DEVELOPMENT OF A POTENCY ASSAY FOR HUMAN DENDRITIC CELLS
Although DCs are known to secrete multiple cytokines, the ability to produce IL-12p70 spontaneously or upon activation with
CD40L, and with or without addition of innate immunity signals (e.g., LPS) is considered essential for optimal in vivo activity. Importantly, the data from animal models of tumor growth indicate that IL-12p70 is the cytokine responsible for
DC-mediated Th1-polarization and for the enhancement of T lymphocyte anti-tumor responses (Table 4).27,28 The ability of DCs to produce and secrete IL-12p70 has been used to develop and standardize a DC potency assay.26
Table 4. Rationale for selecting IL-12 production assay to measure potency of dendritic cells
A potency assay can be defined as a quantitative measure of DCs' biological function(s) assessed in vitro and in vivo. The potency assay for IL-12p70 production, which recently has been introduced and standardized for routine use in the author's
laboratory, consists of two steps (Figure 2). First responder DCs are co-incubated with J588 lymphoma cells stably transfected
with the human CD40 ligand gene as stimulators.26 Second, supernatants are tested from these co-cultures in the Luminex system to determine levels of IL-12p70 secreted by
DCs stimulated with J558/CD40L with or without LPS. Both steps of this potency assay required standardization, and considerable
effort has been invested in defining the assay's performance. This assay has an interassay coefficent of variation of 18.5%
(n = 30), and a broad dynamic range, which facilitates evaluation of various DC products characterized by vastly different
levels of IL-12p70 production. The normal range for the assay established using DC products generated from peripheral blood
monocytes of 13 normal donors and matured in a conventional cytokine cocktail (IL-1b, IL-6, TNF-α, PGE2) was from 8 to 999
pg/mL, with a mean of 270 pg/mL.21 DCs generated from patients with melanoma or a chronic HIV-1 infection and matured in the same cytokines produced vastly
different levels of IL-12p70 (range: 11 to 777 pg/mL and 22 to 1,605 pg/mL, respectively). Not surprisingly, culture conditions
and maturation cytokines used for DC production were the major determining factors in the IL-12p70 production level. Thus,
DC matured in the presence of a cytokine cocktail containing α- or γ-interferons and polyI:C produced high levels of IL-12p70
in subjects with brain cancer (range: 8 to 11,600 pg/mL) or those with Sezary's syndrome (range: 630 to 15,900 pg/mL). The
in vivo therapeutic and immunologic efficacy of DC products that contain cells secreting different levels of the cytokine is currently
unknown and is being evaluated in immunotherapeutic trials to assess the biologic significance of IL-12 secretion levels.
The objective is to establish a correlation between IL-12p70 production levels by DCs used for therapy and clinical endpoints.
Ultimately, this will provide in vivo validation of the IL-12p70 assay. It is expected, but not yet proven, that DC products able to produce high IL-12p70 levels
following ex vivo stimulation will mediate superior priming or antigen-presenting functions, resulting in significant clinical responses.
Figure 2. Diagrammatic representation of a two-step potency assay measuring IL-12p70 production by DC. The assay description
appears in the text.
ASSESSMENTS OF ANTIGEN-SPECIFIC DENDRITIC CELL FUNCTIONS
Once DCs are loaded with antigen, several additional methods are available for evaluating antigen-specific immune responses
in vitro (Table 3).
Assays that measure DCs' ability to induce activation and expansion of antigen-specific T cells are of particular value. Among
them, single-cell T-cell assays,36 which determine the frequency of T cells capable of responding to an epitope presented by DCs by cytokine production (e.g.,
ELISPOT assay or CFC) or MHC-tetramer staining are often used to evaluate DC products. Correlations between the results of
these assays and clinical outcomes have been difficult to demonstrate, however, and there are no standardized approaches across
laboratories for performing these assays. Nevertheless, these single-cell assays could provide the necessary objective data
for optimizing DC-based immunotherapy. ELISPOT assays with antigen-specific T-cell lines as responder cells can evaluate DCs'
ability to present the relevant antigen in vitro, in which a defined tumor antigen or peptide represents the immunogen. However, it is often not possible to perform such
assays in a clinical setting, as antigen-specific or tumor-specific T cells usually are not available.