Immunomodulatory dendritic cells generated from nonfractionated bulk peripheral blood mononuclear cell cultures induce growth of cytotoxic T cells against renal cell carcinoma

Dendritic cells (DCs) loaded with tumor antigens have the potential to beco me a powerful tool for clinical cancer treatment. Recently, the authors sho wed that a tumor-specific immune response can be elicited in culture via st imulation with autologous renal tumor lysate (Tuly)-loaded DCs that were ge nerated from cytokine cultured adherent peripheral blood mononuclear cells (PBMCs). Here, the authors show that immunomodulatory DCs can be generated directly from nonfractionated bulk PBMC cultures. Kinetic studies of DC dif ferentiation and maturation in PBMC cultures were performed by monitoring t he acquisition of DC-associated molecules using fluorescence-activated cell sorting analysis to determine the percentage of positive immunostained cel ls and the mean relative linear fluorescence intensity (MRLFI). Compared wi th conventional adherent CD14(+) cultures, which have mostly natural killer , T, and B cells removed before cytokine culture, bulk PBMC cultures exhibi ted an early loss of CD14(+) cells (day 0 = 78.8%, day 2 = 29.6% versus day 0 = 74%, day 2 = 75%) with an increase in yield of mature DCs (CD19(-) CD8 3(+)) (day 5 = 17%, day 6 = 21%, day 7 = 22% versus day 5 = 11%, day 6 = 15 %, day 7 = 23%). Although a comparable percentage of DCs expressing CD86(+) (B7-2), CD40(+), and HLA-DR+ were detected in both cultures, higher expres sion levels were detected in DCs derived from bulk culture (CD86 = MRLFI 36 65.1 versus 2662.1 on day 6; CD40 = MRLFI 1786 Versus 681.2 on day 6; HLA-D R = MRLFI 6018.2 versus 3444.9 on day 2). Cytokines involved in DC maturati on were determined by polymerase chain reaction demonstrating interleukin-6 (IL-6), IL-12, interferon-gamma, granu locyte-macrophage colony-stimulatin g factor, and tumor necrosis factor-alpha mRNA expression by bulk culture c ells during the entire 9-day culture period, This same cytokine mRNA profil e was not found in the conventional adherent DC culture. Autologous renal T uly (30 mu g prbtein/10(7) PBMCs) enhanced human leukocyte antigen expressi on by DCs (class I = 7367.6 versus 4085.4 MRFLI; class II = 8277.2 versus 6 175.7 MRFLI) and upregulated cytokine mRNAs levels. Concurrently, CD3(+) CD 56(-), CD3(+) CD25(+), and CD3(+) TCR+ cell populations increased and cytot oxicity against autologous renal cell carcinoma tumor target was induced. S pecific cytotoxicity was augmented when cultures were boosted continuously with IL-2 (20 U/mL biological response modifier program) plus Tuly stimulat ion. These results suggest that nonadherent PBMCs may participate in enhanc ing DC maturation. Besides the simplicity of this culture technique, bulk D C cultures potentially may be used with the same efficiency as conventional purified DCs. Furthermore, bulk culture-derived DCs may be used directly i n vivo as a tumor vaccine, or for further ex vivo expansion of co-cultured cytotoxic T cells to be used for adoptive immunotherapy.