RESUMEN
Chimeric antigen receptor (CAR)-T-cell therapeutic efficacy is associated with long-term T-cell persistence and acquisition of memory. Memory-subset formation requires T-cell factor 1 (TCF-1), a master transcription factor for which few regulators have been identified. Here, we demonstrate using an immune-competent mouse model of B-cell acute lymphoblastic leukemia (ALL; B-ALL) that Regnase-1 deficiency promotes TCF-1 expression to enhance CAR-T-cell expansion and memory-like cell formation. This leads to improved CAR-T-mediated tumor clearance, sustained remissions, and protection against secondary tumor challenge. Phenotypic, transcriptional, and epigenetic profiling identified increased tumor-dependent programming of Regnase-1-deficient CAR-T cells into TCF-1+ precursor exhausted T cells (TPEX) characterized by upregulation of both memory and exhaustion markers. Regnase-1 directly targets Tcf7 messenger RNA (mRNA); its deficiency augments TCF-1 expression leading to the formation of TPEX that support long-term CAR-T-cell persistence and function. Regnase-1 deficiency also reduces exhaustion and enhances the activity of TCF-1- CAR-T cells. We further validate these findings in human CAR-T cells, where Regnase-1 deficiency mediates enhanced tumor clearance in a xenograft B-ALL model. This is associated with increased persistence and expansion of a TCF-1+ CAR-T-cell population. Our findings demonstrate the pivotal roles of TPEX, Regnase-1, and TCF-1 in mediating CAR-T-cell persistence and recall responses, and identify Regnase-1 as a modulator of human CAR-T-cell longevity and potency that may be manipulated for improved therapeutic efficacy.
Asunto(s)
Inmunoterapia Adoptiva , Leucemia-Linfoma Linfoblástico de Células Precursoras/inmunología , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Ribonucleasas/metabolismo , Factor 1 de Transcripción de Linfocitos T/metabolismo , Linfocitos T/inmunología , Animales , Antígenos CD19/metabolismo , Línea Celular Tumoral , Reprogramación Celular , Modelos Animales de Enfermedad , Epigénesis Genética , Humanos , Inmunocompetencia/inmunología , Memoria Inmunológica , Ratones Endogámicos C57BL , Ratones Transgénicos , Fenotipo , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologíaRESUMEN
The dimeric cytokine IL-12 is important in the control of various infections but also contributes to the pathology of certain diseases making it a potential target for therapy. However, its specific inhibition with antibodies is complicated by the fact that its two subunits are present in other cytokines: p40 in IL-23 and p35 in IL-35. This has led to erroneous conclusions like the alleged implication of IL-12 in experimental autoimmune encephalomyelitis (EAE). Here, we report the development of a mouse anti-mouse IL-12 vaccine and the production of monoclonal antibodies (mAbs) that do not react with p40 or p35 (in IL-35) but specifically recognize and functionally inhibit the IL-12 heterodimer. Using one of these mAbs, MM12A1.6, that strongly inhibited IFN-γ production and LPS-induced septic shock after viral infection, we demonstrate the critical role played by IL-12 in the rejection of male skin graft by female C57BL/6 syngeneic recipients and in the clearance of an immunogenic mastocytoma tumor variant by DBA/2 mice, but not in a parent to F1 immune aggression model nor in MOG-induced EAE, which was clearly prevented by anti-p40 mAb C17.8. Given this selective inhibition of IL-12, these mAbs provide new options for reassessing IL-12 function in vivo.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Encefalomielitis Autoinmune Experimental/inmunología , Rechazo de Injerto/inmunología , Interleucina-12/metabolismo , Mastocitoma/inmunología , Esclerosis Múltiple/inmunología , Infecciones por Nidovirales/inmunología , Nidovirales/fisiología , Subunidades de Proteína/metabolismo , Sepsis/inmunología , Trasplante de Piel , Animales , Anticuerpos Monoclonales/aislamiento & purificación , Modelos Animales de Enfermedad , Epítopos , Humanos , Hibridomas , Interleucina-12/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Neoplasias Experimentales , Subunidades de Proteína/inmunologíaRESUMEN
IL-6 is a critical driver of acute and chronic inflammation and has been reported to act as a T cell survival factor. The influence of IL-6 on T cell homeostasis is not well resolved. We demonstrate that IL-6 signaling drives T cell expansion under inflammatory conditions but not during normal homeostasis. During inflammation, IL-6Rα-deficient T cells are unable to effectively compete with wild type T cells. IL-6 promotes T cell proliferation, and this is associated with low-level expression of the RORγt transcription factor. T cells upregulate Rorc mRNA at levels substantially diminished from that seen in Th17 cells. Blockade of RORγt through genetic knockout or a small molecule inhibitor leads to T cell expansion defects comparable to those in IL-6Rα-deficient T cells. Our results indicate that IL-6 plays a key role in T cell expansion during inflammation and implicates a role for the transient induction of low-level RORγt.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Diferenciación Celular/inmunología , Interleucina-6/inmunología , Activación de Linfocitos/inmunología , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/inmunología , Animales , Linfocitos T CD4-Positivos/citología , Proliferación Celular/fisiología , Regulación de la Expresión Génica/inmunología , Homeostasis/inmunología , Inflamación/inmunología , Ratones , Ratones Noqueados , Linfocitos T/citología , Linfocitos T/inmunología , Células Th17/citología , Células Th17/inmunologíaRESUMEN
In vivo persistence of chimeric antigen receptor (CAR)-modified T cells correlates with therapeutic efficacy, yet CAR-specific factors that support persistence are not well resolved. Using a CD33-specific CAR in an acute myeloid leukemia (AML) model, we show how CAR expression alters T cell differentiation in a ligand independent manner. Ex vivo expanded CAR-T cells demonstrated decreased naïve and stem memory populations and increased effector subsets relative to vector-transduced control cells. This was associated with reduced in vivo persistence. Decreased persistence was not due to specificity or tumor presence, but to pre-transfer tonic signaling through the CAR CD3ζ ITAMs. We identified activation of the PI3K pathway in CD33 CAR-T cells as responsible. Treatment with a PI3K inhibitor modulated the differentiation program of CAR-T cells, preserved a less differentiated state without affecting T cell expansion, and improved in vivo persistence and reduced tumor burden. These results resolve mechanisms by which tonic signaling of CAR-T cells modulates their fate, and identifies a novel pharmacologic approach to enhance the durability of CAR-T cells for immunotherapy.
Asunto(s)
Inmunoterapia Adoptiva/métodos , Leucemia Mieloide Aguda/terapia , Fosfatidilinositol 3-Quinasas/metabolismo , Receptores Quiméricos de Antígenos/uso terapéutico , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Humanos , Activación de Linfocitos/efectos de los fármacos , Inhibidores de las Quinasa Fosfoinosítidos-3 , Lectina 3 Similar a Ig de Unión al Ácido Siálico/farmacología , Lectina 3 Similar a Ig de Unión al Ácido Siálico/uso terapéutico , Linfocitos T , Carga Tumoral/efectos de los fármacosRESUMEN
Although the TCR repertoire is highly diverse, a small fraction of TCR chains, referred to as public, preferentially form and are shared by most individuals. Prior studies indicated that public TCRß may be preferentially deployed in autoimmunity. We hypothesized that if these TCRß modulate the likelihood of a TCRαß heterodimer productively engaging autoantigen, because they are widely present in the population and often high frequency within individual repertoires, they could also broadly influence repertoire responsiveness to specific autoantigens. We assess this here using a series of public and private TCRß derived from autoimmune encephalomyelitis-associated TCR. Transgenic expression of public, but not private, disease-associated TCRß paired with endogenously rearranged TCRα endowed unprimed T cells with autoantigen reactivity. Further, two of six public, but none of five private TCRß provoked spontaneous early-onset autoimmunity in mice. Our findings indicate that single TCRß are sufficient to confer on TCRαß chains reactivity toward disease-associated autoantigens in the context of diverse TCRα. They further suggest that public TCR can skew autoimmune susceptibility, and that subsets of public TCR sequences may serve as disease- specific biomarkers or therapeutic targets.
Asunto(s)
Enfermedades Autoinmunes/inmunología , Susceptibilidad a Enfermedades , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Secuencia de Aminoácidos , Animales , Enfermedades Autoinmunes/patología , Autoinmunidad , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/patología , Ratones Endogámicos C57BL , Vaina de Mielina/metabolismo , Glicoproteína Mielina-Oligodendrócito/inmunología , Multimerización de Proteína , Receptores de Antígenos de Linfocitos T alfa-beta/química , Linfocitos T/inmunologíaRESUMEN
How the TCR repertoire, in concert with risk-associated MHC, imposes susceptibility for autoimmune diseases is incompletely resolved. Due largely to recombinatorial biases, a small fraction of TCRα or ß-chains are shared by most individuals, or public. If public TCR chains modulate a TCRαß heterodimer's likelihood of productively engaging autoantigen, because they are pervasive and often high frequency, they could also broadly influence disease risk and progression. Prior data, using low-resolution techniques, have identified the heavy use of select public TCR in some autoimmune models. In this study, we assess public repertoire representation in mice with experimental autoimmune encephalomyelitis at high resolution. Saturation sequencing was used to identify >18 × 10(6) TCRß sequences from the CNSs, periphery, and thymi of mice at different stages of autoimmune encephalomyelitis and healthy controls. Analyses indicated the prominent representation of a highly diverse public TCRß repertoire in the disease response. Preferential formation of public TCR implicated in autoimmunity was identified in preselection thymocytes, and, consistently, public, disease-associated TCRß were observed to be commonly oligoclonal. Increased TCR sharing and a focusing of the public TCR response was seen with disease progression. Critically, comparisons of peripheral and CNS repertoires and repertoires from preimmune and diseased mice demonstrated that public TCR were preferentially deployed relative to nonshared, or private, sequences. Our findings implicate public TCR in skewing repertoire response during autoimmunity and suggest that subsets of public TCR sequences may serve as disease-specific biomarkers or influence disease susceptibility or progression.
Asunto(s)
Encefalomielitis Autoinmune Experimental/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Timo/inmunología , Secuencia de Aminoácidos , Animales , Linfocitos T CD8-positivos , Sistema Nervioso Central/citología , Sistema Nervioso Central/inmunología , Femenino , Ratones , Ratones Endogámicos C57BL , Vaina de Mielina/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Timocitos/inmunología , Timo/ultraestructuraRESUMEN
How a large number of cytokines differentially signal through a small number of signal transduction pathways is not well resolved. This is particularly true for IL-6 and IL-10, which act primarily through STAT3 yet induce dissimilar transcriptional programs leading alternatively to pro- and anti-inflammatory effects. Kinetic differences in signaling, sustained to IL-10 and transient to IL-6, are critical to this in macrophages. T cells are also key targets of IL-6 and IL-10, yet how differential signaling in these cells leads to divergent cellular fates is unclear. We show that, unlike for macrophages, signal duration cannot explain the distinct effects of these cytokines in T cells. Rather, naive, activated, activated-rested, and memory CD4(+) T cells differentially express IL-6 and IL-10 receptors in an activation state-dependent manner, and this impacts downstream cytokine effects. We show a dominant role for STAT3 in IL-6-mediated Th17 subset maturation. IL-10 cannot support Th17 differentiation because of insufficient cytokine receptivity rather than signal quality. Enforced expression of IL-10Rα on naive T cells permits an IL-10-generated STAT3 signal equivalent to that of IL-6 and equally capable of promoting Th17 formation. Similarly, naive T cell IL-10Rα expression also allows IL-10 to mimic the effects of IL-6 on both Th1/Th2 skewing and Tfh cell differentiation. Our results demonstrate a key role for the regulation of receptor expression rather than signal quality or duration in differentiating the functional outcomes of IL-6 and IL-10 signaling, and identify distinct signaling properties of these cytokines in T cells compared with myeloid cells.
Asunto(s)
Diferenciación Celular , Interleucina-10/metabolismo , Interleucina-6/metabolismo , Transducción de Señal , Células Th17/citología , Células Th17/metabolismo , Animales , Expresión Génica , Inmunofenotipificación , Interleucina-10/farmacología , Subunidad alfa del Receptor de Interleucina-10/genética , Subunidad alfa del Receptor de Interleucina-10/metabolismo , Interleucina-6/farmacología , Subunidad alfa del Receptor de Interleucina-6/genética , Subunidad alfa del Receptor de Interleucina-6/metabolismo , Ratones , Ratones Transgénicos , Fenotipo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacos , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Células TH1/citología , Células TH1/efectos de los fármacos , Células TH1/inmunología , Células TH1/metabolismo , Células Th17/efectos de los fármacos , Células Th17/inmunología , Células Th2/citología , Células Th2/efectos de los fármacos , Células Th2/inmunología , Células Th2/metabolismoRESUMEN
The heterodimeric IL-12 cytokine family is characterized by the sharing of three α (p19, p28, p35) and two ß (p40 and Ebi3) subunits, and includes IL-12 (p35/p40), IL-23 (p19/p40), IL-27 (p28/Ebi3) and IL-35 (p35/Ebi3). In this study, the dimerization interfaces of IL-12 family members were characterized, with emphasis on IL-35. Ebi3 and p35 subunits from human and mouse paired effectively with each other, indicating there is no species barrier to IL-35 dimerization and suggesting a conserved dimerization interface. Specific p35 residues that contribute to formation of the IL-12 interface were assessed for their contribution to the IL-35 interface, and candidate Ebi3 residues were screened for their contribution to both IL-27 and IL-35 interfaces. Several residues were identified as critical to the IL-12 or IL-27 interfaces. Conversely, no single mutation was identified that completely disrupts p35/Ebi3 pairing. Linear alanine scanning mutagenesis on both p35 and Ebi3 subunits was performed, focusing on residues that are conserved between the mouse and human proteins. Additionally, a structure-based alanine-scanning approach in which mutations were clustered based on proximitiy was performed on the p35 subunit. Both approaches suggest that IL-35 has distinct criteria for subunit pairing and is remarkabley less sensitive to structural perturbation than IL-12 and IL-27. Additionally, studies using a panel of anti-p35 and anti-Ebi3 antibodies indicate differential availability of epitopes within IL-12 family members that share these subunits, suggesting that IL-35 has distinct structural features, relative to IL-12 and IL-27. These results may be useful in future directed therapeutic targeting of IL-12 family members.
Asunto(s)
Interleucina-12/química , Interleucina-12/metabolismo , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia Conservada , Dimerización , Ensayo de Inmunoadsorción Enzimática , Células HEK293 , Humanos , Immunoblotting , Inmunoprecipitación , Subunidad p35 de la Interleucina-12/química , Subunidad p35 de la Interleucina-12/metabolismo , Interleucinas/química , Interleucinas/metabolismo , Ratones , Ratones Endogámicos C57BL , Antígenos de Histocompatibilidad Menor , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Receptores de Citocinas/química , Receptores de Citocinas/metabolismo , Alineación de Secuencia , Especificidad de la EspecieRESUMEN
Production of cytokines by immune cells in response to stimuli and the binding of cytokines to specific receptors on target cells is a central feature of the immune response. The IL-12 cytokine family is particularly influential in determining the fate of T cells and is characterized by the sharing of cytokine and receptor subunits. A thorough understanding of the molecular interactions within this family will be a key to the development of therapeutic inhibitors or enhancers of IL-12 family function. While the current structural and molecular data for IL-12 family members is limited, there is ample information on the structurally related IL-6 cytokine family. This review will summarize the current structural and mutagenesis data within the IL-12 family and will attempt to utilize similarities between the IL-6 and IL-12 families to understand molecular interactions between IL-12 family subunits and with receptor components.
Asunto(s)
Receptores de Interleucina-12 , Receptores de Interleucina-6 , Linfocitos T , Animales , Humanos , Mutagénesis , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Receptores de Interleucina-12/química , Receptores de Interleucina-12/genética , Receptores de Interleucina-12/inmunología , Receptores de Interleucina-12/metabolismo , Receptores de Interleucina-6/química , Receptores de Interleucina-6/genética , Receptores de Interleucina-6/inmunología , Receptores de Interleucina-6/metabolismo , Relación Estructura-Actividad , Linfocitos T/química , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
To understand the mechanisms that govern T cell receptor (TCR)-peptide MHC (pMHC) binding and the role that different regions of the TCR play in affinity and antigen specificity, we have studied the TCR from T cell clone 2C. High-affinity mutants of the 2C TCR that bind QL9-L(d) as a strong agonist were generated previously by site-directed mutagenesis of complementarity determining regions (CDRs) 1beta, 2alpha, 3alpha, or 3beta. We performed isothermal titration calorimetry to assess whether they use similar thermodynamic mechanisms to achieve high affinity for QL9-L(d). Four of the five TCRs examined bound to QL9-L(d) in an enthalpically driven, entropically unfavorable manner. In contrast, the high-affinity CDR1beta mutant resembled the wild-type 2C TCR interaction, with favorable entropy. To assess fine specificity, we measured the binding and kinetics of these mutants for both QL9-L(d) and a single amino acid peptide variant of QL9, called QL9-Y5-L(d). While 2C and most of the mutants had equal or higher affinity for the Y5 variant than for QL9, mutant CDR1beta exhibited 8-fold lower affinity for Y5 compared to QL9. To examine possible structural correlates of the thermodynamic and fine specificity signatures of the TCRs, the structure of unliganded QL9-L(d) was solved and compared to structures of the 2C TCR/QL9-L(d) complex and three high-affinity TCR/QL9-L(d) complexes. Our findings show that the QL9-L(d) complex does not undergo major conformational changes upon binding. Thus, subtle changes in individual CDRs account for the diverse thermodynamic and kinetic binding mechanisms and for the different peptide fine specificities.
Asunto(s)
Oligopéptidos/metabolismo , Receptores de Antígenos de Linfocitos T/química , Receptores de Antígenos de Linfocitos T/metabolismo , Secuencia de Aminoácidos , Animales , Cricetinae , Cinética , Ligandos , Ratones , Modelos Moleculares , Mutación , Oligopéptidos/química , Unión Proteica , Conformación Proteica , Ratas , Receptores de Antígenos de Linfocitos T/genética , Especificidad por Sustrato , Termodinámica , TransfecciónRESUMEN
T cells are known to cross-react with diverse peptide MHC Ags through their alphabeta TCR. To explore the basis of such cross-reactivity, we examined the 2C TCR that recognizes two structurally distinct ligands, SIY-K(b) and alloantigen QL9-L(d). In this study we characterized the cross-reactivity of several high-affinity 2C TCR variants that contained mutations only in the CDR3alpha loop. Two of the TCR lost their ability to cross-react with the reciprocal ligand (SIY-K(b)), whereas another TCR (m67) maintained reactivity with both ligands. Crystal structures of four of the TCRs in complex with QL9-L(d) showed that CDR1, CDR2, and CDR3beta conformations and docking orientations were remarkably similar. Although the CDR3alpha loop of TCR m67 conferred a 2000-fold higher affinity for SIY-K(b), the TCR maintained the same docking angle on QL9-L(d) as the 2C TCR. Thus, CDR3alpha dictated the affinity and level of cross-reactivity, yet it did so without affecting the conserved docking orientation.
Asunto(s)
Regiones Determinantes de Complementariedad/química , Antígenos H-2/metabolismo , Oligopéptidos/metabolismo , Receptores de Antígenos de Linfocitos T alfa-beta/química , Secuencia de Aminoácidos , Animales , Regiones Determinantes de Complementariedad/genética , Regiones Determinantes de Complementariedad/metabolismo , Secuencia Conservada , Reacciones Cruzadas/genética , Reacciones Cruzadas/inmunología , Antígenos H-2/química , Antígenos H-2/genética , Antígeno de Histocompatibilidad H-2D , Complejo Cetoglutarato Deshidrogenasa/genética , Complejo Cetoglutarato Deshidrogenasa/inmunología , Complejo Cetoglutarato Deshidrogenasa/metabolismo , Ratones , Datos de Secuencia Molecular , Oligopéptidos/química , Oligopéptidos/genética , Oligopéptidos/inmunología , Unión Proteica/genética , Unión Proteica/inmunología , Transporte de Proteínas/genética , Transporte de Proteínas/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismoRESUMEN
T cell receptor (TCR) recognition of peptide-MHC (pMHC) is central to the cellular immune response. A large database of TCR-pMHC structures is needed to reveal general structural principles, such as whether the repertoire of TCR/MHC docking modes is dictated by a "recognition code" between conserved elements of the TCR and MHC genes. Although approximately 17 cocrystal structures of unique TCR-pMHC complexes have been determined, cocrystallization of soluble TCR and pMHC remains a major technical obstacle in the field. Here we demonstrate a strategy, based on NMR chemical shift mapping, that permits rapid and reliable analysis of the solution footprint made by a TCR when binding onto the pMHC surface. We mapped the 2C TCR binding interaction with its allogeneic ligand H-2Ld-QL9 and identified a group of NMR-shifted residues that delineated a clear surface of the MHC that we defined as the TCR footprint. We subsequently found that the docking footprint described by NMR shifts was highly accurate compared with a recently determined high-resolution crystal structure of the same complex. The same NMR footprint analysis was done on a high-affinity mutant of the TCR. The current work serves as a foundation to explore the molecular dynamics of pMHC complexes and to rapidly determine the footprints of many Ld-specific TCRs.
Asunto(s)
Antígenos H-2/química , Receptores de Antígenos de Linfocitos T/química , Secuencia de Aminoácidos , Animales , Regiones Determinantes de Complementariedad , Antígeno de Histocompatibilidad H-2D , Espectroscopía de Resonancia Magnética , Ratones , Datos de Secuencia Molecular , Pliegue de ProteínaRESUMEN
alphabeta T cell receptors (TCRs) can crossreact with both self- and foreign- major histocompatibility complex (MHC) proteins in an enigmatic phenomenon termed alloreactivity. Here we present the 2.35 A structure of the 2C TCR complexed with its foreign ligand H-2L(d)-QL9. Surprisingly, we find that this TCR utilizes a different strategy to engage the foreign pMHC in comparison to the manner in which it recognizes a self ligand H-2K(b)-dEV8. 2C engages both shared and polymorphic residues on L(d) and K(b), as well as the unrelated QL9 and dEV8 peptide antigens, in unique pair-wise contacts, resulting in greater structural complementarity with the L(d)-QL9 complex. In the structure of an engineered, high-affinity 2C TCR variant bound to H-2L(d)-QL9, the "wild-type" TCR-MHC binding orientation persists despite modified TCR-CDR3alpha interactions with peptide. Thus, a single TCR recognizes two globally similar, but distinct ligands by divergent mechanisms, indicating that receptor-ligand crossreactivity can occur in the absence of molecular mimicry.
Asunto(s)
Autoantígenos/inmunología , Antígenos H-2/inmunología , Isoantígenos/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Secuencia de Aminoácidos , Autoantígenos/química , Autoantígenos/metabolismo , Regiones Determinantes de Complementariedad/metabolismo , Cristalografía por Rayos X , Antígenos H-2/química , Antígenos H-2/metabolismo , Antígeno de Histocompatibilidad H-2D , Isoantígenos/química , Isoantígenos/metabolismo , Complejo Cetoglutarato Deshidrogenasa/química , Complejo Cetoglutarato Deshidrogenasa/inmunología , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos/química , Péptidos/inmunología , Péptidos/metabolismo , Unión Proteica , Receptores de Antígenos de Linfocitos T alfa-beta/química , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismoRESUMEN
'Superantigens' (SAgs) trigger the massive activation of T cells by simultaneous interactions with MHC and TCR receptors, leading to human diseases. Here we present the first crystal structure, at 2.5-A resolution, of a complete ternary complex between a SAg and its two receptors, HLA-DR1/HA and TCR. The most striking finding is that the SAg Mycoplasma arthritidis mitogen, unlike others, has direct contacts not only with TCR Vbeta but with TCR Valpha.
Asunto(s)
Antígeno HLA-DR1/química , Hemaglutininas/química , Mitógenos/química , Proteínas/química , Receptores de Antígenos de Linfocitos T alfa-beta/química , Superantígenos/química , Secuencia de Aminoácidos , Animales , Antígenos Bacterianos , Cristalografía por Rayos X , Humanos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Mycoplasma arthritidis/inmunología , Péptidos/químicaRESUMEN
The T cell stimulatory activity of peptides is known to be associated with the cell surface stability and lifetime of the peptide-MHC (pepMHC) complex. In this report, soluble high-affinity T cell receptors (TCRs) that are specific for pepMHC complexes recognized by the mouse CD8+ clone 2C were used to monitor the cell surface lifetimes of synthetic agonist peptides. In the 2C system, L(d)-binding peptide p2Ca (LSPFPFDL) has up to 10,000-fold lower activity than peptide QL9 (QLSPFPFDL) even though the 2C TCR binds to p2Ca-L(d) and QL9-L(d) complexes with similar affinities. Unexpectedly, p2Ca-L(d) complexes were found to have a longer cell surface lifetime than QL9-L(d) complexes. However, the strong agonist activity of QL9 correlated with its ability to participate in efficient intracellular delivery followed by cell surface expression of the peptide, resulting in high and persistent surface levels of QL9-L(d). The ability of target cells to take up and present QL9 was observed with TAP-deficient cells and TAP-positive cells, including dendritic cells. The process was brefeldin A-sensitive, indicating a requirement for transport of the pepMHC through the ER and/or golgi. Thus, strong T cell stimulatory activity of some pepMHC complexes can be accomplished not only through long cell surface lifetimes of the ligand, but through a mechanism that leads to delayed presentation of the exogenous antigen after intracellular uptake.
Asunto(s)
Presentación de Antígeno/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Activación de Linfocitos/inmunología , Péptidos/inmunología , Linfocitos T/inmunología , Secuencia de Aminoácidos , Animales , Presentación de Antígeno/efectos de los fármacos , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/inmunología , Brefeldino A/farmacología , Línea Celular Tumoral , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Retículo Endoplásmico/efectos de los fármacos , Aparato de Golgi/efectos de los fármacos , Humanos , Activación de Linfocitos/efectos de los fármacos , Ratones , Datos de Secuencia Molecular , Péptidos/antagonistas & inhibidores , Péptidos/química , Linfocitos T/efectos de los fármacos , Factores de TiempoRESUMEN
The major histocompatibility complex (MHC) is the most polymorphic locus known, with thousands of allelic variants. There is considerable interest in understanding the diversity of structures and peptide-binding features represented by this class of proteins. Although many MHC proteins have been crystallized, others have not been amenable to structural or biochemical studies due to problems with expression or stability. In the present study, yeast display was used to engineer stabilizing mutations into the class I MHC molecule, Ld. The approach was based on previous studies that showed surface levels of yeast-displayed fusion proteins are directly correlated with protein stability. To engineer a more stable Ld, we selected Ld mutants with increased surface expression from randomly mutated yeast display libraries using anti-Ld antibodies or high affinity, soluble T-cell receptors (TCRs). The most stable Ld mutant, Ld-m31, consisted of a single-chain MHC module containing only the alpha1 and alpha2 domains. The enhanced stability was in part due to a single mutation (Trp-97 --> Arg), shown previously to be present in the allele Lq. Mutant Ld-m31 could bind to Ld peptides, and the specific peptide.Ld-m31 complex (QL9.Ld-m31) was recognized by alloreactive TCR 2C. A soluble form of the Ld-m31 protein was expressed in Escherichia coli and refolded from inclusion bodies at high yields. Surface plasmon resonance showed that TCRs bound to peptide.Ld-m31 complexes with affinities similar to those of native full-length Ld. The TCR and QL9.Ld-m31 formed complexes that could be resolved by native gel electrophoresis, suggesting that stabilized alpha1/alpha2 class I platforms may enable various structural studies.