Large T Antigen-Specific Cytotoxic T Cells Protect Against Dendritic Cell Tumors through Perforin-Mediated Mechanisms Independent of CD4 T Cell Help.

Our newly generated murine tumor dendritic cell (MuTuDC) lines, generated from tumors developing in transgenic mice expressing the simian virus 40 large T antigen (SV40LgT) and GFP under the DC specific promoter CD11c, reproduce the phenotypic and functional properties of splenic wild type CD8α(+) conventional DCs. They have an immature phenotype with low co-stimulation molecule expression (CD40, CD70, CD80, and CD86) that is upregulated after activation with toll-like receptor ligands. We observed that after transfer into syngeneic C57BL/6 mice, MuTuDC lines were quickly rejected. Tumors grew efficiently in large T transgene-tolerant mice. To investigate the immune response toward the large T antigen that leads to rejection of the MuTuDC lines, they were genetically engineered by lentiviral transduction to express luciferase and tested for the induction of DC tumors after adoptive transfer in various gene deficient recipient mice. Here, we document that the MuTuDC line was rejected in C57BL/6 mice by a CD4 T cell help-independent, perforin-mediated CD8 T cell response to the SV40LgT without pre-activation or co-injection of adjuvants. Using depleting anti-CD8β antibodies, we were able to induce efficient tumor growth in C57BL/6 mice. These results are important for researchers who want to use the MuTuDC lines for in vivo studies

Interleukin-35-Producing CD8α(+) Dendritic Cells Acquire a Tolerogenic State and Regulate T Cell Function.

Dendritic cells (DCs) play a central role in shaping immunogenic as well as tolerogenic adaptive immune responses and thereby dictate the outcome of adaptive immunity. Here, we report the generation of a CD8α(+) DC line constitutively secreting the tolerogenic cytokine interleukin (IL)-35. IL-35 secretion led to impaired CD4(+) and CD8(+) T lymphocyte proliferation and interfered with their function in vitro and also in vivo. IL-35 was furthermore found to induce a tolerogenic phenotype on CD8α(+) DCs, characterized by the upregulation of CD11b, downregulation of MHC class II, a reduced costimulatory potential as well as production of the immunomodulatory molecule IL-10. Vaccination of mice with IL-35-expressing DCs promoted tumor growth and reduced the severity of autoimmune encephalitis not only in a preventive but also after induction of encephalitogenic T cells. The reduction in experimental autoimmune encephalitis severity was significantly more pronounced when antigen-pulsed IL-35(+) DCs were used. These findings suggest a new, indirect effector mechanism by which IL-35-responding antigen-presenting cells contribute to immune tolerance. Furthermore, IL-35-transfected DCs may be a promising approach for immunotherapy in the context of autoimmune diseases

Establishment and Characterization of a Functionally Competent Type 2 Conventional Dendritic Cell Line.

Dendritic cells (DCs) are the most potent antigen presenting cells and possess an incomparable ability to activate and instruct T cells, which makes them one of the cornerstones in the regulation of the cross-talk between innate and adaptive immunity. Therefore, a deep understanding of DC biology lays the foundations to describe and to harness the mechanisms that regulate the development of the adaptive response, with clear implications in a vast array of fields such as the study of autoimmune diseases and the development of new vaccines. However, the great difficulty to obtain large quantities of viable non-activated DCs for experimentation have considerably hindered the progress of DC research. Several strategies have been proposed to overcome these limitations by promoting an increase of DC abundance in vivo, by inducing DC development from DC progenitors in vitro and by generating stable DC lines. In the past years, we have described a method to derive immortalized stable DC lines, named MutuDCs, from the spleens of Mushi1 mice, a transgenic mouse strain that express the simian virus 40 Large T-oncogene in the DCs. The comparison of these DC lines with the vast variety of DC subsets described in vivo has shown that all the MutuDC lines that we have generated so far have phenotypic and functional features of type 1 conventional DCs (cDC1s). With the purpose of deriving DC lines with characteristics of type 2 conventional DCs (cDC2s), we bred a new Batf3 <sup>-/-</sup> Mushi1 murine line in which the development of the cDC1 subset is severely defective. The new MutuDC line that we generated from Batf3 <sup>-/-</sup> Mushi1 mice was phenotypically and functionally characterized in this work. Our results demonstrated that all the tested characteristics of this new cell line, including the expression of subset-determining transcription factors, the profile of cytokine production and the ability to present antigens, are comparable with the features of splenic CD4 <sup>-</sup> cDC2s. Therefore, we concluded that our new cell line, that we named CD4 <sup>-</sup> MutuDC2 line, represents a valuable model for the CD4 <sup>-</sup> cDC2 subset

Retroviral infection of neonatal Peyer's patch lymphocytes: the mouse mammary tumor virus model.

Mouse mammary tumor virus is known to infect newborn mice via mother's milk. A proposed key step for viral spread to the mammary gland is by the infection of lymphocytes. We show here that although in suckling mice retroviral proteins are found in all epithelial cells of the gut, viral DNA is exclusively detectable in the Peyer's patches. As early as 5 d after birth the infection leads to a superantigen response in the Peyer's patches but not in other lymphoid organs draining the intestine. Viral DNA can be detected before the superantigen response and becomes first evident in the Peyer's patches followed by mesenteric lymph nodes and finally all lymphoid organs

Sirtuin 3 deficiency does not alter host defenses against bacterial and fungal infections.

Sirtuin 3 (SIRT3) is the main mitochondrial deacetylase. SIRT3 regulates cell metabolism and redox homeostasis, and protects from aging and age-associated pathologies. SIRT3 may drive both oncogenic and tumor-suppressive effects. SIRT3 deficiency has been reported to promote chronic inflammation-related disorders, but whether SIRT3 impacts on innate immune responses and host defenses against infections remains essentially unknown. This aspect is of primary importance considering the great interest in developing SIRT3-targeted therapies. Using SIRT3 knockout mice, we show that SIRT3 deficiency does not affect immune cell development and microbial ligand-induced proliferation and cytokine production by splenocytes, macrophages and dendritic cells. Going well along with these observations, SIRT3 deficiency has no major impact on cytokine production, bacterial burden and survival of mice subjected to endotoxemia, Escherichia coli peritonitis, Klebsiella pneumoniae pneumonia, listeriosis and candidiasis of diverse severity. These data suggest that SIRT3 is not critical to fight infections and support the safety of SIRT3-directed therapies based on SIRT3 activators or inhibitors for treating metabolic, oncologic and neurodegenerative diseases without putting patients at risk of infection

β-Catenin Signaling Drives Differentiation and Proinflammatory Function of IRF8-Dependent Dendritic Cells.

β-Catenin signaling has recently been tied to the emergence of tolerogenic dendritic cells (DCs). In this article, we demonstrate a novel role for β-catenin in directing DC subset development through IFN regulatory factor 8 (IRF8) activation. We found that splenic DC precursors express β-catenin, and DCs from mice with CD11c-specific constitutive β-catenin activation upregulated IRF8 through targeting of the Irf8 promoter, leading to in vivo expansion of IRF8-dependent CD8α(+), plasmacytoid, and CD103(+)CD11b(-) DCs. β-Catenin-stabilized CD8α(+) DCs secreted elevated IL-12 upon in vitro microbial stimulation, and pharmacological β-catenin inhibition blocked this response in wild-type cells. Upon infections with Toxoplasma gondii and vaccinia virus, mice with stabilized DC β-catenin displayed abnormally high Th1 and CD8(+) T lymphocyte responses, respectively. Collectively, these results reveal a novel and unexpected function for β-catenin in programming DC differentiation toward subsets that orchestrate proinflammatory immunity to infection

Dendritic Cells Cause Bone Lesions in a New Mouse Model of Histiocytosis.

Langerhans cell histiocytosis (LCH) is a rare disease caused by the clonal accumulation of dendritic Langerhans cells, which is often accompanied by osteolytic lesions. It has been reported that osteoclast-like cells play a major role in the pathogenic bone destruction seen in patients with LCH and these cells are postulated to originate from the fusion of DCs. However, due to the lack of reliable animal models the pathogenesis of LCH is still poorly understood. In this study, we have established a mouse model of histiocytosis- recapitulating human disease for osteolytic lesions seen in LCH patients. At 12 weeks after birth, severe bone lesions were observed in our multisystem histiocytosis (Mushi) model, when CD8α conventional dendritic cells (DCs) are transformed (MuTuDC) and accumulate. Most importantly, our study demonstrates that bone loss in LCH can be accounted for the transdifferentiation of MuTuDCs into functional osteoclasts both in vivo and in vitro. Moreover, we have shown that injected MuTuDCs reverse the osteopetrotic phenotype of oc/oc mice in vivo. In conclusion, our results support a crucial role of DCs in bone lesions in histiocytosis patients. Furthermore, our new model of LCH based on adoptive transfer of MuTuDC lines, leading to bone lesions within 1-2 weeks, will be an important tool for investigating the pathophysiology of this disease and ultimately for evaluating the potential of anti-resorptive drugs for the treatment of bone lesions

Targeted knock-in mice expressing the oxidase-fixed form of xanthine oxidoreductase favor tumor growth.

Xanthine oxidoreductase has been implicated in cancer. Nonetheless, the role played by its two convertible forms, xanthine dehydrogenase (XDH) and oxidase (XO) during tumorigenesis is not understood. Here we produce XDH-stable and XO-locked knock-in (ki) mice to address this question. After tumor transfer, XO ki mice show strongly increased tumor growth compared to wild type (WT) and XDH ki mice. Hematopoietic XO expression is responsible for this effect. After macrophage depletion, tumor growth is reduced. Adoptive transfer of XO-ki macrophages in WT mice increases tumor growth. In vitro, XO ki macrophages produce higher levels of reactive oxygen species (ROS) responsible for the increased Tregs observed in the tumors. Blocking ROS in vivo slows down tumor growth. Collectively, these results indicate that the balance of XO/XDH plays an important role in immune surveillance of tumor development. Strategies that inhibit the XO form specifically may be valuable in controlling cancer growth

Importance of EMT Factor ZEB1 in cDC1 "MutuDC Line" Mediated Induction of Th1 Immune Response.

The role of Epithelial to Mesenchymal Transition (EMT) factor Zeb1 is well defined in metastasis and cancer progression but it's importance in dendritic cells (DCs) is unexplored until now. For the first time we report here that Zeb1 controls immunogenic responses of CD8α <sup>+</sup> conventional Type-I (cDC1) DCs. We found that ZEB1 expression increases significantly after TLR9 stimulation and its depletion impairs activation, co-stimulation and secretion of important cytokines like IL-6, IL-10 and IL-12 in cDC1 MutuDC line. We further confirmed our findings in primary cDC1 DCs derived from bone marrow. Co-culture of these Zeb1 knock down (KD) DCs with OT-II CD4 <sup>+</sup> T helper cells skewed their differentiation toward Th2 subtype. Moreover, adoptive transfer of activated Zeb1 KD DCs cleared intestinal worms in helminth infected mice by increasing Th2 responses in vivo. Integrative genomic analysis showed Zeb1 as an activator of immune response genes in cDC1 MutuDCs as compared to other pathway genes. In addition, differentially regulated genes in Zeb1 KD RNA-seq showed significant enrichment of Th2 activation pathways supporting our in vitro findings. Mechanistically, we showed that decreased IL-12 secreted by Zeb1 KD DCs is the plausible mechanism for increased Th2 differentiation. Collectively our data demonstrate that Zeb1 could be targeted in DCs to modulate T-cell mediated adaptive immune responses