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1.
J Immunol ; 2019 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-31666306

RESUMO

Although the immune adaptor SH2 domain containing leukocyte phosphoprotein of 76 kDa (SLP-76) integrates and propagates the TCR signaling, the regulation of SLP-76 during the TCR signaling is incompletely studied. In this article, we report that SLP-76 interacts with the small ubiquitin-like modifier (SUMO) E2 conjugase Ubc9 and is a substrate for Ubc9-mediated SUMOylation in human and mouse T cells. TCR stimulation promotes SLP-76-Ubc9 binding, accompanied by an increase in SLP-76 SUMOylation. Ubc9 binds to the extreme C terminus of SLP-76 spanning residues 516-533 and SUMOylates SLP-76 at two conserved residues K266 and K284. In addition, SLP-76 and Ubc9 synergizes to augment the TCR-mediated IL-2 transcription by NFAT in a manner dependent of SUMOylation of SLP-76. Moreover, although not affecting the TCR proximal signaling events, the Ubc9-mediated SUMOylation of SLP-76 is required for TCR-induced assembly of Ubc9-NFAT complex for IL-2 transcription. Together, these results suggest that Ubc9 modulates the function of SLP-76 in T cell activation both by direct interaction and by SUMOylation of SLP-76 and that the Ubc9-SLP-76 module acts as a novel regulatory complex in the control of T cell activation.

2.
Adv Exp Med Biol ; 1164: 225-233, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31576552

RESUMO

Immune checkpoint blockade (ICB) has proved successful in the immunotherapeutic treatment of various human cancers. Despite its success, most patients are still not cured while immunogenic cold cancers are still poorly responsive. There is a need for novel clinical interventions in immunotherapy, either alone or in conjunction with ICB. Here, we outline our recent discovery that the intracellular signaling kinase glycogen synthase kinase-3 (GSK-3) is a central regulator of PD-1 in T-cells. We demonstrate the application of small molecule inhibitor (SMI) approaches to down-regulate PD-1 in tumor immunotherapy. GSK-3 SMIs were found as effective as anti-PD-1 in the elimination of melanoma in mouse models. We propose the development of novel SMIs to target co-receptors for the future of immunotherapy.


Assuntos
Regulação Neoplásica da Expressão Gênica , Quinase 3 da Glicogênio Sintase , Imunoterapia , Melanoma , Animais , Modelos Animais de Doenças , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Humanos , Melanoma/terapia , Camundongos , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/metabolismo , Linfócitos T/fisiologia
3.
Semin Immunol ; 42: 101295, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31604533

RESUMO

The past few years have witnessed exciting progress in the application of immune check-point blockade (ICB) for the treatment of various human cancers. ICB was first used against cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) to demonstrate durable anti-tumor responses followed by ICB against programmed cell death-1 (PD-1) or its ligand, PD-L1. Present approaches involve the use of combinations of blocking antibodies against CTLA-4, PD-1 and other inhibitory receptors (IRs) such as TIM3, TIGIT and LAG3. Despite this success, most patients are not cured by ICB therapy and there are limitations to the use of antibodies including cost, tumor penetration, the accessibility of receptors, and clearance from the cell surface as well as inflammatory and autoimmune complications. Recently, we demonstrated that the down-regulation or inhibition of glycogen synthase kinase 3 (GSK-3) down-regulates PD-1 expression in infectious diseases and cancer (Taylor et al., 2016 Immunity 44, 274-86; 2018 Cancer Research 78, 706-717; Krueger and Rudd 2018 Immunity 46, 529-531). In this Review, we outline the use of small molecule inhibitors (SMIs) that target intracellular pathways for co-receptor blockade in cancer immunotherapy.

4.
Semin Immunol ; 42: 101281, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31604538
5.
Nat Commun ; 10(1): 4804, 2019 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-31641113

RESUMO

Immunotherapy involving checkpoint blockades of inhibitory co-receptors is effective in combating cancer. Despite this, the full range of mediators that inhibit T-cell activation and influence anti-tumor immunity is unclear. Here, we identify the GTPase-activating protein (GAP) Rasal1 as a novel TCR-ZAP-70 binding protein that negatively regulates T-cell activation and tumor immunity. Rasal1 inhibits via two pathways, the binding and inhibition of the kinase domain of ZAP-70, and GAP inhibition of the p21ras-ERK pathway. It is expressed in activated CD4 + and CD8 + T-cells, and inhibits CD4 + T-cell responses to antigenic peptides presented by dendritic cells as well as CD4 + T-cell responses to peptide antigens in vivo. Furthermore, siRNA reduction of Rasal1 expression in T-cells shrinks B16 melanoma and EL-4 lymphoma tumors, concurrent with an increase in CD8 + tumor-infiltrating T-cells expressing granzyme B and interferon γ-1. Our findings identify ZAP-70-associated Rasal1 as a new negative regulator of T-cell activation and tumor immunity.

6.
Sci Rep ; 9(1): 10462, 2019 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-31320682

RESUMO

While the immune cell adaptor protein SKAP1 mediates LFA-1 activation induced by antigen-receptor (TCR/CD3) ligation on T-cells, it is unclear whether the adaptor interacts with other mediators of T-cell function. In this context, the serine/threonine kinase, polo-like kinase (PLK1) regulates multiple steps in the mitotic and cell cycle progression of mammalian cells. Here, we show that SKAP1 is phosphorylated by and binds to PLK1 for the optimal cycling of T-cells. PLK1 binds to the N-terminal residue serine 31 (S31) of SKAP1 and the interaction is needed for optimal PLK1 kinase activity. Further, siRNA knock-down of SKAP1 reduced the rate of T-cell division concurrent with a delay in the expression of PLK1, Cyclin A and pH3. Reconstitution of these KD cells with WT SKAP1, but not the SKAP1 S31 mutant, restored normal cell division. SKAP1-PLK1 binding is dynamically regulated during the cell cycle of T-cells. Our findings identify a novel role for SKAP1 in the regulation of PLK1 and optimal cell cycling needed for T-cell clonal expansion in response to antigenic activation.

7.
BMC Res Notes ; 11(1): 869, 2018 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-30522503

RESUMO

OBJECTIVE: Immune cell adaptor protein SKAP1 couples the antigen-receptor (TCR/CD3) with the activation of LFA-1 adhesion in T-cells. Previous work by ourselves and others have shown that SKAP1 can directly bind to other adaptors such as ADAP and RapL. However, it has been unclear whether SKAP1 can form homodimers with itself and the regions within SKAP1 that mediated homodimer formation. RESULTS: Here, we show that SKAP1 and SKAP2 form homodimers in cells. Homodimer formation of immune adaptor protein SKAP1 (SKAP-55) are mediated by residues A17 to L21 in the SKAP1 N-terminal region. SKAP1 dimer formation was not needed for its binding to RapL. These data indicate that the pathway linking SKAP1 to RapL is not dependent on the homo-dimerization of SKAP1.

8.
Front Immunol ; 9: 2737, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30542345

RESUMO

CTLA-4 is a co-receptor on T-cells that controls peripheral tolerance and the development of autoimmunity. Immune check-point blockade (ICB) uses monoclonal antibodies (MAbs) to block the binding of inhibitory receptors (IRs) to their natural ligands. A humanized antibody to CTLA-4 was first approved clinically followed by the use of antibody blockade against PD-1 and its ligand PD-L1. Effective anti-tumor immunity requires the activation of tumor-specific effector T-cells, the blockade of regulatory cells and the migration of T-cells into the tumor. Here, we review data implicating CTLA-4 and PD-1 in the motility of T-cells with a specific reference to the potential exploitation of these pathways for more effective tumor infiltration and eradication.


Assuntos
Antígeno CTLA-4/imunologia , Movimento Celular/imunologia , Neoplasias/imunologia , Neoplasias/terapia , Receptor de Morte Celular Programada 1/imunologia , Linfócitos T Reguladores/imunologia , Animais , Anticorpos Bloqueadores/imunologia , Anticorpos Monoclonais/imunologia , Autoimunidade/imunologia , Humanos , Imunoterapia/métodos
9.
Cancer Res ; 78(3): 706-717, 2018 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-29055015

RESUMO

The impact of PD-1 immune checkpoint therapy prompts exploration of other strategies to downregulate PD-1 for cancer therapy. We previously showed that the serine/threonine kinase, glycogen synthase kinase, GSK-3α/ß, is a central regulator of PD-1 transcription in CD8+ T cells. Here, we show that the use of small-molecule inhibitors of GSK-3α/ß (GSK-3i) to reduce pcdc1 (PD-1) transcription and expression was as effective as anti-PD-1 and PD-L1-blocking antibodies in the control of B16 melanoma, or EL4 lymphoma, in primary tumor and metastatic settings. Furthermore, the conditional genetic deletion of GSK-3α/ß reduced PD-1 expression on CD8+ T cells and limited B16 pulmonary metastasis to the same degree as PD-1 gene deficiency. In each model, GSK-3i inhibited PD-1 expression on tumor-infiltrating lymphocytes, while increasing Tbx21 (T-bet) transcription, and the expression of CD107a+ (LAMP1) and granzyme B (GZMB) on CD8+ T cells. Finally, the adoptive transfer of T cells treated ex vivo with a GSK-3 inhibitor delayed the onset of EL4 lymphoma growth to a similar extent as anti-PD-1 pretreatment. Overall, our findings show how GSK-3 inhibitors that downregulate PD-1 expression can enhance CD8+ T-cell function in cancer therapy to a similar degree as PD-1-blocking antibodies.Significance: These findings show how GSK-3 inhibitors that downregulate PD-1 expression can enhance CD8+ T-cell function in cancer therapy to a similar degree as PD-1 blocking antibodies, offering a next-generation approach in the design of immunotherapeutic approaches for cancer management. Cancer Res; 78(3); 706-17. ©2017 AACR.

10.
Nat Commun ; 8: 16001, 2017 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-28699640

RESUMO

Lymphocyte function-associated antigen 1 (LFA-1) affinity and avidity changes have been assumed to mediate adhesion to intercellular adhesion molecule-1 for T-cell conjugation to dendritic cells (DC). Although the T-cell receptor (TCR) and LFA-1 can generate intracellular signals, the immune cell adaptor protein linker for the activation of T cells (LAT) couples the TCR to downstream events. Here, we show that LFA-1 can mediate both adhesion and de-adhesion, dependent on receptor clustering. Although increased affinity mediates adhesion, LFA-1 cross-linking induced the association and activation of the protein-tyrosine kinases FAK1/PYK1 that phosphorylated LAT selectively on a single Y-171 site for the binding to adaptor complex GRB-2-SKAP1. LAT-GRB2-SKAP1 complexes were distinct from canonical LAT-GADs-SLP-76 complexes. LFA-1 cross-linking increased the presence of LAT-GRB2-SKAP1 complexes relative to LAT-GADs-SLP-76 complexes. LFA-1-FAK1 decreased T-cell-dendritic cell (DC) dwell times dependent on LAT-Y171, leading to reduced DO11.10 T cell binding to DCs and proliferation to OVA peptide. Overall, our findings outline a new model for LFA-1 in which the integrin can mediate both adhesion and de-adhesion events dependent on receptor cross-linking.

11.
Immunity ; 46(4): 529-531, 2017 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-28423334

RESUMO

The identity of PD-1 dependency on other receptors and signaling has been unclear. In a recent issue of Science, Hui et al. (2017) and Kamphorst et al. (2017) now show that CD28 expression is a target of PD-1-associated phosphatases and is needed for T cell expansion in anti-PD-1 immunotherapy.


Assuntos
Antígenos CD28 , Linfócitos T , Ciclo Celular , Humanos , Imunoterapia , Transdução de Sinais
12.
J Biol Chem ; 292(15): 6281-6290, 2017 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-28188290

RESUMO

The adaptor protein Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) plays a crucial role in T cell activation by linking antigen receptor (T cell receptor, TCR) signals to downstream pathways. At its N terminus, SLP-76 has three key tyrosines (Tyr-113, Tyr-128, and Tyr-145, "3Y") as well as a sterile α motif (SAM) domain whose function is unclear. We showed previously that the SAM domain has two binding regions that mediate dimer and oligomer formation. In this study, we have identified SAM domain-carrying non-receptor tyrosine kinase, activated Cdc42-associated tyrosine kinase 1 (ACK1; also known as Tnk2, tyrosine kinase non-receptor 2) as a novel binding partner of SLP-76. Co-precipitation, laser-scanning confocal microscopy, and in situ proximity analysis confirmed the binding of ACK1 to SLP-76. Further, the interaction was induced in response to the anti-TCR ligation and abrogated by the deletion of SLP-76 SAM domain (ΔSAM) or mutation of Tyr-113, Tyr-128, and Tyr-145 to phenylalanine (3Y3F). ACK1 induced phosphorylation of the SLP-76 N-terminal tyrosines (3Y) dependent on the SAM domain. Further, ACK1 promoted calcium flux and NFAT-AP1 promoter activity and decreased the motility of murine CD4+ primary T cells on ICAM-1-coated plates, an event reversed by a small molecule inhibitor of ACK1 (AIM-100). These findings identify ACK1 as a novel SLP-76-associated protein-tyrosine kinase that modulates early activation events in T cells.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Ativação Linfocitária/fisiologia , Fosfoproteínas/metabolismo , Proteínas Tirosina Quinases/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Motivos de Aminoácidos , Substituição de Aminoácidos , Animais , Humanos , Células Jurkat , Camundongos , Mutação de Sentido Incorreto , Fosfoproteínas/genética , Fosfoproteínas/imunologia , Fosforilação/fisiologia , Domínios Proteicos , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/imunologia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Tirosina
13.
Front Immunol ; 8: 1653, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29312284

RESUMO

The rescue of exhausted CD8+ cytolytic T-cells (CTLs) by anti-Programmed Cell Death-1 (anti-PD-1) blockade has been found to require CD28 expression. At the same time, we have shown that the inactivation of the serine/threonine kinase glycogen synthase kinase (GSK)-3α/ß with small-interfering RNAs (siRNAs) and small molecule inhibitors (SMIs) specifically down-regulates PD-1 expression for enhanced CD8+ CTL function and clearance of tumors and viral infections. Despite this, it has been unclear whether the GSK-3α/ß pathway accounts for CD28 costimulation of CD8+ CTL function. In this article, we show that inactivation of GSK-3α/ß through siRNA or by SMIs during priming can substitute CD28 co-stimulation in the potentiation of cytotoxic CD8+ CTL function against the EL-4 lymphoma cells expressing OVA peptide. The effect was seen using several structurally distinct GSK-3 SMIs and was accompanied by an increase in Lamp-1 and GZMB expression. Conversely, CD28 crosslinking obviated the need for GSK-3α/ß inhibition in its enhancement of CTL function. Our findings support a model where GSK-3 is the central cosignal for CD28 priming of CD8+ CTLs in anti-PD-1 immunotherapy.

14.
Mol Cell Biol ; 36(24): 3113-3127, 2016 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-27697866

RESUMO

The clinical potential of transplantation is often reduced by T cell-mediated alloresponses that cause graft rejection or graft-versus-host disease. Integrin-mediated adhesion between alloreactive T cells and antigen-presenting cells is essential for allorejection. The identity of the signaling events needed for the activation of integrins such as LFA-1 is poorly understood. Here, we identified a novel role of the protein tyrosine phosphatase SHP-1 in the regulation of murine LFA-1-mediated adhesion in an allograft setting. Upon alloactivation, SHP-1 activity is reduced, resulting in an increase in LFA-1 adhesion compared to that for syngeneically activated T cells. The importance of these differential activation properties was further indicated by small interfering RNA (siRNA) knockdown of SHP-1 in syngeneically and allogeneically stimulated T cells. Mechanistically, SHP-1 modulated the binding of SLP-76 to ADAP by dephosphorylation of the YDGI tyrosine motif of ADAP, a known docking site for the Src family kinase Fyn. This novel key role of SHP-1 in the regulation of LFA-1-mediated adhesion may provide a new insight into T cell-mediated alloresponses and may pave the way to the development of new immunosuppressive pharmaceutical agents.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Aloenxertos/imunologia , Antígeno-1 Associado à Função Linfocitária/metabolismo , Fosfoproteínas/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Linfócitos T/citologia , Proteínas Adaptadoras de Transdução de Sinal/química , Aloenxertos/metabolismo , Animais , Sítios de Ligação , Adesão Celular , Células Cultivadas , Regulação da Expressão Gênica , Transplante de Células-Tronco Hematopoéticas , Camundongos , Fosforilação , Ligação Proteica , Linfócitos T/metabolismo
15.
J Immunol ; 197(4): 1471-6, 2016 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-27357151

RESUMO

Pancreatic islet transplantation is a promising therapy for diabetes, but acute rejection of the islets by host effector T cells has hindered clinical application. In this study, we addressed the mechanisms of CD8(+) effector T cell migration to islet grafts because interrupting this step is key to preventing rejection. We found that effector T cell migration to revascularized islet transplants in mice is dependent on non-self Ag recognition rather than signaling via Gαi-coupled chemokine receptors. Presentation of non-self Ag by donor cells was necessary for migration, whereas Ag presentation by recipient cells was dispensable. We also observed that deficiency of SKAP1, an immune cell adaptor downstream of the TCR and important for integrin activation, prolongs allograft survival but does not reduce effector T cell migration to the graft. Therefore, effector T cell migration to transplanted islets is Ag driven, not chemokine driven, but SKAP1 does not play a critical role in this process.


Assuntos
Apresentação do Antígeno/imunologia , Linfócitos T CD8-Positivos/imunologia , Quimiotaxia de Leucócito/imunologia , Rejeição de Enxerto/imunologia , Transplante das Ilhotas Pancreáticas/imunologia , Animais , Ilhotas Pancreáticas/imunologia , Camundongos , Modelos Animais
16.
Immunol Lett ; 176: 122-7, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27181093

RESUMO

SKAP1 is an immune cell adaptor that couples the T-cell receptor with the 'inside-out' signalling pathway for LFA-1 mediated adhesion in T-cells. A connection of SKAP1 to the regulation of an autoimmune disorder has not previously been reported. In this study, we show that Skap1-deficient (skap1-/-) mice are highly resistant to the induction of collagen-induced arthritis (CIA), both in terms of incidence or severity. Skap1-/- T-cells were characterised by a selective reduction in the presence IL-17+ (Th17) in response to CII peptide and a marked reduction of joint infiltrating T-cells in Skap1-/- mice. SKAP1 therefore represents a novel connection to Th17 producing T-cells and is new potential target in the therapeutic intervention in autoimmune and inflammatory diseases.


Assuntos
Artrite Experimental/imunologia , Articulações/imunologia , Fosfoproteínas/genética , Células Th17/imunologia , Animais , Movimento Celular , Colágeno Tipo II/imunologia , Suscetibilidade a Doenças , Interleucina-17/metabolismo , Ativação Linfocitária , Antígeno-1 Associado à Função Linfocitária/metabolismo , Camundongos , Camundongos Endogâmicos DBA , Camundongos Knockout , Fosfoproteínas/metabolismo , Transdução de Sinais
18.
Immunity ; 44(2): 274-86, 2016 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-26885856

RESUMO

Despite the importance of the co-receptor PD-1 in T cell immunity, the upstream signaling pathway that regulates PD-1 expression has not been defined. Glycogen synthase kinase 3 (GSK-3, isoforms α and ß) is a serine-threonine kinase implicated in cellular processes. Here, we identified GSK-3 as a key upstream kinase that regulated PD-1 expression in CD8(+) T cells. GSK-3 siRNA downregulation, or inhibition by small molecules, blocked PD-1 expression, resulting in increased CD8(+) cytotoxic T lymphocyte (CTL) function. Mechanistically, GSK-3 inactivation increased Tbx21 transcription, promoting enhanced T-bet expression and subsequent suppression of Pdcd1 (encodes PD-1) transcription in CD8(+) CTLs. Injection of GSK-3 inhibitors in mice increased in vivo CD8(+) OT-I CTL function and the clearance of murine gamma-herpesvirus 68 and lymphocytic choriomeningitis clone 13 and reversed T cell exhaustion. Our findings identify GSK-3 as a regulator of PD-1 expression and demonstrate the applicability of GSK-3 inhibitors in the modulation of PD-1 in immunotherapy.


Assuntos
Aminofenóis/administração & dosagem , Linfócitos T CD8-Positivos/imunologia , Quinase 3 da Glicogênio Sintase/metabolismo , Infecções por Herpesviridae/imunologia , Coriomeningite Linfocítica/imunologia , Vírus da Coriomeningite Linfocítica/fisiologia , Maleimidas/administração & dosagem , Receptor de Morte Celular Programada 1/metabolismo , Rhadinovirus/fisiologia , Proteínas com Domínio T/metabolismo , Linfócitos T Citotóxicos/imunologia , Aminofenóis/efeitos adversos , Animais , Linfócitos T CD8-Positivos/virologia , Células Cultivadas , Citotoxicidade Imunológica/efeitos dos fármacos , Citotoxicidade Imunológica/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Quinase 3 da Glicogênio Sintase/genética , Maleimidas/efeitos adversos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Receptor de Morte Celular Programada 1/genética , RNA Interferente Pequeno/genética , Proteínas com Domínio T/genética , Linfócitos T Citotóxicos/virologia , Carga Viral/efeitos dos fármacos , Carga Viral/genética
19.
Immunol Lett ; 172: 40-6, 2016 04.
Artigo em Inglês | MEDLINE | ID: mdl-26905930

RESUMO

While the cytoskeletal protein talin binds to the ß-chain of LFA-1, the immune cell adaptor SKAP1 (SKAP-55) binds to the α-chain of the same integrin via RapL. Whereas calpain protease cleavage of talin is important for LFA-1 activation, it has been unclear whether SKAP1 can alter the function of talin or its associated adaptor RIAM in T-cells. In this paper, we report that Skap1-/- T-cells showed a reduction in the translocation of talin and RIAM to the contact interface of T-cells with antigenic beads or dendritic cells (DCs) presenting OVA peptide to OT-1 T-cells. In addition, Skap1-/- T-cells show an altered pattern of talin cleavage, while the expression of a cleavage resistant form of talin (L432G) restored the impaired adhesion of OT1 transgenic Skap1-/- T-cells with DCs. SKAP1 therefore can affect the function of talin in T-cells needed for optimal T-cell/DC conjugation.


Assuntos
Células Dendríticas/imunologia , Sinapses Imunológicas/metabolismo , Fosfoproteínas/metabolismo , Linfócitos T/fisiologia , Talina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Células Cultivadas , Antígeno-1 Associado à Função Linfocitária/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfoproteínas/genética , Ligação Proteica , Transporte Proteico , Proteólise , Especificidade por Substrato
20.
J Immunol ; 195(11): 5432-9, 2015 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-26500347

RESUMO

Optimal T cell activation typically requires engagement of both the TCR and costimulatory receptors, such as CD28. Engagement of CD28 leads to tyrosine phosphorylation of its cytoplasmic region and recruitment of cytoplasmic signaling proteins. Although the exact mechanism of CD28 signal transduction is unknown, CD28 triggering has similarities to the TCR, which was proposed to use the kinetic-segregation (KS) mechanism. The KS model postulates that, when small receptors engage their ligands within areas of close (∼15 nm) contact in the T cell/APC interface, this facilitates phosphorylation by segregating the engaged receptor/ligand complex from receptor protein tyrosine phosphatases with large ectodomains, such as CD45. To test this hypothesis, we examined the effect of elongating the extracellular region of the CD28 ligand, CD80, on its ability to costimulate IL-2 production by primary T cells. CD80 elongation reduced its costimulatory effect without abrogating CD28 binding. Confocal microscopy revealed that elongated CD80 molecules were less well segregated from CD45 at the T cell/APC interface. T cells expressing CD28 harboring a key tyrosine-170 mutation were less sensitive to CD80 elongation. In summary, the effectiveness of CD28 costimulation is inversely proportional to the dimensions of the CD28-CD80 complex. Small CD28-CD80 complex dimensions are required for optimal costimulation by segregation from large inhibitory tyrosine phosphatases. These results demonstrate the importance of ligand dimensions for optimal costimulation of IL-2 production by T cells and suggest that the KS mechanism contributes to CD28 signaling.


Assuntos
Antígeno B7-1/imunologia , Antígenos CD28/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Interleucina-2/biossíntese , Animais , Antígeno B7-1/genética , Antígenos CD28/genética , Células CHO , Linhagem Celular , Cricetinae , Cricetulus , Antígenos Comuns de Leucócito/imunologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Complexos Multiproteicos/imunologia , Fosforilação/imunologia , Ligação Proteica , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais/imunologia
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