Results from a novel ex vivo assay of the effects of NK and CD8⁺T-cell responses on HIV laboratory-infected pre-HCT recipient and post-HCT donor-origin CD4⁺T cells. (A) A schema of our assay to determine the effects of allogeneic HSCT on HIV reactivation and effector cell activation and responses is shown. Pre- and post-SCT PBMC were obtained from 30 HIV-uninfected individuals, followed by establishment of latent HIV infection ex vivo in recipient or engrafted donor CD4⁺ T cells. Post-HSCT donor-derived PBMC were then coincubated with HIV-infected recipient pre-SCT (allogeneic experiments) and donor-derived post-SCT CD4⁺ T cells (autologous control experiments). (B) An increase in the percent of HIV-reactivated (GFP expressing) proliferating pre-HCT recipient CD4⁺ T cells was observed in several participant samples following 7 days of coculture (N = 30). Outlier values are shown as individual points on the box plots otherwise representing median and quartile values. Correlations between activated (CD38⁺/HLA-DR⁺) donor CD8⁺ T, NK, and CD3⁺CD56⁺ cells and HIV-iGFP–expressing laboratory infected CD4⁺ T cells on day 7 are shown in panels C (experiments using infected recipient CD4⁺ T cells and PBMCs following donor cell engraftment) and D (control experiments using infected post-HCT engrafted CD4⁺ T cells of donor origin), respectively. Significant correlations between NK and CD3⁺CD56⁺ cells were identified only in the pre-HCT CD4/post-HCT PBMC experiments, whereas only activated CD8+ T cells correlated with HIV-iGFP expression in the post-HCT CD4/post-HCT PBMC control experiments. Dashed lines represent linear regression and P values were obtained using non-parametric Spearman rank correlation analyses.