Figure 6. The autologous CTLs educated by CD40L-sBAFF-B cells enhanced the capability to eliminate the reactivated HIV-1-infected CD4+ T cells isolated from HIV-1-infected individuals receiving suppressive cART.
a. The manufacturing strategy of HIV-1 antigen-specific CTLs. The PBMCs were isolated from ten whole blood samples of HIV-1-infected individuals receiving suppressive cART. B cells were then isolated and co-cultured with the 293T-CD40L-sBAFF cell line in the presence of cytokine cocktails. The activated CD40L-sBAFF-B cells were pulsed with PepMixes containing WF9, TL9, TP9, HA9, and PY9 as described. Then, autologous CD8+ T cells isolated from remaining PBMCs co-cultured at a T: B ratio of 3:1 with irradiated CD40L-sBAFF-B cells and 20 ng/ml IL-2. T cells underwent three weekly stimulations by co-culture with autologous, PepMix-pulsed B cells. b. HIV-1-infected individuals CD4+ T cells were isolated, stimulated with PHA and IL-2 overnight. PepMix-pulsed B cells-educated autologous CD8+ T cells were added into the culture of CD4+ T cells at the effector-to-target ratio 3:1 in the presence of T-20 to prevent further rounds of viral replication. After 5 days of co-culture, the cell-associated HIV-1 gag RNA was quantified by qRT-PCR. c. The fraction of residual p24+ CD4+ T cells was measured and normalized to the control culture without CTLs. Data represent mean ± SD (error bars). The paired t-test was used. P < 0.05 indicates statistically significance difference. * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001.