Purpose: A phase I/II trial was conducted to evaluate clinical and immunologic responses after intralymphatic and intranodal injections of mature dendritic cells. Experimental Design: Fourteen patients with a metastatic melanoma received matured dendritic cells, loaded with Melan-A/MART-1and/or NA17-A peptides and keyhole limpet hemocyanin.The cells were matured overnight with Ribomunyl, a toll-like receptor ligand, and IFN-g, which ensured the production of high levels of interleukin-12p70. Dendritic cells were injected at monthly intervals, first into an afferent lymphatic and then twice intranodally. Immunologic responses were monitored by tetramer staining of circulating CD8 + lymphocytes and delayed-type hypersensitivity tests.

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The present study describes the optimization of an in vitro culture method for generating large amounts of dendritic cells (DC) in serum-free conditions from leukapheresis containing a mixed population of peripheral blood mononuclear cells (PBMC) which are cultured in the presence of GM-CSF and IL-13. Initial comparisons between the generation of DC from bulk and monocyte-enriched leukapheresis products showed that the presence of lymphocytes during the culture favors the differentiation of monocytes into DC. DC yields obtained from mixed mononuclear cell cultures were between 38 and 54% higher than yields obtained from monocyte-enriched cultures. Both types of cultures resulted in the generation of DC with an immature phenotype (CD83- and high phagocytic activity), which have been previously shown to be good stimulators for T cell responses. DC yields of bulk cultures in serum-free conditions were significantly higher than those obtained in the presence of 2% human serum. The cytokines of the supernatants of serum-free cultures comprised a significant content of pro-inflammatory cytokines such as IL-1, IL-12 and TNF-alpha. Maturation of DC generated by this method can be induced by treatment with double-stranded RNA, LPS or TNF-alpha, resulting in enhanced surface expression of CD80, CD86, CD40, CD83 and MHC molecules on the DC. The methodology described here offers the possibility for generating large amounts of clinical grade DC from bulk leukapheresis products, thus avoiding DC precursor purification steps, and thereby minimizing the risks of contamination. This culture process may be applied to cell-based therapeutic approaches for the treatment of cancer or chronic viral infections.

The recent clinical trial in lymphoma using tumor antigenloaded DCs (Hsu et al, Nature Med 1996; 2: 52) demonstrates the efficiency of the use of professional antigen presenting cells (APCs) for taking up, processing and presenting tumor protein in a vaccine strategy in cancer. However, the production of large quantities of clinical grade APCs remains to be resolved. Here, we describe that both dendritic cells (DCs) and macrophages (MØs) can be efficiently differentiated in large numbers from lymphoma patients in spite of their disease and previous therapy. These cells were produced using the VAC and MAK cell processors according to standard operating procedures. DCs and MØs were differentiated from circulating monocytes in gas permeable hydrophobic bags, with 2% autologous serum and in the presence of GM-CSF and IL-13 or GM-CSF alone, respectively. DCs and MØs were then purified by counter flow centrifugation. Phenotypic, morphological and functional analysis showed that cells differentiated from patients with lymphoma present quite similar features to DCs and MØs produced from monocytes of healthy donors. Moreover, we show that MØs, when combined with CD20 antibody (Rituximab), can efficiently engulf tumor cells and propose that a such combination could be used for initiating a clinical trial in lymphoma. Thus, the possibility of producing functional DC and MØs in large amounts in conditions compatible with therapeutic application will allow the development of new immune strategies to eradicate lymphoma.

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