TY - JOUR
T1 - T-cell responses to KSHV infection
T2 - A systematic approach
AU - Roshan, Romin
AU - Labo, Nazzarena
AU - Trivett, Matthew
AU - Miley, Wendell
AU - Marshall, Vickie
AU - Coren, Lori
AU - Castro, Elena M.Cornejo
AU - Perez, Hannah
AU - Holdridge, Benjamin
AU - Davis, Eliza
AU - Matus-Nicodemos, Rodrigo
AU - Ayala, Victor I.
AU - Sowder, Raymond
AU - Wyvill, Kathleen M.
AU - Aleman, Karen
AU - Fennessey, Christine
AU - Lifson, Jeffrey
AU - Polizzotto, Mark N.
AU - Douek, Daniel
AU - Keele, Brandon
AU - Uldrick, Thomas S.
AU - Yarchoan, Robert
AU - Ohlen, Claes
AU - Ott, David
AU - Whitby, Denise
N1 - Publisher Copyright:
© Roshan et al.
PY - 2017
Y1 - 2017
N2 - 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.
AB - 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.
KW - Cell-mediated immunity
KW - ELISpot
KW - KSHV
KW - T-cells
UR - http://www.scopus.com/inward/record.url?scp=85037683856&partnerID=8YFLogxK
U2 - 10.18632/oncotarget.22683
DO - 10.18632/oncotarget.22683
M3 - Article
SN - 1949-2553
VL - 8
SP - 109402
EP - 109416
JO - Oncotarget
JF - Oncotarget
IS - 65
ER -