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1.
J Immunol ; 187(5): 2244-51, 2011 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-21788441

RESUMO

Egress of lymphocytes from lymphoid tissues is a complex process in which Gαi-mediated signals play a decisive role. We show here that although FTY720, an agonist of the sphingosine 1-phosphate (S1P)(1) receptor, induces S1P(1) receptor internalization sufficiently in the presence or absence of Gαi2 or Gαi3, the drug blocks egress of wild-type (WT) and Gαi3-deficent T cells, but not Gαi2-deficient T cells, in both WT and Gαi2-deficient hosts. Intravital imaging of lymph nodes revealed that all three groups of T cells approached and engaged cortical sinusoids similarly in the presence or absence of FTY720. The cells also entered and departed the sinus at an almost identical frequency in the absence of the drug. However, after engagement of the sinus, most WT and Gαi3-deficient T cells retracted and migrated back into the parenchyma in FTY720-treated animals, due to a failure of the cells to establish adhesion on the sinus, whereas Gαi2-deficient T cells adhered firmly on the sinus, which prevented their retraction, facilitating their transmigration of the lymphatic endothelial barrier. These data confirm egress of Gαi2(-/-) T cells independent of S1P-mediated chemotaxis and failure of FTY720 to close lymphatic stromal channels and argue for the first time, to our knowledge, that FTY720 induces lymphopenia in part by impairing T cell adhesion to the sinus in a manner dependent on Gαi2.


Assuntos
Quimiotaxia de Leucócito/efeitos dos fármacos , Imunossupressores/farmacologia , Propilenoglicóis/farmacologia , Esfingosina/análogos & derivados , Linfócitos T/efeitos dos fármacos , Animais , Adesão Celular/efeitos dos fármacos , Adesão Celular/imunologia , Separação Celular , Quimiotaxia de Leucócito/imunologia , Cloridrato de Fingolimode , Citometria de Fluxo , Subunidade alfa Gi2 de Proteína de Ligação ao GTP/genética , Subunidade alfa Gi2 de Proteína de Ligação ao GTP/imunologia , Subunidade alfa Gi2 de Proteína de Ligação ao GTP/metabolismo , Linfonodos/citologia , Linfonodos/efeitos dos fármacos , Linfonodos/imunologia , Camundongos , Camundongos Knockout , Receptores de Lisoesfingolipídeo/agonistas , Esfingosina/farmacologia , Linfócitos T/imunologia , Linfócitos T/metabolismo
2.
Mol Cancer Ther ; 21(6): 986-998, 2022 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-35642431

RESUMO

In the past year, four antibody-drug conjugates (ADC) were approved, nearly doubling the marketed ADCs in oncology. Among other attributes, successful ADCs optimize targeting antibody, conjugation chemistry, and payload mechanism of action. Here, we describe the development of ABBV-011, a novel SEZ6-targeted, calicheamicin-based ADC for the treatment of small cell lung cancer (SCLC). We engineered a calicheamicin conjugate that lacks the acid-labile hydrazine linker that leads to systemic release of a toxic catabolite. We then screened a patient-derived xenograft library to identify SCLC as a tumor type with enhanced sensitivity to calicheamicin ADCs. Using RNA sequencing (RNA-seq) data from primary and xenograft SCLC samples, we identified seizure-related homolog 6 (SEZ6) as a surface-expressed SCLC target with broad expression in SCLC and minimal normal tissue expression by both RNA-seq and IHC. We developed an antibody targeting SEZ6 that is rapidly internalized upon receptor binding and, when conjugated to the calicheamicin linker drug, drives potent tumor regression in vitro and in vivo. These preclinical data suggest that ABBV-011 may provide a novel treatment for patients with SCLC and a rationale for ongoing phase I studies (NCT03639194).


Assuntos
Antineoplásicos , Imunoconjugados , Neoplasias Pulmonares , Carcinoma de Pequenas Células do Pulmão , Antineoplásicos/farmacologia , Calicheamicinas , Ensaios Clínicos Fase I como Assunto , Humanos , Imunoconjugados/farmacologia , Imunoconjugados/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Carcinoma de Pequenas Células do Pulmão/genética
3.
J Biol Chem ; 285(29): 22328-37, 2010 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-20463015

RESUMO

Lymphocyte egress from lymph nodes requires the G-protein-coupled sphingosine 1-phosphate receptor-1 (S1P(1)). The activation antigen CD69 associates with and inhibits the function of S1P(1), inhibiting egress. Here we undertook biochemical characterization of the requirements for S1P(1)-CD69 complex formation. Domain swapping experiments between CD69 and the related type II transmembrane protein, NKRp1A, identified a requirement for the transmembrane and membrane proximal domains for specific interaction. Mutagenesis of S1P(1) showed a lack of requirement for N-linked glycosylation, tyrosine sulfation, or desensitization motifs but identified a requirement for transmembrane helix 4. Expression of CD69 led to a reduction of S1P(1) in cell lysates, likely reflecting degradation. Unexpectedly, the S1P(1)-CD69 complex exhibited a much longer half-life for binding of S1P than S1P(1) alone. In contrast to wild-type CD69, a non-S1P(1) binding mutant of CD69 failed to inhibit T cell egress from lymph nodes. These findings identify an integral membrane interaction between CD69 and S1P(1) and suggest that CD69 induces an S1P(1) conformation that shares some properties of the ligand-bound state, thereby facilitating S1P(1) internalization and degradation.


Assuntos
Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Membrana Celular/metabolismo , Lectinas Tipo C/metabolismo , Receptores de Lisoesfingolipídeo/química , Receptores de Lisoesfingolipídeo/metabolismo , Células 3T3 , Animais , Linhagem Celular , Quimiotaxia de Leucócito , Meia-Vida , Humanos , Imunoprecipitação , Linfócitos/citologia , Linfócitos/metabolismo , Lisofosfolipídeos/metabolismo , Camundongos , Proteínas Mutantes/metabolismo , Mutação/genética , Ligação Proteica , Mapeamento de Interação de Proteínas , Processamento de Proteína Pós-Traducional , Estrutura Secundária de Proteína , Receptores de Lisoesfingolipídeo/genética , Esfingosina/análogos & derivados , Esfingosina/metabolismo
4.
Biochemistry ; 47(47): 12398-408, 2008 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-18973345

RESUMO

To understand the mechanisms that govern T cell receptor (TCR)-peptide MHC (pMHC) binding and the role that different regions of the TCR play in affinity and antigen specificity, we have studied the TCR from T cell clone 2C. High-affinity mutants of the 2C TCR that bind QL9-L(d) as a strong agonist were generated previously by site-directed mutagenesis of complementarity determining regions (CDRs) 1beta, 2alpha, 3alpha, or 3beta. We performed isothermal titration calorimetry to assess whether they use similar thermodynamic mechanisms to achieve high affinity for QL9-L(d). Four of the five TCRs examined bound to QL9-L(d) in an enthalpically driven, entropically unfavorable manner. In contrast, the high-affinity CDR1beta mutant resembled the wild-type 2C TCR interaction, with favorable entropy. To assess fine specificity, we measured the binding and kinetics of these mutants for both QL9-L(d) and a single amino acid peptide variant of QL9, called QL9-Y5-L(d). While 2C and most of the mutants had equal or higher affinity for the Y5 variant than for QL9, mutant CDR1beta exhibited 8-fold lower affinity for Y5 compared to QL9. To examine possible structural correlates of the thermodynamic and fine specificity signatures of the TCRs, the structure of unliganded QL9-L(d) was solved and compared to structures of the 2C TCR/QL9-L(d) complex and three high-affinity TCR/QL9-L(d) complexes. Our findings show that the QL9-L(d) complex does not undergo major conformational changes upon binding. Thus, subtle changes in individual CDRs account for the diverse thermodynamic and kinetic binding mechanisms and for the different peptide fine specificities.


Assuntos
Oligopeptídeos/metabolismo , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/metabolismo , Sequência de Aminoácidos , Animais , Cricetinae , Cinética , Ligantes , Camundongos , Modelos Moleculares , Mutação , Oligopeptídeos/química , Ligação Proteica , Conformação Proteica , Ratos , Receptores de Antígenos de Linfócitos T/genética , Especificidade por Substrato , Termodinâmica , Transfecção
5.
Cancer Res ; 77(14): 3931-3941, 2017 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-28487384

RESUMO

The Notch ligand DLL3 has emerged as a novel therapeutic target expressed in small cell lung cancer (SCLC) and high-grade neuroendocrine carcinomas. Rovalpituzumab teserine (Rova-T; SC16LD6.5) is a first-in-class DLL3-targeted antibody-drug conjugate with encouraging initial safety and efficacy profiles in SCLC in the clinic. Here we demonstrate that tumor expression of DLL3, although orders of magnitude lower in surface protein expression than typical oncology targets of immunoPET, can serve as an imaging biomarker for SCLC. We developed 89Zr-labeled SC16 antibody as a companion diagnostic agent to facilitate selection of patients for treatment with Rova-T based on a noninvasive interrogation of the in vivo status of DLL3 expression using PET imaging. Despite low cell-surface abundance of DLL3, immunoPET imaging with 89Zr-labeled SC16 antibody enabled delineation of subcutaneous and orthotopic SCLC tumor xenografts as well as distant organ metastases with high sensitivity. Uptake of the radiotracer in tumors was concordant with levels of DLL3 expression and, most notably, DLL3 immunoPET yielded rank-order correlation for response to SC16LD6.5 therapy in SCLC patient-derived xenograft models. Cancer Res; 77(14); 3931-41. ©2017 AACR.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/biossíntese , Neoplasias Pulmonares/metabolismo , Proteínas de Membrana/biossíntese , Carcinoma de Pequenas Células do Pulmão/metabolismo , Células A549 , Animais , Linhagem Celular Tumoral , Feminino , Xenoenxertos , Humanos , Imunoconjugados , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Neoplasias Pulmonares/diagnóstico por imagem , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Camundongos , Camundongos Nus , Metástase Neoplásica , Tomografia por Emissão de Pósitrons , Carcinoma de Pequenas Células do Pulmão/diagnóstico por imagem , Carcinoma de Pequenas Células do Pulmão/genética , Carcinoma de Pequenas Células do Pulmão/patologia
6.
J Mol Biol ; 343(4): 1055-65, 2004 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-15476821

RESUMO

It has recently been reported that dipeptidyl aminopeptidase X (DPPX) interacts with the voltage-gated potassium channel Kv4 and that co-expression of DPPX together with Kv4 pore forming alpha-subunits, and potassium channel interacting proteins (KChIPs), reconstitutes properties of native A-type potassium channels in vitro. Here we report the X-ray crystal structure of the extracellular domain of human DPPX determined at 3.0A resolution. This structure reveals the potential for a surface electrostatic change based on the protonation state of histidine. Subtle changes in extracellular pH might modulate the interaction of DPPX with Kv4.2 and possibly with other proteins. We propose models of DPPX interaction with the voltage-gated potassium channel complex. The dimeric structure of DPPX is highly homologous to the related protein DPP-IV. Comparison of the active sites of DPPX and DPP-IV reveals loss of the catalytic serine residue but the presence of an additional serine near the "active" site. However, the arrangement of residues is inconsistent with that of canonical serine proteases and DPPX is unlikely to function as a protease (dipeptidyl aminopeptidase).


Assuntos
Dipeptidil Peptidases e Tripeptidil Peptidases/química , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Processamento Alternativo , Domínio Catalítico , Cistina/química , Cistina/genética , Cistina/metabolismo , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Glicosilação , Humanos , Isoenzimas/química , Isoenzimas/metabolismo , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Canais de Potássio Shal , Eletricidade Estática
7.
Mol Immunol ; 40(14-15): 1033-9, 2004 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-15036907

RESUMO

Degeneracy in immune recognition is usually thought of in terms of the astonishing ability of the T cell receptor to recognize an enormously diverse array of peptides bound to major histocompatibility complex (MHC) molecules. However, in this essay we discuss an alternative aspect of degeneracy in T cell recognition: the notion that peptides can assume different "registers" in the groove of a single MHC molecule, as first suggested and demonstrated by Sercarz and co-workers (reviewed in [J. autoimmun. 16 (2001) 201]). There is now abundant evidence, derived from functional, biochemical and structural studies, that single peptides can assume alternative, unpredictable binding registers by frameshifting within the MHC groove [Nat. Immunol. 3 (2002) 175;; J. Exp. Med. 187 (1998) 1505; J. Mol. Biol. 304 (2000) 177; Biochemistry 38 (1999) 16663; J. Exp. Med. 197 (2003) 1391; Eur. J. Immunol. 19 (1989) 681]. Hence, register shifting adds an additional dimension to the concept of degeneracy. In fact, the possibility of register shifting multiplies the universe of peptide-MHC (pMHC) surfaces that a TCR must recognize by an unknown, perhaps enormous factor. Register shifting also has profound implication for autoimmunity: (1) as a mechanism to "mask" autoantigenic epitopes during thymic education [Immunol. Rev. 169 (1999) 147; Immunity 17 (2002) 83]; and (2) as a possible source for pMHC complexes capable of molecular mimicry.


Assuntos
Antígenos de Histocompatibilidade/metabolismo , Peptídeos/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Animais , Autoimunidade/imunologia , Antígenos de Histocompatibilidade/imunologia , Humanos , Peptídeos/imunologia , Ligação Proteica , Estrutura Terciária de Proteína , Linfócitos T/imunologia
8.
Sci Transl Med ; 7(302): 302ra136, 2015 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-26311731

RESUMO

The high-grade pulmonary neuroendocrine tumors, small cell lung cancer (SCLC) and large cell neuroendocrine carcinoma (LCNEC), remain among the most deadly malignancies. Therapies that effectively target and kill tumor-initiating cells (TICs) in these cancers should translate to improved patient survival. Patient-derived xenograft (PDX) tumors serve as excellent models to study tumor biology and characterize TICs. Increased expression of delta-like 3 (DLL3) was discovered in SCLC and LCNEC PDX tumors and confirmed in primary SCLC and LCNEC tumors. DLL3 protein is expressed on the surface of tumor cells but not in normal adult tissues. A DLL3-targeted antibody-drug conjugate (ADC), SC16LD6.5, comprised of a humanized anti-DLL3 monoclonal antibody conjugated to a DNA-damaging pyrrolobenzodiazepine (PBD) dimer toxin, induced durable tumor regression in vivo across multiple PDX models. Serial transplantation experiments executed with limiting dilutions of cells provided functional evidence confirming that the lack of tumor recurrence after SC16LD6.5 exposure resulted from effective targeting of DLL3-expressing TICs. In vivo efficacy correlated with DLL3 expression, and responses were observed in PDX models initiated from patients with both limited and extensive-stage disease and were independent of their sensitivity to standard-of-care chemotherapy regimens. SC16LD6.5 effectively targets and eradicates DLL3-expressing TICs in SCLC and LCNEC PDX tumors and is a promising first-in-class ADC for the treatment of high-grade pulmonary neuroendocrine tumors.


Assuntos
Anticorpos Monoclonais/imunologia , Antineoplásicos/uso terapêutico , Imunoconjugados/uso terapêutico , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Neoplasias Pulmonares/tratamento farmacológico , Proteínas de Membrana/imunologia , Tumores Neuroendócrinos/tratamento farmacológico , Animais , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Tumores Neuroendócrinos/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Immunol Lett ; 95(2): 185-92, 2004 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-15388259

RESUMO

Antibiotic resistant strains of Staphylococcus aureus pose a growing threat to public health, and immunotherapy offers potential modalities to combat antibiotic resistance. We prepared bispecific monoclonal antibody complexes (heteropolymers, HP), specific for the primate erythrocyte complement C3b receptor (CR1) and type 5 capsular polysaccharide of the T5 isolate of S. aureus. Fluorescence microscopy and flow cytometry revealed that HP promote binding of S. aureus to human erythrocytes. Incubation of erythrocyte-bound, HP-opsonized S. aureus with human monocyte/macrophages or mouse macrophage cell lines led to transfer, internalization and killing of bacteria by macrophages with little erythrocyte loss. This reaction is similar to the process in which C3b-opsonized substrates, bound to erythrocyte CR1 by immune adherence, are transferred to acceptor phagocytes. Our results provide the basis for development of an in vivo paradigm focused on immunotherapeutic approaches for treatment of infections due to antibiotic resistant bacteria.


Assuntos
Anticorpos Biespecíficos/imunologia , Anticorpos Monoclonais/imunologia , Eritrócitos/microbiologia , Macrófagos/imunologia , Fagocitose , Receptores de Complemento 3b/imunologia , Staphylococcus aureus/imunologia , Animais , Células Cultivadas , Eritrócitos/imunologia , Eritrócitos/metabolismo , Humanos , Macrófagos/citologia , Macrófagos/microbiologia , Camundongos , Microscopia de Fluorescência , Receptores de Complemento 3b/metabolismo
10.
J Exp Med ; 206(11): 2469-81, 2009 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-19808259

RESUMO

During a screen for ethylnitrosourea-induced mutations in mice affecting blood natural killer (NK) cells, we identified a strain, designated Duane, in which NK cells were reduced in blood and spleen but increased in lymph nodes (LNs) and bone marrow (BM). The accumulation of NK cells in LNs reflected a decreased ability to exit into lymph. This strain carries a point mutation within Tbx21 (T-bet), which generates a defective protein. Duane NK cells have a 30-fold deficiency in sphingosine-1-phosphate receptor 5 (S1P5) transcript levels, and S1P5-deficient mice exhibit an egress defect similar to Duane. Chromatin immunoprecipitation confirms binding of T-bet to the S1pr5 locus. S1P-deficient mice exhibit a more severe NK cell egress block, and the FTY720-sensitive S1P1 also plays a role in NK cell egress from LNs. S1P5 is not inhibited by CD69, a property that may facilitate trafficking of activated NK cells to effector sites. Finally, the accumulation of NK cells within BM of S1P-deficient mice was associated with reduced numbers in BM sinusoids, suggesting a role for S1P in BM egress. In summary, these findings identify S1P5 as a T-bet-induced gene that is required for NK cell egress from LNs and BM.


Assuntos
Células da Medula Óssea/citologia , Movimento Celular , Células Matadoras Naturais/citologia , Células Matadoras Naturais/metabolismo , Linfonodos/citologia , Receptores de Lisoesfingolipídeo/metabolismo , Proteínas com Domínio T/metabolismo , Animais , Células da Medula Óssea/efeitos dos fármacos , Contagem de Células , Movimento Celular/efeitos dos fármacos , Etilnitrosoureia , Cloridrato de Fingolimode , Células Matadoras Naturais/efeitos dos fármacos , Linfonodos/efeitos dos fármacos , Ativação Linfocitária/efeitos dos fármacos , Lisofosfolipídeos/deficiência , Lisofosfolipídeos/farmacologia , Camundongos , Camundongos Mutantes , Mutação/genética , Propilenoglicóis/farmacologia , Esfingosina/análogos & derivados , Esfingosina/deficiência , Esfingosina/farmacologia
11.
Science ; 316(5822): 291-4, 2007 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-17431183

RESUMO

The pre-B cell receptor (pre-BCR) serves as a checkpoint in B cell development. In the 2.7 angstrom structure of a human pre-BCR Fab-like fragment, consisting of an antibody heavy chain (HC) paired with the surrogate light chain, the "unique regions" of VpreB and lambda5 replace the complementarity-determining region 3 (CDR3) loop of an antibody light chain and appear to "probe" the HC CDR3, potentially influencing the selection of the antibody repertoire. Biochemical analysis indicates that the pre-BCR is impaired in its ability to recognize antigen, which, together with electron microscopic visualization of a pre-BCR dimer, suggests ligand-independent oligomerization as the likely signaling mechanism.


Assuntos
Glicoproteínas de Membrana/química , Receptores de Antígenos de Linfócitos B/química , Animais , Regiões Determinantes de Complementaridade/química , Regiões Determinantes de Complementaridade/fisiologia , Cristalografia por Raios X , Humanos , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas/fisiologia , Cadeias Leves de Imunoglobulina/química , Cadeias Leves de Imunoglobulina/fisiologia , Cadeias Leves Substitutas da Imunoglobulina , Glicoproteínas de Membrana/fisiologia , Glicoproteínas de Membrana/ultraestrutura , Camundongos , Modelos Moleculares , Receptores de Células Precursoras de Linfócitos B , Conformação Proteica , Receptores de Antígenos de Linfócitos B/fisiologia , Receptores de Antígenos de Linfócitos B/ultraestrutura , Proteínas Recombinantes , Relação Estrutura-Atividade
12.
Biochemistry ; 46(33): 9453-61, 2007 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-17655329

RESUMO

The cytokine interleukin-15 (IL-15) signals through the formation of a quaternary receptor complex composed of an IL-15-specific alpha receptor, together with beta and gammac receptors that are shared with interleukin-2 (IL-2). The initiating step in the formation of this signaling complex is the interaction between IL-15 and IL-15Ralpha, which is a single sushi domain bearing strong structural homology to one of the two sushi domains of IL-2Ralpha. The crystal structure of the IL2-Ralpha/IL-2 complex has been determined, however little is known about the analogous IL-15Ralpha/IL-15 binding interaction. Here we show that recombinant IL-15 can be overexpressed as a stable complex in the presence of its high affinity receptor, IL-15Ralpha. We find that this complex is 10-fold more active than IL-15 alone in stimulating proliferation and survival of memory phenotype CD8 T cells. To probe the ligand/receptor interface, we used solution NMR to map chemical shifts on 15N-labeled IL-15Ralpha in complex with unlabeled IL-15. Our results predict that the binding surface on IL-15Ralpha involves strands C and D, similar to IL-2Ralpha. The interface, as predicted here, leaves open the possibility of trans-presentation of IL-15 by IL-15Ralpha on an opposing cell.


Assuntos
Subunidade alfa de Receptor de Interleucina-15/química , Interleucina-15/química , Sítios de Ligação , Linfócitos T CD8-Positivos/imunologia , Humanos , Interleucina-15/genética , Interleucina-15/metabolismo , Subunidade alfa de Receptor de Interleucina-15/genética , Subunidade alfa de Receptor de Interleucina-15/metabolismo , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação Proteica , Mapeamento de Interação de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
13.
Cell ; 129(1): 135-46, 2007 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-17418792

RESUMO

alphabeta T cell receptors (TCRs) can crossreact with both self- and foreign- major histocompatibility complex (MHC) proteins in an enigmatic phenomenon termed alloreactivity. Here we present the 2.35 A structure of the 2C TCR complexed with its foreign ligand H-2L(d)-QL9. Surprisingly, we find that this TCR utilizes a different strategy to engage the foreign pMHC in comparison to the manner in which it recognizes a self ligand H-2K(b)-dEV8. 2C engages both shared and polymorphic residues on L(d) and K(b), as well as the unrelated QL9 and dEV8 peptide antigens, in unique pair-wise contacts, resulting in greater structural complementarity with the L(d)-QL9 complex. In the structure of an engineered, high-affinity 2C TCR variant bound to H-2L(d)-QL9, the "wild-type" TCR-MHC binding orientation persists despite modified TCR-CDR3alpha interactions with peptide. Thus, a single TCR recognizes two globally similar, but distinct ligands by divergent mechanisms, indicating that receptor-ligand crossreactivity can occur in the absence of molecular mimicry.


Assuntos
Autoantígenos/imunologia , Antígenos H-2/imunologia , Isoantígenos/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Sequência de Aminoácidos , Autoantígenos/química , Autoantígenos/metabolismo , Regiões Determinantes de Complementaridade/metabolismo , Cristalografia por Raios X , Antígenos H-2/química , Antígenos H-2/metabolismo , Antígeno de Histocompatibilidade H-2D , Isoantígenos/química , Isoantígenos/metabolismo , Complexo Cetoglutarato Desidrogenase/química , Complexo Cetoglutarato Desidrogenase/imunologia , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/imunologia , Peptídeos/metabolismo , Ligação Proteica , Receptores de Antígenos de Linfócitos T alfa-beta/química , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo
14.
J Immunol ; 179(9): 5907-15, 2007 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-17947664

RESUMO

Several MHC class II alleles linked with autoimmune diseases form unusually low stability complexes with CLIP, leading us to hypothesize that this is an important feature contributing to autoimmune pathogenesis. To investigate cellular consequences of altering class II/CLIP affinity, we evaluated invariant chain (Ii) mutants with varying CLIP affinity for a mouse class II allele, I-E(d), which has low affinity for wild-type CLIP and is associated with a mouse model of spontaneous, autoimmune joint inflammation. Increasing CLIP affinity for I-E(d) resulted in increased cell surface and total cellular abundance and half-life of I-E(d). This reveals a post-endoplasmic reticulum chaperoning capacity of Ii via its CLIP peptides. Quantitative effects on I-E(d) were less pronounced in DM-expressing cells, suggesting complementary chaperoning effects mediated by Ii and DM, and implying that the impact of allelic variation in CLIP affinity on immune responses will be highest in cells with limited DM activity. Differences in the ability of cell lines expressing wild-type or high-CLIP-affinity mutant Ii to present Ag to T cells suggest a model in which increased CLIP affinity for class II serves to restrict peptide loading to DM-containing compartments, ensuring proper editing of antigenic peptides.


Assuntos
Antígenos de Diferenciação de Linfócitos B/imunologia , Antígenos de Diferenciação de Linfócitos B/metabolismo , Antígenos de Histocompatibilidade Classe II/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Alelos , Animais , Apresentação de Antígeno/imunologia , Antígenos de Diferenciação de Linfócitos B/química , Antígenos de Diferenciação de Linfócitos B/genética , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Epitopos/imunologia , Meia-Vida , Antígenos de Histocompatibilidade Classe II/química , Antígenos de Histocompatibilidade Classe II/genética , Camundongos , Modelos Moleculares , Mutação/genética , Ligação Proteica , Estrutura Terciária de Proteína
15.
Proc Natl Acad Sci U S A ; 104(32): 13080-5, 2007 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-17670943

RESUMO

T cell receptor (TCR) recognition of peptide-MHC (pMHC) is central to the cellular immune response. A large database of TCR-pMHC structures is needed to reveal general structural principles, such as whether the repertoire of TCR/MHC docking modes is dictated by a "recognition code" between conserved elements of the TCR and MHC genes. Although approximately 17 cocrystal structures of unique TCR-pMHC complexes have been determined, cocrystallization of soluble TCR and pMHC remains a major technical obstacle in the field. Here we demonstrate a strategy, based on NMR chemical shift mapping, that permits rapid and reliable analysis of the solution footprint made by a TCR when binding onto the pMHC surface. We mapped the 2C TCR binding interaction with its allogeneic ligand H-2Ld-QL9 and identified a group of NMR-shifted residues that delineated a clear surface of the MHC that we defined as the TCR footprint. We subsequently found that the docking footprint described by NMR shifts was highly accurate compared with a recently determined high-resolution crystal structure of the same complex. The same NMR footprint analysis was done on a high-affinity mutant of the TCR. The current work serves as a foundation to explore the molecular dynamics of pMHC complexes and to rapidly determine the footprints of many Ld-specific TCRs.


Assuntos
Antígenos H-2/química , Receptores de Antígenos de Linfócitos T/química , Sequência de Aminoácidos , Animais , Regiões Determinantes de Complementaridade , Antígeno de Histocompatibilidade H-2D , Espectroscopia de Ressonância Magnética , Camundongos , Dados de Sequência Molecular , Dobramento de Proteína
16.
J Biol Chem ; 281(35): 25734-44, 2006 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-16815841

RESUMO

The major histocompatibility complex (MHC) is the most polymorphic locus known, with thousands of allelic variants. There is considerable interest in understanding the diversity of structures and peptide-binding features represented by this class of proteins. Although many MHC proteins have been crystallized, others have not been amenable to structural or biochemical studies due to problems with expression or stability. In the present study, yeast display was used to engineer stabilizing mutations into the class I MHC molecule, Ld. The approach was based on previous studies that showed surface levels of yeast-displayed fusion proteins are directly correlated with protein stability. To engineer a more stable Ld, we selected Ld mutants with increased surface expression from randomly mutated yeast display libraries using anti-Ld antibodies or high affinity, soluble T-cell receptors (TCRs). The most stable Ld mutant, Ld-m31, consisted of a single-chain MHC module containing only the alpha1 and alpha2 domains. The enhanced stability was in part due to a single mutation (Trp-97 --> Arg), shown previously to be present in the allele Lq. Mutant Ld-m31 could bind to Ld peptides, and the specific peptide.Ld-m31 complex (QL9.Ld-m31) was recognized by alloreactive TCR 2C. A soluble form of the Ld-m31 protein was expressed in Escherichia coli and refolded from inclusion bodies at high yields. Surface plasmon resonance showed that TCRs bound to peptide.Ld-m31 complexes with affinities similar to those of native full-length Ld. The TCR and QL9.Ld-m31 formed complexes that could be resolved by native gel electrophoresis, suggesting that stabilized alpha1/alpha2 class I platforms may enable various structural studies.


Assuntos
Antígenos H-2/química , Complexo Principal de Histocompatibilidade , Alelos , Animais , Membrana Celular/metabolismo , Antígeno de Histocompatibilidade H-2D , Humanos , Camundongos , Mutação , Oligopeptídeos/química , Peptídeos/química , Ligação Proteica , Desnaturação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Ressonância de Plasmônio de Superfície
17.
Immunity ; 18(1): 7-11, 2003 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-12530971

RESUMO

Although our structural understanding of T cell recognition has rapidly evolved due to recent crystallographic results, the reality is that detailed answers to many of the most fundamental questions still remain elusive. In this issue, high-resolution insight into the phenomenon of TCR chain bias takes down another brick from the wall.


Assuntos
Receptores de Antígenos de Linfócitos T/química , Sítios de Ligação , Regiões Determinantes de Complementaridade , Antígenos de Histocompatibilidade Classe I/química , Humanos , Substâncias Macromoleculares , Modelos Moleculares , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T/imunologia
18.
Clin Immunol ; 105(1): 36-47, 2002 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-12483992

RESUMO

Immune complexes (IC) bound to human erythrocytes (E) via complement receptor 1 (CR1) are transferred to phagocytes in the liver and spleen. In an in vitro model system using bispecific mAb reagents (antigen-based heteropolymers) to link IC to E, we have made time-lapse movies in which fluorescently labeled IC cross the E-human macrophage interface and remain associated with the macrophage. Both these movies and fixed-time experiments reveal transfer intermediates in which IC hinge E to macrophages. Examination of model macrophages after transfer indicates that the majority of IC are on the surface at short times (2 min) but are internalized at long times (1-4 h). More than half of the surface IC colocalize with CR1 at 2 min. This evidence supports a model in which CR1-bound IC provide a secure linkage between E and macrophages, allowing rearrangements of the macrophage surface necessary for release of CR1, and IC, from the E.


Assuntos
Complexo Antígeno-Anticorpo/imunologia , Membrana Eritrocítica/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Macrófagos/imunologia , Receptores de Complemento 3b/imunologia , Receptores de Complemento/imunologia , Anticorpos Biespecíficos/imunologia , Complexo Antígeno-Anticorpo/metabolismo , DNA/imunologia , Membrana Eritrocítica/metabolismo , Citometria de Fluxo , Humanos , Reação de Imunoaderência , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Macrófagos/metabolismo , Microscopia de Fluorescência , Receptores de Complemento/metabolismo , Receptores Fc/imunologia , Células U937
19.
Mol Cell ; 12(3): 577-89, 2003 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-14527405

RESUMO

Gp130 is a shared cell-surface signaling receptor for at least ten different hematopoietic cytokines, but the basis of its degenerate recognition properties is unknown. We have determined the crystal structure of human leukemia inhibitory factor (LIF) bound to the cytokine binding region (CHR) of gp130 at 2.5 A resolution. Strikingly, we find that the shared binding site on gp130 has an entirely rigid core, while the LIF binding interface diverges sharply in structure and chemistry from that of other gp130 ligands. Dissection of the LIF-gp130 interface, along with comparative studies of other gp130 cytokines, reveal that gp130 has evolved a "thermodynamic plasticity" that is relatively insensitive to ligand structure, to enable crossreactivity. These observations reveal a novel and alternative mechanism for degenerate recognition from that of structural plasticity.


Assuntos
Antígenos CD/química , Citocinas/imunologia , Inibidores do Crescimento/química , Interleucina-6 , Linfocinas/química , Glicoproteínas de Membrana/química , Receptores de Superfície Celular/química , Animais , Antígenos CD/imunologia , Sítios de Ligação/imunologia , Células Cultivadas , Reações Cruzadas/imunologia , Cristalografia por Raios X , Receptor gp130 de Citocina , Células Eucarióticas/imunologia , Células Eucarióticas/metabolismo , Inibidores do Crescimento/imunologia , Imunidade Celular/imunologia , Insetos , Fator Inibidor de Leucemia , Linfocinas/imunologia , Substâncias Macromoleculares , Glicoproteínas de Membrana/imunologia , Modelos Moleculares , Estrutura Molecular , Ligação Proteica/imunologia , Estrutura Terciária de Proteína/fisiologia , Receptores de Superfície Celular/imunologia
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