RESUMO
Vesicular stomatitis virus (VSV) is an oncolytic virus currently being investigated as a promising tool to treat cancer because of its ability to selectively replicate in cancer cells. To enhance the oncolytic property of the nonpathologic laboratory strain of VSV, we generated a recombinant vector [rVSV(MΔ51)-M3] expressing murine gammaherpesvirus M3, a secreted viral chemokine-binding protein that binds to a broad range of mammalian chemokines with high affinity. As previously reported, when rVSV(MΔ51)-M3 was used in an orthotopic model of hepatocellular carcinoma (HCC) in rats, it suppressed inflammatory cell migration to the virus-infected tumor site, which allowed for enhanced intratumoral virus replication leading to increased tumor necrosis and substantially prolonged survival. These encouraging results led to the development of this vector for clinical translation in patients with HCC. However, a scalable current Good Manufacturing Practice (cGMP)-compliant manufacturing process has not been described for this vector. To produce the quantities of high-titer virus required for clinical trials, a process that is amenable to GMP manufacturing and scale-up was developed. We describe here a large-scale (50-liter) vector production process capable of achieving crude titers on the order of 10(9) plaque-forming units (PFU)/ml under cGMP. This process was used to generate a master virus seed stock and a clinical lot of the clinical trial agent under cGMP with an infectious viral titer of approximately 2 × 10(10) PFU/ml (total yield, 1 × 10(13) PFU). The lot has passed all U.S. Food and Drug Administration-mandated release testing and will be used in a phase 1 clinical translational trial in patients with advanced HCC.
Assuntos
Biotecnologia/métodos , Vetores Genéticos , Neoplasias/terapia , Terapia Viral Oncolítica , Vírus da Estomatite Vesicular Indiana , Animais , Técnicas de Cultura de Células , Linhagem Celular , Cromatografia por Troca Iônica , Cricetinae , Vetores Genéticos/genética , Vetores Genéticos/ultraestrutura , Células HEK293 , Humanos , Ultrafiltração , Vírus da Estomatite Vesicular Indiana/genética , Vírus da Estomatite Vesicular Indiana/ultraestruturaRESUMO
Costimulation of T cell activation involves both the B7:CD28 as well as the CD40 ligand (CD40L):CD40 pathway. To determine the importance of these pathways to in vitro and in vivo T cell activation, a direct comparison was made of the responses of TCR transgenic T cells lacking either CD28 or CD40L. In vitro, CD28-/- T cells showed a greater reduction in proliferative responses to Ag than did CD40L-/- T cells. The absence of CD28 resulted in defective Th2 responses, whereas CD40L-/- T cells were defective in Th1 development. In vivo, CD28-/- T cells failed to expand upon immunization, whereas CD40L-/- T cells could not sustain a response. These results suggest that CD28 is critical for initiating T cell responses, whereas CD40L is required for sustained Th1 responses. The different functional roles of these costimulatory pathways may explain why blocking B7:CD28 and CD40L:CD40 interactions has an additive effect in inhibiting T cell responses.
Assuntos
Antígenos CD28/fisiologia , Antígenos CD40/metabolismo , Tolerância Imunológica , Ativação Linfocitária , Glicoproteínas de Membrana/fisiologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Transferência Adotiva , Animais , Antígenos CD28/genética , Ligante de CD40 , Diferenciação Celular/imunologia , Células Cultivadas , Tolerância Imunológica/genética , Ligantes , Ativação Linfocitária/genética , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos Transgênicos , Linfócitos T/citologia , Linfócitos T/transplante , Células Th1/citologia , Células Th1/imunologia , Células Th2/citologia , Células Th2/imunologiaRESUMO
We have determined that certain altered peptide ligands (APLs) can induce T-cells specific for the native peptide myelin basic protein (MBP) p85-99 to secrete Th2-type cytokines such as IL-4 and IL-5 in the absence of significant Th1-type cytokines. However, it is not known whether stimulation with APLs will activate autoreactive T cells or a distinct population of cells. In the present study, 18 T-cell clones that reacted with either MBP p85-99 or one of three APLs of the peptide substituted at TCR contact residues were generated. T-cells were tested functionally for their reactivity to the original stimulating peptide as well as to the MBP APLs. In addition, the T-cell receptor (TCR) alpha and beta chains of each of these clones were sequenced. In a series of T-cell clones isolated from a multiple sclerosis patient, stimulation of T-cells with the APL 93A, which has an alanine for lysine substitution at the TCR contact residue 93, did not induce substantial proliferation of MBPp85-99-specific T-cell clones, indicating that a distinct set of T-cell clones was induced. However, this was not the case for another set of T-cell clones from a different individual in which the 93A peptide induced clonal expansion of T-cells highly reactive with the native MBPp85-99 antigen. Thus, the potential beneficial effect of using APLs to induce downregulatory cytokines appears to depend on the specific T-cell repertoire of the individual patient.
Assuntos
Proteína Básica da Mielina/imunologia , Fragmentos de Peptídeos/imunologia , Linfócitos T/imunologia , Sequência de Aminoácidos , Linhagem Celular , Reações Cruzadas , Humanos , Dados de Sequência Molecular , Receptores de Antígenos de Linfócitos T alfa-beta/genéticaRESUMO
Myelin basic protein (MBP)-specific T cells were isolated directly from peripheral blood by stimulation either with native MBPp85-99 or altered peptide ligands (APLs) in which substitutions of the lysine were made at position 93, a TCR contact residue. We report here that the APL 93A could alter the cytokine profile of some autoreactive MBPp85-99 reactive T cell clones, switching them from a Th0 phenotype secreting high concentrations of IL-4, IL-5, and IFN-gamma into Th2 cells secreting significantly less IFN-gamma. However, in vitro stimulation with the 93A peptide, in some instances, induced T cells that responded better to the native MBPp85-99 peptide. Functionally, the APL 93A was shown to act as an antagonist for IFN-gamma secretion. Based on TCR sequencing and single cell cloning, this alteration in cytokine profile was determined to be the result of the differential activation of individual T cell clones. We discuss the implications of these data for the strength of signal model of T cell activation.
Assuntos
Doenças Autoimunes/imunologia , Epitopos Imunodominantes/imunologia , Interferon gama/metabolismo , Interleucina-4/metabolismo , Interleucina-5/metabolismo , Esclerose Múltipla/imunologia , Proteína Básica da Mielina/imunologia , Fragmentos de Peptídeos/imunologia , Subpopulações de Linfócitos T/imunologia , Células Th2/metabolismo , Doenças Autoimunes/sangue , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Células Clonais , Rearranjo Gênico do Linfócito T , Humanos , Epitopos Imunodominantes/química , Ativação Linfocitária , Esclerose Múltipla/sangue , Proteína Básica da Mielina/química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Relação Estrutura-Atividade , Subpopulações de Linfócitos T/efeitos dos fármacos , Células Th1/metabolismoRESUMO
The influence of costimulation on the T cell response to altered peptide ligands that act as either partial or weak agonists for human CD4+ T cell clones was examined. Using stable Chinese hamster ovary (CHO) cell transfectants expressing DR2 (DRB1*1501) and human B7-1 or B7-2 as APC, presentation of native myelin basic protein (MBP) p85-99 peptide Ag or a partial agonist of MBP p85-99 induced equivalent T cell activation as measured by [3H]TdR incorporation and cytokine secretion. In marked contrast, presentation of cross-reactive peptides of MBP p85-99 that act as weak agonists with B7-1, but not B7-2, costimulation resulted in significant T cell activation as measured by [3H]TdR incorporation and cytokine secretion. These data suggest that decreasing the strength of the signal provided to the TCR allows differences in B7-1 and B7-2 signaling to be observed. Thus, the costimulatory environment during T cell activation may be a mechanism of regulating T cell cross-reactivity in the periphery.
Assuntos
Antígenos CD/fisiologia , Antígeno B7-1/fisiologia , Ativação Linfocitária , Glicoproteínas de Membrana/fisiologia , Linfócitos T/imunologia , Sequência de Aminoácidos , Animais , Doenças Autoimunes/etiologia , Antígeno B7-2 , Células CHO , Células Clonais , Cricetinae , Citocinas/metabolismo , Humanos , Dados de Sequência Molecular , Proteína Básica da Mielina/imunologia , Fragmentos de Peptídeos/imunologiaRESUMO
The frequency of clonally expanded and persistent T cells recognizing the immunodominant autoantigenic peptide of myelin basic protein (MBP)p85-99 was directly measured ex vivo in subjects with typical relapsing remitting multiple sclerosis (MS). T cells expressing mRNA transcripts encoding T cell receptor (TCR)-alpha and -beta chains found in T cell clones previously isolated from these subjects recognizing the MBPp85-99 epitope were examined. In contrast to frequencies of 1 in 10(5)-10(6) as measured by limiting dilution analysis, estimates of the T cell frequencies expressing MBPp85-99-associated TCR chain transcripts were as high as 1 in 300. These high frequencies were confirmed by performing PCR on single T cells isolated by flow cytometry. MBPp85-99 TCR transcripts were present in IL-2 receptor alpha-positive T cells which were induced to undergo Fas-mediated cell death upon antigen stimulation. These data demonstrate that at least a subpopulation of patients with MS can have a very high frequency of activated autoreactive T cells.
Assuntos
Doenças Autoimunes/imunologia , Esclerose Múltipla/imunologia , Linfócitos T/imunologia , Receptor fas/fisiologia , Autoantígenos/imunologia , Morte Celular , Células Clonais , Rearranjo Gênico da Cadeia alfa dos Receptores de Antígenos dos Linfócitos T , Rearranjo Gênico da Cadeia beta dos Receptores de Antígenos dos Linfócitos T , Humanos , Proteína Básica da Mielina/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/genéticaRESUMO
T cells recognize antigen by formation of a trimolecular complex in which the T-cell receptor (TCR) recognizes a specific peptide antigen within the groove of a major histocompatibility complex (MHC) molecule. It has generally been assumed that T-cell recognition of two distinct MHC-antigen complexes is due to similarities in the three-dimensional structure of the complexes. Here we report results of experiments examining the crossreactivity of TCRs recognizing the myelin basic protein peptide MBPp85-99 and several of its analogs in the context of MHC. We demonstrate that single conservative amino acid substitutions of the antigenic peptide at the predominant TCR contact residues at positions 91 and 93 totally abrogate reactivity of specific T-cell clones. Yet, when a conservative substitution is made at position 91 concomitant with a substitution at position 93, the T-cell clones regain reactivity equivalent with that of the original stimulating peptide. Thus, the exact nature of the amino acid side chains engaging one TCR functional pocket may change the apparent selectivity of the other predominant TCR functional pocket, thus suggesting a remarkable degree of receptor plasticity. This ability of the TCR-MHC-peptide complex to undergo conformational changes provides a conceptual framework for reconciling the apparent paradox of the extreme selectivity of the TCR and its remarkable crossreactivity with different MHC-peptide complexes.
Assuntos
Linfócitos B/imunologia , Ativação Linfocitária , Complexo Principal de Histocompatibilidade , Oligopeptídeos/química , Linfócitos T/imunologia , Sequência de Aminoácidos , Células Clonais , Reações Cruzadas , Antígenos HLA-DR/química , Antígenos HLA-DR/imunologia , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteína Básica da Mielina/imunologia , Oligopeptídeos/síntese química , Oligopeptídeos/farmacologia , Receptores de Antígenos de Linfócitos T/imunologia , Relação Estrutura-AtividadeRESUMO
T cell proliferation is potently suppressed by TGF-beta during the G1 phase of the cell cycle. The mechanism appears to involve inhibition of cell cycle kinases that phosphorylate the retinoblastoma protein (pRb), a key regulator of cell cycle progression from G1 to S phase. Although productive infection with the human T cell lymphotropic virus type I (HTLV-I) induces T cell activation, it also, paradoxically, leads to increased production of TGF-beta. To investigate whether infection by HTLV-I conferred resistance to TGF-beta in non-immortalized T cells, we generated T cell clones from patients with HTLV-I myelopathy by direct single cell cloning. Here we report that HTLV-I-infected but not uninfected T cell clones have hyperphosphorylated pRb consistent with viral-induced T cell activation. Furthermore, the HTLV-I-infected T cells were resistant to growth suppression by rTGF-beta 1 and this correlated with the inability of TGF-beta 1 to prevent hyperphosphorylation of pRb. However, when spontaneously proliferating HTLV-I-infected T cell clones were further stimulated by cross-linking of the CD3/TCR complex, the superimposed proliferation was significantly inhibited by TGF-beta 1, suggesting that the TGF-beta 1 signaling pathway was intact. Together these findings suggest that HTLV-I induces T cell activation through a pathway that is insensitive to TGF-beta 1. This may have implications for the altered immune regulation in patients with HTLV-I myelopathy.
Assuntos
Vírus Linfotrópico T Tipo 1 Humano/imunologia , Ativação Linfocitária/efeitos dos fármacos , Linfócitos T/imunologia , Fator de Crescimento Transformador beta/farmacologia , Sequência de Bases , Humanos , Dados de Sequência Molecular , Fosforilação , RNA Mensageiro/análise , RNA Viral/análise , Proteína do Retinoblastoma/metabolismo , Fase S/efeitos dos fármacosRESUMO
Mononuclear cells from subjects infected with human T lymphotrophic virus type I (HTLV-I) display a unique ability to proliferate in vitro in the absence of mitogens or exogenous growth factors. Subjects who have developed an HTLV-I-associated myelopathy (HAM) show an even higher degree of spontaneous proliferation concomitant with transcription of the HTLV-I provirus. The mechanism underlying HTLV-I-induced T cell activation was investigated by characterizing a series of HTLV-I-infected T cell clones generated from the blood of subjects with HAM. Approximately 15% of the T cell clones generated were HTLV-I infected as determined by polymerase chain reaction and Southern blotting. Infected T cell clones displayed altered growth kinetics as they continued to incorporate tritiated thymidine 7 to 14 days after stimulation, a time when noninfected T cell clones had returned to a resting state. This was not due to transformation as all the T cell clones required periodic restimulation with mitogens and feeder cells for continued growth. Although HTLV-I-infected T cell clones showed increased expression of the IL-2 receptor p55 chain, the spontaneous clonal proliferation was not inhibited by anti-IL-2 receptor mAb. Moreover, the spontaneous clonal proliferation was insensitive to cyclosporin A and FK 506 while being highly sensitive to rapamycin, which is known to inhibit IL-2-mediated signaling. Together these results demonstrate that IL-2 is not required for the HTLV-I-induced spontaneous clonal proliferation and further suggest that HTLV-I may induce signaling pathways replacing an IL-2 receptor signal proximal to the site of action of rapamycin.