RESUMO
Studies describing the induction of apoptosis for CD4 mAbs do not delineate between epitope-dependent and Fc-driven epitope cross-linking induced cell death. Keliximab and clenoliximab are two CD4 mAbs that differ only in their heavy chain isotypes, being an IgG1 and a modified IgG4, respectively. These antibodies suppress CD4 T cell responses in vitro and in vivo and have been in human clinical trials for the treatment of RA and asthma. Here we compared the apoptotic activity of these mAbs to differentiate between the contributions of epitope-dependent vs. Fc-driven epitope cross-linking induced cell death in vitro as a link to differential CD4 cell depletion in vivo. We developed a simple flow cytometry procedure that measures apoptosis within intact and compromised subpopulations of PBMCs within a few hours of culture. Attractors software was used to quantitate the percentage of apoptotic CD4 T cells, which generate reactive oxygen species (ROS), express external phosphatidyl serine (PS) and cleaved fluorescein diacetate (FDA), within the intact and compromised lymphocyte populations. Treatment of freshly isolated PBMCs with keliximab resulted in the appearance of characteristic apoptotic condensed CD4 T cells that contained reactive oxygen species, were annexin V positive and had intact esterase activity. Apoptosis was evident within 3 h and continued throughout the 72-h culture period. In contrast, clenoliximab alone did not induce apoptosis. The use of multiparameter flow cytometry and Attractors to analyze subpopulations based on scatter properties and biochemical processes during apoptosis provides a sensitive assay in which to quantitate and characterize the induction of cell death. Depletion of CD4 T cells in vivo by keliximab may reflect, in part, antibody-mediated apoptosis of these cells that is dependent on Fcgamma receptors.
Assuntos
Anticorpos Monoclonais/farmacologia , Apoptose/imunologia , Antígenos CD4/imunologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Citometria de Fluxo/métodos , Anexina A5/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Citometria de Fluxo/estatística & dados numéricos , Fluoresceínas , Humanos , Técnicas In Vitro , Espécies Reativas de Oxigênio/metabolismo , Software , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
CD40-CD154 (CD40 ligand) interactions are essential for the development of protective immunity. Previous studies have described the CD40 binding site as a shallow groove formed between two monomers of CD154. However, these studies have not examined the structure or biological function of the carbohydrate on CD154. Human CD154 contains a single N-linked glycosylation site at asparagine 240. We have characterized the interactions between CD40 and soluble (s) CD154 in which sCD154 contains different types of carbohydrates. Detailed carbohydrate analysis revealed high-mannose structures on sCD154 purified from Pichia pastoris, whereas CD154 purified from Chinese hamster ovary E1A contained heterogeneous populations of complex carbohydrates. sCD154 purified from either system was trimeric, it bound to CD40 with similar affinities of 10-30 nM, and it functionally induced CD69 and CD95 expression on primary B cells. Together, these results indicate that the presence of varied types of N-linked glycans on asparagine 240 of CD154 does not play a significant role in the CD40-CD154 interactions.
Assuntos
Antígenos CD40/química , Ligante de CD40/química , Carboidratos/química , Animais , Asparagina/química , Linfócitos B/imunologia , Antígenos CD40/genética , Antígenos CD40/imunologia , Antígenos CD40/metabolismo , Ligante de CD40/genética , Ligante de CD40/imunologia , Ligante de CD40/metabolismo , Células CHO , Configuração de Carboidratos , Metabolismo dos Carboidratos , Células Cultivadas , Clonagem Molecular , Cricetinae , Humanos , Manose/química , Manose/metabolismo , Pichia/genética , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , SolubilidadeRESUMO
Several CD4 mAbs have entered the clinic for the treatment of autoimmune diseases or transplant rejection. Most of these mAbs caused CD4 cell depletion, and some were murine mAbs which were further hampered by human anti-mouse Ab responses. To obviate these concerns, a primatized CD4 mAb, clenoliximab, was generated by fusing the V domains of a cynomolgus macaque mAb to human constant regions. The heavy chain constant region is a modified IgG4 containing two single residue substitutions designed to ablate residual Fc receptor binding activity and to stabilize heavy chain dimer formation. This study compares and contrasts the in vitro properties of clenoliximab with its matched IgG1 derivative, keliximab, which shares the same variable regions. Both mAbs show potent inhibition of in vitro T cell responses, lack of binding to complement component C1q, and inability to mediate complement-dependent cytotoxicity. However, clenoliximab shows markedly reduced binding to Fc receptors and therefore does not mediate Ab-dependent cell-mediated cytotoxicity or modulation/loss of CD4 from the surface of T cells, except in the presence of rheumatoid factor or activated monocytes. Thus, clenoliximab retains the key immunomodulatory attributes of keliximab without the liability of strong Fcgamma receptor binding. In initial clinical trials, these properties have translated to a reduced incidence of CD4+ T cell depletion.
Assuntos
Anticorpos Monoclonais/fisiologia , Antígenos CD4/imunologia , Imunoglobulina G/metabolismo , Receptores Fc/fisiologia , Animais , Anticorpos Monoclonais/metabolismo , Citotoxicidade Celular Dependente de Anticorpos , Sítios de Ligação de Anticorpos , Antígenos CD4/metabolismo , Linhagem Celular , Testes Imunológicos de Citotoxicidade , Humanos , Hibridomas , Interleucina-2/antagonistas & inibidores , Interleucina-2/biossíntese , Ativação Linfocitária , Teste de Cultura Mista de Linfócitos , Macaca fascicularis , Receptores Fc/metabolismoRESUMO
HO endonuclease-induced double-strand breaks (DSBs) within a direct duplication of Escherichia coli lacZ genes are repaired either by gene conversion or by single-strand annealing (SSA), with > 80% being SSA. Previously it was demonstrated that the RAD52 gene is required for DSB-induced SSA. In the present study, the effects of other genes belonging to the RAD52 epistasis group were analyzed. We show that RAD51, RAD54, RAD55, and RAD57 genes are not required for SSA irrespective of whether recombination occurred in plasmid or chromosomal DNA. In both plasmid and chromosomal constructs with homologous sequences in direct orientation, the proportion of SSA events over gene conversion was significantly elevated in the mutant strains. However, gene conversion was not affected when the two lacZ sequences were in inverted orientation. These results suggest that there is a competition between SSA and gene conversion processes that favors SSA in the absence of RAD51, RAD54, RAD55 and RAD57. Mutations in RAD50 and XRS2 genes do not prevent the completion, but markedly retard the kinetics, of DSB repair by both mechanisms in the lacZ direct repeat plasmid, a result resembling the effects of these genes during mating-type (MAT) switching.
Assuntos
Reparo do DNA , DNA Fúngico , DNA de Cadeia Simples , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Saccharomyces cerevisiae/genética , Cromossomos Fúngicos , Plasmídeos , Proteínas de Saccharomyces cerevisiaeRESUMO
In Saccharomyces cerevisiae, HO endonuclease-induced mating-type (MAT) switching is a specialized mitotic recombination event in which MAT sequences are replaced by those copied from a distant, unexpressed donor (HML or HMR). The donors have a chromatin structure inaccessible for both transcription and HO cleavage. Here we use physical monitoring of DNA to show that MAT switching is completely blocked at an early step in recombination in strains deleted for the DNA repair genes RAD51, RAD52, RAD54, RAD55 or RAD57. We find, however, that only RAD52 is required when the donor sequence is simultaneously not silenced and located on a plasmid. RAD51, RAD54, RAD55 and RAD57 are still required when the same transcribed donor is on the chromosome. We conclude that recombination in vivo occurs between DNA molecules in chromatin, whose structure significantly influences the outcome. RAD51, RAD54, RAD55 and RAD57 are all required to facilitate strand invasion into otherwise inaccessible donor sequences.
Assuntos
DNA Fúngico/genética , Conversão Gênica , Genes Fúngicos , Genes Fúngicos Tipo Acasalamento , Conformação de Ácido Nucleico , Saccharomyces cerevisiae/genética , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Proteínas Fúngicas/genética , Reação em Cadeia da Polimerase , Proteínas de Saccharomyces cerevisiaeRESUMO
A biologically relevant mechanism of generating peripheral tolerance in T cells that have escaped thymic deletion is by the oral administration of soluble antigens. Oral tolerance occurs by two distinct mechanisms. Feeding a single high dose of antigen induces anergy of antigen-specific TH1 cells, while multiple low doses of antigen induce regulatory T cells that mediate suppression by producing immunosuppressive cytokines. Although cytokine production orally tolerant animals has been well studied utilizing in vitro assay systems, semi-quantitative characterization of cytokine production in oral tolerance in vivo has not been carried out. In this paper we have developed a system using semi-quantitative RNA polymerase chain reaction to characterize cytokine gene expression in vivo in mice orally tolerized by feeding either a single high dose or multiple low dosages of antigen. We find that measurable differences in interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) gene expression occurred between the tolerized and non-tolerized groups. Qualitatively, IL-4 mRNA was increased in both orally tolerized groups. However, significant differences in IL-4 gene expression between the two groups in both magnitude and kinetics were found. A large but short-lived increase in IL-4 was produced in mice fed a single high dose, while a relatively more moderate, longer-lived increase was produced in mice fed multiple low doses. The increase in IL-4 gene expression was specific only to the draining lymph node following antigen administration. Expression of IFN-gamma was decreased in both orally tolerant groups. These results indicate that tolerance in TH1 cells is induced by both of these feeding regimens while TH2 responses are intact and amplified upon reexposure to antigen.
Assuntos
Tolerância Imunológica , Interferon gama/genética , Interleucina-4/genética , Administração Oral , Animais , Sequência de Bases , Feminino , Expressão Gênica , Imunização , Interleucina-10/metabolismo , Linfonodos/metabolismo , Camundongos , Dados de Sequência Molecular , RNA Mensageiro/análise , Fator de Crescimento Transformador beta/metabolismoRESUMO
OT is a relevant biological pathway for generating peripheral tolerance against both self and external antigens with minimal side effects (fig. 3). This route might, therefore, contain promising potential for the treatment of autoimmune and allergic diseases in the human (fig. 3). Thus, oral administration of autoantigens suppresses experimental autoimmune diseases (EAE, EAU, AA, collagen-induced arthritis, NOD diabetes) in a disease- and antigen-specific manner, and oral administration of alloantigens has led to increase of allograft survival. OT might be important in treatment of immune complex diseases and food allergies. OT is mediated by T lymphocytes using at least two nonmutually exclusive mechanisms: suppression and anergy. Suppression can be adoptively transferred by CD8+ T lymphocytes which act by releasing TGF-beta and IL-4 following antigen-specific triggering. Antigen-driven tissue-directed suppression occurs following oral administration of an antigen from the target organ, even if it is not the disease-inducing antigen (bystander suppression). Thus, synthetic peptides can induce OT, and tolerogenic epitopes of antigen may be different from the autoreactive epitope. Due to the promising results in animal models, OT is being tested in clinical trials in multiple sclerosis, rheumatoid arthritis and uveitis [193, 194].
Assuntos
Antígenos/imunologia , Tolerância Imunológica , Administração Oral , Animais , Antígenos/administração & dosagem , Autoantígenos/administração & dosagem , Doenças Autoimunes/terapia , Humanos , Hipersensibilidade/terapia , Intestinos/imunologia , Nódulos Linfáticos Agregados/imunologia , Imunologia de Transplantes , Vacinas/administração & dosagem , Vacinas/imunologiaRESUMO
Double-strand breaks (DSBs) in Saccharomyces cerevisiae can be repaired by gene conversions or by deletions resulting from single-strand annealing between direct repeats of homologous sequences. Although rad1 mutants are resistant to x-rays and can complete DSB-mediated mating-type switching, they could not complete recombination when the ends of the break contained approximately 60 base pairs of nonhomology. Recombination was restored when the ends of the break were made homologous to donor sequences. Additionally, the absence of RAD1 led to the frequent appearance of a previously unobserved type of recombination product. These data suggest RAD1 is required to remove nonhomologous DNA from the 3' ends of recombining DNA, a process analogous to the excision of photodimers during repair of ultraviolet-damaged DNA.
Assuntos
Reparo do DNA , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Recombinação Genética , Saccharomyces cerevisiae/genética , DNA Fúngico/genética , Conversão Gênica , Cinética , Proteínas de Saccharomyces cerevisiae , Deleção de Sequência , Raios UltravioletaRESUMO
HO endonuclease-induced double-strand breaks in Saccharomyces cerevisiae can undergo recombination by two distinct and competing pathways. In a plasmid containing a direct repeat, in which one repeat is interrupted by an HO endonuclease cut site, gap repair yields gene conversions while single-strand annealing produces deletions. Consistent with predictions of the single-strand annealing mechanism, deletion formation is not accompanied by the formation of a reciprocal recombination product. Deletions are delayed 60 min when the distance separating the repeats is increased by 4.4 kb. Moreover, the rate of deletion formation corresponds to the time at which complementary regions become single stranded. Gap repair processes are independent of distance but are reduced in rad52 mutants and in G1-arrested cells.