RESUMO
Costimulation by CD28 or lipid-raft-associated CD48 potentiate TCR-induced signals, cytoskeletal reorganization, and IL-2 production. We and others have proposed that costimulators function to construct a raft-based platform(s) especially suited for TCR engagement and sustained and processive signal transduction. Here, we characterize TCR/CD48 and TCR/CD28 costimulation in T cells expressing Lck Src homology 3 (SH3) mutants. We demonstrate that Lck SH3 functions after initiation of TCR-induced tyrosine phosphorylation and concentration of transducers within rafts, to regulate the costimulation-dependent migration of rafts to the TCR contact site. Expression of kinase-active/SH3-impaired Lck mutants disrupts costimulation-dependent raft recruitment, sustained TCR protein tyrosine phosphorylation, and IL-2 production. However, TCR-induced apoptosis, shown only to require "partial" TCR signals, is unaffected by expression of kinase-active/SH3-impaired Lck mutants. Therefore, two distinctly regulated raft reorganization events are required for processive and sustained "complete" TCR signal transduction and T cell activation. Together with recent characterization of CD28 and CD48 costimulatory activities, these findings provide a molecular framework for two signal models of T cell activation.
Assuntos
Proteína Tirosina Quinase p56(lck) Linfócito-Específica/genética , Microdomínios da Membrana/metabolismo , Mutagênese Sítio-Dirigida , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Domínios de Homologia de src/genética , Domínios de Homologia de src/imunologia , Animais , Antígenos CD/fisiologia , Apoptose/genética , Apoptose/imunologia , Antígenos CD28/fisiologia , Antígeno CD48 , Ativação Enzimática/genética , Ativação Enzimática/imunologia , Hibridomas , Interleucina-2/biossíntese , Ligantes , Ativação Linfocitária/genética , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Microdomínios da Membrana/fisiologia , Camundongos , Fosfoproteínas/metabolismo , Fosforilação , Ligação Proteica/genética , Ligação Proteica/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T/fisiologia , Linfócitos T/metabolismo , Domínios de Homologia de src/fisiologiaRESUMO
SIV/HIV-1 (SHIV) chimeric clones, constructed by substituting portions of the pathogenic molecular clone SIVmac239 with counterpart portions from HIV-1 clones, provide a means to analyze functions of selected HIV-1 genes in vivo in nonhuman primates. Our studies focused on SHIVSF33, which contains the vpu, tat, rev, and env genes of the cytopathic, T-cell line tropic clone HIV-1sf33 (subtype-B); this clone has a premature stop codon in the vpu gene. In three juvenile macaques inoculated intravenously with SHIVSF33, low-level persistent infection was established; no disease was observed for a period of >2 years. However, at approximately 16 months p.i., one of four SHIVSF33-infected juvenile macaques exhibited an increase in virus load, depletion of CD4(+) T cells in peripheral blood and lymph nodes, and other symptoms of simian AIDS (SAIDS). Virus recovered from this animal in the symptomatic stage was designated SHIVSF33a (A, adapted); this virus displayed multiple amino acid sequence changes throughout the HIV-1 env gene compared with the input SHIVSF33 clone. Additionally, a mutation in all clones from SHIVSF33a restored the open reading frame for the vpu gene. In vitro evaluations in tissue-culture systems revealed that SHIVSF33a replicated to higher levels and exhibited greater cytopathicity than SHIVSF33. Furthermore cloned env genes for SHIVSF33a were more fusogenic in a cell-fusion assay compared with the env gene of the SHIVSF33. Intravenous inoculation of SHIVsf33a into juvenile and newborn macaques resulted in a rapid decline in CD4(+) T cells to very low levels and development of a fatal AIDS-like disease. A cell-free preparation of this pathogenic chimeric virus also established persistent infection when applied to oral mucosal membranes of juvenile macaques and produced a fatal AIDS-like disease. These studies on pathogenic SHIVSF33a establish the basis for further investigations on the role of the HIV-1 env gene in virus adaptation and in mechanism(s) of immunodeficiency in primates; moreover, the chimeric virus SHIVSF33a can play a role in elucidating mucosal membrane transmission and development of antiviral vaccines in newborns as well as juvenile and adult macaques.