RESUMO
Ultrasound-guided fine needle biopsy, also known as fine needle aspiration, of human axillary lymph nodes is a safe and effective procedure to assess the immune response within the lymph nodes following vaccination. Once acquired, lymph node cells can be characterized via flow cytometric immunophenotyping and/or single-cell RNA sequencing for gene expression and T and B cell receptors. Analysis of the immune cells from the lymph nodes enables the investigation of T and B cells that may interact at this site. These interactions may lead to germinal center formation and expansion, critical for the generation of effective immunity to vaccination. Directly studying the dynamic processes and interaction of the key cells has been challenging in humans due to the anatomically protected location of these cells. Here, we describe the methods involved in ultrasound-guided fine needle biopsy of human axillary lymph nodes in response to vaccination and subsequent analyses of the B cell populations.
Assuntos
Axila , Linfócitos B , Linfonodos , Vacinação , Humanos , Linfonodos/patologia , Linfonodos/imunologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Vacinação/métodos , Citometria de Fluxo/métodos , Imunofenotipagem , Biópsia por Agulha Fina/métodos , Biópsia Guiada por Imagem/métodosRESUMO
T follicular helper (Tfh) cells provide critical help to B cells during the germinal center (GC) reaction to facilitate generation of protective humoral immunity. Accessing the human lymph node (LN) to study the commitment of CD4 T cells to GC Tfh cell differentiation during in vivo vaccine responses is difficult. We used ultrasound guided fine needle biopsy to monitor recall responses in axillary LNs to seasonal influenza vaccination in healthy volunteers. Specific expansion of GC cell subsets occurred exclusively within draining LNs five days postvaccination. Draining LN GC Tfh and precursor-Tfh cells express higher levels of CD38, ICOS, and Ki67, indicating they were significantly more activated, motile, and proliferating, compared to contralateral LN cells. These observations provide insight into the early expansion phase of the human Tfh lineage within LNs during a vaccine induced memory response and highlights early LN immune responses may not be reflected in the periphery.
RESUMO
Accumulating evidence indicates that the immune system does not develop in a linear fashion, but rather as distinct developmental layers formed from sequential waves of hematopoietic stem cells, each giving rise to unique populations of immune cells at different stages of development. Although recent studies have indicated that conventional CD8+ T cells produced in early life persist into adulthood and exhibit distinct roles during infection, the developmental architecture of the peripheral T cell compartment remains undefined. In this study, we used a mouse model to permanently label CD8+ T cells produced during distinct windows of development and traced their history to generate fate maps of CD8+ T cells produced during different stages of life. We then used mathematical modeling to understand the age structure of the CD8+ T cell compartment across the lifespan. Interestingly, we found that survival rate of CD8+ T cells depends on both the age and developmental origin of the cells. Recently produced cells show an initial rapid decay rate, which slows with age of the animal at which the cells were produced. For cells produced at any age, the rate of decay also slows with the age of the cell. We derive a function to describe this and predict the "age distribution" of the CD8+ T cell pool for animals of any given age. These data provide a quantitative framework for understanding the ontogeny of the CD8+ T cell compartment and help to contextualize age-related changes in the CD8+ T cell response to infection.
Assuntos
Envelhecimento/imunologia , Linfócitos T CD8-Positivos/imunologia , Modelos Imunológicos , Envelhecimento/genética , Animais , Linfócitos T CD8-Positivos/citologia , Camundongos , Camundongos TransgênicosRESUMO
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
Despite progress toward understanding the correlates of protective T cell immunity in HIV infection, the optimal approach to Ag delivery by vaccination remains uncertain. We characterized two immunodominant CD8 T cell populations generated in response to immunization of BALB/c mice with a replication-deficient adenovirus serotype 5 vector expressing the HIV-derived Gag and Pol proteins at equivalent levels. The Gag-AI9/H-2K(d) epitope elicited high-avidity CD8 T cell populations with architecturally diverse clonotypic repertoires that displayed potent lytic activity in vivo. In contrast, the Pol-LI9/H-2D(d) epitope elicited motif-constrained CD8 T cell repertoires that displayed lower levels of physical avidity and lytic activity despite equivalent measures of overall clonality. Although low-dose vaccination enhanced the functional profiles of both epitope-specific CD8 T cell populations, greater polyfunctionality was apparent within the Pol-LI9/H-2D(d) specificity. Higher proportions of central memory-like cells were present after low-dose vaccination and at later time points. However, there were no noteworthy phenotypic differences between epitope-specific CD8 T cell populations across vaccine doses or time points. Collectively, these data indicate that the functional and phenotypic properties of vaccine-induced CD8 T cell populations are sensitive to dose manipulation, yet constrained by epitope specificity in a clonotype-dependent manner.
Assuntos
Vacinas contra a AIDS , Linfócitos T CD8-Positivos/imunologia , Epitopos Imunodominantes/metabolismo , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Produtos do Gene pol do Vírus da Imunodeficiência Humana/metabolismo , Adenoviridae/genética , Animais , Linfócitos T CD8-Positivos/virologia , Células Cultivadas , Citotoxicidade Imunológica , Feminino , Vetores Genéticos , Antígenos H-2/metabolismo , Antígeno de Histocompatibilidade H-2D/metabolismo , Humanos , Epitopos Imunodominantes/genética , Memória Imunológica , Camundongos , Camundongos Endogâmicos BALB C , Vacinação , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Produtos do Gene pol do Vírus da Imunodeficiência Humana/genéticaRESUMO
In a rat heart allograft model, preventing T cell costimulation with CD40Ig leads to indefinite allograft survival, which is mediated by the induction of CD8+CD45RClo regulatory T cells (CD8+CD40Ig Tregs) interacting with plasmacytoid dendritic cells (pDCs). The role of TCR-MHC-peptide interaction in regulating Treg activity remains a topic of debate. Here, we identified a donor MHC class II-derived peptide (Du51) that is recognized by TCR-biased CD8+CD40Ig Tregs and activating CD8+CD40Ig Tregs in both its phenotype and suppression of antidonor alloreactive T cell responses. We generated a labeled tetramer (MHC-I RT1.Aa/Du51) to localize and quantify Du51-specific T cells within rat cardiac allografts and spleen. RT1.Aa/Du51-specific CD8+CD40Ig Tregs were the most suppressive subset of the total Treg population, were essential for in vivo tolerance induction, and expressed a biased, restricted Vß11-TCR repertoire in the spleen and the graft. Finally, we demonstrated that treatment of transplant recipients with the Du51 peptide resulted in indefinite prolongation of allograft survival. These results show that CD8+CD40Ig Tregs recognize a dominant donor antigen, resulting in TCR repertoire alterations in the graft and periphery. Furthermore, this allopeptide has strong therapeutic activity and highlights the importance of TCR-peptide-MHC interaction for Treg generation and function.
Assuntos
Rejeição de Enxerto/imunologia , Rejeição de Enxerto/prevenção & controle , Linfócitos T Reguladores/imunologia , Sequência de Aminoácidos , Animais , Apresentação de Antígeno , Antígenos CD8/metabolismo , Transplante de Coração , Tolerância Imunológica , Isoantígenos/genética , Isoantígenos/imunologia , Complexo Principal de Histocompatibilidade , Masculino , Dados de Sequência Molecular , Peptídeos/genética , Peptídeos/imunologia , Ratos , Ratos Endogâmicos Lew , Receptores de Antígenos de Linfócitos T/metabolismo , Proteínas Recombinantes de Fusão/imunologia , Linfócitos T Reguladores/classificaçãoRESUMO
HIV undergoes high rates of mutation and recombination during reverse transcription, but it is not known whether these events occur independently or are linked mechanistically. Here we used a system of silent marker mutations in HIV and a single round of infection in primary T lymphocytes combined with a high-throughput sequencing and mathematical modeling approach to directly estimate the viral recombination and mutation rates. From >7 million nucleotides (nt) of sequences from HIV infection, we observed 4,801 recombination events and 859 substitution mutations (≈1.51 and 0.12 events per 1,000 nt, respectively). We used experimental controls to account for PCR-induced and transfection-induced recombination and sequencing error. We found that the single-cycle virus-induced mutation rate is 4.6 × 10(-5) mutations per nt after correction. By sorting of our data into recombined and nonrecombined sequences, we found a significantly higher mutation rate in recombined regions (P = 0.003 by Fisher's exact test). We used a permutation approach to eliminate a number of potential confounding factors and confirm that mutation occurs around the site of recombination and is not simply colocated in the genome. By comparing mutation rates in recombined and nonrecombined regions, we found that recombination-associated mutations account for 15 to 20% of all mutations occurring during reverse transcription.
Assuntos
Substituição de Aminoácidos , HIV/crescimento & desenvolvimento , HIV/genética , Mutação de Sentido Incorreto , Recombinação Genética , Linfócitos T/virologia , Células Cultivadas , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Modelos TeóricosRESUMO
CD8(+) T cells recognize infected or dysregulated cells via the clonotypically expressed αß TCR, which engages Ag in the form of peptide bound to MHC class I (MHC I) on the target cell surface. Previous studies have indicated that a diverse Ag-specific TCR repertoire can be beneficial to the host, yet the determinants of clonotypic diversity are poorly defined. To better understand the factors that govern TCR repertoire formation, we conducted a comprehensive clonotypic analysis of CD8(+) T cell populations directed against epitopes derived from EBV and CMV. Neither pathogen source nor the restricting MHC I molecule were linked with TCR diversity; indeed, both HLA-A and HLA-B molecules were observed to interact with an overlapping repertoire of expressed TRBV genes. Peptide specificity, however, markedly impacted TCR diversity. In addition, distinct peptides sharing HLA restriction and viral origin mobilized TCR repertoires with distinct patterns of TRBV gene usage. Notably, no relationship was observed between immunodominance and TCR diversity. These findings provide new insights into the forces that shape the Ag-specific TCR repertoire in vivo and highlight a determinative role for the peptide component of the peptide-MHC I complex on the molecular frontline of CD8(+) T cell-mediated immune surveillance.
Assuntos
Antígenos Virais/imunologia , Linfócitos T CD8-Positivos/imunologia , Epitopos/imunologia , Rearranjo Gênico do Linfócito T/imunologia , Antígenos HLA-A/imunologia , Antígenos HLA-B/imunologia , Fragmentos de Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Especificidade do Receptor de Antígeno de Linfócitos T/imunologia , Sequência de Aminoácidos , Linfócitos T CD8-Positivos/metabolismo , Seleção Clonal Mediada por Antígeno , Citomegalovirus/imunologia , Herpesvirus Humano 4/imunologia , Humanos , Epitopos Imunodominantes/imunologia , Vigilância Imunológica , Interferon gama/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/imunologiaRESUMO
Defects in T cell responses against pathogens and reduced diversity of TCRs have been described at both extremes of the life span. Yet, we still lack information on how Ag-specific T cell populations are maintained and/or altered from birth to old age. In this study, for the first time to our knowledge, we provide insight into Ag-specific TCR repertoire changes over the life span at the single-cell level. We have examined the TCR diversity of the primary CD8(+) T cell response to the immunodominant HSV-1 epitope HSV glycoprotein B 495-502 (HSV gB(498-505); SSIEFARL) (gB-8p) in neonatal, adult, and old C57BL/6 mice. The global distinctive features of the gB-8p-specific TCR repertoire were preserved in mice of different ages. However, both old and especially neonatal mice exhibited significant decreases in TCR diversity compared with that of adult mice. Still, although the neonatal Ag-specific repertoire comprised expectedly shorter germline-biased CDR3ß lengths, the repertoire was surprisingly complex, and only a minority of responding cells lacked random nucleotide additions. Changes with aging included increased use of the already dominant TCRVß10 family, a trend for lower content of the TCR containing the germline WG motif in the CDR3, and a remarkable sharing of one dominant clonotype between individual old mice, implying operation of selective mechanisms. Implications for the rational design of vaccines for neonates and the elderly are discussed.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Senescência Celular/imunologia , Epitopos de Linfócito T/imunologia , Evolução Molecular , Epitopos Imunodominantes/imunologia , Longevidade/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/biossíntese , Animais , Animais Recém-Nascidos , Linfócitos T CD8-Positivos/virologia , Senescência Celular/genética , Células Clonais , Epitopos de Linfócito T/genética , Herpesvirus Humano 1/imunologia , Epitopos Imunodominantes/genética , Região Variável de Imunoglobulina/biossíntese , Região Variável de Imunoglobulina/genética , Longevidade/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fragmentos de Peptídeos/biossíntese , Fragmentos de Peptídeos/genética , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Vaccinia virus/genética , Vaccinia virus/imunologia , Proteínas do Envelope Viral/biossínteseRESUMO
Different human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) vaccine vectors expressing the same viral antigens can elicit disparate T-cell responses. Within this spectrum, replicating variable vaccines, like SIVmac239Δnef, appear to generate particularly efficacious CD8(+) T-cell responses. Here, we sequenced T-cell receptor ß-chain (TRB) gene rearrangements from immunodominant Mamu-A 01-restricted Tat(28-35)SL8-specific CD8(+) T-cell populations together with the corresponding viral epitope in four rhesus macaques during acute SIVmac239Δnef infection. Ultradeep pyrosequencing showed that viral variants arose with identical kinetics in SIVmac239Δnef and pathogenic SIVmac239 infection. Furthermore, distinct Tat(28-35)SL8-specific T-cell receptor (TCR) repertoires were elicited by SIVmac239Δnef compared to those observed following a DNA/Ad5 prime-boost regimen, likely reflecting differences in antigen sequence stability.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Produtos do Gene nef/imunologia , Imunização Secundária/métodos , Vacinas contra a SAIDS/imunologia , Vírus da Imunodeficiência Símia/imunologia , Vacinação/métodos , Adenoviridae/genética , Adenovírus Humanos , Animais , Portadores de Fármacos/administração & dosagem , Vetores Genéticos , Sequenciamento de Nucleotídeos em Larga Escala , Macaca mulatta , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Vacinas contra a SAIDS/administração & dosagem , Subpopulações de Linfócitos T/imunologia , Vacinas de DNA/administração & dosagem , Vacinas de DNA/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologiaRESUMO
Despite the â¼10(18) αß T cell receptor (TCR) structures that can be randomly manufactured by the human thymus, some surface more frequently than others. The pinnacles of this distortion are public TCRs, which exhibit amino acid-identical structures across different individuals. Public TCRs are thought to result from both recombinatorial bias and antigen-driven selection, but the mechanisms that underlie inter-individual TCR sharing are still largely theoretical. To examine this phenomenon at the atomic level, we solved the co-complex structure of one of the most widespread and numerically frequent public TCRs in the human population. The archetypal AS01 public TCR recognizes an immunodominant BMLF1 peptide, derived from the ubiquitous Epstein-Barr virus, bound to HLA-A*0201. The AS01 TCR was observed to dock in a diagonal fashion, grasping the solvent exposed peptide crest with two sets of complementarity-determining region (CDR) loops, and was fastened to the peptide and HLA-A*0201 platform with residue sets found only within TCR genes biased in the public response. Computer simulations of a random V(D)J recombination process demonstrated that both TCRα and TCRß amino acid sequences could be manufactured easily, thereby explaining the prevalence of this receptor across different individuals. Interestingly, the AS01 TCR was encoded largely by germline DNA, indicating that the TCR loci already comprise gene segments that specifically recognize this ancient pathogen. Such pattern recognition receptor-like traits within the αß TCR system further blur the boundaries between the adaptive and innate immune systems.
Assuntos
Antígenos Virais/imunologia , Antígenos HLA-A/imunologia , Infecções por Herpesviridae/imunologia , Herpesvirus Humano 4/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/química , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Sequência de Aminoácidos , Linfócitos T CD8-Positivos , Simulação por Computador , Cristalização , Cristalografia por Raios X , Citotoxicidade Imunológica , Antígeno HLA-A2 , Infecções por Herpesviridae/metabolismo , Infecções por Herpesviridae/virologia , Humanos , Tolerância Imunológica , Dados de Sequência Molecular , Conformação Proteica , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Recombinação Genética , Homologia de Sequência de Aminoácidos , Ressonância de Plasmônio de SuperfícieRESUMO
A novel T-cell vaccine strategy designed to deal with the enormity of HIV-1 variation is described and tested for the first time in macaques to inform and complement approaching clinical trials. T-cell immunogen HIVconsv, which directs vaccine-induced responses to the most conserved regions of the HIV-1, proteome and thus both targets diverse clades in the population and reduces the chance of escape in infected individuals, was delivered using six different vaccine modalities: plasmid DNA (D), attenuated human (A) and chimpanzee (C) adenoviruses, modified vaccinia virus Ankara (M), synthetic long peptides, and Semliki Forest virus replicons. We confirmed that the initial DDDAM regimen, which mimics one of the clinical schedules (DDDCM), is highly immunogenic in macaques. Furthermore, adjuvanted synthetic long peptides divided into sub-pools and delivered into anatomically separate sites induced T-cell responses that were markedly broader than those elicited by traditional single-open-reading-frame genetic vaccines and increased by 30% the overall response magnitude compared with DDDAM. Thus, by improving both the HIV-1-derived immunogen and vector regimen/delivery, this approach could induce stronger, broader, and theoretically more protective T-cell responses than vaccines previously used in humans.
Assuntos
Vacinas contra a AIDS , Antígenos HIV/administração & dosagem , HIV-1/imunologia , Fragmentos de Peptídeos/administração & dosagem , Linfócitos T/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Sequência Conservada/genética , Sistemas de Liberação de Medicamentos , Mapeamento de Epitopos/métodos , Epitopos de Linfócito T/genética , Vetores Genéticos , Antígenos HIV/genética , Humanos , Imunização , Ativação Linfocitária/efeitos dos fármacos , Macaca mulatta , Fragmentos de Peptídeos/genética , Biblioteca de Peptídeos , Especificidade do Receptor de Antígeno de Linfócitos T/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/patologiaRESUMO
The diversity of the pathogen-specific T cell repertoire is believed to be important in allowing recognition of different pathogen epitopes and their variants and thereby reducing the opportunities for mutation-driven pathogen escape. However, the extent to which the TCR repertoire can be manipulated by different vaccine strategies so as to obtain broad diversity and optimal protection is incompletely understood. We have investigated the influence of the infectious/inflammatory context on the TCR diversity of the CD8(+) T cell response specific for the immunodominant epitope in C57BL/6 mice, derived from glycoprotein B of HSV-1. To that effect, we compared TCR V segment utilization, CDR3 length, and sequence diversity of the response to natural HSV-1 infection with those elicited by either Listeria monocytogenes or vaccinia virus expressing the immunodominant epitope in C57BL/6 mice. We demonstrate that although the type of infection in which the epitope was encountered can influence the magnitude of the CD8(+) T cell responses, TCR beta-chain repertoires did not significantly differ among the three infections. These results suggest that widely different live vaccine vectors may have little impact upon the diversity of the induced CTL response, which has important implications for the design of live CTL vaccine strategies against acute and chronic infections.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Epitopos Imunodominantes/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/biossíntese , Sequência de Aminoácidos , Animais , Linfócitos T CD8-Positivos/microbiologia , Linfócitos T CD8-Positivos/virologia , Sistemas de Liberação de Medicamentos , Vacinas contra o Vírus do Herpes Simples/administração & dosagem , Vacinas contra o Vírus do Herpes Simples/imunologia , Herpesvirus Humano 1/imunologia , Epitopos Imunodominantes/metabolismo , Listeria monocytogenes/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Vacínia/imunologia , Vacínia/metabolismo , Vacínia/virologia , Vaccinia virus/imunologia , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/metabolismoRESUMO
Despite the pressing need for an AIDS vaccine, the determinants of protective immunity to HIV remain concealed within the complexity of adaptive immune responses. We dissected immunodominant virus-specific CD8(+) T cell populations in Mamu-A*01(+) rhesus macaques with primary SIV infection to elucidate the hallmarks of effective immunity at the level of individual constituent clonotypes, which were identified according to the expression of distinct T cell receptors (TCRs). The number of public clonotypes, defined as those that expressed identical TCR beta-chain amino acid sequences and recurred in multiple individuals, contained within the acute phase CD8(+) T cell population specific for the biologically constrained Gag CM9 (CTPYDINQM; residues 181-189) epitope correlated negatively with the virus load set point. This independent molecular signature of protection was confirmed in a prospective vaccine trial, in which clonotype engagement was governed by the nature of the antigen rather than the context of exposure and public clonotype usage was associated with enhanced recognition of epitope variants. Thus, the pattern of antigen-specific clonotype recruitment within a protective CD8(+) T cell population is a prognostic indicator of vaccine efficacy and biological outcome in an AIDS virus infection.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Produtos do Gene gag/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Animais , Produtos do Gene gag/genética , Variação Genética , Imunização , Macaca mulatta , Fenótipo , Vírus da Imunodeficiência Símia , Linfócitos T/imunologia , Vacinação/veterinária , Vacinas Virais/imunologiaRESUMO
The CD8(+) TCR repertoires specific for many immunogenic epitopes of CMV and EBV are dominated by a few TCR clonotypes and involve public TCRs that are shared between many MHC-matched individuals. In previous studies, we demonstrated that the observed sharing of epitope-specific TCRbeta chains between individuals is strongly associated with TCRbeta production frequency, and that a process of convergent recombination facilitates the more efficient production of some TCRbeta sequences. In this study, we analyzed a total of 2836 TCRbeta sequences from 23 CMV-infected and 10 EBV-infected individuals to investigate the factors that influence the sharing of TCRbeta sequences in the CD8(+) T cell responses to two immunodominant HLA-A*0201-restricted epitopes from these viruses. The most shared TCRbeta amino acid sequences were found to have two features that indicate efficient TCRbeta production, as follows: 1) they required fewer nucleotide additions, and 2) they were encoded by a greater variety of nucleotide sequences. We used simulations of random V(D)J recombination to demonstrate that the in silico TCRbeta production frequency was predictive of the extent to which both TCRbeta nucleotide and amino acid sequences were shared in vivo. These results suggest that TCRbeta production frequency plays an important role in the interindividual sharing of TCRbeta sequences within CD8(+) T cell responses specific for CMV and EBV.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Infecções por Citomegalovirus/imunologia , Citomegalovirus/imunologia , Epitopos de Linfócito T/imunologia , Infecções por Vírus Epstein-Barr/imunologia , Rearranjo Gênico do Linfócito T/imunologia , Genes Codificadores da Cadeia beta de Receptores de Linfócitos T/genética , Antígenos HLA-A/genética , Herpesvirus Humano 4/imunologia , Infecções por Citomegalovirus/genética , Epitopos de Linfócito T/genética , Infecções por Vírus Epstein-Barr/genética , Rearranjo Gênico do Linfócito T/genética , Genes Codificadores da Cadeia beta de Receptores de Linfócitos T/imunologia , Antígenos HLA-A/imunologia , Antígeno HLA-A2 , HumanosRESUMO
Virus-immune CD8(+) TCR repertoires specific for particular peptide-MHC class I complexes may be substantially shared between (public), or unique to, individuals (private). Because public TCRs can show reduced TdT-mediated N-region additions, we analyzed how TdT shapes the heavily public (to D(b)NP(366)) and essentially private (to D(b)PA(224)) CTL repertoires generated following influenza A virus infection of C57BL/6 (B6, H2(b)) mice. The D(b)NP(366)-specific CTL response was virtually clonal in TdT(-/-) B6 animals, with one of the three public clonotypes prominent in the wild-type (wt) response consistently dominating the TdT(-/-) set. Furthermore, this massive narrowing of TCR selection for D(b)NP(366) reduced the magnitude of D(b)NP(366)-specific CTL response in the virus-infected lung. Conversely, the D(b)PA(224)-specific responses remained comparable in both magnitude and TCR diversity within individual TdT(-/-) and wt mice. However, the extent of TCR diversity across the total population was significantly reduced, with the consequence that the normally private wt D(b)PA(224)-specific repertoire was now substantially public across the TdT(-/-) mouse population. The key finding is thus that the role of TdT in ensuring enhanced diversity and the selection of private TCR repertoires promotes optimal CD8(+) T cell immunity, both within individuals and across the species as a whole.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Citotoxicidade Imunológica , DNA Nucleotidilexotransferase/fisiologia , Epitopos de Linfócito T/imunologia , Rearranjo Gênico da Cadeia beta dos Receptores de Antígenos dos Linfócitos T , Vírus da Influenza A Subtipo H3N2/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/biossíntese , Animais , Linfócitos T CD8-Positivos/enzimologia , Linfócitos T CD8-Positivos/virologia , Células Clonais , Citotoxicidade Imunológica/genética , DNA Nucleotidilexotransferase/deficiência , DNA Nucleotidilexotransferase/genética , Epitopos de Linfócito T/biossíntese , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infecções por Orthomyxoviridae/enzimologia , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Linfócitos T Citotóxicos/enzimologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/virologiaRESUMO
T-cell receptors (TCRs) govern the specificity, efficacy, and cross-reactivity of CD8 T cells. Here, we studied CD8 T-cell clonotypes from Mane-A*10(+) pigtail macaques responding to the simian immunodeficiency virus (SIV) Gag KP9 epitope in a setting of vaccination and subsequent viral challenge. We observed a diverse TCR repertoire after DNA, recombinant poxvirus, and live attenuated virus vaccination, with none of 59 vaccine-induced KP9-specific TCRs being identical between macaques. The KP9-specific TCR repertoires remained diverse after SIV or simian-human immunodeficiency virus challenge but, remarkably, exhibited substantially different clonotypic compositions compared to the corresponding populations prechallenge. Within serial samples from individual pigtail macaques, only a small subset (33.9%) of TCRs induced by vaccination were maintained or expanded after challenge. Most (66.1%) of the TCRs induced by vaccination were not detectable after challenge. Our results suggest that some CD8 T cells induced by vaccination are more efficient than others at responding to a viral challenge. These findings have implications for future AIDS virus vaccine studies, which should consider the "fitness" of vaccine-induced T cells in order to generate robust responses in the face of virus exposure.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Vacinas contra a SAIDS/imunologia , Vírus da Imunodeficiência Símia/imunologia , Animais , Epitopos/imunologia , Frequência do Gene , Produtos do Gene gag/imunologia , Macaca nemestrina , Dados de Sequência Molecular , Análise de Sequência de DNARESUMO
Recent advances in the field of molecular clonotype analysis have enabled detailed repertoire characterization of viably isolated antigen-specific T cell populations directly ex vivo. However, in the absence of a biologically contained FACS facility, peripheral blood mononuclear cell (PBMC) preparations derived from patients infected with agents such as HIV must be formaldehyde fixed to inactivate the pathogen; this procedure adversely affects nucleic acid template quality. Here, we developed and validated a method to amplify and sequence mRNA species derived from formaldehyde fixed PBMC specimens. Antigen-specific CD8+ cytotoxic T-lymphocyte populations were identified with standard fluorochrome-conjugated peptide-major histocompatibility complex class I tetramers refolded around synthetic peptides representing immunodominant epitopes from HIV p24 Gag (KRWII[M/L]GLNK/HLA B*2705) and CMV pp65 (NLVPMVATV/HLA A*0201 and TPRVTGGGAM/HLA B*0702), and acquired in separate laboratories with or without fixation. In the presence of proteinase K pre-treatment, the observed antigen-specific CD8+ T-cell repertoire determined by molecular clonotype analysis was statistically no different whether derived from fixed or unfixed PBMC. However, oligo-dT recovery methods were not suitable for use with fixed tissue as significant skewing of clonotypic representation was observed. Thus, we have developed a reliable RNA-based method for molecular clonotype analysis that is compatible with formaldehyde fixation and therefore suitable for use with primary human samples isolated by FACS outside the context of a biological safety level 3 containment facility.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Fixadores , Formaldeído , Genes Codificadores da Cadeia beta de Receptores de Linfócitos T , Imunofenotipagem , RNA/análise , Animais , Linfócitos T CD8-Positivos/virologia , Separação Celular , Células Cultivadas , Células Clonais , Regiões Determinantes de Complementaridade/genética , Citometria de Fluxo , HIV-1/imunologia , HIV-1/metabolismo , Células HeLa , Humanos , Camundongos , RNA/sangue , RNA/metabolismo , Inativação de VírusRESUMO
Analysis of T cell receptor (TCR) data has become a crucial element in many studies aimed at better understanding the evolution of the T cell repertoire and the role of TCR diversity in immune responses. In this paper we focus on comparing the diversity between samples of the TCR repertoire. We discuss some of the limitations and potential problems inherent in some of the more popular approaches to comparing samples of the TCR repertoire and we suggest alternate methods that both avoid these problems and enrich the analysis of TCR data. Examples from published studies of the CD8(+) T cell responses to the influenza A virus D(b)NP(366) and D(b)PA(224) epitopes in mice are used to demonstrate the implementation of these methods. One example involves a comparison between the central and effector memory T cell subsets, defined on the basis of CD62L expression, and the other examines changes in the TCR repertoire over time.
Assuntos
Variação Antigênica , Memória Imunológica , Receptores de Antígenos de Linfócitos T/análise , Projetos de Pesquisa , Subpopulações de Linfócitos T/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Regiões Determinantes de Complementaridade/análise , Interpretação Estatística de Dados , Vírus da Influenza A/imunologia , Selectina L/análise , Camundongos , Camundongos Endogâmicos C57BL , Modelos Estatísticos , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/virologia , Fragmentos de Peptídeos/imunologia , Tamanho da Amostra , Fatores de Tempo , Proteínas do Core Viral/imunologiaRESUMO
Many current-generation human immunodeficiency virus (HIV) vaccines induce specific T cells to control acute viremia, but their utility following infection with escape mutant virus is unclear. We studied reversion to wild type of an escape mutant simian-HIV in major histocompatibility complex-matched vaccinated pigtail macaques. High levels of vaccine-induced CD8+ T cells strongly correlated with maintenance of escape mutant virus during acute infection. Interestingly, in animals with lower CD8+ T-cell levels, transient reversion to wild-type virus resulted in better postacute control of viremia. Killing of wild-type virus facilitated by transient reversion outweighs the benefit of a larger CD8+ T-cell response that only maintains the less fit escape mutant virus. These findings have important implications for the further development of T-cell-based HIV vaccines where exposure to escape mutant viruses is common.