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Review
. 2013:2013:726239.
doi: 10.1155/2013/726239. Epub 2013 Sep 30.

Immune monitoring in cancer vaccine clinical trials: critical issues of functional flow cytometry-based assays

Iole Macchia  1 , Francesca Urbani, Enrico Proietti
Affiliations
Review

Immune monitoring in cancer vaccine clinical trials: critical issues of functional flow cytometry-based assays

Iole Macchia et al. Biomed Res Int. 2013.

Abstract

The development of immune monitoring assays is essential to determine the immune responses against tumor-specific antigens (TSAs) and tumor-associated antigens (TAAs) and their possible correlation with clinical outcome in cancer patients receiving immunotherapies. Despite the wide range of techniques used, to date these assays have not shown consistent results among clinical trials and failed to define surrogate markers of clinical efficacy to antitumor vaccines. Multiparameter flow cytometry- (FCM-) based assays combining different phenotypic and functional markers have been developed in the past decade for informative and longitudinal analysis of polyfunctional T-cells. These technologies were designed to address the complexity and functional heterogeneity of cancer biology and cellular immunity and to define biomarkers predicting clinical response to anticancer treatment. So far, there is still a lack of standardization of some of these immunological tests. The aim of this review is to overview the latest technologies for immune monitoring and to highlight critical steps involved in some of the FCM-based cellular immune assays. In particular, our laboratory is focused on melanoma vaccine research and thus our main goal was the validation of a functional multiparameter test (FMT) combining different functional and lineage markers to be applied in clinical trials involving patients with melanoma.

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Figures

Figure 1
Figure 1
Comparison of intracellular and cell surface markers after treatment of cells with two different fixation/permeabilization buffers. Assessment of cytokine secretion and cytotoxic factor expression in CD8+ T-cells. Briefly, thawed PBMC from a healthy donor was cultured (1 hour at 37°C) in presence of anti-CD107a and Staphylococcus enterotoxin B (SEB; Sigma-Aldrich, Munich, Germany, used at 2 μg/mL) or PHA (HA16, Murex Biotech, Dartford, UK, used at 1.5 μg/mL) in presence of costimulatory antibodies (CD28 and CD49d). After the addition of brefeldin A (Golgi Plug) and monensin (Golgi stop) (Becton Dickinson, San Jose, CA, USA), cells were incubated for additional 5 hours. Following stimulation, final 2 mM EDTA was added to each well and incubated for 15 minutes. Cells were then incubated for 30 min at 4°C with surface antibodies (CD8), fixed, and permeabilized with the previously mentioned lysing/permeabilization buffers and stained with fluorescently labelled antibodies directed against IL-2 and TNF-α. Samples were then acquired on a FacsCanto flow cytometer instrument (BD Biosciences) and analyzed by FACSDiva and/or FlowJo software (Tree Star, Ashland). (a) Bar graph showing the percentages of total CD107a+, TNF-α +, and IL-2+ analyzed within CD8+ gated cells. (b) Bar graph showing the polyfunctionality of CD8+ T-cells upon SEB stimulation (Boolean analysis). As negative controls, we included untreated cell (only costimuli).
Figure 2
Figure 2
Comparison of different MHC multimers for detection of antigen-specific T-cells: dot plots representing percentages of CD8+ MART-1+ tetramer+/pentamer+/dextramers+ cells, analyzed within the singlets-live gate of a CD8+ expanded line obtained from a healthy donor.
Figure 3
Figure 3
Validation of FMT (representative example of a melanoma patient). (a) Sequential gating strategy. Representative example dot plots and FACSDIVA analysis. Forward-scatter (FSC) area versus FSC height parameters were used to exclude cell doublets; cells were gated by forward and side scatter for lymphocytes; gated populations are plotted as CD8 (horizontal axis) versus tetramer staining (vertical axis). Direct ex vivo analysis cytokine production (IFNα, TNFγ, IL-2) and degranulation CD107a/LAMP-1) within CD8+ population or CD8+/MART-1 tetramer gated T-cells after stimulation with Melan-A/MART-1 peptide. (b) Histogram plots representing the individual functional combinations as a proportion of the total responding cells after stimulation with Melan-A/MART-1 peptide. Mutual exclusion, red bars: Percentage of cells expressing a certain combination of parameters (+) and not expressing the parameter indicated as (−). No mutual exclusion, blue bars: percentage of cells expressing a certain combination of parameters (+) independently from the expression of the parameter indicated with (−). The pie slices indicate the average proportion of the response producing 1, 2, 3, or 4 functions (regarding in this case “no mutual exclusion” variables). Each slice indicates one of the functions.
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