CD4⁺ T cells reactive against autologous EBV B-LCL cells may be efficiently induced by repeated stimulations with LCL lysate-pulsed mature autologous DCs.

CD4⁺ T cells freshly isolated from EBV-seropositive donor IP1 were stimulated on a weekly regimen (days 0, 7, and 14) with autologous mature DCs prepulsed with freeze–thaw lysate (10 kd or larger) prepared from autologous EBV B-LCL. Freshly isolated (day 0) CD4⁺ T cells and T lymphocyte responders harvested on day 21 of culture (both seeded in triplicates at 10⁵ and 10⁴ cells per well) were analyzed in IFN-γ and IL-5 Elispot assays. T-cell reactivity was screened against intact autologous EBV B-LCL (●); against autologous mature DCs preloaded with freeze–thaw lysates (10 kd or larger) isolated from autologous EBV B-LCL (○), B-cell blasts (▾), or T-cell blasts (▪); and against autologous mature DCs pulsed with naturally processed peptides acid-eluted from affinity-purified HLA-DR complexes of the autologous EBV B-LCL (■). Resulting spots were developed and evaluated as described in Figure 1. Spot production observed in microwells where CD4⁺ (responder) lymphocytes were seeded with the autologous EBV B-LCL or with mature DCs loaded with the EBV B-LCL lysate was completely blocked by the addition of the anti-HLA-DR (class II) antibody L243 (100 μg/mL) but not by the anti-HLA class I antibody W6/32 (100 μg/mL). Results were confirmed in 2 independent experiments.