RESUMEN
MHC restriction, which describes the binding of TCRs from CD4+ T cells to class II MHC proteins and TCRs from CD8+ T cells to class I MHC proteins, is a hallmark of immunology. Seemingly rare TCRs that break this paradigm exist, but mechanistic insight into their behavior is lacking. TIL1383I is a prototypical class-mismatched TCR, cloned from a CD4+ T cell but recognizing the tyrosinase tumor antigen presented by the class I MHC HLA-A2 in a fully functional manner. Here we find that TIL1383I binds this class I target with a highly atypical geometry. Despite unorthodox binding, TCR signaling, antigen specificity, and the ability to use CD8 are maintained. Structurally, a key feature of TIL1383I is an exceptionally long CDR3ß loop that mediates functions that are traditionally performed separately by hypervariable and germline loops in canonical TCR structures. Our findings thus expand the range of known TCR binding geometries compatible with normal function and specificity, provide insight into the determinants of MHC restriction, and may help guide TCR selection and engineering for immunotherapy.
Asunto(s)
Linfocitos T CD8-positivos , Receptores de Antígenos de Linfocitos T , Membrana Celular , Ingeniería , Antígeno HLA-A2/genéticaRESUMEN
Monoclonal antibodies are at the vanguard of the most promising cancer treatments. Whereas traditional therapeutic antibodies have been limited to extracellular antigens, T cell receptor mimic (TCRm) antibodies can target intracellular antigens presented by cell surface major histocompatibility complex (MHC) proteins. TCRm antibodies can therefore target a repertoire of otherwise undruggable cancer antigens. However, the consequences of off-target peptide/MHC recognition with engineered T cell therapies are severe, and thus there are significant safety concerns with TCRm antibodies. Here we explored the specificity and safety profile of a new TCRm-based T cell therapy for hepatocellular carcinoma (HCC), a solid tumor for which no effective treatment exists. We targeted an alpha-fetoprotein peptide presented by HLA-A*02 with a highly specific TCRm, which crystallographic structural analysis showed binds directly over the HLA protein and interfaces with the full length of the peptide. We fused the TCRm to the γ and δ subunits of a TCR, producing a signaling AbTCR construct. This was combined with an scFv/CD28 co-stimulatory molecule targeting glypican-3 for increased efficacy towards tumor cells. This AbTCR + co-stimulatory T cell therapy showed potent activity against AFP-positive cancer cell lines in vitro and an in an in vivo model and undetectable activity against AFP-negative cells. In an in-human safety assessment, no significant adverse events or cytokine release syndrome were observed and evidence of efficacy was seen. Remarkably, one patient with metastatic HCC achieved a complete remission after nine months and ultimately qualified for a liver transplant.
Asunto(s)
Carcinoma Hepatocelular , Neoplasias Hepáticas , Anticuerpos Monoclonales , Carcinoma Hepatocelular/tratamiento farmacológico , Humanos , Inmunoterapia , Neoplasias Hepáticas/tratamiento farmacológico , Péptidos , Receptores de Antígenos de Linfocitos T/genética , alfa-FetoproteínasRESUMEN
Using a variety of activating and inhibitory receptors, natural killer (NK) cells protect against disease by eliminating cells that have downregulated class I major histocompatibility complex (MHC) proteins, such as in response to cell transformation or viral infection. The inhibitory murine NK receptor Ly49C specifically recognizes the class I MHC protein H-2Kb. Unusual among NK receptors, Ly49C exhibits a peptide-dependent sensitivity to H-2Kb recognition, which has not been explained despite detailed structural studies. To gain further insight into Ly49C peptide sensitivity, we examined Ly49C recognition biochemically and through the lens of dynamic allostery. We found that the peptide sensitivity of Ly49C arises through small differences in H-2Kb-binding affinity. Although molecular dynamics simulations supported a role for peptide-dependent protein dynamics in producing these differences in binding affinity, calorimetric measurements indicated an enthalpically as opposed to entropically driven process. A quantitative linkage analysis showed that this emerges from peptide-dependent dynamic tuning of electrostatic interactions across the Ly49C-H-2Kb interface. We propose a model whereby different peptides alter the flexibility of H-2Kb, which in turn changes the strength of electrostatic interactions across the protein-protein interface. Our results provide a quantitative assessment of how peptides alter Ly49C-binding affinity, suggest the underlying mechanism, and demonstrate peptide-driven allostery at work in class I MHC proteins. Lastly, our model provides a solution for how dynamic allostery could impact binding of some, but not all, class I MHC partners depending on the structural and chemical composition of the interfaces.
Asunto(s)
Células Asesinas Naturales/metabolismo , Subfamilia A de Receptores Similares a Lectina de Células NK/metabolismo , Regulación Alostérica , Animales , Cinética , Ratones , Modelos Moleculares , Simulación de Dinámica Molecular , Subfamilia A de Receptores Similares a Lectina de Células NK/química , Unión Proteica , Dominios Proteicos , Especificidad por SustratoRESUMEN
Presentation of peptides by class I MHC proteins underlies T cell immune responses to pathogens and cancer. The association between peptide binding affinity and immunogenicity has led to the engineering of modified peptides with improved MHC binding, with the hope that these peptides would be useful for eliciting cross-reactive immune responses directed toward their weak binding, unmodified counterparts. Increasing evidence, however, indicates that T cell receptors (TCRs) can perceive such anchor-modified peptides differently than wild-type (WT) peptides, although the scope of discrimination is unclear. We show here that even modifications at primary anchors that have no discernible structural impact can lead to substantially stronger or weaker T cell recognition depending on the TCR. Surprisingly, the effect of peptide anchor modification can be sensed by a TCR at regions distant from the site of modification, indicating a through-protein mechanism in which the anchor residue serves as an allosteric modulator for TCR binding. Our findings emphasize caution in the use and interpretation of results from anchor-modified peptides and have implications for how anchor modifications are accounted for in other circumstances, such as predicting the immunogenicity of tumor neoantigens. Our data also highlight an important need to better understand the highly tunable dynamic nature of class I MHC proteins and the impact this has on various forms of immune recognition.
Asunto(s)
Antígeno HLA-A2/química , Péptidos/química , Receptores de Antígenos de Linfocitos T alfa-beta/química , Células Th2/inmunología , Regulación Alostérica , Sitios de Unión , Clonación Molecular , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Antígeno HLA-A2/genética , Antígeno HLA-A2/inmunología , Humanos , Células Jurkat , Cinética , Modelos Moleculares , Péptidos/genética , Péptidos/inmunología , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Ingeniería de Proteínas , Dominios y Motivos de Interacción de Proteínas , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Células Th2/citología , TermodinámicaRESUMEN
T cell receptors (TCRs) orchestrate cellular immunity by recognizing peptides presented by a range of major histocompatibility complex (MHC) proteins. Naturally occurring TCRs bind the composite peptide/MHC surface, recognizing peptides that are structurally and chemically compatible with the TCR binding site. Here we describe a molecularly evolved TCR variant that binds the human class I MHC protein HLA-A2 independent of the bound peptide, achieved by a drastic perturbation of the TCR binding geometry that places the molecule far from the peptide binding groove. This unique geometry is unsupportive of normal T cell signaling. A substantial divergence between affinity measurements in solution and in two dimensions between proximal cell membranes leads us to attribute the lack of signaling to steric hindrance that limits binding in the confines of a cell-cell interface. Our results provide an example of how receptor binding geometry can impact T cell function and provide further support for the view that germline-encoded residues in TCR binding loops evolved to drive productive TCR recognition and signaling.
Asunto(s)
Receptores de Antígenos de Linfocitos T/metabolismo , Sitios de Unión , Antígenos HLA-A/metabolismo , Humanos , Complejo Mayor de Histocompatibilidad/genética , Complejo Mayor de Histocompatibilidad/fisiología , Unión Proteica , Conformación ProteicaRESUMEN
T cell receptor (TCR) recognition of antigenic peptides bound and presented by class I major histocompatibility complex (MHC) proteins underlies the cytotoxic immune response to diseased cells. Crystallographic structures of TCR-peptide/MHC complexes have demonstrated how TCRs simultaneously interact with both the peptide and the MHC protein. However, it is increasingly recognized that, beyond serving as a static platform for peptide presentation, the physical properties of class I MHC proteins are tuned by different peptides in ways that are not always structurally visible. These include MHC protein motions, or dynamics, which are believed to influence interactions with a variety of MHC-binding proteins, including not only TCRs, but other activating and inhibitory receptors as well as components of the peptide loading machinery. Here, we investigated the mechanisms by which peptides tune the dynamics of the common class I MHC protein HLA-A2. By examining more than 50 lengthy molecular dynamics simulations of HLA-A2 presenting different peptides, we identified regions susceptible to dynamic tuning, including regions in the peptide binding domain as well as the distal α3 domain. Further analyses of the simulations illuminated mechanisms by which the influences of different peptides are communicated throughout the protein, and involve regions of the peptide binding groove, the ß2-microglobulin subunit, and the α3 domain. Overall, our results demonstrate that the class I MHC protein is a highly tunable peptide sensor whose physical properties vary considerably with bound peptide. Our data provides insight into the underlying principles and suggest a role for dynamically driven allostery in the immunological function of MHC proteins.
Asunto(s)
Antígeno HLA-A2/metabolismo , Péptidos/metabolismo , Escherichia coli/genética , Antígeno HLA-A2/química , Antígeno HLA-A2/genética , Simulación de Dinámica Molecular , Péptidos/química , Unión Proteica , Proteínas Recombinantes/metabolismoRESUMEN
T cell receptors (TCRs) have emerged as a new class of immunological therapeutics. However, though antigen specificity is a hallmark of adaptive immunity, TCRs themselves do not possess the high specificity of monoclonal antibodies. Although a necessary function of T cell biology, the resulting cross-reactivity presents a significant challenge for TCR-based therapeutic development, as it creates the potential for off-target recognition and immune toxicity. Efforts to enhance TCR specificity by mimicking the antibody maturation process and enhancing affinity can inadvertently exacerbate TCR cross-reactivity. Here we demonstrate this concern by showing that even peptide-targeted mutations in the TCR can introduce new reactivities against peptides that bear similarity to the original target. To counteract this, we explored a novel structure-guided approach for enhancing TCR specificity independent of affinity. Tested with the MART-1-specific TCR DMF5, our approach had a small but discernible impact on cross-reactivity toward MART-1 homologs yet was able to eliminate DMF5 cross-recognition of more divergent, unrelated epitopes. Our study provides a proof of principle for the use of advanced structure-guided design techniques for improving TCR specificity, and it suggests new ways forward for enhancing TCRs for therapeutic use.
Asunto(s)
Receptores de Antígenos de Linfocitos T/metabolismo , Inmunidad Adaptativa/fisiología , Anticuerpos Monoclonales/inmunología , Humanos , Antígeno MART-1/inmunología , Estructura Secundaria de Proteína , Resonancia por Plasmón de Superficie , Especificidad del Receptor de Antígeno de Linfocitos TRESUMEN
T cell receptor cross-reactivity allows a fixed T cell repertoire to respond to a much larger universe of potential antigens. Recent work has emphasized the importance of peptide structural and chemical homology, as opposed to sequence similarity, in T cell receptor cross-reactivity. Surprisingly, though, T cell receptors can also cross-react between ligands with little physiochemical commonalities. Studying the clinically relevant receptor DMF5, we demonstrate that cross-recognition of such divergent antigens can occur through mechanisms that involve heretofore unanticipated rearrangements in the peptide and presenting MHC protein, including binding-induced peptide register shifts and extensions from MHC peptide binding grooves. Moreover, cross-reactivity can proceed even when such dramatic rearrangements do not translate into structural or chemical molecular mimicry. Beyond demonstrating new principles of T cell receptor cross-reactivity, our results have implications for efforts to predict and control T cell specificity and cross-reactivity and highlight challenges associated with predicting T cell reactivities.
Asunto(s)
Oligopéptidos/química , Receptores de Antígenos de Linfocitos T/química , Antígenos/química , Autoinmunidad , Reacciones Cruzadas , Cristalografía por Rayos X , Epítopos/química , Humanos , Cinética , Ligandos , Imitación Molecular , Unión Proteica , Dominios Proteicos , Retroviridae , Resonancia por Plasmón de Superficie , Linfocitos T/químicaRESUMEN
The use of T cell receptor (TCR) gene-modified T cells in adoptive cell transfer has had promising clinical success, but often, simple preclinical evaluation does not necessarily accurately predict treatment efficacy or safety. Preclinical studies generally evaluate one or a limited number of type 1 cytokines to assess antigen recognition. However, recent studies have implicated other "typed" T cells in effective anti-tumor/viral immunity, and limited functional evaluations may underestimate cross-reactivity. In this study, we use an altered peptide ligand (APL) model and multi-dimensional flow cytometry to evaluate polyfunctionality of TCR gene-modified T cells. Evaluating six cytokines and the lytic marker CD107a on a per cell basis revealed remarkably diverse polyfunctional phenotypes within a single T cell culture and among peripheral blood lymphocyte (PBL) donors. This polyfunctional assessment identified unexpected phenotypes, including cells producing both type 1 and type 2 cytokines, and highlighted interferon γneg (IFNγneg) antigen-reactive populations overlooked in our previous studies. Additionally, APLs skewed functional phenotypes to be less polyfunctional, which was not necessarily related to changes in TCR-peptide-major histocompatibility complex (pMHC) affinity. A better understanding of gene-modified T cell functional diversity may help identify optimal therapeutic phenotypes, predict clinical responses, anticipate off-target recognition, and improve the design and delivery of TCR gene-modified T cells.
Asunto(s)
Péptidos/metabolismo , Fenotipo , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Biomarcadores , Células Cultivadas , Citocinas/metabolismo , Citotoxicidad Inmunológica , Antígenos de Histocompatibilidad/química , Antígenos de Histocompatibilidad/inmunología , Antígenos de Histocompatibilidad/metabolismo , Humanos , Inmunofenotipificación , Inmunoterapia Adoptiva/métodos , Ligandos , Activación de Linfocitos , Modelos Moleculares , Péptidos/química , Unión Proteica , Conformación Proteica , Receptores de Antígenos de Linfocitos T/química , Relación Estructura-Actividad , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismoRESUMEN
Malignant melanoma incidence has been increasing for over 30 years, and despite promising new therapies, metastatic disease remains difficult to treat. We describe preliminary results from a Phase I clinical trial (NCT01586403) of adoptive cell therapy in which three patients received autologous CD4+ and CD8+ T cells transduced with a lentivirus carrying a tyrosinase-specific TCR and a marker protein, truncated CD34 (CD34t). This unusual MHC Class I-restricted TCR produces functional responses in both CD4+ and CD8+ T cells. Parameters monitored on transduced T cells included activation (CD25, CD69), inhibitory (PD-1, TIM-3, CTLA-4), costimulatory (OX40), and memory (CCR7) markers. For the clinical trial, T cells were activated, transduced, selected for CD34t+ cells, then re-activated, and expanded in IL-2 and IL-15. After lymphodepleting chemotherapy, patients were given transduced T cells and IL-2, and were followed for clinical and biological responses. Transduced T cells were detected in the circulation of three treated patients for the duration of observation (42, 523, and 255 days). Patient 1 tolerated the infusion well but died from progressive disease after 6 weeks. Patient 2 had a partial response by RECIST criteria then progressed. After progressing, Patient 2 was given high-dose IL-2 and subsequently achieved complete remission, coinciding with the development of vitiligo. Patient 3 had a mixed response that did not meet RECIST criteria for a clinical response and developed vitiligo. In two of these three patients, adoptive transfer of tyrosinase-reactive TCR-transduced T cells into metastatic melanoma patients had clinical and/or biological activity without serious adverse events.
Asunto(s)
Antígenos de Neoplasias/inmunología , Melanoma/terapia , Receptores de Antígenos de Linfocitos T/inmunología , Neoplasias Cutáneas/terapia , Subgrupos de Linfocitos T/trasplante , Adulto , Anciano , Humanos , Masculino , Melanoma/inmunología , Melanoma/patología , Persona de Mediana Edad , Metástasis de la Neoplasia , Pronóstico , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/secundario , Subgrupos de Linfocitos T/inmunología , Trasplante AutólogoRESUMEN
The authors would like to make the following corrections to the published article.
RESUMEN
T-cell receptor (TCR) allorecognition is often presumed to be relatively nonspecific, attributable to either a TCR focus on exposed major histocompatibility complex (MHC) polymorphisms or the degenerate recognition of allopeptides. However, paradoxically, alloreactivity can proceed with high peptide and MHC specificity. Although the underlying mechanisms remain unclear, the existence of highly specific alloreactive TCRs has led to their use as immunotherapeutics that can circumvent central tolerance and limit graft-versus-host disease. Here, we show how an alloreactive TCR achieves peptide and MHC specificity. The HCV1406 TCR was cloned from T cells that expanded when a hepatitis C virus (HCV)-infected HLA-A2- individual received an HLA-A2+ liver allograft. HCV1406 was subsequently shown to recognize the HCV nonstructural protein 3 (NS3):1406-1415 epitope with high specificity when presented by HLA-A2. We show that NS3/HLA-A2 recognition by the HCV1406 TCR is critically dependent on features unique to both the allo-MHC and the NS3 epitope. We also find cooperativity between structural mimicry and a crucial peptide "hot spot" and demonstrate its role, along with the MHC, in directing the specificity of allorecognition. Our results help explain the paradox of specificity in alloreactive TCRs and have implications for their use in immunotherapy and related efforts to manipulate TCR recognition, as well as alloreactivity in general.
Asunto(s)
Receptores de Antígenos de Linfocitos T/metabolismo , Secuencia de Aminoácidos , Línea Celular , Reacciones Cruzadas , Cristalografía por Rayos X , Epítopos/metabolismo , Células HEK293 , Antígeno HLA-A2/química , Antígeno HLA-A2/genética , Antígeno HLA-A2/metabolismo , Hepacivirus/química , Hepacivirus/genética , Hepacivirus/inmunología , Humanos , Inmunoterapia , Isoantígenos/metabolismo , Células Jurkat , Complejo Mayor de Histocompatibilidad , Modelos Moleculares , Imitación Molecular/genética , Imitación Molecular/inmunología , Péptidos/inmunología , Dominios Proteicos , Linfocitos T/inmunología , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/inmunologíaRESUMEN
T-cell receptor (TCR)-pMHC affinity has been generally accepted to be the most important factor dictating antigen recognition in gene-modified T-cells. As such, there is great interest in optimizing TCR-based immunotherapies by enhancing TCR affinity to augment the therapeutic benefit of TCR gene-modified T-cells in cancer patients. However, recent clinical trials using affinity-enhanced TCRs in adoptive cell transfer (ACT) have observed unintended and serious adverse events, including death, attributed to unpredicted off-tumor or off-target cross-reactivity. It is critical to re-evaluate the importance of other biophysical, structural, or cellular factors that drive the reactivity of TCR gene-modified T-cells. Using a model for altered antigen recognition, we determined how TCR-pMHC affinity influenced the reactivity of hepatitis C virus (HCV) TCR gene-modified T-cells against a panel of naturally occurring HCV peptides and HCV-expressing tumor targets. The impact of other factors, such as TCR-pMHC stabilization and signaling contributions by the CD8 co-receptor, as well as antigen and TCR density were also evaluated. We found that changes in TCR-pMHC affinity did not always predict or dictate IFNγ release or degranulation by TCR gene-modified T-cells, suggesting that less emphasis might need to be placed on TCR-pMHC affinity as a means of predicting or augmenting the therapeutic potential of TCR gene-modified T-cells used in ACT. A more complete understanding of antigen recognition by gene-modified T-cells and a more rational approach to improve the design and implementation of novel TCR-based immunotherapies is necessary to enhance efficacy and maximize safety in patients.
Asunto(s)
Traslado Adoptivo/métodos , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Linfocitos T/trasplante , Animales , Unión Competitiva/inmunología , Línea Celular , Línea Celular Tumoral , Técnicas de Cocultivo , Citometría de Flujo , Células HEK293 , Células Hep G2 , Humanos , Interferón gamma/inmunología , Interferón gamma/metabolismo , Células Jurkat , Ratones , Péptidos/genética , Péptidos/inmunología , Péptidos/metabolismo , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/metabolismo , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/inmunología , Proteínas no Estructurales Virales/metabolismoRESUMEN
A complex link exists between cell-wall recycling/repair and the manifestation of resistance to ß-lactam antibiotics in many Enterobacteriaceae and Pseudomonas aeruginosa. This process is mediated by specific cell-wall-derived muropeptide products. These muropeptides are internalized into the cytoplasm and bind to the transcriptional regulator AmpR, which controls the cytoplasmic events that lead to expression of ß-lactamase, an antibiotic-resistance determinant. The effector-binding domain (EBD) of AmpR was purified to homogeneity. We document that the EBD exists exclusively as a dimer, even at a concentration as low as 1 µM. The EBD binds to the suppressor ligand UDP-N-acetyl-ß-d-muramyl-l-Ala-γ-d-Glu-meso-DAP-d-Ala-d-Ala and binds to two activator muropeptides, N-acetyl-ß-d-glucosamine-(1â4)-1,6-anhydro-N-acetyl-ß-d-muramyl-l-Ala-γ-d-Glu-meso-DAP-d-Ala-d-Ala and 1,6-anhydro-N-acetyl-ß-d-muramyl-l-Ala-γ-d-Glu-meso-DAP-d-Ala-d-Ala, as assessed by non-denaturing mass spectrometry. The EBD does not bind to 1,6-anhydro-N-acetyl-ß-d-muramyl-l-Ala-γ-d-Glu-meso-DAP. This binding selectivity revises the dogma in the field. The crystal structure of the EBD dimer was solved to 2.2 Å resolution. The EBD crystallizes in a "closed" conformation, in contrast to the "open" structure required to bind the muropeptides. Structural issues of this ligand recognition are addressed by molecular dynamics simulations, which reveal significant differences among the complexes with the effector molecules.
Asunto(s)
Proteínas Bacterianas/química , Péptidos/química , Pseudomonas aeruginosa/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Péptidos/metabolismo , Dominios Proteicos , Pseudomonas aeruginosa/metabolismoRESUMEN
T-cell receptors (TCRs) have emerged as a new class of therapeutics, most prominently for cancer where they are the key components of new cellular therapies as well as soluble biologics. Many studies have generated high affinity TCRs in order to enhance sensitivity. Recent outcomes, however, have suggested that fine manipulation of TCR binding, with an emphasis on specificity may be more valuable than large affinity increments. Structure-guided design is ideally suited for this role, and here we studied the generality of structure-guided design as applied to TCRs. We found that a previous approach, which successfully optimized the binding of a therapeutic TCR, had poor accuracy when applied to a broader set of TCR interfaces. We thus sought to develop a more general purpose TCR design framework. After assembling a large dataset of experimental data spanning multiple interfaces, we trained a new scoring function that accounted for unique features of each interface. Together with other improvements, such as explicit inclusion of molecular flexibility, this permitted the design new affinity-enhancing mutations in multiple TCRs, including those not used in training. Our approach also captured the impacts of mutations and substitutions in the peptide/MHC ligand, and recapitulated recent findings regarding TCR specificity, indicating utility in more general mutational scanning of TCR-pMHC interfaces.
Asunto(s)
Ingeniería de Proteínas/métodos , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Biología Computacional , Antígeno HLA-A2/genética , Antígeno HLA-A2/metabolismo , Simulación de Dinámica Molecular , Mutación , Unión Proteica , Estructura Secundaria de Proteína , Receptores de Antígenos de Linfocitos T/químicaRESUMEN
Measurements of thermal stability by circular dichroism (CD) spectroscopy have been widely used to assess the binding of peptides to MHC proteins, particularly within the structural immunology community. Although thermal stability assays offer advantages over other approaches such as IC50 measurements, CD-based stability measurements are hindered by large sample requirements and low throughput. Here we demonstrate that an alternative approach based on differential scanning fluorimetry (DSF) yields results comparable to those based on CD for both class I and class II complexes. As they require much less sample, DSF-based measurements reduce demands on protein production strategies and are amenable for high throughput studies. DSF can thus not only replace CD as a means to assess peptide/MHC thermal stability, but can complement other peptide-MHC binding assays used in screening, epitope discovery, and vaccine design. Due to the physical process probed, DSF can also uncover complexities not observed with other techniques. Lastly, we show that DSF can also be used to assess peptide/MHC kinetic stability, allowing for a single experimental setup to probe both binding equilibria and kinetics.
Asunto(s)
Fluorometría/métodos , Antígeno HLA-A2/metabolismo , Antígeno HLA-DR1/metabolismo , Calor , Péptidos/metabolismo , Dicroismo Circular , Antígeno HLA-A2/química , Antígeno HLA-DR1/química , Humanos , Cinética , Péptidos/química , Unión Proteica , Desnaturalización Proteica , Estabilidad ProteicaRESUMEN
A major obstacle hindering the development of effective immunity against viral infections, their associated disease, and certain cancers is their inherent genomic instability. Accumulation of mutations can alter processing and presentation of antigens recognized by antibodies and T cells that can lead to immune escape variants. Use of an agent that can intrinsically combat rapidly mutating viral or cancer-associated antigens would be quite advantageous in developing effective immunity against such disease. We propose that T cells harboring cross-reactive TCRs could serve as a therapeutic agent in these instances. With the use of hepatitis C virus, known for its genomic instability as a model for mutated antigen recognition, we demonstrate cross-reactivity against immunogenic and mutagenic nonstructural protein 3:1406-1415 and nonstructural protein 3:1073-1081 epitopes in PBL-derived, TCR-gene-modified T cells. These single TCR-engineered T cells can CD8-independently recognize naturally occurring and epidemiologically relevant mutant variants. TCR-peptide MHC modeling data allow us to rationalize how TCR structural properties accommodate recognition of certain mutated epitopes and how these substitutions impact the requirement of CD8 affinity enhancement for recognition. A better understanding of such TCRs' promiscuous behavior may allow for exploitation of these properties to develop novel, adoptive T cell-based therapies for viral infections and cancers exhibiting similar genomic instability.
Asunto(s)
Epítopos de Linfocito T/inmunología , Inestabilidad Genómica , Hepacivirus/inmunología , Hepatitis C/prevención & control , Antígenos de Histocompatibilidad Clase I/inmunología , Inmunoterapia , Receptores de Antígenos de Linfocitos T/inmunología , Antígenos Virales/inmunología , Linfocitos T CD8-positivos/inmunología , Reacciones Cruzadas , Hepacivirus/genética , Hepatitis C/etiología , HumanosRESUMEN
The Munc13 family of exocytosis regulators has multiple Ca(2+)-binding, C2 domains. Here, we probed the mechanism by which Munc13-4 regulates in vitro membrane fusion and platelet exocytosis. We show that Munc13-4 enhances in vitro soluble NSF attachment protein receptor (SNARE)-dependent, proteoliposome fusion in a Ca(2+)- and phosphatidylserine (PS)-dependent manner that was independent of SNARE concentrations. Munc13-4-SNARE interactions, under the conditions used, were minimal in the absence or presence of Ca(2+). However, Munc13-4 was able to bind and cluster liposomes harbouring PS in response to Ca(2+). Interestingly, Ca(2+)-dependent liposome binding/clustering and enhancement of proteoliposome fusion required both Munc13-4 C2 domains, but only the Ca(2+)-liganding aspartate residues of the C2B domain. Analytical ultracentrifugation (AUC) measurements indicated that, in solution, Munc13-4 was a monomeric prolate ellipsoid with dimensions consistent with a molecule that could bridge two fusing membranes. To address the potential role of Munc13-4 as a tethering protein in platelets, we examined mepacrine-stained, dense granule mobility and secretion in platelets from wild-type and Munc13-4 null (Unc13d(Jinx)) mice. In the absence of Munc13-4, dense granules were highly mobile in both resting and stimulated platelets, and stimulation-dependent granule release was absent. These observations suggest that dense granules are stably docked in resting platelets awaiting stimulation and that Munc13-4 plays a vesicle-stabilizing or tethering role in resting platelets and also in activated platelets in response to Ca(2+). In summary, we show that Munc13-4 conveys Ca(2+) sensitivity to platelet SNARE-mediated membrane fusion and reveal a potential mechanism by which Munc13-4 bridges and stabilizes apposing membranes destined for fusion.
Asunto(s)
Plaquetas/fisiología , Proteínas de la Membrana/metabolismo , Animales , Plaquetas/ultraestructura , Calcio/metabolismo , Fusión Celular , Exocitosis , Humanos , Liposomas , Proteínas de la Membrana/genética , Ratones Noqueados , Mutación , Fosfatidilserinas/metabolismo , Ratas , Proteínas SNARE/metabolismo , Vesículas Secretoras/fisiología , Vesículas Secretoras/ultraestructuraRESUMEN
Tumor-associated myeloid cells, including dendritic cells (DCs) and macrophages, are immune suppressive. This study demonstrates a novel mechanism involving FOXO3 and NF-κB RelA that controls myeloid cell signaling and impacts their immune-suppressive nature. We find that FOXO3 binds NF-κB RelA in the cytosol, impacting both proteins by preventing FOXO3 degradation and preventing NF-κB RelA nuclear translocation. The location of protein-protein interaction was determined to be near the FOXO3 transactivation domain. In turn, NF-κB RelA activation was restored upon deletion of the same sequence in FOXO3 containing the DNA binding domain. We have identified for the first time, to our knowledge, a direct protein-protein interaction between FOXO3 and NF-κB RelA in tumor-associated DCs. These detailed biochemical interactions provide the foundation for future studies to use the FOXO3-NF-κB RelA interaction as a target to enhance tumor-associated DC function to support or enhance antitumor immunity.
Asunto(s)
Adenocarcinoma/inmunología , Células Dendríticas/inmunología , Factores de Transcripción Forkhead/metabolismo , Macrófagos/inmunología , Melanoma Experimental/inmunología , Células Mieloides/inmunología , FN-kappa B/metabolismo , Neoplasias de la Próstata/inmunología , Animales , Proteína Forkhead Box O3 , Factores de Transcripción Forkhead/genética , Masculino , Ratones , Ratones Noqueados , Terapia Molecular Dirigida , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Transducción de Señal , Activación TranscripcionalRESUMEN
Glycogen is the major mammalian glucose storage cache and is critical for energy homeostasis. Glycogen synthesis in neurons must be tightly controlled due to neuronal sensitivity to perturbations in glycogen metabolism. Lafora disease (LD) is a fatal, congenital, neurodegenerative epilepsy. Mutations in the gene encoding the glycogen phosphatase laforin result in hyperphosphorylated glycogen that forms water-insoluble inclusions called Lafora bodies (LBs). LBs induce neuronal apoptosis and are the causative agent of LD. The mechanism of glycogen dephosphorylation by laforin and dysfunction in LD is unknown. We report the crystal structure of laforin bound to phosphoglucan product, revealing its unique integrated tertiary and quaternary structure. Structure-guided mutagenesis combined with biophysical and biochemical analyses reveal the basis for normal function of laforin in glycogen metabolism. Analyses of LD patient mutations define the mechanism by which subsets of mutations disrupt laforin function. These data provide fundamental insights connecting glycogen metabolism to neurodegenerative disease.