We describe use of ImageStream technology (www.amnis.com), which combines quantitative flow cytometry with simultaneous high-resolution digital imaging, to quantify cellular mechanisms of primary immune cells from well-defined patient cohorts. Our studies provide a blueprint for translational investigations to quantify lineage specific cellular responses in small samples from subject cohorts.
Individual variations in immune status determine responses to infection and contribute to disease severity and outcome. Aging is associated with an increased susceptibility to viral and bacterial infections and decreased responsiveness to vaccines with a well-documented decline in humoral as well as cell-mediated immune responses1,2. We have recently assessed the effects of aging on Toll-like receptors (TLRs), key components of the innate immune system that detect microbial infection and trigger antimicrobial host defense responses3. In a large cohort of healthy human donors, we showed that peripheral blood monocytes from the elderly have decreased expression and function of certain TLRs4 and similar reduced TLR levels and signaling responses in dendritic cells (DCs), antigen-presenting cells that are pivotal in the linkage between innate and adaptive immunity5. We have shown dysregulation of TLR3 in macrophages and lower production of IFN by DCs from elderly donors in response to infection with West Nile virus6,7.
Paramount to our understanding of immunosenescence and to therapeutic intervention is a detailed understanding of specific cell types responding and the mechanism(s) of signal transduction. Traditional studies of immune responses through imaging of primary cells and surveying cell markers by FACS or immunoblot have advanced our understanding significantly, however, these studies are generally limited technically by the small sample volume available from patients and the inability to conduct complex laboratory techniques on multiple human samples. ImageStream combines quantitative flow cytometry with simultaneous high-resolution digital imaging and thus facilitates investigation in multiple cell populations contemporaneously for an efficient capture of patient susceptibility. Here we demonstrate the use of ImageStream in DCs to assess TLR7/8 activation-mediated increases in phosphorylation and nuclear translocation of a key transcription factor, NF-κB, which initiates transcription of numerous genes that are critical for immune responses8. Using this technology, we have also recently demonstrated a previously unrecognized alteration of TLR5 signaling and the NF-κB pathway in monocytes from older donors that may contribute to altered immune responsiveness in aging9.
1. Isolation and Stimulation of Immune Cells
2. Labeling of Cells
3. ImageStream Analysis
4. Representative Results
We have quantified signaling pathways in response to a model viral ligand by stimulating PBMCs with the TLR7/8 ligand, R848 (Fig. 1). We collected both cellular localization images and population statistics in our sample groups (Fig. 2). ImageStream allows us to gate cell subsets directly from PBMCs and image cell responses from gated, but unsorted cell populations. Here we quantified the effect of TLR signaling on nuclear localization of NF-κB (p65) in Lin1-, CD4 dim, CD11c+ myeloid DCs (mDCs). At baseline, we observed a high percentage of unstimulated cells with the transcription factor NF-κB (p65) in the cytoplasm. After stimulation, treated cells have a significantly higher similarity score of NF-B (p65) with nuclear stain PI, indicating that NF-κB (p65) translocated into the nucleus after stimulation (median similarity scores are 1.52 and 3.01, respectively, Fig. 2). Similar results are noted in TLR5-stimulated monocytes9.
Figure 1. Staining and Gating Strategy to quantify effects of stimulation on human mDCs. The NF-κB transcription factor regulates expression of numerous immune system genes. This study measures the nuclear localization of NF-κB (p65) in mDCs from one representative subject after TLR7/8 ligand stimulation. In-focus single cells were identified by gating on PI positive events with high nuclear aspect ratios (R1). mDCs (Lin1-, CD4dim, CD11c+) were gated in R3. Nuclear localization of NF-κB (p65) is plotted in R4. Click here to view larger figure.
Figure 2. Translocation of NF-κB in mDCs after stimulation. The translocation of NF-κB (p65) into the nucleus after stimulation (R4) is depicted in populations of untreated and stimulated mDCs; the median similarity score is 1.52 in the untreated sample and 3.01 in the stimulated sample (A). Digital images collected simultaneously of the untreated or R848-stimulated cell populations show representative cells and the intensity of NF-B translocated into the cell nucleus (B). Click here to view larger figure.
The critical steps in use of ImageStream for translational studies are the selection of relevant comparison groups and optimization and validation of antibody specificity. Differences between subject groups in the labeled target will be readily apparent through histograms and cell images. In combination with a thorough analysis of changes in relevant receptors and signaling pathways, these data will provide valuable insight into mechanisms that underlie immune dysregulation in specialized cohorts. The technique will be limited by availability of specific antibodies of sufficient affinity for relevant targets. In addition, the instrument is most suitable for a multi-user facility and the analysis software requires some care to master.
This technology is a powerful technique for translational investigations of human diseases. Advances in genome sequencing and array technology have allowed identification of variants implicated in many diseases, but rarely identify the mechanism of action of the nominated gene. Our studies provide a blueprint for translational investigations in individual subjects such as the nuclear to cytoplasmic ratio of NF-κB after stimulation. From a small sample of blood, we can identify differential processing or signaling in a lineage specific manner in a subject’s own cells. Using this method, we have quantified cellular responses in monocytes of a large cohort of young and elderly subjects and demonstrated alterations in cell signaling in aging9. ImageStream may be applied more broadly to highlight key mechanisms in cells in many investigations such as therapy responders and non-responders, or for an individual subject before and after treatment, or during flare and remission. With abundant modifications possible for choice of subject groups under study, cell types, and cellular mechanisms to investigate, the future applications of ImageStream are numerous and should allow significant advances in many translational areas.
The authors have nothing to disclose.
This work was supported in part by the NIH (N01 HHSN272201100019C, U19 AI 089992, and the NCRR/GCRC Program M01-RR00125). The authors declare no competing financial interests and thank Dr. Mark Shlomchik, director of the Yale Cell Sorter Core Facility.
Name of the reagent | Company | Catalogue number |
Ficoll-Paque Plus | GE Healthcare | 17-1440-03 |
R848 | Invivogen | tlrl-r848 |
Paraformaldehyde (16%) | Electron Microscopy Sciences | 15710 |
BD Perm/Wash | BD Biosciences | 554723 |
CD14-PE | eBioscience | 12-0149-41 |
CD4-PacBlue | BD Pharmingen | 68436 |
Lineage cocktail 1 (lin 1)-FITC | BD Biosciences | 340546 |
CD11c-PE | BD Biosciences | 347637 |
CD123-PE | BD Biosciences | 340545 |
rabbit anti-NF-κB (p65) | SantaCruz | SC-372 |
Alexa647 F(ab’)2 goat anti-rabbit IgG | Invitrogen | A21246 |
DAPI | Invitrogen | D21490 |
ImageStream X | Amnis |
Table 1. Specific reagents and equipment.
Cell type | FITC | PE | Alexa 647 | PacBlue |
Monocyte | CD14 | NF-κB (p65) | ||
mDC | Lin1 | CD11c | NF-κB (p65) | CD4 |
pDC | Lin1 | CD123 | NF-κB (p65) | CD4 |
Table 2. Antibody panel for cell lineage staining of signaling pathways
Monocyte: CD14+
mDC: Lin1-, CD4dim, CD11c+
pDC: Lin1-, CD4dim, CD123+
Lin1 (lineage) marker includes CD3, CD14, CD19, CD20, CD56 markers for monocytes, NK cells, T & B cells.