van Doorn, A. | bioRxiv Science

bioRxiv2021

Measuring host immune response status by simultaneous quantitative measurement of activity of signal transduction pathways that coordinate functional activity of immune cells from innate and adaptive immune system

For many diseases, including cancer, viral infections such as COVID-19, bacterial infections, and auto-immune diseases, the immune response is a major determinant of progression, response to therapy, and clinical outcome. Innate and adaptive immune response are controlled by coordinated activity of multiple immune cell types. The functional activity state of immune cells is determined by cellular signal transduction pathways (STPs). A novel mRNA-based signaling pathway assay platform has been developed to quantitatively measure relevant STP activities in all types of immune cells and mixed immune cell samples for experimental and diagnostic purposes. We generated a STP activity profile, termed Immune-Pathway Activity Profile (I-PAP), for a variety of immune cell types in resting and activated state, and provide a first example for use in patient samples. MethodsThe technology to measure STP activity has been described for androgen and estrogen receptor, PI3K, MAPK, TGF{beta}, Notch, NF{kappa}B, JAK-STAT1/2, and JAK-STAT3 pathways. STP activity was measured on Affymetrix expression microarray data from preclinical studies containing public data from different types of immune cells, resting/naive or immune-activated in vitro, to establish I-PAPs. Subsequently data from a clinical study on rheumatoid arthritis were analyzed. ResultsI-PAPs of naive/resting and immune-activated CD4+ and CD8+ T cells, T helper cells, B cells, NK cells, monocytes, macrophages, and dendritic cells were established and in agreement with known experimental immunobiology. In whole blood samples of rheumatoid arthritis patients TGF{beta} pathway activity was increased; JAK-STAT3 pathway activity was selectively increased in female patients. In naive CD4+ Tregs TGF{beta} pathway activity was increased, while in memory T effector cells JAK-STAT3 pathway activity tended to increase, suggesting that these immune cell types contributed to whole blood analysis results. ConclusionSTP assay technology (currently being converted to qPCR-based assays) makes it possible to directly measure functional activity of cells of the innate and adaptive immune response enabling quantitative assessment of the immune response of an individual patient. Envisioned utility lies in (1) prediction and monitoring of response to immunomodulatory treatments for a variety of immune-mediated diseases, including RA; (2) uncovering novel treatment targets; (3) improvement and standardization of in vitro immunology research and drug development.

immunology↗

bioRxiv2020

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

Cancer immunotolerance can be reversed by checkpoint blockade immunotherapy in some patients, but response prediction remains a challenge. CD4+ T cells play an important role in activating adaptive immune responses against cancer. Conversion to an immune suppressive state impairs the anti-cancer immune response and is mainly effected by CD4+ Treg cells. A number of signal transduction pathways activate and control functions of CD4+ T cell subsets. As previously described, assays have been developed which enable quantitative measurement of the activity of signal transduction pathways (e.g. TGF{beta}, NF{kappa}B, PI3K-FOXO, JAK-STAT1/2, JAK-STAT3, Notch) in a cell or tissue sample. Using these assays, pathway activity profiles for various CD4+ T cell subsets were defined and cellular mechanisms underlying breast cancer-induced immunotolerance investigated in vitro. Results were used to measure the immune response state in a clinical breast cancer study. MethodsSignal transduction pathway activity scores were measured on Affymetrix expression microarray data of resting and immune-activated CD4+ T cells, immune-activated CD4+ T cells incubated with breast cancer tissue supernatants, CD4+ Th1, Th2, and Treg cells, and of clinical study samples in which CD4+ T cells were derived from blood, lymph node and cancer tissue from primary breast cancer patients (n=10). ResultsIn vitro CD4+ T cell activation induced PI3K, NF{kappa}B, JAK-STAT1/2, and JAK-STAT3 pathway activity. Simultaneous incubation with primary cancer supernatant reduced PI3K and NF{kappa}B, and partly reduced JAK-STAT3, pathway activity, while simultaneously increasing TGF{beta} pathway activity; characteristic of an immune tolerant state. CD4+ Th1, Th2, and Treg cells all had a specific pathway activity profile, with activated immune suppressive Treg cells characterized by high NF{kappa}B, JAK-STAT3, TGF{beta}, and Notch pathway activity scores. An immune tolerant pathway profile was identified in CD4+ T cells from tumor infiltrate of a subset of primary breast cancer patients which could be contributed to activated Treg cells. A Treg pathway profile was also identified in blood samples. ConclusionSignaling pathway assays can be used to quantitatively measure the functional immune response state of lymphocyte subsets in vitro and in vivo. Clinical results suggest that in primary breast cancer the adaptive immune response of CD4+ T cells has frequently been replaced by immunosuppressive Treg cells, potentially causing resistance to checkpoint inhibition. In vitro study results suggest that this effect is mediated by soluble factors from cancer tissue (e.g. TGF{beta}). Signaling pathway activity analysis on TIL and/or blood samples is expected to improve predicting and monitoring response to checkpoint inhibitor immunotherapy.

cancer biology↗