RESUMO
Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have promising potential for opening new avenues in regenerative medicine. However, since the tumorigenic potential of undifferentiated pluripotent stem cells (PSCs) is a major safety concern for clinical transplantation, inducible Caspase-9 (iC9) is under consideration for use as a fail-safe system. Here, we used targeted gene editing to introduce the iC9 system into human iPSCs, and then interrogated the efficiency of inducible apoptosis with normal iPSCs as well as diseased iPSCs derived from patients with acute myeloid leukemia (AML-iPSCs). The iC9 system induced quick and efficient apoptosis to iPSCs in vitro. More importantly, complete eradication of malignant cells without AML recurrence was shown in disease mouse models by using AML-iPSCs. In parallel, it shed light on several limitations of the iC9 system usage. Our results suggest that careful use of the iC9 system will serve as an important countermeasure against posttransplantation adverse events in stem cell transplantation therapies.
Assuntos
Células-Tronco Pluripotentes Induzidas , Células-Tronco Pluripotentes , Animais , Apoptose , Caspase 9/genética , Caspase 9/metabolismo , Diferenciação Celular , Linhagem Celular , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Células-Tronco Pluripotentes/metabolismoRESUMO
During selection of the T cell repertoire, the immune system navigates the subtle distinction between self-restriction and self-tolerance, yet how this is achieved is unclear. Here we describe how self-tolerance toward a trans-HLA (human leukocyte antigen) allotype shapes T cell receptor (TCR) recognition of an Epstein-Barr virus (EBV) determinant (FLRGRAYGL). The recognition of HLA-B8-FLRGRAYGL by two archetypal TCRs was compared. One was a publicly selected TCR, LC13, that is alloreactive with HLA-B44; the other, CF34, lacks HLA-B44 reactivity because it arises when HLA-B44 is coinherited in trans with HLA-B8. Whereas the alloreactive LC13 TCR docked at the C terminus of HLA-B8-FLRGRAYGL, the CF34 TCR docked at the N terminus of HLA-B8-FLRGRAYGL, which coincided with a polymorphic region between HLA-B8 and HLA-B44. The markedly contrasting footprints of the LC13 and CF34 TCRs provided a portrait of how self-tolerance shapes the specificity of TCRs selected into the immune repertoire.
Assuntos
Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/imunologia , Tolerância a Antígenos Próprios/imunologia , Sequência de Aminoácidos , Antígenos Virais/química , Antígenos Virais/imunologia , Cristalografia por Raios X , Antígeno HLA-B8/química , Antígeno HLA-B8/imunologia , Herpesvirus Humano 4/química , Herpesvirus Humano 4/imunologia , Humanos , Modelos Moleculares , Peptídeos/química , Peptídeos/imunologia , Estrutura Quaternária de Proteína , Homologia Estrutural de Proteína , Ressonância de Plasmônio de SuperfícieRESUMO
T cells often alloreact with foreign human leukocyte antigens (HLA). Here we showed the LC13 T cell receptor (TCR), selected for recognition on self-HLA-B( *)0801 bound to a viral peptide, alloreacts with B44 allotypes (HLA-B( *)4402 and HLA-B( *)4405) bound to two different allopeptides. Despite extensive polymorphism between HLA-B( *)0801, HLA-B( *)4402, and HLA-B( *)4405 and the disparate sequences of the viral and allopeptides, the LC13 TCR engaged these peptide-HLA (pHLA) complexes identically, accommodating mimicry of the viral peptide by the allopeptide. The viral and allopeptides adopted similar conformations only after TCR ligation, revealing an induced-fit mechanism of molecular mimicry. The LC13 T cells did not alloreact against HLA-B( *)4403, and the single residue polymorphism between HLA-B( *)4402 and HLA-B( *)4403 affected the plasticity of the allopeptide, revealing that molecular mimicry was associated with TCR specificity. Accordingly, molecular mimicry that is HLA and peptide dependent is a mechanism for human T cell alloreactivity between disparate cognate and allogeneic pHLA complexes.
Assuntos
Antígenos Nucleares do Vírus Epstein-Barr/imunologia , Antígenos HLA-B/imunologia , Mimetismo Molecular/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Linhagem Celular , Antígeno HLA-B8 , Humanos , Células Jurkat , Ativação Linfocitária/imunologia , Peptídeos/química , Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/metabolismo , TransfecçãoRESUMO
Ligation of the alphabeta T cell receptor (TCR) by a specific peptide-loaded major histocompatibility complex (pMHC) molecule initiates T cell signaling via the CD3 complex. However, the initial events that link antigen recognition to T cell signal transduction remain unclear. Here we show, via fluorescence-based experiments and structural analyses, that MHC-restricted antigen recognition by the alphabeta TCR results in a specific conformational change confined to the A-B loop within the alpha chain of the constant domain (Calpha). The apparent affinity constant of this A-B loop movement mirrored that of alphabeta TCR-pMHC ligation and was observed in two alphabeta TCRs with distinct pMHC specificities. The Ag-induced A-B loop conformational change could be inhibited by fixing the juxtapositioning of the constant domains and was shown to be reversible upon pMHC disassociation. Notably, the loop movement within the Calpha domain, although specific for an agonist pMHC ligand, was not observed with a pMHC antagonist. Moreover, mutagenesis of residues within the A-B loop impaired T cell signaling in an in vitro system of antigen-specific TCR stimulation. Collectively, our findings provide a basis for the earliest molecular events that underlie Ag-induced T cell triggering.
Assuntos
Antígenos/química , Receptores de Antígenos de Linfócitos T alfa-beta/química , Linfócitos T/imunologia , Animais , Antígenos/imunologia , Humanos , Complexo Principal de Histocompatibilidade/imunologia , Mutação/genética , Peptídeos/química , Peptídeos/imunologia , Ligação Proteica/imunologia , Estrutura Terciária de Proteína , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/imunologiaRESUMO
The nonclassical HLA molecule MHC-related protein 1 (MR1) presents metabolites of the vitamin B synthesis pathways to mucosal-associated invariant T (MAIT) cells and other MR1-restricted T cells. This new class of Ags represents a variation on the classical paradigm of self/non-self discrimination because these T cells are activated through their TCR by small organic compounds generated during microbial vitamin B2 synthesis. Beyond the fundamental significance, the invariant nature of MR1 across the human population is a tantalizing feature for the potential development of universal immune therapeutic and diagnostic tools. However, many aspects of MR1 Ag presentation and MR1-restricted T cell biology remain unknown, and the ubiquitous expression of MR1 across tissues and cell lines can be a confounding factor for experimental purposes. In this study, we report the development of a novel CRISPR/Cas9 genome editing lentiviral system and its use to efficiently disrupt MR1 expression in A459, THP-1, and K562 cell lines. We generated isogenic MR1(-/-) clonal derivatives of the A549 lung carcinoma and THP-1 monocytic cell lines and used these to study T cell responses to intracellular pathogens. We confirmed that MAIT cell clones were unable to respond to MR1(-/-) clones infected with bacteria whereas Ag presentation by classical and other nonclassical HLAs was unaffected. This system represents a robust and efficient method to disrupt the expression of MR1 and should facilitate investigations into the processing and presentation of MR1 Ags as well as into the biology of MAIT cells.
Assuntos
Apresentação de Antígeno/imunologia , Edição de Genes/métodos , Antígenos de Histocompatibilidade Classe I/imunologia , Ativação Linfocitária/imunologia , Antígenos de Histocompatibilidade Menor/imunologia , Linfócitos T/imunologia , Linhagem Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Citometria de Fluxo , Vetores Genéticos , Humanos , Lentivirus , Mutagênese Sítio-Dirigida , Reação em Cadeia da Polimerase , Subpopulações de Linfócitos T/imunologiaRESUMO
Human leukocyte antigens (HLAs) are highly polymorphic proteins that initiate immunity by presenting pathogen-derived peptides to T cells. HLA polymorphisms mostly map to the antigen-binding cleft, thereby diversifying the repertoire of self-derived and pathogen-derived peptide antigens selected by different HLA allotypes. A growing number of immunologically based drug reactions, including abacavir hypersensitivity syndrome (AHS) and carbamazepine-induced Stevens-Johnson syndrome (SJS), are associated with specific HLA alleles. However, little is known about the underlying mechanisms of these associations, including AHS, a prototypical HLA-associated drug reaction occurring exclusively in individuals with the common histocompatibility allele HLA-B*57:01, and with a relative risk of more than 1,000 (refs 6, 7). We show that unmodified abacavir binds non-covalently to HLA-B*57:01, lying across the bottom of the antigen-binding cleft and reaching into the F-pocket, where a carboxy-terminal tryptophan typically anchors peptides bound to HLA-B*57:01. Abacavir binds with exquisite specificity to HLA-B*57:01, changing the shape and chemistry of the antigen-binding cleft, thereby altering the repertoire of endogenous peptides that can bind HLA-B*57:01. In this way, abacavir guides the selection of new endogenous peptides, inducing a marked alteration in 'immunological self'. The resultant peptide-centric 'altered self' activates abacavir-specific T-cells, thereby driving polyclonal CD8 T-cell activation and a systemic reaction manifesting as AHS. We also show that carbamazepine, a widely used anti-epileptic drug associated with hypersensitivity reactions in HLA-B*15:02 individuals, binds to this allotype, producing alterations in the repertoire of presented self peptides. Our findings simultaneously highlight the importance of HLA polymorphism in the evolution of pharmacogenomics and provide a general mechanism for some of the growing number of HLA-linked hypersensitivities that involve small-molecule drugs.
Assuntos
Apresentação de Antígeno/efeitos dos fármacos , Autoimunidade/efeitos dos fármacos , Autoimunidade/imunologia , Didesoxinucleosídeos/farmacologia , Antígenos HLA-B/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Sítios de Ligação , Doadores de Sangue , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Carbamazepina/farmacologia , Hipersensibilidade a Drogas , Antígenos HLA-B/química , Humanos , Modelos Moleculares , Conformação Proteica , SíndromeRESUMO
The CD8 co-receptor engages peptide-major histocompatibility complex class I (pMHCI) molecules at a largely invariant site distinct from the T-cell receptor (TCR)-binding platform and enhances the sensitivity of antigen-driven activation to promote effective CD8+ T-cell immunity. A small increase in the strength of the pMHCI/CD8 interaction (~1.5-fold) can disproportionately amplify this effect, boosting antigen sensitivity by up to two orders of magnitude. However, recognition specificity is lost altogether with more substantial increases in pMHCI/CD8 affinity (~10-fold). In this study, we used a panel of MHCI mutants with altered CD8-binding properties to show that TCR-mediated antigen specificity is delimited by a pMHCI/CD8 affinity threshold. Our findings suggest that CD8 can be engineered within certain biophysical parameters to enhance the therapeutic efficacy of adoptive T-cell transfer irrespective of antigen specificity.
Assuntos
Antígenos CD8/metabolismo , Linfócitos T CD8-Positivos/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Membrana Celular/metabolismo , Humanos , Ativação Linfocitária/imunologia , Mutação/genética , Peptídeos/metabolismoRESUMO
UNLABELLED: Reconstitution of T cell immunity is absolutely critical for the effective control of virus-associated infectious complications in hematopoietic stem cell transplant (HSCT) recipients. Coinfection with genetic variants of human cytomegalovirus (CMV) in transplant recipients has been linked to clinical disease manifestation; however, how these genetic variants impact T cell immune reconstitution remains poorly understood. In this study, we have evaluated dynamic changes in the emergence of genetic variants of CMV in HSCT recipients and correlated these changes with reconstitution of antiviral T cell responses. In an analysis of single nucleotide polymorphisms within sequences encoding HLA class I-restricted CMV epitopes from the immediate early 1 gene of CMV, coinfection with genetically distinct variants of CMV was detected in 52% of patients. However, in spite of exposure to multiple viral variants, the T cell responses in these patients were preferentially directed to a limited repertoire of HLA class I-restricted CMV epitopes, either conserved, variant, or cross-reactive. More importantly, we also demonstrate that long-term control of CMV infection after HSCT is primarily mediated through the efficient induction of stable antiviral T cell immunity irrespective of the nature of the antigenic target. These observations provide important insights for the future design of antiviral T cell-based immunotherapeutic strategies for transplant recipients, emphasizing the critical impact of robust immune reconstitution on efficient control of viral infection. IMPORTANCE: Infection and disease caused by human cytomegalovirus (CMV) remain a significant burden in patients undergoing hematopoietic stem cell transplantation (HSCT). The establishment of efficient immunological control, primarily mediated by cytotoxic T cells, plays a critical role in preventing CMV-associated disease in transplant recipients. Recent studies have also begun to investigate the impact genetic variation in CMV has upon disease outcome in transplant recipients. In this study, we sought to investigate the role T cell immunity plays in recognizing and controlling genetic variants of CMV. We demonstrate that while a significant proportion of HSCT recipients may be exposed to multiple genetic variants of CMV, this does not necessarily lead to immune control mediated via recognition of this genetic variation. Rather, immune control is associated with the efficient establishment of a stable immune response predominantly directed against immunodominant conserved T cell epitopes.
Assuntos
Infecções por Citomegalovirus/imunologia , Infecções por Citomegalovirus/virologia , Citomegalovirus/imunologia , Genótipo , Transplante de Células-Tronco Hematopoéticas , Linfócitos T/imunologia , Transplantados , Coinfecção/imunologia , Coinfecção/virologia , Citomegalovirus/classificação , Citomegalovirus/genética , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Genes Precoces , Humanos , Polimorfismo de Nucleotídeo ÚnicoRESUMO
T cell cross-reactivity underpins the molecular mimicry hypothesis in which microbial peptides sharing structural features with host peptides stimulate T cells that cross-react with self-peptides, thereby initiating and/or perpetuating autoimmune disease. EBV represents a potentially important factor in the pathogenesis of several T cell-mediated autoimmune disorders, with molecular mimicry a likely mechanism. In this study, we describe a human self-peptide (DELEIKAY) that is a homolog of a highly immunogenic EBV T cell epitope (SELEIKRY) presented by HLA-B*18:01. This self-peptide was shown to bind stably to HLA-B*18:01, and peptide elution/mass spectrometric studies showed it is naturally presented by this HLA molecule on the surface of human cells. A significant proportion of CD8(+) T cells raised from some healthy individuals against this EBV epitope cross-reacted with the self-peptide. A diverse array of TCRs was expressed by the cross-reactive T cells, with variable functional avidity for the self-peptide, including some T cells that appeared to avoid autoreactivity by a narrow margin, with only 10-fold more of the self-peptide required for equivalent activation as compared with the EBV peptide. Structural studies revealed that the self-peptide-HLA-B*18:01 complex is a structural mimic of the EBV peptide-HLA-B*18:01 complex, and that the strong antiviral T cell response is primarily dependent on the alanine/arginine mismatch at position 7. To our knowledge, this is the first report confirming the natural presentation of a self-peptide cross-recognized in the context of self-HLA by EBV-reactive CD8(+) T cells. These results illustrate how aberrant immune responses and immunopathological diseases could be generated by EBV infection.
Assuntos
Antígenos Virais/imunologia , Autoantígenos/imunologia , Autoimunidade/imunologia , Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Herpesvirus Humano 4/imunologia , Apresentação de Antígeno/imunologia , Cromatografia Líquida , Reações Cruzadas/imunologia , Infecções por Vírus Epstein-Barr/imunologia , Antígenos HLA-B/imunologia , Humanos , Mimetismo Molecular/imunologia , Espectrometria de Massas em TandemRESUMO
Evidence indicates that autoimmunity can be triggered by virus-specific CD8(+) T cells that crossreact with self-derived peptide epitopes presented on the cell surface by major histocompatibility complex class I (MHCI) molecules. Identification of the associated viral pathogens is challenging because individual T-cell receptors can potentially recognize up to a million different peptides. Here, we generate peptide length-matched combinatorial peptide library (CPL) scan data for a panel of virus-specific CD8(+) T-cell clones spanning different restriction elements and a range of epitope lengths. CPL scan data drove a protein database search limited to viruses that infect humans. Peptide sequences were ranked in order of likelihood of recognition. For all anti-viral CD8(+) T-cell clones examined in this study, the index peptide was either the top-ranked sequence or ranked as one of the most likely sequences to be recognized. Thus, we demonstrate that anti-viral CD8(+) T-cell clones are highly focused on their index peptide sequence and that 'CPL-driven database searching' can be used to identify the inciting virus-derived epitope for a given CD8(+) T-cell clone. Moreover, to augment access to CPL-driven database searching, we have created a publicly accessible webtool. Application of these methodologies in the clinical setting may clarify the role of viral pathogens in the etiology of autoimmune diseases.
Assuntos
Antígenos de Histocompatibilidade Classe I/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Linfócitos T CD8-Positivos/imunologia , Bases de Dados de Proteínas , HIV-1/imunologia , Herpesvirus Humano 4/imunologia , Humanos , Ligantes , Biblioteca de Peptídeos , Peptídeos/química , Peptídeos/metabolismo , Reprodutibilidade dos Testes , Especificidade da EspécieRESUMO
UNLABELLED: Polymorphism in the human leukocyte antigen (HLA) loci ensures that the CD8(+) T cell response to viruses is directed against a diverse range of antigenic epitopes, thereby minimizing the impact of virus escape mutation across the population. The BZLF1 antigen of Epstein-Barr virus is an immunodominant target for CD8(+) T cells, but the response has been characterized only in the context of a limited number of HLA molecules due to incomplete epitope mapping. We have now greatly expanded the number of defined CD8(+) T cell epitopes from BZLF1, allowing the response to be evaluated in a much larger proportion of the population. Some regions of the antigen fail to be recognized by CD8(+) T cells, while others include clusters of overlapping epitopes presented by different HLA molecules. These highly immunogenic regions of BZLF1 include polymorphic sequences, such that up to four overlapping epitopes are impacted by a single amino acid variation common in different regions of the world. This focusing of the immune response to limited regions of the viral protein could be due to sequence similarity to human proteins creating "immune blind spots" through self-tolerance. This study significantly enhances the understanding of the immune response to BZLF1, and the precisely mapped T cell epitopes may be directly exploited in vaccine development and adoptive immunotherapy. IMPORTANCE: Epstein-Barr virus (EBV) is an important human pathogen, associated with several malignancies, including nasopharyngeal carcinoma and Hodgkin lymphoma. T lymphocytes are critical for virus control, and clinical trials aimed at manipulating this arm of the immune system have demonstrated efficacy in treating these EBV-associated diseases. These trials have utilized information on the precise location of viral epitopes for T cell recognition, for either measuring or enhancing responses. In this study, we have characterized the T cell response to the highly immunogenic BZLF1 antigen of EBV by greatly expanding the number of defined T cell epitopes. An unusual clustering of epitopes was identified, highlighting a small region of BZLF1 that is targeted by the immune response of a high proportion of the world's population. This focusing of the immune response could be utilized in developing vaccines/therapies with wide coverage, or it could potentially be exploited by the virus to escape the immune response.
Assuntos
Epitopos de Linfócito T/imunologia , Herpesvirus Humano 4/imunologia , Transativadores/imunologia , Linfócitos T CD8-Positivos/imunologia , Mapeamento de Epitopos , HumanosRESUMO
To follow the fate of CD8+ T cells responsive to Plasmodium berghei ANKA (PbA) infection, we generated an MHC I-restricted TCR transgenic mouse line against this pathogen. T cells from this line, termed PbT-I T cells, were able to respond to blood-stage infection by PbA and two other rodent malaria species, P. yoelii XNL and P. chabaudi AS. These PbT-I T cells were also able to respond to sporozoites and to protect mice from liver-stage infection. Examination of the requirements for priming after intravenous administration of irradiated sporozoites, an effective vaccination approach, showed that the spleen rather than the liver was the main site of priming and that responses depended on CD8α+ dendritic cells. Importantly, sequential exposure to irradiated sporozoites followed two days later by blood-stage infection led to augmented PbT-I T cell expansion. These findings indicate that PbT-I T cells are a highly versatile tool for studying multiple stages and species of rodent malaria and suggest that cross-stage reactive CD8+ T cells may be utilized in liver-stage vaccine design to enable boosting by blood-stage infections.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Imunização Secundária/métodos , Estágios do Ciclo de Vida/imunologia , Malária/prevenção & controle , Plasmodium berghei/imunologia , Receptores de Antígenos de Linfócitos T/genética , Esporozoítos/imunologia , Transferência Adotiva , Animais , Anopheles , Sangue/parasitologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/patologia , Células Cultivadas , Fígado/imunologia , Fígado/parasitologia , Malária/sangue , Malária/imunologia , Malária/parasitologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Plasmodium berghei/crescimento & desenvolvimento , Plasmodium chabaudi , Plasmodium yoelii , Receptores de Antígenos de Linfócitos T/imunologiaRESUMO
T cells that express clonally distributed αß T-cell receptors (TCRs) corecognize antigenic peptides (p) bound to major histocompatibility complex class I (MHC-I) and class II molecules (MHC-II). Extensive human leukocyte antigen (HLA) polymorphism enables HLA molecules from different haplotypes to capture an array of self- and microbe-derived peptide antigens that is fundamental to adaptive immunity. T cells developing in the thymus are selected for weak binding to self-peptide-HLA complexes generating a vast repertoire of clonally distinct T cells in the periphery. Indeed, diversity within germline loci and the finally assembled TCR genes, coupled with inherent TCR cross-reactivity, enables CD8(+) T cells to survey the multitude of pHLA-I landscapes. Precisely how does the TCR ligate to pHLA-I, and how does knowledge of the detailed structural interactions inform immunobiology? A recent number of our structural studies concerning the TCR-pMHC-I axis, alongside others in the field, have provided insight into HLA-I polymorphism, pMHC-I flexibility, TCR bias, TCR polymorphism, maintenance of self-tolerance, T-cell cross-reactivity, and alloreactivity. Collectively, the data also provide an opportunity to address the structural correlates of MHC-I restriction. Here, we provide our perspective concerning these advances in the field. Although much key information has been gleaned, the structural data show that some of the key concepts surrounding the TCR-pMHC-I interaction remain controversial and unresolved.
Assuntos
Antígenos HLA/química , Antígenos de Histocompatibilidade Classe I/química , Complexo Principal de Histocompatibilidade/imunologia , Peptídeos/química , Receptores de Antígenos de Linfócitos T alfa-beta/química , Linfócitos T/imunologia , Imunidade Adaptativa , Animais , Sítios de Ligação , Reações Cruzadas , Antígenos HLA/imunologia , Antígenos HLA/metabolismo , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Camundongos , Modelos Moleculares , Peptídeos/imunologia , Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Linfócitos T/citologia , Linfócitos T/metabolismoRESUMO
Mutations within T cell epitopes represent a common mechanism of viral escape from the host protective immune response. The diverse T cell repertoire and the extensive human leukocyte antigen (HLA) polymorphism across populations is the evolutionary response to viral mutation. However, the molecular basis underpinning the interplay between HLA polymorphism, the T cell repertoire, and viral escape is unclear. Here we investigate the T cell response to a HLA-B*35:01- and HLA-B*35:08-restricted (407)HPVGEADYFEY(417) epitope from Epstein-Barr virus and naturally occurring variants at positions 4 and 5 thereof. Each viral variant differently impacted on the epitope's flexibility and conformation when bound to HLA-B*35:08 or HLA-B*35:01. We provide a molecular basis for understanding how the single residue polymorphism that discriminates between HLA-B*35:01/08 profoundly impacts on T cell receptor recognition. Surprisingly, one viral variant (P5-Glu to P5-Asp) effectively changed restriction preference from HLA-B*35:01 to HLA-B*35:08. Collectively, our study portrays the interplay between the T cell response, viral escape, and HLA polymorphism, whereby HLA polymorphism enables altered presentation of epitopes from different strains of Epstein-Barr virus.
Assuntos
Antígeno HLA-B35/química , Herpesvirus Humano 4/genética , Polimorfismo de Nucleotídeo Único , Receptores de Antígenos de Linfócitos T/química , Alelos , Sequência de Aminoácidos , Epitopos/química , Epitopos/genética , Antígeno HLA-B35/genética , Herpesvirus Humano 4/imunologia , Humanos , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Conformação Proteica , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T/imunologiaRESUMO
Basic parameters of the naive antigen (Ag)-specific T-cell repertoire in humans remain poorly defined. Systematic characterization of this 'ground state' immunity in comparison with memory will allow a better understanding of clonal selection during immune challenge. Here, we used high-definition cell isolation from umbilical cord blood samples to establish the baseline frequency, phenotype and T-cell antigen receptor (TCR) repertoire of CD8(+) T-cell precursor populations specific for a range of viral and self-derived Ags. Across the board, these precursor populations were phenotypically naive and occurred with hierarchical frequencies clustered by Ag specificity. The corresponding patterns of TCR architecture were highly ordered and displayed partial overlap with adult memory, indicating biased structuring of the T-cell repertoire during Ag-driven selection. Collectively, these results provide new insights into the complex nature and dynamics of the naive T-cell compartment.
Assuntos
Autoantígenos/imunologia , Linfócitos T CD8-Positivos/imunologia , Seleção Clonal Mediada por Antígeno , Sangue Fetal/imunologia , Células-Tronco Hematopoéticas/imunologia , Fosfoproteínas/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Especificidade do Receptor de Antígeno de Linfócitos T , Proteínas da Matriz Viral/imunologia , Adulto , Envelhecimento/imunologia , Dasatinibe/farmacologia , Sangue Fetal/citologia , Citometria de Fluxo , Antígenos HLA/imunologia , Humanos , Memória Imunológica , Separação Imunomagnética , Imunofenotipagem , Recém-Nascido , Fragmentos de Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/genéticaRESUMO
αß-TCRs expressed at the CD8(+) T-cell surface interact with short peptide fragments (p) bound to MHC class I molecules (pMHCI). The TCR/pMHCI interaction is pivotal in all aspects of CD8(+) T-cell immunity. However, the rules that govern the outcome of TCR/pMHCI engagement are not entirely understood, and this is a major barrier to understanding the requirements for both effective immunity and vaccination. In the present study, we discovered an unexpected feature of the TCR/pMHCI interaction by showing that any given TCR exhibits an explicit preference for a single MHCI-peptide length. Agonists of nonpreferred length were extremely rare, suboptimal, and often entirely distinct in sequence. Structural analysis indicated that alterations in peptide length have a major impact on antigenic complexity, to which individual TCRs are unable to adapt. This novel finding demonstrates that the outcome of TCR/pMHCI engagement is determined by peptide length in addition to the sequence identity of the MHCI-bound peptide. Accordingly, the effective recognition of pMHCI Ag, which is a prerequisite for successful CD8(+) T-cell immunity and protective vaccination, can only be achieved by length-matched Ag-specific CD8(+) T-cell clonotypes.
Assuntos
Antígenos de Histocompatibilidade Classe I/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Sequência de Aminoácidos , Apresentação de Antígeno , Antígenos/química , Antígenos/genética , Antígenos/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Clonais , Humanos , Imunidade Celular , Modelos Moleculares , Oligopeptídeos/química , Oligopeptídeos/genética , Oligopeptídeos/imunologia , Fragmentos de Peptídeos/genética , Biblioteca de PeptídeosRESUMO
Class I HLAs generally present peptides of 8-10 aa in length, although it is unclear whether peptide length preferences are affected by HLA polymorphism. In this study, we investigated the CD8(+) T cell response to the BZLF1 Ag of EBV, which includes overlapping sequences of different size that nevertheless conform to the binding motif of the large and abundant HLA-B*44 supertype. Whereas HLA-B*18:01(+) individuals responded strongly and exclusively to the octamer peptide (173)SELEIKRY(180), HLA-B*44:03(+) individuals responded to the atypically large dodecamer peptide (169)EECDSELEIKRY(180), which encompasses the octamer peptide. Moreover, the octamer peptide bound more stably to HLA-B*18:01 than did the dodecamer peptide, whereas, conversely, HLA-B*44:03 bound only the longer peptide. Furthermore, crystal structures of these viral peptide-HLA complexes showed that the Ag-binding cleft of HLA-B*18:01 was more ideally suited to bind shorter peptides, whereas HLA-B*44:03 exhibited characteristics that favored the presentation of longer peptides. Mass spectrometric identification of > 1000 naturally presented ligands revealed that HLA-B*18:01 was more biased toward presenting shorter peptides than was HLA-B*44:03. Collectively, these data highlight a mechanism through which polymorphism within an HLA class I supertype can diversify determinant selection and immune responses by varying peptide length preferences.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Antígeno HLA-B18/imunologia , Antígeno HLA-B44/imunologia , Fragmentos de Peptídeos/imunologia , Sítios de Ligação de Anticorpos , Células Cultivadas , Epitopos de Linfócito T/imunologia , Antígeno HLA-B18/genética , Antígeno HLA-B44/genética , Humanos , Leucócitos Mononucleares/imunologia , Polimorfismo de Nucleotídeo Único , Estrutura Terciária de Proteína , Transativadores/imunologiaRESUMO
Human leukocyte antigen (HLA)-I molecules can present long peptides, yet the mechanisms by which T-cell receptors (TCRs) recognize featured pHLA-I landscapes are unclear. We compared the binding modes of three distinct human TCRs, CA5, SB27, and SB47, complexed with a "super-bulged" viral peptide (LPEPLPQGQLTAY) restricted by HLA-B*35:08. The CA5 and SB27 TCRs engaged HLA-B*35:08(LPEP) similarly, straddling the central region of the peptide but making limited contacts with HLA-B*35:08. Remarkably, the CA5 TCR did not contact the α1-helix of HLA-B*35:08. Differences in the CDR3ß loop between the CA5 and SB27 TCRs caused altered fine specificities. Surprisingly, the SB47 TCR engaged HLA-B*35:08(LPEP) using a completely distinct binding mechanism, namely "bypassing" the bulged peptide and making extensive contacts with the extreme N-terminal end of HLA-B*35:08. This docking footprint included HLA-I residues not observed previously as TCR contact sites. The three TCRs exhibited differing patterns of alloreactivity toward closely related or distinct HLA-I allotypes. Thus, the human T-cell repertoire comprises a range of TCRs that can interact with "bulged" pHLA-I epitopes using unpredictable strategies, including the adoption of atypical footprints on the MHC-I.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Antígeno HLA-B35/imunologia , Herpesvirus Humano 4/imunologia , Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Proteínas Virais/imunologia , Linfócitos T CD8-Positivos/citologia , Regiões Determinantes de Complementaridade/genética , Regiões Determinantes de Complementaridade/imunologia , Antígeno HLA-B35/genética , Herpesvirus Humano 4/genética , Peptídeos/genética , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptores de Antígenos de Linfócitos T/genética , Proteínas Virais/genéticaRESUMO
T-box transcription factors T-bet (Tbx21) and Eomesodermin (Eomes) are critical players in CD8(+) cytotoxic T lymphocyte effector function and differentiation, but how the expression of these transcription factors is regulated remains poorly defined. Here we show that dominant T cells directed toward human CMV, expressing significantly higher levels of T-bet with graded loss of Eomes expression (T-bet(hi)Eomes(hi/lo)), are more efficient in recognizing endogenously processed peptide-major histocompatibility complexes (pMHC) compared with subdominant virus-specific T cells expressing lower levels of T-bet and high levels of Eomes (T-bet(int)Eomes(hi)). Paradoxically, the T-bet(hi)Eomes(hi/lo) dominant populations that efficiently recognized endogenous antigen demonstrated lower intrinsic avidity for pMHC, whereas T-bet(int)Eomes(hi) subdominant populations were characterized by higher pMHC avidity and less efficient recognition of virus-infected cells. Importantly, differential endogenous viral antigen recognition by CMV-specific CD8(+) T cells also correlated with the differentiation status and expression of perforin, granzyme B and K. Furthermore, we demonstrate that the expression of T-bet correlates with clonal expansion, differentiation status, and expression of perforin, granzyme B and K in antigen-specific T cells. These findings illustrate how endogenous viral antigen presentation during persistent viral infection may influence the transcriptional program of virus-specific T cells and their functional profile in the peripheral blood of humans.
Assuntos
Apresentação de Antígeno/imunologia , Linfócitos T CD8-Positivos/imunologia , Proteínas com Domínio T/metabolismo , Linfócitos T Citotóxicos/imunologia , Antígenos Virais/imunologia , Linfócitos T CD8-Positivos/metabolismo , Diferenciação Celular , Células Cultivadas , Citocinas/metabolismo , Citomegalovirus/imunologia , Infecções por Citomegalovirus/imunologia , Infecções por Citomegalovirus/metabolismo , Infecções por Citomegalovirus/microbiologia , Citometria de Fluxo , Granzimas/metabolismo , Humanos , Interferon gama/metabolismo , Ativação Linfocitária , Complexo Principal de Histocompatibilidade/imunologia , Perforina/metabolismo , Proteínas com Domínio T/imunologiaRESUMO
BACKGROUND: Patients with multiple sclerosis (MS) have a deficiency of circulating CD8+ T cells, which might impair control of Epstein-Barr virus (EBV) and predispose to MS by allowing EBV-infected autoreactive B cells to accumulate in the central nervous system. Based on the expression of CD45RA and CD62L, CD4+ T cells and CD8+ T cells can be subdivided into four subsets with distinct homing and functional properties, namely: naïve, central memory, effector memory (EM) and effector memory re-expressing CD45RA (EMRA) cells. OBJECTIVE: Our aim was to determine which memory subsets are involved in the CD8+ T cell deficiency and how these relate to clinical course. METHODS: We used flow cytometry to analyze the memory phenotypes of T cells in the blood of 118 MS patients and 112 healthy subjects. RESULTS: MS patients had a decreased frequency of EM (CD45RA(-)CD62L(-)) and EMRA (CD45RA(+)CD62L(-)) CD8+ T cells, which was present at the onset of disease and persisted throughout the clinical course. The frequencies of CD4+ EM and EMRA T cells were normal. CONCLUSION: Deficiency of effector memory CD8+ T cells is an early and persistent feature of MS and might underlie the impaired CD8+ T cell control of EBV.