Mediana Edad , Factores de TiempoRESUMEN
The specificity of the influenza nucleoprotein-induced T-cell proliferative response by mouse strains differing in either H-2- or non-H-2-linked background genes was compared by using a panel of synthetic peptides covering 90% of the nucleoprotein molecule. The results showed, as expected, that H-2 genes strongly influenced the major regions of the molecules recognized by T cells, as the response was focused on different peptides in mice of different H-2 haplotypes. However, some regions of the molecule (e.g. 260-283) were recognized by several different haplotypes, with overlapping but distinct minimal determinants. The lymph node proliferative response appeared to be predominantly restricted by the I-A molecule, as expression of I-E in mice did not result in any detectable recognition of additional epitopes. In the majority of cases the same T-cell epitopes were recognized by mouse strains sharing the same H-2 haplotype but differing in many background genes. Low responsiveness was however observed to p55-77 by DBA/2 and p127-141 by AKR mice to which other H-2d or H-2k strains were high responders. Low responsiveness is therefore unlikely to be a consequence of failure of these peptides to bind to the relevant major histocompatibility complex class II molecule. In addition antigen-presenting cells from the DBA/2 low responder strain was able to process and present whole influenza virus or nucleoprotein as well as antigen-presenting cells from the high responder BALB/c strain. It is therefore suggested that low responsiveness to the peptide p55-77 may be due to a 'hole in the T-cell repertoire', caused perhaps by expression of Mls-1a in the DBA/2 strain. This is supported by the observation that low responsiveness to this epitope appears to be dominant in F1 (low x high) mice.
Asunto(s)
Antígenos Virales/inmunología , Linfocitos T CD4-Positivos/inmunología , Genes MHC Clase II/inmunología , Antígenos H-2/genética , Virus de la Influenza A/inmunología , Nucleoproteínas/inmunología , Proteínas de Unión al ARN , Proteínas del Núcleo Viral/inmunología , Animales , Células Presentadoras de Antígenos/inmunología , Técnicas de Cultivo de Célula , División Celular/inmunología , Epítopos/inmunología , Femenino , Antígenos de Histocompatibilidad Clase II/genética , Ratones , Ratones Endogámicos , Mutación , Proteínas de la Nucleocápside , Fragmentos de Péptidos/inmunologíaRESUMEN
A fully humanized anti-CD4 antibody was studied for its effects on resting and activated CD4 T cells. Whereas the antibody was poorly lytic, it induced dramatic down-modulation of CD4 expression on both types of cell. In order to down-modulate CD4 on resting, normal CD4 T cells there was an absolute requirement for FcR-mediated cross-linking of the anti-CD4 antibody, and only CD4 levels were affected. When activated cloned T-cell lines were studied there was no requirement for cross-linking and several other cell surface markers were also affected. Although the total cellular CD4 was reduced in the down-modulated cells, as judged by Western blot analysis, that CD4 which remained was associated with p56lck. The results are discussed in relation to the potential use of humanized anti-CD4 antibodies in the therapy of autoimmune disease and the choice of antibody isotype for such a therapeutic antibody.
Asunto(s)
Antígenos CD4/inmunología , Linfocitos T CD4-Positivos/inmunología , Activación de Linfocitos/inmunología , Citotoxicidad Celular Dependiente de Anticuerpos , Western Blotting , Células Cultivadas , Citotoxicidad Inmunológica , Regulación hacia Abajo/inmunología , Humanos , Inmunoglobulina G/inmunología , Leucocitos Mononucleares/inmunología , Receptores Fc/inmunologíaRESUMEN
The CAMPATH-1 (CD52) antigen is a 21-28 kDa glycopeptide which is highly expressed on lymphocytes and macrophages and is coupled to the membrane by a glycosylphosphatidylinositol (GPI) anchoring structure. The function of this molecule is unknown. However, it is an extremely good target for complement-mediated attack and antibody-mediated cellular cytotoxicity. The humanized CAMPATH-1H antibody, which is directed against CD52, is very efficient at mediating lymphocyte depletion in vivo, and is currently being used in clinical trials for lymphoid malignancy and rheumatoid arthritis. It is therefore important to examine the functional effects of this antibody on different lymphocyte sub-populations. Because several other GPI-linked molecules expressed on the surface of T lymphocytes are capable of signal transduction resulting in cell proliferation, we have investigated whether the CAMPATH-1 antigen can also mediate these effects. In the presence of phorbol esters and cross-linking anti-Ig antibodies, mAbs specific for CD52 induced proliferation and lymphokine production in highly purified resting CD4+ and CD8+ T lymphocytes. The rat IgG2c YTH 361.10 anti-CD52 antibody, however, was able to activate resting CD4+ and CD8+ T cells directly without cross-linking or phorbol myristate acetate in the absence of Fc-bearing cells. Anti-CD52 antibodies also augmented the anti-CD3 mediated proliferative response of CD4+ and CD8+ T cells when the two antibodies were co-immobilized onto the same surface or cross-linked in solution by the same second antibody. Both CD4+ CD45RA and CD4+ CD45RO T cells were stimulated to proliferate by anti-CD52 antibodies in the presence of appropriate co-stimulatory factors. Anti-CD52 mAbs did not, however, synergize with anti-CD2 or CD28 mAb to induce CD4+ T cell proliferation. The activation of CD4+ T cells by anti-CD52 antibodies was inhibited by cyclosporin A, suggesting a role for the calcineurin-dependent signal transduction pathways. Although CD52 could transduce a signal in T cells, anti-CD52 antibodies did not inhibit antigen-specific or polyclonal T cell responses, suggesting this molecule does not play an essential co-stimulatory role in normal T cell activation.
Asunto(s)
Antígenos CD/fisiología , Antígenos de Neoplasias , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Glicoproteínas , Activación de Linfocitos , Anticuerpos Monoclonales/inmunología , Antígeno CD52 , Ciclosporina/farmacología , Humanos , Técnicas In Vitro , Antígenos Comunes de Leucocito/análisis , Activación de Linfocitos/efectos de los fármacos , Agregación de Receptores , Receptores Inmunológicos/fisiología , Transducción de Señal , Subgrupos de Linfocitos T/inmunologíaRESUMEN
We report a detailed comparison of two commonly used stable, amplifiable mammalian expression systems (Chinese Hamster Ovary cells/dihydrofolate reductase and Mouse NSO myeloma/glutamine synthetase) used to express a humanized IgG1 monoclonal antibody. We compare copy number and steady state mRNA levels of both the selectable marker and heavy chain of the antibody throughout the selection and amplification process. In both cell lines, copy number and steady state levels of heavy chain and selectable marker increased during selection and were further increased during amplification. As expected, an increase in steady state mRNA levels of heavy chain correlated with an increase in expression of antibody whilst an increase in the steady state levels of mRNA of the selectable marker correlated with increased resistance to the selective agent. In NSO and CHO cells producing equivalent amounts of antibody, the copy number of the antibody genes and selectable marker was significantly higher in the CHO cells than in the NSO cells. However, the steady state mRNA levels of the heavy chain of the antibody were virtually identical. Rates of protein secretion in the two cell lines were also compared and found to be very similar. When the antibody purified from both systems was compared in a number of functional assays they behaved identically.
Asunto(s)
Anticuerpos Monoclonales/genética , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/farmacología , Antígenos CD4/inmunología , Células CHO , Cricetinae , Expresión Génica , Marcadores Genéticos , Vectores Genéticos , Glutamato-Amoníaco Ligasa/genética , Humanos , Activación de Linfocitos , Macrófagos/inmunología , Ratones , ARN/genética , ARN/metabolismo , Linfocitos T/inmunología , Tetrahidrofolato Deshidrogenasa/genética , Transfección , Células Tumorales Cultivadas/inmunologíaRESUMEN
The influence of different antigen delivery systems on antibody isotype and lymphokine profile has been investigated using influenza nucleoprotein as a model antigen system. Mice exposed to live or inactivated influenza virus produced antibody against whole virus or recombinant nucleoprotein (rNP), which was predominantly of the IgG2a isotype. Spleen or lymph node cells from these mice rapidly produced large amounts of interferon-gamma (IFN-gamma), but no detectable interleukin-5 (IL-5) when stimulated in vitro with specific antigen. In contrast, after primary immunization with rNP or p206-229 in different adjuvants (CFA, quil A or alhydrogel), specific antibody was predominantly of the IgG1 isotype and relatively lower amounts of IFN-gamma but no IL-5 were detected following in vitro antigenic stimulation. Secondary immunization, however, resulted in detection of IgG2a antibodies and increased levels of IFN-gamma. IL-5 was only detected after secondary immunization with peptide in adjuvant. Mice infected with aro A- Salmonella typhimurium expressing NP produced antibody of both IgG1 and IgG2a isotypes and large amounts of IFN-gamma and no IL-5, following in vitro antigenic stimulation, and therefore parallelled the pattern seen with whole virus more closely than that seen following primary immunization with protein or peptide in conventional adjuvants. The results suggest that the antigen delivery vehicle influences both quantitative and qualitative differences in the type of immune response elicited, which may be important in determining the potency of protective immunity induced.
Asunto(s)
Antígenos Virales/administración & dosificación , Citocinas/biosíntesis , Inmunoglobulina G/biosíntesis , Nucleoproteínas/inmunología , Proteínas de Unión al ARN , Proteínas del Núcleo Viral/inmunología , Vacunas Virales/administración & dosificación , Animales , Antígenos Virales/inmunología , División Celular/inmunología , Interferón gamma/biosíntesis , Interleucina-5/biosíntesis , Cinética , Ganglios Linfáticos/inmunología , Ratones , Ratones Endogámicos BALB C , Proteínas de la Nucleocápside , Linfocitos T/inmunología , Vacunas Virales/inmunologíaRESUMEN
The invasin proteins of Yersinia spp. are outer membrane proteins which are involved in the penetration of these bacteria into mammalian cells (Cell 1990. 60: 861). Invasin binds to several different beta 1 integrins with extremely high affinity, the integrin-binding domain of invasin has been mapped to the C-terminal 192 amino-acids of the molecule (J. Biol. Chem. 1991. 266:24367). Expression of this fragment alone on the cell surface of non-invasive bacteria is enough to confer the invasive phenotype on these strains (EMBO J. 1990. 9: 1979). Here we show that the carboxy-terminal 192 amino acids of invasin expressed as a fusion protein with the maltose binding protein of E. coli is capable of delivering co-stimulatory signals to human T cells through the beta 1 integrins. Co-stimulation was assayed by the ability of invasin to augment the response of highly purified CD4+ and CD8+ T cells to co-immobilized anti-CD3 antibody. Antibody blocking studies indicated that the co-stimulation was mediated through beta 1 integrins. The proliferation induced by co-stimulation of CD4+ T cells was accompanied by the synthesis of the cytokines tumor necrosis factor-alpha and interferon-gamma, whereas the activation of CD8+ T cells led to the generation of cytotoxic effectors. The region of the invasin molecule involved in T cell activation was further mapped using synthetic peptides. A region of the invasin molecule containing the residues TAKSKKFPSY could substitute for invasin in T cell activation. The co-stimulation by peptide could also be inhibited by anti-integrin antibodies. The observation that an outer membrane protein of a bacterium which is associated with reactive arthritis and other autoimmune spondyloarthropathies can act as a T cell co-stimulus may have implications for the etiology of these diseases.
Asunto(s)
Adhesinas Bacterianas , Proteínas Bacterianas/inmunología , Integrinas , Activación de Linfocitos , Subgrupos de Linfocitos T/inmunología , Yersinia/inmunología , Secuencia de Aminoácidos , Secuencia de Bases , Linfocitos T CD4-Positivos/inmunología , Antígenos CD8/inmunología , Citocinas/biosíntesis , Citotoxicidad Inmunológica , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/química , Péptidos/química , Péptidos/inmunología , Proteínas Recombinantes de Fusión/inmunología , Transducción de SeñalRESUMEN
A recombinant baculovirus-expressed hybrid protein containing epitopes for the C-terminal fragment of the Plasmodium falciparum precursor to the major merozoite surface antigens (PMMSA) and the tetrapeptide repeats of the circumsporozoite protein (CSP) was assessed for its immunogenicity. Murine MHC-II restriction of the antibody response to the CSP repeats was not overcome by the PMMSA component, the response to which showed no restriction. In an adjuvant trial the highest antibody titres in rabbits to both components of the hybrid were obtained using Freund's adjuvant. Lack of a boosting antibody response to the CSP repeats appeared to be linked to the conformation of the PMMSA component. Formulation of the hybrid protein into Iscoms gave antibody titres of only short duration to both components.
Asunto(s)
Anticuerpos Antiprotozoarios/biosíntesis , Antígenos de Protozoos/inmunología , Plasmodium falciparum/inmunología , Vacunas Antiprotozoos/inmunología , Proteínas Recombinantes de Fusión/inmunología , Adyuvantes Inmunológicos , Secuencia de Aminoácidos , Animales , Antígenos de Superficie/inmunología , Línea Celular , ISCOMs/inmunología , Inmunización , Inmunización Secundaria , Activación de Linfocitos , Proteína 1 de Superficie de Merozoito , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Endogámicos , Datos de Secuencia Molecular , Precursores de Proteínas/inmunología , Proteínas Protozoarias/inmunología , Conejos , Linfocitos T/inmunologíaRESUMEN
Rationally attenuated strains of Salmonella expressing foreign proteins represent a potentially important vaccine delivery system. The characteristics of Ag presentation of influenza nucleoprotein expressed in an AroA- strain of Salmonella typhimurium (SL3262-pNP-2) have therefore been compared with those of soluble purified nucleoprotein (NP) and infectious influenza virus. This represents three distinct modes of internalization of the same protein into APC. Human monocytes and the monocytic leukemia cell line THP-1 infected with SL3261-pNP-2 were found to present several different epitopes from NP to human CD4+ class II-restricted T lymphocytes. Ag presentation to these T cell clones was enhanced by pretreatment of THP-1 cells with IFN-gamma but not TNF-alpha. Bacterial phagocytosis and Ag presentation of NP were increased after opsonization of Salmonella with immune serum. Macrophages infected with SL3261-pNP-2 were unable to present NP to class I-restricted T cells. In contrast, cells infected with live influenza virus, although recognized by NP-specific class I-restricted CTL, were inefficiently recognized by NP-specific class II-restricted T cells. Ag presentation to CD4+ T cell clones by monocytes of SL3261-pNP-2, purified recombinant NP, and live influenza virus, but not the synthetic peptide 206-229, was inhibited by chloroquine and the protease inhibitors pepstatin A and leupeptin, suggesting that the major route of processing in each case was via the exogenous pathway. T cell recognition of NP via all of these Ag delivery systems was also abrogated by cycloheximide and brefeldin A treatment, indicating a requirement for recently synthesized MHC class II molecules in presentation of whole NP after processing but not for the corresponding synthetic peptide.
Asunto(s)
Transferasas Alquil y Aril , Células Presentadoras de Antígenos/inmunología , Proteínas Bacterianas/inmunología , Virus de la Influenza A/inmunología , Nucleoproteínas/inmunología , Proteínas de Unión al ARN , Salmonella typhimurium/inmunología , Proteínas del Núcleo Viral/inmunología , 3-Fosfoshikimato 1-Carboxiviniltransferasa , Animales , Antígenos de Diferenciación de Linfocitos T/inmunología , Brefeldino A , Cicloheximida/farmacología , Ciclopentanos/farmacología , Epítopos/inmunología , Femenino , Vectores Genéticos , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Virus de la Influenza A/patogenicidad , Cinética , Macrófagos/inmunología , Ratones , Ratones Endogámicos BALB C , Monocitos/inmunología , Monocitos/microbiología , Proteínas de la Nucleocápside , Fagocitosis , Inhibidores de Proteasas/farmacología , Salmonella typhimurium/genética , Salmonella typhimurium/patogenicidad , Células Tumorales Cultivadas , VirulenciaRESUMEN
The P.69 Bordetella pertussis protective antigen was expressed by use of the trc promoter from the chromosome of a Salmonella typhimurium aro vaccine strain, BRD509, by integrating the prn gene, encoding the 93-kDa precursor of this protein, into the aroC locus. P.69 was detected on the cell surface of the S. typhimurium strain (BRD640) by agglutination and immunoelectron microscopy. BALB/c mice immunized orally or intravenously with BRD640 showed a significant level of protection against an aerosol challenge with virulent B. pertussis, compared with control animals. No anti-P.69 antibodies in the serum or anti-P.69 antibody-secreting cells in the lungs were detected in BRD640-vaccinated animals, although cells isolated from spleens showed a P.69-dependent cell proliferative response. In contrast, low levels of anti-P.69 antibodies in the serum and anti-P.69 antibody-secreting cells in the lungs were detected in immunized mice following a B. pertussis challenge.
Asunto(s)
Adhesinas Bacterianas , Antígenos Bacterianos/inmunología , Bordetella pertussis/inmunología , Vacuna contra la Tos Ferina/inmunología , Salmonella typhimurium/inmunología , Factores de Virulencia de Bordetella/inmunología , Administración Oral , Animales , Anticuerpos Antibacterianos/análisis , Femenino , Hemaglutininas/genética , Hemaglutininas/inmunología , Ratones , Ratones Endogámicos BALB C , Salmonella typhimurium/genética , Vacunación , Factores de Virulencia de Bordetella/biosíntesisRESUMEN
To address the question of whether Salmonella-infected nonphagocytic cells could serve as target cells for recognition by antigen-specific, major histocompatibility complex class I-restricted cytotoxic T lymphocytes (CTL), four recombinant Salmonella typhimurium constructs that expressed full-length, or fragments of, influenza A virus nucleoprotein (NP) were made. The bacteria were shown to infect Chinese hamster ovary (CHO) cells. Appropriate major histocompatibility complex restriction molecules, HLA-B27 and H-2 Db, were transfected into CHO cells, which were then infected with recombinant S. typhimurium and used as targets for NP-specific CTL. The cells in which NP was expressed by intracellularly replicating bacteria were not lysed by NP-specific CTL, although they were killed when appropriate influenza A virus or peptides were used. Thus, S.typhimurium bacteria within nonphagocytic cells were resistant to CTL recognition. In contrast to these results, mice infected with recombinant S.typhimurium that expressed fragments of NP in the periplasm were primed for NP-specific CTL responses. The results indicate that CTL responses specific to Salmonella antigens can be generated, but the bacteria may be safe from the CTL attack once they have entered the nonphagocytic cells.
Asunto(s)
Proteínas de Unión al ARN , Salmonella typhimurium/inmunología , Linfocitos T Citotóxicos/inmunología , Animales , Secuencia de Bases , Células CHO , Cricetinae , Ratones , Datos de Secuencia Molecular , Proteínas de la Nucleocápside , Nucleoproteínas/biosíntesis , Nucleoproteínas/genética , Nucleoproteínas/inmunología , Recombinación Genética , Salmonella typhimurium/genética , Transfección , Proteínas del Núcleo Viral/biosíntesis , Proteínas del Núcleo Viral/genética , Proteínas del Núcleo Viral/inmunologíaRESUMEN
Potent immunological adjuvants are urgently required to complement recombinant and synthetic vaccines. However, it has not been possible to derive new principles for the design of vaccine adjuvants from knowledge of the mechanism of immunogenicity. Carbonyl-amino condensations, which are essential to the inductive interaction between antigen-presenting cells and T helper cells, were tested as a target for the enhancement of immune responses. Enzymic oxidation of cell-surface galactose to increase aminereactive carbonyl groups on murine lymphocytes and antigen-presenting cells provided a potent, noninflammatory method of enhancing the immunogenicity of viral, bacterial, and protozoal subunit vaccines in mice.
Asunto(s)
Adyuvantes Inmunológicos , Galactosa Oxidasa/administración & dosificación , Galactosa/metabolismo , Linfocitos T/inmunología , Vacunación/métodos , Animales , Formación de Anticuerpos , Citotoxicidad Inmunológica , Proteína gp120 de Envoltorio del VIH/inmunología , Activación de Linfocitos , Ratones , Neuraminidasa/administración & dosificación , Oxidación-Reducción , Péptidos/inmunologíaRESUMEN
We have described a new class of live attenuated salmonella vaccines harbouring lesions in htrA, a stress protein gene previously. The virulence and invasiveness of Salmonella htrA mutants was investigated in three models of increased susceptibility to Salmonella infection. These included BALB/c mice, either given sublethal whole body irradiation (350 R) or administered rabbit anti-TNF alpha antiserum, and (CBA/NfemaleXBALB/cmale)F1 male mice which express the xid sex-linked B cell defect of CBA/N mice and are more susceptible to salmonellae than female littermates. Salmonella typhimurium htrA mutants derived from virulent strains, C5046 (C5 htrA::TnphoA) and BRD726 (SL1344 delta htrA) were not more invasive in immunosuppressed mice than in normal controls in the three mouse models of defective immunity. The results indicate that susceptibility to S. typhimurium htrA vaccines derived from virulent parents is not enhanced by conditions of impaired resistance to infection.