Figure 2.
Figure 2. Further rounds of T cell activation induce additional proviruses to produce replication-competent virus. (a) Fraction of initially seeded wells becoming p24+ after each of four consecutive stimulations with PHA. For each subject (n = 12), a total of 64–72 wells were initially seeded. The asterisk indicates that cells from subject 1 only received three rounds of PHA stimulation. (b) Fraction of total p24+ wells from each subject (n = 12) that were detected for each round of PHA stimulation. The denominator is the total number of initially seeded wells. (c) Frequency of cells in the culture at the time of stimulation that are induced to produce replication-competent virus by the indicated round of stimulation. Frequency in infectious units per million (IUPM) cells was determined for each subject (n = 12) from mean cell counts before each stimulation and the number of wells turning p24+ after stimulation using a maximum likelihood estimation of infected cell frequency (Rosenbloom et al., 2015). (d) Expression of TIM-3 before stimulation (black line) and 7 d after the indicated round of stimulation. Results are presentative of exhaustion marker analysis in three subjects. (e) Frequency of latently infected cells among the initially plated cells from each subject (n = 12) as detected after a single round of PHA stimulation and after a total of four consecutive rounds of stimulation. (f) Schematic representation of the relative frequency of infected resting CD4+ T cells detected by different assays. The VOA result presents the mean frequency detected after a single round of PHA stimulation in cultures from 12 subjects studied here. The MS-VOA result represents the mean frequency among the initially plated cells of latently infected cells as detected by a total of four consecutive rounds of PHA stimulation. The frequencies of cells with intact (nondefective) proviruses and the total frequency of infected cells as measured by PCR with gag primers were estimated from the VOA results and the ratios described by Bruner et al. (2016). The true frequency of latently infected cells is between the frequency measured in the MS-VOA and the frequency of cells with intact proviruses.

Further rounds of T cell activation induce additional proviruses to produce replication-competent virus. (a) Fraction of initially seeded wells becoming p24+ after each of four consecutive stimulations with PHA. For each subject (n = 12), a total of 64–72 wells were initially seeded. The asterisk indicates that cells from subject 1 only received three rounds of PHA stimulation. (b) Fraction of total p24+ wells from each subject (n = 12) that were detected for each round of PHA stimulation. The denominator is the total number of initially seeded wells. (c) Frequency of cells in the culture at the time of stimulation that are induced to produce replication-competent virus by the indicated round of stimulation. Frequency in infectious units per million (IUPM) cells was determined for each subject (n = 12) from mean cell counts before each stimulation and the number of wells turning p24+ after stimulation using a maximum likelihood estimation of infected cell frequency (Rosenbloom et al., 2015). (d) Expression of TIM-3 before stimulation (black line) and 7 d after the indicated round of stimulation. Results are presentative of exhaustion marker analysis in three subjects. (e) Frequency of latently infected cells among the initially plated cells from each subject (n = 12) as detected after a single round of PHA stimulation and after a total of four consecutive rounds of stimulation. (f) Schematic representation of the relative frequency of infected resting CD4+ T cells detected by different assays. The VOA result presents the mean frequency detected after a single round of PHA stimulation in cultures from 12 subjects studied here. The MS-VOA result represents the mean frequency among the initially plated cells of latently infected cells as detected by a total of four consecutive rounds of PHA stimulation. The frequencies of cells with intact (nondefective) proviruses and the total frequency of infected cells as measured by PCR with gag primers were estimated from the VOA results and the ratios described by Bruner et al. (2016). The true frequency of latently infected cells is between the frequency measured in the MS-VOA and the frequency of cells with intact proviruses.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal