T cell receptor (TCR) signal strength determines the differentiation outcome of naïve CD4+ T cells: low signal strength favors Foxp3pos regulatory T cells (Treg) whereas high TCR signals are required to induce IL-2-producing helper T cells (Th). To better understand the signaling requirements for this cell-fate decision, we constructed a logic circuit model of the TCR signaling pathways. A major feature of this model is an incoherent feed-forward loop involving activation of Foxp3 and its inhibition by mTOR, which leads to transient appearance of Foxp3pos cells under simulation conditions that drive IL-2 producing Th cells. This behavior along with the predicted ability of TGF-β to induce Treg despite continued activation of the Akt/mTOR pathway were confirmed experimentally. The latter provides a possible reason for the observed instability of TGF-β-induced Treg. The model also predicted and experiments confirmed that transient high dose Ag stimulation results in three stable T cell fates (Th, Treg, and non-activated) with relative proportions depending on the duration of stimulation. Experimental analysis of the cell population at the time of Ag removal identified three distinct populations based on CD25 abundance and Akt/mTOR activation that correlated with these T cell fates. Further analysis of corresponding simulation trajectories implicated a negative feedback loop involving Foxp3, PTEN, and Akt/mTOR. Taken together, these results suggest that there is a critical period following TCR stimulation during which heterogeneity in the differentiating population leads to increased plasticity of cell fate.

Signaling via the Akt/mTOR pathway influences CD4+ T cell differentiation; low levels favor Treg induction and high levels favor Th induction. Although the lipid phosphatase PTEN suppresses Akt activity the control of PTEN activity is poorly studied in T cells. Here, we identify multiple mechanisms that regulate PTEN expression. During Th induction, PTEN function is suppressed via lower mRNA levels, lower protein levels and an increase in C terminal phosphorylation. Conversely, during Treg induction, PTEN function is maintained through the stabilization of PTEN mRNA transcription and sustained protein levels. We demonstrate that differential Akt/mTOR signaling regulates PTEN transcription via the FoxO1 transcription factor. A mathematical model that includes multiple modes of PTEN regulation recapitulates our experimental findings and demonstrates how several feedback loops determine differentiation outcomes. Together, this work provides novel mechanistic insights into how differential regulation of PTEN controls alternate CD4+ T cell fate outcomes.

Patients with ulcerative colitis have an increased risk of developing colitis-associated colon cancer (CACC). Changes in glycosylation of the oncoprotein MUC1 commonly occur in chronic inflammation, including ulcerative colitis, and this abnormally glycosylated MUC1 promotes cancer development and progression. It is not known what causes changes in glycosylation of MUC1. Gene expression profiling of myeloid cells in inflamed and malignant colon tissues showed increased expression levels of inflammatory macrophage–associated cytokines compared with normal tissues. We analyzed the involvement of macrophage-associated cytokines in the induction of aberrant MUC1 glycoforms. A coculture system was used to examine the effects of M1 and M2 macrophages on glycosylation-related enzymes in colon cancer cells. M2-like macrophages induced the expression of the glycosyltransferase ST6GALNAC1, an enzyme that adds sialic acid to O-linked GalNAc residues, promoting the formation of tumor-associated sialyl-Tn (sTn) O-glycans. Immunostaining of ulcerative colitis and CACC tissue samples confirmed the elevated number of M2-like macrophages as well as high expression of ST6GALNAC1 and the altered MUC1-sTn glycoform on colon cells. Cytokine arrays and blocking antibody experiments indicated that the macrophage-dependent ST6GALNAC1 activation was mediated by IL13 and CCL17. We demonstrated that IL13 promoted phosphorylation of STAT6 to activate transcription of ST6GALNAC1. A computational model of signaling pathways was assembled and used to test IL13 inhibition as a possible therapy. Our findings revealed a novel cellular cross-talk between colon cells and macrophages within the inflamed and malignant colon that contributes to the pathogenesis of ulcerative colitis and CACC. See related Spotlight on p. 160

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Abstract. Biomedical research results are being published at a high rate, and with existing search engines, the vast amount of published work is usually easily accessible. However, reproducing published results, either experimental data or observations is often not viable. In this work, we propose a framework to overcome some of the issues of reproducing previous research, and to ensure re-usability of published information. We present here a framework that utilizes the results from state-of-theart biomedical literature mining, biological system modeling and analysis techniques, and provides means to scientists to assemble and reason about information from voluminous, fragmented and sometimes inconsistent literature. The overall process of automated reading, assembly and reasoning can speed up discoveries from the order of decades to the order of hours or days. Our framework described here allows for rapidly conducting thousands of in silico experiments that are designed as part of this process.