RESUMEN
The signal peptide peptidase (SPP) is an intramembrane cleaving aspartyl protease involved in release of leader peptide remnants from the endoplasmic reticulum membrane, hence its name. We now found a new activity of SPP that mediates liberation of C-terminal peptides. In our search for novel proteolytic enzymes involved in MHC class I (MHC-I) presentation, we found that SPP generates the C-terminal peptide-epitope of a ceramide synthase. The display of this immunogenic peptide-MHC-I complex at the cell surface was independent of conventional processing components like proteasome and peptide transporter TAP. Absence of TAP activity even increased the MHC-I presentation of this Ag. Mutagenesis studies revealed the crucial role of the C-terminal location of the epitope and "helix-breaking" residues in the transmembrane region just upstream of the peptide, indicating that SPP directly liberated the minimal 9-mer peptide. Moreover, silencing of SPP and its family member SPPL2a led to a general reduction of surface peptide-MHC-I complexes, underlining the involvement of these enzymes in Ag processing and presentation.
Asunto(s)
Presentación de Antígeno , Ácido Aspártico Endopeptidasas/metabolismo , Antígenos de Histocompatibilidad Clase I/inmunología , Proteínas de la Membrana/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Ácido Aspártico Endopeptidasas/genética , Ácido Aspártico Endopeptidasas/inmunología , Línea Celular Tumoral , Retículo Endoplásmico/metabolismo , Células HEK293 , Células HeLa , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Ratones , Mutación , Oxidorreductasas/metabolismo , Péptidos/inmunología , Interferencia de ARN , ARN Interferente PequeñoRESUMEN
In the last two decades, antisense oligonucleotides (AONs) that induce corrective exon skipping have matured as promising therapies aimed at tackling the dystrophin deficiency that underlies the severe and progressive muscle fiber degeneration in Duchenne muscular dystrophy (DMD) patients. Pioneering first generation exon 51 skipping AONs like drisapersen and eteplirsen have more recently been followed up by AONs for exons 53 and 45, with, to date, a total of four exon skipping AON drugs having reached (conditional) regulatory US Food and Drug Administration (FDA) approval for DMD. Nonetheless, considering the limited efficacy of these drugs, there is room for improvement. The aim of this study was to develop more efficient [2'-O-methyl-modified phosphorothioate (2'OMePS) RNA] AONs for DMD exon 51 skipping by implementing precision chemistry as well as identifying a more potent target binding site. More than a hundred AONs were screened in muscle cell cultures, followed by a selective comparison in the hDMD and hDMDdel52/mdx mouse models. Incorporation of 5-methylcytosine and position-specific locked nucleic acids in AONs targeting the drisapersen/eteplirsen binding site resulted in 15-fold higher exon 51 skipping levels compared to drisapersen in hDMDdel52/mdx mice. However, with similarly modified AONs targeting an alternative site in exon 51, 65-fold higher skipping levels were obtained, restoring dystrophin up to 30% of healthy control. Targeting both sites in exon 51 with a single AON further increased exon skipping (100-fold over drisapersen) and dystrophin (up to 40%) levels. These dystrophin levels allowed for normalization of creatine kinase (CK) and lactate dehydrogenase (LDH) levels, and improved motor function in hDMDdel52/mdx mice. As no major safety observation was obtained, the improved therapeutic index of these next generation AONs is encouraging for further (pre)clinical development.
Asunto(s)
Distrofia Muscular de Duchenne , Ratones , Animales , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Distrofina/genética , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/farmacología , Oligonucleótidos Antisentido/uso terapéutico , Ratones Endogámicos mdx , Terapia Genética/métodos , Exones/genéticaRESUMEN
We recently described a category of TAP-independent peptide-epitopes that are selectively presented by cells with processing defects in the classical MHC class I (MHC-I) pathway. Here, we studied the ER-resident ceramide synthase Trh4 as a prototypic example of these neo-antigens and found that moderate inhibition of TAP permits cell surface presentation of the Trh4 peptide. The absence of this peptide from WT cells was not related to the binding or stability of the Trh4/D(b) complexes, or to the availability of MHC-I heavy chains, but rather to the limited expression of the antigen. Strongly elevated antigen levels were needed to reach comparable peptide display on WT as on TAP-deficient cells. Our data suggest that the normal influx of TAP-transported peptides in the ER during routine processing creates an efficient barrier for peptides from alternative processing routes. Impairment of TAP function, as commonly found in cancers and virus-infected cells, lowers this resistance allowing for MHC-I presentation of other peptide sources.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/inmunología , Presentación de Antígeno/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Activación de Linfocitos/inmunología , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Separación Celular , Citometría de Flujo , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Reacción en Cadena de la Polimerasa , Linfocitos T Citotóxicos/inmunologíaRESUMEN
Target cell recognition by CTLs depends on the presentation of peptides by HLA class I molecules. Tumors and herpes viruses have adopted strategies to greatly hamper this peptide presentation at the important bottleneck, the peptide transporter TAP. Previously, we described the existence of a CD8(+) CTL subpopulation that selectively recognizes such TAP-deficient cells in mouse models. In this study, we show that the human counterpart of this CTL subset is readily detectable in healthy subjects. Autologous PBMC cultures were initiated with dendritic cells rendered TAP-impaired by gene transfer of the viral evasion molecule UL49.5. Strikingly, specific reactivity to B-LCLs expressing one of the other viral TAP-inhibitors (US6, ICP47, or BNLF2a) was already observed after three rounds of stimulation. These short-term T cell cultures and isolated CD8(+) CTL clones derived thereof did not recognize the normal B-LCL, indicating that the cognate peptide-epitopes emerge at the cell surface upon an inhibition in the MHC class I processing pathway. A diverse set of TCRs was used by the clones, and the cellular reactivity was TCR-dependent and HLA class I-restricted, implying the involvement of a broad antigenic peptide repertoire. Our data indicate that the human CD8(+) T cell pool comprises a diverse reactivity to target cells with impairments in the intracellular processing pathway, and these might be exploited for cancers that are associated with such defects and for infections with immune-evading herpes viruses.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/fisiología , Presentación de Antígeno/inmunología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Transportadoras de Casetes de Unión a ATP/genética , Presentación de Antígeno/genética , Linfocitos T CD8-positivos/virología , Línea Celular , Células Cultivadas , Células Clonales , Técnicas de Cocultivo , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Células Dendríticas/virología , Epítopos de Linfocito T/inmunología , Epítopos de Linfocito T/aislamiento & purificación , Técnicas de Transferencia de Gen , Infecciones por Herpesviridae/inmunología , Infecciones por Herpesviridae/patología , Herpesvirus Bovino 1/genética , Herpesvirus Bovino 1/inmunología , Humanos , Evasión Inmune/genética , Infecciones Tumorales por Virus/inmunología , Infecciones Tumorales por Virus/patología , Proteínas Estructurales Virales/genéticaRESUMEN
BACKGROUND: HIV-1 infects macrophages and microglia in the brain and can cause neurological disorders in infected patients. We and others have shown that brain-derived envelope glycoproteins (Env) have lower CD4 dependence and higher avidity for CD4 than those from peripheral isolates, and we have also observed increased fusogenicity and reduced sensitivity to the fusion inhibitor T-1249. Due to the genetic differences between brain and spleen env from one individual throughout gp120 and in gp41's heptad repeat 2 (HR2), we investigated the viral determinants for the phenotypic differences by performing functional studies with chimeric and mutant Env. RESULTS: Chimeric Env showed that the V1/V2-C2-V3 region in brain's gp120 determines the low CD4 dependence and high avidity for CD4, as well as macrophage tropism and reduced sensitivity to the small molecule BMS-378806. Changes in brain gp41's HR2 region did not contribute to the increased fusogenicity or to the reduced sensitivity to T-1249, since a T-1249-based peptide containing residues found in brain's but not in spleen's HR2 had similar potency than T-1249 and interacted similarly with an immobilized heptad repeat 1-derived peptide in surface plasmon resonance analysis. However, the increased fusogenicity and reduced T-1249 sensitivity of brain and certain chimeric Env mostly correlated with the low CD4 dependence and high avidity for CD4 determined by brain's V1-V3 region. Remarkably, most but not all of these low CD4-dependent, macrophage tropic envelopes glycoproteins also had increased sensitivity to the novel allosteric entry inhibitor HNG-105. The gp120's C2 region asparagine 283 (N283) has been previously associated with macrophage tropism, brain infection, lower CD4 dependence and higher CD4 affinity. Therefore, we introduced the N283T mutation into an env clone from a brain-derived isolate and into a brain tissue-derived env clone, and the T283N change into a spleen-derived env from the same individual; however, we found that their phenotypes were not affected. CONCLUSION: We have identified that the V1-V3 region of a brain-derived envelope glycoprotein seems to play a crucial role in determining not only the low CD4 dependence and increased macrophage tropism, but also the augmented fusogenicity and reduced sensitivity to T-1249 and BMS-378806. By contrast, increased sensitivity to HNG-105 mostly correlated with low CD4 dependence and macrophage tropism but was not determined by the presence of the brain's V1-V3 region, confirming that viral determinants of phenotypic changes in brain-derived envelope glycoproteins are likely complex and context-dependent.
Asunto(s)
Encéfalo/virología , Antígenos CD4/inmunología , Proteína gp120 de Envoltorio del VIH/inmunología , Inhibidores de Fusión de VIH/farmacología , Infecciones por VIH/inmunología , VIH-1/fisiología , Macrófagos/virología , Internalización del Virus/efectos de los fármacos , Secuencias de Aminoácidos , Animales , Encéfalo/efectos de los fármacos , Encéfalo/inmunología , Fusión Celular , Línea Celular , Células Cultivadas , Proteína gp120 de Envoltorio del VIH/química , Proteína gp120 de Envoltorio del VIH/genética , Infecciones por VIH/metabolismo , Infecciones por VIH/virología , VIH-1/efectos de los fármacos , VIH-1/genética , Humanos , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Especificidad de Órganos , Codorniz , Receptores CCR5/inmunologíaRESUMEN
Cancers frequently evade immune-recognition by lowering peptide:MHC-I complexes on their cell surface. Limited peptide supply due to TAP-deficiency results in such MHC-Ilow immune-escape variants. Previously, we reported on a category of TAP-independent self-peptides, called TEIPP, with selective presentation by these tumors. Here we demonstrate that in contrast to T cells specific for conventional tumor antigens, TEIPP-directed T cells remain naïve in mice bearing immune-escaped tumors. This unaffected state was caused by low levels of MHC-I on the tumors and the failure to cross-present low levels of antigenic protein by host APCs. Importantly, increased levels of MHC-I, antigen or co-stimulation resulted in potent activation of TEIPP-specific T cells via direct presentation. Genetic knockdown by CRISPR/Cas9 technology of the relevant MHC-I allele in tumor cells indeed abrogated T cell activation. Vaccine-mediated priming of TEIPP-specific T cells induced efficient homing to MHC-Ilow tumors and subsequently protected mice against outgrowth of their MHC-Ilow tumor. Thus, our data open up the search of TEIPP-specific T cells in cancer patients to explore their application against MHC-Ilow tumor cells.
RESUMEN
The HLA-E homolog in the mouse (Qa-1b) is a conserved MHC class Ib molecule presenting monomorphic peptides to germline-encoded natural killer receptor CD94/NKG2A. Previously, we demonstrated the replacement of this canonical peptide by a diverse peptidome upon deficiency of the TAP peptide transporter. Analysis of this Qa-1b-restricted T cell repertoire against these non-mutated neoantigens revealed characteristics of conventional hypervariable CD8+ T cells, but also of invariant T cell receptor (TCR)αß T cells. A shared TCR Vα chain was used by this subset in combination with a variety of Vß chains. The TCRs target peptide ligands that are conserved between mouse and man, like the identified peptide derived from the transcriptional cofactor Med15. The thymus selection was studied in a TCR-transgenic mouse and emerging naïve CD8+ T cells displayed a slightly activated phenotype, as witnessed by higher CD122 and Ly6C expression. Moreover, the Qa-1b protein was dispensable for thymus selection. Importantly, no self-reactivity was observed as reported for other MHC class Ib-restricted subsets. Naïve Qa-1b restricted T cells expanded, contracted, and formed memory cells in vivo upon peptide vaccination in a similar manner as conventional CD8+ T cells. Based on these data, the Qa-1b restricted T cell subset might be positioned closest to conventional CD8+ T cells of all MHC class Ib populations.
Asunto(s)
Proteínas Portadoras/metabolismo , Antígenos de Histocompatibilidad Clase I/inmunología , Lipoproteínas/metabolismo , Péptidos/inmunología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Transactivadores/metabolismo , Animales , Transporte Biológico , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Proteínas Portadoras/genética , Línea Celular , Secuencia Conservada , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Ligandos , Lipoproteínas/genética , Activación de Linfocitos , Ratones , Ratones Noqueados , Ratones Transgénicos , Péptidos/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Timo/inmunología , Timo/metabolismo , Transactivadores/genéticaRESUMEN
Tumor cells frequently escape from CD8+ T cell recognition by abrogating MHC-I antigen presentation. Deficiency in processing components, like the transporter associated with antigen processing (TAP), results in strongly decreased surface display of peptide/MHC-I complexes. We previously identified a class of hidden self-antigens known as T cell epitopes associated with impaired peptide processing (TEIPP), which emerge on tumor cells with such processing defects. In the present study, we analyzed thymus selection and peripheral behavior of T cells with specificity for the prototypic TEIPP antigen, the "self" TRH4 peptide/Db complex. TEIPP T cells were efficiently selected in the thymus, egressed with a naive phenotype, and could be exploited for immunotherapy against immune-escaped, TAP-deficient tumor cells expressing low levels of MHC-I (MHC-Ilo). In contrast, overt thymus deletion and functionally impaired TEIPP T cells were observed in mice deficient for TAP1 due to TEIPP antigen presentation on all body cells in these mice. Our results strongly support the concept that TEIPPs derive from ubiquitous, nonmutated self-antigens and constitute a class of immunogenic neoantigens that are unmasked during tumor immune evasion. These data suggest that TEIPP-specific CD8+ T cells are promising candidates in the treatment of tumors that have escaped from conventional immunotherapies.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/inmunología , Presentación de Antígeno , Antígenos de Neoplasias/inmunología , Autoantígenos/inmunología , Epítopos de Linfocito T/inmunología , Neoplasias Experimentales/inmunología , Péptidos/inmunología , Escape del Tumor , Transportador de Casetes de Unión a ATP, Subfamilia B, Miembro 2 , Transportadoras de Casetes de Unión a ATP/genética , Animales , Antígenos de Neoplasias/genética , Autoantígenos/genética , Línea Celular Tumoral , Epítopos de Linfocito T/genética , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/inmunología , Ratones , Ratones Noqueados , Neoplasias Experimentales/genética , Neoplasias Experimentales/patología , Péptidos/genéticaRESUMEN
Tumors frequently display defects in the MHC-I antigen processing machinery, such as deficiency of the peptide transporter TAP. Interestingly, the residual peptide repertoire contains neo-antigens which are not presented by processing-proficient cells. We termed these immunogenic peptides TEIPP ('T-cell epitopes associated with impaired peptide processing') and were interested to unravel their TAP-independent processing pathways. With an array of chemical inhibitors we assessed the participation of numerous proteases to TAP-independent peptides and found that the previously described catalytic enzymes signal peptidase and furin contributed in a cell-type and MHC-I allele-specific way. In addition, a dominant role for the proteasome and metallopeptidases was observed. These findings raised the question how these proteasome products get access to MHC-I molecules. A novel TEIPP peptide-epitope that represented this intracellular route revealed that the lysosomal peptide transporter ABCB9 ('TAP-like') was dispensable for its presentation. Interestingly, prevention of endolysosomal vesicle acidification by bafilomycin enhanced the surface display of this TEIPP peptide, suggesting that this proteasome-dependent pathway intersects endolysosomes and that these antigens are merely destroyed there. In conclusion, the proteasome has a surprisingly dominant role in shaping the TAP-independent MHC-I peptide repertoire and some of these antigens might be targeted to the endocytic vesicular pathway.
Asunto(s)
Presentación de Antígeno , Antígenos de Histocompatibilidad Clase I/inmunología , Metaloproteasas/fisiología , Complejo de la Endopetidasa Proteasomal/fisiología , Transportadoras de Casetes de Unión a ATP/fisiología , Animales , Línea Celular Tumoral , Epítopos/química , Epítopos/inmunología , Células HEK293 , Antígenos de Histocompatibilidad Clase I/química , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
The nonclassical major histocompatibility complex (MHC) Qa-1b accommodates monomorphic leader peptides and functions as a ligand for germ line receptors CD94/NKG2, which are expressed by natural killer cells and CD8+ T cells. We here describe that the conserved peptides are replaced by a novel peptide repertoire of surprising diversity as a result of impairments in the antigen-processing pathway. This novel peptide repertoire represents immunogenic neoantigens for CD8+ T cells, as we found that these Qa-1b-restricted T cells dominantly participated in the response to tumors with processing deficiencies. A surprisingly wide spectrum of target cells, irrespective of transformation status, MHC background, or type of processing deficiency, was recognized by this T cell subset, complying with the conserved nature of Qa-1b. Target cell recognition depended on T cell receptor and Qa-1b interaction, and immunization with identified peptide epitopes demonstrated in vivo priming of CD8+ T cells. Our data reveal that Qa-1b, and most likely its human homologue human leukocyte antigen-E, is important for the defense against processing-deficient cells by displacing the monomorphic leader peptides, which relieves the inhibition through CD94/NKG2A on lymphocytes, and by presenting a novel repertoire of immunogenic peptides, which recruits a subset of cytotoxic CD8+ T cells.
Asunto(s)
Presentación de Antígeno/inmunología , Linfocitos T CD8-positivos/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Vigilancia Inmunológica/inmunología , Subgrupos Linfocitarios/inmunología , Péptidos/inmunología , Animales , Presentación de Antígeno/genética , Linfocitos T CD8-positivos/citología , Línea Celular Tumoral , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Vigilancia Inmunológica/genética , Células Asesinas Naturales/citología , Células Asesinas Naturales/inmunología , Subgrupos Linfocitarios/citología , Ratones , Ratones Noqueados , Subfamília D de Receptores Similares a Lectina de las Células NK/genética , Subfamília D de Receptores Similares a Lectina de las Células NK/inmunología , Neoplasias/genética , Neoplasias/inmunología , Péptidos/genéticaRESUMEN
The AIDS epidemic continues to spread at an alarming rate worldwide, especially in developing countries. One approach to solving this problem is the generation of anti-human immunodeficiency virus (HIV) compounds with inhibition spectra broad enough to include globally prevailing forms of the virus. We have examined the HIV type 1 (HIV-1) envelope specificity of a recently identified entry inhibitor candidate, HNG-105, using surface plasmon resonance spectroscopy and pseudovirus inhibition assays. The combined results suggest that the HNG-105 molecule may be effective across the HIV-1 subtypes, and they highlight its potential as a lead for developing therapeutic and microbicidal agents to help combat the spread of AIDS.