RESUMO
Although several cancer immunotherapy strategies are based on the use of analog peptides and on the modulation of the TCR affinity of adoptively transferred T cells, it remains unclear whether tumor-specific T cell activation by strong and weak TCR stimuli evoke different Ca(2+) signatures from the Ca(2+) intracellular stores and whether the amplitude of Ca(2+) release from the endoplasmic reticulum (ER) can be further modulated by coreceptor binding to peptide/MHC. In this study, we combined functional, structural, and kinetic measurements to correlate the intensity of Ca(2+) signals triggered by the stimulation of the 1G4 T cell clone specific to the tumor epitope NY-ESO-1(157-165). Two analogs of the NY-ESO-1(157-165) peptide, having similar affinity to HLA-A2 molecules, but a 6-fold difference in binding affinity for the 1G4 TCR, resulted in different Ca(2+) signals and T cell activation. 1G4 stimulation by the stronger stimulus emptied the ER of stored Ca(2+), even in the absence of CD8 binding, resulting in sustained Ca(2+) influx. In contrast, the weaker stimulus induced only partial emptying of stored Ca(2+), resulting in significantly diminished and oscillatory Ca(2+) signals, which were enhanced by CD8 binding. Our data define the range of TCR/peptide MHC affinities required to induce depletion of Ca(2+) from intracellular stores and provide insights into the ability of T cells to tailor the use of the CD8 coreceptor to enhance Ca(2+) release from the ER. This, in turn, modulates Ca(2+) influx from the extracellular environment, ultimately controlling T cell activation.
Assuntos
Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Epitopos de Linfócito T/imunologia , Proteínas de Neoplasias/imunologia , Fragmentos de Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Células Clonais , Cristalografia por Raios X , Grânulos Citoplasmáticos/imunologia , Grânulos Citoplasmáticos/metabolismo , Testes Imunológicos de Citotoxicidade , Retículo Endoplasmático/imunologia , Epitopos de Linfócito T/metabolismo , Humanos , Ativação Linfocitária/imunologia , Proteínas de Neoplasias/metabolismo , Fragmentos de Peptídeos/metabolismo , Ligação Proteica/imunologia , Isoformas de Proteínas/imunologia , Isoformas de Proteínas/metabolismo , Receptores de Antígenos de Linfócitos T/fisiologiaRESUMO
HLA-A*6801 exhibits several unusual features. First, it is known to bind weakly to CD8 due to the presence of an A245V substitution in the alpha3 domain. Second, it is able to accommodate unusually long peptides as a result of peptide 'kinking' in the binding groove. Third, CD8+ cytotoxic T lymphocytes that recognise HLA-A*6801-restricted antigens can tolerate substantial changes in the peptide sequence without apparent loss of recognition. In addition, it has been suggested that HLA-A68-restricted TCR might bind with higher affinity than other TCR due to their selection in the presence of a decreased contribution from CD8. Here we (1) examine monoclonal T cell recognition of an HLA-A*6801-restricted HIV-1 Tat-derived 11-amino acid peptide (ITKGLGISYGR) and natural variant sequences thereof; (2) measure the affinity and kinetics of a TCR/pHLA-A68 interaction biophysically for the first time, showing that equilibrium binding occurs within the range previously determined for non-HLA-A68-restricted TCR (KD approx. 7 microM); and (3) show that "normalization" of the non-canonical HLA-A*6801 CD8-binding domain enhances recognition of agonist peptides without inducing non-specific activation. This latter effect may provide a fundamental new mechanism with which to enhance T cell immunity to specific antigens.
Assuntos
Antígenos HIV/imunologia , Infecções por HIV/imunologia , Antígenos HLA-A/imunologia , Receptores de Antígenos de Linfócitos T/biossíntese , Receptores de Antígenos de Linfócitos T/imunologia , Adulto , Antígenos CD8/imunologia , Antígenos HIV/genética , HIV-1/imunologia , Antígenos HLA-A/biossíntese , Humanos , Ativação Linfocitária/imunologia , Masculino , Reação em Cadeia da Polimerase , Ressonância de Plasmônio de Superfície , Linfócitos T Citotóxicos/imunologiaRESUMO
CD8(+) cytotoxic T lymphocytes (CTL) are key determinants of immunity to intracellular pathogens and neoplastic cells. Recognition of specific antigens in the form of peptide-MHC class I complexes (pMHCI) presented on the target cell surface is mediated by T cell receptor (TCR) engagement. The CD8 coreceptor binds to invariant domains of pMHCI and facilitates antigen recognition. Here, we investigate the biological effects of a Q115E substitution in the alpha2 domain of human leukocyte antigen (HLA)-A*0201 that enhances CD8 binding by approximately 50% without altering TCR/pMHCI interactions. Soluble and cell surface-expressed forms of Q115E HLA-A*0201 exhibit enhanced recognition by CTL without loss of specificity. These CD8-enhanced antigens induce greater CD3 zeta chain phosphorylation in cognate CTL leading to substantial increases in cytokine production, proliferation and priming of naive T cells. This effect provides a fundamental new mechanism with which to enhance cellular immunity to specific T cell antigens.
Assuntos
Apresentação de Antígeno/imunologia , Antígenos CD8/metabolismo , Antígenos HLA-A/genética , Antígenos HLA-A/metabolismo , Ativação Linfocitária/imunologia , Linfócitos T Citotóxicos/imunologia , Sequência de Aminoácidos , Apresentação de Antígeno/genética , Antígenos CD8/química , Antígenos CD8/imunologia , Epitopos de Linfócito T/imunologia , Antígenos HLA-A/imunologia , Antígeno HLA-A2 , Humanos , Dados de Sequência Molecular , Mutação , Estrutura Quaternária de Proteína , Receptores de Antígenos de Linfócitos T/imunologia , Ressonância de Plasmônio de Superfície , TransfecçãoRESUMO
The off-rate (k(off)) of the T cell receptor (TCR)/peptide-major histocompatibility complex class I (pMHCI) interaction, and hence its half-life, is the principal kinetic feature that determines the biological outcome of TCR ligation. However, it is unclear whether the CD8 coreceptor, which binds pMHCI at a distinct site, influences this parameter. Although biophysical studies with soluble proteins show that TCR and CD8 do not bind cooperatively to pMHCI, accumulating evidence suggests that TCR associates with CD8 on the T cell surface. Here, we titrated and quantified the contribution of CD8 to TCR/pMHCI dissociation in membrane-constrained interactions using a panel of engineered pMHCI mutants that retain faithful TCR interactions but exhibit a spectrum of affinities for CD8 of >1,000-fold. Data modeling generates a "stabilization factor" that preferentially increases the predicted TCR triggering rate for low affinity pMHCI ligands, thereby suggesting an important role for CD8 in the phenomenon of T cell cross-reactivity.
Assuntos
Membrana Celular/metabolismo , Genes MHC Classe I/genética , Receptores de Antígenos de Linfócitos T/genética , Antígenos/química , Biofísica/métodos , Biotinilação , Antígenos CD8/biossíntese , Antígenos CD8/química , Relação Dose-Resposta a Droga , Epitopos/química , Citometria de Fluxo , Engenharia Genética , HIV-1/metabolismo , Herpesvirus Humano 4/metabolismo , Humanos , Cinética , Ligantes , Modelos Químicos , Mutação , Ligação Proteica , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/metabolismo , Fatores de TempoRESUMO
Cytotoxic T lymphocytes recognize short peptides presented in association with MHC class I (MHCI) molecules on the surface of target cells. The Ag specificity of T lymphocytes is conferred by the TCR, but invariable regions of the peptide-MHCI (pMHCI) molecule also interact with the cell surface glycoprotein CD8. The distinct binding sites for CD8 and the TCR allow pMHCI to be bound simultaneously by both molecules. Even before it was established that the TCR recognized pMHCI, it was shown that CTL exhibit clonal heterogeneity in their ability to activate in the presence of anti-CD8 Abs. These Ab-based studies have since been interpreted in the context of the interaction between pMHCI and CD8 and have recently been extended to show that anti-CD8 Ab can affect the cell surface binding of multimerized pMHCI Ags. In this study, we examine the role of CD8 further using point-mutated pMHCI Ag and show that anti-CD8 Abs can either enhance or inhibit the activation of CTL and the stable cell surface binding of multimerized pMHCI, regardless of whether there is a pMHCI/CD8 interaction. We further demonstrate that multimerized pMHCI Ag can recruit CD8 in the absence of a pMHCI/CD8 interaction and that anti-CD8 Abs can generate an intracellular activation signal resulting in CTL effector function. These results question many previous assumptions as to how anti-CD8 Abs must function and indicate that CD8 has multiple roles in CTL activation that are not necessarily dependent on an interaction with pMHCI.
Assuntos
Adjuvantes Imunológicos/farmacologia , Anticorpos Bloqueadores/farmacologia , Sítios de Ligação de Anticorpos , Antígenos CD8/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Imunossupressores/farmacologia , Ativação Linfocitária/imunologia , Peptídeos/metabolismo , Animais , Ligação Competitiva/imunologia , Antígenos CD8/metabolismo , Linhagem Celular , Linhagem Celular Transformada , Linhagem Celular Tumoral , Membrana Celular/imunologia , Membrana Celular/metabolismo , Células Clonais , Reagentes de Ligações Cruzadas/metabolismo , Citotoxicidade Imunológica/imunologia , Antígenos H-2/genética , Antígenos H-2/metabolismo , Antígeno HLA-A2/metabolismo , Humanos , Camundongos , Receptores de Antígenos de Linfócitos T/antagonistas & inibidores , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismoRESUMO
T lymphocytes recognize peptides presented in the context of major histocompatibility complex (MHC) molecules on the surface of antigen presenting cells. Recognition specificity is determined by the alphabeta T cell receptor (TCR). The T lymphocyte surface glycoproteins CD8 and CD4 enhance T cell antigen recognition by binding to MHC class I and class II molecules, respectively. Biophysical measurements have determined that equilibrium binding of the TCR with natural agonist peptide-MHC (pMHC) complexes occurs with KD values of 1-50 microm. The pMHCI/CD8 and pMHCII/CD4 interactions are significantly weaker than this (KD >100 microm), and the relative roles of TCR/pMHC and pMHC/coreceptor affinity in T cell activation remain controversial. Here, we engineer mutations in the MHCI heavy chain and beta2-microglobulin that further reduce or abolish the pMHCI/CD8 interaction to probe the significance of pMHC/coreceptor affinity in T cell activation. We demonstrate that the pMHCI/CD8 coreceptor interaction retains the vast majority of its biological activity at affinities that are reduced by over 15-fold (KD > 2 mm). In contrast to previous reports, we observe that the weak interaction between HLA A68 and CD8, which falls within this spectrum of reduced affinities, retains substantial functional activity. These findings are discussed in the context of current concepts of coreceptor dependence and the mechanism by which TCR coreceptors facilitate T cell activation.
Assuntos
Antígenos CD8/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Apresentação de Antígeno/genética , Antígenos CD8/genética , Antígenos CD8/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Ativação Linfocitária/genéticaRESUMO
The CD8 coreceptor of cytotoxic T lymphocytes binds to a conserved region of major histocompatibility complex class I molecules during recognition of peptide-major histocompatibility complex (MHC) class I antigens on the surface of target cells. This event is central to the activation of cytotoxic T lymphocyte (CTL) effector functions. The contribution of the MHC complex class I light chain, beta(2)-microglobulin, to CD8alphaalpha binding is relatively small and is mediated mainly through the lysine residue at position 58. Despite this, using molecular modeling, we predict that its mutation should have a dramatic effect on CD8alphaalpha binding. The predictions are confirmed using surface plasmon resonance binding studies and human CTL activation assays. Surprisingly, the charge-reversing mutation, Lys(58) --> Glu, enhances beta(2)m-MHC class I heavy chain interactions. This mutation also significantly reduces CD8alphaalpha binding and is a potent antagonist of CTL activation. These results suggest a novel approach to CTL-specific therapeutic immunosuppression.