Tumor-Shed PGE2 Impairs IL2Rγc-Signaling to Inhibit CD4+ T Cell Survival: Regulation by Theaflavins

BACKGROUND:Many tumors are associated with decreased cellular immunity and elevated levels of prostaglandin E2 (PGE2), a known inhibitor of CD4+ T cell activation and inducer of type-2 cytokine bias. However, the role of this immunomodulator in the survival of T helper cells remained unclear. Since CD4+ T cells play critical roles in cell-mediated immunity, detail knowledge of the effect tumor-derived PGE2 might have on CD4+ T cell survival and the underlying mechanism may, therefore, help to overcome the overall immune deviation in cancer. METHODOLOGY/PRINCIPAL FINDINGS:By culturing purified human peripheral CD4+ T cells or Jurkat cells with spent media of theaflavin- or celecoxib-pre-treated MCF-7 cells, we show that tumor-shed PGE2 severely impairs interleukin 2 receptor gammac (IL2Rgammac)-mediated survival signaling in CD4+ T cells. Indeed, tumor-shed PGE2 down-regulates IL2Rgammac expression, reduces phosphorylation as well as activation of Janus kinase 3 (Jak-3)/signal transducer and activator of transcription 5 (Stat-5) and decreases Bcl-2/Bax ratio thereby leading to activation of intrinsic apoptotic pathway. Constitutively active Stat-5A (Stat-5A1 6) over-expression efficiently elevates Bcl-2 levels in CD4+ T cells and protects them from tumor-induced death while dominant-negative Stat-5A over-expression fails to do so, indicating the importance of Stat-5A-signaling in CD4+ T cell survival. Further support towards the involvement of PGE2 comes from the results that (a) purified synthetic PGE2 induces CD4+ T cell apoptosis, and (b) when knocked out by small interfering RNA, cyclooxygenase-2 (Cox-2)-defective tumor cells fail to initiate death. Interestingly, the entire phenomena could be reverted back by theaflavins that restore cytokine-dependent IL2Rgammac/Jak-3/Stat-5A signaling in CD4+ T cells thereby protecting them from tumor-shed PGE2-induced apoptosis. CONCLUSIONS/SIGNIFICANCE:These data strongly suggest that tumor-shed PGE2 is an important factor leading to CD4+ T cell apoptosis during cancer and raise the possibility that theaflavins may have the potential as an effective immunorestorer in cancer-bearer

The Janus kinases (Jaks)

The Janus kinase (Jak) family is one of ten recognized families of non-receptor tyrosine kinases. Mammals have four members of this family, Jak1, Jak2, Jak3 and Tyrosine kinase 2 (Tyk2). Birds, fish and insects also have Jaks. Each protein has a kinase domain and a catalytically inactive pseudo-kinase domain, and they each bind cytokine receptors through amino-terminal FERM (Band-4.1, ezrin, radixin, moesin) domains. Upon binding of cytokines to their receptors, Jaks are activated and phosphorylate the receptors, creating docking sites for signaling molecules, especially members of the signal transducer and activator of transcription (Stat) family. Mutations of the Drosophila Jak (Hopscotch) have revealed developmental defects, and constitutive activation of Jaks in flies and humans is associated with leukemia-like syndromes. Through the generation of Jak-deficient cell lines and gene-targeted mice, the essential, nonredundant functions of Jaks in cytokine signaling have been established. Importantly, deficiency of Jak3 is the basis of human autosomal recessive severe combined immunodeficiency (SCID); accordingly, a selective Jak3 inhibitor has been developed, forming a new class of immunosuppressive drugs

Rift Valley Fever Virus NSs Protein Promotes Post-Transcriptional Downregulation of Protein Kinase PKR and Inhibits eIF2α Phosphorylation

Rift Valley fever virus (RVFV) (genus Phlebovirus, family Bunyaviridae) is a negative-stranded RNA virus with a tripartite genome. RVFV is transmitted by mosquitoes and causes fever and severe hemorrhagic illness among humans, and fever and high rates of abortions in livestock. A nonstructural RVFV NSs protein inhibits the transcription of host mRNAs, including interferon-β mRNA, and is a major virulence factor. The present study explored a novel function of the RVFV NSs protein by testing the replication of RVFV lacking the NSs gene in the presence of actinomycin D (ActD) or α-amanitin, both of which served as a surrogate of the host mRNA synthesis suppression function of the NSs. In the presence of the host-transcriptional inhibitors, the replication of RVFV lacking the NSs protein, but not that carrying NSs, induced double-stranded RNA-dependent protein kinase (PKR)–mediated eukaryotic initiation factor (eIF)2α phosphorylation, leading to the suppression of host and viral protein translation. RVFV NSs promoted post-transcriptional downregulation of PKR early in the course of the infection and suppressed the phosphorylated eIF2α accumulation. These data suggested that a combination of RVFV replication and NSs-induced host transcriptional suppression induces PKR-mediated eIF2α phosphorylation, while the NSs facilitates efficient viral translation by downregulating PKR and inhibiting PKR-mediated eIF2α phosphorylation. Thus, the two distinct functions of the NSs, i.e., the suppression of host transcription, including that of type I interferon mRNAs, and the downregulation of PKR, work together to prevent host innate antiviral functions, allowing efficient replication and survival of RVFV in infected mammalian hosts

Impaired chemokine-induced migration during T-cell development in the absence of Jak 3

The arrival of bone marrow T-cell progenitors to the thymus, and the directed migration of thymocytes, are thought to be regulated by the expression of chemokines and their receptors. Recent data has shown that the Jak\Stat signalling pathway is involved in chemokine receptor signalling. We have investigated the role of Jak 3 in chemokine-mediated signalling in the thymus using Jak 3(–\–) mice. These mice show defects in T-cell development, as well as in peripheral T-cell function, resulting in a hypoplastic thymus and an altered T-cell homeostasis. Here we demonstrate, for the first time, that bone marrow progenitors and thymocytes from Jak 3(–\–) mice have decreased chemotactic responses to CXCL12 and CCL25. We also show that Jak 3 is involved in signalling through CCR9 and CXCR4, and that specific inhibition of Jak 3 in wild-type progenitors and thymocytes decreases their chemotactic responses towards CCL25 and CXCL12. Finally, quantitative reverse transcription–polymerase chain reaction analysis showed that thymocytes from Jak 3(–\–) mice express similar levels of CXCR4 and CCR9 compared to wild-type mice. Altogether, deficient CCL25- and CXCL12-induced migration could result in a homing defect of T-cell progenitors to the thymus, as well as in a deficient thymocyte migration through the thymic stroma. Our results strongly suggest that the absence of Jak 3 affects T-cell development, not only through an impaired interleukin-7 receptor (IL-7R)-mediated signalling, but also through impaired chemokine-mediated responses, which are crucial for thymocyte migration and differentiation

Janus kinase 3-deficient T lymphocytes have an intrinsic defect in CCR7-mediated homing to peripheral lymphoid organs

Chemokine-mediated signalling involves the activation of a Janus kinase (Jak) pathway. We have previously shown that Jak3 mediates CCR9 and CXCR4 signalling in response to CCL25 and CXCL12 in BM progenitors and thymocytes. The lack of peripheral lymph nodes and Peyer's patches observed in Jak3–/– mice suggested a possible role of Jak3 in CCR7-mediated homing to these organs. Here, we demonstrate phosphorylation of Jak3 in peripheral lymphocytes in response CCL19 and CCL21. In addition, Jak3–/– naïve T cells and pharmacologically inhibited Jak3+/+ T lymphocytes have impaired chemotactic responses towards these ligands. Interestingly, CCR7 expression was higher in Jak3–/– thymocytes compared to their Jak3+/– littermates, indicating that the impaired migration must be caused by impaired CCR7-mediated signalling, in the absence of Jak3. In addition, adoptive transfer experiments showed that Jak3+/+ mice reconstituted with Jak3–/– green fluorescent protein (GFP)+ bone marrow progenitors had reduced T-lymphocyte homing to peripheral and mesenteric lymph nodes, compared to reconstitution with Jak3+/+ GFP+ progenitors. Furthermore, reciprocal transfer experiments indicated that Jak3–/– stromal cells were not responsible for the deficient T-cell homing. Finally, we performed direct competitive homing assays and demonstrated that Jak3–/– T lymphocytes have a clear defect in homing to peripheral and mesenteric lymph nodes, while migration to spleen was moderately impaired. Our data demonstrates that Jak3–/– T lymphocytes have an intrinsic defect in CCR7-mediated homing to peripheral lymphoid organs

Essential biphasic role for JAK3 catalytic activity in IL-2 receptor signaling

To drive lymphocyte proliferation and differentiation, common γ-chain (γ(c)) cytokine receptors require hours to days of sustained stimulation. While JAK1 and JAK3 kinases are found together in all γ(c)-receptor complexes, it is not known how their respective catalytic activities contribute to signaling over time. Here, we dissect the temporal requirements for JAK3 kinase activity with a selective covalent inhibitor (JAK3i). By monitoring STAT5 phosphorylation over 20 hours in IL-2-stimulated CD4+ T cells, we document a previously unappreciated second wave of signaling that is much more sensitive to JAK3i than the first wave. Selective inhibition of this second wave is sufficient to block cyclin expression and S-phase entry. An inhibitor-resistant JAK3 mutant (Cys905Ser) rescued all effects of JAK3i in isolated T cells and in mice. Our chemical genetic toolkit elucidates a biphasic requirement for JAK3 kinase activity in IL-2-driven T-cell proliferation and will find broad utility in studies of γ(c)-receptor signaling