The priming of an immune response against a major histocompatibility complex class I-restricted antigen expressed by nonhematopoietic cells involves the transfer of that antigen to a host bone marrow-derived antigen presenting cell (APC) for presentation to CD8+ T lymphocytes. Dendritic cells (DC), as bone marrow-derived APC, are first candidates for presentation of tumor-associated antigens (TAA). The aim of this study was to see whether DC are able to prime in vivo antigen-specific cytotoxic T lymphocytes after exposure to a soluble protein antigen in vitro. Lacking a well-defined murine TAA, we took advantage of beta-galactosidase (beta-gal)-transduced tumor cell lines as a model in which beta-gal operationally functions as TAA. For in vivo priming both a DC line, transduced or not transduced with the gene coding for murine GM-CSF, and fresh bone marrow-derived DC (bm-DC), loaded in vitro with soluble beta-gal, were used. Priming with either granulocyte macrophage colony-stimulating factor-transduced DC line or fresh bm-DC but not with untransduced DC line generated CTL able to lyse beta-gal-transfected target cells. Furthermore, GM-CSF was necessary for the DC line to efficiently present soluble beta-gal as an H-2Ld-restricted peptide to a beta-gal-specific CTL clone. Data also show that a long-lasting immunity against tumor challenge can be induced using beta-gal-pulsed bm-DC as vaccine. These results indicate that effector cells can be recruited and activated in vivo by antigen-pulsed DC, providing an efficient immune reaction against tumors.

Antineutrophil cytoplasmic antibodies (ANCAs) target proteins normally retained within neutrophils, indicating that cell death is involved in the autoimmunity process. Still, ANCA pathogenesis remains obscure. ANCAs activate neutrophils inducing their respiratory burst and a peculiar form of cell death, named NETosis, characterized by formation of neutrophil extracellular traps (NETs), decondensed chromatin threads decorated with cytoplasmic proteins endorsed with antimicrobial activity. NETs have been consistently detected in ANCA-associated small-vessel vasculitis, and this association prompted us to test whether the peculiar structure of NET favors neutrophil proteins uploading into myeloid dendritic cells and the induction of ANCAs and associated autoimmunity. Here we show that myeloid DCs uploaded with and activated by NET components induce ANCA and autoimmunity when injected into naive mice. DC uploading and autoimmunity induction are prevented by NET treatment with DNAse, indicating that NET structural integrity is needed to maintain the antigenicity of cytoplasmic proteins. We found NET intermingling with myeloid dendritic cells also positive for neutrophil myeloperoxidase in myeloperoxidase-ANCA-associated microscopic poliangiitis providing a potential correlative picture in human pathology. These data provide the first demonstration that NET structures are highly immunogenic such to trigger adaptive immune response relevant for autoimmunity.

In tumor-bearing mice, cyclic fasting or fasting-mimicking diets (FMD) enhance the activity of antineoplastic treatments by modulating systemic metabolism and boosting antitumor immunity. Here we conducted a clinical trial to investigate the safety and biological effects of cyclic, five-day FMD in combination with standard antitumor therapies. In 101 patients, the FMD was safe, feasible, and resulted in a consistent decrease of blood glucose and growth factor concentration, thus recapitulating metabolic changes that mediate fasting/FMD anticancer effects in preclinical experiments. Integrated transcriptomic and deep-phenotyping analyses revealed that FMD profoundly reshapes anticancer immunity by inducing the contraction of peripheral blood immunosuppressive myeloid and regulatory T-cell compartments, paralleled by enhanced intratumor Th1/cytotoxic responses and an enrichment of IFNγ and other immune signatures associated with better clinical outcomes in patients with cancer. Our findings lay the foundations for phase II/III clinical trials aimed at investigating FMD antitumor efficacy in combination with standard antineoplastic treatments. Significance: Cyclic FMD is well tolerated and causes remarkable systemic metabolic changes in patients with different tumor types and treated with concomitant antitumor therapies. In addition, the FMD reshapes systemic and intratumor immunity, finally activating several antitumor immune programs. Phase II/III clinical trials are needed to investigate FMD antitumor activity/efficacy. This article is highlighted in the In This Issue feature, p. 1

Progressing tumors in man and mouse are often infiltrated by dendritic cells (DCs). Deficient antitumor immunity could be related to a lack of tumor-associated antigen (TAA) presentation by tumor-infiltrating DCs (TIDCs) or to a functional defect of TIDCs. Here we investigated the phenotype and function of TIDCs in transplantable and transgenic mouse tumor models. Although TIDCs could encompass various known DC subsets, most had an immature phenotype. We observed that TIDCs were able to present TAA in the context of major histocompatibility complex class I but that they were refractory to stimulation with the combination of lipopolysaccharide, interferon γ, and anti-CD40 antibody. We could revert TIDC paralysis, however, by in vitro or in vivo stimulation with the combination of a CpG immunostimulatory sequence and an anti-interleukin 10 receptor (IL-10R) antibody. CpG or anti–IL-10R alone were inactive in TIDCs, whereas CpG triggered activation in normal DCs. In particular, CpG plus anti–IL-10R enhanced the TAA-specific immune response and triggered de novo IL-12 production. Subsequently, CpG plus anti–IL-10R treatment showed robust antitumor therapeutic activity exceeding by far that of CpG alone, and elicited antitumor immune memory.

Transgenic female mice expressing the transforming rat oncogene c-erbB-2 (HER-2/neu) under the mouse mammary tumor virus (MMTV) promoter (BALB-neuT) spontaneously develop mammary carcinomas with a progression resembling that of human breast cancer. In these mice, activating antitumor immunotherapy fails to induce T cell–mediated cytotoxicity, suggesting a suppression of the immune response. We found a direct correlation between tumor multiplicity and an increased proportion of Gr-1⁺ (Ly6G)/Mac-1⁺(CD11b)/ER-MP12⁺(CD31) immature myeloid cells in the peripheral blood (PB) and spleen, suggesting that tumor load profoundly affects overall BALB-neuT hematopoiesis. In fact, myeloid colony formation was increased in bone marrow (BM) and spleen. The immature myeloid cells displayed suppressive activity on host T lymphocytes, which progressively failed to respond to alloantigens and CD3 triggering, while maintaining the ability to proliferate in response to nonspecific mitogens. Transplantation of normal BM into BALB-neuT mice readily resulted in hypertrophic hematopoiesis with myeloid cell expansion. This persistent influence of the tumor was mediated through the release of vascular endothelial growth factor (VEGF) but not granulocyte-macrophage colony-stimulating factor (GM-CSF), and was down-modulated when tumor load was reduced but not when BM was transplanted. Together, the data obtained in the BALB-neuT model of naturally occurring carcinogenesis show that tumor-associated immune suppression is secondary to a more general alteration of host hematopoiesis, conditioned by tumor-secreted soluble factors.