Evaluation of Dendritic Cell Products Generated for Human Therapy and Post-Treatment Immune Monitoring - - BioPharm International

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Evaluation of Dendritic Cell Products Generated for Human Therapy and Post-Treatment Immune Monitoring


BioPharm International
Volume 21, Issue 3

SINGLE-CELL ASSAYS IN MONITORING DC-BASED VACCINATION TRIALS

Single-cell assays that detect the frequency of epitope-specific effector T lymphocytes have been used to evaluate immune responses to DC-based cancer vaccines. Among these, CFC, tetramer binding, and ELISPOT measure the frequency of these lymphocytes in the mononuclear cell specimens obtained from the peripheral circulation of vaccinated individuals.42–44 All three assays are based on TCR recognition of cognate peptides presented by MHC Class I or Class II molecules on the surface of DC-presenting antigens to the responder T cells. No consensus exists, however, as to which of the three assays should be used to monitor vaccination results. The perception that these assays are equivalent (i.e., that they provide the same results) may not be correct. The authors have compared the performance of the assays in monitoring the frequency of melanoma peptide-specific CD8+ T cells in the peripheral circulation of subjects with metastatic melanoma who had received multi-epitope DC-based vaccines.45 Concordance among the three assays was estimated using a 3 x 3 scatter plot matrix design constructed for each of the four peptides tested in all three assays before and after vaccination therapy was completed. The three single-cell assays were not found to be concordant in measuring the frequency of immune effector cells in the peripheral blood of vaccinated subjects. The results for tetramer staining were consistently higher than those obtained with the ELISPOT or CFC assays,40 indicating either that tetramers bound to peptide-specific T cells unable to secrete cytokines or that they bound to nonspecific peptide T cells. Similar observations have been reported by others.46

Advantages and Disadvantages of Single-cell Assays

The ELISPOT assay measures the production of cytokines (most commonly IFN-γ or IL-5) by individual T cells in the plated population, with a theoretical detection sensitivity of 1/100,000 cells.47 CFC identifies single-responding T cells (1/50,000) with expression of a cytokine in the Golgi zone. Tetramer binding detects peptide-specific T cells expressing the relevant TCR with a theoretical detection sensitivity of 1/10,000 cells. The assays not only have different sensitivities of detection, but also differ in specificity. ELISPOT and CFC are antibody-based and are highly specific. In contrast, tetramers, which are complexes of peptides sitting in grooves of four MHC molecules held together by a streptavidin-biotin scaffold,48 bind to T lymphocytes expressing the relevant TCR with variable affinity. Tetramers might easily dissociate or non-specifically bind to B cells, monocytes, or apoptotic cells.49,50 Tetramer specificity needs to be carefully controlled. T cells that bind tetramers may not be functional, as TCR signaling could be compromised, as often happens in cancer.46,51 This reduces the tetramer-binding assay to a phenotypic category because the assay detects T cells that bind tetramers but may not be functional.46 CFC measures cytokine expression in a cell and not its secretion (although it is commonly assumed that the expressed cytokine would be secreted). Cell permeabilization necessary for intercellular staining of a cytokine in the Golgi zone might introduce problems with immunodetection in CFC assays.

ELISPOT is based on a similar principle as CFC, but it measures cytokine secretion from stimulated responder cells that are plated as a monolayer of individual cells on a nitrocellulose membrane to avoid cell-to-cell contact and allow for adequate spot display. ELISPOT measures the function of individual responder cells by identifying those that produce and secrete the measured cytokine. ELISPOT does not require cell permeabilization or the use of a flow cytometer for cytokine detection.43 Measuring function rather than phenotype is preferable, making ELISPOT an assay of choice.

However, CFC and tetramer binding are flow cytometry-based assays and allow for surface labeling of responder cells and their identification. It is possible to select CD8+ or CD4+ T-cell subsets on antibody-charged columns before ELISPOT,52 and two-color ELISPOT now available offers the possibility of identifying T cells simultaneously producing two cytokines.53 In addition, supernatants from ELISPOT wells can be collected and tested for cytokine levels in multiplex assays. On the other hand, tetramer binding can be combined with surface staining to determine cellular phenotype and intracytoplasmic staining to detect cytokine production.46 While very informative, especially in situations when some tetramer-binding cells do not express cytokines, this technology is time-consuming and labor-intensive and thus not the best choice for serial monitoring. The recommended solution would be to monitor by ELISPOT or CFC (but not both), depending on sample numbers, time, labor, cost, and access to a flow cytometer, and then use tetramer binding as a confirmatory assay in situations where it is important to demonstrate a functional deficiency of tetramer-binding T cells. ELISPOT performed under strictly controlled, standardized conditions provides accurate estimates of the frequency of functionally competent effector T cells in batched, serial samples obtained from subjects enrolled in clinical studies with DC-based vaccines. Compared to CFC and tetramer binding, the cost of ELISPOT permits its use in a high-volume testing. However, the ELISPOT assay is not easy to standardize, and responder-stimulator interactions might result in unacceptably high background spot counts, making the assay uninterpretable.


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