RESUMO
Cancer immunotherapy using T cell receptor-engineered T cells (TCR-Ts) represents a promising treatment option. However, technologies for pre-clinical safety assessment are incomplete or inaccessible to most laboratories. Here, TCR-T off-target reactivity was assessed in five steps: (1) Mapping target amino acids necessary for TCR-T recognition, followed by (2) a computational search for, and (3) reactivity screening against, candidate cross-reactive peptides in the human proteome. Natural processing and presentation of recognized peptides was evaluated using (4) short mRNAs, and (5) full-length proteins. TCR-Ts were screened for recognition of unintended HLA alleles, and as proxy for off-target reactivity in vivo, a syngeneic, HLA-A*02:01-transgenic mouse model was used. Validation demonstrated importance of studying recognition of full-length candidate off-targets, and that the clinically applied 1G4 TCR has a hitherto unknown reactivity to unintended HLA alleles, relevant for patient selection. This widely applicable strategy should facilitate evaluation of candidate therapeutic TCRs and inform clinical decision-making.
RESUMO
The emergence of SARS-CoV-2 variants of concern (VOC) is driven by mutations that mediate escape from neutralizing antibodies. There is also evidence that mutations can cause loss of T cell epitopes. However, studies on viral escape from T cell immunity have been hampered by uncertain estimates of epitope prevalence. Here, we map and quantify CD8 T cell responses to SARS-CoV-2-specific minimal epitopes in blood drawn from April to June 2020 from 83 COVID-19 convalescents. Among 37 HLA ligands eluted from five prevalent alleles and an additional 86 predicted binders, we identify 29 epitopes with an immunoprevalence ranging from 3% to 100% among individuals expressing the relevant HLA allele. Mutations in VOC are reported in 10.3% of the epitopes, while 20.6% of the non-immunogenic peptides are mutated in VOC. The nine most prevalent epitopes are conserved in VOC. Thus, comprehensive mapping of epitope prevalence does not provide evidence that mutations in VOC are driven by escape of T cell immunity.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Linfócitos T CD8-Positivos , COVID-19/imunologia , Epitopos de Linfócito T/genética , Epitopos Imunodominantes/genética , SARS-CoV-2/genéticaRESUMO
Unlike chimeric antigen receptors, T-cell receptors (TCRs) can recognize intracellular targets presented on human leukocyte antigen (HLA) molecules. Here we demonstrate that T cells expressing TCRs specific for peptides from the intracellular lymphoid-specific enzyme terminal deoxynucleotidyl transferase (TdT), presented in the context of HLA-A*02:01, specifically eliminate primary acute lymphoblastic leukemia (ALL) cells of T- and B-cell origin in vitro and in three mouse models of disseminated B-ALL. By contrast, the treatment spares normal peripheral T- and B-cell repertoires and normal myeloid cells in vitro, and in vivo in humanized mice. TdT is an attractive cancer target as it is highly and homogeneously expressed in 80-94% of B- and T-ALLs, but only transiently expressed during normal lymphoid differentiation, limiting on-target toxicity of TdT-specific T cells. TCR-modified T cells targeting TdT may be a promising immunotherapy for B-ALL and T-ALL that preserves normal lymphocytes.
Assuntos
DNA Nucleotidilexotransferase , Linfócitos T , Animais , Células-Tronco Hematopoéticas , Linfócitos , Camundongos , Receptores de Antígenos de Linfócitos T/genéticaRESUMO
Interferon regulatory factors (IRFs) are crucial for transcription during innate immune responses. We have previously shown that the tyrosine kinase c-Src enhances IRF-3-dependent transcription in response to viral double-stranded RNA. In this study, we show that c-Src has distinct roles in Toll-like receptor (TLR)-mediated activation of IRF-5 and IRF-3. Surprisingly, c-Src inhibition markedly enhanced IRF-5 activation after treatment with unmethylated CpG, while suppressing IRF-3 activation. Also, CpG-elicited interleukin-6 mRNA production was increased, whereas IP10 mRNA synthesis was reduced in cells deficient in c-Src. Interestingly, c-Src regulated TLR-stimulated induction of activating transcription factor 3 (ATF3), a transcriptional repressor. Depletion of ATF3 by small interfering RNA markedly enhanced interleukin-6 production after CpG treatment, whereas IP10 production was reduced. These results demonstrate functional specificity for c-Src in TLR-stimulated responses and suggest that c-Src modulation and ATF3 activity may contribute to differential regulation of IRF-3- versus IRF-5-mediated gene expression.
Assuntos
Proteínas Tirosina Quinases/metabolismo , Receptores Toll-Like/metabolismo , Fator 3 Ativador da Transcrição/metabolismo , Animais , Proteína Tirosina Quinase CSK , Linhagem Celular , Ilhas de CpG , Humanos , Fator Regulador 3 de Interferon/metabolismo , Fatores Reguladores de Interferon/metabolismo , Interleucina-6/metabolismo , Camundongos , Modelos Biológicos , Interferência de RNA , Transdução de Sinais , Quinases da Família srcRESUMO
TLR9 is critical in parasite recognition and host resistance to experimental infection with Trypanosoma cruzi. However, no information is available regarding nucleotide sequences and cellular events involved on T. cruzi recognition by TLR9. In silico wide analysis associated with in vitro screening of synthetic oligonucleotides demonstrates that the retrotransposon VIPER elements and mucin-like glycoprotein (TcMUC) genes in the T. cruzi genome are highly enriched for CpG motifs that are immunostimulatory for mouse and human TLR9, respectively. Importantly, infection with T. cruzi triggers high levels of luciferase activity under NF-kappaB-dependent transcription in HEK cells cotransfected with human TLR9, but not in control (cotransfected with human MD2/TLR4) HEK cells. Further, we observed translocation of TLR9 to the lysosomes during invasion/uptake of T. cruzi parasites by dendritic cells. Consistently, potent proinflammatory activity was observed when highly unmethylated T. cruzi genomic DNA was delivered to the endo-lysosomal compartment of host cells expressing TLR9. Thus, together our results indicate that the unmethylated CpG motifs found in the T. cruzi genome are likely to be main parasite targets and probably become available to TLR9 when parasites are destroyed in the lysosome-fused vacuoles during parasite invasion/uptake by phagocytes.
Assuntos
Células Dendríticas/imunologia , Células Dendríticas/parasitologia , Lisossomos/imunologia , NF-kappa B/metabolismo , Receptor Toll-Like 9/metabolismo , Trypanosoma cruzi/imunologia , Animais , Linhagem Celular , Ilhas de CpG/imunologia , Células Dendríticas/citologia , Interações Hospedeiro-Parasita , Humanos , Lisossomos/parasitologia , Camundongos , Camundongos Knockout , NF-kappa B/imunologia , Oligodesoxirribonucleotídeos/imunologia , Retroelementos , Receptor Toll-Like 9/imunologia , Trypanosoma cruzi/genéticaRESUMO
Toll-like receptor 2 (TLR2) is a signaling receptor for a variety of microbial products, including bacterial lipoproteins and peptidoglycan, and is central in initiating immune responses toward Gram-positive bacteria, spirochetes, and mycobacteria. The mechanisms behind regulation of TLR2 protein expression are still not well understood. By using a newly developed monoclonal antibody against mouse TLR2, we detected TLR2 protein expression on macrophages, neutrophils, and dendritic cells. Endogenous macrophage TLR2 localized mostly to the cell membrane, with particular accumulation around phagosomes containing zymosan. Treatment of macrophages with the TLR2 antibody diminished cellular response to lipoproteins and down-regulated membrane TLR2. Marked up-regulation of surface TLR2 was observed on macrophages in response to whole bacteria, lipoproteins, lipopolysaccharide, poly(I-C) (double-stranded RNA), R848, and CpG DNA, and this up-regulation appeared to be a very sensitive marker for the presence of microbial products. Up-regulation of TLR2 in response to stimuli correlated with an increased response to secondary lipoprotein exposure following a low concentration of primary lipoprotein challenge. By comparison, exposure to a larger primary challenge induced a hyporeactive state. Most interestingly, lipopolysaccharide- and double-stranded RNA-induced up-regulation of surface TLR2 in macrophages was found to be MyD88-independent, whereas the up-regulation in response to lipoproteins, R848, and CpG DNA was absent in MyD88-deficient cells. We conclude that complex mechanisms regulate expression and signaling via TLR2. Up-regulation of TLR2 in the presence of low, yet clinically relevant amounts of microbial products may be an important mechanism by which the immune system boosts its response to a beginning infection.
Assuntos
Antígenos de Diferenciação/metabolismo , Lipopolissacarídeos/farmacologia , Glicoproteínas de Membrana/metabolismo , RNA de Cadeia Dupla/farmacologia , Receptores de Superfície Celular/metabolismo , Receptores Imunológicos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Anticorpos Monoclonais , Células Dendríticas/metabolismo , Regulação para Baixo/imunologia , Citometria de Fluxo , Ligantes , Macrófagos Peritoneais/metabolismo , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C3H , Fator 88 de Diferenciação Mieloide , Neutrófilos/metabolismo , Receptores de Superfície Celular/imunologia , Receptor 2 Toll-Like , Receptores Toll-Like , Regulação para Cima/imunologiaRESUMO
Microbial DNA sequences containing unmethylated CpG dinucleotides activate Toll-like receptor 9 (TLR9). We have found that TLR9 is localized to the endoplasmic reticulum (ER) of dendritic cells (DCs) and macrophages. Because there is no precedent for immune receptor signaling in the ER, we investigated how TLR9 is activated. We show that CpG DNA binds directly to TLR9 in ligand-binding studies. CpG DNA moves into early endosomes and is subsequently transported to a tubular lysosomal compartment. Concurrent with the movement of CpG DNA in cells, TLR9 redistributes from the ER to CpG DNA-containing structures, which also accumulate MyD88. Our data indicate a previously unknown mechanism of cellular activation involving the recruitment of TLR9 from the ER to sites of CpG DNA uptake, where signal transduction is initiated.
Assuntos
Ilhas de CpG/imunologia , Proteínas de Ligação a DNA/imunologia , Retículo Endoplasmático/imunologia , Lisossomos/imunologia , Glicoproteínas de Membrana/imunologia , Receptores de Superfície Celular/imunologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/imunologia , Transporte Biológico Ativo , Linhagem Celular , Proteínas de Ligação a DNA/genética , Células Dendríticas/imunologia , Humanos , Técnicas In Vitro , Proteínas Luminescentes/genética , Proteínas Luminescentes/imunologia , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide , Receptores de Superfície Celular/genética , Receptores Imunológicos/deficiência , Receptores Imunológicos/genética , Receptores Imunológicos/imunologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Transdução de Sinais , Receptor Toll-Like 9 , Receptores Toll-LikeRESUMO
Bacterial and synthetic DNAs containing CpG dinucleotides in specific sequence contexts activate the vertebrate immune system through Toll-like receptor 9 (TLR9). In the present study, we used a synthetic nucleoside with a bicyclic heterobase [1-(2'-deoxy-beta-d-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine; R] to replace the C in CpG, resulting in an RpG dinucleotide. The RpG dinucleotide was incorporated in mouse- and human-specific motifs in oligodeoxynucleotides (oligos) and 3'-3-linked oligos, referred to as immunomers. Oligos containing the RpG motif induced cytokine secretion in mouse spleen-cell cultures. Immunomers containing RpG dinucleotides showed activity in transfected-HEK293 cells stably expressing mouse TLR9, suggesting direct involvement of TLR9 in the recognition of RpG motif. In J774 macrophages, RpG motifs activated NF-kappa B and mitogen-activated protein kinase pathways. Immunomers containing the RpG dinucleotide induced high levels of IL-12 and IFN-gamma, but lower IL-6 in time- and concentration-dependent fashion in mouse spleen-cell cultures costimulated with IL-2. Importantly, immunomers containing GTRGTT and GARGTT motifs were recognized to a similar extent by both mouse and human immune systems. Additionally, both mouse- and human-specific RpG immunomers potently stimulated proliferation of peripheral blood mononuclear cells obtained from diverse vertebrate species, including monkey, pig, horse, sheep, goat, rat, and chicken. An immunomer containing GTRGTT motif prevented conalbumin-induced and ragweed allergen-induced allergic inflammation in mice. We show that a synthetic bicyclic nucleotide is recognized in the C position of a CpG dinucleotide by immune cells from diverse vertebrate species without bias for flanking sequences, suggesting a divergent nucleotide motif recognition pattern of TLR9.