Fig. 3: CCR5 ablation in primary CD4⁺ T cells prevents HIV spread in vitro.

PBMCs from 3 healthy adult donors were stimulated with PHA for 3 days, electroporated with indicated gRNAs complexed with Cas9, and challenged with HIV_JRCSF at a 0.5 multiplicity of infection 2 days after electroporation and in triplicate for each donor. HIV spread in culture was assessed at 2-day intervals by intracellular staining for Gag p24 antigen. A Kinetics of HIV spread following HIV_JRCSF infection of either mock edited (GFP gRNA, green) or CCR5 edited CD4⁺ T cells. Error bars represent ± SD for 3 technical replicates. B A box and whisker plot depicts the average initial infection rate 2 days after infection for all biological and technical replicates (n = 9) for each gRNA. Error bars denote minimum and maximum values, the line denotes median, and box bounds the 25th and 75th percentiles. C A box and whisker plot depicts the cumulative infection rate calculated as the area-under-the-curve (AUC) for all biological and technical replicates (n = 9) for each gRNA. Error bars denote minimum and maximum values, the line denotes median, and box bounds the 25th and 75th percentiles. Dotted horizontal lines represent the average background Gag p24 staining in mock-infected CD4⁺ T cells. D Plot depicting the association between the log₁₀-transformed total AUC percentage of CCR5⁺ CD4⁺ T cells and the log₁₀-transformed total AUC percentage of p24⁺ CD8^- T cells per PBMC donor and gRNA condition. Different symbols represent distinct PBMC donors and represent the mean of 3 technical replicates. Error bars denote ± SD. B, C ****denotes p < 0.0001, ***denotes p < 0.001, **denotes p < 0.01, *denotes p < 0.05. Statistics were generated from the Mann-Whitney test and p values are two-tailed. D Statistics were generated from a two-tailed Spearman rank correlation (p < 0.0001).