Regulatory T cells and human myeloid dendritic cells promote tolerance via programmed death ligand-1.

Immunotherapy using regulatory T cells (Treg) has been proposed, yet cellular and molecular mechanisms of human Tregs remain incompletely characterized. Here, we demonstrate that human Tregs promote the generation of myeloid dendritic cells (DC) with reduced capacity to stimulate effector T cell responses. In a model of xenogeneic graft-versus-host disease (GVHD), allogeneic human DC conditioned with Tregs suppressed human T cell activation and completely abrogated posttransplant lethality. Tregs induced programmed death ligand-1 (PD-L1) expression on Treg-conditioned DC; subsequently, Treg-conditioned DC induced PD-L1 expression in vivo on effector T cells. PD-L1 blockade reversed Treg-conditioned DC function in vitro and in vivo, thereby demonstrating that human Tregs can promote immune suppression via DC modulation through PD-L1 up-regulation. This identification of a human Treg downstream cellular effector (DC) and molecular mechanism (PD-L1) will facilitate the rational design of clinical trials to modulate alloreactivity.

Host-based Th2 cell therapy for prolongation of cardiac allograft viability.

Donor T cell transfusion, which is a long-standing approach to prevent allograft rejection, operates indirectly by alteration of host T cell immunity. We therefore hypothesized that adoptive transfer of immune regulatory host Th2 cells would represent a novel intervention to enhance cardiac allograft survival. Using a well-described rat cardiac transplant model, we first developed a method for ex vivo manufacture of rat host-type Th2 cells in rapamycin, with subsequent injection of such Th2.R cells prior to class I and class II disparate cardiac allografting. Second, we determined whether Th2.R cell transfer polarized host immunity towards a Th2 phenotype. And third, we evaluated whether Th2.R cell therapy prolonged allograft viability when used alone or in combination with a short-course of cyclosporine (CSA) therapy. We found that host-type Th2.R cell therapy prior to cardiac allografting: (1) reduced the frequency of activated T cells in secondary lymphoid organs; (2) shifted post-transplant cytokines towards a Th2 phenotype; and (3) prolonged allograft viability when used in combination with short-course CSA therapy. These results provide further support for the rationale to use "direct" host T cell therapy for prolongation of allograft viability as an alternative to "indirect" therapy mediated by donor T cell infusion.

Rapamycin generates anti-apoptotic human Th1/Tc1 cells via autophagy for induction of xenogeneic GVHD.

Murine T cells exposed to rapamycin maintain flexibility towards Th1/Tc1 differentiation, thereby indicating that rapamycin promotion of regulatory T cells (Tregs) is conditional. The degree to which rapamycin might inhibit human Th1/Tc1 differentiation has not been evaluated. In the presence of rapamycin, T cell costimulation and polarization with IL-12 or IFN-α permitted human CD4+ and CD8+ T cell differentiation towards a Th1/Tc1 phenotype; activation of STAT1 and STAT4 pathways essential for Th1/Tc1 polarity was preserved during mTOR blockade but instead abrogated by PI3 kinase inhibition. Such rapamycin-resistant human Th1/Tc1 cells: (1) were generated through autophagy (increased LC3BII expression; phenotype reversion by autophagy inhibition via 3-MA or siRNA for Beclin1); (2) expressed anti-apoptotic bcl-2 family members (reduced Bax, Bak; increased phospho-Bad); (3) maintained mitochondrial membrane potentials; and (4) displayed reduced apoptosis. In vivo, type I polarized and rapamycin-resistant human T cells caused increased xenogeneic graft-versus-host disease (x-GVHD). Murine recipients of rapamycin-resistant human Th1/Tc1 cells had: (1) persistent T cell engraftment; (2) increased T cell cytokine and cytolytic effector function; and (3) T cell infiltration of skin, gut, and liver. Rapamycin therefore does not impair human T cell capacity for type I differentiation. Rather, rapamycin yields an anti-apoptotic Th1/Tc1 effector phenotype by promoting autophagy.

Twist is transcriptionally induced by activation of STAT3 and mediates STAT3 oncogenic function.

To explore the basis of metastasis, we compared the human breast cancer lines MCF-7 and MDA-MB453, which have low invasive ability, with their sublines MCF7-I4 and MDA-MB453-I4 with high invasive ability for gene expression and signaling pathways. We previously showed that the I4 lines had dramatically elevated levels of Twist compared with their parental lines. In this study, we observed significantly increased STAT3 Tyr(705) phosphorylation, but not the STAT3 protein levels, in the I4 lines. Activation of STAT3 by interleukin-6 or expression of activated Src induced Twist expression at protein and mRNA levels. Inhibiting STAT3 by a small molecule inhibitor, JSI-124, STAT3 small hairpin RNAs, or dominant negative STAT3 resulted in significant reduction of Twist protein and mRNA expression. STAT3 directly bound to the second proximal STAT3-binding site on the human Twist promoter and activated its transcriptional activity. Inhibition of STAT3 reduced migration, invasion, and colony formation of the I4 cells. Ectopic expression of Twist significantly rescued those phenotypes. Ten normal and 46 tumor specimens of breast tissues were examined for activation of STAT3 and expression of Twist. There was a strong correlation between Tyr(705) p-STAT3 and Twist level in the late stage tumor tissues. Our results indicate that activated STAT3 transcriptionally induces Twist, which plays an important role in promoting migration, invasion, and anchorage-independent growth. Together with our previous observation that Twist transcriptionally induces AKT2 to mediate Twist-promoted oncogenic functions, we conclude that STAT3, Twist, and AKT2 form a functional signaling axis to regulate pivotal oncogenic properties of cancer cells.