Virus-associated activation of innate immunity induces rapid disruption of Peyer's patches in mice.

Early in the course of infection, detection of pathogen-associated molecular patterns by innate immune receptors can shape the subsequent adaptive immune response. Here we investigate the influence of virus-associated innate immune activation on lymphocyte distribution in secondary lymphoid organs. We show for the first time that virus infection of mice induces rapid disruption of the Peyer's patches but not of other secondary lymphoid organs. The observed effect was not dependent on an active infectious process, but due to innate immune activation and could be mimicked by virus-associated molecular patterns such as the synthetic double-stranded RNA poly(I:C). Profound histomorphologic changes in Peyer's patches were associated with depletion of organ cellularity, most prominent among the B-cell subset. We demonstrate that the disruption is entirely dependent on type I interferon (IFN). At the cellular level, we show that virus-associated immune activation by IFN-α blocks B-cell trafficking to the Peyer's patches by downregulating expression of the homing molecule α4ß7-integrin. In summary, our data identify a mechanism that results in type I IFN-dependent rapid but reversible disruption of intestinal lymphoid organs during systemic viral immune activation. We propose that such rerouted lymphocyte trafficking may impact the development of B-cell immunity to systemic viral pathogens.

TLR activation excludes circulating naive CD8+ T cells from gut-associated lymphoid organs in mice.

The trafficking of effector T cells is tightly regulated by the expression of site-specific sets of homing molecules. In contrast, naive T cells are generally assumed to express a uniform pattern of homing molecules and to follow a random distribution within the blood and secondary lymphoid organs. In this study, we demonstrate that systemic infection fundamentally modifies the trafficking of circulating naive CD8(+) T cells. We show that on naive CD8(+) T cells, the constitutive expression of the integrin α4ß7 that effects their entry into GALT is downregulated following infection of mice with Salmonella typhimurium. We further show that this downregulation is dependent on TLR signaling, and that the TLR-activated naive CD8(+) T cells are blocked from entering GALT. This contrasts strongly with Ag-experienced effector T cells, for which TLR costimulation in the GALT potently upregulates α4ß7 and enhances trafficking to intestinal tissues. Thus, TLR activation leads to opposite effects on migration of naive and effector CD8(+) T cells. Our data identify a mechanism that excludes noncognate CD8(+) T cells from selected immune compartments during TLR-induced systemic inflammation.

Cellular immunostimulation by CpG-sequence-coated DNA origami structures.

To investigate the potential of DNA origami constructs as programmable and noncytotoxic immunostimulants, we tested the immune responses induced by hollow 30-helix DNA origami tubes covered with up to 62 cytosine-phosphate-guanine (CpG) sequences in freshly isolated spleen cells. Unmethylated CpG sequences that are highly specific for bacterial DNA are recognized by a specialized receptor of the innate immune system localized in the endosome, the Toll-like receptor 9 (TLR9). When incubated with oligonucleotides containing CpGs, immune cells are stimulated through TLR9 to produce and secrete cytokine mediators such as interleukin-6 (IL-6) and interleukin-12p70 (IL-12p70), a process associated with the initiation of an immune response. In our studies, the DNA origami tube built from an 8634 nt long variant of the commonly used single-stranded DNA origami scaffold M13mp18 and 227 staple oligonucleotides decorated with 62 CpG-containing oligonucleotides triggered a strong immune response, characterized by cytokine production and immune cell activation, which was entirely dependent on TLR9 stimulation. Such decorated origami tubes also triggered higher immunostimulation than equal amounts of CpG oligonucleotides associated with a standard carrier system such as Lipofectamine. In the absence of CpG oligonucleotides, cytokine production induced by the origami tubes was low and was not related to TLR9 recognition. Fluorescent microscopy revealed localization of CpG-containing DNA origami structures in the endosome. The DNA constructs showed in contrast to Lipofectamine no detectable toxicity and did not affect the viability of splenocytes. We thus demonstrate that DNA origami constructs represent a delivery system for CpG oligonucleotides that is both efficient and nontoxic.

Efficient eradication of subcutaneous but not of autochthonous gastric tumors by adoptive T cell transfer in an SV40 T antigen mouse model.

In stomach cancer, there is a need for new therapeutic strategies, in particular for the treatment of unresectable tumors and micrometastases. We investigated the efficacy of immunotherapy in an autochthonous model of gastric cancer, the CEA424-SV40 T Ag (TAg) transgenic mice. Treatment efficacy against both the autochthonous tumors and s.c. tumors induced by the derived cell line mGC3 were assessed. In wild-type mice, a dendritic cell vaccine loaded with irradiated tumor cells combined with CpG oligonucleotides induced efficient cytotoxic T cell and memory responses against mGC3 s.c. tumors. In contrast, neither s.c. nor autochthonous tumors responded to vaccination in CEA424-SV40 TAg mice, indicating tolerance to the SV40 TAg. To examine whether tumors in these mice were principally accessible to immunotherapy, splenocytes from immune wild-type mice were adoptively transferred into CEA424-SV40 TAg transgenic mice. Treated mice showed complete regression of the s.c. tumors associated with intratumoral infiltrates of CD8 and CD4 T cells. In contrast, the autochthonous gastric tumors in the same mice were poorly infiltrated and did not regress. Thus, even in the presence of an active anti-tumoral T cell response, autochthonous gastric tumors do not respond to immunotherapy. This is the first comparison of the efficacy of adoptive T cell transfer between transplanted s.c. tumors and autochthonous tumors in the same animals. Our results suggest that in gastric cancer patients, even a strong anti-tumor T cell response will not efficiently penetrate the tumor in the absence of additional therapeutic strategies targeting the tumor microenvironment.