Quantitative measurement of activity of JAK-STAT signaling pathways in blood samples and immune cells to predict innate and adaptive cellular immune response to viral infection and accelerate vaccine development

Abstract

The host immune response determines the clinical course of a viral infection, for example in case of COVID-19 infection. The effectiveness of vaccination also depends on the induced immune response. Currently there is no method to measure the cellular immune response in blood samples. The functional activity of cells of innate and adaptive immune system is determined by coordinated activity of signaling pathways, especially the JAK-STAT pathways. Using a previously described approach we developed mRNA-based tests to measure activity of these signaling Page2 pathways, and show that they can be used to measure in a quantitative manner the cellular innate and adaptive immune response to a viral infection or vaccine in whole blood, PBMC, and specific immune cell type samples. Pathway activity level and range in healthy individuals was established, enabling interpretation of a pathway activity score on a patient sample without the need for a reference sample. Evidence is presented that the pathway activity analysis may also be useful for in vitro vaccine development and assessment of vaccine immunogenicity. Other envisioned applications lie in development of immunomodulatory drugs and drug response prediction and monitoring. Tests are expected to be of value in the COVID-19 crisis. In addition to the described Affymetrix microarray-based pathway tests for measuring host immune response, qPCR-based versions are in development; the latter can in principle be performed within three hours in routine hospital labs.

Characterization of immunoactive and immunotolerant CD4+ T cells in breast cancer by measuring activity of signaling pathways that determine immune cell function

“…Subsequently, the comparison was made with CD4+ T cell samples from breast cancer patients to identify the CD4+ T cell subset responsible for immunotolerance in TIL, and to investigate whether the immunotolerant CD4+ T cell pathway profile can also be detected in blood samples from breast cancer patients. The signal transduction pathway assays used in the current study have been biologically validated on multiple cell types, including immune cell types [12]– [14], [16]. Affymetrix expression microarray data were used for the signaling pathway analysis, however, it is good to keep in mind that with this assay technology Affymetrix microarrays are only used as a method to measure a carefully preselected set of mRNA levels, and not to discover a new gene profile.…”

“…Affymetrix expression microarray data were used for the signaling pathway analysis, however, it is good to keep in mind that with this assay technology Affymetrix microarrays are only used as a method to measure a carefully preselected set of mRNA levels, and not to discover a new gene profile. From Affymetrix whole transcriptome data, expression data of only 20-30 pathway target genes per signaling pathway are used to calculate a signaling pathway activity score, the respective genes have been described [12]–[14], [16], [18].…”

“…Tests to quantitatively measure functional activity of PI3K, NFκB, TGFβ, JAK-STAT, and Notch signal transduction pathways on Affymetrix Human Genome U133 Plus 2.0 expression microarrays data have been described before [13], [14], [16], [18]. In brief, the pathway assays are based on the concept of a Bayesian network computational model which calculates from mRNA levels of a selected set, usually between 20 and 30, target genes of the pathway-associated transcription factor a probability score for pathway activity, which is translated to a log2 value of the transcription factor odds, i.e.…”

“…the PI3K, JAK-STAT1/2, JAK-STAT3, NFκB, TGFβ, and Notch pathways [6]–[11]. Recently, assays have been developed which enable quantitative measurement of the activity of these signal transduction pathways in tissue and blood samples [12]–[16].…”

Development of a Notch pathway assay and quantification of functional Notch pathway activity in T-cell acute lymphoblastic leukemia

“…Measurement of the functional activity of these pathways in tumor biopsies from individual patients is expected to improve the prediction of therapy response. We have previously described a novel approach to quantitatively measure activity levels of individual signal transduction pathways in various cell and tissue types [22][23][24][25] . In addition to development of assays to measure activity of the estrogen and androgen receptor pathways, the PI3K, JAK-STAT3, Wnt, Hedgehog, TGFβ, NFκB and JAK-STAT1/2 pathways, we now report the development and biological validation of a quantitative Notch pathway activity assay.…”