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Chapter 40 : Monitoring of Immune Response Using Cytokine Flow Cytometry
This chapter describes the optimized methods for cytokine flow cytometry (CFC) that offer increased throughput and robustness. The basic principle of the CFC assay is that whole blood or peripheral blood mononuclear cells (PBMCs) are activated with a specific antigen in the presence of a secretion inhibitor such as brefeldin A (BFA) or monensin for a short duration. The samples in the plates can be acquired using a multiwell-plate loader connected to the flow cytometer. Dynamic gating strategies that account for sample-to-sample staining variability can simplify data analysis and improve the reproducibility of the results. The procedural details discussed in the chapter include recommendations for sample type and handling, choice of antigen(s) and plates, and the gating strategy for this improved CFC format using cytomegalovirus (CMV) pp65 peptide mix as a model antigen. Gating of flow cytometry data for analysis is a frequent source of assay variation, especially in rare-event assays such as CFC where antigen-specific responses may be as low as 0.1%. In situations where large numbers of samples need to be evaluated, e.g., in vaccine immune response-monitoring studies, it is desirable to minimize time and errors that may occur in assay setup and processing. The authors have optimized the process of using lyophilized antigens and lyophilized antibody cocktails for CFC assays of both PBMC and whole blood.
Flow chart of plate-based CFC assay. PBMC or whole blood is added to appropriate plate(s) as described in the text. After 6 h of incubation at 37°C, the cells are processed as described in the protocol. After staining with mAb cocktails for CFC, the cells are acquired on a flow cytometer using a plate loader and the data are batch analyzed. Ag, antigen; FACS Lyse, BD FACS, lysing solution; FACS Perm 2, BD FACS permeabilizing solution 2.
Representative four-color flow cytometric analysis of plate-based CFC. PBMC 106 (A), 200 μl of whole blood (B), or 1 ml of whole blood (C) from a CMV-seropositive donor was stimulated (or not) with the pp65 peptide mix in a 96-well shallow-well plate, a 96-well deep-well plate, or a 24-well deep-well block, respectively. Samples were processed as described in the “Procedure.” The plots were gated on CD3+ CD8+ lymphocytes. Responses and staining intensities of IFN7+ CD69+ cells were comparable in all three assays. R4, snap-to region to identify double-negative population; R5, manually drawn region around cytokine+ CD69+ cells.
Example of CFC data analysis using dynamic gating. Analysis templates are typically defined using a positive control sample (e.g., SEB-activated PBMC or blood). R1 (lymphocytes based on forward- and right-angle light scatter), R2 (CD3+ CD8+ cells), and R3 (CD3+ CD8-, i.e., CD3+ CD4+ cells) are all Snap-To regions. The sizes and the movement of R2 and R3 are maximized to automatically include activated cells that have down-regulated CD3 and CD8 (bold dots) and to track the “roaming” populations that may be a result of variations in reagents, donors, and instrument settings. In the lower left plot that is gated on CD3+ CD8+ cells (R1 and R2), R4 is another Snap-To region (double-negative population) that is tethered to manually drawn region R5 that identifies cytokine-positive events (rare-event response region). The plot on the lower right is gated on CD3+ CD8- cells (R1 and R3), and regions R4 and R5 are selected, copied, and pasted onto this plot. The percentage of gated events in R5 is reported.
Performance of lyophilized reagents in CFC. Whole blood from a CMV-seropositive donor was activated with either liquid (column A) or lyophilized (column B) SEB or the pp65 pep-tide mix. The cells were processed as per the procedure described in the text. Cells were stained with either liquid (column A) or lyophilized (column B) cocktail containing IFN-γ–FITC, CD69-PE, CD8-PerCP-Cy5.5, and CD3-APC. Comparable frequencies (percent positive) and staining intensities (mean fluorescence intensity [MFI]) of CD3, CD8, IFN-γ, and CD69 were observed with both sets of reagents. Significant correlation between liquid and lyophilized reagents was observed in terms of percent positive (C) and staining intensities (D) of responding cells (CD4+ and CD8+ gated; n = 8). R2, region to identify CD8+ T cells; R4, region to identify cytokine+ CD69+ cells; lyo, lyophilized; Ag, antigen; Ab, antibody.
Use of lyophilized cells as process controls. CMV pp65 peptide mix-activated whole blood was lysed or fixed and permeabilized according to the protocol, and three aliquots were processed as described below. (A) Nonlyophilized. The first aliquot was stained with IFN-γ–FITC-CD69-PE-CD8-peridinin chlorophyll protein-Cy5.5-CD3-allophycocyanin, washed twice, and analyzed immediately. (B) Lyophilized then stained. The second aliquot was lyophilized, reconstituted in wash buffer, washed once, and then stained as described above. (C) Stained then lyophilized. The third aliquot was stained as described for panel, A, lyophilized, reconstituted in wash buffer, washed once, and then analyzed. Equivalent frequencies and staining intensities of responding cells were obtained for all three sets of cells.
Example of stimulus preparation per well