RESUMEN
The fate of developing T cells is determined by the strength of T cell receptor (TCR) signal they receive in the thymus. This process is finely regulated through the tuning of positive and negative regulators in thymocytes. The Family with sequence similarity 49 member B (Fam49b) protein is a newly discovered negative regulator of TCR signaling that has been shown to suppress Rac-1 activity in vitro in cultured T cell lines. However, the contribution of Fam49b to the thymic development of T cells is unknown. To investigate this important issue, we generated a novel mouse line deficient in Fam49b (Fam49b-KO). We observed that Fam49b-KO double positive (DP) thymocytes underwent excessive negative selection, whereas the positive selection stage was unaffected. Fam49b deficiency impaired the survival of single positive thymocytes and peripheral T cells. This altered development process resulted in significant reductions in CD4 and CD8 single-positive thymocytes as well as peripheral T cells. Interestingly, a large proportion of the TCRγδ+ and CD8αα+TCRαß+ gut intraepithelial T lymphocytes were absent in Fam49b-KO mice. Our results demonstrate that Fam49b dampens thymocytes TCR signaling in order to escape negative selection during development, uncovering the function of Fam49b as a critical regulator of the selection process to ensure normal thymocyte development and peripheral T cells survival.
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Receptores de Antígenos de Linfocitos T , Transducción de Señal , Timocitos , Animales , Ratones , Supervivencia Celular , Ratones Noqueados , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores de Antígenos de Linfocitos T/genética , Linfocitos T/inmunología , Timocitos/metabolismo , Timocitos/citologíaRESUMEN
MHC-E restricted CD8 T cells show promise in vaccine settings, but their development and specificity remain poorly understood. Here we focus on a CD8 T cell population reactive to a self-peptide (FL9) bound to mouse MHC-E (Qa-1b) that is presented in response to loss of the MHC I processing enzyme ERAAP, termed QFL T cells. We find that mature QFL thymocytes are predominantly CD8αß+CD4-, show signs of agonist selection, and give rise to both CD8αα and CD8αß intraepithelial lymphocytes (IEL), as well as memory phenotype CD8αß T cells. QFL T cells require the MHC I subunit ß-2 microglobulin (ß2m), but do not require Qa1b or classical MHC I for positive selection. However, QFL thymocytes do require Qa1b for agonist selection and full functionality. Our data highlight the relaxed requirements for positive selection of an MHC-E restricted T cell population and suggest a CD8αß+CD4- pathway for development of CD8αα IELs.
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Linfocitos T CD8-positivos , Receptores de Antígenos de Linfocitos T alfa-beta , Animales , Ratones , Péptidos/metabolismo , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Timocitos/metabolismo , Genes MHC Clase IIRESUMEN
CD4+ T cells have been found to play critical roles in the control of both acute and chronic Toxoplasma infection. Previous studies identified a protective role for the Toxoplasma CD4+ T cell-eliciting peptide AS15 (AVEIHRPVPGTAPPS) in C57BL/6J mice. Herein, we found that immunizing mice with AS15 combined with GLA-SE, a TLR-4 agonist in emulsion adjuvant, can be either helpful in protecting male and female mice at early stages against Type I and Type II Toxoplasma parasites or harmful (lethal with intestinal, hepatic, and spleen pathology associated with a storm of IL6). Introducing the universal CD4+ T cell epitope PADRE abrogates the harmful phenotype of AS15. Our findings demonstrate quantitative and qualitative features of an effective Toxoplasma-specific CD4+ T cell response that should be considered in testing next-generation vaccines against toxoplasmosis. Our results also are cautionary that individual vaccine constituents can cause severe harm depending on the company they keep.
RESUMEN
The endoplasmic reticulum aminopeptidase associated with antigen processing (ERAAP) plays a crucial role in shaping the peptide-major histocompatibility complex (MHC) class I repertoire and maintaining immune surveillance. While murine cytomegalovirus (MCMV) has multiple strategies for manipulating the antigen processing pathway to evade immune responses, the host has also developed ways to counter viral immune evasion. In this study, we find that MCMV modulates ERAAP and induces an interferon γ (IFN-γ)-producing CD8+ T cell effector response that targets uninfected ERAAP-deficient cells. We observe that ERAAP downregulation during infection leads to the presentation of the self-peptide FL9 on non-classical Qa-1b, thereby eliciting Qa-1b-restricted QFL T cells to proliferate in the liver and spleen of infected mice. QFL T cells upregulate effector markers upon MCMV infection and are sufficient to reduce viral load after transfer to immunodeficient mice. Our study highlights the consequences of ERAAP dysfunction during viral infection and provides potential targets for anti-viral therapies.
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Presentación de Antígeno , Muromegalovirus , Animales , Ratones , Aminopeptidasas/metabolismo , Linfocitos T CD8-positivos , Retículo Endoplásmico/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Leucil Aminopeptidasa/metabolismo , Péptidos/metabolismoRESUMEN
Introduction: Critical steps in Major Histocompatibility Complex Class I (MHC-I) antigen presentation occur in the endoplasmic reticulum (ER). In general, peptides that enter the ER are longer than the optimal length for MHC-I binding. The final trimming of MHC-I epitopes is performed by two related aminopeptidases, ERAP1 and ERAP2 in humans that possess unique and complementary substrate trimming specificities. While ERAP1 efficiently trims peptides longer than 9 residues, ERAP2 preferentially trims peptides shorter than 9 residues. Materials and Methods: Using a combination of biochemical and proteomic studies followed by biological verification. Results: We demonstrate that the optimal ligands for either enzyme act as inhibitors of the other enzyme. Specifically, the presence of octamers reduced the trimming of long peptides by ERAP1, while peptides longer than nonomers inhibit ERAP2 activity. Discussion: We propose a mechanism for how ERAP1 and ERAP2 synergize to modulate their respective activities and shape the MHC-I peptidome by generating optimal peptides for presentation.
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Aminopeptidasas , Proteómica , Humanos , Aminopeptidasas/genética , Antígenos de Histocompatibilidad Clase I , Péptidos , Retículo Endoplásmico/metabolismo , Antígenos de Histocompatibilidad Menor/genética , Antígenos de Histocompatibilidad Menor/metabolismoRESUMEN
Chronic inflammation plays a critical role in the development of obesity-associated metabolic disorders such as insulin resistance. Obesity alters the microenvironment of adipose tissue and the intestines from anti-inflammatory to pro-inflammatory, which promotes low grade systemic inflammation and insulin resistance in obese mice. Various T cell subsets either help maintain metabolic homeostasis in healthy states or contribute to obesity-associated metabolic syndromes. In this review, we will discuss the T cell subsets that reside in adipose tissue and intestines and their role in the development of obesity-induced systemic inflammation.
RESUMEN
The peptide repertoire presented by MHC class I molecules on the cell surface is essential for the immune surveillance of intracellular pathogens and transformed cells. The generation of this peptide repertoire is critically dependent on the endoplasmic reticulum aminopeptidase associated with antigen processing (ERAAP). Loss of ERAAP function leads to the generation of a profoundly disrupted peptide repertoire including many novel and immunogenic peptides. Strikingly, a large fraction of these novel peptides on ERAAP-KO cells are presented by the nonclassical MHC Ib molecule, Qa-1b. One immunodominant Qa-1b-restricted novel peptide is recognized by a unique CD8+ T cell population showing features of both conventional cytotoxic T cells and unconventional innate-like T cells. While much remains to be uncovered, here we summarize the latest discoveries of our lab on the important immune surveillance of ERAAP function mediated by nonclassical MHC Ib molecules and their unusual cognate T cells.
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Presentación de Antígeno/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Vigilancia Inmunológica/inmunología , Animales , HumanosRESUMEN
Immune surveillance of infected or tumor cells by CD8+ T cells requires that MHC class I molecules present a diverse repertoire of peptides on the cell surface. Even a few copies of individual peptides among this mixture are sufficient for recognition by the antigen receptors of appropriate CD8 T cells. Here we describe methods for biochemical analysis of the naturally processed peptides and their precursors in living cells. The peptides are fractionated using reverse phase high performance liquid chromatography and detected by the activation of CD8+ T cell hybridomas. The results provide information on the structure and amount of the peptides and yield insights into the mechanisms that generate the naturally processed peptides.
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Presentación de Antígeno/inmunología , Bioensayo/métodos , Antígenos de Histocompatibilidad Clase I/metabolismo , Péptidos/metabolismo , Animales , Linfocitos T CD8-positivos/inmunología , Línea Celular , Cromatografía Líquida de Alta Presión , Cromatografía de Fase Inversa , Humanos , Ratones , TransfecciónRESUMEN
Most studies of T lymphocytes focus on recognition of classical major histocompatibility complex (MHC) class I or II molecules presenting oligopeptides, yet there are numerous variations and exceptions of biological significance based on recognition of a wide variety of nonclassical MHC molecules. These include αß and γδ T cells that recognize different class Ib molecules (CD1, MR-1, HLA-E, G, F, et al.) that are nearly monomorphic within a given species. Collectively, these T cells can be considered "unconventional," in part because they recognize lipids, metabolites, and modified peptides. Unlike classical MHC-specific cells, unconventional T cells generally exhibit limited T-cell antigen receptor (TCR) repertoires and often produce innate immune cell-like rapid effector responses. Exploiting this system in new generation vaccines for human immunodeficiency virus (HIV), tuberculosis (TB), other infectious agents, and cancer was the focus of a recent workshop, "Immune Surveillance by Non-classical MHC Molecules: Improving Diversity for Antigens," sponsored by the National Institute of Allergy and Infectious Diseases. Here, we summarize salient points presented regarding the basic immunobiology of unconventional T cells, recent advances in methodologies to measure unconventional T-cell activity in diseases, and approaches to harness their considerable clinical potential.
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Vigilancia Inmunológica/inmunología , Complejo Mayor de Histocompatibilidad/inmunología , Animales , Antígenos , Antígenos HLA , Antígenos de Histocompatibilidad Clase I/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Humanos , Receptores de Antígenos de Linfocitos T , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Linfocitos T/inmunologíaRESUMEN
Why does cancer develop in situations where the immune system is perfectly capable of eliminating it?
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Células Clonales , Sistema Inmunológico , Humanos , Neoplasias , PéptidosRESUMEN
ERAAP is an intracellular amino-peptidase that plays a central role in determining the repertoire of peptides displayed by cells by MHC class I molecules, and dysfunctions in ERAAP are linked to a variety of diseases. There is therefore great interest in developing probes that can image ERAAP in cells. In this report we present a fluorescent probe, termed Ep, that can image ERAAP activity in live cells. Ep is composed of a 10 amino acid ERAAP substrate that has a donor quencher pair conjugated to it, composed of BODIPY and dinitro-toluene. Ep undergoes a 20-fold increase in fluorescence after ERAAP cleavage, and was able to image ERAAP activity in cell culture via fluorescence microscopy. In addition, we used Ep to develop a high throughput screen for ERAAP inhibitors, and screened an electrophile library containing 1460 compounds. From this Ep based screen we identified aromatic alkyne-ketone as a lead fragment that can irreversibly inhibit ERAAP activity. We anticipate numerous applications of Ep given its unique ability to image ERAAP within cells.
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Colorantes Fluorescentes/química , Ensayos Analíticos de Alto Rendimiento , Leucil Aminopeptidasa/análisis , Imagen Óptica , Péptidos/química , Animales , Leucil Aminopeptidasa/deficiencia , Leucil Aminopeptidasa/metabolismo , Ratones , Ratones Noqueados , Microscopía FluorescenteRESUMEN
Toxoplasma gondii, an obligate intracellular protozoan parasite, establishes a chronic infection by forming cysts preferentially in the brain. Up to one third of the human population worldwide is estimated to be chronically infected with this parasite. However, there is currently no drug effective against the cyst form of the parasite. In addition, the protective immunity against the cysts remains largely unknown. We analyzed the molecular mechanisms by which the immune system detects host cells harboring the cysts to eliminate the latent stage of the parasite using mice with the H-2d haplotype, which are genetically resistant to the infection. Our study revealed that CD8+ immune T cells bearing TCR Vß8.1, 8.2 chain have a potent activity to remove T. gondii cysts from the brain. Our studies also uncovered that H-2Ld is the major Ag-presenting molecule to CD8+ T cells for initiating cyst elimination, and that CD8+Vß8.1, 8.2+ immune T cells recognize the N-terminal region (aa 41-152) of dense granule protein 6 (GRA6Nt) of the parasite presented by the H-2Ld molecule. Furthermore, CD8+ immune T cells induced by immunization with recombinant GRA6Nt were eventually capable of removing the cysts from the brain when transferred to infected immunodeficient mice lacking T cells. Thus, GRA6Nt is a novel and potent Ag to activate CD8+ T cells capable of removing T. gondii cysts. These observations offer a basis for immunological intervention to combat chronic infection with T. gondii by targeting the persistent cysts of the parasite.
Asunto(s)
Antígenos de Protozoos/inmunología , Linfocitos T CD8-positivos/inmunología , Proteínas Protozoarias/inmunología , Toxoplasma/inmunología , Toxoplasmosis Animal/inmunología , Animales , Antígenos de Protozoos/química , Encéfalo/inmunología , Encéfalo/parasitología , Ratones , Proteínas Protozoarias/química , Toxoplasmosis Animal/parasitología , Toxoplasmosis Cerebral/inmunología , Toxoplasmosis Cerebral/parasitologíaRESUMEN
Ag processing in the endoplasmic reticulum (ER) by the ER aminopeptidase associated with Ag processing (ERAAP) is central to presentation of a normal peptide-MHC class I (MHC I) repertoire. Alternations in ERAAP function cause dramatic changes in the MHC I-presented peptides, which elicit potent immune responses. An unusual subset of CD8+ T cells monitor normal Ag processing by responding to a highly conserved FL9 peptide that is presented by Qa-1b, a nonclassical MHC Ib molecule (QFL) in ERAAP-deficient cells. To understand the structural basis for recognition of the conserved ligand, we analyzed the αß TCRs of QFL-specific T cells. Individual cells in normal wild-type and TCRß-transgenic mice were assessed for QFL-specific TCR α- and ß-chains. The QFL-specific cells expressed a predominant semi-invariant TCR generated by DNA rearrangement of TRAV9d-3-TRAJ21 α-chain and TRBV5-TRBD1-TRBJ2-7 ß-chain gene segments. Furthermore, the CDR3 regions of the α- as well as ß-chains were required for QFL ligand recognition. Thus, the αß TCRs used to recognize the peptide-Qa-1 ligand presented by ERAAP-deficient cells are semi-invariant and likely reflect a conserved mechanism for monitoring the fidelity of Ag processing in the ER.
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Presentación de Antígeno , Linfocitos T CD8-positivos/inmunología , Retículo Endoplásmico/metabolismo , Leucil Aminopeptidasa/metabolismo , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Animales , Antígenos/metabolismo , Células Cultivadas , Secuencia Conservada , Variación Genética , Antígenos de Histocompatibilidad Clase I/metabolismo , Leucil Aminopeptidasa/genética , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Péptidos/metabolismo , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Análisis de la Célula IndividualRESUMEN
Cytolytic T cells eliminate infected or cancer cells by recognizing peptides presented by MHC class I molecules on the cell surface. The antigenic peptides are derived primarily from newly synthesized proteins including those produced by cryptic translation mechanisms. Previous studies have shown that cryptic translation can be initiated by distinct mechanisms at non-AUG codons in addition to conventional translation initiated at the canonical AUG start codon. In this study, we show that presentation of endogenously translated cryptic peptides is enhanced by TLR signaling pathways involved in pathogen recognition as well as by infection with different viruses. This enhancement of cryptic peptides was caused by proinflammatory cytokines, secreted in response to microbial infection. Furthermore, blocking these cytokines abrogated the enhancement of cryptic peptide presentation in response to infection. Thus, presentation of cryptic peptides is selectively enhanced during inflammation and infection, which could allow the immune system to detect intracellular pathogens that might otherwise escape detection because of inhibition of conventional host translation mechanisms.
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Presentación de Antígeno , Infecciones por Herpesviridae/inmunología , Inflamación/inmunología , Macrófagos/inmunología , Muromegalovirus/inmunología , Animales , Antígenos Virales/inmunología , Células Cultivadas , Citocinas/metabolismo , Antígenos de Histocompatibilidad Clase II , Mediadores de Inflamación/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Péptidos/inmunología , Biosíntesis de Proteínas/inmunología , Transducción de Señal , Receptores Toll-Like/metabolismoRESUMEN
The peptide repertoire presented by classical as well as nonclassical MHC class I (MHC I) molecules is altered in the absence of the endoplasmic reticulum aminopeptidase associated with Ag processing (ERAAP). To characterize the extent of these changes, peptides from cells lacking ERAAP were eluted from the cell surface and analyzed by high-throughput mass spectrometry. We found that most peptides found in wild-type (WT) cells were retained in the absence of ERAAP. In contrast, a subset of "ERAAP-edited" peptides was lost in WT cells, and ERAAP-deficient cells presented a unique "unedited" repertoire. A substantial fraction of MHC-associated peptides from ERAAP-deficient cells contained N-terminal extensions and had a different molecular composition than did those from WT cells. We found that the number and immunogenicity of peptides associated with nonclassical MHC I was increased in the absence of ERAAP. Conversely, only peptides presented by classical MHC I were immunogenic in ERAAP-sufficient cells. Finally, MHC I peptides were also derived from different intracellular sources in ERAAP-deficient cells.
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Presentación de Antígeno/inmunología , Autoinmunidad/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Leucil Aminopeptidasa/inmunología , Fragmentos de Péptidos/inmunología , Animales , Ensayos Analíticos de Alto Rendimiento , Leucil Aminopeptidasa/metabolismo , Activación de Linfocitos/inmunología , Espectrometría de Masas , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Linfocitos T/inmunologíaRESUMEN
We generated a CD8 T-cell receptor (TCR) transnuclear (TN) mouse specific to the Ld -restricted immunodominant epitope of GRA6 from Toxoplasma gondii as a source of cells to facilitate further investigation into the CD8 T-cell-mediated response against this pathogen. The TN T cells bound Ld -Gra6 tetramer and proliferated upon unspecific and peptide-specific stimulation. The TCR beta sequence of the Gra6-specific TN CD8 T cells is identical in its V- and J-region to the TCR-ß harboured by a hybridoma line generated in response to Gra6 peptide. Adoptively transferred Gra6 TN CD8 T cells proliferated upon Toxoplasma infection in vivo and exhibited an activated phenotype similar to host CD8 T cells specific to Gra6. The brain of Toxoplasma-infected mice carried Gra6 TN cells already at day 8 post-infection. Both Gra6 TN mice as well as adoptively transferred Gra6 TN cells were able to significantly reduce the parasite burden in the acute phase of Toxoplasma infection. Overall, the Gra6 TN mouse represents a functional tool to study the protective and immunodominant specific CD8 T-cell response to Toxoplasma in both the acute and the chronic phases of infection.
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Antígenos de Protozoos/metabolismo , Linfocitos T CD8-positivos/fisiología , Epítopos Inmunodominantes/metabolismo , Proteínas Protozoarias/metabolismo , Toxoplasma/fisiología , Toxoplasmosis/inmunología , Enfermedad Aguda , Traslado Adoptivo , Animales , Antígenos de Protozoos/inmunología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Antígenos de Histocompatibilidad Clase I/metabolismo , Hibridomas , Epítopos Inmunodominantes/inmunología , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Proteínas Protozoarias/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Especificidad del Receptor de Antígeno de Linfocitos TRESUMEN
Highly functional CD8(+) effector T (Teff) cells can persist in large numbers during controlled persistent infections, as exemplified by rare HIV-infected individuals who control the virus. Here we examined the cellular mechanisms that maintain ongoing T effector responses using a mouse model for persistent Toxoplasma gondii infection. In mice expressing the protective MHC-I molecule, H-2L(d), a dominant T effector response against a single parasite antigen was maintained without a contraction phase, correlating with ongoing presentation of the dominant antigen. Large numbers of short-lived Teff cells were continuously produced via a proliferative, antigen-dependent intermediate (Tint) population with a memory-effector hybrid phenotype. During an acute, resolved infection, decreasing antigen load correlated with a sharp drop in the Tint cell population and subsequent loss of the ongoing effector response. Vaccination approaches aimed at the development of Tint populations might prove effective against pathogens that lead to chronic infection.
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Linfocitos T CD8-positivos/inmunología , Diferenciación Celular , Subgrupos Linfocitarios/inmunología , Toxoplasma/inmunología , Toxoplasmosis/inmunología , Animales , Presentación de Antígeno , Antígenos de Protozoos/inmunología , Antígenos de Protozoos/metabolismo , Linfocitos T CD8-positivos/parasitología , Proliferación Celular , Células Cultivadas , Enfermedad Crónica , Citotoxicidad Inmunológica , Antígenos de Histocompatibilidad Clase I/metabolismo , Epítopos Inmunodominantes/inmunología , Epítopos Inmunodominantes/metabolismo , Memoria Inmunológica , Subgrupos Linfocitarios/parasitología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptores de Antígenos de Linfocitos T/genéticaRESUMEN
Effective immune surveillance by CD8(+) cytotoxic T cells of intracellular microbes and cancer depends on the antigen presentation pathway. This pathway produces an optimal peptide repertoire for presentation by major histocompatibility (MHC) class I molecules (pMHCs I) on the cell surface. We have known for years that the pMHC I repertoire is a reflection of the intracellular protein pool. However, many studies have revealed that pMHCs I present peptides not only from precursors encoded in open-reading frames of mRNA transcripts but also cryptic peptides encoded in apparently 'untranslated' regions. These sources vastly increase the availability of peptides for presentation and immune evasion. Here, we review studies on the composition of the cryptic pMHC I repertoire, the immunological significance of these pMHC I, and the novel translational mechanisms that generate cryptic peptides from unusual sources.
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Antígenos de Neoplasias/metabolismo , Linfocitos T CD8-positivos/inmunología , Vigilancia Inmunológica , Neoplasias/inmunología , Fragmentos de Péptidos/metabolismo , Animales , Presentación de Antígeno , Antígenos de Neoplasias/inmunología , Citotoxicidad Inmunológica , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Fragmentos de Péptidos/inmunología , Biosíntesis de Proteínas , Sitios de Empalme de ARN/genéticaRESUMEN
Translated regions distinct from annotated coding sequences have emerged as essential elements of the proteome. This includes upstream open reading frames (uORFs) present in mRNAs controlled by the integrated stress response (ISR) that show "privileged" translation despite inhibited eukaryotic initiation factor 2-guanosine triphosphate-initiator methionyl transfer RNA (eIF2·GTP·Met-tRNA(i )(Met)). We developed tracing translation by T cells to directly measure the translation products of uORFs during the ISR. We identified signature translation events from uORFs in the 5' untranslated region of binding immunoglobulin protein (BiP) mRNA (also called heat shock 70-kilodalton protein 5 mRNA) that were not initiated at the start codon AUG. BiP expression during the ISR required both the alternative initiation factor eIF2A and non-AUG-initiated uORFs. We propose that persistent uORF translation, for a variety of chaperones, shelters select mRNAs from the ISR, while simultaneously generating peptides that could serve as major histocompatibility complex class I ligands, marking cells for recognition by the adaptive immune system.