Transforming growth factor beta (TGF-␤) is a pleiotropic cytokine known to regulate cell growth, differentiation, and motility and is a potent modulator of immune function. TGF-␤ consequently plays a central role in carcinogenesis, and a dampened TGF-␤2 response by Theileria annulata-infected monocytes/macrophages underpins disease resistance to tropical theileriosis. Here, we show that concomitant with the loss of TGF-␤2 production, there is ablated expression of COX2 and EP4, which leads to a drop in cyclic AMP (cAMP) levels and, consequently, reduced activation of protein kinase A (PKA) and EPAC. This ablated phenotype can be rescued in attenuated macrophages by the addition of exogenous TGF-␤2, which reactivates the expression of COX2 and EP4 while repressing that of protein kinase inhibitor gamma (PKIG) to the levels in virulent macrophages. TGF-␤2 therefore promotes the adhesion and invasiveness of virulent macrophages by modulating COX2, EP4, and PKIG transcription to initiate a prostaglandin E 2 (PGE2)-driven autostimulatory loop that augments PKA and EPAC activities. A virulence phenotype stemming from the double activation of PKA and EPAC is the induction of a CREB-mediated transcriptional program and the upregulation of JAM-L-and integrin 4␣␤1-mediated adhesion of Theileria-infected macrophages.T heileria annulata is a tick-borne apicomplexan parasite and the causative agent of the cattle disease tropical theileriosis, which is of major economic importance in countries in Northern Africa and Asia. T. annulata transforms B cells and monocytes/macrophages, and infected leukocytes display many characteristics of cancer cells, such as heightened migratory and invasive capacities (1, 2). However, and importantly, the tumor-like phenotype is reversed upon drug-induced parasite death (3). Moreover, virulent macrophages can be attenuated by multiple in vitro passages, and upon attenuation, they lose both adhesion and invasiveness (4). The similarities in tumor hyperinvasiveness between Theileria-transformed leukocytes and human leukemias argue that studying Theileria-induced transformation can give insights into generally applicable mechanisms involved in tumor virulence.Transforming growth factor beta (TGF-␤) is a pleiotropic cytokine involved in numerous critical processes such as cell proliferation, differentiation, and survival or apoptosis. In normal cells and cells in early stages of cancer, TGF-␤ acts as a tumor suppressor and inhibits cell growth, while in late stages of cancer, it plays a contrasting role, acting as a tumor promoter enhancing metastasis and invasion. TGF-␤2 is strongly expressed in highly aggressive and malignant tumors (5). We have demonstrated that following infection, Theileria-transformed Holstein-Friesian (HF) (Bos taurus) macrophages produce more TGF-␤2 than do infected Sahiwal (Bos indicus) macrophages, and augmented TGF-␤2 output underpins the greater invasive capacity of infected HF macrophages. Moreover, attenuated Ode HF macrophages used as a live vaccine against tropical ...

Theileria-infected macrophages display many features of cancer cells such as heightened invasive capacity; however, the tumor-like phenotype is reversible by killing the parasite. Moreover, virulent macrophages can be attenuated by multiple in vitro passages and so provide a powerful model to elucidate mechanisms related to transformed macrophage virulence. Here, we demonstrate that in two independent Theileria-transformed macrophage cell lines Grb2 expression is down-regulated concomitant with loss of tumor virulence. Using peptidimer-c to ablate SH2 and SH3 interactions of Grb2 we identify TGF-receptor II and the p85 subunit of PI3-K, as Grb2 partners in virulent macrophages. Ablation of Grb2 interactions reduces PI3-K recruitment to TGF-RII and decreases PIP3 production, and dampens JNK phosphorylation and AP-1-driven transcriptional activity down to levels characteristic of attenuated macrophages. Loss of TGF-R>PI3-K>JNK>AP-1 signaling negatively impacts on virulence traits such as reduced JAM-L/ITG4A and Fos-B/MMP9 expression that contribute to virulent macrophage adhesion and invasiveness.