STING-dependent cytosolic DNA sensing mediates innate immune recognition of immunogenic tumors.

Spontaneous T cell responses against tumors occur frequently and have prognostic value in patients. The mechanism of innate immune sensing of immunogenic tumors leading to adaptive T cell responses remains undefined, although type I interferons (IFNs) are implicated in this process. We found that spontaneous CD8(+) T cell priming against tumors was defective in mice lacking stimulator of interferon genes complex (STING), but not other innate signaling pathways, suggesting involvement of a cytosolic DNA sensing pathway. In vitro, IFN-? production and dendritic cell activation were triggered by tumor-cell-derived DNA, via cyclic-GMP-AMP synthase (cGAS), STING, and interferon regulatory factor 3 (IRF3). In the tumor microenvironment in vivo, tumor cell DNA was detected within host antigen-presenting cells, which correlated with STING pathway activation and IFN-? production. Our results demonstrate that a major mechanism for innate immune sensing of cancer occurs via the host STING pathway, with major implications for cancer immunotherapy.

Therapeutic activity of high-dose intratumoral IFN-ß requires direct effect on the tumor vasculature.

Endogenous type I IFN production after innate immune recognition of tumor cells is critical for generating natural adaptive immune responses against tumors in vivo. We recently have reported that targeting low doses of IFN-ß to the tumor microenvironment using tumor-specific mAbs can facilitate antitumor immunity, which could be augmented further with PD-L1/PD-1 blockade. However, sustained high doses of type I IFNs in the tumor microenvironment, which are potently therapeutic alone, may function through distinct mechanisms. In the current report, we demonstrate that high-dose intratumoral type I IFNs indeed exerted a profound therapeutic effect in the murine B16 model, which unexpectedly did not increase T cell responses. Moreover, bone marrow chimeras revealed a role for type I IFN signaling on nonhematopoietic cells, and most of the therapeutic effect was retained in mice deficient in T, B, and NK cells. Rather, the tumor vasculature was ablated with high-dose intratumoral IFN-ß, and conditional deletion of IFN-α/ßR in Tie2-positive vascular endothelial cells eliminated most of the antitumor activity. Therefore, the major component of the antitumor activity of sustained high doses of type I IFNs occurs through a direct antiangiogenic effect. Our data help resolve conditions under which distinct antitumor mechanisms of type I IFNs are operational in vivo.

Increasing hydrophobicity of residues in an anti-HIV-1 Env peptide synergistically improves potency.

T-20/Fuzeon/Enfuvirtide (ENF), a peptide inhibitor of HIV-1 infection, targets the grooves created by heptad repeat 2 (HR2) of Env's coiled-coil, but mutants resistant to ENF emerge. In this study, ENF-resistant mutants--V38A, N43D, N43D/S138A, Q40H/L45M--were combined with modified inhibitory peptides to identify what we believe to be novel ways to improve peptide efficacy. V38A did not substantially reduce infectivity, but was relatively resistant to inhibitory peptides. N43D was more resistant to inhibitory peptides than wild-type, but infectivity was reduced. The additional mutation S138A (N43D/S138A) increased infectivity and further reduced peptide inhibitory potency. It is concluded that S138A increased binding of HR2/ENF into grooves and that S138A compensated for electrostatic repulsion between N43D and HR2. The six-helix bundle structure indicated that E148A should increase hydrophobic interactions between the coiled-coil and peptide. Importantly, the modifications S138A and E148A in the same peptide retained potency against ENF-escape mutants. The double mutant's increase in potency was greater than the increases from the sum of S138A and E148A individually, showing that these two altered residues synergistically contributed to peptide binding. Isothermal titration calorimetry established that hydrophobic substitutions at positions S138 and E148 improved potency of inhibitory peptides against escape mutants by increasing enthalpic release of energy upon peptide binding.

Mammary gland-specific secretion of biologically active immunosuppressive agent cytotoxic-T-lymphocyte antigen 4 human immunoglobulin fusion protein (CTLA4Ig) in milk by transgenesis.

A major challenge in the field of transplantation is to prevent graft rejection and prolong graft survival. Tolerance induction is a promising way to achieve long-term graft survival without the need for potent immunosuppression and its associated side effects. The recent success of co-stimulatory blockade by the chimeric protein CTLA4Ig in the modulation of the recipient's immune system and the prolongation of graft survival in animal models suggests a possible application of CTLA4Ig in clinical transplantation. To produce sufficient amounts of CTLA4Ig for future clinical application, we sought to use the mammary gland as a bioreactor and produce CTLA4Ig in the milk of transgenic farm animals. Prior to the generation of transgenic farm animals, we tested our strategy in mice. Using the promoter of the sheep beta-lactoglobulin gene, we expressed our CTLA4Ig chimeric gene in the mammary gland of transgenic mice. The yield of CTLA4Ig was fivefold higher in transgenic milk than that from transfected cells. Purified milk-derived CTLA4Ig is biologically active and suppresses T cell activation. We showed that the production of CTLA4Ig in the milk has no adverse immunosuppression effect on the transgenic animals and the offsprings that were fed with the transgenic milk. The findings suggest that the approach to produce CTLA4Ig in milk by transgenesis is feasible; further studies involving farm animals are warranted.