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1.
Cancer Immunol Immunother ; 73(8): 150, 2024 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-38832948

RESUMO

Hotspot driver mutations presented by human leukocyte antigens might be recognized by anti-tumor T cells. Based on their advantages of tumor-specificity and immunogenicity, neoantigens derived from hotspot mutations, such as PIK3CAH1047L, may serve as emerging targets for cancer immunotherapies. NetMHCpan V4.1 was utilized for predicting neoepitopes of PIK3CA hotspot mutation. Using in vitro stimulation, antigen-specific T cells targeting the HLA-A*11:01-restricted PIK3CA mutation were isolated from healthy donor-derived peripheral blood mononuclear cells. T cell receptors (TCRs) were cloned using single-cell PCR and sequencing. Their functionality was assessed through T cell activation markers, cytokine production and cytotoxic response to cancer cell lines pulsed with peptides or transduced genes of mutant PIK3CA. Immunogenic mutant antigens from PIK3CA and their corresponding CD8+ T cells were identified. These PIK3CA mutation-specific CD8+ T cells were subsequently enriched, and their TCRs were isolated. The TCR clones exhibited mutation-specific and HLA-restricted reactivity, demonstrating varying degrees of functional avidity. Identified TCR genes were transferred into CD8+ Jurkat cells and primary T cells deficient of endogenous TCRs. TCR-expressing cells demonstrated specific recognition and reactivity against the PIK3CAH1047L peptide presented by HLA-A*11:01-expressing K562 cells. Furthermore, mutation-specific TCR-T cells demonstrated an elevation in cytokine production and profound cytotoxic effects against HLA-A*11:01+ malignant cell lines harboring PIK3CAH1047L. Our data demonstrate the immunogenicity of an HLA-A*11:01-restricted PIK3CA hotspot mutation and its targeting therapeutic potential, together with promising candidates of TCR-T cell therapy.


Assuntos
Classe I de Fosfatidilinositol 3-Quinases , Mutação , Neoplasias , Receptores de Antígenos de Linfócitos T , Humanos , Classe I de Fosfatidilinositol 3-Quinases/genética , Classe I de Fosfatidilinositol 3-Quinases/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/genética , Neoplasias/imunologia , Neoplasias/terapia , Neoplasias/genética , Imunoterapia/métodos , Antígeno HLA-A11/genética , Antígeno HLA-A11/imunologia , Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Epitopos de Linfócito T/genética , Antígenos de Neoplasias/imunologia , Antígenos de Neoplasias/genética , Linhagem Celular Tumoral
2.
Cell Immunol ; 403-404: 104863, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39186873

RESUMO

For adoptive therapy with T cell receptor engineered T (TCR-T) cells, the quantity and quality of the final cell product directly affect their anti-tumor efficacy. The post-transfer efficacy window of TCR-T cells is keen to optimizing attempts during the manufacturing process. Cbl-b is a E3 ubiquitin ligase previously shown with critical negative impact in T cell functions. This study investigated whether strategic inclusion of a commercially available small inhibitor targeting Cbl-b (Cbl-b-IN-1) prior to T cell activation could enhance the quality of the final TCR-T cell product. Examination with both PBMCs and TCR-T cells revealed that Cbl-b-IN-1 treatment promoted TCR expression efficiency, T cell proliferation potential and, specifically, cell survival capability post antigenic stimulation. Cbl-b-IN-1 exposure facilitated T cells in maintaining less differentiated states with enhanced cytokine production. Further, we found that Cbl-b-IN-1 effectively augmented the activation of TCR signaling, shown by increased phosphorylation levels of Zeta-chain-associated protein kinase 70 (ZAP70) and phospholipase c-γ1 (PLCγ1). In conclusion, our results evidence that the inclusion of Cbl-b inhibitor immediately prior to TCR-T cell activation may enhance their proliferation, survival, and function potentials, presenting an applicable optimization strategy for immunotherapy with adoptive cell transfer.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Diferenciação Celular , Proliferação de Células , Citocinas , Ativação Linfocitária , Proteínas Proto-Oncogênicas c-cbl , Receptores de Antígenos de Linfócitos T , Transdução de Sinais , Linfócitos T , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Humanos , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Linfócitos T/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Citocinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Proliferação de Células/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fosfolipase C gama/metabolismo , Proteína-Tirosina Quinase ZAP-70/metabolismo , Fosforilação/efeitos dos fármacos , Imunoterapia Adotiva/métodos , Fenótipo , Sobrevivência Celular/efeitos dos fármacos
3.
Int J Mol Sci ; 25(3)2024 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-38339043

RESUMO

Programmed ribosomal frameshifting (PRF) exists in all branches of life that regulate gene expression at the translational level. The eukaryotic translation initiation factor 5A (eIF5A) is a highly conserved protein essential in all eukaryotes. It is identified initially as an initiation factor and functions broadly in translation elongation and termination. The hypusination of eIF5A is specifically required for +1 PRF at the shifty site derived from the ornithine decarboxylase antizyme 1 (OAZ1) in Saccharomyces cerevisiae. However, whether the regulation of +1 PRF by yeast eIF5A is universal remains unknown. Here, we found that Sc-eIF5A depletion decreased the putrescine/spermidine ratio. The re-introduction of Sc-eIF5A in yeast eIF5A mutants recovered the putrescine/spermidine ratio. In addition, the Sc-eIF5A depletion decreases +1 PRF during the decoding of Ty1 retrotransposon mRNA, but has no effect on -1 PRF during the decoding of L-A virus mRNA. The re-introduction of Sc-eIF5A in yeast eIF5A mutants restored the +1 PRF rate of Ty1. The inhibition of the hypusine modification of yeast eIF5A by GC7 treatment or by mutating the hypusination site Lys to Arg caused decreases of +1 PRF rates in the Ty1 retrotransposon. Furthermore, mutational studies of the Ty1 frameshifting element support a model where the efficient removal of ribosomal subunits at the first Ty1 frame 0 stop codon is required for the frameshifting of trailing ribosomes. This dependency is likely due to the unique position of the frame 0 stop codon distance from the slippery sequence of Ty1. The results showed that eIF5A is a trans-regulator of +1 PRF for Ty1 retrotransposon and could function universally in yeast.


Assuntos
Mudança da Fase de Leitura do Gene Ribossômico , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Espermidina/metabolismo , Putrescina/metabolismo , Retroelementos/genética , Códon de Terminação/genética , Códon de Terminação/metabolismo , Fatores de Iniciação de Peptídeos/genética , Fatores de Iniciação de Peptídeos/metabolismo
4.
Int J Biol Macromol ; 254(Pt 1): 127743, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38287569

RESUMO

Programmed ribosomal frameshifting (PRF) exists in all branches of life that regulate gene expression at the translational level. The single-celled eukaryote Euplotes exhibit high frequency of PRF. However, the molecular mechanism of modulating Euplotes PRF remains largely unknown. Here, we identified two novel eIF5A genes, eIF5A1 and eIF5A2, in Euplotes octocarinatus and found that the Eo-eIF5A2 gene requires a -1 PRF to produce complete protein product. Although both Eo-eIF5As showed significant structural similarity with yeast eIF5A, neither of them could functionally replace yeast eIF5A. Eo-eIF5A knockdown inhibited +1 PRF of the η-tubulin gene. Using an in vitro reconstituted translation system, we found that hypusinated Eo-eIF5A (Eo-eIF5AH) can promote +1 PRF at the canonical AAA_UAA frameshifting site of Euplotes. The results showed eIF5A is a novel trans-regulator of PRF in Euplotes and has an evolutionary conserved role in regulating +1 PRF in eukaryotes.


Assuntos
Euplotes , Mudança da Fase de Leitura do Gene Ribossômico , Mudança da Fase de Leitura do Gene Ribossômico/genética , Euplotes/genética , Euplotes/metabolismo , Saccharomyces cerevisiae/genética
5.
Science ; 383(6681): 413-421, 2024 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-38271512

RESUMO

Age-associated B cells (ABCs) accumulate during infection, aging, and autoimmunity, contributing to lupus pathogenesis. In this study, we screened for transcription factors driving ABC formation and found that zinc finger E-box binding homeobox 2 (ZEB2) is required for human and mouse ABC differentiation in vitro. ABCs are reduced in ZEB2 haploinsufficient individuals and in mice lacking Zeb2 in B cells. In mice with toll-like receptor 7 (TLR7)-driven lupus, ZEB2 is essential for ABC formation and autoimmune pathology. ZEB2 binds to +20-kb myocyte enhancer factor 2b (Mef2b)'s intronic enhancer, repressing MEF2B-mediated germinal center B cell differentiation and promoting ABC formation. ZEB2 also targets genes important for ABC specification and function, including Itgax. ZEB2-driven ABC differentiation requires JAK-STAT (Janus kinase-signal transducer and activator of transcription), and treatment with JAK1/3 inhibitor reduces ABC accumulation in autoimmune mice and patients. Thus, ZEB2 emerges as a driver of B cell autoimmunity.


Assuntos
Autoimunidade , Linfócitos B , Diferenciação Celular , Regulação da Expressão Gênica , Lúpus Eritematoso Sistêmico , Homeobox 2 de Ligação a E-box com Dedos de Zinco , Animais , Humanos , Camundongos , Autoimunidade/genética , Linfócitos B/citologia , Linfócitos B/metabolismo , Diferenciação Celular/genética , Linhagem da Célula/genética , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/imunologia , Fatores de Transcrição MEF2/genética , Fatores de Transcrição MEF2/metabolismo , Homeobox 2 de Ligação a E-box com Dedos de Zinco/genética , Homeobox 2 de Ligação a E-box com Dedos de Zinco/metabolismo , Haploinsuficiência , Envelhecimento/imunologia , Modelos Animais de Doenças , Feminino
6.
Arthritis Rheumatol ; 75(7): 1203-1215, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-36575806

RESUMO

OBJECTIVE: Emerging evidence indicates that a distinct CD11c+T-bet+ B cell subset, termed age/autoimmune-associated B cells (ABCs), is the major pathogenic autoantibody producer in lupus. Human lupus is associated with significant metabolic alterations, but how ABCs orchestrate their typical transcription factors and metabolic programs to meet specific functional requirements is unclear. We undertook this study to characterize the metabolism of ABCs and to identify the regulators of their metabolic pathways in an effort to develop new therapies for ABC-mediated autoimmunity. METHODS: We developed a T-bet-tdTomato reporter mouse strain to trace live T-bet+ B cells and adoptively transferred CD4+ T cells from bm12 mice to induce lupus. We next sorted CD11c+tdTomato+ B cells and conducted RNA sequencing and an extracellular flux assay. A metabolic restriction to constrain ABC formation was tested in human and mouse B cells. We used a bm12-induced lupus mouse model to conduct the metabolic intervention. RESULTS: ABCs exhibited a hypermetabolic state with enhanced glycolytic capacity. The increased glycolytic rate in ABCs was promoted by interferon-γ (IFNγ) signaling. T-bet, a downstream transcription factor of IFNγ, regulated the gene program of the glycolysis pathway in ABCs by repressing the expression of Bcl6. Functionally, glycolysis restriction could impair ABC formation. The engagement of glycolysis promoted survival and terminal differentiation of antibody-secreting cells. Administration of a glycolysis inhibitor ameliorated ABC accumulation and autoantibody production in the lupus-induced bm12 mouse model. CONCLUSION: T-bet can couple immune signals and metabolic programming to establish pathogenic ABC formation and functional capacities. Modulation of ABCs favored a metabolic program that could be a novel therapeutic approach for lupus.


Assuntos
Subpopulações de Linfócitos B , Lúpus Eritematoso Sistêmico , Humanos , Animais , Camundongos , Autoimunidade , Proteínas com Domínio T , Subpopulações de Linfócitos B/metabolismo , Autoanticorpos , Interferon gama/metabolismo , Metabolismo Energético , Fatores de Transcrição/metabolismo
7.
Arthritis Rheumatol ; 75(8): 1381-1394, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-36862399

RESUMO

OBJECTIVE: Disruption of B cell homeostasis and subsequent dominance of effector B cell subsets are critical for the development of systemic lupus erythematosus (SLE). Revealing the key intrinsic regulators involved in the homeostatic control of B cells has important therapeutic value for SLE. This study was undertaken to determine the regulatory role of the transcription factor Pbx1 in B cell homeostasis and lupus pathogenesis. METHODS: We constructed mice with B cell-specific deletion of Pbx1. T cell-dependent and T cell-independent humoral responses were induced by intraperitoneal injection of nitrophenyl-containing hapten (NP) conjugated to keyhole limpet hemocyanin or NP-Ficoll. The regulatory effects of Pbx1 on autoimmunity were observed in a Bm12-induced lupus murine model. We investigated mechanisms of Pbx1 using RNA sequencing, the cleavage under targets and tagmentation assay, and chromatin immunoprecipitation-quantitative polymerase chain reaction assay. We transduced B cells from SLE patients with plasmids that overexpressed PBX1 to explore the in vitro therapeutic efficacy of PBX1. RESULTS: Pbx1 was specifically down-regulated in autoimmune B cells and negatively correlated with disease activity. The deficiency of Pbx1 in B cells resulted in excessive humoral responses following immunization. In the Bm12-induced lupus model, mice with B cell-specific Pbx1 deficiency displayed enhancements in germinal center responses, plasma cell differentiation, and autoantibody production. Pbx1-deficient B cells had increased survival and proliferative advantages after activation. Pbx1 regulated genetic programs by directly targeting critical components of the proliferation and apoptosis pathways. In SLE patients, PBX1 expression was negatively correlated with effector B cell expansion; when PBX1 expression was enforced, the survival and proliferative capacity of SLE B cells were attenuated. CONCLUSION: Our study reveals the regulatory function and mechanism of Pbx1 in adjusting B cell homeostasis and highlights Pbx1 as a therapeutic target in SLE.


Assuntos
Autoimunidade , Lúpus Eritematoso Sistêmico , Camundongos , Animais , Fatores de Transcrição/genética , Regulação da Expressão Gênica , Linfócitos B , Fator de Transcrição 1 de Leucemia de Células Pré-B/genética , Fator de Transcrição 1 de Leucemia de Células Pré-B/metabolismo
8.
Biomed Res Int ; 2018: 9619253, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30627584

RESUMO

As a common disorder, chronic kidney disease (CKD) poses a great threat to human health. Chronic kidney disease-mineral and bone disorder (CKD-MBD) is a complication of CKD characterized by disturbances in the levels of calcium, phosphorus, parathyroid hormone (PTH), and vitamin D; abnormal bone formation affecting the mineralization and linear growth of bone; and vascular and soft tissue calcification. PTH reflects the function of the parathyroid gland and also takes part in the metabolism of minerals. The accurate measurement of PTH plays a vital role in the clinical diagnosis, treatment, and prognosis of patients with secondary hyperparathyroidism (SHPT). Previous studies have shown that there are different fragments of PTH in the body's circulation, causing antagonistic effects on bone and the kidney. Here we review the metabolism of PTH fragments; the progress being made in PTH measurement assays; the effects of PTH fragments on bone, kidney, and the cardiovascular system in CKD; and the predictive value of PTH measurement in assessing the effectiveness of parathyroidectomy (PTX). We hope that this review will help to clarify the value of accurate PTH measurements in CKD-MBD and promote the further development of multidisciplinary diagnosis and treatment.


Assuntos
Doenças Ósseas Metabólicas , Hiperparatireoidismo , Hormônio Paratireóideo , Peptídeos , Insuficiência Renal Crônica , Animais , Doenças Ósseas Metabólicas/sangue , Doenças Ósseas Metabólicas/diagnóstico , Doenças Ósseas Metabólicas/tratamento farmacológico , Humanos , Hiperparatireoidismo/sangue , Hiperparatireoidismo/diagnóstico , Hiperparatireoidismo/tratamento farmacológico , Glândulas Paratireoides/metabolismo , Hormônio Paratireóideo/sangue , Hormônio Paratireóideo/uso terapêutico , Peptídeos/sangue , Peptídeos/uso terapêutico , Insuficiência Renal Crônica/sangue , Insuficiência Renal Crônica/diagnóstico , Insuficiência Renal Crônica/tratamento farmacológico
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