TLR2/6 agonists and interferon-gamma induce human melanoma cells to produce CXCL10.

Clinical approaches to treat advanced melanoma include immune therapies, whose benefits depend on tumor-reactive T-cell infiltration of metastases. However, most tumors lack significant immune infiltration prior to therapy. Selected chemokines promote T-cell migration into tumors; thus, agents that induce these chemokines in the tumor microenvironment (TME) may improve responses to systemic immune therapy. CXCL10 has been implicated as a critical chemokine supporting T-cell infiltration into the TME. Here, we show that toll-like receptor (TLR) agonists can induce chemokine production directly from melanoma cells when combined with IFNγ treatment. We find that TLR2 and TLR6 are widely expressed on human melanoma cells, and that TLR2/6 agonists (MALP-2 or FSL-1) synergize with interferon-gamma (IFNγ) to induce production of CXCL10 from melanoma cells. Furthermore, melanoma cells and immune cells from surgical specimens also respond to TLR2/6 agonists and IFNγ by upregulating CXCL10 production, compared to treatment with either agent alone. Collectively, these data identify a novel mechanism for inducing CXCL10 production directly from melanoma cells, with TLR2/6 agonists +IFNγ and raise the possibility that intratumoral administration of these agents may improve immune signatures in melanoma and have value in combination with other immune therapies, by supporting T-cell migration into melanoma metastases.

Mauldin IS ，Wang E ，Deacon DH ，Olson WC ，Bao Y ，Slingluff CL Jr ... -  《-》

Intratumoral interferon-gamma increases chemokine production but fails to increase T cell infiltration of human melanoma metastases.

Mauldin IS ，Wages NA ，Stowman AM ，Wang E ，Smolkin ME ，Olson WC ，Deacon DH ，Smith KT ，Galeassi NV ，Chianese-Bullock KA ，Dengel LT ，Marincola FM ，Petroni GR ，Mullins DW ，Slingluff CL Jr ... -  《-》

Stimulation of pulmonary immune responses by the TLR2/6 agonist MALP-2 and effect on melanoma metastasis to the lung.

Given that metastasized melanoma is a fatal disease in most cases, it is tempting to develop strategies to a priori prevent metastasis. We have stimulated the pulmonary innate immune system by macrophage-activating lipopeptide-2 (MALP-2), a specific agonist at Toll-like receptor (TLR) 2/6, and investigated its impact on experimental melanoma metastasis. In C57BL/6 mice, intratracheal application of MALP-2 induced a profound influx of neutrophils and macrophages into the lung, which peaked after 24 h (sixfold increase) and returned to baseline within 72 h. Further analysis revealed that MALP-2 also markedly induced VCAM-1 expression on pulmonary blood vessels. In vitro experiments demonstrated that this adhesion molecule mediates binding of B16F10 melanoma cells. Furthermore, in vivo or in vitro treatment with MALP-2 did not significantly affect the ability of immune cells to lyse melanoma cells. As a consequence, notwithstanding the profound pulmonary immune response induction and in contrast to conclusions drawn from some previous publications, the net extent of experimental metastasis did not change significantly, regardless of the application regimen of MALP-2 prior to, concomitant with or after tumor cell inoculation. Melanoma cells stably transfected with green fluorescent protein allowed tracking of early events after tumor cell dissemination and showed that MALP-2-mediated TLR2/6 activation did not interfere with pulmonary melanoma cell arrest. Likewise, boosting the immune induction after establishment of metastases did not change the clinical outcome. These unexpected results vividly counsel caution regarding predictions of immunomodulating therapies, as multiple intertwined effects may influence the net outcome.

Schill T ，Schön MP ，Pletz N ，Emmert S ，Schön M ... -  《-》

Microbial Toll-like receptor ligands differentially regulate CXCL10/IP-10 expression in fibroblasts and mononuclear leukocytes in synergy with IFN-gamma and provide a mechanism for enhanced synovial chemokine levels in septic arthritis.

Proost P ，Vynckier AK ，Mahieu F ，Put W ，Grillet B ，Struyf S ，Wuyts A ，Opdenakker G ，Van Damme J ... -  《EUROPEAN JOURNAL OF IMMUNOLOGY》

Toll like receptor 2 agonists lipoteichoic acid and peptidoglycan are able to enhance antigen specific IFNγ release in whole blood during recall antigen responses.

Interferon gamma release assays (IGRA) have been developed to support the diagnosis of diseases like tuberculosis, which lack robust serological test systems. IGRAs focus on cellular immunity especially memory T cells and thus complement serological testing. However, the low frequency of antigen-specific memory T cells in peripheral blood limits IFNγ production to minute amounts and constitutes a major challenge for downstream test systems. We hypothesized that certain toll like receptor (TLR) agonists might enhance IFNγ production in IGRAs after antigen challenge without inducing background cytokine production. In addition, we investigated the potential use of IL2 release after TLR agonist application as another surrogate marker in cytokine release assays. 176 healthy controls (HC) were tested for IFNγ- and IL2-secretion in whole blood in the presence of different TLR agonists with and without antigen challenge by ELISA. The selected TLR agonists were lipopolysaccharide (LPS ≙ TLR4), lipoteichoic acid (LTA ≙ TLR2), peptidoglycan (PGN ≙ TLR2), zymosan (Zym ≙ TLR2 and 6), polyinosinic-polycytidylic acid (Poly I:C ≙ TLR3), flagellin (Fla ≙ TLR5), R848 (≙TLR7 and 8), loxoribine (Lox ≙ TLR7) and bropirimine (Bro ≙ TLR7). TLR2 agonists LTA and PGN increased IFNγ secretion after antigen challenge nearly twofold (740 vs. 443 pg/ml for LTA and 969 vs. 469 pg/ml for PGN, respectively) without eliciting higher background expression. TLR3 agonist Poly(I:C) and TLR5 agonist Fla also induced a twofold increase in IFNγ synthesis (2.230 vs. 1.085 pg/ml for Poly(I:C) and 518 vs. 278 pg/ml for Fla, respectively), but background expression was slightly increased (114 vs. 7 pg/ml for Poly(I:C) and 47 vs. 12 pg/ml for Fla, respectively). IL2 production was not increased after antigen challenge in the presence of LTA, PGN, Poly(I:C) or Fla. The agonists LPS, Zym, R848, Lox and Bro did not raise cytokine synthesis after antigen challenge or they generated high levels of cytokines by themselves. Of all tested agonists TLR2-specific LTA and PGN met the requirements to increase IFNγ synthesis in whole blood after challenge with recall antigens without heightening basal cytokine levels alone. Thus, they constitute a potential costimulating reagent for IGRAs. IL2 did not show any potential as a surrogate marker in cytokine release assays in combination with TLR agonists.

Dammermann W ，Wollenberg L ，Bentzien F ，Lohse A ，Lüth S ... -  《-》