A critical role for mast cells and mast cell-derived IL-6 in TLR2-mediated inhibition of tumor growth.

Several TLR agonists are effective in tumor immunotherapy, but their early innate mechanisms of action, particularly those of TLR2 agonists, are unclear. Mast cells are abundant surrounding solid tumors where they are often protumorigenic and enhance tumor angiogenesis. However, antitumor roles for mast cells have also been documented. The impact of mast cells may be dependent on their activation status and mediator release in different tumors. Using an orthotopic melanoma model in wild-type C57BL/6 and mast cell-deficient Kit(W-sh/W-sh) mice and a complementary Matrigel-tumor model in C57BL/6 mice, mast cells were shown to be crucial for TLR2 agonist (Pam(3)CSK(4))-induced tumor inhibition. Activation of TLR2 on mast cells reversed their well-documented protumorigenic role. Tumor growth inhibition after peritumoral administration of Pam(3)CSK(4) was restored in Kit(W-sh/W-sh) mice by local reconstitution with wild-type, but not TLR2-deficient, mast cells. Mast cells secrete multiple mediators after Pam(3)CSK(4) activation, and in vivo mast cell reconstitution studies also revealed that tumor growth inhibition required mast cell-derived IL-6, but not TNF. Mast cell-mediated anticancer properties were multifaceted. Direct antitumor effects in vitro and decreased angiogenesis and recruitment of NK and T cells in vivo were observed. TLR2-activated mast cells also inhibited the growth of lung cancer cells in vivo. Unlike other immune cells, mast cells are relatively radioresistant making them attractive candidates for combined treatment modalities. This study has important implications for the design of immunotherapeutic strategies and reveals, to our knowledge, a novel mechanism of action for TLR2 agonists in vivo.

Potential of resveratrol analogues as antagonists of osteoclasts and promoters of osteoblasts.

The plant phytoalexin resveratrol was previously demonstrated to inhibit the differentiation and bone resorbing activity of osteoclasts, to promote the formation of osteoblasts from mesenchymal precursors in cultures, and inhibit myeloma cell proliferation, when used at high concentrations. In the current study, we screened five structurally modified resveratrol analogues for their ability to modify the differentiation of osteoclasts and osteoblasts and proliferation of myeloma cells. Compared to resveratrol, analogues showed an up to 5,000-fold increased potency to inhibit osteoclast differentiation. To a lesser extent, resveratrol analogues also promoted osteoblast maturation. However, they did not antagonize the proliferation of myeloma cells. The potency of the best-performing candidate in vitro was tested in vivo in an ovariectomy-induced model of osteoporosis, but an effect on bone loss could not be detected. Based on their powerful antiresorptive activity in vitro, resveratrol analogues might be attractive modulators of bone remodeling. However, further studies are required to establish their efficacy in vivo.

Treatment failure of a TLR-7 agonist occurs due to self-regulation of acute inflammation and can be overcome by IL-10 blockade.

Multiple TLR agonists have been shown to have antitumor effects in animal models. However, the therapeutic efficacy of TLR agonist monotherapy in cancer treatment has been limited, and the mechanisms of failure remain unknown. We demonstrate that topical treatment with a TLR-7 agonist, imiquimod, can elicit significant regression of spontaneous breast cancers in neu transgenic mice, a model of human HER-2/neu(+) breast cancer. However, tumor growth progressed once imiquimod therapy was ended. Gene expression analysis using tumor-derived RNA demonstrated that imiquimod induced high levels of IL-10 in addition to TNF-alpha and IFN-gamma. Elevated levels of circulating IL-10 were also detected in sera from imiquimod-treated mice. Elevated serum IL-10 appeared to be derived from IL-10 and dual cytokine secreting (IFN-gamma(+) and IL-10(+)) CD4(+) T cells rather than CD4(+)CD25(+)Foxp3(+) T regulatory cells, which were also induced by imiquimod treatment. Blockade of IL-10, but not TGF-beta, enhanced the antitumor effect of imiquimod by significantly prolonging survival in treated mice. These data suggest that the excessive inflammation induced by TLR agonists may result in a self-regulatory immunosuppression via IL-10 induction and that blocking IL-10 could enhance the therapeutic efficacy of these agents.

A2A adenosine receptor may allow expansion of T cells lacking effector functions in extracellular adenosine-rich microenvironments.

Immunosuppressive signaling via the A2A adenosine receptor (A2AR) provokes a mechanism that protects inflamed tissues from excessive damage by immune cells. This mechanism is desirable not only for preventing uncontrolled tissue destruction by overactive immune responses, but also for protecting tumor tissues from antitumor immune responses. In aforementioned circumstances, T cell priming may occur in an environment containing high concentrations of extracellular adenosine. To examine qualitative changes in T cells activated in the presence of adenosine, we asked whether different functional responses of T cells are equally susceptible to A2AR agonists. In this study, we demonstrate that A2AR signaling during T cell activation strongly inhibited development of cytotoxicity and cytokine-producing activity in T cells, whereas the inhibition of T cell proliferation was only marginal. Both CD8(+) and CD4(+) T cells proliferated well in the presence of A2AR agonists, but their IFN-gamma-producing activities were susceptible to inhibition by cAMP-elevating A2AR. Importantly, the impaired effector functions were maintained in T cells even after removal of the A2AR agonist, reflecting T cell memory of the immunoregulatory effect of adenosine. Thus, although the adenosine-rich environment may allow for the expansion of T cells, the functional activation of T cells may be critically impaired. This physiological mechanism could explain the inefficiency of antitumor T cells in the tumor microenvironment.

Role of TLR in B cell development: signaling through TLR4 promotes B cell maturation and is inhibited by TLR2.

The role of TLR4 in mature B cell activation is well characterized. However, little is known about TLR4 role in B cell development. Here, we analyzed the effects of TLR4 and TLR2 agonists on B cell development using an in vitro model of B cell maturation. Highly purified B220(+)IgM(-) B cell precursors from normal C57BL/6 mouse were cultured for 72 h, and B cell maturation in the presence of the TLR agonists was evaluated by expression of IgM, IgD, CD23, and AA4. The addition of LPS or lipid A resulted in a marked increase in the percentage of CD23(+) B cells, while Pam3Cys had no effect alone, but inhibited the increase of CD23(+) B cell population induced by lipid A or LPS. The TLR4-induced expression of CD23 is not accompanied by full activation of the lymphocyte, as suggested by the absence of activation Ag CD69. Experiments with TLR2-knockout mice confirmed that the inhibitory effects of Pam3Cys depend on the expression of TLR2. We studied the effects of TLR-agonists on early steps of B cell differentiation by analyzing IL-7 responsiveness and phenotype of early B cell precursors: we found that both lipid A and Pam3Cys impaired IL-7-dependent proliferation; however, while lipid A up-regulates B220 surface marker, consistent with a more mature phenotype of the IgM(-) precursors, Pam3Cys keeps the precursors on a more immature stage. Taken together, our results suggest that TLR4 signaling favors B lymphocyte maturation, whereas TLR2 arrests/retards that process, ascribing new roles for TLRs in B cell physiology.

A positive regulatory role for suppressor of cytokine signaling 1 in IFN-gamma-induced MHC class II expression in fibroblasts.

Suppressor of cytokine signaling 1 (SOCS1) is rapidly induced following stimulation by several cytokines. SOCS1 negatively regulates cytokine receptor signal transduction by inhibiting Janus family tyrosine kinases. Lack of such feedback regulation underlies the premature death of SOCS1(-/-) mice due to unbridled IFN-gamma signaling. We used mouse embryo fibroblasts derived from SOCS1(-/-) mice to investigate the role of SOCS1 in IFN-gamma signaling pathways. SOCS1(-/-) fibroblasts were exquisitely sensitive to the IFN-gamma-mediated growth arrest and showed sustained STAT1 phosphorylation. However, SOCS1(-/-) fibroblasts were inefficient in MHC class II surface expression following IFN-gamma stimulation, despite a marked induction of the MHC class II transactivator and MHC class II gene expression. Retroviral transduction of wild-type SOCS1 relieved the growth-inhibitory effects of IFN-gamma in SOCS1(-/-) fibroblasts by inhibiting STAT1 activation. SOCS1R105K, carrying a mutation within the phosphotyrosine-binding pocket of the Src homology 2 domain, did not inhibit STAT1 phosphorylation, yet considerably inhibited IFN-gamma-mediated growth arrest. Strikingly, expression of SOCS1R105K restored the IFN-gamma-induced MHC class II expression in SOCS1(-/-) cells, indicating that expression of SOCS1 facilitates MHC class II expression in fibroblasts. Our results show that SOCS1, in addition to its negative regulatory role of inhibiting Janus kinases, has an unanticipated positive regulatory function in retarding the degradation of IFN-gamma-induced MHC class II proteins in fibroblasts.

Glycine inhibits growth of T lymphocytes by an IL-2-independent mechanism.

Previously, it was shown that glycine prevented increases in intracellular calcium ([Ca2+]i) in Kupffer cells. Since Kupffer cells and T lymphocytes are derived from the same pluripotent stem cell, it was hypothesized that glycine would prevent increases in [Ca2+]i in lymphocytes and inhibit cell proliferation. Lymphocyte proliferation was measured in one-way MLC with spleen cells from DA and Lewis rats and in enriched T lymphocyte preparations stimulated by immobilized anti-CD3 Ab. Glycine caused a dose-dependent decrease in cell proliferation to about 40% of control. Con A caused a dose-dependent increase in [Ca2+]i in Jurkat cells which was blunted maximally with 0.6 mM glycine. The effect of glycine was dependent on extracellular chloride and reversed by strychnine, an antagonist of the glycine-gated chloride channel. Similar results were obtained with rat T lymphocytes stimulated by anti-CD3 Ab. Surprisingly, glycine had no effect on IL-2 production in the mixed lymphocyte culture; therefore, the effect of glycine on IL-2-dependent proliferation was tested. Glycine and rapamycin caused dose-dependent decreases in IL-2-stimulated growth of Ctll-2 cells to about 60% and 40%, respectively, of control. Moreover, glycine also inhibited the IL-2-stimulated growth of rat splenic lymphocytes. It is concluded that glycine blunts proliferation in an IL-2-independent manner. This is consistent with the hypothesis that glycine activates a glycine-gated chloride channel and hyperpolarizes the cell membrane-blunting increases in [Ca2+]i that are required for transcription of factors necessary for cell proliferation.

Complex conserved organization of the mammalian leukemia inhibitory factor gene: regulated expression of intracellular and extracellular cytokines.

Leukemia inhibitory factor (LIF) is a member of the IL-6 family of pleiotropic cytokines, which are extensively involved in modulating hematopoiesis and immunity. We have undertaken a detailed analysis of LIF genomic organization and gene transcription and investigated the proteins expressed from alternate transcripts. Previously unidentified LIF transcripts, containing alternate first exons spliced onto common second and third exons, were cloned from murine embryonic stem cells, human embryonal carcinoma cells, and primary porcine fibroblasts. Based on sequence homology and position within the genomic sequence, this confirmed the existence of the LIF-M transcript in species other than the mouse and identified a new class of transcript, designated LIF-T. Thus, a complex genomic organization of the LIF gene, conserved among eutherian mammals, results in the expression of three LIF transcripts (LIF-D, LIF-M, and LIF-T) differentially expressed from alternate promoters. The first exon of the LIF-T transcript contained no in-frame AUG, causing translation to initiate downstream of the secretory signal sequence at the first AUG in exon two, producing a truncated LIF protein that was localized within the cell. Enforced secretion of this protein demonstrated that it could act as a LIF receptor agonist. Regulated expression of biologically active intracellular and extracellular LIF cytokine could thus provide alternate mechanisms for the modulation of hematopoiesis and immune system function.