Evaluation of a quantitative real-time PCR assay to measure HIV-specific mucosal CD8+ T Cell responses in the cervix
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Date
2010Author
Chege, Duncan
Chai, Yijie
Huibner, Sanja
McKinnon, Lyle
Wachihi, Charles
Kimani, Makubo
Jaoko Walter G.
Kimani, Joshua
Ball, T. Blake
Plummer, Francis A.
Kaul, Rupert
Rebbapragada, Anuradha
Type
ArticleLanguage
enMetadata
Show full item recordAbstract
Several candidate HIV vaccines aim to induce virus-specific cellular immunity particularly in the genital tract, typically the
initial site of HIV acquisition. However, standardized and sensitive methods for evaluating HIV-specific immune responses at
the genital level are lacking. Therefore we evaluated real-time quantitative PCR (qPCR) as a potential platform to measure
these responses. b-Actin and GAPDH were identified as the most stable housekeeping reference genes in peripheral blood
mononuclear cells (PBMCs) and cervical mononuclear cells (CMCs) respectively and were used for normalizing transcript
mRNA expression. HIV-specific cellular T cell immune responses to a pool of optimized CD8+ HIV epitopes (HIV epitope
pool) and Staphylococcal enterotoxin B (SEB) superantigen control were assayed in HIV infected PBMC by qPCR, with
parallel assessment of cytokine protein production. Peak HIV-specific mRNA expression of IFNc, IL-2 and TNFa occurred after
3, 5 and 12 hours respectively. PBMCs were titrated to cervical appropriate cell numbers to determine minimum required
assay input cell numbers; qPCR retained sensitivity with input of at least 2.56104 PBMCs. This optimized qPCR assay was
then used to assess HIV-specific cellular T cell responses in cytobrush-derived cervical T cells from HIV positive individuals.
SEB induced IFNc mRNA transcription was detected in CMCs and correlated positively with IFNc protein production.
However, qPCR was unable to detect HIV-induced cytokine mRNA production in the cervix of HIV-infected women despite
robust detection of gene induction in PBMCs. In conclusion, although qPCR can be used to measure ex vivo cellular immune
responses to HIV in blood, HIV-specific responses in the cervix may fall below the threshold of qPCR detection. Nonetheless,
this platform may have a potential role in measuring mitogen-induced immune responses in the genital tract.
URI
doi:10.1371/journal.pone.0013077http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2951338/
http://erepository.uonbi.ac.ke:8080/xmlui/handle/123456789/9898
Citation
http://www.ncbi.nlm.nih.gov/pubmed/20949096Collections
- Faculty of Health Sciences (FHS) [10377]