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2.
Anticancer Res ; 41(7): 3439-3448, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34230139

RESUMO

BACKGROUND/AIM: The role of immune cells and PD-L1 in cutaneous squamous carcinogenesis is unclear. This study examines T-cell populations, Langerhans cells (LCs) and PD-L1 in invasive squamous cell carcinoma (inSCC), adjacent precursors and normal skin (NS) to investigate their participation in tumorigenesis. MATERIALS AND METHODS: Cases of cutaneous inSCC with adjacent precursors (n=125) were selected. In situ SCC (isSCC) and actinic keratosis (AK) were observed in 53 and 123 cases, respectively, whereas NS was present in 123 lesions. Immunohistochemistry was performed for CD3, CD8, Foxp3, CD1a and PD-L1. RESULTS: T-cells, LCs and PD-L1 gradually increase during the evolution from AK to isSCC and inSCC, with statistical significance between all lesions, except for CD3+ and CD8+ cells between isSCC and inSCC. Epithelial PD-L1 expression correlates with tumor diameter and thickness. CONCLUSION: The progressive increase of T-cells, LCs and PD-L1 in cutaneous squamous carcinogenesis provides rationale for immunotherapy and identification of predictive biomarkers.


Assuntos
Antígeno B7-H1/metabolismo , Carcinoma de Células Escamosas/imunologia , Carcinoma de Células Escamosas/metabolismo , Células de Langerhans/metabolismo , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/metabolismo , Subpopulações de Linfócitos T/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Complexo CD3/imunologia , Linfócitos T CD8-Positivos/imunologia , Carcinoma de Células Escamosas/patologia , Feminino , Humanos , Imuno-Histoquímica/métodos , Células de Langerhans/imunologia , Células de Langerhans/patologia , Masculino , Pessoa de Meia-Idade , Neoplasias Cutâneas/patologia , Subpopulações de Linfócitos T/imunologia , Microambiente Tumoral/imunologia
3.
Front Immunol ; 12: 667897, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34108968

RESUMO

A therapy that includes an oral vaccine for type 1 diabetes (T1D) using live attenuated Salmonella MvP728 (ΔhtrA/ΔpurD), cytokines (IL10 and TGFß) and preproinsulin (PPI) antigen in combination with a sub-therapeutic dose of anti-CD3 mAb was developed by our team. The vaccine combination therapy reduced insulitis and prevented and reversed diabetes in non-obese diabetic (NOD) mice. Here, we show the effectiveness of an alternative Salmonella mutant (ΔmsbB) as a carrier strain, which is anticipated to have lower risks of an inflammatory response and septicemia as a result of modification in the lipopolysaccharide (LPS) via detoxification of lipid A. This mutant strain proved to have highly reduced pathogenic side effects. Salmonella strain ΔmsbB expressed autoantigens and in combination with cytokines and anti-CD3 mAb, successfully prevented and reversed T1D to levels comparable to the previously used carrier strain ΔhtrA/ΔpurD. Additionally, the Salmonella msbB mutant resulted in higher rates of host cell infection. These results further demonstrate the potential of an oral Salmonella-based combined therapy in the treatment of early T1D.


Assuntos
Aciltransferases/genética , Proteínas de Bactérias/genética , Glicemia/metabolismo , Diabetes Mellitus Tipo 1/prevenção & controle , Vetores Genéticos , Mutação , Salmonella/genética , Vacinas de DNA/administração & dosagem , Administração Oral , Animais , Anticorpos Monoclonais/administração & dosagem , Biomarcadores/sangue , Complexo CD3/antagonistas & inibidores , Complexo CD3/imunologia , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/imunologia , Modelos Animais de Doenças , Feminino , Insulina/administração & dosagem , Insulina/genética , Interleucina-10/administração & dosagem , Interleucina-10/genética , Camundongos , Camundongos Endogâmicos NOD , Precursores de Proteínas/administração & dosagem , Precursores de Proteínas/genética , Células RAW 264.7 , Salmonella/imunologia , Salmonella/patogenicidade , Fator de Crescimento Transformador beta1/administração & dosagem , Fator de Crescimento Transformador beta1/genética , Vacinas Atenuadas/administração & dosagem , Vacinas de DNA/genética , Vacinas de DNA/imunologia
5.
Theranostics ; 11(13): 6393-6406, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33995664

RESUMO

Rationale: Endoglin, also known as CD105, is a homo-dimeric membrane glycoprotein required for angiogenesis and serves as a marker for cancer vasculature. In this study, we constructed a bispecific T-cell engager (BiTE) antibody that targets human endoglin and CD3 (hEND-CD3/BiTE). We examined BiTE binding to endoglin-expressing cells and its effects on the cytolytic activity of T cells and cancer development. Methods: The in vitro effects of hEND-CD3/BiTE, including binding to target cells, T-cell activation, proliferation, and cytotoxicity, were examined in endoglin-expressing 293T cells, human umbilical vascular endothelial cells, tumor-derived endothelial cells, and CD3+ T cells. An in vivo xenograft tumor model was established using A549 human lung cancer cells. The therapeutic efficacy of hEND-CD3/BiTE was assessed by monitoring tumor growth, angiogenesis, and mouse survival. Results: hEND-CD3/BiTE specifically bound to endoglin-expressing cells and CD3+ T cells in vitro and stimulated T-cell activation, proliferation, and Th1 cytokine secretion, and promoted T-cell-mediated cytolysis of endoglin-expressing cells. The hEND-CD3/BiTE in vivo caused minimal toxicity to major organs, reduced tumor neoangiogenesis, inhibited tumor growth, and significantly improved mouse survival. Conclusions: Our study demonstrated the therapeutic potential of hEND-CD3/BiTE and provided a novel approach to clinical cancer treatment.


Assuntos
Anticorpos Biespecíficos/uso terapêutico , Antineoplásicos Imunológicos/uso terapêutico , Complexo CD3/imunologia , Endoglina/imunologia , Células Endoteliais/imunologia , Linfócitos T/imunologia , Células A549 , Sequência de Aminoácidos , Inibidores da Angiogênese/uso terapêutico , Animais , Anticorpos Biespecíficos/genética , Anticorpos Biespecíficos/imunologia , Sequência de Bases , Citocinas/metabolismo , Citotoxicidade Imunológica , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/imunologia , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/imunologia , Células Th1/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Scand J Immunol ; 94(1): e13049, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33934376

RESUMO

To investigate whether serum-soluble PD-L1 (sPD-L1) is a potential biomarker for identifying sepsis. This study enrolled 64 septic patients, 29 patients with acute appendicitis, 33 patients with acute pancreatitis and 30 healthy volunteers. Sepsis was defined according to the Sepsis 3.0 criteria.[1] The associated clinical parameters were recorded, blood samples were collected on the first day of diagnosis, and serum sPD-L1 levels were measured using enzyme-linked immunosorbent assays. Compared with the control group, a significant increase in sPD-L1 levels was observed in patients with sepsis (n = 64). Increased sPD-L1 expression correlated strongly with increased clinical inflammatory values (CRP, PCT and WBC) and decreased immunological functional parameters (CD3+ , CD4+ and CD8+ cell counts). The area under the ROC curve (AUC) for sPD-L1 in combination with the sequential organ failure assessment (SOFA) score was superior to the AUC for either sPD-L1 or SOFA score in regard to the diagnosis of sepsis. sPD-L1 may represent a valuable biomarker for the diagnosis of sepsis.


Assuntos
Antígeno B7-H1/sangue , Biomarcadores/sangue , Sepse/sangue , Doença Aguda , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Área Sob a Curva , Antígeno B7-H1/imunologia , Complexo CD3/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Escores de Disfunção Orgânica , Pancreatite/sangue , Pancreatite/imunologia , Prognóstico , Curva ROC , Sepse/imunologia , Adulto Jovem
7.
Aging (Albany NY) ; 13(6): 7713-7722, 2021 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-33714947

RESUMO

If age boundaries are arbitrarily or roughly defined, age-related analyses can result in questionable findings. Here, we aimed to delineate the uniquely age-dependent immune features of coronavirus disease 2019 (COVID-19) in a retrospective study of 447 patients, stratified according to age distributions of COVID-19 morbidity statistics into well-defined age-cohorts (2-25y, 26-38y, 39-57y, 58-68y, and 69-79y). Age-dependent susceptibilities and severities of the disease were observed in COVID-19 patients. A comparison of the lymphocyte counts among the five age-groups indicated that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection led to age-dependent lymphopenia. Among the lymphocyte subsets, the CD8+ T cell count alone was significantly and age-dependently decreased (520, 385, 320, 172, and 139 n/µl in the five age-groups, respectively). In contrast, the CD4+ T cell, B cell, and natural killer cell counts did not differ among age-cohorts. Age and CD8+ T cell counts (r=‒0.435, p<0.0001) were negatively correlated in COVID-19 patients. Moreover, SARS-CoV-2 infection age-dependently increased the plasma C-reactive protein concentrations (2.0, 5.0, 9.0, 11.6, and 36.1 mg/L in the five age-groups, respectively). These findings can be used to elucidate the role of CD8+ T cells in age-related pathogenesis and to help develop therapeutic strategies for COVID-19.


Assuntos
Distribuição por Idade , Complexo CD3/imunologia , Linfócitos T CD8-Positivos/imunologia , COVID-19/complicações , Linfopenia/complicações , Admissão do Paciente , Adolescente , Adulto , Idoso , COVID-19/virologia , Criança , Pré-Escolar , Estudos de Coortes , Feminino , Humanos , Contagem de Linfócitos , Linfopenia/imunologia , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , SARS-CoV-2/isolamento & purificação , Adulto Jovem
8.
Res Vet Sci ; 136: 310-317, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33756379

RESUMO

Oral probiotics are used to induce immune responses in the intestines to protect against infection. However, oral probiotics may also affect immune responses in other mucosal tissues such as in the respiratory tract. To examine this possibility, we explored the potential of immunocytes to home to the respiratory system after oral administration of Bacillus subtilis. The results showed that B. subtilis could promote intestinal development and not cause pathological changes in the respiratory tract. Following the oral administration with B. subtilis, the number of IgA-secreting cells and CD3+ T cells not only significantly increased in the intestinal tracts but also in respiratory tracts (P < 0.01). Moreover, the levels of secretory IgA were significantly higher in the trachea, lungs, ileum, and jejunum after oral B. subtilis administration than in the control groups (P < 0.05). The mRNA expression of interleukin (IL)-1ß, IL-5, IL-6, tumor necrosis factor-α, B cell activating factor, and IgA-inducing protein increased following B. subtilis administration (P < 0.01) in the trachea, lungs, ileum, and jejunum. These data suggest that B. subtilis administration regulates the immune response not only in the intestine but also in the respiratory tract of piglets. Our work highlights a potentially new strategy for promoting respiratory mucosal immunity and may contribute to the design of vaccines with B. subtilis as a mucosal adjuvant.


Assuntos
Bacillus subtilis/química , Vacinas Bacterianas/imunologia , Complexo CD3/imunologia , Imunidade nas Mucosas , Sistema Respiratório/imunologia , Linfócitos T/imunologia , Adjuvantes Imunológicos/administração & dosagem , Administração Oral , Animais , Animais Recém-Nascidos , Imunoglobulina A Secretora/imunologia , Masculino , Sus scrofa
9.
PLoS One ; 16(3): e0247669, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33667236

RESUMO

To study the dysregulated host immune response to infection in sepsis, gene expression profiles from the Gene Expression Omnibus (GEO) datasets GSE54514, GSE57065, GSE64456, GSE95233, GSE66099 and GSE72829 were selected. From the Kyoto Encyclopedia of Genes and Genomes (KEGG) immune system pathways, 998 unique genes were selected, and genes were classified as follows based on gene annotation from KEGG, Gene Ontology, and Reactome: adaptive immunity, antigen presentation, cytokines and chemokines, complement, hematopoiesis, innate immunity, leukocyte migration, NK cell activity, platelet activity, and signaling. After correlation matrix formation, correlation coefficient of 0.8 was selected for network generation and network analysis. Total transcriptome was analyzed for differentially expressed genes (DEG), followed by gene set enrichment analysis. The network topological structure revealed that adaptive immunity tended to form a prominent and isolated cluster in sepsis. Common genes within the cluster from the 6 datasets included CD247, CD8A, ITK, LAT, and LCK. The clustering coefficient and modularity parameters were increased in 5/6 and 4/6 datasets in the sepsis group that seemed to be associated with functional aspect of the network. GSE95233 revealed that the nonsurvivor group showed a prominent and isolated adaptive immunity cluster, whereas the survivor group had isolated complement-coagulation and platelet-related clusters. T cell receptor signaling (TCR) pathway and antigen processing and presentation pathway were down-regulated in 5/6 and 4/6 datasets, respectively. Complement and coagulation, Fc gamma, epsilon related signaling pathways were up-regulated in 5/6 datasets. Altogether, network and gene set enrichment analysis showed that adaptive-immunity-related genes along with TCR pathway were down-regulated and isolated from immune the network that seemed to be associated with unfavorable prognosis. Prominence of platelet and complement-coagulation-related genes in the immune network was associated with survival in sepsis. Complement-coagulation pathway was up-regulated in the sepsis group that was associated with favorable prognosis. Network and gene set enrichment analysis supported elucidation of sepsis pathogenesis.


Assuntos
Imunidade Adaptativa , Regulação da Expressão Gênica/imunologia , Redes Reguladoras de Genes/imunologia , Sepse/genética , Transcriptoma/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Apresentação do Antígeno/genética , Complexo CD3/genética , Complexo CD3/imunologia , Antígenos CD8/genética , Antígenos CD8/imunologia , Proteínas do Sistema Complemento/genética , Proteínas do Sistema Complemento/imunologia , Biologia Computacional/métodos , Conjuntos de Dados como Assunto , Perfilação da Expressão Gênica , Ontologia Genética , Humanos , Imunidade Inata , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/genética , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/imunologia , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Anotação de Sequência Molecular , Prognóstico , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/imunologia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Sepse/diagnóstico , Sepse/imunologia , Sepse/mortalidade , Transdução de Sinais , Análise de Sobrevida
10.
PLoS One ; 16(2): e0245917, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33596227

RESUMO

Pre-clinical murine models are critical for translating drug candidates from the bench to the bedside. There is interest in better understanding how anti-human CD3 therapy works based on recent longitudinal studies of short-term administration. Although several models have been created in this pursuit, each have their own advantages and disadvantages in Type-1 diabetes. In this study, we report a murine genetic knock-in model which expresses both a murine and a humanized-CD3ε-exon, rendering it sensitive to manipulation with anti-human CD3. These huCD3εHET mice are viable and display no gross abnormalities. Specifically, thymocyte development and T cell peripheral homeostasis is unaffected. We tested immune functionality of these mice by immunizing them with T cell-dependent antigens and no differences in antibody titers compared to wild type mice were recorded. Finally, we performed a graft-vs-host disease model that is driven by effector T cell responses and observed a wasting disease upon transfer of huCD3εHET T cells. Our results show a viable humanized CD3 murine model that develops normally, is functionally engaged by anti-human CD3 and can instruct on pre-clinical tests of anti-human CD3 antibodies.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Complexo CD3/genética , Complexo CD3/imunologia , Técnicas de Introdução de Genes , Animais , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Linfócitos T/citologia , Linfócitos T/imunologia , Timócitos/citologia , Timócitos/imunologia
11.
Proc Natl Acad Sci U S A ; 118(9)2021 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-33627407

RESUMO

Anticytotoxic T lymphocyte-associated protein 4 (CTLA4) antibodies have shown potent antitumor activity, but systemic immune activation leads to severe immune-related adverse events, limiting clinical usage. We developed novel, conditionally active biologic (CAB) anti-CTLA4 antibodies that are active only in the acidic tumor microenvironment. In healthy tissue, this binding is reversibly inhibited by a novel mechanism using physiological chemicals as protein-associated chemical switches (PaCS). No enzymes or potentially immunogenic covalent modifications to the antibody are required for activation in the tumor. The novel anti-CTLA4 antibodies show similar efficacy in animal models compared to an analog of a marketed anti-CTLA4 biologic, but have markedly reduced toxicity in nonhuman primates (in combination with an anti-PD1 checkpoint inhibitor), indicating a widened therapeutic index (TI). The PaCS encompass mechanisms that are applicable to a wide array of antibody formats (e.g., ADC, bispecifics) and antigens. Examples shown here include antibodies to EpCAM, Her2, Nectin4, CD73, and CD3. Existing antibodies can be engineered readily to be made sensitive to PaCS, and the inhibitory activity can be optimized for each antigen's varying expression level and tissue distribution. PaCS can modulate diverse physiological molecular interactions and are applicable to various pathologic conditions, enabling differential CAB antibody activities in normal versus disease microenvironments.


Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais/farmacologia , Anticorpos Antineoplásicos/farmacologia , Antígeno B7-H1/antagonistas & inibidores , Antígeno CTLA-4/antagonistas & inibidores , Neoplasias do Colo/terapia , Imunoterapia/métodos , 5'-Nucleotidase/antagonistas & inibidores , 5'-Nucleotidase/genética , 5'-Nucleotidase/imunologia , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais Humanizados/química , Anticorpos Antineoplásicos/química , Antígeno B7-H1/genética , Antígeno B7-H1/imunologia , Bicarbonatos/química , Complexo CD3/antagonistas & inibidores , Complexo CD3/genética , Complexo CD3/imunologia , Antígeno CTLA-4/genética , Antígeno CTLA-4/imunologia , Moléculas de Adesão Celular/antagonistas & inibidores , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/imunologia , Neoplasias do Colo/genética , Neoplasias do Colo/imunologia , Neoplasias do Colo/patologia , Molécula de Adesão da Célula Epitelial/antagonistas & inibidores , Molécula de Adesão da Célula Epitelial/genética , Molécula de Adesão da Célula Epitelial/imunologia , Proteínas Ligadas por GPI/antagonistas & inibidores , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/imunologia , Expressão Gênica , Humanos , Sulfeto de Hidrogênio/química , Concentração de Íons de Hidrogênio , Macaca fascicularis , Camundongos , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/imunologia , Engenharia de Proteínas/métodos , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/genética , Receptor ErbB-2/imunologia , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/patologia , Carga Tumoral/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Br J Cancer ; 124(6): 1037-1048, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33469153

RESUMO

The development of bispecific antibodies that redirect the cytotoxic activity of CD3+ T cells to tumours is a promising immunotherapeutic strategy for the treatment of haematological malignancies and solid cancers. Since the landmark FDA approval at the end of 2014 of the anti-CD3 × anti-CD19 bispecific antibody blinatumomab (Blincyto®) for the treatment of relapsed/refractory B-cell acute lymphoblastic leukaemia, ~100 clinical trials investigating the safety and efficacy of CD3+ bispecific T-cell redirectors for cancer have been initiated. However, despite early success, numerous challenges pertaining to CD3+ T-cell redirection in the context of cancer exist, including the recruitment of counterproductive CD3+ T-cell subsets, the release of systemic cytokines, the expansion of immune checkpoint molecules, the presence of an immunosuppressive tumour microenvironment, tumour antigen loss/escape, on-target off-tumour toxicity and suboptimal potency. The aim of the present review is to discuss novel approaches to overcome the key challenges associated with CD3+ bispecific T-cell redirection in order to achieve an optimal balance of anti-tumour activity and safety.


Assuntos
Anticorpos Biespecíficos/uso terapêutico , Antígenos CD19/imunologia , Antineoplásicos/uso terapêutico , Complexo CD3/imunologia , Neoplasias/tratamento farmacológico , Microambiente Tumoral/imunologia , Animais , Humanos , Neoplasias/imunologia , Neoplasias/patologia
13.
MAbs ; 13(1): 1850395, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33459147

RESUMO

We report here the discovery and optimization of a novel T cell retargeting anti-GUCY2C x anti-CD3ε bispecific antibody for the treatment of solid tumors. Using a combination of hybridoma, phage display and rational design protein engineering, we have developed a fully humanized and manufacturable CD3 bispecific antibody that demonstrates favorable pharmacokinetic properties and potent in vivo efficacy. Anti-GUCY2C and anti-CD3ε antibodies derived from mouse hybridomas were first humanized into well-behaved human variable region frameworks with full retention of binding and T-cell mediated cytotoxic activity. To address potential manufacturability concerns, multiple approaches were taken in parallel to optimize and de-risk the two antibody variable regions. These approaches included structure-guided rational mutagenesis and phage display-based optimization, focusing on improving stability, reducing polyreactivity and self-association potential, removing chemical liabilities and proteolytic cleavage sites, and de-risking immunogenicity. Employing rapid library construction methods as well as automated phage display and high-throughput protein production workflows enabled efficient generation of an optimized bispecific antibody with desirable manufacturability properties, high stability, and low nonspecific binding. Proteolytic cleavage and deamidation in complementarity-determining regions were also successfully addressed. Collectively, these improvements translated to a molecule with potent single-agent in vivo efficacy in a tumor cell line adoptive transfer model and a cynomolgus monkey pharmacokinetic profile (half-life>4.5 days) suitable for clinical development. Clinical evaluation of PF-07062119 is ongoing.


Assuntos
Anticorpos Biespecíficos/imunologia , Complexo CD3/imunologia , Imunoterapia Adotiva/métodos , Neoplasias/terapia , Receptores de Enterotoxina/imunologia , Animais , Anticorpos Biespecíficos/farmacocinética , Anticorpos Biespecíficos/uso terapêutico , Linhagem Celular Tumoral , Feminino , Humanos , Hibridomas , Macaca fascicularis/imunologia , Macaca fascicularis/metabolismo , Camundongos Endogâmicos BALB C , Neoplasias/imunologia , Neoplasias/metabolismo , Engenharia de Proteínas/métodos , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/farmacocinética , Anticorpos de Cadeia Única/uso terapêutico , Linfócitos T/imunologia , Linfócitos T/metabolismo
14.
J Enzyme Inhib Med Chem ; 36(1): 175-182, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33404266

RESUMO

Recent findings have shown that nanovesicles preparations from either primary immune cells culture supernatants or plasma contain immunoglobulins, suggesting that a natural way of antibody production may be through exosome release. To verify this hypothesis, we used the OKT3 hybridoma clone, which produces a murine IgG2a monoclonal antibody used to reduce rejection in patients undergoing organ transplantation. We showed exosome-associated immunoglobulins in hybridoma supernatants, by Western blot, nanoscale flow cytometry and immunocapture-based ELISA. The OKT3-exo was also being able to trigger cytokines production in both CD4 and CD8 T cells. These results show that nanovesicles contain immunoglobulin and could be used for immunotherapy. These data could lead to a new approach to improve the effectiveness of therapeutic antibodies by exploiting their natural property to be expressed on nanovesicle membrane, that probably render them more stable and as a consequence more capable to interact with their specific ligand in the best way.


Assuntos
Linfócitos B/imunologia , Exossomos/imunologia , Hibridomas/imunologia , Imunoglobulina G/biossíntese , Muromonab-CD3/imunologia , Linfócitos T/imunologia , Animais , Antígenos de Superfície/genética , Antígenos de Superfície/imunologia , Linfócitos B/citologia , Complexo CD3/genética , Complexo CD3/imunologia , Linhagem Celular Tumoral , Citocinas/biossíntese , Citocinas/imunologia , Exossomos/química , Exossomos/genética , Expressão Gênica , Humanos , Hibridomas/química , Imunoglobulina G/imunologia , Ativação Linfocitária , Macrófagos/citologia , Macrófagos/imunologia , Camundongos , Mieloma Múltiplo/imunologia , Muromonab-CD3/genética , Neoplasias Experimentais/imunologia , Cultura Primária de Células , Baço/citologia , Baço/imunologia , Linfócitos T/citologia
15.
PLoS Pathog ; 17(1): e1008748, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33465149

RESUMO

The biomolecular mechanisms controlling latent HIV-1 infection, despite their importance for the development of a cure for HIV-1 infection, are only partially understood. For example, ex vivo studies have recently shown that T cell activation only triggered HIV-1 reactivation in a fraction of the latently infected CD4+ T cell reservoir, but the molecular biology of this phenomenon is unclear. We demonstrate that HIV-1 infection of primary T cells and T cell lines indeed generates a substantial amount of T cell receptor (TCR)/CD3 activation-inert latently infected T cells. RNA-level analysis identified extensive transcriptomic differences between uninfected, TCR/CD3 activation-responsive and -inert T cells, but did not reveal a gene expression signature that could functionally explain TCR/CD3 signaling inertness. Network analysis suggested a largely stochastic nature of these gene expression changes (transcriptomic noise), raising the possibility that widespread gene dysregulation could provide a reactivation threshold by impairing overall signal transduction efficacy. Indeed, compounds that are known to induce genetic noise, such as HDAC inhibitors impeded the ability of TCR/CD3 activation to trigger HIV-1 reactivation. Unlike for transcriptomic data, pathway enrichment analysis based on phospho-proteomic data directly identified an altered TCR signaling motif. Network analysis of this data set identified drug targets that would promote TCR/CD3-mediated HIV-1 reactivation in the fraction of otherwise TCR/CD3-reactivation inert latently HIV-1 infected T cells, regardless of whether the latency models were based on T cell lines or primary T cells. The data emphasize that latent HIV-1 infection is largely the result of extensive, stable biomolecular changes to the signaling network of the host T cells harboring latent HIV-1 infection events. In extension, the data imply that therapeutic restoration of host cell responsiveness prior to the use of any activating stimulus will likely have to be an element of future HIV-1 cure therapies.


Assuntos
Complexo CD3/metabolismo , Linfócitos T CD4-Positivos/imunologia , Infecções por HIV/imunologia , Proteoma , Receptores de Antígenos de Linfócitos T/metabolismo , Transcriptoma , Latência Viral , Complexo CD3/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/virologia , Regulação Viral da Expressão Gênica , Redes Reguladoras de Genes , Infecções por HIV/genética , Infecções por HIV/metabolismo , Infecções por HIV/virologia , HIV-1/fisiologia , Humanos , Ativação Linfocitária , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais , Ativação Viral , Replicação Viral
16.
Eur J Immunol ; 51(2): 342-353, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33169379

RESUMO

The immunological synapse is a transient junction that occurs when the plasma membrane of a T cell comes in close contact with an APC after recognizing a peptide from the antigen-MHC. The interaction starts when CRAC channels embedded in the T cell membrane open, flowing calcium ions into the cell. To counterbalance the ion influx and subsequent depolarization, Kv 1.3 and KCa3.1 channels are recruited to the immunological synapse, increasing the extracellular K+ concentration. These processes are crucial as they initiate gene expression that drives T cell activation and proliferation. The T cell-specific function of the K2P channel family member TASK2 channels and their role in autoimmune processes remains unclear. Using mass spectrometry analysis together with epifluorescence and super-resolution single-molecule localization microscopy, we identified TASK2 channels as novel players recruited to the immunological synapse upon stimulation. TASK2 localizes at the immunological synapse, upon stimulation with CD3 antibodies, likely interacting with these molecules. Our findings suggest that, together with Kv 1.3 and KCa3.1 channels, TASK2 channels contribute to the proper functioning of the immunological synapse, and represent an interesting treatment target for T cell-mediated autoimmune disorders.


Assuntos
Sinapses Imunológicas/imunologia , Canais de Potássio de Domínios Poros em Tandem/imunologia , Animais , Doenças Autoimunes/imunologia , Complexo CD3/imunologia , Cálcio/imunologia , Linhagem Celular Tumoral , Membrana Celular/imunologia , Células Cultivadas , Feminino , Expressão Gênica/imunologia , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/imunologia , Células Jurkat , Canal de Potássio Kv1.3/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T/imunologia
17.
Cancer Immunol Immunother ; 70(6): 1569-1581, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33225419

RESUMO

Targeted cancer therapy with monoclonal antibodies has proven successful for different cancer types but is limited by the availability of suitable antibody targets. CD43s, a unique sialylated form of CD43 expressed by hematologic malignancies, is a recently identified target and antibodies interacting with CD43s may have therapeutic potential against acute myeloid leukemia (AML) and myelodysplastic syndrome. CD43s is recognized by the human antibody AT1413, that was derived from a high-risk AML patient who successfully cleared leukemia after allogeneic stem cell transplantation. Here we observed that AT1413 binds also to certain non-hematopoietic tumor cells, particularly melanoma and breast cancer. AT1413 immune precipitated CD43s from melanoma cells confirming that it recognizes the same target on melanoma as on AML. AT1413 induced antibody-dependent cellular cytotoxicity against short-term cultured patient-derived melanoma samples. However, AT1413 was unable to affect the growth of melanoma cells in vivo. To increase the efficacy of AT1413 as a therapeutic antibody, we generated two different formats of bispecific T-cell engaging antibodies (TCEs): one binding bivalently (bTCE) and the other monovalently (knob-in-hole; KiH) to both CD43s and CD3ε. In vitro, these TCEs redirected T-cell cytotoxicity against melanoma cells with differences in potencies. To investigate their effects in vivo, we grafted mice that harbor a human immune system with the melanoma cell line A375. Treatment with both AT1413 bTCE and AT1413 KiH significantly reduced tumor outgrowth in these mice. These data indicate a broad therapeutic potential of AT1413 that includes AML and CD43s-expressing solid tumors that originate from CD43-negative tissues.


Assuntos
Anticorpos Biespecíficos/farmacologia , Anticorpos Monoclonais/farmacologia , Antineoplásicos Imunológicos/farmacologia , Complexo CD3/imunologia , Leucossialina/imunologia , Melanoma/terapia , Ácido N-Acetilneuramínico/química , Linfócitos T/imunologia , Animais , Apoptose , Proliferação de Células , Citotoxicidade Imunológica , Feminino , Humanos , Técnicas In Vitro , Melanoma/imunologia , Melanoma/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Eur J Immunol ; 51(4): 848-863, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33345332

RESUMO

Upon generation of monoclonal antibodies to the T cell antigen receptor/CD3 (TCR/CD3) complex, we isolated mAb MT3, whose reactivity correlates inversely with the production of IFN-γ by human peripheral blood T lymphocytes. Using eukaryotic expression cloning, we identified the MT3 antigen as myelin-and-lymphocyte (MAL) protein. Flow cytometry analysis demonstrates high surface expression of MAL on all naïve CD4+ T cells whereas MAL expression is diminished on central memory- and almost lost on effector memory T cells. MAL- T cells proliferate strongly in response to stimulation with CD3/CD28 antibodies, corroborating that MAL+ T cells are naïve and MAL- T cells memory subtypes. Further, resting MAL- T cells harbor a larger pool of Ser59- and Tyr394- double phosphorylated lymphocyte-specific kinase (Lck), which is rapidly increased upon in vitro restimulation. Previously, lack of MAL was reported to prevent transport of Lck, the key protein tyrosine kinase of TCR/CD3 signaling to the cell membrane, and to result in strongly impaired human T cell activation. Here, we show that knocking out MAL did not significantly affect Lck membrane localization and immune synapse recruitment, or transcriptional T cell activation. Collectively, our results indicate that loss of MAL is associated with activation-induced differentiation of human T cells but not with impaired membrane localization of Lck or TCR signaling capacity.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Diferenciação Celular/imunologia , Ativação Linfocitária/imunologia , Proteínas Proteolipídicas Associadas a Linfócitos e Mielina/imunologia , Animais , Antígenos CD28/imunologia , Antígenos CD28/metabolismo , Complexo CD3/imunologia , Complexo CD3/metabolismo , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Diferenciação Celular/genética , Linhagem Celular Tumoral , Citometria de Fluxo , Expressão Gênica/imunologia , Humanos , Interferon gama/imunologia , Interferon gama/metabolismo , Células Jurkat , Ativação Linfocitária/genética , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/genética , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/imunologia , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Camundongos , Proteínas Proteolipídicas Associadas a Linfócitos e Mielina/genética , Proteínas Proteolipídicas Associadas a Linfócitos e Mielina/metabolismo , Fosforilação , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismo
19.
Prostate ; 81(1): 50-57, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32986884

RESUMO

BACKGROUND: Characterization of markers of both immune suppression and activation may provide more prognostic information than assessment of single markers in localized prostate cancer. We therefore sought to determine the association between CD8 and PD-L1 expression in localized prostate tumors and biochemical recurrence (BCR) and metastasis-free survival (MFS). METHODS: Tissue microarrays were constructed on 109 men undergoing radical prostatectomy (RP) for localized prostate cancer at Dana-Farber Cancer Institute between 1991 and 2008. Fluorescence immunohistochemistry was used to evaluate the expression of six immune markers (CD3, CD4, CD8, PD-1, PD-L1, FOXP3). Quantitative multispectral imaging analysis was used to calculate the density of each marker, which was dichotomized by the median as "high" or "low." Cox proportional hazards regression models and Kaplan-Meier analyses were used to analyze associations between immune marker densities and time to BCR and MFS. RESULTS: Over a median follow-up of 8.1 years, 55 (51%) and 39 (36%) men developed BCR and metastases, respectively. Median time to BCR was shorter in men with low CD8 (hazard ratio [HR] = 2.27 [1.27-4.08]) and high PD-L1 expression (HR = 2.03 [1.17-3.53]). While neither low CD8 or high PD-L1 alone were independent predictors of BCR or MFS on multivariable analysis, men with low CD8 and/or high PD-L1 had a significantly shorter time to BCR (median 3.5 years vs. NR) and MFS (median 10.8 vs. 18.4 years) compared to those with high CD8 and low PD-L1 expression. The main limitation is the retrospective and singe-center nature of the study. CONCLUSION: The presence of higher CD8 and lower PD-L1 expression in prostatectomy specimens was associated a low risk of biochemical relapse and metastatic disease. These findings are hypothesis-generating and further study is needed.


Assuntos
Antígeno B7-H1/biossíntese , Antígenos CD8/biossíntese , Neoplasias da Próstata/imunologia , Antígeno B7-H1/imunologia , Complexo CD3/biossíntese , Complexo CD3/imunologia , Antígenos CD8/imunologia , Estudos de Coortes , Fatores de Transcrição Forkhead/biossíntese , Fatores de Transcrição Forkhead/imunologia , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Recidiva Local de Neoplasia/imunologia , Receptor de Morte Celular Programada 1/biossíntese , Receptor de Morte Celular Programada 1/imunologia , Modelos de Riscos Proporcionais , Prostatectomia , Neoplasias da Próstata/cirurgia , Estudos Retrospectivos , Análise Serial de Tecidos
20.
Int J Mol Sci ; 21(23)2020 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-33255436

RESUMO

Bispecific antibodies (bsAbs) have emerged as promising therapeutics. A bispecific diabody (bsDb) is a small bsAb consisting of two distinct chimeric single-chain components, with two possible arrangements of the domains. We previously reported the effect of domain order on the function of a humanized bsDb targeting the epidermal growth factor receptor (EGFR) on cancer cells, and CD3 on T cells. Notably, the co-localization of a T-cell receptor (TCR) with CD3 is bulky, potentially affecting the cross-linking ability of bsDbs, due to steric hindrance. Here, we constructed and evaluated humanized bsDbs, with different domain orders, targeting EGFR and CD16 on natural killer (NK) cells (hEx16-Dbs). We predicted minimal effects due to steric hindrance, as CD16 lacks accessory molecules. Interestingly, one domain arrangement displayed superior cytotoxicity in growth inhibition assays, despite similar cross-linking abilities for both domain orders tested. In hEx16-Dbs specifically, domain order might affect the agonistic activity of the anti-CD16 portion, which was supported by a cytokine production test, and likely contributed to the superiority of one of the hEx16-Dbs. Our results indicate that both the target antigen and mode of action of an antibody must be considered in the construction of highly functional bsAbs.


Assuntos
Anticorpos Biespecíficos/imunologia , Células Matadoras Naturais/imunologia , Neoplasias/terapia , Receptores de IgG/imunologia , Anticorpos Biespecíficos/farmacologia , Antineoplásicos/imunologia , Antineoplásicos/farmacologia , Complexo CD3/imunologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/imunologia , Humanos , Imunoterapia , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Neoplasias/imunologia , Neoplasias/patologia , Receptores de IgG/antagonistas & inibidores , Linfócitos T/imunologia
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