Evaluation of a Quantitative Real-Time PCR Assay to Measure HIV-Specific Mucosal CD8+T Cell Responses in the Cervix

被引：14

作者：

Chege, Duncan ^{[1
]}

Chai, Yijie ^{[1
]}

Huibner, Sanja ^{[1
]}

McKinnon, Lyle ^{[1
,5
,7
]}

Wachihi, Charles ^{[7
]}

Kimani, Makubo ^{[7
]}

Jaoko, Walter ^{[7
]}

Kimani, Joshua ^{[5
,6
,7
]}

Ball, T. Blake ^{[5
,6
,7
]}

Plummer, Francis A. ^{[5
,6
,7
]}

Kaul, Rupert ^{[1
,2
,7
]}

Rebbapragada, Anuradha ^{[3
,4
]}

机构：

[1] Univ Toronto, Dept Med, Toronto, ON, Canada

[2] Univ Hlth Network, Dept Med, Toronto, ON, Canada

[3] Ontario Agcy Hlth Protect & Promot, Publ Hlth Lab Toronto, Toronto, ON, Canada

[4] Univ Toronto, Dept Lab Med & Pathobiol, Toronto, ON, Canada

[5] Univ Manitoba, Dept Med Microbiol, Winnipeg, MB, Canada

[6] Publ Hlth Agcy Canada, Natl HIV & Retrovirol Labs, Lab HIV Immunol, Winnipeg, MB, Canada

[7] Univ Nairobi, Dept Med Microbiol, Nairobi, Kenya

来源：

PLOS ONE | 2010年 / 5卷 / 10期

基金：

加拿大健康研究院;

关键词：

HUMAN-IMMUNODEFICIENCY-VIRUS; CYTOTOXIC T-LYMPHOCYTES; FEMALE GENITAL-TRACT; MONONUCLEAR-CELLS; CYTOKINE EXPRESSION; INFECTED WOMEN; BLOOD; IMMUNITY; DNA; QUANTIFICATION;

D O I：

10.1371/journal.pone.0013077

中图分类号：

O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];

学科分类号：

07 ; 0710 ; 09 ;

摘要：

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. beta-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 IFN gamma, IL-2 and TNF alpha 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 2.5x10(4) 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 IFN gamma 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.

引用

页数：8