T-cell allostimulatory capacity of ^HGFMo. Allogeneic CD4⁺CD25^-T cells were first labeled with the fluorescent dye CFSE and then cultured for 7 days at increasing stimulator-to-responder (S/R) ratios with cytokine-differentiated monocytes (primary MLR). (A) CFSE-labeled T cells activated as detailed were counterstained with PE-conjugated anti-CD62L mAb prior to flow cytometry analysis of CFSE dye dilution. Results from 1 representative experiment of 9 with similar results are shown. (B) Expression levels of CD25 on DC-challenged CD4⁺ T cells. Markers were set according to the proper isotypic control. The percentage of CD4⁺CD25⁺ T cells in 1 representative experiment of 9 with similar results is indicated. (C) Effect of exogenous IL-2 on T-cell hyporesponsiveness. CFSE-labeled T cells initially challenged with ^HGFMo were recovered from the primary MLR and treated with 100 IU/mL IL-2 for an additional 72 hours. The proliferation index (PI) was calculated as previously detailed.²⁷  Results (mean ± SD) are representative of 3 independent experiments performed in duplicate. T-cell proliferation in response to GM4-DCs is shown as control. ^*P < .01 compared with T cells initially activated with ^HGFMo and subsequently exposed to IL-2. (D) Ag-specific restimulation of DC-activated T cells. T cells initially challenged with cytokine-differentiated DCs in the primary MLR were recovered and left untouched (□) or restimulated either with same-donor GM4-DCs (○), with ^HGFMo (•), or with monocytes that were cryopreserved at the start of the experiment (▪). Control cultures were established with T cells activated with GM4-DCs both in the primary and in the secondary MLR (▴). Results (mean ± SD) are representative of 4 independent experiments performed in duplicate. ^*P < .01 compared with T cells restimulated with ^HGFMo. (E) Secondary polyclonal stimulation of DC-activated T cells. T cells initially challenged with ^HGFMo in the primary MLR (•) were recovered and restimulated for 72 hours with 12.5 μL Dynabeads CD3/CD28 per 1 × 10⁶cells in the presence of 10 IU/mL IL-2. Control cultures consisted of T cells initially activated with GM4-DCs (□) and then restimulated under the same experimental conditions. The proliferation of T cells in response to ^HGFMo in the primary MLR is also shown (○).