Characterization of Immunoactive and Immunotolerant CD4+ T Cells in Breast Cancer by Measuring Activity of Signaling Pathways That Determine Immune Cell Function

SIMPLE SUMMARY: Immunotherapy enhances the immune response against cancer and is potentially curative. Unfortunately, few patients with breast cancer benefit from this therapy. It is not possible to predict which patients will benefit. A blood cell, called CD4+ T-cell, plays a role in the immune response and in resistance to immunotherapy. Its function is determined by activity of biochemical processes, called signal transduction pathways (STPs). We developed a new technology to measure activity of these STPs, which was used to investigate whether CD4+ T cells function abnormally in breast cancer patients. We show that in CD4+ T-cells from most of the investigated breast cancer patients a number of these STPs are overactive. The abnormal activity of a few notable STPs (Notch and TGFβ) suggests that CD4+ T-cells have changed into regulatory T-cells, which inhibit the immune response against cancer and have been associated with resistance to immunotherapy. We also provide evidence that this change in the CD4+ T- cells is caused by a factor produced by breast cancer cells. We conclude that this new technology can be used to measure STP activity in blood of patients with cancer and has the potential to better identify patients who will benefit from immunotherapy. 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.