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. 2011 Aug 5;6(8):e23202. doi: 10.1371/journal.pone.0023202

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This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration, which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.

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Ex vivo PBMCs from two viremic HIV-infected subjects (PS05 with 38,165 vRNA copies/ml and PS07 with 35,201 vRNA copies/ml) were stained with mAbs for surface expression of CD3, CD8, and total LFA-1, as well as for intracellular p24 antigen after permeabilization. The cells were subjected to flow cytometric analyses, and the data analyzed by the FlowJo software. The dot plots (top) show the percentages of p24+ cells among CD4 T cells (CD3+ CD8-) from subjects PS05 and PS07. The p24+ gating were determined based on p24 staining of HIV-seronegative PBMCs (Fig. S3). The histograms (bottom) compare LFA-1 expression on p24+ and p24- CD4 T cells; the mean fluorescence intensity (mfi) for p24+ and p24- CD4 T cells are 462 and 376 for PS05, and 311 and 228 for PS07. (B) Expression of active LFA-1 on p24+ versus p24- CD4 T cells. PBMCs from viremic HIV-infected subjects (PS15 with 5,075 vRNA copies/ml and PS16 with 7,077 vRNA copies/ml) were stained with mAb NKI-L16 and secondary fluorescent anti-mouse antibodies, followed with direct staining with fluorescent mAbs to CD3, CD8 and p24. The histograms compare mAb NKI-L16 staining on p24+ and p24- CD4 T cells; the mfi for p24+ and p24- cells are 405 and 95 for PS15 and 835 and 86 for PS16. p24+ CD4 T cells treated with the secondary antibody (no NKI-L16) were also shown for control (mfi = 19 and 18). (C) Reduction of viral DNA in HIV-infected PBMCs due to LtxA cytotoxicity. PBMCs from two viremic HIV-infected subjects (PS05 with 38,165 vRNA copies/ml and CD4 count of 814 and PS14 with 21,815 vRNA copies/ml and CD4 count of 494) were treated with LtxA (7.8 µg/ml) for 20 hrs. The viral DNA were quantified by real time PCR with the specific primers. Averages and standard deviation from epeat experiments are presented. Statistical analysis was done by one-sided Student's t test.