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

Abstract

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. TGFβ, NFκB, 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, immune-activated, and immune-activated CD4+ T cells incubated with breast cancer tissue supernatants, and of CD4+ Th1, Th2, and Treg cells, and in a clinical study 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, NFκB, JAK-STAT1/2, and JAK-STAT3 pathway activity. Simultaneous incubation with primary cancer supernatant reduced PI3K and NFκB, and partly reduced JAK-STAT3, pathway activity, while simultaneously increasing TGFβ 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 NFκB, JAK-STAT3, TGFβ, and Notch pathway activity. 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. TGFβ). Signaling pathway activity analysis on TIL and/or blood samples is expected to improve predicting and monitoring response to checkpoint inhibitor immunotherapy.

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

“…Our observation that PI3K activity in CD4+ and CD8+ T cells increased upon activation is in line with the role of this pathway to induce clonal proliferation of activated T cells, (Han et al, 2012). The observed inverse relationship of TGFβ pathway activity with CD4+ and CD8+ T cell activation supports our earlier findings that this pathway becomes active in CD4+ T cells upon exposure to breast cancer supernatant, inducing immunotolerance (Wesseling-Rozendaal et al, 2020). The immunosuppressive role of the TGFβ pathway in diseases such as cancer is well known (Li et al, 2007), (Yang, 2015), (Batlle and Massagué, 2019).…”

“…Development and validation of Affymetrix-based assays for measuring activity of the estrogen (ER) and androgen receptor (AR), PI3K-FOXO, MAPK, NFκB, TGFβ, Notch, JAK-STAT1/2, and JAK-STAT3 pathways has been described before, and can in principle be used to analyze pathway activity on any cell type (Bouwman et al, 2020), (Verhaegh et al, 2014), (Stolpe et al, 2019), (Canté-Barrett et al, 2020), (Wesseling-Rozendaal et al, 2021), (van Ooijen et al, 2018) including blood cells (Bouwman et al, 2020), (Wesseling-Rozendaal et al, 2020),(van de , (Bouwman et al, 2021). For the current study, quantitative pathway activity scores (PAS) were measured on Affymetrix expression microarray data.…”

Androgen receptor pathway activity assay for sepsis diagnosis and prediction of favorable prognosis

“…The copyright holder for this preprint this version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.07.21259768 doi: medRxiv preprint [9], [10], [11], [12]. Measuring combined activity of these STPs is expected to enable quantitative assessment of the innate and adaptive immune response in an individual patient [7], [13].…”

“…The functional state of immune cells is determined by a small number of so-called cellular signal transduction pathways (STPs) [4], [5], [6], [7], [8]. Recently, novel assays have been developed to quantitatively measure activity of STPs in cell and tissue samples, including blood samples [9], [10], [11], [12].…”

“…Recently, novel assays have been developed to quantitatively measure activity of STPs in cell and tissue samples, including blood samples [9], [10], [11], [12]. Measuring combined activity of these STPs is expected to enable quantitative assessment of the innate and adaptive immune response in an individual patient [7], [13].…”