RESUMEN
CD8+ T cell-mediated recognition of peptide-major histocompatibility complex class I (pMHCI) molecules involves cooperative binding of the T cell receptor (TCR), which confers antigen specificity, and the CD8 coreceptor, which stabilizes the TCR/pMHCI complex. Earlier work has shown that the sensitivity of antigen recognition can be regulated in vitro by altering the strength of the pMHCI/CD8 interaction. Here, we characterized two CD8 variants with moderately enhanced affinities for pMHCI, aiming to boost antigen sensitivity without inducing non-specific activation. Expression of these CD8 variants in model systems preferentially enhanced pMHCI antigen recognition in the context of low-affinity TCRs. A similar effect was observed using primary CD4+ T cells transduced with cancer-targeting TCRs. The introduction of high-affinity CD8 variants also enhanced the functional sensitivity of primary CD8+ T cells expressing cancer-targeting TCRs, but comparable results were obtained using exogenous wild-type CD8. Specificity was retained in every case, with no evidence of reactivity in the absence of cognate antigen. Collectively, these findings highlight a generically applicable mechanism to enhance the sensitivity of low-affinity pMHCI antigen recognition, which could augment the therapeutic efficacy of clinically relevant TCRs.
Asunto(s)
Antígenos CD8 , Linfocitos T CD8-positivos , Antígenos de Histocompatibilidad Clase I , Activación de Linfocitos , Antígenos de Histocompatibilidad Clase I/metabolismo , Péptidos/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , HumanosRESUMEN
CD8+ T cells are inherently cross-reactive and recognize numerous peptide antigens in the context of a given major histocompatibility complex class I (MHCI) molecule via the clonotypically expressed T cell receptor (TCR). The lineally expressed coreceptor CD8 interacts coordinately with MHCI at a distinct and largely invariant site to slow the TCR/peptide-MHCI (pMHCI) dissociation rate and enhance antigen sensitivity. However, this biological effect is not necessarily uniform, and theoretical models suggest that antigen sensitivity can be modulated in a differential manner by CD8. We used two intrinsically controlled systems to determine how the relationship between the TCR/pMHCI interaction and the pMHCI/CD8 interaction affects the functional sensitivity of antigen recognition. Our data show that modulation of the pMHCI/CD8 interaction can reorder the agonist hierarchy of peptide ligands across a spectrum of affinities for the TCR.
Asunto(s)
Antígenos CD8/inmunología , Péptidos/agonistas , Péptidos/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Antígenos/química , Antígenos/inmunología , Linfocitos T CD8-positivos/inmunología , Reacciones Cruzadas , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Cinética , Ligandos , Activación de Linfocitos , Modelos Inmunológicos , MutaciónRESUMEN
The capacity of the immune system to adapt to rapidly evolving viruses is a primary feature of effective immunity, yet its molecular basis is unclear. Here, we investigated protective HIV-1-specific CD8+ T cell responses directed against the immunodominant p24 Gag-derived epitope KK10 (KRWIILGLNK263-272) presented by human leukocyte antigen (HLA)-B∗2705. We found that cross-reactive CD8+ T cell clonotypes were mobilized to counter the rapid emergence of HIV-1 variants that can directly affect T cell receptor (TCR) recognition. These newly recruited clonotypes expressed TCRs that engaged wild-type and mutant KK10 antigens with similar affinities and almost identical docking modes, thereby accounting for their antiviral efficacy in HLA-B∗2705+ individuals. A protective CD8+ T cell repertoire therefore encompasses the capacity to control TCR-accessible mutations, ultimately driving the development of more complex viral escape variants that disrupt antigen presentation.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Infecciones por VIH/inmunología , VIH-1/inmunología , Antígeno HLA-B27/inmunología , Secuencia de Aminoácidos , Presentación de Antígeno/inmunología , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/virología , Células Clonales/inmunología , Células Clonales/metabolismo , Células Clonales/virología , Cristalografía por Rayos X , Epítopos de Linfocito T/química , Epítopos de Linfocito T/inmunología , Epítopos de Linfocito T/metabolismo , Proteína p24 del Núcleo del VIH/genética , Proteína p24 del Núcleo del VIH/inmunología , Proteína p24 del Núcleo del VIH/metabolismo , Infecciones por VIH/virología , VIH-1/genética , VIH-1/metabolismo , Antígeno HLA-B27/química , Antígeno HLA-B27/metabolismo , Humanos , Epítopos Inmunodominantes/química , Epítopos Inmunodominantes/inmunología , Epítopos Inmunodominantes/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Unión Proteica/inmunología , Estructura Terciaria de Proteína , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismoRESUMEN
Tuberculosis (TB), caused by Mycobacterium tuberculosis, remains a major human pandemic. Germline-encoded mycolyl lipid-reactive (GEM) T cells are donor-unrestricted and recognize CD1b-presented mycobacterial mycolates. However, the molecular requirements governing mycolate antigenicity for the GEM T cell receptor (TCR) remain poorly understood. Here, we demonstrate CD1b expression in TB granulomas and reveal a central role for meromycolate chains in influencing GEM-TCR activity. Meromycolate fine structure influences T cell responses in TB-exposed individuals, and meromycolate alterations modulate functional responses by GEM-TCRs. Computational simulations suggest that meromycolate chain dynamics regulate mycolate head group movement, thereby modulating GEM-TCR activity. Our findings have significant implications for the design of future vaccines that target GEM T cells.
Asunto(s)
Antígenos CD1/inmunología , Mycobacterium tuberculosis/inmunología , Mycobacterium tuberculosis/metabolismo , Ácidos Micólicos/inmunología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Tuberculosis/inmunología , Antígenos Bacterianos/inmunología , Antígenos Bacterianos/metabolismo , Antígenos CD1/química , Antígenos CD1/genética , Expresión Génica , Granuloma/inmunología , Granuloma/metabolismo , Granuloma/microbiología , Granuloma/patología , Humanos , Inmunohistoquímica , Activación de Linfocitos/inmunología , Modelos Moleculares , Conformación Molecular , Ácidos Micólicos/química , Ácidos Micólicos/metabolismo , Unión Proteica , Receptores de Antígenos de Linfocitos T/metabolismo , Tuberculosis/microbiologíaRESUMEN
Antigen recognition by the T-cell receptor (TCR) is a hallmark of the adaptive immune system. When the TCR engages a peptide bound to the restricting major histocompatibility complex molecule (pMHC), it transmits a signal via the associated CD3 complex. How the extracellular antigen recognition event leads to intracellular phosphorylation remains unclear. Here, we used single-molecule localization microscopy to quantify the organization of TCR-CD3 complexes into nanoscale clusters and to distinguish between triggered and nontriggered TCR-CD3 complexes. We found that only TCR-CD3 complexes in dense clusters were phosphorylated and associated with downstream signaling proteins, demonstrating that the molecular density within clusters dictates signal initiation. Moreover, both pMHC dose and TCR-pMHC affinity determined the density of TCR-CD3 clusters, which scaled with overall phosphorylation levels. Thus, TCR-CD3 clustering translates antigen recognition by the TCR into signal initiation by the CD3 complex, and the formation of dense signaling-competent clusters is a process of antigen discrimination.
Asunto(s)
Antígenos/inmunología , Complejo CD3/inmunología , Complejo Mayor de Histocompatibilidad/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Inmunidad Adaptativa/genética , Animales , Antígenos/genética , Humanos , Ratones , Péptidos/inmunología , Fosforilación/inmunología , Transducción de Señal , Imagen Individual de MoléculaRESUMEN
The CD8 co-receptor engages peptide-major histocompatibility complex class I (pMHCI) molecules at a largely invariant site distinct from the T-cell receptor (TCR)-binding platform and enhances the sensitivity of antigen-driven activation to promote effective CD8+ T-cell immunity. A small increase in the strength of the pMHCI/CD8 interaction (~1.5-fold) can disproportionately amplify this effect, boosting antigen sensitivity by up to two orders of magnitude. However, recognition specificity is lost altogether with more substantial increases in pMHCI/CD8 affinity (~10-fold). In this study, we used a panel of MHCI mutants with altered CD8-binding properties to show that TCR-mediated antigen specificity is delimited by a pMHCI/CD8 affinity threshold. Our findings suggest that CD8 can be engineered within certain biophysical parameters to enhance the therapeutic efficacy of adoptive T-cell transfer irrespective of antigen specificity.
Asunto(s)
Antígenos CD8/metabolismo , Linfocitos T CD8-positivos/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Membrana Celular/metabolismo , Humanos , Activación de Linfocitos/inmunología , Mutación/genética , Péptidos/metabolismoRESUMEN
The inherent resistance of cancer stem cells (CSCs) to existing therapies has largely hampered the development of effective treatments for advanced malignancy. To help develop novel immunotherapy approaches that efficiently target CSCs, an experimental model allowing reliable distinction of CSCs and non-CSCs was set up to study their interaction with non-MHC-restricted γδ T cells and antigen-specific CD8+ T cells. Stable lines with characteristics of breast CSC-like cells were generated from ras-transformed human mammary epithelial (HMLER) cells as confirmed by their CD44hi CD24lo GD2+ phenotype, their mesenchymal morphology in culture and their capacity to form mammospheres under non-adherent conditions, as well as their potent tumorigenicity, self-renewal and differentiation in xenografted mice. The resistance of CSC-like cells to γδ T cells could be overcome by inhibition of farnesyl pyrophosphate synthase (FPPS) through pretreatment with zoledronate or with FPPS-targeting short hairpin RNA. γδ T cells induced upregulation of MHC class I and CD54/ICAM-1 on CSC-like cells and thereby increased the susceptibility to antigen-specific killing by CD8+ T cells. Alternatively, γδ T-cell responses could be specifically directed against CSC-like cells using the humanised anti-GD2 monoclonal antibody hu14.18K322A. Our findings identify a powerful synergism between MHC-restricted and non-MHC-restricted T cells in the eradication of cancer cells including breast CSCs. Our research suggests that novel immunotherapies may benefit from a two-pronged approach combining γδ T-cell and CD8+ T-cell targeting strategies that triggers effective innate-like and tumour-specific adaptive responses.
Asunto(s)
Neoplasias de la Mama/inmunología , Neoplasias de la Mama/patología , Linfocitos T CD8-positivos/inmunología , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Animales , Anticuerpos/farmacología , Mama/patología , Citotoxicidad Inmunológica , Difosfonatos/farmacología , Células Epiteliales/metabolismo , Epítopos/inmunología , Femenino , Humanos , Imidazoles/farmacología , Inmunidad Innata , Interferón gamma/metabolismo , Complejo Mayor de Histocompatibilidad , Ratones , Fenotipo , Ácido Zoledrónico , Proteínas ras/metabolismoRESUMEN
T cells engrafted with chimeric AgRs (CAR) are showing exciting potential for targeting B cell malignancies in early-phase clinical trials. To determine whether the second-generation CAR was essential for optimal antitumor activity, two CD28-based CAR constructs targeting CD19 were tested for their ability to redirect mouse T cell function against established B cell lymphoma in a BALB/c syngeneic model system. T cells armed with either CAR eliminated A20 B cell lymphoma in vivo; however, one construct induced a T cell dose-dependent acute toxicity associated with a raised serum Th1 type cytokine profile on transfer into preconditioned mice. Moreover, a chronic toxicity manifested as granuloma-like formation in spleen, liver, and lymph nodes was observed in animals receiving T cells bearing either CD28 CAR, albeit with different kinetics dependent upon the specific receptor used. This phenotype was associated with an expansion of CD4+ CAR+ T cells and CD11b+ Gr-1(+) myeloid cells and increased serum Th2-type cytokines, including IL-10 and IL-13. Mouse T cells engrafted with a first-generation CAR failed to develop such autotoxicity, whereas toxicity was not apparent when T cells bearing the same receptors were transferred into C57BL/6 or C3H animals. In summary, the adoptive transfer of second-generation CD19-specific CAR T cells can result in a cell dose-dependent acute toxicity, whereas the prolonged secretion of high levels of Th2 cytokines from these CAR T cells in vivo drives a granulomatous reaction resulting in chronic toxicity. Strategies that prevent a prolonged Th2-cytokine biased CAR T cell response are clearly warranted.
Asunto(s)
Antígenos CD19/inmunología , Citocinas/metabolismo , Citotoxicidad Inmunológica , Receptores de Antígenos de Linfocitos T/metabolismo , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Traslado Adoptivo/efectos adversos , Animales , Antígenos CD19/genética , Análisis por Conglomerados , Perfilación de la Expresión Génica , Humanos , Inmunofenotipificación , Linfoma de Células B/genética , Linfoma de Células B/inmunología , Linfoma de Células B/metabolismo , Linfoma de Células B/terapia , Ratones , Ratones Endogámicos BALB C , Fenotipo , Receptores de Antígenos de Linfocitos T/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Subgrupos de Linfocitos T/trasplante , Células TH1/inmunología , Células TH1/metabolismo , Células Th2/inmunología , Células Th2/metabolismoRESUMEN
Killer Ig-like receptors (KIRs) control the activation of human NK cells via interactions with peptide-laden HLAs. KIR3DL1 is a highly polymorphic inhibitory receptor that recognizes a diverse array of HLA molecules expressing the Bw4 epitope, a group with multiple polymorphisms incorporating variants within the Bw4 motif. Genetic studies suggest that KIR3DL1 variation has functional significance in several disease states, including HIV infection. However, owing to differences across KIR3DL1 allotypes, HLA-Bw4, and associated peptides, the mechanistic link with biological outcome remains unclear. In this study, we elucidated the impact of KIR3DL1 polymorphism on peptide-laden HLA recognition. Mutational analysis revealed that KIR residues involved in water-mediated contacts with the HLA-presented peptide influence peptide binding specificity. In particular, residue 282 (glutamate) in the D2 domain underpins the lack of tolerance of negatively charged C-terminal peptide residues. Allotypic KIR3DL1 variants, defined by neighboring residue 283, displayed differential sensitivities to HLA-bound peptide, including the variable HLA-B*57:01-restricted HIV-1 Gag-derived epitope TW10. Residue 283, which has undergone positive selection during the evolution of human KIRs, also played a central role in Bw4 subtype recognition by KIR3DL1. Collectively, our findings uncover a common molecular regulator that controls HLA and peptide discrimination without participating directly in peptide-laden HLA interactions. Furthermore, they provide insight into the mechanics of interaction and generate simple, easily assessed criteria for the definition of KIR3DL1 functional groupings that will be relevant in many clinical applications, including bone marrow transplantation.
Asunto(s)
Antígenos HLA-B/inmunología , Péptidos/inmunología , Receptores KIR3DL1/inmunología , Secuencia de Aminoácidos , Sitios de Unión/genética , Sitios de Unión/inmunología , Epítopos/genética , Epítopos/inmunología , Células HEK293 , Antígenos HLA-B/química , Antígenos HLA-B/genética , Proteínas del Virus de la Inmunodeficiencia Humana/genética , Proteínas del Virus de la Inmunodeficiencia Humana/inmunología , Humanos , Células Jurkat , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Modelos Moleculares , Mutación , Péptidos/química , Péptidos/genética , Polimorfismo Genético , Unión Proteica/inmunología , Multimerización de Proteína , Estructura Terciaria de Proteína , Receptores KIR3DL1/química , Receptores KIR3DL1/genéticaRESUMEN
Transfer of autologous tumor infiltrating lymphocytes (TIL) to patients with refractory melanoma has shown clinical efficacy in a number of trials. However, extending the clinical benefit to patients with other cancers poses a challenge. Inefficient costimulation in the tumor microenvironment can lead to T cell anergy and exhaustion resulting in poor anti-tumor activity. Here, we describe a chimeric costimulatory antigen receptor (CoStAR) comprised of FRα-specific scFv linked to CD28 and CD40 intracellular signaling domains. CoStAR signaling alone does not activate T cells, while the combination of TCR and CoStAR signaling enhances T cell activity resulting in less differentiated T cells, and augmentation of T cell effector functions, including cytokine secretion and cytotoxicity. CoStAR activity resulted in superior T cell proliferation, even in the absence of exogenous IL-2. Using an in vivo transplantable tumor model, CoStAR was shown to improve T cell survival after transfer, enhanced control of tumor growth, and improved host survival. CoStAR could be reliably engineered into TIL from multiple tumor indications and augmented TIL activity against autologous tumor targets both in vitro and in vivo. CoStAR thus represents a general approach to improving TIL therapy with synthetic costimulation.
Asunto(s)
Melanoma , Receptores Quiméricos de Antígenos , Humanos , Linfocitos T , Antígenos CD28 , Linfocitos Infiltrantes de Tumor , Receptor 1 de Folato , Receptores Quiméricos de Antígenos/genética , Antígenos CD40 , Microambiente TumoralRESUMEN
The molecular rules that govern MHC restriction, and allow T-cells to differentiate between peptides derived from healthy cells and those from diseased cells, remain poorly understood. Here we provide an overview of the structural constraints that enable the T-cell receptor (TCR) to discriminate between self and non-self peptides, and summarize studies that have attempted to correlate the biophysical parameters of TCR/peptide-major histocompatibility complex (pMHC) binding with T-cell activation. We further review how the antigenic origin of peptide epitopes affects TCR binding parameters and the 'quality' of a T-cell response. Understanding the principles that govern pMHC recognition by T-cells will unlock pathways to the rational development of immunotherapeutic approaches for the treatment of infectious disease, cancer and autoimmunity.
Asunto(s)
Autoantígenos/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Autoinmunidad/inmunología , Antígenos CD4/inmunología , Antígenos CD8/inmunología , Humanos , Activación de Linfocitos/inmunología , Complejo Mayor de Histocompatibilidad/genética , Complejo Mayor de Histocompatibilidad/inmunología , Péptidos/inmunología , Conformación Proteica , Receptores de Antígenos de Linfocitos T alfa-beta/química , Receptores de Antígenos de Linfocitos T alfa-beta/genéticaRESUMEN
Chimeric Ag receptors (CARs) expressed in T cells permit the redirected lysis of tumor cells in an MHC-unrestricted manner. In the Jurkat T cell model system, expression of a carcinoembryonic Ag-specific CD3zeta CAR (MFEzeta) resulted in an increased sensitivity of the transduced Jurkat cell to generate cytokines when stimulated through the endogenous TCR complex. This effect was driven through two key characteristics of the MFEzeta CAR: 1) receptor dimerization and 2) the interaction of the CAR with the endogenous TCR complex. Mutations of the CAR transmembrane domain that abrogated these interactions resulted in a reduced functional capacity of the MFEzeta CAR to respond to carcinoembryonic Ag protein Ag. Taken together, these results indicate that CARs containing the CD3zeta transmembrane domain can form a complex with the endogenous TCR that may be beneficial for optimal T cell activation. This observation has potential implications for the future design of CARs for cancer therapy.
Asunto(s)
Complejo CD3/inmunología , Activación de Linfocitos/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Animales , Western Blotting , Complejo CD3/metabolismo , Antígeno Carcinoembrionario/inmunología , Separación Celular , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Inmunoprecipitación , Células Jurkat , Ratones , Receptores de Antígenos de Linfocitos T/metabolismo , Regulación hacia ArribaRESUMEN
Nonselected autologous tumor-infiltrating lymphocytes (TILs) may provide advantages over other treatments for solid tumors, including checkpoint inhibitor-refractory melanoma. This retrospective analysis reports a single-center experience of nonselected autologous TILs derived from digested tumors for compassionate use treatment of advanced cutaneous melanoma, including after programmed cell death protein 1 (PD-1) inhibition. Patients with histologically confirmed metastatic cutaneous melanoma and no standard-of-care treatment options underwent tumor resection for TIL product manufacturing. Patients received lymphodepleting chemotherapy with cyclophosphamide for 2 days and fludarabine for 5 days, followed by a single TIL infusion and post-TIL high-dose interleukin (IL)-2. Safety assessments included clinically significant adverse events (AEs). Efficacy assessments included overall response rate (ORR), complete response (CR) rate, disease control rate (DCR), and overall survival. Between October 2011 and August 2019, 21 patients underwent treatment (median follow-up time, 52.2 months from TIL infusion). Among all treated patients, median age was 45 years, median number of disease sites was 4, 100% had M1c or M1d disease, and 90% received prior checkpoint inhibitor. Twelve patients received TILs after prior PD-1 inhibition. The safety profile among all treated patients and the prior PD-1 inhibitor subgroup was generally consistent with lymphodepletion and high-dose IL-2. No treatment-related deaths occurred. Among all patients, the ORR was 67%, CR rate was 19%, and the DCR was 86%, which was consistent with that observed in the prior PD-1 inhibitor subgroup (58%, 8%, and 75%, respectively). Median overall survival in all treated patients and the prior PD-1 inhibitor subgroup was 21.3 months. In total, 5 patients (24%) had durable ongoing responses (>30 months post-TIL infusion) at data cutoff, and all patients who achieved CR remained alive and disease free. To further illustrate how TIL therapy may integrate into established treatment paradigms, several case studies of patients treated in this series were included. Overall, these data demonstrate that manufacturing of nonselected autologous TILs from tumor digests is feasible and resulted in high rates of durable response in poor-risk patient populations, which may address significant unmet medical need.
RESUMEN
Co-immunoprecipitation is the classical approach for investigating protein-protein interactions. Analysis is generally conducted using the Western blot approach. We set out to investigate whether flow cytometry was a feasible alternative to Western blotting. Using the TCR-CD3 complex as a model for intermolecular interactions in the MA5.8 cell line, FLAG-tagged CD3zeta-scFv fusion proteins could be captured on anti-FLAG coupled beads and associated TCRbeta molecules could be detected by flow cytometry. This association was abrogated by mutations to the CD3zeta transmembrane domain. Using multicolor flow cytometry, TCRbeta, CD3epsilon, and the scFv region of the CD3zeta fusion molecule could all be detected from a single sample. This multicolor analysis was then applied to demonstrate the importance of correct lysis conditions for extraction of the TCR complex. In summary, this flow cytometric immunoprecipitation technique is a feasible alternative to classical co-immunoprecipitation analysis technique and offers many potential advantages including rapid analysis with increased target sensitivity, reduced technical demands, amenable to multiple protein analysis from a single sample, and provides a framework that may facilitate the development of high throughput analytical assays investigating protein-protein interactions.
Asunto(s)
Citometría de Flujo/métodos , Inmunoprecipitación/métodos , Receptores de Antígenos de Linfocitos T/metabolismo , Complejo CD3/metabolismo , Extractos Celulares , Línea Celular , Membrana Celular/metabolismo , Color , Humanos , Región Variable de Inmunoglobulina/inmunología , Unión Proteica , Proteínas Recombinantes de Fusión/metabolismo , Transducción GenéticaRESUMEN
With the advent of immunotherapy as a realistic and promising option for cancer treatment, adoptive cellular therapies are gaining significant interest in the clinic. Whilst the recent successes of chimeric antigen receptor T-cell therapies for haematological malignancies are widely known, they have yet to show great success in solid cancers. However, immune cells transduced with T-cell receptors have been shown to traffic to and exert anti-cancer effects on solid tumour cells with some great successes. In this review, we explore the field of transgenic T-cell receptor immunotherapy, highlighting some of the key clinical trials which have paved the way for this type of cellular immunotherapy. Some trials have shown amazing clinical results, including long-term remissions and minimal toxicity, and can be looked at as an exemplar for this adoptive cell therapy. There have also been key trials where unexpected, fatal, off-tumour toxicity has occurred, and these trials have also been instrumental in shaping safer clinical trials, particularly regarding preclinical testing. In addition to previous trials, we analysed the current clinical trial space for T-cell receptor T-cell therapy, showing which trials are dominating in the clinic and which targets are being prioritised by researchers around the world. By looking at both past and current trials, we have been able to identify key drivers in developing transgenic T-cell receptor immunotherapy for the future.
RESUMEN
T cells engineered to express antigen-specific T cell receptors (TCRs) are potent therapies for viral infections and cancer. However, efficient identification of clinical candidate TCRs is complicated by the size and complexity of T cell repertoires and the challenges of working with primary T cells. Here we present a high-throughput method to identify TCRs with high functional avidity from diverse human T cell repertoires. The approach used massively parallel microfluidics to generate libraries of natively paired, full-length TCRαß clones, from millions of primary T cells, which were then expressed in Jurkat cells. The TCRαß-Jurkat libraries enabled repeated screening and panning for antigen-reactive TCRs using peptide major histocompatibility complex binding and cellular activation. We captured more than 2.9 million natively paired TCRαß clonotypes from six healthy human donors and identified rare (<0.001% frequency) viral-antigen-reactive TCRs. We also mined a tumor-infiltrating lymphocyte sample from a patient with melanoma and identified several tumor-specific TCRs, which, after expression in primary T cells, led to tumor cell killing.
Asunto(s)
Antígenos/análisis , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Linfocitos T/citología , Ingeniería Celular , Biblioteca de Genes , Humanos , Células Jurkat , Linfocitos Infiltrantes de Tumor/inmunología , Melanoma/inmunología , Linfocitos T/inmunología , Virus/inmunologíaRESUMEN
OBJECTIVES: Vaccines that prime Wilms' tumor 1 (WT1)-specific CD8+ T cells are attractive cancer immunotherapies. However, immunogenicity and clinical response rates may be enhanced by delivering WT1 to CD141+ dendritic cells (DCs). The C-type lectin-like receptor CLEC9A is expressed exclusively by CD141+ DCs and regulates CD8+ T-cell responses. We developed a new vaccine comprising a human anti-CLEC9A antibody fused to WT1 and investigated its capacity to target human CD141+ DCs and activate naïve and memory WT1-specific CD8+ T cells. METHODS: WT1 was genetically fused to antibodies specific for human CLEC9A, DEC-205 or ß-galactosidase (untargeted control). Activation of WT1-specific CD8+ T-cell lines following cross-presentation by CD141+ DCs was quantified by IFNγ ELISPOT. Humanised mice reconstituted with human immune cell subsets, including a repertoire of naïve WT1-specific CD8+ T cells, were used to investigate naïve WT1-specific CD8+ T-cell priming. RESULTS: The CLEC9A-WT1 vaccine promoted cross-presentation of WT1 epitopes to CD8+ T cells and mediated priming of naïve CD8+ T cells more effectively than the DEC-205-WT1 and untargeted control-WT1 vaccines. CONCLUSIONS: Delivery of WT1 to CD141+ DCs via CLEC9A stimulates CD8+ T cells more potently than either untargeted delivery or widespread delivery to all Ag-presenting cells via DEC-205, suggesting that cross-presentation by CD141+ DCs is sufficient for effective CD8+ T-cell priming in humans. The CLEC9A-WT1 vaccine is a promising candidate immunotherapy for malignancies that express WT1.
RESUMEN
L-selectin on T-cells is best known as an adhesion molecule that supports recruitment of blood-borne naïve and central memory cells into lymph nodes. Proteolytic shedding of the ectodomain is thought to redirect activated T-cells from lymph nodes to sites of infection. However, we have shown that activated T-cells re-express L-selectin before lymph node egress and use L-selectin to locate to virus-infected tissues. Therefore, we considered other roles for L-selectin proteolysis during T cell activation. In this study, we used T cells expressing cleavable or non-cleavable L-selectin and determined the impact of L-selectin proteolysis on T cell activation in virus-infected mice. We confirm an essential and non-redundant role for ADAM17 in TCR-induced proteolysis of L-selectin in mouse and human T cells and show that L-selectin cleavage does not regulate T cell activation measured by CD69 or TCR internalisation. Following virus infection of mice, L-selectin proteolysis promoted early clonal expansion of cytotoxic T cells resulting in an 8-fold increase over T cells unable to cleave L-selectin. T cells unable to cleave L-selectin showed delayed proliferation in vitro which correlated with lower CD25 expression. Based on these results, we propose that ADAM17-dependent proteolysis of L-selectin should be considered a regulator of T-cell activation at sites of immune activity.
Asunto(s)
Proteína ADAM17/metabolismo , Células Clonales/inmunología , Selectina L/metabolismo , Linfocitos T Citotóxicos/inmunología , Virosis/metabolismo , Proteína ADAM17/genética , Animales , Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos T/metabolismo , Movimiento Celular , Células Cultivadas , Humanos , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Selectina L/genética , Lectinas Tipo C/metabolismo , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Mutación , Proteolisis , Virosis/inmunologíaRESUMEN
Serial accumulation of mutations to fixation in the SLYNTVATL (SL9) immunodominant, HIV p17 Gag-derived, HLA A2-restricted cytotoxic T lymphocyte epitope produce the SLFNTIAVL triple mutant "ultimate" escape variant. These mutations in solvent-exposed residues are believed to interfere with TCR recognition, although confirmation has awaited structural verification. Here, we solved a TCR co-complex structure with SL9 and the triple escape mutant to determine the mechanism of immune escape in this eminent system. We show that, in contrast to prevailing hypotheses, the main TCR contact residue is 4N and the dominant mechanism of escape is not via lack of TCR engagement. Instead, mutation of solvent-exposed residues in the peptide destabilise the peptide-HLA and reduce peptide density at the cell surface. These results highlight the extraordinary lengths that HIV employs to evade detection by high-affinity TCRs with a broad peptide-binding footprint and necessitate re-evaluation of this exemplar model of HIV TCR escape.