Table 3. Assays for functional evaluation of dendritic cell products*
|
Functional assays. A variety of functional assays for newly generated DC products are available (Table 3). One popular assay is the mixed lymphocyte
reaction (MLR), in which allogeneic T cells serve as responders and DCs—titered into the co-culture at various T cell–DC ratios—serve
as stimulators.29 The assay measures levels of T-cell responses to alloantigens presented by DCs, and is not directly relevant to the response
elicited by tumor antigens. A requirement for the panel of T cells that can recognize a variety of HLA specificities represents
a considerable practical limitation for a routine use of this assay. Therefore, flow cytometry-based assays have largely replaced
MLR for functional assessments of DCs. Among these, endocytosis of FITC-labeled dextran 30 combined with measurement of mannose
receptor expression with anti-CD206 antibody is often used.25 This assay measures an entirely different function of DCs, one that is essential for antigen uptake and that potentially
discriminates between a receptor-mediated antigen internalization versus endocytosis. These two pathways of antigen uptake
may have different consequences for antigen processing and presentation.31 Therefore, it might be important to determine their use by tumor-derived antigens relative to the type of T-cell response
these antigens elicit in vivo.
Flow cytometry assays for DCs. Flow cytometry-based assays measure the state of DC maturation. After five to six days of culture, immature DCs are CD40+,
CD80+, CD86+, and HLA-DR+, but have low expression levels of CD83. Upon maturation, DCs typically express higher levels of
HLA molecules and co-stimulatory molecules (CD80, CD83, and CD86), in addition to enhanced expression of the chemokine receptor,
CCR7. The presence of CD206 (the mannose receptor) and absence of CD14 (a monocyte marker) on the DC surface may be helpful
for defining the state of DC differentiation. Of the utmost importance is full ex vivo activation of DCs, which can be achieved by various approaches (e.g., combinations of cytokines, toll-receptor ligands, or
the CD40 ligand) and can be evaluated by upregulation of surface markers and by functional assays.
More recently, methods able to quantitate expression levels of Class I and Class II major histocompatibility complex (MHC)
molecules, ICAM, or individual components of APM have been introduced to assess DCs' potential for T-cell stimulation.32 These flow-based assays proved to be quite informative, as DCs derived from monocytes of patients with cancer were shown
to have aberrant expression of APM components, which was related to inadequate T-cell stimulation, at least in vitro.
32
The ability to evaluate DC-mediated Th1/Th2 polarization (e.g., expression of interferon (IFN)-gamma or IL-4) following co-culture
of purified CD3+CD45RA+ lymphocytes with DC and re-stimulation with PMA/ionomycin also has been used in some studies.33
Measuring co-stimulatory capacity. In addition to processing antigens and presenting individual epitopes to epitope-specific T cells, DCs exhibit a strong co-stimulatory
capacity, which is much higher than that of other cells. In fact, expression of co-stimulatory molecules on DC has been used
as a surrogate marker for their functional potential.34 Because co-stimulation plays a critical role in the induction of tumor-specific immunity, this assay may be relevant to
the ability of DCs to function in stimulating T cells specific for tumor antigens. As such, the assay for co-stimulatory molecule
expression, called the costimulatory (COSTIM) assay, might be especially useful in assessing the functional potential of ex vivo-generated DCs in anticancer vaccines. It has been reported to work with both allogeneic and autologous DC products.35
General remarks on DC release criteria. While the various functional assays listed in Table 3 provide useful information about DC properties, none has been validated
as a tool for measuring the potency of DCs generated for human therapy. These assays might be standardized, but a formal validation
would require establishing a correlation with the in vivo performance of the cells. It appears that none of the listed assays has been correlated with the clinical efficacy of the
administered DC products. Nevertheless, the availability of several assays that measure diverse DC functions provides an opportunity
for an improved control of the variability of DC products and for establishing a clinically meaningful functional profile
for DCs. A functional DC profile based on results of these assays is likely to give a more reliable estimate of DCs' therapeutic
potential than any single assay and may prove to be a better surrogate of their clinical efficacy.
|
