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 Clinical results suggest that in primary breast cancer the adaptive immune response of CD4+ T cells has frequently been replaced by immunosuppressive Treg cells, potentially causing resistance to checkpoint inhibition.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.

Thin films were made by spinning a dispersion of tin-doped indium oxide particles, having an average diameter of 14 nm, onto glass substrates. As-deposited thin films displayed a resistivity ρ of 0.3 Ω m and some optical absorption. Annealing in vacuum at 200–400̂C for 2 h, and subsequently in air at 500̂C for 2 h, produced films with ρ ≈ 10−3 Ω m and a visible transmittance exceeding 90%. Leaving out the vacuum treatment yielded higher resistivity.

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.

Tumor cells can induce immunotolerance, which is reversed by checkpoint blockade immunotherapy in some patients, although response prediction remains a challenge. CD4+ T cells play an important role in activating adaptive immune responses with their conversion to a suppressed state impairing anti-tumor immune responses. CD4+ T cells function by activating and controlling various signal transduction pathways. Over the past decade we have developed tests that quantitatively measure functional activities of signal transduction pathways (e.g. Hedgehog, Wnt, TGFβ Notch, NFκB, PI3K, JAK-STAT 1/2 and 3, and MAPK). They are based on Bayesian computational model inference of pathway activities from measurements (expression microarray, qPCR) of mRNA levels of target genes of the transcription factor associated with the respective signalling pathways1,2. These tests were extensively biologically validated, including on immune cells, and can be used to characterize their functional activity status. In the present study, this approach was used to investigate cellular mechanisms underlying breast cancer-induced immunosuppression of CD4+ T cells. Method: Generation of Affymetrix gene expression data has been previously described (J Clin Invest 2013;123(7):2873-92) and data is publically available (GEO dataset GSE36766). Briefly, breast cancer tissue sections from fresh surgical specimens were mechanically dissociated in X-VIVO 20. Following activation with anti-CD3/CD28, CD4+ T cells from healthy donor blood were incubated with primary tumor supernatants (SN) and compared to controls. Signaling pathway activities were measured using Affymetrix expression data from the individual CD4+ T cell treatment groups. Results: CD4+ T cell activation resulted in induction of PI3K, NFkB, JAK-STAT1/2, JAK-STAT3, Notch, and parallel decrease in TGFβ pathway activities. Incubation with primary tumor SN did not affect pathway activity in non-activated CD4+ T cells, but reduced activity of PI3K, NFκB, JAK-STAT1/2, JAK-STAT3, Notch, while increasing TGFβ pathway activity in activated CD4+ T cells. Conclusion: A soluble factor(s) from breast tumor tissues increases TGFβ and reduces effector immune pathway activity in activated CD4+ T cells and thereby can induce an immunotolerant state. Investigation into the nature of this soluble factor(s) is in progress. These data demonstrate that signaling pathway assays can be used to quantitatively measure the functional state of immune responses in CD4+ lymphocytes. The ultimate goal is to apply this approach for predicting and monitoring immunotherapy responses and identifying novel drug targets that can reverse tumor-induced immunosuppression. Ref: 1. Verhaegh W, et al. Cancer Res 2014;74(11):2936-45; 2. Ooijen H. van, et al. Am J Pathol 2018;188(9):1956-1972. Citation Format: Anja Van De Stolpe, Wim Verhaegh, Arie van Doorn, Grégory Noël, Chunyan Gu-Trantien, Karen Willard-Gallo. Breast cancer induces tolerogenic state of healthy activated CD4+ lymphocytes, characterized by reduced PI3K, NFκB, JAK-STAT, Notch, and increased TGFβpathway activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2371.

Thin transparent and electrically conductive films of tin doped indium oxide (ITO) were made by sintering of nanoparticle dispersions. The resistivity decreased to 1. 10-2 Qcm upon treatment at 800'C, while the luminous transmittance remained high. The property evolution was connected with sintering and densification as studied by Scanning Electron Microscopy, X-ray Diffraction, X-ray Fluorescence and Elastic Recoil Detection Analysis.

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. Methods. The technology to measure STP activity has been described for androgen and estrogen receptor, PI3K, MAPK, TGFB;, Notch, NFkB, 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. Results. I-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 TGFβ pathway activity was increased; JAK-STAT3 pathway activity was selectively increased in female patients. In naive CD4+ Tregs TGFB; 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. Conclusion. STP 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.

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