RESUMO
Antigen-activated lymphocytes undergo extraordinarily rapid cell division in the course of immune responses. We hypothesized that this unique aspect of lymphocyte biology leads to unusual genomic stress in recently antigen-activated lymphocytes and that targeted manipulation of DNA damage-response (DDR) signaling pathways would allow for selective therapeutic targeting of pathological T cells in disease contexts. Consistent with these hypotheses, we found that activated mouse and human T cells display a pronounced DDR in vitro and in vivo. Upon screening a variety of small-molecule compounds, we found that potentiation of p53 (via inhibition of MDM2) or impairment of cell cycle checkpoints (via inhibition of CHK1/2 or WEE1) led to the selective elimination of activated, pathological T cells in vivo. The combination of these strategies [which we termed "p53 potentiation with checkpoint abrogation" (PPCA)] displayed therapeutic benefits in preclinical disease models of hemophagocytic lymphohistiocytosis and multiple sclerosis, which are driven by foreign antigens or self-antigens, respectively. PPCA therapy targeted pathological T cells but did not compromise naive, regulatory, or quiescent memory T-cell pools, and had a modest nonimmune toxicity profile. Thus, PPCA is a therapeutic modality for selective, antigen-specific immune modulation with significant translational potential for diverse immune-mediated diseases.
Assuntos
Dano ao DNA/imunologia , Doenças do Sistema Imunitário/terapia , Animais , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/imunologia , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/terapia , Etoposídeo/administração & dosagem , Humanos , Doenças do Sistema Imunitário/imunologia , Ativação Linfocitária , Linfo-Histiocitose Hemofagocítica/imunologia , Linfo-Histiocitose Hemofagocítica/terapia , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Esclerose Múltipla/imunologia , Esclerose Múltipla/terapia , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-mdm2/imunologia , Transdução de Sinais/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/patologia , Proteína Supressora de Tumor p53/imunologiaRESUMO
Hemophagocytic lymphohistiocytosis (HLH) is an inborn disorder of immune regulation caused by mutations affecting perforin-dependent cytotoxicity. Defects in this pathway impair negative feedback between cytotoxic lymphocytes and APCs, leading to prolonged and pathologic activation of T cells. Etoposide, a widely used chemotherapeutic drug that inhibits topoisomerase II, is the mainstay of treatment for HLH, although its therapeutic mechanism remains unknown. We used a murine model of HLH, involving lymphocytic choriomeningitis virus infection of perforin-deficient mice, to study the activity and mechanism of etoposide for treating HLH and found that it substantially alleviated all symptoms of murine HLH and allowed prolonged survival. This therapeutic effect was relatively unique among chemotherapeutic agents tested, suggesting distinctive effects on the immune response. We found that the therapeutic mechanism of etoposide in this model system involved potent deletion of activated T cells and efficient suppression of inflammatory cytokine production. This effect was remarkably selective; etoposide did not exert a direct anti-inflammatory effect on macrophages or dendritic cells, and it did not cause deletion of quiescent naive or memory T cells. Finally, etoposide's immunomodulatory effects were similar in wild-type and perforin-deficient animals. Thus, etoposide treats HLH by selectively eliminating pathologic, activated T cells and may have usefulness as a novel immune modulator in a broad array of immunopathologic disorders.
Assuntos
Etoposídeo/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Linfo-Histiocitose Hemofagocítica/tratamento farmacológico , Linfo-Histiocitose Hemofagocítica/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Animais , Apresentação de Antígeno/efeitos dos fármacos , Apresentação de Antígeno/imunologia , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Proteínas de Ligação a DNA/genética , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Modelos Animais de Doenças , Etoposídeo/administração & dosagem , Interferon gama/biossíntese , Vírus da Coriomeningite Linfocítica , Linfo-Histiocitose Hemofagocítica/genética , Ativação de Macrófagos/efeitos dos fármacos , Ativação de Macrófagos/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Camundongos Knockout , Inibidores da Topoisomerase II/administração & dosagem , Inibidores da Topoisomerase II/farmacologiaRESUMO
Mammalian cell-surface receptors typically display N- or O-linked glycans added post-translationally. Plant lectins such as phytohemagluttinin (PHA) can activate the T cell receptor (TCR) and other cell-surface receptors by binding to glycans and initiating receptor cross-linking. Pathogenic microorganisms such as Bordetella pertussis also express proteins with lectin-like activities. Similar to plant lectins, pertussis toxin (PTx) can activate the TCR and bind to a variety of glycans. However, whether the lectin-like activity of PTx is responsible for its ability to activate TCR signaling has not been formally proven. Here we examined the ability of PTx and a panel of lectins to activate the TCR or a CD8α/CD3ζ chimeric receptor (termed CD8ζ). We demonstrate that CD8ζ rescues PTx-induced signaling events lacking in TCR null cells. This result indicates that CD8ζ can substitute for TCR and supports the hypothesis that PTxB (functioning as a lectin) stimulates signaling via receptor cross-linking rather than by binding to a specific epitope on the TCR. Moreover, PTx is able to activate signaling by binding either N-linked or O-linked glycan-modified receptors as the TCR displays N-linked glycans while CD8ζ displays O-linked glycans. Finally, studies with a diverse panel of lectins indicate that the signaling activity of the lectins does not always correlate with the biochemical reports of ligand preferences. Comparison of lectin signaling through TCR or CD8ζ allows us to better define the structural and functional properties of lectin-glycan interactions using a biologically based signaling readout.
Assuntos
Complexo CD3/química , Antígenos CD8/química , Toxina Pertussis/química , Lectinas de Plantas/química , Linfócitos T/química , Complexo CD3/genética , Complexo CD3/imunologia , Antígenos CD8/genética , Antígenos CD8/imunologia , Engenharia Genética , Humanos , Células Jurkat , Toxina Pertussis/imunologia , Lectinas de Plantas/imunologia , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/imunologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/imunologia , Transdução de Sinais/imunologia , Linfócitos T/imunologiaRESUMO
Binding of pertussis toxin (PTx) was examined by a glycan microarray; 53 positive hits fell into four general groups. One group represents sialylated biantennary compounds with an N-glycan core terminating in alpha2-6-linked sialic acid. The second group consists of multiantennary compounds with a canonical N-glycan core, but lacking terminal sialic acids, which represents a departure from the previous understanding of PTx binding to N-glycans. The third group consists of Neu5Acalpha2-3(lactose or N-acetyllactosamine) forms that lack the branched mannose core found in N-glycans; thus, their presentation is more similar to that of O-linked glycans and glycolipids. The fourth group of compounds consists of Neu5Acalpha2-8Neu5Acalpha2-8Neu5Ac, which is seen in the c series gangliosides and some N-glycans. Quantitative analysis by surface plasmon resonance of the relative affinities of PTx for terminal Neu5Acalpha2-3 versus Neu5Acalpha2-6, as well as the affinities for the trisaccharide Neu5Acalpha2-8Neu5Acalpha2-8Neu5Ac versus disaccharide, revealed identical global affinities, even though the amount of bound glycan varied by 4-5-fold. These studies suggest that the conformational space occupied by a glycan can play an important role in binding, independent of affinity. Characterization of N-terminal and C-terminal binding sites on the S2 and S3 subunits by mutational analysis revealed that binding to all sialylated compounds was mediated by the C-terminal binding sites, and binding to nonsialylated N-linked glycans is mediated by the N-terminal sites present on both the S2 and S3 subunits. A detailed understanding of the glycans recognized by pertussis toxin is essential to understanding which cells are targeted in clinical disease.
Assuntos
Polissacarídeos/química , Polissacarídeos/metabolismo , Amino Açúcares , Sequência de Carboidratos , Carboidratos , Ligantes , Ácido N-Acetilneuramínico/química , Ácido N-Acetilneuramínico/metabolismo , Toxina Pertussis/metabolismo , Ligação Proteica , Ácidos Siálicos , Ressonância de Plasmônio de Superfície , TrissacarídeosRESUMO
Whooping cough due to Bordetella pertussis is increasing in incidence, in part due to accumulation of mutations which increase bacterial fitness in highly vaccinated populations. Polymorphisms in the pertussis toxin, ptxA and ptxB genes, and the pertactin, prn genes of clinical isolates of Bordetella pertussis collected in Cincinnati from 1989 through 2005 were examined. While the ptxA and prn genotypes were variable, all 48 strains had the ptxB2 genotype; ptxB1 encodes glycine at amino acid 18 of the S2 subunit of pertussis toxin, while ptxB2 encodes serine. We investigated antigenic and functional differences of PtxB1 and PtxB2. The S2 protein was not very immunogenic. Only a few vaccinated or individuals infected with B. pertussis developed antibody responses to the S2 subunit, and these sera recognized both polymorphic forms equally well. Amino acid 18 of S2 is in a glycan binding domain, and the PtxB forms displayed differences in receptor recognition and toxicity. PtxB1 bound better to the glycoprotein, fetuin, and Jurkat T cells in vitro, but the two forms were equally effective at promoting CHO cell clustering. To investigate in vivo activity of Ptx, one µg of Ptx was administered to DDY mice and blood was collected on 4 days after injection. PtxB2 was more effective at promoting lymphocytosis in mice.
Assuntos
Substituição de Aminoácidos , Toxina Pertussis/genética , Toxina Pertussis/imunologia , Polimorfismo Genético , Alelos , Animais , Sequência de Bases , Células CHO , Cricetinae , Cricetulus , Feminino , Humanos , Camundongos , Modelos Moleculares , Toxina Pertussis/química , Toxina Pertussis/toxicidade , Multimerização Proteica , Estrutura Quaternária de ProteínaRESUMO
Pertussis toxin (PTx) is the major virulence factor of Bordetella pertussis. The enzymatic or active (A) subunit inactivates host G protein coupled receptor (GPCR) signaling pathways. The non-enzymatic binding (B) subunit also mediates biological effects due to lectin-like binding characteristics, including the induction of T cell receptor (TCR) signaling and subsequent down-regulation of chemokine receptor expression. Here we report another activity attributable to PTxB, facilitating transfer of membrane material between mammalian cells. This activity does not require the TCR, and does not require cell-to-cell contact or cellular aggregation. Rather, membrane vesicles are transferred from donor to recipient cells in a toxin-dependent fashion. Membrane transfer occurs in different cell types, including cultured human T cells, CHO cells, and human primary peripheral blood mononuclear cells. Transfer involves both lipid and integral membrane proteins, as evidenced by the transfer of T and B cell-specific receptor molecules to other PBMCs. Interestingly, membrane transfer activity is a property that PTx shares with some, but not all, cell-aggregating lectins that are mitogenic for human T cells, and appears to be related to the ability to bind certain host cell glycolipids. This phenomenon may represent another mechanism by which pertussis toxin disrupts mammalian intra- and inter-cellular signaling.
Assuntos
Metabolismo dos Lipídeos , Proteínas de Membrana/metabolismo , Toxina Pertussis/farmacologia , Animais , Células CHO , Cricetulus , Humanos , Células Jurkat , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Antibody-dependent complement killing of Bordetella pertussis after immunization with a three-component acellular pertussis vaccine was characterized. Postimmunization activity was unchanged for about half of the adult vaccine recipients. The responses of the other individuals were complex, with evidence of both beneficial and antagonistic responses occurring, sometimes in the same individual.
Assuntos
Bordetella pertussis/imunologia , Proteínas do Sistema Complemento/fisiologia , Vacina contra Coqueluche/imunologia , Anticorpos Antibacterianos/sangue , Atividade Bactericida do Sangue , Método Duplo-Cego , Humanos , Imunização , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Estudos Prospectivos , Vacinas Acelulares/imunologiaRESUMO
Antibody-mediated neutralization of pertussis toxin-induced proliferation of human peripheral blood mononuclear cells (PBMC) was assessed using alamarBlue and compared with results from the Chinese hamster ovary (CHO) cell assay using sera from vaccinated adults and convalescent children. Neutralization values for the CHO assay were similar for vaccinated and convalescent subjects; however. the convalescent group had higher titers in the PBMC assay. Results for pertussis toxin neutralization with the CHO assay appear to be distinct from those with the PBMC assay.