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. 2019 Mar 13;9:4373. doi: 10.1038/s41598-019-40760-x

Figure 6.

Figure 6

HMBA synergizes with PRO at upregulating ULBP2 on HIV-1-infected T cells that exit from viral latency. (A–D) To establish viral latency, freshly isolated CD4+ T cells were cultivated in the presence of CCL19 for 1–3 days, infected or not with HIV-1, and further cultured for 3 days, as described in details in Materials and Methods. Then, cells were treated with 5 mM HMBA or 1 μM PRO, alone or in combination, stimulated with 10 μg/ml PHA or not stimulated (ns), further cultivated for 3 days and finally analyzed by 2-color flow cytometry for the expression of intracellular p24 and cell-surface ULBP2. (A) Representative dot plots show the frequency of reactivated p24+ cells gated by setting non-infected PHA-stimulated control cells at 0%. (B) Percentage of p24+ cells was determined as shown in panel A in three independent experiments and normalized to HIV-infected PHA-treated cultures (mean ± SEM). Each symbol represents 1 donor. (C) Histograms show ULBP2 fluorescence on gated p24− (top panels) and p24+ (bottom panels) cell populations measured in a representative experiment on ns, HMBA-, PRO-, and HMBA + PRO-stimulated cell samples. Signals obtained with control IgG (filled histograms) and the percentage of ligand-positive cells are shown. (D) ULBP2 expression (mean ± SEM), both % of positive cells and MFI, was determined in p24− (grey bars) and p24+ (black bars) cells as shown in panel C in 3 independent experiments. *P < 0.05 by two-tailed paired t test.