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

Re-confirmation of PGE₂ as the molecule behind tumor-induced perturbation in CD4⁺ T cell survival signaling.

<p>A, MCF-7 cells were treated with theaflavins or celecoxib or transfected with Cox-2-siRNA and the levels of Cox-2 and GAPDH (internal control) mRNA were determined by RT-PCR (<i>upper panel</i>). Western blot analysis was performed for the determination of levels of Cox-2 or α-Actin (internal control) proteins (<i>middle panel</i>). In parallel experiments the amount of tumor-secreted PGE₂ in the cell-free supernatant was determined by ELISA (<i>lower panel</i>). B, Purified CD4⁺ T cells were cultured in the presence of media alone or MCF-7-spent media (tumors were either pre-treated with 25 µg/ml theaflavins/3.5 ng/ml PGE₂/50 µM celecoxib or transfected with 300pmole Cox-2-siRNA) for 48 h. Expression levels of IL2Rγc and Bcl-2 as well as phosphorylation status of Jak-3/-Stat-5 were determined by Western blotting in which α-Actin was used as internal control (<i>upper panel</i>). In parallel experiments, flowcytometric determination of percent cell death (<i>lower panel</i>) was established. Values are mean±S.E.M. of three independent experiments.</p

Tumor-shed PGE₂ is responsible for CD4⁺ T cell apoptosis.

<p>A, Tumor-secreted PGE₂ over time in cell-free spent media of MCF-7 cells (control (○), Cox-2-siRNA-transfected (Δ) or theaflavin-treated (•) was determined by ELISA. B, Percent CD4⁺ T cell death (Annexin-V-PE⁺/7AAD⁺), induced by the spent media as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007382#pone-0007382-g002" target="_blank">Fig. 2A</a>, was plotted over time. Values are mean±S.E.M. of three independent sets of experiments.</p

Stat-5A transfection confers resistance to CD4⁺ T cells from tumor-induced death.

<p>A, Stat-5A (<i>left panel</i>) and Stat-5B (<i>right panel</i>) isoforms were immunoprecipitated from cell lysates using specific antibodies and then Western blotted with anti-phospho-tyrosine or anti-Stat-5A/Stat-5B antibodies to determine phosphorylation status of specific proteins. B, Jurkat T cells were transfected with control vector, wild-type <i>Stat-5A/Stat-5B</i>, C-terminal truncated <i>Stat-5A</i>₇₁₃/<i>Stat-5B</i>₇₁₈ or constitutively active <i>Stat-5A1*6</i> genes and were cultured in the presence of media alone or MCF-7 spent media (±theaflavins) for 48 h. Percent cell death (Annexin-V-PE⁺/7AAD⁺) was determined flow cytometrically. Values are mean±S.E.M. of three independent sets of experiments.</p

Cell free tumor supernatant leads to CD4⁺ T cell depletion by inducing apoptosis.

<p>A, Purified human peripheral CD4⁺ T cells were cultured in the presence of media alone or cell-free MCF-7-spent media (±theaflavins, doses from 6.25 µg/ml to 50 µg/ml). After 48 hours, viable cell numbers were scored by Trypan Blue exclusion method. B, Graphical representation of percent apoptosis of CD4⁺ T cells (<i>left panel</i>) and Jurkat T cells (<i>right panel</i>). CD4⁺ T cells labelled with Annexin V-PE and 7AAD were analyzed flow cytometrically. Annexin V/7AAD-positive cells were regarded as apoptotic cells. Values are mean±S.E.M. of five independent sets of experiments.</p

Consensus guidelines for the definition, detection and interpretation of immunogenic cell death

Cells succumbing to stress via regulated cell death (RCD) can initiate an adaptive immune response associated with immunological memory, provided they display sufficient antigenicity and adjuvanticity. Moreover, multiple intracellular and microenvironmental features determine the propensity of RCD to drive adaptive immunity. Here, we provide an updated operational definition of immunogenic cell death (ICD), discuss the key factors that dictate the ability of dying cells to drive an adaptive immune response, summarize experimental assays that are currently available for the assessment of ICD in vitro and in vivo, and formulate guidelines for their interpretation

Consensus guidelines for the definition, detection and interpretation of immunogenic cell death

Cells succumbing to stress via regulated cell death (RCD) can initiate an adaptive immune response associated with immunological memory, provided they display sufficient antigenicity and adjuvanticity. Moreover, multiple intracellular and microenvironmental features determine the propensity of RCD to drive adaptive immunity. Here, we provide an updated operational definition of immunogenic cell death (ICD), discuss the key factors that dictate the ability of dying cells to drive an adaptive immune response, summarize experimental assays that are currently available for the assessment of ICD in vitro and in vivo, and formulate guidelines for their interpretation.Peer reviewe