RESUMEN
T cells in jawed vertebrates comprise two lineages, αß T-cells and γδ T-cells, defined by the antigen receptors they express, i.e., αß and γδ T-cell receptors (TCRs), respectively. The two lineages have different immunological roles, requiring γδ TCRs to recognize more structurally-diverse ligands1. Nevertheless, the receptors use shared CD3 subunits to initiate signaling. Whereas the structural organization of αß TCRs is understood2,3, the architecture of γδ TCRs is unknown. Here, we used cryogenic electron microscopy to determine the structure of a fully-assembled, MR1-reactive human Vδ3Vγ8 TCR/CD3δγε2ζ2 complex bound by anti-CD3ε antibody Fab fragments4,5. The arrangement of CD3 subunits in γδ and αß TCRs is conserved and, although the transmembrane α-helices of the TCR-γδ and -αß subunits differ markedly in sequence, the packing of the eight transmembrane-helix bundles is similar6. However, in contrast to the apparently rigid αß TCR2,3,6, the γδ TCR exhibits considerable conformational heterogeneity, owing to the ligand-binding TCR-γδ subunits being tethered to the CD3 subunits by their transmembrane regions only. Reducing this conformational heterogeneity by transferring the Vδ3Vγ8 TCR variable domains to an αß TCR enhanced receptor signaling, suggesting that γδ TCR organization reflects a compromise between efficient signaling and the ability to engage structurally-diverse ligands. Our findings reveal the remarkable structural plasticity of the TCR on evolutionary timescales, and recast it as a highly versatile receptor capable of initiating signaling as either a rigid or flexible structure.
RESUMEN
Antibodies can block immune receptor engagement or trigger the receptor machinery to initiate signaling. We hypothesized that antibody agonists trigger signaling by sterically excluding large receptor-type protein tyrosine phosphatases (RPTPs) such as CD45 from sites of receptor engagement. An agonist targeting the costimulatory receptor CD28 produced signals that depended on antibody immobilization and were sensitive to the sizes of the receptor, the RPTPs, and the antibody itself. Although both the agonist and a non-agonistic anti-CD28 antibody locally excluded CD45, the agonistic antibody was more effective. An anti-PD-1 antibody that bound membrane proximally excluded CD45, triggered Src homology 2 domain-containing phosphatase 2 recruitment, and suppressed systemic lupus erythematosus and delayed-type hypersensitivity in experimental models. Paradoxically, nivolumab and pembrolizumab, anti-PD-1-blocking antibodies used clinically, also excluded CD45 and were agonistic in certain settings. Reducing these agonistic effects using antibody engineering improved PD-1 blockade. These findings establish a framework for developing new and improved therapies for autoimmunity and cancer.
Asunto(s)
Proteínas Tirosina Fosfatasas , Transducción de Señal , Proteínas Tirosina Fosfatasas/metabolismo , Antígenos CD28 , Receptores InmunológicosRESUMEN
African swine fever virus is a complex DNA virus that causes high fatality in pigs and wild boar and has a great socio-economic impact. An attenuated genotype II strain was constructed by replacing the gene for wildtype CD2v protein with versions in which single or double amino acid substitutions were introduced to reduce or abrogate the binding to red blood cells and reduce virus persistence in blood. The mutant CD2v proteins were expressed at similar levels to the wildtype protein on the surface of infected cells. Three recombinant viruses also had K145R, EP153R, and in one virus DP148R genes deleted. Following immunization of pigs, the virus with a single amino acid substitution in CD2v, Q96R, induced moderate levels of replication, and 100% protection against virulent ASFV. Two additional recombinant viruses had two amino acid substitutions in CD2v, Q96R, and K108D, and induced no binding to red blood cells in vitro. In immunized pigs, reduced levels of virus in blood and strong early ASFV-specific antibody and cellular responses were detected. After challenge low to moderate replication of challenge virus was observed. Reduced clinical signs post-challenge were observed in pigs immunized with the virus from which DP148R gene was deleted. Protection levels of 83-100% were maintained across a range of doses. Further experiments with virus GeorgiaΔDP148RΔK145RΔEP153R-CD2v_mutantQ96R/K108D showed low levels of virus dissemination in tissue and transient clinical signs at high doses. The results support further evaluation of GeorgiaΔDP148RΔK145RΔEP153R-CD2v_mutantQ96R/K108D as a vaccine candidate.
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Virus de la Fiebre Porcina Africana , Fiebre Porcina Africana , Vacunas Virales , Porcinos , Animales , Virus de la Fiebre Porcina Africana/fisiología , Fiebre Porcina Africana/prevención & control , Proteínas Virales/genética , Genotipo , Anticuerpos AntiviralesRESUMEN
The T cell receptor (TCR) expressed by T lymphocytes initiates protective immune responses to pathogens and tumors. To explore the structural basis of how TCR signaling is initiated when the receptor binds to peptide-loaded major histocompatibility complex (pMHC) molecules, we used cryogenic electron microscopy to determine the structure of a tumor-reactive TCRαß/CD3δγε2ζ2 complex bound to a melanoma-specific human class I pMHC at 3.08 Å resolution. The antigen-bound complex comprises 11 subunits stabilized by multivalent interactions across three structural layers, with clustered membrane-proximal cystines stabilizing the CD3-εδ and CD3-εγ heterodimers. Extra density sandwiched between transmembrane helices reveals the involvement of sterol lipids in TCR assembly. The geometry of the pMHC/TCR complex suggests that efficient TCR scanning of pMHC requires accurate pre-positioning of T cell and antigen-presenting cell membranes. Comparisons of the ligand-bound and unliganded receptors, along with molecular dynamics simulations, indicate that TCRs can be triggered in the absence of spontaneous structural rearrangements.
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Neoplasias , Receptores de Antígenos de Linfocitos T , Humanos , Complejo Mayor de Histocompatibilidad , Péptidos/química , Unión Proteica , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores de Antígenos de Linfocitos T alfa-beta/química , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismoRESUMEN
Native and non-native ligands of the T cell receptor (TCR), including antibodies, have been proposed to induce signaling in T cells via intra- or intersubunit conformational rearrangements within the extracellular regions of TCR complexes. We have investigated whether any signatures can be found for such postulated structural changes during TCR triggering induced by antibodies, using crystallographic and mutagenesis-based approaches. The crystal structure of murine CD3ε complexed with the mitogenic anti-CD3ε antibody 2C11 enabled the first direct structural comparisons of antibody-liganded and unliganded forms of CD3ε from a single species, which revealed that antibody binding does not induce any substantial rearrangements within CD3ε. Saturation mutagenesis of surface-exposed CD3ε residues, coupled with assays of antibody-induced signaling by the mutated complexes, suggests a new configuration for the complex within which CD3ε is highly exposed and reveals that no large new CD3ε interfaces are required to form during antibody-induced signaling. The TCR complex therefore appears to be a structure that is capable of initiating intracellular signaling in T cells without substantial structural rearrangements within or between the component subunits. Our findings raise the possibility that signaling by native ligands might also be initiated in the absence of large structural rearrangements in the receptor.
Asunto(s)
Complejo CD3 , Receptores de Antígenos de Linfocitos T , Transducción de Señal/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Complejo CD3/química , Complejo CD3/genética , Complejo CD3/inmunología , Cristalografía por Rayos X , Dimerización , Epítopos de Linfocito T/inmunología , Humanos , Fragmentos Fab de Inmunoglobulinas/inmunología , Células Jurkat , Ratones , Mutagénesis Sitio-Dirigida , Conformación Proteica , Estructura Terciaria de Proteína , Receptores de Antígenos de Linfocitos T/química , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Relación Estructura-ActividadRESUMEN
The Fc-α receptor (FcαR/CD89) is involved in IgA complex formation and may affect the development of IgA nephropathy (IgAN). In this study, we tested the genetic variations of the CD89 gene in relation to disease susceptibility in IgAN and the expression of soluble CD89 (sCD89) in sera of patients with IgAN and in controls. There was a significant difference between the levels of sCD89-IgA complexes, measured by sandwich enzyme-linked immunosorbent assay (ELISA), in 177 patients with IgAN with and without disease progression at the time of first diagnosis. No such difference was found in 42 patients with other renal diseases. The patients with IgAN without disease progression had stable but high levels of sCD89 over 5-15 years of follow-up in contrast to stable but low levels of sCD89 in the disease progression group. Moreover, levels of sCD89 complexes were correlated with one of the five CD89 genetic variants in 212 patients with IgAN and 477 healthy Caucasians; the single-nucleotide polymorphism (SNP) rs11084377 was significantly associated with a lower expression of sCD89. However, no association between CD89 gene polymorphisms and susceptibility to IgAN was detected. Thus, we found an association between the levels of sCD89-IgA complexes in serum and the severity of IgAN, and a possible genetic component in regulating the production or expression of sCD89.
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Antígenos CD/sangre , Antígenos CD/genética , Progresión de la Enfermedad , Predisposición Genética a la Enfermedad/genética , Glomerulonefritis por IGA/sangre , Glomerulonefritis por IGA/genética , Receptores Fc/sangre , Receptores Fc/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Complejo Antígeno-Anticuerpo/metabolismo , Biomarcadores/sangre , Estudios de Casos y Controles , Femenino , Genotipo , Humanos , Inmunoglobulina A/metabolismo , Fallo Renal Crónico/sangre , Fallo Renal Crónico/diagnóstico , Fallo Renal Crónico/epidemiología , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple/genética , Pronóstico , Factores de Riesgo , Índice de Severidad de la Enfermedad , Adulto JovenRESUMEN
CD8 engagement with class I major histocompatibility antigens greatly enhances T-cell activation, but it is not clear how this is achieved. We address the question of whether or not the antibody-mediated ligation of CD8 alone induces transcriptional remodeling in a T-cell clone, using serial analysis of gene expression. Even though it fails to induce overt phenotypic changes, we find that CD8 ligation profoundly alters transcription in the T-cell clone, at a scale comparable to that induced by antibody-mediated ligation of CD3. The character of the resulting changes is distinct, however, with the net effect of CD8 ligation being substantially inhibitory. We speculate that ligating CD8 induces weak, T-cell receptor (TCR)-mediated inhibitory signals reminiscent of the effects of TCR antagonists. Our results imply that CD8 ligation alone is incapable of activating the T-cell clone because it fails to fully induce NFAT-dependent transcription.
Asunto(s)
Complejo CD3/metabolismo , Antígenos CD8/metabolismo , Perfilación de la Expresión Génica , Transducción de Señal , Linfocitos T/metabolismo , Anticuerpos/farmacología , Células Clonales , Regulación de la Expresión Génica/efectos de los fármacos , Biblioteca de Genes , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal/efectos de los fármacos , Linfocitos T/efectos de los fármacosRESUMEN
BACKGROUND: Deep transcriptome analysis will underpin a large fraction of post-genomic biology. 'Closed' technologies, such as microarray analysis, only detect the set of transcripts chosen for analysis, whereas 'open' e.g. tag-based technologies are capable of identifying all possible transcripts, including those that were previously uncharacterized. Although new technologies are now emerging, at present the major resources for open-type analysis are the many publicly available SAGE (serial analysis of gene expression) and MPSS (massively parallel signature sequencing) libraries. These technologies have never been compared for their utility in the context of deep transcriptome mining. RESULTS: We used a single LongSAGE library of 503,431 tags and a "classic" MPSS library of 1,744,173 tags, both prepared from the same T cell-derived RNA sample, to compare the ability of each method to probe, at considerable depth, a human cellular transcriptome. We show that even though LongSAGE is more error-prone than MPSS, our LongSAGE library nevertheless generated 6.3-fold more genome-matching (and therefore likely error-free) tags than the MPSS library. An analysis of a set of 8,132 known genes detectable by both methods, and for which there is no ambiguity about tag matching, shows that MPSS detects only half (54%) the number of transcripts identified by SAGE (3,617 versus 1,955). Analysis of two additional MPSS libraries shows that each library samples a different subset of transcripts, and that in combination the three MPSS libraries (4,274,992 tags in total) still only detect 73% of the genes identified in our test set using SAGE. The fraction of transcripts detected by MPSS is likely to be even lower for uncharacterized transcripts, which tend to be more weakly expressed. The source of the loss of complexity in MPSS libraries compared to SAGE is unclear, but its effects become more severe with each sequencing cycle (i.e. as MPSS tag length increases). CONCLUSION: We show that MPSS libraries are significantly less complex than much smaller SAGE libraries, revealing a serious bias in the generation of MPSS data unlikely to have been circumvented by later technological improvements. Our results emphasize the need for the rigorous testing of new expression profiling technologies.