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T-cell responses to KSHV infection: a systematic approach

Prior studies of T-cell responses to KSHV have included relatively few participants and focused on relatively few KSHV antigens. To provide a more comprehensive analysis, we investigated T-cell responses to the whole KSHV proteome using IFN-γ ELISpot. Using ∼7,500 overlapping 15mer peptides we gener...

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Autores principales: Roshan, Romin, Labo, Nazzarena, Trivett, Matthew, Miley, Wendell, Marshall, Vickie, Coren, Lori, Cornejo Castro, Elena M., Perez, Hannah, Holdridge, Benjamin, Davis, Eliza, Matus-Nicodemos, Rodrigo, Ayala, Victor I., Sowder, Raymond, Wyvill, Kathleen M., Aleman, Karen, Fennessey, Christine, Lifson, Jeffrey, Polizzotto, Mark N., Douek, Daniel, Keele, Brandon, Uldrick, Thomas S., Yarchoan, Robert, Ohlen, Claes, Ott, David, Whitby, Denise
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Impact Journals LLC 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5752530/
https://www.ncbi.nlm.nih.gov/pubmed/29312617
http://dx.doi.org/10.18632/oncotarget.22683
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author Roshan, Romin
Labo, Nazzarena
Trivett, Matthew
Miley, Wendell
Marshall, Vickie
Coren, Lori
Cornejo Castro, Elena M.
Perez, Hannah
Holdridge, Benjamin
Davis, Eliza
Matus-Nicodemos, Rodrigo
Ayala, Victor I.
Sowder, Raymond
Wyvill, Kathleen M.
Aleman, Karen
Fennessey, Christine
Lifson, Jeffrey
Polizzotto, Mark N.
Douek, Daniel
Keele, Brandon
Uldrick, Thomas S.
Yarchoan, Robert
Ohlen, Claes
Ott, David
Whitby, Denise
author_facet Roshan, Romin
Labo, Nazzarena
Trivett, Matthew
Miley, Wendell
Marshall, Vickie
Coren, Lori
Cornejo Castro, Elena M.
Perez, Hannah
Holdridge, Benjamin
Davis, Eliza
Matus-Nicodemos, Rodrigo
Ayala, Victor I.
Sowder, Raymond
Wyvill, Kathleen M.
Aleman, Karen
Fennessey, Christine
Lifson, Jeffrey
Polizzotto, Mark N.
Douek, Daniel
Keele, Brandon
Uldrick, Thomas S.
Yarchoan, Robert
Ohlen, Claes
Ott, David
Whitby, Denise
author_sort Roshan, Romin
collection PubMed
description Prior studies of T-cell responses to KSHV have included relatively few participants and focused on relatively few KSHV antigens. To provide a more comprehensive analysis, we investigated T-cell responses to the whole KSHV proteome using IFN-γ ELISpot. Using ∼7,500 overlapping 15mer peptides we generated one to three peptide pools for each of the 82 KSHV ORFs. IFN-γ ELISpot analysis of PBMCs from 19 patients with a history of KSHV-associated disease and 24 healthy donors (11 KSHV seropositive) detected widely varied responses. Fifty six of the 82 ORFs were recognized by at least one individual but there was little overlap between participants. Responses to at least one ORF pool were observed in all 19 patients and in 7 seropositive donors. Four seropositive donors and 10 seronegative donors had no detectable responses while 3 seronegative donors had weak responses to one ORF. Patients recognised more ORFs than the donors (p=0.04) but the response intensity (spot forming units: SFU per million cells) was similar in the two groups. In four of the responding donors, individual peptides eliciting the predominant responses were identified: three donors responded to only one peptide per ORF, while one recognized five. Using intracellular cytokine staining in four participant samples, we detected peptide-induced IFN-γ, MIP1-β, and TNF-α as well as CD107a degranulation, consistent with multifunctional effector responses in CD8+ and CD4+ T cells. Sequence analysis of TCRs present in peptide specific T-cell clones generated from two participants showed both mono- and multi-clonotypic responses. Finally, we molecularly cloned the KSHV specific TCRs and incorporated the sequences into retroviral vectors to transfer the specificities to fresh donor cells for additional studies. This study suggests that KSHV infected individuals respond to diverse KSHV antigens, consistent with a lack of shared immunodominance and establishes useful tools to facilitate KSHV immunology studies.
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spelling pubmed-57525302018-01-08 T-cell responses to KSHV infection: a systematic approach Roshan, Romin Labo, Nazzarena Trivett, Matthew Miley, Wendell Marshall, Vickie Coren, Lori Cornejo Castro, Elena M. Perez, Hannah Holdridge, Benjamin Davis, Eliza Matus-Nicodemos, Rodrigo Ayala, Victor I. Sowder, Raymond Wyvill, Kathleen M. Aleman, Karen Fennessey, Christine Lifson, Jeffrey Polizzotto, Mark N. Douek, Daniel Keele, Brandon Uldrick, Thomas S. Yarchoan, Robert Ohlen, Claes Ott, David Whitby, Denise Oncotarget Research Paper Prior studies of T-cell responses to KSHV have included relatively few participants and focused on relatively few KSHV antigens. To provide a more comprehensive analysis, we investigated T-cell responses to the whole KSHV proteome using IFN-γ ELISpot. Using ∼7,500 overlapping 15mer peptides we generated one to three peptide pools for each of the 82 KSHV ORFs. IFN-γ ELISpot analysis of PBMCs from 19 patients with a history of KSHV-associated disease and 24 healthy donors (11 KSHV seropositive) detected widely varied responses. Fifty six of the 82 ORFs were recognized by at least one individual but there was little overlap between participants. Responses to at least one ORF pool were observed in all 19 patients and in 7 seropositive donors. Four seropositive donors and 10 seronegative donors had no detectable responses while 3 seronegative donors had weak responses to one ORF. Patients recognised more ORFs than the donors (p=0.04) but the response intensity (spot forming units: SFU per million cells) was similar in the two groups. In four of the responding donors, individual peptides eliciting the predominant responses were identified: three donors responded to only one peptide per ORF, while one recognized five. Using intracellular cytokine staining in four participant samples, we detected peptide-induced IFN-γ, MIP1-β, and TNF-α as well as CD107a degranulation, consistent with multifunctional effector responses in CD8+ and CD4+ T cells. Sequence analysis of TCRs present in peptide specific T-cell clones generated from two participants showed both mono- and multi-clonotypic responses. Finally, we molecularly cloned the KSHV specific TCRs and incorporated the sequences into retroviral vectors to transfer the specificities to fresh donor cells for additional studies. This study suggests that KSHV infected individuals respond to diverse KSHV antigens, consistent with a lack of shared immunodominance and establishes useful tools to facilitate KSHV immunology studies. Impact Journals LLC 2017-11-25 /pmc/articles/PMC5752530/ /pubmed/29312617 http://dx.doi.org/10.18632/oncotarget.22683 Text en Copyright: © 2017 Roshan et al. http://creativecommons.org/licenses/by/3.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/) 3.0 (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Paper
Roshan, Romin
Labo, Nazzarena
Trivett, Matthew
Miley, Wendell
Marshall, Vickie
Coren, Lori
Cornejo Castro, Elena M.
Perez, Hannah
Holdridge, Benjamin
Davis, Eliza
Matus-Nicodemos, Rodrigo
Ayala, Victor I.
Sowder, Raymond
Wyvill, Kathleen M.
Aleman, Karen
Fennessey, Christine
Lifson, Jeffrey
Polizzotto, Mark N.
Douek, Daniel
Keele, Brandon
Uldrick, Thomas S.
Yarchoan, Robert
Ohlen, Claes
Ott, David
Whitby, Denise
T-cell responses to KSHV infection: a systematic approach
title T-cell responses to KSHV infection: a systematic approach
title_full T-cell responses to KSHV infection: a systematic approach
title_fullStr T-cell responses to KSHV infection: a systematic approach
title_full_unstemmed T-cell responses to KSHV infection: a systematic approach
title_short T-cell responses to KSHV infection: a systematic approach
title_sort t-cell responses to kshv infection: a systematic approach
topic Research Paper
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5752530/
https://www.ncbi.nlm.nih.gov/pubmed/29312617
http://dx.doi.org/10.18632/oncotarget.22683
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