Figure 1. MS-VOA. (a) A transwell... | Rockefeller University Press

Figure 1.

$Figure 1. MS-VOA. (a) A transwell co-culture system (Ho et al., 2013) was used to allow separate manipulation of patient CD4+ T cells (green) and MOLT4/CCR5 cells (red). Purified resting CD4+ T cells from subjects on ART were plated at a limiting dilution for viral outgrowth and stimulated with PHA and irradiated allogeneic PBMCs (not depicted) as previously described (Finzi et al., 1997, 1999; Siliciano et al., 2003; Laird et al., 2016). After 24 h, PHA was removed, and 106 MOTL4/CCR5 cells were added. Outgrowth in this system is equivalent to standard co-cultures (Ho et al., 2013). (b) Assay time course. At 8 d after the initial PHA stimulation, half the volume from the top and bottom chambers of each transwell was transferred to a new set of transwell plates for a second round of PHA stimulation. The initial plates were cultured without further stimulation for a total of 21 d. The restimulated plates were cultured for 8 d after the second round of PHA stimulation and then split as above to generate a third set of plates, which received a third round of stimulation. Similarly, these plates were split 8 d later to generate the fourth set of plates, which received a fourth round of stimulation. A p24 ELISA was performed 21 d after each respective round of PHA stimulation to quantify viral outgrowth (dashed lines). In this hypothetical example, each round of PHA stimulation induced outgrowth from an additional well (black circles). (c) PHA stimulation induces uniform proliferation of resting CD4+ T cells. Immediately before the first PHA stimulation, an aliquot of resting CD4+ T cells was stained with CFSE. CFSE dilution was quantitated by flow cytometry 7 d after stimulation to determine the fraction of cells that had proliferated (red histogram). Cells that did not receive PHA stimulation served as controls (blue histograms). The histogram is representative of CFSE dilution from three subjects. (d) Increase in cell number after each round of stimulation. Results for three representative subjects are shown. (e) Activation marker expression induced by each round of PHA stimulation. Cells were stained with antibodies to CD4 (x axis) and the activation markers CD25, CD69, and HLA-DR (y axis) 7 d after the indicated round of PHA stimulation (red dot plots). Cells that did not receive the most recent round of PHA stimulation were analyzed in parallel (blue dot plots). Results are shown for subject 10 and are representative of two other subjects analyzed.$

MS-VOA. (a) A transwell co-culture system (Ho et al., 2013) was used to allow separate manipulation of patient CD4⁺ T cells (green) and MOLT4/CCR5 cells (red). Purified resting CD4⁺ T cells from subjects on ART were plated at a limiting dilution for viral outgrowth and stimulated with PHA and irradiated allogeneic PBMCs (not depicted) as previously described (Finzi et al., 1997, 1999; Siliciano et al., 2003; Laird et al., 2016). After 24 h, PHA was removed, and 10⁶ MOTL4/CCR5 cells were added. Outgrowth in this system is equivalent to standard co-cultures (Ho et al., 2013). (b) Assay time course. At 8 d after the initial PHA stimulation, half the volume from the top and bottom chambers of each transwell was transferred to a new set of transwell plates for a second round of PHA stimulation. The initial plates were cultured without further stimulation for a total of 21 d. The restimulated plates were cultured for 8 d after the second round of PHA stimulation and then split as above to generate a third set of plates, which received a third round of stimulation. Similarly, these plates were split 8 d later to generate the fourth set of plates, which received a fourth round of stimulation. A p24 ELISA was performed 21 d after each respective round of PHA stimulation to quantify viral outgrowth (dashed lines). In this hypothetical example, each round of PHA stimulation induced outgrowth from an additional well (black circles). (c) PHA stimulation induces uniform proliferation of resting CD4⁺ T cells. Immediately before the first PHA stimulation, an aliquot of resting CD4⁺ T cells was stained with CFSE. CFSE dilution was quantitated by flow cytometry 7 d after stimulation to determine the fraction of cells that had proliferated (red histogram). Cells that did not receive PHA stimulation served as controls (blue histograms). The histogram is representative of CFSE dilution from three subjects. (d) Increase in cell number after each round of stimulation. Results for three representative subjects are shown. (e) Activation marker expression induced by each round of PHA stimulation. Cells were stained with antibodies to CD4 (x axis) and the activation markers CD25, CD69, and HLA-DR (y axis) 7 d after the indicated round of PHA stimulation (red dot plots). Cells that did not receive the most recent round of PHA stimulation were analyzed in parallel (blue dot plots). Results are shown for subject 10 and are representative of two other subjects analyzed.

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