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 may be reversed by checkpoint inhibitor immunotherapy; however, only a subset of patients responds to immunotherapy. The prediction of clinical response in the individual patient remains a challenge. CD4+ T cells play a role in activating adaptive immune responses against cancer, while the conversion to immunosuppression is mainly caused by CD4+ regulatory T cell (Treg) cells. Signal transduction pathways (STPs) control the main functions of immune cells. A novel previously described assay technology enables the quantitative measurement of activity of multiple STPs in individual cell and tissue samples. The activities of the TGFβ, NFκB, PI3K-FOXO, JAK-STAT1/2, JAK-STAT3, and Notch STPs were measured in CD4+ T cell subsets and used to investigate cellular mechanisms underlying breast cancer-induced immunotolerance. Methods: STP activity scores were measured on Affymetrix expression microarray data of the following: (1) resting and immune-activated CD4+ T cells; (2) CD4+ T-helper 1 (Th1) and T-helper 2 (Th2) cells; (3) CD4+ Treg cells; (4) immune-activated CD4+ T cells incubated with breast cancer tissue supernatants; and (5) CD4+ T cells from blood, lymph nodes, and cancer tissue of 10 primary breast cancer patients. Results: CD4+ T cell activation induced PI3K, NFκB, JAK-STAT1/2, and JAK-STAT3 STP activities. Th1, Th2, and Treg cells each showed a typical pathway activity profile. The incubation of activated CD4+ T cells with cancer supernatants reduced the PI3K, NFκB, and JAK-STAT3 pathway activities and increased the TGFβ pathway activity, characteristic of an immunotolerant state. Immunosuppressive Treg cells were characterized by high NFκB, JAK-STAT3, TGFβ, and Notch pathway activity scores. An immunotolerant pathway activity profile was identified in CD4+ T cells from tumor infiltrate and blood of a subset of primary breast cancer patients, which was most similar to the pathway activity profile in immunosuppressive Treg cells. Conclusion: Signaling 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 may be frequently replaced by immunosuppressive Treg cells, potentially causing resistance to checkpoint inhibition. In vitro study results suggest that this is mediated by soluble factors from cancer tissue. Signaling pathway activity analysis on TIL and/or blood samples may improve response prediction and monitoring response to checkpoint inhibitors and may provide new therapeutic targets (e.g., the Notch pathway) to reduce resistance to immunotherapy.

Improved diagnosis of inflammatory bowel disease and prediction and monitoring of response to anti-TNF alpha treatment based on measurement of signal transduction pathway activity

“…We previously reported a completely new method to quantitatively measure activity of clinically relevant signal transduction pathways (STP) on transcriptome data of individual cell-containing samples ( 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 ), ( Bouwman et al, 2020 ), ( Wesseling-Rozendaal et al, 2022 ), ( Bouwman et al, 2021 ). Using this technology to analyze RNA expression data of colon mucosa biopsy samples, signaling pathway activity was investigated in CD and UC, and related to pathophysiology of IBD and the clinical response to anti-TNFα treatment.…”

“…Development and validation of computational models to quantitatively measure activity of the androgen receptor (AR), MAPK, NFκB, WNT, TGFβ, JAK-STAT1/2, and JAK-STAT3 pathways have been described before and is simplified shown in Supplementary Figure S1 . The models can in principle be used to analyze pathway activity on any cell type, based on analysis of transcriptome data ( 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 ), ( Bouwman et al, 2020 ), ( Wesseling-Rozendaal et al, 2022 ), ( Bouwman et al, 2021 ). For the current study, pathway activity scores (PAS) were measured on publicly available Affymetrix expression microarray data from preclinical and clinical IBD studies.…”

A Metabolic Gene Signature to Predict Breast Cancer Prognosis

“…Some metabolic proteins have been found to be overexpressed on intratumoral Tregs of patients with BC ( Malla et al, 2022 ) . In primary BC, the adaptive immune response of CD4 + T cells may often be replaced by immunosuppressive Treg cells, which may induce resistance to checkpoint inhibition ( Wesseling-Rozendaal et al, 2022 ).…”

The Notch Signaling Pathway Contributes to Angiogenesis and Tumor Immunity in Breast Cancer

“…146 TNBC is sensitive to PF-03084014, and the Notch inhibitor PF-03084014 combined with the AKT 149 Nevertheless, GISs cannot target specific receptors; thus, they also bring about adverse effects such as nausea and vomiting, which appear to be very limiting as monotherapy drugs. 140 The development of antibodies that specifically inhibit members of the Notch family indicates a step toward a solution to this problem. There are currently two main classes of such antibodies.…”

“… 107 Recently, analytical methods capable of quantitatively measuring the activity of signal transduction pathways in tissue and blood samples have been developed, and the pathway analysis revealed that Treg cells in breast cancer have high Notch pathway activity scores, and Notch is expected to be a new target for the characterization of immune activity and immunosuppressive therapy in BC. 107 , 140 In summary, Notch signaling has important implications for Th17/Treg in BC.…”