The slow indolent nature of B-CLL makes it eminently suitable for immune-based treatment strategies. Currently, there exist very few CLL-associated, defined antigen which can be used in vaccination approaches. The use of whole tumor preparations in conjunction with dendritic cells (DC) as cellular adjuvants presents an alternative method for generating therapeutic T-cell responses in CLL patients. We have examined various strategies of loading DC with whole tumor antigens; viz. tumor-DC hybrids, endocytosed apoptotic bodies, RNA or lysate from B-CLL cells. Dendritic cells were generated in vitro, using GM-CSF and IL-4 from immunomagnetically purified, CD14+ monocyte precursors obtained from the peripheral blood of B-CLL patients. The immature DC were loaded with whole tumor antigen using the above methods and matured using TNFα. The tumor antigen-loaded DC were compared for their ability to stimulate autologous T-cell responses. Among all the antigen-loading methods tested, DC that had endocytosed apoptotic bodies (Apo-DC) consistently generated the greatest numbers and magnitude of reactive T-cells as quantified in proliferation and ELISPOT assays. RT-PCR analysis for cytokine mRNA revealed that T-cells stimulated by Apo-DC resulted in an immune response that was almost entirely of the TH1 type as manifested by the production of mRNA for IFN-γ and TNFα. In contrast, other methods of loading antigen resulted in a mixed TH1-TH2 population with varying amounts of TH2 cytokines like IL-4 and IL-10.

In a separate series of experiments we compared the T-cell stimulatory capacities of cryopreserved and thawed, with fresh antigen-loaded DC. The results of these studies are essential for the development of a protocol for clinical therapy of B-CLL patients using Apo-DC. Our results indicated that cryopreserved DC could be recovered with high viability. A comparison of functional abilities demonstrated that no significant differences in T-cell stimulatory capacity between cryopreserved and fresh Apo-DC could be noted over a wide variety of immunological assays. Cumulatively, our results suggest that Apo-DC may be a suitable vaccine candidate for immunotherapy of B-CLL patients.

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