RESUMO
Adoptive T cell therapy with T cells expressing affinity-enhanced TCRs has shown promising results in phase 1/2 clinical trials for solid and hematological tumors. However, depth and durability of responses to adoptive T cell therapy can suffer from an inhibitory tumor microenvironment. A common immune-suppressive agent is TGF-ß, which is secreted by tumor cells and cells recruited to the tumor. We investigated whether human T cells could be engineered to be resistant to inhibition by TGF-ß. Truncating the intracellular signaling domain from TGF-ß receptor (TGFßR) II produces a dominant-negative receptor (dnTGFßRII) that dimerizes with endogenous TGFßRI to form a receptor that can bind TGF-ß but cannot signal. We previously generated specific peptide enhanced affinity receptor TCRs recognizing the HLA-A*02-restricted peptides New York esophageal squamous cell carcinoma 1 (NY-ESO-1)157-165/l-Ag family member-1A (TCR: GSK3377794, formerly NY-ESO-1c259) and melanoma Ag gene A10254-262 (TCR: ADP-A2M10, formerly melanoma Ag gene A10c796). In this article, we show that exogenous TGF-ß inhibited in vitro proliferation and effector functions of human T cells expressing these first-generation high-affinity TCRs, whereas inhibition was reduced or abolished in the case of second-generation TCRs coexpressed with dnTGFßRII (e.g., GSK3845097). TGF-ß isoforms and a panel of TGF-ß-associated genes are overexpressed in a range of cancer indications in which NY-ESO-1 is commonly expressed, particularly in synovial sarcoma. As an example, immunohistochemistry/RNAscope identified TGF-ß-positive cells close to T cells in tumor nests and stroma, which had low frequencies of cells expressing IFN-γ in a non-small cell lung cancer setting. Coexpression of dnTGFßRII may therefore improve the efficacy of TCR-transduced T cells.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Carcinoma de Células Escamosas/terapia , Neoplasias Hematológicas/terapia , Imunoterapia Adotiva/métodos , Melanoma/terapia , Receptor do Fator de Crescimento Transformador beta Tipo II/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos Quiméricos/metabolismo , Sarcoma Sinovial/terapia , Fator de Crescimento Transformador beta/metabolismo , Antígenos de Neoplasias/imunologia , Carcinoma de Células Escamosas/imunologia , Linhagem Celular Tumoral , Engenharia Genética , Antígeno HLA-A2/metabolismo , Neoplasias Hematológicas/imunologia , Humanos , Tolerância Imunológica , Melanoma/imunologia , Proteínas de Membrana/imunologia , Proteínas de Neoplasias/imunologia , Fragmentos de Peptídeos/imunologia , Receptor do Fator de Crescimento Transformador beta Tipo II/genética , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos Quiméricos/genética , Sarcoma Sinovial/imunologia , Especificidade do Receptor de Antígeno de Linfócitos T , Microambiente TumoralRESUMO
Patients with hepatocellular carcinoma (HCC) have a poor prognosis and limited therapeutic options. Alpha-fetoprotein (AFP) is often expressed at high levels in HCC and is an established clinical biomarker of the disease. Expression of AFP in nonmalignant liver can occur, particularly in a subset of progenitor cells and during chronic inflammation, at levels typically lower than in HCC. This cancer-specific overexpression indicates that AFP may be a promising target for immunotherapy. We verified expression of AFP in normal and diseased tissue and generated an affinity-optimized T-cell receptor (TCR) with specificity to AFP/HLA-A*02+ tumors. Expression of AFP was investigated using database searches, by qPCR, and by immunohistochemistry (IHC) analysis of a panel of human tissue samples, including normal, diseased, and malignant liver. Using in vitro mutagenesis and screening, we generated a TCR that recognizes the HLA-A*02-restricted AFP158-166 peptide, FMNKFIYEI, with an optimum balance of potency and specificity. These properties were confirmed by an extension of the alanine scan (X-scan) and testing TCR-transduced T cells against normal and tumor cells covering a variety of tissues, cell types, and human leukocyte antigen (HLA) alleles. Conclusion: We have used a combination of physicochemical, in silico, and cell biology methods for optimizing a TCR for improved affinity and function, with properties that are expected to allow TCR-transduced T cells to differentiate between antigen levels on nonmalignant and cancer cells. T cells transduced with this TCR constitute the basis for a trial of HCC adoptive T-cell immunotherapy.
Assuntos
Carcinoma Hepatocelular/imunologia , Antígeno HLA-A2/metabolismo , Neoplasias Hepáticas/imunologia , Receptores de Antígenos de Linfócitos T/uso terapêutico , alfa-Fetoproteínas/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/terapia , Células Hep G2 , Humanos , Imunoterapia/métodos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/terapia , Receptores de Antígenos de Linfócitos T/imunologiaRESUMO
UNLABELLED: Epstein-Barr Virus (EBV) persists for the lifetime of the infected host despite eliciting strong immune responses. This persistence requires a fine balance between the host immune system and EBV immune evasion. Accumulating evidence suggests an important role for natural killer (NK) cells in this balance. NK cells can kill EBV-infected cells undergoing lytic replication in vitro, and studies in both humans and mice with reconstituted human immune systems have shown that NK cells can limit EBV replication and prevent infectious mononucleosis. We now show that NK cells, via NKG2D and DNAM-1 interactions, recognize and kill EBV-infected cells undergoing lytic replication and that expression of a single EBV lytic gene, BZLF1, is sufficient to trigger sensitization to NK cell killing. We also present evidence suggesting the possibility of the existence of an as-yet-unidentified DNAM-1 ligand which may be particularly important for killing lytically infected normal B cells. Furthermore, while cells entering the lytic cycle become sensitized to NK cell killing, we observed that cells in the late lytic cycle are highly resistant. We identified expression of the vBcl-2 protein, BHRF1, as one effective mechanism by which EBV mediates this protection. Thus, contrary to the view expressed in some reports, EBV has evolved the ability to evade NK cell responses. IMPORTANCE: This report extends our understanding of the interaction between EBV and host innate responses. It provides the first evidence that the susceptibility to NK cell lysis of EBV-infected B cells undergoing lytic replication is dependent upon the phase of the lytic cycle. Induction of the lytic cycle is associated with acquired sensitization to NK cell killing, while progress through the late lytic cycle is associated with acquired resistance to killing. We provide mechanistic explanations for this novel observation, indicating important roles for the BZLF1 immediate early transactivator, the BHRF1 vBcl-2 homologue, and a novel ligand for the DNAM-1 NK cell receptor.
Assuntos
Linfócitos B/imunologia , Linfócitos B/virologia , Herpesvirus Humano 4/fisiologia , Evasão da Resposta Imune , Células Matadoras Naturais/imunologia , Transativadores/metabolismo , Ativação Viral , Células Cultivadas , Herpesvirus Humano 4/imunologia , Humanos , Replicação ViralRESUMO
UNLABELLED: The ability of Epstein-Barr virus (EBV) to spread and persist in human populations relies on a balance between host immune responses and EBV immune evasion. CD8(+) cells specific for EBV late lytic cycle antigens show poor recognition of target cells compared to immediate early and early antigen-specific CD8(+) cells. This phenomenon is due in part to the early EBV protein BILF1, whose immunosuppressive activity increases with lytic cycle progression. However, published data suggest the existence of a hitherto unidentified immune evasion protein further enhancing protection against late EBV antigen-specific CD8(+) cells. We have now identified the late lytic BDLF3 gene as the missing link accounting for efficient evasion during the late lytic cycle. Interestingly, BDLF3 also contributes to evasion of CD4(+) cell responses to EBV. We report that BDLF3 downregulates expression of surface major histocompatibility complex (MHC) class I and class II molecules in the absence of any effect upon other surface molecules screened, including CD54 (ICAM-1) and CD71 (transferrin receptor). BDLF3 both enhanced internalization of surface MHC molecules and reduced the rate of their appearance at the cell surface. The reduced expression of surface MHC molecules correlated with functional protection against CD8(+) and CD4(+) T cell recognition. The molecular mechanism was identified as BDLF3-induced ubiquitination of MHC molecules and their subsequent downregulation in a proteasome-dependent manner. IMPORTANCE: Immune evasion is a necessary feature of viruses that establish lifelong persistent infections in the face of strong immune responses. EBV is an important human pathogen whose immune evasion mechanisms are only partly understood. Of the EBV immune evasion mechanisms identified to date, none could explain why CD8(+) T cell responses to late lytic cycle genes are so infrequent and, when present, recognize lytically infected target cells so poorly relative to CD8(+) T cells specific for early lytic cycle antigens. The present work identifies an additional immune evasion protein, BDLF3, that is expressed late in the lytic cycle and impairs CD8(+) T cell recognition by targeting cell surface MHC class I molecules for ubiquitination and proteasome-dependent downregulation. Interestingly, BDLF3 also targets MHC class II molecules to impair CD4(+) T cell recognition. BDLF3 is therefore a rare example of a viral protein that impairs both the MHC class I and class II antigen-presenting pathways.
Assuntos
Herpesvirus Humano 4/imunologia , Herpesvirus Humano 4/fisiologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Evasão da Resposta Imune , Glicoproteínas de Membrana/metabolismo , Ubiquitinação , Proteínas Virais/metabolismo , Linhagem Celular , Regulação para Baixo , Humanos , Complexo Principal de Histocompatibilidade , Proteínas de Membrana/metabolismo , Linfócitos T/imunologiaRESUMO
CD8+ T cell responses to Epstein-Barr virus (EBV) lytic cycle expressed antigens display a hierarchy of immunodominance, in which responses to epitopes of immediate-early (IE) and some early (E) antigens are more frequently observed than responses to epitopes of late (L) expressed antigens. It has been proposed that this hierarchy, which correlates with the phase-specific efficiency of antigen presentation, may be due to the influence of viral immune-evasion genes. At least three EBV-encoded genes, BNLF2a, BGLF5 and BILF1, have the potential to inhibit processing and presentation of CD8+ T cell epitopes. Here we examined the relative contribution of these genes to modulation of CD8+ T cell recognition of EBV lytic antigens expressed at different phases of the replication cycle in EBV-transformed B-cells (LCLs) which spontaneously reactivate lytic cycle. Selective shRNA-mediated knockdown of BNLF2a expression led to more efficient recognition of immediate-early (IE)- and early (E)-derived epitopes by CD8+ T cells, while knock down of BILF1 increased recognition of epitopes from E and late (L)-expressed antigens. Contrary to what might have been predicted from previous ectopic expression studies in EBV-negative model cell lines, the shRNA-mediated inhibition of BGLF5 expression in LCLs showed only modest, if any, increase in recognition of epitopes expressed in any phase of lytic cycle. These data indicate that whilst BNLF2a interferes with antigen presentation with diminishing efficiency as lytic cycle progresses (IE>E>>L), interference by BILF1 increases with progression through lytic cycle (IEAssuntos
Apresentação de Antígeno/imunologia
, Linfócitos T CD8-Positivos/imunologia
, Infecções por Vírus Epstein-Barr/imunologia
, Evasão da Resposta Imune/imunologia
, Western Blotting
, Desoxirribonucleases/imunologia
, Técnicas de Silenciamento de Genes
, Herpesvirus Humano 4/imunologia
, Humanos
, Reação em Cadeia da Polimerase em Tempo Real
, Receptores Acoplados a Proteínas G/imunologia
, Proteínas da Matriz Viral/imunologia
, Proteínas Virais/imunologia
RESUMO
EBV elicits primary CD8(+) T cell responses that, by T cell cloning from infectious mononucleosis (IM) patients, appear skewed toward immediate early (IE) and some early (E) lytic cycle proteins, with late (L) proteins rarely targeted. However, L Ag-specific responses have been detected regularly in polyclonal T cell cultures from long-term virus carriers. To resolve this apparent difference between responses to primary and persistent infection, 13 long-term carriers were screened in ex vivo IFN-γ ELISPOT assays using peptides spanning the two IE, six representative E, and seven representative L proteins. This revealed memory CD8 responses to 44 new lytic cycle epitopes that straddle all three protein classes but, in terms of both frequency and size, maintain the IE > E > L hierarchy of immunodominance. Having identified the HLA restriction of 10 (including 7 L) new epitopes using memory CD8(+) T cell clones, we looked in HLA-matched IM patients and found such reactivities but typically at low levels, explaining why they had gone undetected in the original IM clonal screens. Wherever tested, all CD8(+) T cell clones against these novel lytic cycle epitopes recognized lytically infected cells naturally expressing their target Ag. Surprisingly, however, clones against the most frequently recognized L Ag, the BNRF1 tegument protein, also recognized latently infected, growth-transformed cells. We infer that BNRF1 is also a latent Ag that could be targeted in T cell therapy of EBV-driven B-lymphoproliferative disease.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Herpesvirus Humano 4/imunologia , Mononucleose Infecciosa/imunologia , Sequência de Aminoácidos , Linfócitos T CD8-Positivos/virologia , Células Cultivadas , ELISPOT , Antígenos HLA/metabolismo , Humanos , Epitopos Imunodominantes/imunologia , Epitopos Imunodominantes/metabolismo , Interferon gama/metabolismo , Dados de Sequência Molecular , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/metabolismo , Latência Viral/imunologiaRESUMO
Regulating appropriate activation of the immune response in the healthy host despite continual immune surveillance dictates that immune responses must be either self-limiting and therefore negatively regulated following their activation or prevented from developing inappropriately. In the case of antigen-specific T cells, their response is attenuated by several mechanisms, including ligation of CTLA-4 and PD-1. Through the study of the viral OX2 (vOX2) immunoregulator encoded by Kaposi's sarcoma-associated herpesvirus (KSHV), we have identified a T cell-attenuating role both for this protein and for CD200, a cellular orthologue of the viral vOX2 protein. In vitro, antigen-presenting cells (APC) expressing either native vOX2 or CD200 suppressed two functions of cognate antigen-specific T cell clones: gamma interferon (IFN-γ) production and mobilization of CD107a, a cytolytic granule component and measure of target cell killing ability. Mechanistically, vOX2 and CD200 expression on APC suppressed the phosphorylation of ERK1/2 mitogen-activated protein kinase in responding T cells. These data provide the first evidence for a role of both KSHV vOX2 and cellular CD200 in the negative regulation of antigen-specific T cell responses. They suggest that KSHV has evolved to harness the host CD200-based mechanism of attenuation of T cell responses to facilitate virus persistence and dissemination within the infected individual. Moreover, our studies define a new paradigm in immune modulation by viruses: the provision of a negative costimulatory signal to T cells by a virus-encoded orthologue of CD200.
Assuntos
Antígenos CD/metabolismo , Herpesvirus Humano 8/imunologia , Herpesvirus Humano 8/patogenicidade , Tolerância Imunológica , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Neuropeptídeos/metabolismo , Linfócitos T/imunologia , Proteínas Virais/metabolismo , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/virologia , Antígenos CD/imunologia , Humanos , Interferon gama/metabolismo , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , Receptores de Orexina , Receptores Acoplados a Proteínas G/imunologia , Receptores de Neuropeptídeos/imunologia , Proteínas Virais/imunologia , Fatores de Virulência/imunologia , Fatores de Virulência/metabolismoRESUMO
EBV, a B lymphotropic herpesvirus, encodes two immediate early (IE)-, >30 early (E)-, and >30 late (L)-phase proteins during its replication (lytic) cycle. Despite this, lytic Ag-induced CD8 responses are strongly skewed toward IE and a few E proteins only, all expressed before HLA I presentation is blocked in lytically infected cells. For comparison, we examined CD4(+) T cell responses to eight IE, E, or L proteins, screening 14 virus-immune donors to overlapping peptide pools in IFN-γ ELISPOT assays, and established CD4(+) T cell clones against 12 defined epitopes for target-recognition assays. We found that the lytic Ag-specific CD4(+) T cell response differs radically from its CD8 counterpart in that it is widely distributed across IE, E, and L Ag targets, often with multiple reactivities detectable per donor and with IE, E, or L epitope responses being numerically dominant, and that all CD4(+) T cell clones, whether IE, E, or L epitope-specific, show strong recognition of EBV-transformed B cell lines, despite the lines containing only a small fraction of lytically infected cells. Efficient recognition occurs because lytic Ags are released into the culture and are acquired and processed by neighboring latently infected cells. These findings suggested that lytic Ag-specific CD4 responses are driven by a different route of Ag display than drives CD8 responses and that such CD4 effectors could be therapeutically useful against EBV-driven lymphoproliferative disease lesions, which contain similarly small fractions of EBV-transformed cells entering the lytic cycle.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , Testes Imunológicos de Citotoxicidade/métodos , Regulação Viral da Expressão Gênica/imunologia , Herpesvirus Humano 4/imunologia , Replicação Viral/imunologia , Antígenos Virais/biossíntese , Antígenos Virais/imunologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD8-Positivos/citologia , Células Cultivadas , Células Clonais , Mapeamento de Epitopos/métodos , Epitopos de Linfócito T/imunologia , Herpesvirus Humano 4/crescimento & desenvolvimento , Humanos , Proteínas Imediatamente Precoces/biossíntese , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/imunologia , Interferon gama/metabolismo , Fosfoproteínas/imunologia , Transativadores/biossíntese , Transativadores/genética , Transativadores/imunologiaRESUMO
Adoptive cell therapy with T cells expressing affinity-enhanced T-cell receptors (TCRs) is a promising treatment for solid tumors. Efforts are ongoing to further engineer these T cells to increase the depth and durability of clinical responses and broaden efficacy toward additional indications. In the present study, we investigated one such approach: T cells were transduced with a lentiviral vector to coexpress an affinity-enhanced HLA class I-restricted TCR directed against MAGE-A4 alongside a CD8α coreceptor. We hypothesized that this approach would enhance CD4 + T-cell helper and effector functions, possibly leading to a more potent antitumor response. Activation of transduced CD4 + T cells was measured by detecting CD40 ligand expression on the surface and cytokine and chemokine secretion from CD4 + T cells and dendritic cells cultured with melanoma-associated antigen A4 + tumor cells. In addition, T-cell cytotoxic activity against 3-dimensional tumor spheroids was measured. Our data demonstrated that CD4 + T cells coexpressing the TCR and CD8α coreceptor displayed enhanced responses, including CD40 ligand expression, interferon-gamma secretion, and cytotoxic activity, along with improved dendritic cell activation. Therefore, our study supports the addition of the CD8α coreceptor to HLA class I-restricted TCR-engineered T cells to enhance CD4 + T-cell functions, which may potentially improve the depth and durability of antitumor responses in patients.
Assuntos
Antineoplásicos , Ligante de CD40 , Humanos , Linfócitos T CD4-Positivos , Linfócitos T Auxiliares-Indutores , Receptores de Antígenos de Linfócitos T/metabolismoRESUMO
Despite triggering strong immune responses, Epstein-Barr virus (EBV) has colonized more than 90% of the adult human population. Successful persistence of EBV depends on the establishment of a balance between host immune responses and viral immune evasion. Here we have extended our studies on the EBV-encoded BILF1 protein, which was recently identified as an immunoevasin that functions by enhancing degradation of major histocompatibility complex class I (MHC-I) antigens via lysosomes. We now demonstrate that disruption of the EKT signaling motif of BILF1 by a K122A mutation impairs the ability of BILF1 to enhance endocytosis of surface MHC-I molecules, while subsequent lysosomal degradation was impaired by deletion of the 21-residue C-terminal tail of BILF1. Furthermore, we identified another mechanism of BILF1 immunomodulation: it targets newly synthesized MHC-I/peptide complexes en route to the cell surface. Importantly, although the diversion of MHC-I on the exocytic pathway caused a relatively modest reduction in cell surface MHC-I, presentation of endogenously processed target peptides to immune CD8(+) effector T cells was reduced by around 65%. The immune-modulating functions of BILF1 in the context of the whole virus were confirmed in cells lytically infected with a recombinant EBV in which BILF1 was deleted. This study therefore extends our initial observations on BILF1 to show that this immunoevasin can target MHC-I antigen presentation via both the exocytic and endocytic trafficking pathways. The results also emphasize the merits of including functional T cell recognition assays to gain a more complete picture of immunoevasin effects on the antigen presentation pathway.
Assuntos
Apresentação de Antígeno/imunologia , Endocitose/imunologia , Exocitose/imunologia , Herpesvirus Humano 4/patogenicidade , Antígenos de Histocompatibilidade Classe I/imunologia , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Virais/metabolismo , Animais , Linhagem Celular , Regulação da Expressão Gênica/imunologia , Células HEK293 , Herpesvirus Humano 4/fisiologia , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Camundongos , Transdução de Sinais , Linfócitos T Citotóxicos/imunologiaRESUMO
A substantial obstacle to the success of adoptive T cell-based cancer immunotherapy is the sub-optimal affinity of T-cell receptors (TCRs) for most tumor antigens. Genetically engineered TCRs that have enhanced affinity for specific tumor peptide-MHC complexes may overcome this barrier. However, this enhancement risks increasing weak TCR cross-reactivity to other antigens expressed by normal tissues, potentially leading to clinical toxicities. To reduce the risk of such adverse clinical outcomes, we have developed an extensive preclinical testing strategy, involving potency testing using 2D and 3D human cell cultures and primary tumor material, and safety testing using human primary cell and cell-line cross-reactivity screening and molecular analysis to predict peptides recognized by the affinity-enhanced TCR. Here, we describe this strategy using a developmental T-cell therapy, ADP-A2M4, which recognizes the HLA-A2-restricted MAGE-A4 peptide GVYDGREHTV. ADP-A2M4 demonstrated potent anti-tumor activity in the absence of major off-target cross-reactivity against a range of human primary cells and cell lines. Identification and characterization of peptides recognized by the affinity-enhanced TCR also revealed no cross-reactivity. These studies demonstrated that this TCR is highly potent and without major safety concerns, and as a result, this TCR is now being investigated in two clinical trials (NCT03132922, NCT04044768).