RESUMO
BACKGROUND AIMS: Amyotrophic lateral sclerosis (ALS) is a fatal disease associated with motor neuron degeneration, accumulation of aggregated misfolded proteins and neuroinflammation in motor regions of the central nervous system (CNS). Clinical trials using regulatory T cells (Tregs) are ongoing because of Tregs' immunomodulatory function, ability to traffic to the CNS, high numbers correlating with slower disease in ALS and disease-modifying activity in ALS mouse models. In the current study, a chimeric antigen receptor (CAR) was developed and characterized in human Tregs to enhance their immunomodulatory activity when in contact with an ALS protein aggregate. METHODS: A CAR (DG05-28-3z) consisting of a human superoxide dismutase 1 (hSOD1)-binding single-chain variable fragment, CD28 hinge, transmembrane and co-stimulatory domain and CD3ζ signaling domain was created and expressed in human Tregs. Human Tregs were isolated by either magnetic enrichment for CD4+CD25hi cells (Enr-Tregs) or cell sorting for CD4+CD25hiCD127lo cells (FP-Tregs), transduced and expanded for 17 days. RESULTS: The CAR bound preferentially to the ALS mutant G93A-hSOD1 protein relative to the wild-type hSOD1 protein. The CAR Tregs produced IL-10 when cultured with aggregated G93A-hSOD1 proteins or spinal cord explants from G93A-hSOD1 transgenic mice. Co-culturing DG05-28-3z CAR Tregs with human monocytes/macrophages inhibited production of tumor necrosis factor alpha and reactive oxygen species. Expanded FP-Tregs resulted in more robust Tregs compared with Enr-Tregs. FP-Tregs produced similar IL-10 and less interferon gamma, had lower Treg-specific demethylated region methylation and expressed higher FoxP3 and CD39. CONCLUSIONS: Taken together, this study demonstrates that gene-modified Tregs can be developed to target an aggregated ALS-relevant protein to elicit CAR-mediated Treg effector functions and provides an approach for generating Treg therapies for ALS with the goal of enhanced disease site-specific immunomodulation.
Assuntos
Esclerose Lateral Amiotrófica , Receptores de Antígenos Quiméricos , Camundongos , Animais , Humanos , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismo , Superóxido Dismutase-1/uso terapêutico , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/terapia , Receptores de Antígenos Quiméricos/uso terapêutico , Interleucina-10/genética , Superóxido Dismutase/metabolismo , Camundongos Transgênicos , Linfócitos T CD4-Positivos/metabolismo , Imunomodulação , Modelos Animais de DoençasRESUMO
Chimeric-antigen receptor (CAR) T-cell therapy has shown remarkable efficacy against hematologic tumors. Yet, CAR T-cell therapy has had little success against solid tumors due to obstacles presented by the tumor microenvironment (TME) of these cancers. Here, we show that CAR T cells armored with the engineered IL-2 superkine Super2 and IL-33 were able to promote tumor control as a single-agent therapy. IFNγ and perforin were dispensable for the effects of Super2- and IL-33-armored CAR T cells. Super2 and IL-33 synergized to shift leukocyte proportions in the TME and to recruit and activate a broad repertoire of endogenous innate and adaptive immune cells including tumor-specific T cells. However, depletion of CD8+ T cells or NK cells did not disrupt tumor control, suggesting that broad immune activation compensated for loss of individual cell subsets. Thus, we have shown that Super2 and IL-33 CAR T cells can promote antitumor immunity in multiple solid tumor models and can potentially overcome antigen loss, highlighting the potential of this universal CAR T-cell platform for the treatment of solid tumors.
Assuntos
Neoplasias , Microambiente Tumoral , Humanos , Imunoterapia Adotiva , Interleucina-2 , Interleucina-33RESUMO
The NKG2D ligand MHC class I chain-related protein A (MICA) is expressed on many varieties of malignant cells but is absent from most normal tissues, and thus represents a potential target for chimeric Ag receptor (CAR) T cell-based therapeutics. However, there are more than 100 alleles of MICA, so the ability to target a conserved site is needed for a therapy to be used in most patients. In this study, we describe a fully human anti-MICA CAR created by fusing the single-chain fragment variable B2 to the full length DAP10 protein and the traditional CD3ζ signaling domain. Human T cells expressing the B2 CAR killed MICA-positive tumor cells, produced IFN-γ when in contact with MICA-positive tumor cells or plate-bound MICA protein, and inhibited PANC-1 growth in a mouse xenograft model. To localize B2's epitope on MICA, we used novel computational methods to model potential binding modes and to design mutational variants of MICA testing these hypotheses. Flow cytometry using a commercial anti-MICA/MICB Ab indicated that the variant proteins were expressed at high levels on transduced P815 cell lines. One variant protein (R38S/K40T/K57E) showed reduced staining with a B2-IgG1 fusion protein compared with controls and did not induce IFN-γ production by human T cells expressing the B2 CAR. These results show antitumor activity of MICA-specific CAR T cells and indicate an essential role for a conserved site in the exposed loop involving aa 38-57 of MICA. This study describes a novel MICA-specific CAR and discusses its potential use as a cancer therapeutic.
Assuntos
Anticorpos Monoclonais/farmacologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Receptores de Antígenos Quiméricos/imunologia , Linfócitos T/imunologia , Animais , Linhagem Celular Tumoral , Citometria de Fluxo , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Interferon gama/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Subfamília K de Receptores Semelhantes a Lectina de Células NK , Transdução de Sinais , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Chimeric antigen receptors (CARs) are genetically engineered proteins that combine an extracellular antigen-specific recognition domain with one or several intracellular T-cell signaling domains. When expressed in T cells, these CARs specifically trigger T-cell activation upon antigen recognition. While the clinical proof of principle of CAR T-cell therapy has been established in hematological cancers, CAR T cells are only at the early stages of being explored to tackle solid cancers. This special report discusses the concept of exploiting natural killer cell receptors as an approach that could broaden the specificity of CAR T cells and potentially enhance the efficacy of this therapy against solid tumors. New data demonstrating feasibility of this approach in humans and supporting the ongoing clinical trial are also presented.
Assuntos
Antígenos de Neoplasias/imunologia , Imunoterapia Adotiva , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Neoplasias/imunologia , Neoplasias/terapia , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Animais , Antígenos de Neoplasias/metabolismo , Ensaios Clínicos como Assunto , Citotoxicidade Imunológica , Avaliação Pré-Clínica de Medicamentos , Humanos , Imunoterapia Adotiva/métodos , Subfamília K de Receptores Semelhantes a Lectina de Células NK/genética , Neoplasias/metabolismo , Receptores de Antígenos de Linfócitos T/genética , Resultado do TratamentoRESUMO
Targeting cancer through the use of effector T cells bearing chimeric Ag receptors (CARs) leads to elimination of tumors in animals and patients, but recognition of normal cells or excessive activation can result in significant toxicity and even death. CAR T cells based on modified NKG2D receptors are effective against many types of tumors, and their efficacy is mediated through direct cytotoxicity and cytokine production. Under certain conditions, their ligands can be expressed on nontumor cells, so a better understanding of the potential off-tumor activity of these NKG2D CAR T cells is needed. Injection of very high numbers of activated T cells expressing CARs based on murine NKG2D or DNAM1 resulted in increased serum cytokines (IFN-γ, IL-6, and MCP-1) and acute toxicity similar to cytokine release syndrome. Acute toxicity required two key effector molecules in CAR T cells-perforin and GM-CSF. Host immune cells also contributed to this toxicity, and mice with severe immune cell defects remained healthy at the highest CAR T cell dose. These data demonstrate that specific CAR T cell effector mechanisms and the host immune system are required for this cytokine release-like syndrome in murine models.
Assuntos
Vacinas Anticâncer/imunologia , Imunoterapia Adotiva/métodos , Linfoma de Células T/terapia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Linfócitos T/fisiologia , Animais , Complexo CD3/genética , Linhagem Celular Tumoral , Citotoxicidade Imunológica , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Ativação Linfocitária , Linfoma de Células T/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Subfamília K de Receptores Semelhantes a Lectina de Células NK/genética , Neoplasias Experimentais , Perforina/metabolismo , Proteínas Recombinantes de Fusão/genética , Linfócitos T/transplanteRESUMO
Iron oxide nanoparticles (IONPs) hold great potential for cancer therapy. Actively targeting IONPs to tumor cells can further increase therapeutic efficacy and decrease off-target side effects. To target tumor cells, a natural killer (NK) cell activating receptor, NKG2D, was utilized to develop pan-tumor targeting IONPs. NKG2D ligands are expressed on many tumor types and its ligands are not found on most normal tissues under steady state conditions. The data showed that mouse and human fragment crystallizable (Fc)-fusion NKG2D (Fc-NKG2D) coated IONPs (NKG2D/NPs) can target multiple NKG2D ligand positive tumor types in vitro in a dose dependent manner by magnetic cell sorting. Tumor targeting effect was robust even under a very low tumor cell to normal cell ratio and targeting efficiency correlated with NKG2D ligand expression level on tumor cells. Furthermore, the magnetic separation platform utilized to test NKG2D/NP specificity has the potential to be developed into high throughput screening strategies to identify ideal fusion proteins or antibodies for targeting IONPs. In conclusion, NKG2D/NPs can be used to target multiple tumor types and magnetic separation platform can facilitate the proof-of-concept phase of tumor targeting IONP development.
Assuntos
Nanopartículas de Magnetita/química , Subfamília K de Receptores Semelhantes a Lectina de Células NK/química , Neoplasias/tratamento farmacológico , Animais , Humanos , Nanopartículas de Magnetita/uso terapêutico , Camundongos , Subfamília K de Receptores Semelhantes a Lectina de Células NK/uso terapêuticoRESUMO
The methylotrophic yeast, Pichia pastoris, is an important organism used for the production of therapeutic proteins. Previously, we have reported the glycoengineering of this organism to produce human-like N-linked glycans but up to now no one has addressed engineering the O-linked glycosylation pathway. Typically, O-linked glycans produced by wild-type P. pastoris are linear chains of four to five α-linked mannose residues, which may be capped with ß- or phospho-mannose. Previous genetic engineering of the N-linked glycosylation pathway of P. pastoris has eliminated both of these two latter modifications, resulting in O-linked glycans which are linear α-linked mannose structures. Here, we describe a method for the co-expression of an α-1,2-mannosidase, which reduces these glycans to primarily a single O-linked mannose residue. In doing so, we have reduced the potential of these glycans to interact with carbohydrate-binding proteins, such as dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin. Furthermore, the introduction of the enzyme protein-O-linked-mannose ß-1,2-N-acetylglucosaminyltransferase 1, resulted in the capping of the single O-linked mannose residues with N-acetylglucosamine. Subsequently, this glycoform was extended into human-like sialylated glycans, similar in structure to α-dystroglycan-type glycoforms. As such, this represents the first example of sialylated O-linked glycans being produced in yeast and extends the utility of the P. pastoris production platform beyond N-linked glycosylated biotherapeutics to include molecules possessing O-linked glycans.
Assuntos
Manose/biossíntese , Engenharia Metabólica/métodos , Pichia/metabolismo , alfa-Manosidase/metabolismo , Pichia/crescimento & desenvolvimento , Engenharia de Proteínas , alfa-Manosidase/genéticaRESUMO
The methylotrophic yeast, Pichiapastoris, is an important organism used for the production of therapeutic proteins. However, the presence of fungal-like glycans, either N-linked or O-linked, can elicit an immune response or enable the expressed protein to bind to mannose receptors, thus reducing their efficacy. Previously we have reported the elimination of ß-linked glycans in this organism. In the current report we have focused on reducing the O-linked mannose content of proteins produced in P. pastoris, thereby reducing the potential to bind to mannose receptors. The initial step in the synthesis of O-linked glycans in P. pastoris is the transfer of mannose from dolichol-phosphomannose to a target protein in the yeast secretory pathway by members of the protein-O-mannosyltransferase (PMT) family. In this report we identify and characterize the members of the P. pastoris PMT family. Like Candida albicans, P. pastoris has five PMT genes. Based on sequence homology, these PMTs can be grouped into three sub-families, with both PMT1 and PMT2 sub-families possessing two members each (PMT1 and PMT5, and PMT2 and PMT6, respectively). The remaining sub-family, PMT4, has only one member (PMT4). Through gene knockouts we show that PMT1 and PMT2 each play a significant role in O-glycosylation. Both, by gene knockouts and the use of Pmt inhibitors we were able to significantly reduce not only the degree of O-mannosylation, but also the chain-length of these glycans. Taken together, this reduction of O-glycosylation represents an important step forward in developing the P. pastoris platform as a suitable system for the production of therapeutic glycoproteins.
Assuntos
Manosiltransferases/genética , Pichia/enzimologia , Pichia/genética , Candida albicans/enzimologia , Candida albicans/genética , Técnicas de Inativação de Genes , Genes Fúngicos , Glicosilação , Manosiltransferases/metabolismo , Filogenia , Pichia/metabolismo , Polissacarídeos/metabolismoRESUMO
Myeloid-derived suppressor cells (MDSCs) have been characterized in several disease settings, especially in many tumor systems. Compared to their involvement in tumor microenvironments, however, MDSCs have been less well studied in their responses to infectious disease processes, in particular to retroviruses that induce immunodeficiency. Here, we demonstrate for the first time the development of a highly immunosuppressive MDSC population that is dependent on infection by the LP-BM5 retrovirus, which causes murine acquired immunodeficiency. These MDSCs express a cell surface marker signature (CD11b(+) Gr-1(+) Ly6C(+)) characteristic of monocyte-type MDSCs. Such MDSCs profoundly inhibit immune responsiveness by a cell dose- and substantially inducible nitric oxide synthase (iNOS)-dependent mechanism that is independent of arginase activity, PD-1-PD-L1 expression, and interleukin 10 (IL-10) production. These MDSCs display levels of immunosuppressive function in parallel with the extent of disease in LP-BM5-infected wild-type (w.t.) versus knockout mouse strains that are differentially susceptible to pathogenesis. These MDSCs suppressed not only T-cell but also B-cell responses, which are an understudied target for MDSC inhibition. The MDSC immunosuppression of B-cell responses was confirmed by the use of purified B responder cells, multiple B-cell stimuli, and independent assays measuring B-cell expansion. Retroviral load measurements indicated that the suppressive Ly6G(low/±) Ly6C(+) CD11b(+)-enriched MDSC subset was positive for LP-BM5, albeit at a significantly lower level than that of nonfractionated splenocytes from LP-BM5-infected mice. These results, including the strong direct MDSC inhibition of B-cell responsiveness, are novel for murine retrovirus-induced immunosuppression and, as this broadly suppressive function mirrors that of the LP-BM5-induced disease syndrome, support a possible pathogenic effector role for these retrovirus-induced MDSCs.
Assuntos
Síndrome da Imunodeficiência Adquirida/virologia , Linfócitos B/imunologia , Tolerância Imunológica , Monócitos/imunologia , Retroviridae/patogenicidade , Linfócitos T/imunologia , Síndrome da Imunodeficiência Adquirida/imunologia , Animais , Antígenos Ly/análise , Antígeno CD11b/análise , Imunofenotipagem , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Quimiocinas/análise , Retroviridae/imunologiaRESUMO
Pathology due to the immune system's response to viral infections often represents a delicate balance between inhibition of viral pathogenesis and regulation of protective immunity. In susceptible C57BL/6 (B6) mice, the murine retroviral isolate LP-BM5 induces splenomegaly, hypergammaglobulinemia, profound B- and T-cell immunodeficiency, and increased susceptibility to opportunistic pathogens and terminal B-cell lymphomas. Here, we report that B6.PD-1 (programmed death-1) and B6.IL-10 knockout mice are substantially more susceptible to LP-BM5-induced disease than wild-type B6 mice. LP-BM5-infected B6.PD-1(-/-) mice developed more severe splenomegaly, hypergammaglobulinemia, and immunodeficiency than infected B6 mice: PD-1(-/-) mice are more susceptible to lower doses of LP-BM5 and show more exaggerated disease early postinfection. LP-BM5-infected B6.IL-10(-/-) mice also develop exaggerated LP-BM5-induced disease, compared to B6 mice, without a significant change in the retroviral load. By reciprocal reconstitution experiments, comparing wild-type versus PD-1(-/-) sources of the requisite cells for LP-BM5 pathogenesis-CD4 T and B cells, PD-1(+) B cells appear to be crucial in the normal limitation of LP-BM5-induced disease in B6 mice. Also, infected B6 mice have increased CD11b(+) spleen cells that express interleukin-10 (IL-10). However, PD-1(-/-) mice, though showing an even greater expansion of CD11b(+) cells after LP-BM5 inoculation, did not show an equivalent increase in IL-10-producing cells. Thus, it appears that PD-1/PD-L interactions and IL-10 are primarily important in moderating the effects of LP-BM5-induced disease in B6 mice.
Assuntos
Proteínas Reguladoras de Apoptose/fisiologia , Regulação para Baixo , Síndromes de Imunodeficiência/fisiopatologia , Interleucina-10/fisiologia , Retroviridae/fisiologia , Animais , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Análise de Sequência com Séries de OligonucleotídeosRESUMO
The recruitment of tumor necrosis factor receptor-associated factors (TRAFs) 1, 2, 3, 5, and 6 to the CD40 cytoplasmic tail upon CD40 trimerization results in downstream signaling events that ultimately lead to CD40-dependent, thymus-dependent (TD) humoral immune responses. Previously, we have shown signaling through the C-terminal tail of CD40 in the absence of canonical TRAF-binding sites is capable of signaling through an alternative TRAF2-binding site. Here, we demonstrate that B cells from mice harboring CD40 with only the C-terminal tail can activate both canonical and noncanonical NFkappaB signaling pathways. Moreover, while lacking germinal center formation, several hallmarks of humoral immune responses including clonal B-cell activation/expansion, antibody isotype switching, and affinity maturation remain normal. This study demonstrates a new functional domain in CD40 that controls critical aspects of B-cell immunity in an in vivo setting.
Assuntos
Linfócitos B/citologia , Antígenos CD40/fisiologia , Fator 2 Associado a Receptor de TNF/metabolismo , Animais , Formação de Anticorpos , Sítios de Ligação , Antígenos CD40/química , Antígenos CD40/imunologia , Diferenciação Celular , Proliferação de Células , Camundongos , NF-kappa B/metabolismo , Transdução de SinaisRESUMO
As the fastest growing class of therapeutic proteins, monoclonal antibodies (mAbs) represent a major potential drug class. Human antibodies are glycosylated in their native state and all clinically approved mAbs are produced by mammalian cell lines, which secrete mAbs with glycosylation structures that are similar, but not identical, to their human counterparts. Glycosylation of mAbs influences their interaction with immune effector cells that kill antibody-targeted cells. Here we demonstrate that human antibodies with specific human N-glycan structures can be produced in glycoengineered lines of the yeast Pichia pastoris and that antibody-mediated effector functions can be optimized by generating specific glycoforms. Glycoengineered P. pastoris provides a general platform for producing recombinant antibodies with human N-glycosylation.
Assuntos
Anticorpos Monoclonais/biossíntese , Melhoramento Genético/métodos , Imunoglobulina G/biossíntese , Imunoglobulina G/genética , Pichia/genética , Pichia/metabolismo , Engenharia de Proteínas/métodos , Anticorpos Monoclonais/genética , Glicosilação , Humanos , Proteínas Recombinantes/biossínteseRESUMO
C57BL/6 (H-2b), but not spontaneous virus-expressing AKR.H-2b congenic, mice generate retrovirus-specific CD8+ CTL responses to the immunodominant Kb-restricted epitope, KSPWFTTL. AKR.H-2b non-responsiveness is mediated by a peripheral tolerance mechanism. When co-cultured with primed B6 antiviral pCTL, AKR.H-2b splenocytes are recognized by the antiviral TcR as "veto" cells, which inhibit by an exquisitely virus-specific, MHC-restricted, veto cell FasL/responder T cell Fas, mediated apoptotic mechanism. Here, AKR.H-2b thymus, lymph node, and bone marrow cells are also shown to inhibit antiviral CTL generation. Purified AKR.H-2b CD4+ and CD8+ T cells, and B cells, served effectively as FasL-dependent veto cells. In contrast, AKR.H-2b dendritic cells (DC) did not efficiently veto antiviral CTL responses, despite expressing sufficient MHC class I/viral peptide complexes for TcR recognition. AKR.H-2b DC also expressed FasL mRNA and cell surface protein, albeit at a lower level than AKR.H-2b T and B cells. These findings suggest a fail-safe escape mechanism by virus-infected cells for escape from CTL-mediated immunity.
Assuntos
Apoptose , Linfócitos B/imunologia , Citotoxicidade Imunológica , Vírus da Leucemia Murina/imunologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T/imunologia , Vírus AKR da Leucemia Murina/imunologia , Animais , Antígenos Virais/imunologia , Linfócitos B/virologia , Linhagem Celular , Células Cultivadas , Células Dendríticas/imunologia , Células Dendríticas/virologia , Proteína Ligante Fas , Citometria de Fluxo , Expressão Gênica , Antígenos H-2/imunologia , Masculino , Glicoproteínas de Membrana/fisiologia , Proteínas de Membrana/análise , Proteínas de Membrana/imunologia , Camundongos , RNA Mensageiro/análise , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/virologia , Fatores de Necrose Tumoral/fisiologia , Receptor fas/fisiologiaRESUMO
Inflammatory cytokines and infiltrating T cells are readily detected in herpes simplex virus (HSV) infected mouse cornea and trigeminal ganglia (TG) during the acute phase of infection, and certain cytokines continue to be expressed at lower levels in infected TG during the subsequent latent phase. Recent results have shown that HSV infection activates Toll-like receptor signaling. Thus, we hypothesized that chemokines may be broadly expressed at both primary sites and latent sites of HSV infection for prolonged periods of time. Real-time reverse transcriptase-polymrease chain reaction (RT-PCR) to quantify expression levels of transcripts encoding chemokines and their receptors in cornea and TG following corneal infection. RNAs encoding the inflammatory-type chemokine receptors CCR1, CCR2, CCR5, and CXCR3, which are highly expressed on activated T cells, macrophages and most immature dendritic cells (DC), and the more broadly expressed CCR7, were highly expressed and strongly induced in infected cornea and TG at 3 and 10 days postinfection (dpi). Elevated levels of these RNAs persisted in both cornea and TG during the latent phase at 30 dpi. RNAs for the broadly expressed CXCR4 receptor was induced at 30 dpi but less so at 3 and 10 dpi in both cornea and TG. Transcripts for CCR3 and CCR6, receptors that are not highly expressed on activated T cells or macrophages, also appeared to be induced during acute and latent phases; however, their very low expression levels were near the limit of our detection. RNAs encoding the CCR1 and CCR5 chemokine ligands MIP-1alpha, MIP-1beta and RANTES, and the CCR2 ligand MCP-1 were also strongly induced and persisted in cornea and TG during the latent phase. These and other recent results argue that HSV antigens or DNA can stimulate expression of chemokines, perhaps through activation of Toll-like receptors, for long periods of time at both primary and latent sites of HSV infection. These chemokines recruit activated T cells and other immune cells, including DC, that express chemokine receptors to primary and secondary sites of infection. Prolonged activation of chemokine expression could provide mechanistic explanations for certain aspects of HSV biology and pathogenesis.
Assuntos
Quimiocinas/genética , Regulação da Expressão Gênica , Herpes Simples/genética , Herpes Simples/virologia , Herpesvirus Humano 1/fisiologia , Receptores de Quimiocinas/genética , Latência Viral , Animais , Córnea/metabolismo , Gânglios/metabolismo , Leucócitos/metabolismo , Camundongos , Camundongos Endogâmicos ICR , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
LP-BM5 retrovirus-infected C57BL/6 mice develop splenomegaly, lymphadenopathy, hypergammaglobulinemia, and immunodeficiency; thus, this disease has been named mouse AIDS. In this syndrome, CD154/CD40 interactions are required for but do not mediate disease by upregulation of CD80 or CD86. We report here that there is nonetheless a necessity for CD40 signaling competence, specifically an intact tumor necrosis factor receptor-associated factor 6 (TRAF 6) binding site.
Assuntos
Antígenos CD40/fisiologia , Síndrome de Imunodeficiência Adquirida Murina/etiologia , Proteínas/fisiologia , Animais , Sítios de Ligação , Camundongos , Camundongos Transgênicos , Fator 6 Associado a Receptor de TNFRESUMO
Long-lived humoral immunity is manifested by the ability of bone marrow plasma cells (PCs) to survive for extended periods of time. Recent studies have underscored the importance of BLyS and APRIL as factors that can support the survival of B lineage lymphocytes. We show that BLyS can sustain PC survival in vitro, and this survival can be further enhanced by interleukin 6. Selective up-regulation of Mcl-1 in PCs by BLyS suggests that this alpha-apoptotic gene product may play an important role in PC survival. Blockade of BLyS, via transmembrane activator and cyclophilin ligand interactor-immunoglobulin treatment, inhibited PC survival in vitro and in vivo. Heightened expression of B cell maturation antigen (BCMA), and lowered expression of transmembrane activator and cyclophilin ligand interactor and BAFF receptor in PCs relative to resting B cells suggests a vital role of BCMA in PC survival. Affirmation of the importance of BCMA in PC survival was provided by studies in BCMA-/- mice in which the survival of long-lived bone marrow PCs was impaired compared with wild-type controls. These findings offer new insights into the molecular basis for the long-term survival of PCs.
Assuntos
Células da Medula Óssea/citologia , Sobrevivência Celular/fisiologia , Plasmócitos/citologia , Receptores do Fator de Necrose Tumoral/fisiologia , Animais , Fator Ativador de Células B , Antígeno de Maturação de Linfócitos B , Sequência de Bases , Células da Medula Óssea/fisiologia , Primers do DNA , Proteínas de Membrana/deficiência , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Plasmócitos/fisiologia , Receptores do Fator de Necrose Tumoral/deficiência , Receptores do Fator de Necrose Tumoral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Necrose Tumoral alfa/deficiência , Fator de Necrose Tumoral alfa/fisiologiaRESUMO
Tumor necrosis factor receptor-associated factors (TRAFs) belong to a family of adapter proteins that are involved in tumor necrosis factor receptor superfamily signaling. It has been shown that the recruitment of TRAFs to the CD40 cytoplasmic tail is essential for CD40-mediated B cell responses. However, it has also been shown that some early B cell responses, such as up-regulation of cell surface molecules and B cell proliferation are only marginally impaired by the disruption of previously defined TRAF binding sites (Ahonen, C., Manning, E., Erickson, L. D., O'Connor, B. P., Lind, E. F., Pullen, S. S., Kehry, M. R., and Noelle, R. J. (2002) Nat. Immunol. 3, 451-456; and Manning, E., Pullen, S. S., Souza, D. J., Kehry, M., and Noelle, R. J. (2002) Eur. J. Immunol. 32, 39-49). In this report, we identify a second TRAF2 binding site in the CD40 C terminus. The binding motif "SVQE" fits into the major TRAF2 binding consensus sequence, and its disruption resulted in the loss of remaining CD40 functions. Hence, like CD30, the CD40 cytoplasmic tail contains two distinct and functionally important TRAF2 binding sites.
Assuntos
Antígenos CD40/metabolismo , Proteínas/metabolismo , Transdução de Sinais , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sítios de Ligação , Western Blotting , Ligante de CD40/biossíntese , Divisão Celular , Linhagem Celular , Citoplasma/metabolismo , DNA/metabolismo , Antígeno Ki-1/biossíntese , Camundongos , Modelos Genéticos , Dados de Sequência Molecular , Mutação , Testes de Precipitina , Ligação Proteica , Estrutura Terciária de Proteína , Fator 2 Associado a Receptor de TNF , Transfecção , Técnicas do Sistema de Duplo-Híbrido , Regulação para CimaRESUMO
The persistence of herpes simplex virus (HSV) and the diseases that it causes in the human population can be attributed to the maintenance of a latent infection within neurons in sensory ganglia. Little is known about the effects of latent infection on the host neuron. We have addressed the question of whether latent HSV infection affects neuronal gene expression by using microarray transcript profiling of host gene expression in ganglia from latently infected versus mock-infected mouse trigeminal ganglia. (33)P-labeled cDNA probes from pooled ganglia harvested at 30 days postinfection or post-mock infection were hybridized to nylon arrays printed with 2,556 mouse genes. Signal intensities were acquired by phosphorimager. Mean intensities (n = 4 replicates in each of three independent experiments) of signals from mock-infected versus latently infected ganglia were compared by using a variant of Student's t test. We identified significant changes in the expression of mouse neuronal genes, including several with roles in gene expression, such as the Clk2 gene, and neurotransmission, such as genes encoding potassium voltage-gated channels and a muscarinic acetylcholine receptor. We confirmed the neuronal localization of some of these transcripts by using in situ hybridization. To validate the microarray results, we performed real-time reverse transcriptase PCR analyses for a selection of the genes. These studies demonstrate that latent HSV infection can alter neuronal gene expression and might provide a new mechanism for how persistent viral infection can cause chronic disease.
Assuntos
Neurônios Aferentes/fisiologia , Neurônios Aferentes/virologia , Simplexvirus/patogenicidade , Animais , Expressão Gênica , Perfilação da Expressão Gênica , Genes MHC da Classe II , Genes Virais , Herpes Simples/etiologia , Herpes Simples/genética , Herpes Simples/fisiopatologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Simplexvirus/genéticaRESUMO
Murine AIDS (MAIDS) develops in susceptible mouse strains after infection with the LP-BM5 murine leukemia virus (MuLV) complex that contains a mixture of defective (BM5def) and replication-competent viruses. While the BM5def virus is the causative agent in MAIDS, the replication-competent viruses in LP-BM5, including ecotropic MuLV (BM5eco), are required for BM5def propagation and thus function as helper viruses. We describe quantitative real-time RT-PCR assays for RNA encoded by the BM5def and BM5eco components of LP-BM5. The assays were used to standardize better the input doses of LP-BM5 viruses across viral preparations and to quantify BM5def and BM5eco gag RNA levels in spleen and blood cells from MAIDS-susceptible and -insusceptible infected mice. Spleens of MAIDS-susceptible infected mice harbored approximately similar levels of BM5def gag RNA as infected spleens of mice that are insusceptible to MAIDS due to lack of CD40. In contrast, the same infected spleens of CD40-deficient mice contained substantially higher (up to 10-fold) levels of BM5eco gag RNA compared with susceptible controls. Similar to that seen in spleen, infected blood of CD40-deficient mice contained similar levels of BM5def gag as susceptible strains, but increased levels (up to threefold) of BM5eco gag RNA. The assays described below can be used to characterize better the contributions of different functional viral components of the LP-BM5 mixture to the development of MAIDS.
Assuntos
Vírus da Leucemia Murina/genética , RNA Viral/análise , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Virologia/métodos , Animais , Sequência de Bases , Primers do DNA/genética , Expressão Gênica , Genes gag , Vírus Auxiliares/genética , Vírus da Leucemia Murina/patogenicidade , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Síndrome de Imunodeficiência Adquirida Murina/virologia , RNA Viral/sangue , Baço/virologiaRESUMO
C57BL/6 (B6) mice infected with LP-BM5 retroviruses develop disease, including an immunodeficiency similar to AIDS. This disease, murine AIDS (MAIDS), is inhibited by in vivo anti-CD154 monoclonal antibody treatment. The similar levels of insusceptibility of CD40(-/-) and CD154(-/-) B6 mice indicate that CD154/CD40 molecular interactions are required for MAIDS. CD4(+) T and B cells, respectively, provide the CD154 and CD40 expression needed for MAIDS induction. Here, the required CD154/CD40 interaction is shown to be independent of CD80 and CD86 expression: CD80/CD86(-/-) B6 mice develop MAIDS after LP-BM5 infection.