Bone metastases, present in 70% of patients with metastatic breast cancer, lead to skeletal disease, fractures and intense pain, which are all believed to be mediated by tumor cells. Engraftment of tumor cells is supposed to be preceded by changes in the target tissue to create a permissive microenvironment, the pre-metastatic niche, for the establishment of the metastatic foci. In bone metastatic niche, metastatic cells stimulate bone consumption resulting in the release of growth factors that feed the tumor, establishing a vicious cycle between the bone remodeling system and the tumor itself. Yet, how the pre-metastatic niches arise in the bone tissue remains unclear. Here we show that tumor-specific T cells induce osteolytic bone disease before bone colonization. T cells pro-metastatic activity correlate with a pro-osteoclastogenic cytokine profile, including RANKL, a master regulator of osteoclastogenesis. In vivo inhibition of RANKL from tumor-specific T cells completely blocks bone loss and metastasis. Our results unveil an unexpected role for RANKL-derived from T cells in setting the pre-metastatic niche and promoting tumor spread. We believe this information can bring new possibilities for the development of prognostic and therapeutic tools based on modulation of T cell activity for prevention and treatment of bone metastasis.

Acute graft-versus-host disease (aGVHD) is the main complication of allogeneic hematopoietic stem cell transplantation, and many efforts have been made to overcome this important limitation. We showed previously that G-CSF treatment generates low-density splenic granulocytes that inhibit experimental aGVHD. In this article, we show that aGVHD protection relies on incoming IL-10 neutrophils from G-CSF-treated donor spleen (G-Neutrophils). These G-Neutrophils have high phagocytic capacity, high peroxide production, low myeloperoxidase activity, and low cytoplasmic granule content, which accounts for their low density. Furthermore, they have low expression of MHC class II, costimulatory molecules, and low arginase1 expression. Also, they have low IFN-γ, IL-17F, IL-2, and IL-12 levels, with increased IL-10 production and NO synthase 2 expression. These features are in accordance with the modulatory capacity of G-Neutrophils on regulatory T cell (Treg) generation. In vivo, CD25 Treg depletion shortly after transplantation with splenic cells from G-CSF-treated donors blocks suppression of aGVHD, suggesting Treg involvement in the protection induced by the G-Neutrophils. The immunocompetence and specificity of the semiallogeneic T cells, long-term after the bone marrow transplant using G-Neutrophils, were confirmed by third-party skin graft rejection; importantly, a graft-versus-leukemia assay showed that T cell activity was maintained, and all of the leukemic cells were eliminated. We conclude that G-CSF treatment generates a population of activated and suppressive G-Neutrophils that reduces aGVHD in an IL-10- and Treg-dependent manner, while maintaining immunocompetence and the graft versus leukemia effect.

Allogeneic hematopietic stem cell transplantation (aHSCT) is widely used for the treatment of hematologic malignancies. Although aHSCT provides a good response against the malignant cells (graft-versus-leukemia [GVL]), it also leads to the development of graft-versus-host disease (GVHD), a severe disease with high mortality and morbidity rates. Therapy for GVHD is commonly based on nonspecific immunosupression of the transplanted recipient, resulting in the concomitant inhibition of the GVL effect. In this study, we propose an alternative approach to specifically suppress GVHD while sparing the GVL, based on oral treatment of transplant donors with recipient Ags, associated with the intake of probiotic Lactococcus lactis as tolerogenic adjuvant (combined therapy). We show that treatment of C57BL/6 donor mice with combined therapy before the transplant protects the recipients F1 (C57BL/6 × BAL/c) mice from clinical and pathological manifestations of disease, resulting in 100% survival rate. Importantly, the animals keep the immunological competence maintaining the GVL response as well as the response to third-party Ags. The protection is specific, long lasting and dependent on donor IL-10–sufficient B cells activity, which induces regulatory T cells in the host. These data suggest that combined therapy is a promising strategy for prevention of GVHD with preservation of GVL, opening new possibilities to treat human patients subjected to transplantation.

Regulatory T cells (Treg) deficiency leads to a severe, systemic, and lethal disease, as showed in immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome patients, and scurfy mouse. Postneonatal thymectomy autoimmune gastritis has also been attributed to the absence of Tregs. In this case however, disease is mild, organ-specific, and, more important, it is not an obligatory outcome. We addressed this paradox comparing T cell compartments in gastritis-susceptible and resistant animals. We found that neonatal thymectomy-induced gastritis is not caused by the absence of Tregs. Instead of this, it is the presence of gastritogenic T cell clones that determines susceptibility to disease. The expansion of such clones under lymphopenic conditions results in a reduced Treg:effector T cell ratio that is not enough to control gastritis development. Finally, the presence of gastritogenic clones is determined by the amount of gastric Ag expressed in the neonatal thymus, emphasizing the importance of effector repertoire variability, present even in genetically identical subjects, to organ-specific autoimmune disease susceptibility.

The eighth author's name was omitted from the article. The corrected author and affiliation lines are shown below.

Background. Allogeneic hematopietic stem cell transplantation (aHSCT) is widely used for the treatment of hematologic malignancies. Although aHSCT provides a good response against the malignant cells (GVL), it also leads to the development of graft-versus-host disease (GVHD), a severe disease with high mortality and morbidity rates. Therapy for GVHD is commonly based on non-specific immunosupression of the transplanted recipient, resulting in the concomitant inhibition of the GVL effect. Material and Methods. Here we propose an alternative approach to specifically suppress GVHD, while sparing the GVL, based on oral treatment of transplant donors with recipient antigens, associated with the intake of probiotic Lactococcus lactis as tolerogenic adjuvant (combined therapy). Results. We show that treatment of C57Bl/6 donor mice with combined therapy before the transplant protects the recipients F1 (C57Bl/6XBAL/c) mice from clinical and pathological manifestations of disease, resulting in 100% survival rate. Importantly, the animals keep the immunological competence maintaining the GVL response as well as the response to third party antigens. The protection is specific, long lasting and dependent on donor IL-10 sufficient B cells activity which induces regulatory T cells in the host. Conclusion. These data suggest that combined therapy is a promising strategy for prevention of GVHD with preservation of GVL, opening new possibilities to treat human patients subjected transplantation. Note: This abstract was not presented at the meeting. Citation Format: Ana Carolina T. Mercadante, Suelen Perobelli, Ana Paula G. Alves, Triciana Gonçalves-Silva, Wallace Mello, Ana C Gomes-Santos, Anderson Miyoshi, Ana Maria C. Faria, Adriana Bonomo. Oral combined therapy with probiotics and alloantigen induces B cell dependent long lasting specific tolerance. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2590. doi:10.1158/1538-7445.AM2014-2590

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Allogeneic hematopoietic stem cell transplantation (HSCT) is used to treat a series of hematological diseases. While the presence of T cells mediates the graft-versus-leukemia (GVL) effect and improves engraftment, it is also responsible for the graft-versus-host disease (GVHD), which is the main barrier of HSCT. Patients transplanted with cells from G-CSF treated donors show an unexpected low rate of acute GVHD given the high numbers of T cells present. We have previously shown that blood from G-CSF treated donors have high numbers of inhibitory granulocytes. Experimentally these granulocytes are able to prevent aGVHD in a semi-allogeneic mouse model. Here, our goal is to characterize the granulocytes and its mechanism of action. When we compare this granulocytes from the spleens of 5 day G-CSF treated donors and its untreated control, we observed on flow cytometry that the percentage of this granulocytes/neutrophils Ly6G+ increases from about 2% to about 20%. These cells have some differences among themselves after treatment as increased burst oxidative (measured by DHR) and phagocytosis of E.coli, as well as differences in the pattern of expression of some important surface molecules, such as MHC-II. Levels of myeloperoxidase are lower after treatment with G-CSF, and we confirmed by transmission electron microscopy that these neutrophils have a degranulated profile, with 83% fewer cytoplasmic granules than controls. Indeed, the spleens from 5 day G-CSF treated donors together with control bone marrow cells (G-B6) injected into irradiated F1(B6XBALB/c) mice shows survival rate of 100%, against 10% in non-G-B6 transplants. This protection depends on Gr1+ cells and presents milder pathological and clinical manifestations in gastrointestinal mucosa, skin and liver. However, when these G-B6 neutrophils are from IL-10 KO mice, protection is abolished both in survival curve and in clinical score. Surprisingly, 25 days after transplantation the percentage of the subtype of T regulatory Foxp3+ cells in protected chimeras is increased in all lymphoid organs analyzed and this can be associated with the long term specific tolerance observed. Finally, the anti-tumoral effect in G-B6 chimeras is as potent as in the B6 control chimera and this effect was maintained for more than 20 weeks after HSCT. So we can conclude that treatment with G-CSF generates degranulated neutrophils that reduce GVHD in an IL-10 dependent manner while keeping GVL effect opening a promising road in the prevention of human aGVHD. Financial Support: FAF, INCA, Swiss Bridge, INCT Cancer, FAPERJ, CNPQ, CAPES, Fiocruz. Citation Format: Suelen Martins Perobelli, Ana Carolina Terra Mercadante, Triciana Gonçalves-Silva, Rômulo Galvani, Antônio Pereira-Neves, Marlene Benchimol, Alberto Nobrega, Adriana Bonomo. Neutrophils G-CSF stimulated promotes specific protection against graft vs. host disease and keeps the graft vs. leukemia effect. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B39.