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1.
Adv Exp Med Biol ; 1172: 63-78, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31628651

RESUMO

The co-stimulation and co-inhibition signal pathways, immune checkpoints, are among the central mechanisms to regulate the T-cell immunity. Optimal signals involve intricate interactions of numerous ligands and receptors. Manipulation of these signals offers great clinical opportunities and has revolutionized the cancer treatment therapies. The 2018 Nobel Prize in Physiology or Medicine was awarded to James P. Allison and Tasuku Honjo in recognition of their discovery of cancer immunotherapy by inhibition of immune checkpoint molecules. Despite the landmark discovery in cancer immunotherapy, the efforts to harness immunity against cancer are also restricted by the limited knowledge on the co-stimulation and co-inhibition signaling networks. Understanding the structures of these molecules, in particular, tackling the interaction paradigms from the structural perspective, help to provide more accurate insights into the signaling mechanisms, which may further facilitate the development of novel biologics and improve the efficacy of the existing biologics against these targets. Here we review our current understanding on the structures of these co-stimulatory and co-inhibitory molecules. Specifically, we focus on the structural basis of several checkpoint molecules among the CD28-B7 family and discuss the therapeutic drugs against these targets for the treatment of human cancers, autoimmune disorders, and transplantation.


Assuntos
Antígenos CD28 , Linfócitos T , Doenças Autoimunes , Antígenos CD28/química , Antígenos CD28/imunologia , Humanos , Imunoterapia , Neoplasias/terapia , Transplante de Órgãos , Transdução de Sinais/imunologia , Linfócitos T/imunologia
2.
Fish Shellfish Immunol ; 72: 95-103, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29074133

RESUMO

Interaction of CD28 with CD80 or CD86 molecules provides a costimulatory signals required in T cell activation. In this study, we cloned and analyzed a CD28 gene (On-CD28) and a CD80/86 gene (On-CD80/86) from Nile tilapia (Oreochromis niloticus). Sequence analysis revealed the typical characteristics of On-CD28 protein; for instance, the proline-based motif (117TYPPPL122) is essential in binding of CD28 to CD80/86 ligands. Moreover, an extracellular Ig domain was found in On-CD80/86; this domain is responsible in binding of CD28 to CD80/86 receptors. Subcellular localization analysis showed that both On-CD28 and On-CD80/86 were distributed predominantly in the cytomembrane. Yeast two-hybrid assay showed that On-CD28 directly interacted with On-CD80/86. On-CD28 and On-CD80/86 transcripts were detected in all the examined tissues of healthy Nile tilapia, and the highest expression levels of On-CD28 and On-CD80/86 were detected in the brain and heart, respectively. Following a bacterial challenge using Streptococcus agalactiae in vivo, On-CD28 and On-CD80/86 were upregulated in head kidney, spleen, intestines, and brain. However, they showed different expression profiles in response to stimulation with inactivated S. agalactiae in vitro. These findings indicated that the interaction of On-CD28 with On-CD80/86 provides a costimulatory signals that possibly play an important role in T cell activation during S. agalactiae infection.


Assuntos
Imunidade Adaptativa/genética , Ciclídeos/genética , Ciclídeos/imunologia , Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Sequência de Aminoácidos , Animais , Antígeno B7-1/química , Antígeno B7-1/genética , Antígeno B7-1/imunologia , Antígeno B7-2/química , Antígeno B7-2/genética , Antígeno B7-2/imunologia , Antígenos CD28/química , Antígenos CD28/genética , Antígenos CD28/imunologia , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Proteínas de Membrana/química , Filogenia , Alinhamento de Sequência/veterinária , Infecções Estreptocócicas/imunologia , Streptococcus agalactiae/fisiologia
3.
J Clin Invest ; 127(9): 3462-3471, 2017 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-28805662

RESUMO

BACKGROUND: Targeting CD30 with monoclonal antibodies in Hodgkin lymphoma (HL) and anaplastic large cell lymphoma (ALCL) has had profound clinical success. However, adverse events, mainly mediated by the toxin component of the conjugated antibodies, cause treatment discontinuation in many patients. Targeting CD30 with T cells expressing a CD30-specific chimeric antigen receptor (CAR) may reduce the side effects and augment antitumor activity. METHODS: We conducted a phase I dose escalation study in which 9 patients with relapsed/refractory HL or ALCL were infused with autologous T cells that were gene-modified with a retroviral vector to express the CD30-specific CAR (CD30.CAR-Ts) encoding the CD28 costimulatory endodomain. Three dose levels, from 0.2 × 108 to 2 × 108 CD30.CAR-Ts/m2, were infused without a conditioning regimen. All other therapy for malignancy was discontinued at least 4 weeks before CD30.CAR-T infusion. Seven patients had previously experienced disease progression while being treated with brentuximab. RESULTS: No toxicities attributable to CD30.CAR-Ts were observed. Of 7 patients with relapsed HL, 1 entered complete response (CR) lasting more than 2.5 years after the second infusion of CD30.CAR-Ts, 1 remained in continued CR for almost 2 years, and 3 had transient stable disease. Of 2 patients with ALCL, 1 had a CR that persisted 9 months after the fourth infusion of CD30.CAR-Ts. CD30.CAR-T expansion in peripheral blood peaked 1 week after infusion, and CD30.CAR-Ts remained detectable for over 6 weeks. Although CD30 may also be expressed by normal activated T cells, no patients developed impaired virus-specific immunity. CONCLUSION: CD30.CAR-Ts are safe and can lead to clinical responses in patients with HL and ALCL, indicating that further assessment of this therapy is warranted. TRIAL REGISTRATION: ClinicalTrials.gov NCT01316146. FUNDING: National Cancer Institute (3P50CA126752, R01CA131027 and P30CA125123), National Heart, Lung, and Blood Institute (R01HL114564), and Leukemia and Lymphoma Society (LLSTR 6227-08).


Assuntos
Doença de Hodgkin/terapia , Antígeno Ki-1/metabolismo , Linfoma Anaplásico de Células Grandes/terapia , Receptores de Antígenos de Linfócitos T/química , Linfócitos T/citologia , Adulto , Antineoplásicos/química , Antígenos CD28/química , Progressão da Doença , Relação Dose-Resposta a Droga , Feminino , Doença de Hodgkin/imunologia , Humanos , Imunoconjugados/administração & dosagem , Imunoconjugados/uso terapêutico , Imunofenotipagem , Linfoma Anaplásico de Células Grandes/imunologia , Masculino , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Recidiva Local de Neoplasia , Condicionamento Pré-Transplante , Resultado do Tratamento , Adulto Jovem
4.
Methods Mol Biol ; 1584: 291-306, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28255708

RESUMO

The immune synapse has emerged as a compelling example of structural complexity within cell-cell interfaces. This chapter focuses on the use of microcontact printing to isolate and investigate how spatial organization of signaling molecules drives the function of immune cells. In the process detailed here, multiple rounds of microcontact printing are combined to create patterned surfaces that control the relative spatial localization of CD3 and CD28 signaling in T cells, effectively replacing an antigen presenting cell with an engineered surface. A set of approaches used to address key issues of T cell activation are described and discussed.


Assuntos
Antígenos CD28/química , Complexo CD3/química , Sinapses Imunológicas/química , Transdução de Sinais , Linfócitos T/química , Animais , Antígenos CD28/imunologia , Complexo CD3/imunologia , Humanos , Sinapses Imunológicas/imunologia , Linfócitos T/imunologia
5.
J Biol Chem ; 292(3): 1052-1060, 2017 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-27927989

RESUMO

Full activation of T cells and differentiation into effector T cells are essential for many immune responses and require co-stimulatory signaling via the CD28 receptor. Extracellular ligand binding to CD28 recruits protein-tyrosine kinases to its cytoplasmic tail, which contains a YMNM motif. Following phosphorylation of the tyrosine, the proteins growth factor receptor-bound protein 2 (Grb2), Grb2-related adaptor downstream of Shc (Gads), and p85 subunit of phosphoinositide 3-kinase may bind to pYMNM (where pY is phosphotyrosine) via their Src homology 2 (SH2) domains, leading to downstream signaling to distinct immune pathways. These three adaptor proteins bind to the same site on CD28 with variable affinity, and all are important for CD28-mediated co-stimulatory function. However, the mechanism of how these proteins recognize and compete for CD28 is unclear. To visualize their interactions with CD28, we have determined the crystal structures of Gads SH2 and two p85 SH2 domains in complex with a CD28-derived phosphopeptide. The high resolution structures obtained revealed that, whereas the CD28 phosphopeptide bound to Gads SH2 is in a bent conformation similar to that when bound to Grb2 SH2, it adopts a more extended conformation when bound to the N- and C-terminal SH2 domains of p85. These differences observed in the peptide-protein interactions correlated well with the affinity and other thermodynamic parameters for each interaction determined by isothermal titration calorimetry. The detailed insight into these interactions reported here may inform the development of compounds that specifically inhibit the association of CD28 with these adaptor proteins to suppress excessive T cell responses, such as in allergies and autoimmune diseases.


Assuntos
Antígenos CD28/química , Fosfopeptídeos/química , Domínios de Homologia de src/fisiologia , Antígenos CD28/genética , Antígenos CD28/metabolismo , Humanos , Fosfopeptídeos/genética , Fosfopeptídeos/metabolismo , Ligação Proteica/fisiologia , Linfócitos T/química , Linfócitos T/metabolismo , Termodinâmica
6.
Sci Signal ; 9(445): rs10, 2016 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-27625306

RESUMO

Dimethyl fumarate (DMF) is an electrophilic drug that is used to treat autoimmune conditions, including multiple sclerosis and psoriasis. The mechanism of action of DMF is unclear but may involve the covalent modification of proteins or DMF serving as a prodrug that is converted to monomethyl fumarate (MMF). We found that DMF, but not MMF, blocked the activation of primary human and mouse T cells. Using a quantitative, site-specific chemical proteomic platform, we determined the DMF sensitivity of >2400 cysteine residues in human T cells. Cysteines sensitive to DMF, but not MMF, were identified in several proteins with established biochemical or genetic links to T cell function, including protein kinase Cθ (PKCθ). DMF blocked the association of PKCθ with the costimulatory receptor CD28 by perturbing a CXXC motif in the C2 domain of this kinase. Mutation of these DMF-sensitive cysteines also impaired PKCθ-CD28 interactions and T cell activation, designating the C2 domain of PKCθ as a key functional, electrophile-sensing module important for T cell biology.


Assuntos
Fumarato de Dimetilo/química , Proteoma/química , Proteômica , Linfócitos T/química , Animais , Antígenos CD28/química , Antígenos CD28/imunologia , Cisteína/química , Cisteína/imunologia , Humanos , Ativação Linfocitária/fisiologia , Camundongos , Camundongos Knockout , Proteína Quinase C/química , Proteína Quinase C/imunologia , Proteoma/imunologia , Linfócitos T/imunologia
7.
Integr Biol (Camb) ; 7(11): 1442-53, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26400012

RESUMO

T cells are key mediators of adaptive immunity. However, the overall immune response is often directed by minor subpopulations of this heterogeneous family of cells, owing to specificity of activation and amplification of functional response. Knowledge of differences in signaling and function between T cell subtypes is far from complete, but is clearly needed for understanding and ultimately leveraging this branch of the adaptive immune response. This report investigates differences in cell response to micropatterned surfaces by conventional and regulatory T cells. Specifically, the ability of cells to respond to the microscale geometry of TCR/CD3 and CD28 engagement is made possible using a magnetic-microfluidic device that overcomes limitations in imaging efficiency associated with conventional microscopy equipment. This device can be readily assembled onto micropatterned surfaces while maintaining the activity of proteins and other biomolecules necessary for such studies. In operation, a target population of cells is tagged using paramagnetic beads, and then trapped in a divergent magnetic field within the chamber. Following washing, the target cells are released to interact with a designated surface. Characterization of this system with mouse CD4(+) T cells demonstrated a 50-fold increase in target-to-background cell purity, with an 80% collection efficiency. Applying this approach to CD4(+)CD25(+) regulatory T cells, it is then demonstrated that these rare cells respond less selectively to micro-scale features of anti-CD3 antibodies than CD4(+)CD25(-) conventional T cells, revealing a difference in balance between TCR/CD3 and LFA-1-based adhesion. PKC-θ localized to the distal pole of regulatory T cells, away from the cell-substrate interface, suggests a mechanism for differential regulation of TCR/LFA-1-based adhesion. Moreover, specificity of cell adhesion to anti-CD3 features was dependent on the relative position of anti-CD28 signaling within the cell-substrate interface, revealing an important role for coincidence of TCR and costimulatory pathway in triggering regulatory T cell function.


Assuntos
Dispositivos Lab-On-A-Chip , Linfócitos T Reguladores/citologia , Imunidade Adaptativa , Animais , Antígenos CD28/química , Complexo CD3/química , Linfócitos T CD4-Positivos/citologia , Adesão Celular , Moléculas de Adesão Celular , Movimento Celular , Subunidade alfa de Receptor de Interleucina-2/química , Ativação Linfocitária/imunologia , Camundongos , Microfluídica , Microscopia , Transdução de Sinais
9.
Cell Mol Life Sci ; 72(14): 2739-48, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25725801

RESUMO

The CD28 costimulatory receptor has a pivotal role in T cell biology as this molecule amplifies T cell receptor (TCR) signals to provide an efficient immune T cell response. There is a large debate about how CD28 mediates these signals. Here, we designed a CD28 gene-targeted knock-in mouse strain lacking the cytoplasmic tail of CD28. As is the case in CD28-deficient (CD28 knock-out) mice, regulatory T cell homeostasis and T cell activation are altered in these CD28 knock-in mice. Unexpectedly, the presence of a CD28 molecule deprived of its cytoplasmic tail could partially induce some early activation events in T cells such as signaling events or expression of early activation markers. These results unravel a new mechanism of T cell costimulation by CD28, independent of its cytoplasmic tail.


Assuntos
Antígenos CD28/fisiologia , Ativação Linfocitária/fisiologia , Linfócitos T/imunologia , Animais , Antígenos CD28/química , Antígenos CD28/genética , Técnicas de Introdução de Genes , Ativação Linfocitária/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Estrutura Terciária de Proteína , Febre Q/imunologia , Transdução de Sinais
10.
Clin Cancer Res ; 21(10): 2359-66, 2015 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-25549724

RESUMO

PURPOSE: HHLA2 (B7H7/B7-H5/B7y) is a newly identified B7 family member that regulates human T-cell functions. However, its protein expression in human organs and significance in human diseases are unknown. The objective of this study was to analyze HHLA2 protein expression in normal human tissues and cancers, as well as its prognostic significance, to explore mechanisms regulating HHLA2 expression, and to identify candidate HHLA2 receptors. EXPERIMENTAL DESIGN: An immunohistochemistry protocol and a flow cytometry assay with newly generated monoclonal antibodies were developed to examine HHLA2 protein. HHLA2 gene copy-number variation was analyzed from cancer genomic data. The combination of bioinformatics analysis and immunologic approaches was established to explore HHLA2 receptors. RESULTS: HHLA2 protein was detected in trophoblastic cells of the placenta and the epithelium of gut, kidney, gallbladder, and breast, but not in most other organs. In contrast, HHLA2 protein was widely expressed in human cancers from the breast, lung, thyroid, melanoma, pancreas, ovary, liver, bladder, colon, prostate, kidney, and esophagus. In a cohort of 50 patients with stage I-III triple-negative breast cancer, 56% of patients had aberrant expression of HHLA2 on their tumors, and high HHLA2 expression was significantly associated with regional lymph node metastasis and stage. The Cancer Genome Atlas revealed that HHLA2 copy-number gains were present in 29% of basal breast cancers, providing a potential mechanism for increased HHLA2 protein expression in breast cancer. Finally, Transmembrane and Immunoglobulin Domain Containing 2 (TMIGD2) was identified as one of the receptors for HHLA2. CONCLUSIONS: Wide expression of HHLA2 in human malignancies, together with its association with poor prognostic factors and its T-cell coinhibitory capability, suggests that the HHLA2 pathway represents a novel immunosuppressive mechanism within the tumor microenvironment and an attractive target for human cancer therapy.


Assuntos
Imunoglobulinas/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Células 3T3 , Sequência de Aminoácidos , Animais , Antígenos CD28/química , Antígenos CD28/metabolismo , Linhagem Celular Tumoral , Variações do Número de Cópias de DNA , Feminino , Expressão Gênica , Glicosilação , Humanos , Imunoglobulinas/genética , Metástase Linfática , Camundongos , Pessoa de Meia-Idade , Dados de Sequência Molecular , Processamento de Proteína Pós-Traducional , Neoplasias de Mama Triplo Negativas/patologia
11.
Cytokine ; 71(2): 289-95, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25484350

RESUMO

The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) regulates a battery of antioxidant, detoxification, and cell stress genes. It is activated by oxidative stress and a number of exogenous compounds, one of which is tert-butylhydroquinone (tBHQ), a widely used food preservative. Nrf2 modulates immune responses in numerous rodent models of inflammation, but its effects on human immune cells are not well characterized. The purpose of these studies was to evaluate the effects of the Nrf2 activator tBHQ on early events of T cell activation in primary human cells. Treatment with tBHQ induced mRNA expression of the Nrf2 target genes HMOX-1, GCLC, and NQO1, and also increased NRF2 mRNA expression, albeit to a lesser extent than the other target genes. tBHQ decreased production of the cytokines IL-2 and IFN-γ at both the protein and mRNA levels after stimulation with anti-CD3/anti-CD28 in human peripheral blood mononuclear cells and to an even greater extent in isolated CD4 T cells. Likewise, tBHQ decreased induction of CD25 and CD69 in peripheral blood mononuclear cells (PBMCs) and this decrease was even more marked in isolated CD4 T cells. In addition, tBHQ inhibited induction of NFκB DNA binding in anti-CD3/anti-CD28-activated PBMCs. Collectively, these data suggest that tBHQ inhibits activation of primary human CD4 T cells, which correlates with activation of Nrf2 and inhibition of NFκB DNA binding. Although these studies suggest the food additive tBHQ negatively impacts T cell activation, further studies will be needed to fully elucidate the effect of tBHQ on human immune responses.


Assuntos
Hidroquinonas/química , Fator 2 Relacionado a NF-E2/metabolismo , NF-kappa B/metabolismo , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Antioxidantes/metabolismo , Antígenos CD28/química , Complexo CD3/química , Linfócitos T CD4-Positivos/citologia , Citocinas/metabolismo , DNA/química , Relação Dose-Resposta a Droga , Citometria de Fluxo , Humanos , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Lectinas Tipo C/metabolismo , Leucócitos Mononucleares/citologia , Ativação Linfocitária , Estresse Oxidativo/efeitos dos fármacos , Ligação Proteica
12.
J Immunol ; 194(3): 1323-33, 2015 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-25539813

RESUMO

Phosphatidylinositol 4,5-biphosphate (PIP2) is a cell membrane phosphoinositide crucial for cell signaling and activation. Indeed, PIP2 is a pivotal source for second messenger generation and controlling the activity of several proteins regulating cytoskeleton reorganization. Despite its critical role in T cell activation, the molecular mechanisms regulating PIP2 turnover remain largely unknown. In human primary CD4(+) T lymphocytes, we have recently demonstrated that CD28 costimulatory receptor is crucial for regulating PIP2 turnover by allowing the recruitment and activation of the lipid kinase phosphatidylinositol 4-phosphate 5-kinase (PIP5Kα). We also identified PIP5Kα as a key modulator of CD28 costimulatory signals leading to the efficient T cell activation. In this study, we extend these data by demonstrating that PIP5Kα recruitment and activation is essential for CD28-mediated cytoskeleton rearrangement necessary for organizing a complete signaling compartment leading to downstream signaling functions. We also identified Vav1 as the linker molecule that couples the C-terminal proline-rich motif of CD28 to the recruitment and activation of PIP5Kα, which in turn cooperates with Vav1 in regulating actin polymerization and CD28 signaling functions.


Assuntos
Actinas/metabolismo , Antígenos CD28/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas Proto-Oncogênicas c-vav/metabolismo , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Antígenos CD28/química , Antígenos CD28/genética , Comunicação Celular , Linhagem Celular , Ativação Enzimática , Expressão Gênica , Humanos , Mutação , Proteínas Oncogênicas/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Domínios Proteicos Ricos em Prolina , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo
13.
ACS Chem Biol ; 10(2): 485-92, 2015 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-25372624

RESUMO

Dendritic cells (DCs) are antigen-presenting cells that play an essential role in T cell activation. Recent efforts in cancer immunotherapy have been directed at the development of artificial antigen presenting cells (aAPCs) loaded with tumor antigens. These aAPCs are designed to mimic DCs with the goal of triggering an efficient and specific T cell response directed against the tumor. We have designed a novel synthetic dendritic cell (sDC) that possesses the essential features of natural DCs. Our sDC is based on a semiflexible poly(isocyano peptide) polymer and carries anti-CD3 antibodies (αCD3) for triggering the T cell receptor/CD3 complex as well as anti-CD28 antibodies (αCD28) as a co-stimulatory signal. Multiple copies of both antibodies facilitate multivalent binding similar to natural DCs. The high mobility of these polymer-bound antibodies, reminiscent of protein motility in a natural plasma membrane, enables receptor rearrangements to occur during T cell activation. We show that our bifunctional αCD3/αCD28-sDC triggers T cell activation at significantly lower antibody concentrations than freely soluble antibodies. This superior performance is further demonstrated in comparison to a mixture of monofunctional αCD3-sDC and αCD28-sDC. The presence of both antibodies on the same polymer not only reduces the threshold for T cell activation but, more importantly, critically shapes the specificity of the T cell response. αCD3/αCD28-sDC is a far more efficient activator of multifunctional killer cells. These findings demonstrate the potential of multifunctional polymers for mimicking natural DCs, paving the way for their exploitation in immunotherapeutic strategies.


Assuntos
Células Dendríticas , Polímeros/síntese química , Subpopulações de Linfócitos T/fisiologia , Antígenos CD28/química , Complexo CD3/química , Vacinas Anticâncer/química , Humanos , Estrutura Molecular
14.
J Infect Dis ; 211(6): 995-1003, 2015 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-25305323

RESUMO

BACKGROUND: Severe gram-negative bacterial infections and sepsis are major causes of morbidity and mortality. Dysregulated, excessive proinflammatory cytokine expression contributes to the pathogenesis of sepsis. A CD28 mimetic peptide (AB103; previously known as p2TA) that attenuates CD28 signaling and T-helper type 1 cytokine responses was tested for its ability to increase survival in models of polymicrobial infection and gram-negative sepsis. METHODS: Mice received AB103, followed by an injection of Escherichia coli 0111:B4 lipopolysaccharide (LPS); underwent induction E. coli 018:K1 peritonitis induction, followed by treatment with AB103; or underwent cecal ligation and puncture (CLP), followed by treatment with AB103. The effects of AB103 on factors associated with and the lethality of challenge infections were analyzed. RESULTS: AB103 strongly attenuated induction of tumor necrosis factor α and interleukin 6 (IL-6) by LPS in human peripheral blood mononuclear cells. Receipt of AB103 following intraperitoneal injection of LPS resulted in survival among 73% of CD1 mice (11 of 15), compared with 20% of controls (3 of 15). Suboptimal doses of antibiotic alone protected 20% of mice (1 of 5) from E. coli peritonitis, whereas 100% (15 of 15) survived when AB103 was added 4 hours following infection. Survival among mice treated with AB103 12 hours after CLP was 100% (8 of 8), compared with 17% among untreated mice (1 of 6). In addition, receipt of AB103 12 hours after CLP attenuated inflammatory cytokine responses and neutrophil influx into tissues and promoted bacterial clearance. Receipt of AB103 24 hours after CLP still protected 63% of mice (5 of 8). CONCLUSIONS: Single-dose AB103 reduces mortality in experimental models of polymicrobial and gram-negative bacterial infection and sepsis, warranting further studies of this agent in clinical trials.


Assuntos
Antibacterianos/uso terapêutico , Antígenos CD28/química , Infecções por Escherichia coli/prevenção & controle , Peritonite/prevenção & controle , Sepse/prevenção & controle , Animais , Animais não Endogâmicos , Antibacterianos/farmacologia , Antígenos CD28/uso terapêutico , Células Cultivadas , Quimiocinas/metabolismo , Infecções por Escherichia coli/tratamento farmacológico , Feminino , Humanos , Lipopolissacarídeos/farmacologia , Camundongos Endogâmicos BALB C , Mimetismo Molecular , Infiltração de Neutrófilos/efeitos dos fármacos , Peritonite/tratamento farmacológico , Peritonite/imunologia , Domínios e Motivos de Interação entre Proteínas , Sepse/tratamento farmacológico
15.
Bioanalysis ; 6(18): 2371-83, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25384590

RESUMO

AIM: To support drug development of a PEGylated anti-CD28 domain antibody, a sensitive and robust LC-MS/MS assay was developed for the first in-human multiple ascending dose study. MATERIALS & METHODS: The procedure consists of a protein precipitation with acidified acetonitrile, followed by trypsin digestion of the supernatant. A surrogate peptide from the complementarity determining region was quantified with an LC-MS/MS assay using a stable isotope-labeled internal standard with flanking amino acids. An acid dissociation step was found to be essential to achieve full analyte recovery in the presence of antidrug antibodies and soluble target CD28. RESULTS & CONCLUSION: The fully validated LC-MS/MS assay demonstrates good accuracy (% deviation ≤6.3) and precision (%CV ≤5.2) with an lower limit of quantitation of 10 ng/ml.


Assuntos
Análise Química do Sangue/métodos , Antígenos CD28/imunologia , Cromatografia Líquida/métodos , Preparações Farmacêuticas , Polietilenoglicóis/química , Anticorpos de Domínio Único/sangue , Espectrometria de Massas em Tandem/métodos , Acetonitrilos/química , Análise Química do Sangue/normas , Antígenos CD28/química , Calibragem , Precipitação Química , Estabilidade de Medicamentos , Feminino , Humanos , Limite de Detecção , Masculino , Proteólise , Padrões de Referência , Anticorpos de Domínio Único/imunologia , Anticorpos de Domínio Único/isolamento & purificação , Anticorpos de Domínio Único/metabolismo , Solubilidade , Tripsina/metabolismo
16.
J Immunol ; 193(10): 5315-26, 2014 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-25320284

RESUMO

Adoptive transfer of T cells that are gene engineered to express a defined TCR represents a feasible and promising therapy for patients with tumors. However, TCR gene therapy is hindered by the transient presence and effectiveness of transferred T cells, which are anticipated to be improved by adequate T cell costimulation. In this article, we report the identification and characterization of a novel two-chain TCR linked to CD28 and CD3ε (i.e., TCR:28ε). This modified TCR demonstrates enhanced binding of peptide-MHC and mediates enhanced T cell function following stimulation with peptide compared with wild-type TCR. Surface expression of TCR:28ε depends on the transmembrane domain of CD28, whereas T cell functions depend on the intracellular domains of both CD28 and CD3ε, with IL-2 production showing dependency on CD28:LCK binding. TCR:28ε, but not wild-type TCR, induces detectable immune synapses in primary human T cells, and such immune synapses show significantly enhanced accumulation of TCR transgenes and markers of early TCR signaling, such as phosphorylated LCK and ERK. Importantly, TCR:28ε does not show signs of off-target recognition, as evidenced by lack of TCR mispairing, as well as preserved specificity. Notably, when testing TCR:28ε in immune-competent mice, we observed a drastic increase in T cell survival, which was accompanied by regression of large melanomas with limited recurrence. Our data argue that TCR transgenes that contain CD28, and, thereby, may provide T cell costimulation in an immune-suppressive environment, represent candidate receptors to treat patients with tumors.


Assuntos
Antígenos CD28/imunologia , Complexo CD3/imunologia , Melanoma/terapia , Receptores de Antígenos de Linfócitos T/imunologia , Neoplasias Cutâneas/terapia , Linfócitos T/imunologia , Animais , Antígenos CD28/química , Antígenos CD28/genética , Complexo CD3/química , Complexo CD3/genética , Linhagem Celular Tumoral , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/imunologia , Expressão Gênica , Regulação da Expressão Gênica , Humanos , Sinapses Imunológicas , Interleucina-2/genética , Interleucina-2/imunologia , Ativação Linfocitária , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/genética , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/imunologia , Melanoma/genética , Melanoma/imunologia , Melanoma/mortalidade , Camundongos , Recidiva Local de Neoplasia/prevenção & controle , Transplante de Neoplasias , Ligação Proteica , Engenharia de Proteínas , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Transdução de Sinais , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/mortalidade , Análise de Sobrevida , Linfócitos T/metabolismo , Linfócitos T/transplante , Carga Tumoral
17.
PLoS One ; 9(7): e101161, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25054224

RESUMO

The goal of this study was to elucidate the action of the CD28 mimetic peptide p2TA (AB103) that attenuates an excessive inflammatory response in mitigating radiation-induced inflammatory injuries. BALB/c and A/J mice were divided into four groups: Control (C), Peptide (P; 5 mg/kg of p2TA peptide), Radiation (R; total body irradiation with 8 Gy γ-rays), and Radiation + Peptide (RP; irradiation followed by p2TA peptide 24 h later). Gastrointestinal tissue damage was evaluated by analysis of jejunum histopathology and immunohistochemistry for cell proliferation (Cyclin D1) and inflammation (COX-2) markers, as well as the presence of macrophages (F4/80). Pro-inflammatory cytokines IL-6 and KC as well as fibrinogen were quantified in plasma samples obtained from the same mice. Our results demonstrated that administration of p2TA peptide significantly reduced the irradiation-induced increase of IL-6 and fibrinogen in plasma 7 days after exposure. Seven days after total body irradiation with 8 Gy of gamma rays numbers of intestinal crypt cells were reduced and villi were shorter in irradiated animals compared to the controls. The p2TA peptide delivery 24 h after irradiation led to improved morphology of villi and crypts, increased Cyclin D1 expression, decreased COX-2 staining and decreased numbers of macrophages in small intestine of irradiated mice. Our study suggests that attenuation of CD28 signaling is a promising therapeutic approach for mitigation of radiation-induced tissue injury.


Assuntos
Antígenos CD28/antagonistas & inibidores , Trato Gastrointestinal/efeitos dos fármacos , Inflamação/prevenção & controle , Peptídeos/farmacologia , Trombose/prevenção & controle , Sequência de Aminoácidos , Animais , Antígenos de Diferenciação/metabolismo , Antígenos CD28/química , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Ciclina D1/metabolismo , Ciclo-Oxigenase 2/metabolismo , Fibrinogênio/metabolismo , Raios gama , Trato Gastrointestinal/patologia , Trato Gastrointestinal/efeitos da radiação , Imuno-Histoquímica , Inflamação/etiologia , Inflamação/metabolismo , Interleucina-6/sangue , Jejuno/efeitos dos fármacos , Jejuno/patologia , Jejuno/efeitos da radiação , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/efeitos da radiação , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos , Lesões Experimentais por Radiação/complicações , Protetores contra Radiação/farmacologia , Trombose/sangue , Trombose/etiologia , Irradiação Corporal Total
18.
J Immunol ; 192(8): 3465-9, 2014 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-24639356

RESUMO

CD28 is a critical regulator of T cell function, augmenting proliferation, cytokine secretion, and cell survival. Our previous work using knockin mice expressing point mutations in CD28 demonstrated that the distal proline motif was primarily responsible for much of CD28 function, whereas in marked contrast to prior studies, mutation of the PI3K-binding motif had little discernible effect. In this study, we examined the phenotype of mice in which both motifs are simultaneously mutated. We found that mutation of the PYAP motif unmasks a critical role for the proximal tyrosine motif in regulating T cell proliferation and expression of Bcl-xL but not cytokine secretion. In addition, we demonstrated that, although function is more severely impaired in the double mutant than in either single mutant, there remained residual CD28-dependent responses, definitively establishing that additional motifs can partially mediate CD28 function.


Assuntos
Motivos de Aminoácidos , Antígenos CD28/genética , Antígenos CD28/imunologia , Mutação , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Proteína bcl-X/genética , Sequência de Aminoácidos , Animais , Antígenos CD28/química , Proliferação de Células , Citocinas/biossíntese , Ativação Linfocitária , Camundongos , Camundongos Transgênicos , Proteína bcl-X/metabolismo
19.
PLoS One ; 8(9): e74482, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24098653

RESUMO

Src homology 2 (SH2) domains play a critical role in cellular signal transduction. They bind to peptides containing phosphotyrosine (pY) with various specificities that depend on the flanking amino-acid residues. The SH2 domain of growth-factor receptor-bound protein 2 (Grb2) specifically recognizes pY-X-N-X, whereas the SH2 domains in phosphatidylinositol 3-kinase (PI3K) recognize pY-X-X-M. Binding of the pY site in CD28 (pY-M-N-M) by PI3K and Grb2 through their SH2 domains is a key step that triggers the CD28 signal transduction for T cell activation and differentiation. In this study, we determined the crystal structure of the Grb2 SH2 domain in complex with a pY-containing peptide derived from CD28 at 1.35 Å resolution. The peptide was found to adopt a twisted U-type conformation, similar to, but distinct from type-I ß-turn. In all previously reported crystal structures, the peptide bound to the Grb2 SH2 domains adopts a type-I ß-turn conformation, except those with a proline residue at the pY+3 position. Molecular modeling also suggests that the same peptide bound to PI3K might adopt a very different conformation.


Assuntos
Proteína Adaptadora GRB2/química , Modelos Moleculares , Conformação Proteica , Domínios de Homologia de src/genética , Antígenos CD28/química , Cristalografia , Fosfopeptídeos/química
20.
Methods Cell Biol ; 117: 181-96, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24143978

RESUMO

Fluorescence correlation spectroscopy (FCS) performed using a laser scanning confocal microscope is a technique with single-molecule sensitivity that is becoming more accessible to cell biologists. In this chapter, we describe the use of FCS for the analysis of diffusion coefficients and receptor-receptor interactions in live cells in culture. In particular, we describe a protocol to collect fluorescence fluctuation data from fluorescence-tagged receptors as they diffuse into an out of a small laser-illuminated observation volume using a commercially available system such as the Zeiss ConfoCor 3 or LSM-780 microscope. Autocorrelation analysis of the fluctuations in fluorescence intensity provides information about the diffusion time and number of fluorescent molecules in the observation volume. A photon-counting histogram can be used to examine the relationship between fluorescence intensity and the number of fluorescent molecules to estimate the average molecular brightness of the sample. Since molecular brightness is directly proportional to the number of fluorescent molecules, it can be used to monitor receptor-receptor interactions and to decode the number of receptor monomers present in an oligomeric complex.


Assuntos
Antígeno B7-2/química , Proteínas de Bactérias/química , Proteínas de Fluorescência Verde/química , Proteínas Luminescentes/química , Fótons , Receptores Adrenérgicos beta 2/química , Espectrometria de Fluorescência/estatística & dados numéricos , Animais , Antígeno B7-2/genética , Antígeno B7-2/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Antígenos CD28/química , Antígenos CD28/genética , Antígenos CD28/metabolismo , Células CHO , Cricetulus , Difusão , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Imagem Molecular , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/metabolismo , Espectrometria de Fluorescência/métodos , Coloração e Rotulagem , Transfecção
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