PEGylated IL-10 Activates Kupffer Cells to Control Hypercholesterolemia.

Interleukin-10 (IL-10) is a multifunctional cytokine that exerts potent context specific immunostimulatory and immunosuppressive effects. We have investigated the mechanism by which PEGylated rIL-10 regulates plasma cholesterol in mice and humans. In agreement with previous work on rIL-10, we report that PEGylated rIL-10 harnesses the myeloid immune system to control total plasma cholesterol levels. We have discovered that PEG-rMuIL-10's dramatic lowering of plasma cholesterol is dependent on phagocytotic cells. In particular, PEG-rHuIL-10 enhances cholesterol uptake by Kupffer cells. In addition, removal of phagocytotic cells dramatically increases plasma cholesterol levels, suggesting for the first time that immunological cells are implicitly involved in regulating total cholesterol levels. These data suggest that treatment with PEG-rIL-10 potentiates endogenous cholesterol regulating cell populations not currently targeted by standard of care therapeutics. Furthermore, we show that IL-10's increase of Kupffer cell cholesterol phagocytosis is concomitant with decreases in liver cholesterol and triglycerides. This leads to the reversal of early periportal liver fibrosis and facilitates the restoration of liver health. These data recommend PEG-rIL-10 for evaluation in the treatment of fatty liver disease and preventing its progression to non-alcoholic steatohepatitis. In direct confirmation of our in vivo findings in the treatment of hypercholesterolemic mice with PEG-rMuIL-10, we report that treatment of hypercholesterolemic cancer patients with PEG-rHuIL-10 lowers total plasma cholesterol by up to 50%. Taken together these data suggest that PEG-rIL-10's cholesterol regulating biology is consistent between mice and humans.

PEG-rIL-10 treatment decreases FoxP3(+) Tregs despite upregulation of intratumoral IDO.

IL-10 has been classically defined as a broad-spectrum immunosuppressant and is thought to facilitate the development of regulatory CD4(+) T cells. IL-10 is believed to represent one of the major suppressive factors secreted by IDO(+)FoxP3(+)CD4(+) Tregs. Contrary to this view, we have previously reported that PEGylated recombinant IL-10 (PEG-rIL-10) treatment of mice induces potent IFNγ and CD8(+) T-cell-dependent antitumor immunity. This hypothesis is currently being tested in clinical trials and we have reported that treatment of cancer patients with PEG-rHuIL-10 results in inhibition and regression of tumor growth as well as increased serum IFNγ. We have continued to assess PEG-rIL-10's pleiotropic effects and report that treatment of tumor-bearing mice and humans with PEG-rIL-10 increases intratumoral indoleamine 2, 3-dioxygenase (IDO) in an IFNγ-dependent manner. This should result in an increase in Tregs, but paradoxically our data illustrate that PEG-rIL-10 treatment of mice reduces intratumoral FoxP3(+)CD4(+) T cells in an IDO-independent manner. Additional investigation indicates that PEG-rIL-10 inhibits TGFß/IL-2-dependent in vitro polarization of FoxP3(+)CD4(+) Tregs and potentiates IFNγ(+)T-bet(+)CD4(+) T cells. These data suggest that rather than acting as an immunosuppressant, PEG-rIL-10 may counteract the FoxP3(+)CD4(+) Treg suppressive milieu in tumor-bearing mice and humans, thereby further facilitating PEG-rIL-10's potent antitumor immunity.

The Potentiation of IFN-γ and Induction of Cytotoxic Proteins by Pegylated IL-10 in Human CD8 T Cells.

Interleukin-10 (IL-10) exerts both immunosuppressive and immunostimulatory effects. While the immunosuppressive effects are widely known, it has only been recently reported that pegylated recombinant human IL-10 (PEG-rHuIL-10) elicits potent interferon-γ (IFN-γ) and CD8 T-cell-dependent antitumor effects in murine tumor models. In this study, we show that PEG-rHuIL-10 exerts immune inhibitory effects on human peripheral blood mononuclear cell (PBMC) bulk cultures and stimulatory effects in CD8 T cells within the same culture. Also, in isolated CD8 T cells, PEG-rHuIL-10 potentiates prototypic Tc1 cytokine IFN-γ expression and induces perforin and granzyme B secretion. IFN-γ and granzyme B secretion is dependent on T-cell receptor ligation and is therefore not indiscriminately released by PEG-rHuIL-10 treatment. STAT3, NF-κB, AP1, and MEK inhibition blocks IFN-γ potentiation, while perforin induction is impeded by AP1 inhibition, and granzyme B induction is blocked by both AP1 and MEK inhibition. These results extend previous pegylated IL-10 preclinical findings to human CD8 T cells and implicate a strong degree of translation for pegylated IL-10 use in cancer therapy.

A clinical biomarker assay to quantitate thymic stromal lymphopoietin in human plasma at sub-pg/ml level.

BACKGROUND: Thymic stromal lymphopoietin (TSLP) is an attractive therapeutic target for the treatment of allergic diseases, and plasma TSLP is a potential patient selection marker in the development of therapeutic agents. RESULTS: We developed and validated an ultrasensitive electrochemiluminescence assay for measurement of TSLP in plasma with a lower limit of quantitation of 0.12 pg/ml, which allowed the quantitation of TSLP in approximately 90% of human plasma samples tested. The assay demonstrated excellent performance characteristics, including precision, accuracy, sensitivity and dilution linearity. Stability and biological variability of TSLP in plasma were also assessed for clinical sample analysis and data interpretation. CONCLUSION: The validated TSLP assay enables assessment of circulating TSLP as a patient selection marker in the development of therapeutics to treat atopic diseases.