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1.
Front Immunol ; 13: 867962, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35432373

RESUMO

Antigen-specific TRM persist and protect against skin or female reproductive tract (FRT) HSV infection. As the pathogenesis of HSV differs between humans and model organisms, we focus on humans with well-characterized recurrent genital HSV-2 infection. Human CD8+ TRM persisting at sites of healed human HSV-2 lesions have an activated phenotype but it is unclear if TRM can be cultivated in vitro. We recovered HSV-specific TRM from genital skin and ectocervix biopsies, obtained after recovery from recurrent genital HSV-2, using ex vivo activation by viral antigen. Up to several percent of local T cells were HSV-reactive ex vivo. CD4 and CD8 T cell lines were up to 50% HSV-2-specific after sorting-based enrichment. CD8 TRM displayed HLA-restricted reactivity to specific HSV-2 peptides with high functional avidities. Reactivity to defined peptides persisted locally over several month and was quite subject-specific. CD4 TRM derived from biopsies, and from an extended set of cervical cytobrush specimens, also recognized diverse HSV-2 antigens and peptides. Overall we found that HSV-2-specific TRM are abundant in the FRT between episodes of recurrent genital herpes and maintain competency for expansion. Mucosal sites are accessible for clinical monitoring during immune interventions such as therapeutic vaccination.


Assuntos
Herpes Genital , Herpes Simples , Antígenos Virais , Feminino , Herpesvirus Humano 2 , Humanos , Memória Imunológica , Masculino , Peptídeos
2.
JCI Insight ; 2022 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-35439166

RESUMO

BACKGROUND: Measuring the immune response to SARS-CoV-2 enables assessment of past infection and protective immunity. SARS-CoV-2 infection induces humoral and T-cell responses, but these responses vary with disease severity and individual characteristics. METHODS: A T-cell receptor (TCR) immunosequencing assay was conducted using small-volume blood samples from 302 individuals recovered from COVID-19. Correlations between the magnitude of the T-cell response and neutralizing antibody (nAb) titers or indicators of disease severity were evaluated. Sensitivity of T-cell testing was assessed and compared to serologic testing. RESULTS: SARS-CoV-2-specific T-cell responses were significantly correlated with nAb titers and clinical indicators of disease severity, including hospitalization, fever, and difficulty breathing. Despite modest declines in depth and breadth of T-cell responses during convalescence, high sensitivity was observed until at least 6 months after infection, with overall sensitivity ~5% greater than serology tests for identifying prior SARS-CoV-2 infection. Improved performance of T-cell testing was most apparent in recovered, non-hospitalized individuals sampled >150 days after initial illness, suggesting greater sensitivity than serology at later timepoints and in individuals with less severe disease. T-cell testing identified SARS-CoV-2 infection in 68% (55/81) of samples with undetectable nAb titers (<1:40) and in 37% (13/35) of samples negative by 3 antibody assays. CONCLUSION: These results support TCR-based testing as a scalable, reliable measure of past SARS-CoV-2 infection with clinical value beyond serology. FUNDING: Adaptive Biotechnologies, Frederick National Laboratory for Cancer Research, National Institutes of Allergy and Infectious Diseases, Fred Hutchinson Joel Meyers Endowment; Fast Grants, American Society for Transplantation and Cell Therapy.

3.
JCI Insight ; 7(6)2022 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-35133988

RESUMO

SARS-CoV-2 provokes a robust T cell response. Peptide-based studies exclude antigen processing and presentation biology, which may influence T cell detection studies. To focus on responses to whole virus and complex antigens, we used intact SARS-CoV-2 and full-length proteins with DCs to activate CD8 and CD4 T cells from convalescent people. T cell receptor (TCR) sequencing showed partial repertoire preservation after expansion. Resultant CD8 T cells recognize SARS-CoV-2-infected respiratory tract cells, and CD4 T cells detect inactivated whole viral antigen. Specificity scans with proteome-covering protein/peptide arrays show that CD8 T cells are oligospecific per subject and that CD4 T cell breadth is higher. Some CD4 T cell lines enriched using SARS-CoV-2 cross-recognize whole seasonal coronavirus (sCoV) antigens, with protein, peptide, and HLA restriction validation. Conversely, recognition of some epitopes is eliminated for SARS-CoV-2 variants, including spike (S) epitopes in the Alpha, Beta, Gamma, and Delta variant lineages.

4.
medRxiv ; 2022 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-35118477

RESUMO

SARS-CoV-2 provokes a brisk T cell response. Peptide-based studies exclude antigen processing and presentation biology and may influence T cell detection studies. To focus on responses to whole virus and complex antigens, we used intact SARS-CoV-2 and full-length proteins with DC to activate CD8 and CD4 T cells from convalescent persons. T cell receptor (TCR) sequencing showed partial repertoire preservation after expansion. Resultant CD8 T cells recognize SARS-CoV-2-infected respiratory cells, and CD4 T cells detect inactivated whole viral antigen. Specificity scans with proteome-covering protein/peptide arrays show that CD8 T cells are oligospecific per subject and that CD4 T cell breadth is higher. Some CD4 T cell lines enriched using SARS-CoV-2 cross-recognize whole seasonal coronavirus (sCoV) antigens, with protein, peptide, and HLA restriction validation. Conversely, recognition of some epitopes is eliminated for SARS-CoV-2 variants, including spike (S) epitopes in the alpha, beta, gamma, and delta variant lineages.

5.
Nat Commun ; 13(1): 78, 2022 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-35013257

RESUMO

T cells recognize mycobacterial glycolipid (mycolipid) antigens presented by CD1b molecules, but the role of CD4 and CD8 co-receptors in mycolipid recognition is unknown. Here we show CD1b-mycolipid tetramers reveal a hierarchy in which circulating T cells expressing CD4 or CD8 co-receptor stain with a higher tetramer mean fluorescence intensity than CD4-CD8- T cells. CD4+ primary T cells transduced with mycolipid-specific T cell receptors bind CD1b-mycolipid tetramer with a higher fluorescence intensity than CD8+ primary T cells. The presence of either CD4 or CD8 also decreases the threshold for interferon-γ secretion. Co-receptor expression increases surface expression of CD3ε, suggesting a mechanism for increased tetramer binding and activation. Targeted transcriptional profiling of mycolipid-specific T cells from individuals with active tuberculosis reveals canonical markers associated with cytotoxicity among CD8+ compared to CD4+ T cells. Thus, expression of co-receptors modulates T cell receptor avidity for mycobacterial lipids, leading to in vivo functional diversity during tuberculosis disease.


Assuntos
Antígenos CD1/imunologia , Glicolipídeos/imunologia , Mycobacterium tuberculosis/imunologia , Tuberculose/imunologia , Antígenos CD1/genética , Complexo CD3/genética , Complexo CD3/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/microbiologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/microbiologia , Citotoxicidade Imunológica , Expressão Gênica , Glicolipídeos/metabolismo , Humanos , Interferon gama/genética , Interferon gama/imunologia , Ativação Linfocitária , Mycobacterium tuberculosis/crescimento & desenvolvimento , Cultura Primária de Células , Ligação Proteica , Multimerização Proteica , Transdução Genética , Tuberculose/genética , Tuberculose/microbiologia
6.
Commun Biol ; 5(1): 133, 2022 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-35173258

RESUMO

Pre-existing pathogen-specific memory T cell responses can contribute to multiple adverse outcomes including autoimmunity and drug hypersensitivity. How the specificity of the T cell receptor (TCR) is subverted or seconded in many of these diseases remains unclear. Here, we apply abacavir hypersensitivity (AHS) as a model to address this question because the disease is linked to memory T cell responses and the HLA risk allele, HLA-B*57:01, and the initiating insult, abacavir, are known. To investigate the role of pathogen-specific TCR specificity in mediating AHS we performed a genome-wide screen for HLA-B*57:01 restricted T cell responses to Epstein-Barr virus (EBV), one of the most prevalent human pathogens. T cell epitope mapping revealed HLA-B*57:01 restricted responses to 17 EBV open reading frames and identified an epitope encoded by EBNA3C. Using these data, we cloned the dominant TCR for EBNA3C and a previously defined epitope within EBNA3B. TCR specificity to each epitope was confirmed, however, cloned TCRs did not cross-react with abacavir plus self-peptide. Nevertheless, abacavir inhibited TCR interactions with their cognate ligands, demonstrating that TCR specificity may be subverted by a drug molecule. These results provide an experimental road map for future studies addressing the heterologous immune responses of TCRs including T cell mediated adverse drug reactions.


Assuntos
Infecções por Vírus Epstein-Barr , Herpesvirus Humano 4 , Didesoxinucleosídeos , Epitopos de Linfócito T , Antígenos HLA-B , Herpesvirus Humano 4/genética , Humanos , Receptores de Antígenos de Linfócitos T/genética , Receptores de Complemento 3d
7.
Front Immunol ; 12: 735643, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34552595

RESUMO

Tissue-resident-memory T cells (TRM) populate the body's barrier surfaces, functioning as frontline responders against reencountered pathogens. Understanding of the mechanisms by which CD8TRM achieve effective immune protection remains incomplete in a naturally recurring human disease. Using laser capture microdissection and transcriptional profiling, we investigate the impact of CD8TRM on the tissue microenvironment in skin biopsies sequentially obtained from a clinical cohort of diverse disease expression during herpes simplex virus 2 (HSV-2) reactivation. Epithelial cells neighboring CD8TRM display elevated and widespread innate and cell-intrinsic antiviral signature expression, largely related to IFNG expression. Detailed evaluation via T-cell receptor reconstruction confirms that CD8TRM recognize viral-infected cells at the specific HSV-2 peptide/HLA level. The hierarchical pattern of core IFN-γ signature expression is well-conserved in normal human skin across various anatomic sites, while elevation of IFI16, TRIM 22, IFITM2, IFITM3, MX1, MX2, STAT1, IRF7, ISG15, IFI44, CXCL10 and CCL5 expression is associated with HSV-2-affected asymptomatic tissue. In primary human cells, IFN-γ pretreatment reduces gene transcription at the immediate-early stage of virus lifecycle, enhances IFI16 restriction of wild-type HSV-2 replication and renders favorable kinetics for host protection. Thus, the adaptive immune response through antigen-specific recognition instructs innate and cell-intrinsic antiviral machinery to control herpes reactivation, a reversal of the canonical thinking of innate activating adaptive immunity in primary infection. Communication from CD8TRM to surrounding epithelial cells to activate broad innate resistance might be critical in restraining various viral diseases.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Células Epiteliais/imunologia , Herpes Genital/imunologia , Herpesvirus Humano 2/imunologia , Imunidade Inata , Memória Imunológica , Pele/imunologia , Imunidade Adaptativa/genética , Adulto , Idoso , Antígenos Virais/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/virologia , Células Cultivadas , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Feminino , Perfilação da Expressão Gênica , Herpes Genital/genética , Herpes Genital/metabolismo , Herpes Genital/virologia , Herpesvirus Humano 2/patogenicidade , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata/genética , Interferon gama/genética , Interferon gama/metabolismo , Masculino , /virologia , Pessoa de Meia-Idade , Fenótipo , Pele/metabolismo , Pele/virologia , Transcriptoma
8.
J Clin Invest ; 131(3)2021 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-33320842

RESUMO

BACKGROUNDSARS-CoV-2-specific antibodies may protect from reinfection and disease, providing rationale for administration of plasma containing SARS-CoV-2-neutralizing antibodies (nAbs) as a treatment for COVID-19. Clinical factors and laboratory assays to streamline plasma donor selection, and the durability of nAb responses, are incompletely understood.METHODSPotential convalescent plasma donors with virologically documented SARS-CoV-2 infection were tested for serum IgG against SARS-CoV-2 spike protein S1 domain and against nucleoprotein (NP), and for nAb.RESULTSAmong 250 consecutive persons, including 27 (11%) requiring hospitalization, who were studied a median of 67 days since symptom onset, 97% were seropositive on 1 or more assays. Sixty percent of donors had nAb titers ≥1:80. Correlates of higher nAb titers included older age (adjusted OR [AOR] 1.03 per year of age, 95% CI 1.00-1.06), male sex (AOR 2.08, 95% CI 1.13-3.82), fever during illness (AOR 2.73, 95% CI 1.25-5.97), and disease severity represented by hospitalization (AOR 6.59, 95% CI 1.32-32.96). Receiver operating characteristic analyses of anti-S1 and anti-NP antibody results yielded cutoffs that corresponded well with nAb titers, with the anti-S1 assay being slightly more predictive. nAb titers declined in 37 of 41 paired specimens collected a median of 98 days (range 77-120) apart (P < 0.001). Seven individuals (2.8%) were persistently seronegative and lacked T cell responses.CONCLUSIONnAb titers correlated with COVID-19 severity, age, and sex. SARS-CoV-2 IgG results can serve as useful surrogates for nAb testing. Functional nAb levels declined, and a small proportion of convalescent individuals lacked adaptive immune responses.FUNDINGThe project was supported by the Frederick National Laboratory for Cancer Research with support from the NIAID under contract number 75N91019D00024, and was supported by the Fred Hutchinson Joel Meyers Endowment, Fast-Grants, a New Investigator award from the American Society for Transplantation and Cellular Therapy, and NIH contracts 75N93019C0063, 75N91019D00024, and HHSN272201800013C, and NIH grants T32-AI118690, T32-AI007044, K08-AI119142, and K23-AI140918.


Assuntos
Anticorpos Neutralizantes , Anticorpos Antivirais , Doadores de Sangue , COVID-19/terapia , Imunoglobulina G , SARS-CoV-2 , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , COVID-19/sangue , COVID-19/imunologia , Feminino , Humanos , Imunização Passiva , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Masculino , Pessoa de Meia-Idade , SARS-CoV-2/imunologia , SARS-CoV-2/metabolismo
9.
medRxiv ; 2020 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-33052361

RESUMO

BACKGROUND: SARS-CoV-2-specific antibodies may protect from reinfection and disease, providing the rationale for administration of plasma containing SARS-CoV-2 neutralizing antibodies (nAb) as a treatment for COVID-19. The clinical factors and laboratory assays to streamline plasma donor selection, and the durability of nAb responses, are incompletely understood. METHODS: Adults with virologically-documented SARS-CoV-2 infection in a convalescent plasma donor screening program were tested for serum IgG to SARS-CoV-2 spike protein S1 domain, nucleoprotein (NP), and for nAb. RESULTS: Amongst 250 consecutive persons studied a median of 67 days since symptom onset, 243/250 (97%) were seropositive on one or more assays. Sixty percent of donors had nAb titers ≥1:80. Correlates of higher nAb titer included older age (adjusted OR [AOR] 1.03/year of age, 95% CI 1.00-1.06), male sex (AOR 2.08, 95% CI 1.13-3.82), fever during acute illness (AOR 2.73, 95% CI 1.25-5.97), and disease severity represented by hospitalization (AOR 6.59, 95% CI 1.32-32.96). Receiver operating characteristic (ROC) analyses of anti-S1 and anti-NP antibody results yielded cutoffs that corresponded well with nAb titers, with the anti-S1 assay being slightly more predictive. NAb titers declined in 37 of 41 paired specimens collected a median of 98 days (range, 77-120) apart (P<0.001). Seven individuals (2.8%) were persistently seronegative and lacked T cell responses. CONCLUSIONS: Nab titers correlated with COVID-19 severity, age, and sex. Standard commercially available SARS-CoV-2 IgG results can serve as useful surrogates for nAb testing. Functional nAb levels were found to decline and a small proportion of COVID-19 survivors lack adaptive immune responses.

10.
Cancer Immunol Res ; 8(5): 648-659, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32179557

RESUMO

Merkel cell carcinoma (MCC) is often caused by persistent expression of Merkel cell polyomavirus (MCPyV) T-antigen (T-Ag). These non-self proteins comprise about 400 amino acids (AA). Clinical responses to immune checkpoint inhibitors, seen in about half of patients, may relate to T-Ag-specific T cells. Strategies to increase CD8+ T-cell number, breadth, or function could augment checkpoint inhibition, but vaccines to augment immunity must avoid delivery of oncogenic T-antigen domains. We probed MCC tumor-infiltrating lymphocytes (TIL) with an artificial antigen-presenting cell (aAPC) system and confirmed T-Ag recognition with synthetic peptides, HLA-peptide tetramers, and dendritic cells (DC). TILs from 9 of 12 (75%) subjects contained CD8+ T cells recognizing 1-8 MCPyV epitopes per person. Analysis of 16 MCPyV CD8+ TIL epitopes and prior TIL data indicated that 97% of patients with MCPyV+ MCC had HLA alleles with the genetic potential that restrict CD8+ T-cell responses to MCPyV T-Ag. The LT AA 70-110 region was epitope rich, whereas the oncogenic domains of T-Ag were not commonly recognized. Specific recognition of T-Ag-expressing DCs was documented. Recovery of MCPyV oncoprotein-specific CD8+ TILs from most tumors indicated that antigen indifference was unlikely to be a major cause of checkpoint inhibition failure. The myriad of epitopes restricted by diverse HLA alleles indicates that vaccination can be a rational component of immunotherapy if tumor immune suppression can be overcome, and the oncogenic regions of T-Ag can be modified without impacting immunogenicity.


Assuntos
Antígenos Virais de Tumores/imunologia , Linfócitos T CD8-Positivos/imunologia , Carcinoma de Célula de Merkel/imunologia , Epitopos/imunologia , Linfócitos do Interstício Tumoral/imunologia , Poliomavírus das Células de Merkel/imunologia , Neoplasias Cutâneas/imunologia , Adulto , Idoso , Antígenos Virais de Tumores/metabolismo , Carcinogênese/imunologia , Carcinoma de Célula de Merkel/terapia , Feminino , Humanos , Imunoterapia/métodos , Masculino , Pessoa de Meia-Idade , Neoplasias Cutâneas/terapia , Adulto Jovem
11.
J Virol ; 94(9)2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32051273

RESUMO

Pharmacological HIV-1 reactivation to reverse latent infection has been extensively studied. However, HIV-1 reactivation also occurs naturally, as evidenced by occasional low-level viremia ("viral blips") during antiretroviral treatment (ART). Clarifying where blips originate from and how they happen could provide clues to stimulate latency reversal more effectively and safely or to prevent viral rebound following ART cessation. We studied HIV-1 reactivation in the female genital tract, a dynamic anatomical target for HIV-1 infection throughout all disease stages. We found that primary endocervical epithelial cells from several women reactivated HIV-1 from latently infected T cells. The endocervical cells' HIV-1 reactivation capacity further increased upon Toll-like receptor 3 stimulation with poly(I·C) double-stranded RNA or infection with herpes simplex virus 2 (HSV-2). Notably, acyclovir did not eliminate HSV-2-induced HIV-1 reactivation. While endocervical epithelial cells secreted large amounts of several cytokines and chemokines, especially tumor necrosis factor alpha (TNF-α), CCL3, CCL4, and CCL20, their HIV-1 reactivation capacity was almost completely blocked by TNF-α neutralization alone. Thus, immunosurveillance activities by columnar epithelial cells in the endocervix can cause endogenous HIV-1 reactivation, which may contribute to viral blips during ART or rebound following ART interruption.IMPORTANCE A reason that there is no universal cure for HIV-1 is that the virus can hide in the genome of infected cells in the form of latent proviral DNA. This hidden provirus is protected from antiviral drugs until it eventually reactivates to produce new virions. It is not well understood where in the body or how this reactivation occurs. We studied HIV-1 reactivation in the female genital tract, which is often the portal of HIV-1 entry and which remains a site of infection throughout the disease. We found that the columnar epithelial cells lining the endocervix, the lower part of the uterus, are particularly effective in reactivating HIV-1 from infected T cells. This activity was enhanced by certain microbial stimuli, including herpes simplex virus 2, and blocked by antibodies against the inflammatory cytokine TNF-α. Avoiding HIV-1 reactivation could be important for maintaining a functional HIV-1 cure when antiviral therapy is stopped.


Assuntos
HIV-1/fisiologia , Ativação Viral/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Aciclovir/farmacologia , Antirretrovirais/uso terapêutico , Antivirais/farmacologia , Linfócitos T CD4-Positivos/virologia , Linhagem Celular , Colo do Útero/patologia , Células Epiteliais/patologia , Feminino , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Infecções por HIV/virologia , Soropositividade para HIV/tratamento farmacológico , HIV-1/patogenicidade , Humanos , Cultura Primária de Células , Viremia/tratamento farmacológico , Latência Viral/efeitos dos fármacos , Replicação Viral/fisiologia
12.
J Virol ; 93(14)2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-31043533

RESUMO

Human herpesvirus 6 (HHV-6) and cytomegalovirus (CMV) are population-prevalent betaherpesviruses with intermittent lytic replication that can be pathogenic in immunocompromised hosts. Elucidation of the adaptive immune response is valuable for understanding pathogenesis and designing novel treatments. Knowledge of T-cell antigens has reached the genome-wide level for CMV and other human herpesviruses, but study of HHV-6 is at an earlier stage. Using rare-cell enrichment combined with an HLA-agnostic, proteome-wide approach, we queried HHV-6B-specific CD4 T cells from 18 healthy donors with each known HHV-6B protein. We detected a low abundance of HHV-6-specific CD4 T cells in blood; however, the within-person CD4 T-cell response is quite broad: the median number of open reading frame (ORF) products recognized was nine per person. Overall, the data expand the number of documented HHV-6B CD4 T-cell antigens from approximately 11 to 60. Epitopes in the proteins encoded by U14, U90, and U95 were mapped with synthetic peptides, and HLA restriction was defined for some responses. Intriguingly, CD4 T-cell antigens newly described in this report are among the most population prevalent, including U73, U72, U95, and U30. Our results indicate that selection of HHV-6B ORFs for immunotherapy should consider this expanded panel of HHV-6B antigens.IMPORTANCE Human herpesvirus 6 is highly prevalent and maintains chronic infection in immunocompetent individuals, with the potential to replicate widely in settings of immunosuppression, leading to clinical disease. Antiviral compounds may be ineffective and/or pose dose-limiting toxicity, and therefore, immune-based therapies have garnered increased interest in recent years. Attempts at addressing this unmet medical need begin with understanding the cellular response to HHV-6 at the individual and population levels. The present study provides a comprehensive assessment of HHV-6-specific T-cell responses that may inform the development of cell-based therapies directed at this virus.


Assuntos
Antígenos Virais/imunologia , Linfócitos T CD4-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Herpesvirus Humano 6/imunologia , Fases de Leitura Aberta/imunologia , Infecções por Roseolovirus/imunologia , Antígenos Virais/genética , Linhagem Celular , Mapeamento de Epitopos , Epitopos de Linfócito T/genética , Estudo de Associação Genômica Ampla , Herpesvirus Humano 6/genética , Humanos , Infecções por Roseolovirus/genética
13.
Immunogenetics ; 71(7): 465-478, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31123763

RESUMO

Invariant NKT (iNKT) cells in both humans and non-human primates are activated by the glycolipid antigen, α-galactosylceramide (α-GalCer). However, the extent to which the molecular mechanisms of antigen recognition and in vivo phenotypes of iNKT cells are conserved among primate species has not been determined. Using an evolutionary genetic approach, we found a lack of diversifying selection in CD1 genes over 45 million years of evolution, which stands in stark contrast to the history of the MHC system for presenting peptide antigens to T cells. The invariant T cell receptor (TCR)-α chain was strictly conserved across all seven primate clades. Invariant NKT cells from rhesus macaques (Macaca mulatta) bind human CD1D-α-GalCer tetramer and are activated by α-GalCer-loaded human CD1D transfectants. The dominant TCR-ß chain cloned from a rhesus-derived iNKT cell line is nearly identical to that found in the human iNKT TCR, and transduction of the rhesus iNKT TCR into human Jurkat cells show that it is sufficient for binding human CD1D-α-GalCer tetramer. Finally, we used a 20-color flow cytometry panel to probe tissue phenotypes of iNKT cells in a cohort of rhesus macaques. We discovered several tissue-resident iNKT populations that have not been previously described in non-human primates but are known in humans, such as TCR-γδ iNKTs. These data reveal a diversity of iNKT cell phenotypes despite convergent evolution of the genes required for lipid antigen presentation and recognition in humans and non-human primates.


Assuntos
Antígenos CD1/genética , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Primatas/genética , Sequência de Aminoácidos , Animais , Antígenos CD1/metabolismo , Sequência Conservada , Evolução Molecular , Feminino , Humanos , Células Jurkat , Macaca mulatta/imunologia , Masculino , Fenótipo , Primatas/imunologia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo
14.
PLoS Pathog ; 15(3): e1007650, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30870532

RESUMO

Varicella zoster virus (VZV) is a lymphotropic alpha-herpesvirinae subfamily member that produces varicella on primary infection and causes zoster, vascular disease and vision loss upon reactivation from latency. VZV-infected peripheral blood mononuclear cells (PBMCs) disseminate virus to distal organs to produce clinical disease. To assess immune evasion strategies elicited by VZV that may contribute to dissemination of infection, human PBMCs and VZV-specific CD8+ T cells (V-CD8+) were mock- or VZV-infected and analyzed for immunoinhibitory protein PD-1, PD-L1, PD-L2, CTLA-4, LAG-3 and TIM-3 expression using flow cytometry. All VZV-infected PBMCs (monocytes, NK, NKT, B cells, CD4+ and CD8+ T cells) and V-CD8+ showed significant elevations in PD-L1 expression compared to uninfected cells. VZV induced PD-L2 expression in B cells and V-CD8+. Only VZV-infected CD8+ T cells, NKT cells and V-CD8+ upregulated PD-1 expression, the immunoinhibitory receptor for PD-L1/PD-L2. VZV induced CTLA-4 expression only in V-CD8+ and no significant changes in LAG-3 or TIM-3 expression were observed in V-CD8+ or PBMC T cells. To test whether PD-L1, PD-L2 or CTLA-4 regulates V-CD8+ effector function, autologous PBMCs were VZV-infected and co-cultured with V-CD8+ cells in the presence of blocking antibodies against PD-L1, PD-L2 or CTLA-4; ELISAs revealed significant elevations in IFNγ only upon blocking of PD-L1. Together, these results identified additional immune cells that are permissive to VZV infection (monocytes, B cells and NKT cells); along with a novel mechanism for inhibiting CD8+ T cell effector function through induction of PD-L1 expression.


Assuntos
Antígeno B7-H1/genética , Linfócitos T CD8-Positivos/metabolismo , Herpesvirus Humano 3/metabolismo , Anticorpos Antivirais , Antígenos CD , Antígeno B7-H1/imunologia , Antígeno B7-H1/metabolismo , Linfócitos T CD4-Positivos , Antígeno CTLA-4 , Técnicas de Cocultura , Regulação da Expressão Gênica , Receptor Celular 2 do Vírus da Hepatite A , Herpes Zoster/metabolismo , Herpes Zoster/virologia , Herpesvirus Humano 3/patogenicidade , Humanos , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/virologia , Proteína 2 Ligante de Morte Celular Programada 1 , Receptor de Morte Celular Programada 1 , Viroses
15.
J Infect Dis ; 219(7): 1058-1066, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30383234

RESUMO

BACKGROUND: Orolabial herpes simplex virus type 1 (HSV-1) infection has a wide spectrum of severity in immunocompetent persons. To study the role of viral genotype and host immunity, we characterized oral HSV-1 shedding rates and host cellular response, and genotyped viral strains, in monozygotic (MZ) and dizygotic (DZ) twins. METHODS: A total of 29 MZ and 22 DZ HSV-1-seropositive twin pairs were evaluated for oral HSV-1 shedding for 60 days. HSV-1 strains from twins were genotyped as identical or different. CD4+ T-cell responses to HSV-1 proteins were studied. RESULTS: The median per person oral HSV shedding rate was 9% of days that a swab was obtained (mean, 10.2% of days). A positive correlation between shedding rates was observed within all twin pairs, and in the MZ and DZ twins. In twin subsets with sufficient HSV-1 DNA to genotype, 15 had the same strain and 14 had different strains. Viral shedding rates were correlated for those with the same but not different strains. The median number of HSV-1 open reading frames recognized per person was 16. The agreement in the CD4+ T-cell response to specific HSV-1 open reading frames was greater between MZ twins than between unrelated persons (P = .002). CONCLUSION: Viral strain characteristics likely contribute to oral HSV-1 shedding rates.


Assuntos
Herpes Labial/imunologia , Herpes Labial/virologia , Herpesvirus Humano 1/genética , Eliminação de Partículas Virais/genética , Adulto , Idoso , Linfócitos T CD4-Positivos/imunologia , Feminino , Genótipo , Herpes Labial/classificação , Herpesvirus Humano 1/fisiologia , Humanos , Masculino , Pessoa de Meia-Idade , Boca/virologia , Fases de Leitura Aberta/genética , Fases de Leitura Aberta/imunologia , Filogenia , Gêmeos Dizigóticos , Gêmeos Monozigóticos , Adulto Jovem
16.
Front Immunol ; 9: 2811, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30619245

RESUMO

Infection and vaccination can lead to activation of autoreactive T cells, including the activation of cross-reactive T cells. However, detecting these cross-reactive T cells and identifying the non-self and self-antigen epitopes is difficult. The current study demonstrates the utility of a novel approach that effectively accomplishes both. We utilized surface expression of CD38 on newly activated CD4 memory T cells as a strategy to identify type 1 diabetes associated autoreactive T cells activated by influenza vaccination in healthy subjects. We identified an influenza A matrix protein (MP) specific CD4+ T cell clone that cross-recognizes an immunodominant epitope from Glutamic Acid Decarboxylase 65 (GAD65) protein. The sequences of the MP and GAD65 peptides are rather distinct, with only 2 identical amino acids within the HLA-DR binding region. This result suggests that activation of autoreactive T cells by microbial infection under certain physiological conditions can occur amongst peptides with minimum amino acid sequence homology. This novel strategy also provides a new research pathway in which to examine activation of autoreactive CD4+ T cells after vaccination or natural infection.


Assuntos
Antígenos Virais/imunologia , Linfócitos T CD4-Positivos/imunologia , Reações Cruzadas/imunologia , Epitopos de Linfócito T/imunologia , Epitopos Imunodominantes/imunologia , ADP-Ribosil Ciclase 1/imunologia , Sequência de Aminoácidos , Linfócitos T CD4-Positivos/virologia , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/virologia , Glutamato Descarboxilase/imunologia , Antígenos HLA-DR/imunologia , Humanos , Vírus da Influenza A/imunologia , Vacinas contra Influenza/imunologia , Influenza Humana/imunologia , Peptídeos/imunologia
18.
Front Microbiol ; 8: 2342, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29259582

RESUMO

Herpes simplex virus 1 and 2 (HSV-1/2) similarly initiate infection in mucosal epithelia and establish lifelong neuronal latency. Anogenital HSV-2 infection augments the risk for sexual human immunodeficiency virus (HIV) transmission and is associated with higher HIV viral loads. However, whether oral HSV-1 infection contributes to oral HIV susceptibility, viremia, or oral complications of HIV infection is unknown. Appropriate non-human primate (NHP) models would facilitate this investigation, yet there are no published studies of HSV-1/SIV co-infection in NHPs. Thus, we performed a pilot study for an oral HSV-1 infection model in SIV-infected rhesus macaques to describe the feasibility of the modeling and resultant immunological changes. Three SIV-infected, clinically healthy macaques became HSV-1-infected by inoculation with 4 × 108 pfu HSV-1 McKrae on buccal, tongue, gingiva, and tonsils after gentle abrasion. HSV-1 DNA was shed in oral swabs for up to 21 days, and shedding recurred in association with intra-oral lesions after periods of no shedding during 56 days of follow up. HSV-1 DNA was detected in explant cultures of trigeminal ganglia collected at euthanasia on day 56. In the macaque with lowest baseline SIV viremia, SIV plasma RNA increased following HSV-1 infection. One macaque exhibited an acute pro-inflammatory response, and all three animals experienced T cell activation and mobilization in blood. However, T cell and antibody responses to HSV-1 were low and atypical. Through rigorous assessesments, this study finds that the virulent HSV-1 strain McKrae resulted in a low level HSV-1 infection that elicited modest immune responses and transiently modulated SIV infection.

19.
J Virol ; 91(19)2017 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-28701399

RESUMO

Herpes simplex virus (HSV) infection is restricted to epithelial cells and neurons and is controlled by CD8 T cells. These cells both traffic to epithelial sites of recurrent lytic infection and to ganglia and persist at the dermal-epidermal junction for up to 12 weeks after lesion resolution. We previously showed that cutaneous lymphocyte-associated antigen (CLA), a functional E-selectin ligand (ESL), is selectively expressed on circulating HSV-2-specific CD8 T cells. CLA/ESL mediates adhesion of T cells to inflamed vascular endothelium. Later stages in T-cell homing involve chemokines (Ch) and lymphocyte chemokine receptors (ChR) for vascular wall arrest and diapedesis. Several candidate ChR have been implicated in skin homing. We measured cell surface ChR on HSV-specific human peripheral blood CD8 T cells and extended our studies to HSV-1. We observed preferential cell surface expression of CCR10 and CXCR3 by HSV-specific CD8 T cells compared to CD8 T cells specific for control viruses, Epstein-Barr virus (EBV) and cytomegalovirus (CMV), and compared to bulk memory CD8 T cells. CXCR3 ligand mRNA levels were selectively increased in skin biopsy specimens from persons with recurrent HSV-2, while the mRNA levels of the CCR10 ligand CCL27 were equivalent in lesion and control skin. Our data are consistent with a model in which CCL27 drives baseline recruitment of HSV-specific CD8 T cells expressing CCR10, while interferon-responsive CXCR3 ligands recruit additional cells in response to virus-driven inflammation.IMPORTANCE HSV-2 causes very localized recurrent infections in the skin and genital mucosa. Virus-specific CD8 T cells home to the site of recurrent infection and participate in viral clearance. The exit of T cells from the blood involves the use of chemokine receptors on the T-cell surface and chemokines that are present in infected tissue. In this study, circulating HSV-2-specific CD8 T cells were identified using specific fluorescent tetramer reagents, and their expression of several candidate skin-homing-associated chemokine receptors was measured using flow cytometry. We found that two chemokine receptors, CXCR3 and CCR10, are upregulated on HSV-specific CD8 T cells in blood. The chemokines corresponding to these receptors are also expressed in infected tissues. Vaccine strategies to prime CD8 T cells to home to HSV lesions should elicit these chemokine receptors if possible to increase the homing of vaccine-primed cells to sites of infection.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Quimiocina CCL27/imunologia , Herpes Simples/imunologia , Herpesvirus Humano 2/imunologia , Ativação Linfocitária/imunologia , Receptores CCR10/imunologia , Receptores CXCR3/imunologia , Antígenos de Diferenciação de Linfócitos T/imunologia , Linfócitos T CD8-Positivos/metabolismo , Quimiocina CCL27/genética , Citomegalovirus/imunologia , Feminino , Citometria de Fluxo , Herpes Simples/virologia , Herpesvirus Humano 4/imunologia , Humanos , Memória Imunológica/imunologia , Masculino , Glicoproteínas de Membrana/imunologia , RNA Mensageiro/genética , Receptores CCR10/biossíntese , Receptores CCR10/genética , Receptores CXCR3/biossíntese , Receptores CXCR3/genética , Pele/virologia
20.
Pediatr Infect Dis J ; 36(8): 741-744, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28471862

RESUMO

BACKGROUND: Herpes simplex encephalitis (HSE) after primary herpes simplex virus (HSV)-1 infection can occur in children due to inborn errors of cell-intrinsic immunity in the central nervous system. Paradoxically, symptomatic mucocutaneous HSV-1 recurrences are rare survivors of childhood HSE. T-cell-acquired immunity is thought to be involved in control of recurrent mucocutaneous HSV infection. We thus tested HSV-1-specific immunity in HSE survivors. METHODS: We obtained serum and peripheral blood mononuclear cells (PBMCs) from participants a median of 13.5 years after HSE. HSV-1 and HSV-2 IgG was detected by type-specific immunoblot. PBMCs from subjects passing quality control criteria were tested using enzyme-linked immunospot assay for CD4 interferon-γ responses with an HSV-1 lysate and for CD8 responses using pooled synthetic HSV-1 peptide CD8 T-cell epitopes. Healthy adult PBMCs were used to standardize assays and as comparators. RESULTS: All participants were HSV-1 seropositive. Most (23/24) HSE survivors had human leukocyte antigen class I types matching the human leukocyte antigen restriction of the pooled peptides. We detected HSV-specific CD8 T-cell responses in 14 of 24 (58%) HSE survivors and in 9 of 9 healthy HSV-1 seropositive adults. HSV-specific CD4 T-cell responses were present in all 5 HSE subjects tested and in 8 of 9 healthy adults. Response magnitudes were overlapping between subject groups. CONCLUSIONS: The defects in cell-intrinsic immunity leading to failure to control primary central nervous system HSV-1 infection do not preclude the acquisition of specific immunity or the control of recurrent mucocutaneous HSV infections. The rarity and lack of severe or recurrent mucocutaneous HSV infection in survivors of childhood HSE corresponds with intact adaptive T-cell immunity.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Encefalite por Herpes Simples/imunologia , Herpesvirus Humano 1/imunologia , Adolescente , Linfócitos T CD4-Positivos/imunologia , Criança , Pré-Escolar , Estudos de Coortes , ELISPOT , Epitopos/imunologia , Humanos , Lactente , Proteínas Virais/imunologia
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