Polychromatic flow cytometric high-throughput assay to analyze the innate immune response to Toll-like receptor stimulation

Abstract

Polychromatic flow cytometry allows the capture of multidimensional data, providing the technical tool to assess complex immune responses. Interrogation of the adaptive T cell response to infection or vaccination already has benefited greatly from standardized protocols for polychromatic flow cytometric analysis. The innate immune system plays an important role in health and disease, and presents potentially important therapeutic and diagnostic modalities. We describe here a high-throughput polychromatic flow cytometry-based platform that enables the rapid interrogation and large scale screening of human blood antigen presenting cell responses to Toll-like receptor (TLR) ligands and other innate immune modulators. Using this assay, we found that for certain stimuli (e.g., TLR9 and TLR3 ligands), the general protocol for intracellular cytokine cytometry had to be significantly modified to allow response detection. Furthermore, high concentrations of TLR7/8 and TLR4 stimuli caused substantial changes in lineage markers, potentially confounding analysis if one were to use a conventional "lineage-negative" cocktail. The assay we developed is reproducible and has been used to show that a given individual's TLR response pattern is relatively stable over at least several months. This protocol is in strict compliance with published guidelines for polychromatic flow cytometry, provides a common platform for scientists to compare their results directly, and may be applicable to the diagnostic evaluation of Toll-like receptor function and the rapid screening of promising therapeutic innate immune modulators.

Figures

Figure 1. Gating Strategy & Staining Profile for 8-color flow cytometry

Shown is a PBMC sample in A, C, and D, and a whole blood sample in B. Shown in A and B on an unstimulated sample is the gating strategy to identify various innate immune cell subsets: monocytes (MHC-IIpos, CD14pos), B cells (MHC-IIpos, CD19pos), myeloid DCs (MHCIIpos, CD14neg/low, CD123neg, CD11cpos) and plasmacytoid DCs (MHCIIpos, CD14neg/low, CD11cneg, CD123pos). C Shown is a PBMC sample from the same adult subject, either unstimulated (red) or stimulated with 10 uM of the TLR7/8 ligand 3M-003 (blue) for 6 hours in the presence of BFA. Innate immune cell subsets were identified as indicated above, then analyzed for expression of IL-6, IL-12, and IFN-α with TNF-α. C. Shown is a PBMC sample from the same adult subject, either unstimulated (red) or stimulated with TLR7/8 ligand 3M-003 (blue) for 18 hours without any Golgi-blocker. Innate immune cell subsets were identified as indicated above, then analyzed for expression of CD86, CCR7 and CD40.

Figure 2. Permeabilization Impacts Surface Marker Detection

Adult PBMC were either left unstimulated (red) or stimulated with 10 µM of the TLR7/8 ligand 3M-003 (blue) for 18 hours without any Golgi-blocker. Subsequently, samples were either processed as described for intracellular cytokine cytometry (i.e., permeabilized) or left untreated. Both samples were then stained to identify innate immune cell subsets as indicated in Figure 1A. The impact of permeabilization on expression of CD86 and CD40 by monocytes, mDCs and pDCs is shown in the left panels and the impact of permeabilization on CD19 expression is shown on the far-right panel.

Figure 3. Timing of Brefeldin A Addition has Significant Impact on Responses to CpGs

A. Adult PBMC were either left unstimulated (top row), or stimulated with 10 ug/ml of the TLR9 ligand CpGA (lower row) for 6 hours. The Golgi-blocker Brefeldin A (BFA) was added hourly right from the start (0 hours) or up to 5 hours later. All cells were incubated for the same total time of 6 hours, and processed further for intracellular expression of cytokines in the various innate immune cell subsets. Shown is the co-expression of TNF-α and IFN-α in plasmacytoid DCs. B. Graph summarizing co-expression of TNF-α and IFN-α in plasmacytoid DCs in three different adult subjects with the bars indicating the standard deviation.

Figure 4. Brefeldin A is the better Golgi Blocking Reagent for ntracellular Cytokine Analysis in Innate Immune Cells

Adult PBMC were either left unstimulated (top row) or were stimulated with 10 µg/ml of the TLR9 ligand CpGA (lower row) for 6 hours. The Golgi-blockers Brefeldin A (BFA) or Monensin were added 3 hours into the incubation period. Cells were incubated for another 3 hours, then processed further for intracellular expression of cytokines. Shown is the co-expression of TNF-α and IFN-α in plasmacytoid DCs.

Figure 5. Dose Response Profiles for 3M-003 and LPS

Adult PBMC were either left unstimulated or stimulated with different concentrations of the TLR7/8 ligand 3M-003 (A) or the TLR4 ligand LPS (B). C. Adult whole blood samples were either left unstimulated or stimulated with different concentrations of the TLR4 ligand LPS. Shown in all graphs is the result of intracellular cytokine detection in samples stimulated for 6 hours in the presence of BFA. These graphs depict the % cytokine positive cells for each of the cytokines analyzed from 5 different adult subjects (n=5).

Figure 6. Lineage Marker Expression is Altered with TLR stimulation

Adult whole blood (top row) or PBMC (bottom row) were either left unstimulated or stimulated for 6 hours with the indicated concentrations of the TLR4 ligand LPS in the presence of the Golgi-blocker BFA. Samples were processed as described for intracellular cytokine cytometry and analyzed for the surface expression of CD11c and CD14.

Figure 7. Optimal Time Point of Total Incubation Length

Adult PBMC were either left unstimulated or stimulated with 10µM of the TLR7/8 ligand 3M-003. Shown is the result of intracellular cytokine detection in myeloid DC, Monocytes or plasmacytoid DC stimulated for 3 to 9 hours in the presence of BFA. The graphs depict the % cytokine positive cells for each of the cytokines analyzed. This graph is representative of results obtained with 3 different adult subjects.

Figure 8. Precision of the Assay

PBMC from 3 different adults were either left unstimulated or stimulated with 10 µM of the TLR7/8 ligand 3M-003 or 10ng/ml of the TLR4 ligand LPS for 6 hours in the presence of BFA. Triplicate wells were assayed either within the same plate (Same Plate) or across three different plates (Separate Plates). Furthermore, samples from these 3 adult subjects were taken three months apart between the assay using the Same Plate (time = 0 months) and those taken to assay triplicates across 3 different plates (time = 3 months), to estimate changes in a given subject over time. Shown is the result of intracellular TNF-α detection in monocytes (A) and mDCs (B), and for INF-α in pDCs (C); error bars = standard deviation.

Figure 9. Summary of the protocol