Careful manipulation of blood-derived DC precursors using a cocktail of cytokines to generate DC-like cells in vitro
has been shown to generate efficient antigen-specific T cell immune responses [42
]. Advanced understanding of the technologies required to generate human DC, load DC with antigens of interest, and demonstrate a DC-mediated cytotoxic T cell response has enabled the execution of a number of Phase I clinical cancer vaccine trials[43
]. However, lack of standardization of the source of DC precursors (e.g., fresh vs. cryopreserved), and the type of DC (e.g., immature vs. mature) utilized for therapy make it difficult to compare the outcomes across trials in order to develop better therapeutic strategies[45
In the present report, monocytes were used as precursors to generate DC because they do not require mobilization and can generate enriched populations of DC in vitro in 7 days. The effect of cryopreservation on differentiation of precursors into DC-like cells was assessed by performing a cross-sectional comparison of MDDC derived from fresh and cryopreserved PBMC of healthy donors. In addition, cryopreserved PBMC-derived MDDC from cancer patients were compared to cryopreserved PBMC-derived MDDC from healthy donors to evaluate their phenotypic and functional differences. The mature MDDC from cryopreserved PBMC of healthy donors show reduced functional ability compared to the fresh healthy group. However, this observation could also be partially attributed to differences in the donors used in these two groups. The marker expression pattern of mature MDDC from cryopreserved PBMC of cancer patients is at least equivalent to that associated with cryopreserved PBMC of healthy donors.
MDDC generated from all three sources of precursors were morphologically identical, being large in size and having a round or oval nucleus (data not shown). The number of CD209+ MDDC from cryopreserved PBMC of healthy donors was higher, although not significantly, when compared to that of cancer patients (data not shown). However, the immature as well as mature cultures from the cryopreserved healthy donor group contained significantly higher numbers of CD209+ DC compared to the fresh healthy donor group (immature cells, 45.6% [cryopreserved] versus 13.6% [fresh], p < 0.01; mature cells, 53.8% [cryopreserved] versus 24.3% [fresh], p < 0.01). These differences in yields could be due to the effect of cryopreservation, or to blood sample collection by CPT versus leukapheresis, or to differences in donors used for this comparison.
Loss of CD14 expression is a characteristic feature of mature MDDC. MDDC from all the three groups were very low or negative (MFI and percent positive) in their CD14 expression. Consistent with an earlier report, the cytokine/PGE-2 maturation cocktail used in this study provided strong maturation signals for cancer-patient derived DC[15
]. In the healthy donor group, CD86 expression was higher on cryopreserved PBMC-derived MDDC compared to those derived from fresh PBMC. This observation suggests that higher expression levels of CD86 on cryopreserved PBMC-derived MDDC could be related to non-specific activation due to components of the freezing medium, such as albumin or DMSO, or the freezing process itself. However, there were no significant differences between CD86 expression on cryopreserved PBMC-derived MDDC from the healthy donors and cancer patients. We also compared the expression of HLA-DR and CD83, both of which are markers of activated and mature DC. MDDC from cancer patients expressed significantly higher levels of HLA-DR and CD83 compared to healthy donors (cryopreserved), confirming their activated and mature phenotype. This increased expression of activation and/or maturation markers on MDDC generated from cryopreserved PBMC of healthy donors and cancer patients is either endogenous condition or could also be due to the uptake of dead cells that may be generated during the freezing/thawing and subsequent culture process.
IL-12 and COX-2 were selected as markers to compare the functional capacity of MDDC. The ability to produce IL-12, which drives the Th1 helper T cell response, is considered to be one of the most important functions of DC because IL-12 secretion appears to correlate with therapeutic efficacy in clinical trials [47
]. In our study, although there were no significant differences in the frequency of IL-12+
mature MDDC from fresh versus cryopreserved PBMC of healthy donors, culture supernatants from fresh PBMC-derived mature MDDC contained higher levels of secreted IL-12 (range of 5–25 pg/mL/0.5 million cells). The fact that the actual levels of the secreted cytokines were low may be related to the observation that only 23%–54% of the heterogeneous cell population was actually CD209+
DC. The low levels of secreted IL-12 could also be related to the presence of PGE-2 in the maturation cocktail: PGE-2 is a potent inducer of IL-10 and an inhibitor of IL-12 production by APC, including DC. In our study, comparable amounts of IL-10 (median = 11.5 pg/ml) and IL-12 (median = 9.5 pg/ml) were secreted by fresh PBMC-derived mature MDDC from healthy donors. This observation differs from an earlier report showing the absence of IL-12 and presence of IL-10 in cancer patient-derived MDDC culture supernatants[5
] and may be associated with differences in the timing of addition of maturation stimuli and harvest of DC culture supernatants. Significantly higher frequencies of IL-12+
cells were observed in mature MDDC cultures derived from cryopreserved PBMC of cancer patients when compared to those from cryopreserved PBMC of healthy donors. However, actual IL-12 secretion by mature MDDC from these two groups was below the limit of detection (<5 pg/ml). This suggests that despite the use of cryopreserved PBMC as a precursor source and the use of PGE-2 for maturation, MDDC from cancer patients could nonetheless still produce intracellular IL-12.
COX-2 is over-expressed in a variety of pre-malignant and malignant conditions. In spite of the demonstrated association of COX-2 with immuno-modulation of APC function in cancer, there are no reports comparing COX-2 expression in DC from healthy donors to those from cancer patients. Other studies have used mRNA expression, immunohistochemistry, or western blot to detect COX-2 in various cells including DC [52
]. Here, we report the use of flow cytometry to detect COX-2 expressing DC in response to inflammatory stimulation. Mature MDDC from cryopreserved PBMC of healthy donors contained significantly lower numbers of COX-2+
cells compared to those derived from fresh PBMC, indicating that cryopreservation of precursors may adversely affect some functionality of mature MDDC. Mature MDDC derived from cryopreserved PBMC of cancer patients, conversely, showed a trend towards higher numbers of COX-2+
cells compared to those derived from cryopreserved PBMC of healthy donors, suggesting a more activated or inflamed phenotype of cells from cancer patients. These cells may be producing PGE-2 endogenously and thereby regulating DC function, i.e., maturation and IL-12 production in vivo
]. It is of interest to note that when LPS-stimulated MDDC were simultaneously stained for intracellular expression of IL-12 and COX-2, about 50–80% of IL-12+
cells also expressed COX-2. Higher frequency of COX-2+
cells and lower amounts of IL-12 production by MDDC matured in presence of PGE-2 may warrant further studies to evaluate whether PGE-2 could be eliminated from maturation cocktail.
Phenotypic and functional deficiencies and decreased in vitro
T cell stimulatory capacity of DC from patients with chronic myeloid leukemia and breast cancer have been reported [56, 57]. However, it is evident from our data that the expression of co-stimulatory molecules and intracellular functional markers relevant for T cell interaction and activation are largely preserved in MDDC from cancer patients. Consistent with these observations, MDDC in our study were also able to stimulate both allogeneic and antigen-specific autologous T cells. Our autologous T cell stimulation results are in agreement with those reported earlier for advanced breast cancer patients[5
] and pancreatic carcinoma patients[12
] but different from those described for patients with operable or early stage breast cancer [7, 14, 57]. The differences in these reports could be related to the disease stage or the techniques used in culturing the DC or measuring the response.
It is of interest that MDDC from healthy donors in our study stimulated responses to several cancer antigens. Fresh PBMC-derived DC-driven CD4+ T cell proliferation in response to Her2/neu and CEA was significantly higher compared to that driven by cryopreserved PBMC-derived DC. Whereas there were no differences in the DC-driven CD4+ T cell proliferative responses of these two groups to SEB, pp65 and MAGE antigens. These results suggest that healthy donors are able to make T cell responses to certain cancer antigens, and some of these antigen-specific responses are sensitive to cryopreservation. Cancer-antigen-specific intracellular cytokine expression in T cells has also been observed in a fresh PBMC healthy donor cohort (M. Inokuma, manuscript in preparation). Not surprisingly, the median T cell responses to DC pulsed with cancer antigens were higher in cancer patients compared to those from healthy donors, although the difference was not statistically significant. All of these observations indicate that although cryopreservation affects some functional responses in healthy donors, which could be partially attributed to differences in the donor pool, MDDC from cancer patients are at least as functionally equivalent as those from healthy donors. It is important to note that although the cancer patient cohort used in this study consisted of breast, colon, and lung cancers, the characteristics of the MDDC did not appear to segregate based on the type of cancer. Thus, for example, MDDC from breast cancer patients behaved similarly to those from colon cancer patients. However, a larger number of patients may be required to investigate any cancer-specific differences.
Although altered DC function and differentiation have been proposed as a fundamental mechanism by which tumors evade the immune system, DC from the cancer patients used in the present study appear to possess basic functionality associated with generating efficient T cell responses. The failure of immune surveillance in these patients may more likely be associated with the tumoral environment than with DC functional capacity itself. Thus, tumor-derived immunosuppressive factors, such as vascular endothelial growth factor [58, 59], PGE-2[54
], spermine [6
], and mechanisms such as apoptosis of DC and T cells [60, 61], Fas/FasL interaction , TLR-4 mediated resistance of tumor cells to CTL attack , as well as defective maturation of hematopoetic cells  may obstruct effective in vivo
immune responses by inhibiting endogenous DC function. This suggests that the negative influence of endogenously-growing tumors on DC function may be partially responsible for the mixed success of clinical trials reported so far. Increased understanding of tumor-host interactions may help uncover these phenomena and allow better harnessing of the immune system for effective cancer immunotherapy.