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Dendritic cell (DC) fine-tunes inflammatory versus tolerogenic responses to protect from immune-pathology. However, the role of co-regulators in maintaining this balance is unexplored. NCoR1-mediated repression of DC immune-tolerance has been recently reported. Here we found that depletion of NCoR1 paralog SMRT (NCoR2) enhanced cDC1 activation and expression of IL-6, IL-12 and IL-23 while concomitantly decreasing IL-10 expression/secretion. Consequently, co-cultured CD4 + and CD8 + T-cells depicted enhanced Th1/Th17 frequency and cytotoxicity, respectively. Comparative genomic and transcriptomic analysis demonstrated differential regulation of IL-10 by SMRT and NCoR1. SMRT depletion represses mTOR-STAT3-IL10 signaling in cDC1 by down-regulating NR4A1. Besides, Nfkbia and Socs3 were down-regulated in Ncor2 ( Smrt ) depleted cDC1, supporting increased production of inflammatory cytokines. Moreover, studies in mice showed, adoptive transfer of SMRT depleted cDC1 in OVA-DTH induced footpad inflammation led to increased Th1/Th17 and reduced tumor burden after B16 melanoma injection by enhancing oncolytic CD8 + T-cell frequency, respectively. We also depicted decreased Ncor2 expression in Rheumatoid Arthritis, a Th1/Th17 disease., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Jha, Ahad, Mishra, Sen, Smita, Minz, Biswas, Tripathy, Senapati, Gupta, Acha-Orbea and Raghav.)

Dendritic cells (DCs) are professional antigen-presenting cells involved in the initiation of immune responses. We generated a tolerogenic DC (tolDC) line that constitutively secretes interleukin-10 (IL10-DCs), expressed lower levels of co-stimulatory and MHCII molecules upon stimulation, and induced antigen-specific proliferation of T cells. Vaccination with IL10-DCs combined with another tolDC line that secretes IL-35, reduced antigen-specific local inflammation in a delayed-type hypersensitivity assay independently on regulatory T cell differentiation. In an autoimmune model of rheumatoid arthritis, vaccination with the combined tolDCs after the onset of the disease impaired disease development and promoted recovery of mice. After stable memory was established, the tolDCs promoted CD4 downregulation and induced lymphocyte activation gene 3 (LAG-3) expression in reactivated memory T cells, reducing T cell activation. Taken together, our findings indicate the benefits of combining anti-inflammatory cytokines in an antigen-specific context to treat excessive inflammation when memory is already established., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Koga, Engel, Pigni, Lavanchy, Stevanin, Laversenne, Schneider and Acha-Orbea.)

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 + 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 + 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.

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 -/- Mushi1 murine line in which the development of the cDC1 subset is severely defective. The new MutuDC line that we generated from Batf3 -/- 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 - cDC2s. Therefore, we concluded that our new cell line, that we named CD4 - MutuDC2 line, represents a valuable model for the CD4 - cDC2 subset.

Sirtuin 2 (SIRT2) is one of the seven members of the family of NAD + -dependent histone deacetylases. Sirtuins target histones and non-histone proteins according to their subcellular localization, influencing various biological processes. SIRT2 resides mainly in the cytoplasm and regulates cytoskeleton dynamics, cell cycle, and metabolic pathways. As such, SIRT2 has been implicated in the pathogenesis of neurodegenerative, metabolic, oncologic, and chronic inflammatory disorders. This motivated the development of SIRT2-directed therapies for clinical purposes. However, the impact of SIRT2 on antimicrobial host defense is largely unknown. Here, we address this question using SIRT2 knockout mice. We show that SIRT2 is the most highly expressed sirtuin in myeloid cells, especially macrophages. SIRT2 deficiency does not affect immune cell development and marginally impacts on intracellular signaling and cytokine production by splenocytes and macrophages. However, SIRT2 deficiency enhances bacterial phagocytosis by macrophages. In line with these observations, in preclinical models, SIRT2 deficiency increases survival of mice with chronic staphylococcal infection, while having no effect on the course of toxic shock syndrome toxin-1, LPS or TNF-induced shock, fulminant Escherichia coli peritonitis, sub-lethal Klebsiella pneumoniae pneumonia, and chronic candidiasis. Altogether, these data support the safety profile of SIRT2 inhibitors under clinical development in terms of susceptibility to infections.

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.

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.

Activin A, a member of the TGFβ superfamily, is involved in physiological processes such as cell differentiation, tissue homeostasis, wound healing, reproduction, and in pathological conditions, such as fibrosis, cancer, and asthma. Activin enhances mast cell maturation, as well as regulatory T-cell and Langerhans cell differentiation. In this study we investigated the potential role of activin in epicutaneous sensitization with ovalbumin (OVA), notably with respect to its effect on known Th2-polarization. For this purpose, transgenic mice overexpressing activin in keratinocytes and their wild-type (WT) controls were sensitized epicutaneously with OVA. Skin biopsies were analyzed with regard to histopathological features and mRNA expression of pro-inflammatory and Th1/Th2 cytokines, and Ig levels were measured in the serum. Unexpectedly, activin overexpressing animals were protected from Th2-cytokine expression and induction of OVA-specific IgE levels compared to WT animals. On the other hand, transgenic mice were more susceptible to inflammation compared to WT littermates after tape-stripping and saline (vehicle) or OVA application, as shown by increased pro-inflammatory cytokine mRNA levels and neutrophil accumulation at the site of the treatment. We conclude that activin protects from antigen-induced cutaneous Th2-polarization through modulation of the immune response. These findings highlight the role of activin in cutaneous sensitization, allergy, and in skin homeostasis.

Research in vitro facilitates discovery, screening, and pilot experiments, often preceding research in vivo. Several technical difficulties render Dendritic Cell (DC) research particularly challenging, including the low frequency of DC in vivo, thorough isolation requirements, and the vulnerability of DC ex vivo. Critically, there is not as yet a widely accepted human or murine DC line and in vitro systems of DC research are limited. In this study, we report the generation of new murine DC lines, named MutuDC, originating from cultures of splenic CD8α conventional DC (cDC) tumors. By direct comparison to normal WT splenic cDC subsets, we describe the phenotypic and functional features of the MutuDC lines and show that they have retained all the major features of their natural counterpart in vivo, the splenic CD8α cDC. These features include expression of surface markers Clec9A, DEC205, and CD24, positive response to TLR3 and TLR9 but not TLR7 stimuli, secretion of cytokines, and chemokines upon activation, as well as cross-presentation capacity. In addition to the close resemblance to normal splenic CD8α cDC, a major advantage is the ease of derivation and maintenance of the MutuDC lines, using standard culture medium and conditions, importantly without adding supplementary growth factors or maturation-inducing stimuli to the medium. Furthermore, genetically modified MutuDC lines have been successfully obtained either by lentiviral transduction or by culture of DC tumors originating from genetically modified mice. In view of the current lack of stable and functional DC lines, these novel murine DC lines have the potential to serve as an important auxiliary tool for DC research.

Coordinated interactions between T and B cells are crucial for inducing physiological B cell responses. Mutant mice in which tyrosine 136 of linker for activation of T cell (LAT) is replaced by a phenylalanine (Lat(Y136F)) exhibit a strong CD4(+) T cell proliferation in the absence of intended immunization. The resulting effector T cells produce high amounts of T(H)2 cytokines and are extremely efficient at inducing polyclonal B cell activation. As a consequence, these Lat(Y136F) mutant mice showed massive germinal center formations and hypergammaglobulinemia. Here, we analyzed the involvement of different costimulators and their ligands in such T-B interactions both in vitro and in vivo, using blocking antibodies, knockout mice, and adoptive transfer experiments. Surprisingly, we showed in vitro that although B cell activation required contact with T cells, CD40, and inducible T cell costimulator molecule-ligand (ICOSL) signaling were not necessary for this process. These observations were further confirmed in vivo, where none of these molecules were required for the unfolding of the LAT CD4(+) T cell expansion and the subsequent polyclonal B cell activation, although, the absence of CD40 led to a reduction of the follicular B cell response. These results indicate that the crucial functions played by CD40 and ICOSL in germinal center formation and isotype switching in physiological humoral responses are partly overcome in Lat(Y136F) mice. By comparison, the absence of CD80-CD86 was found to almost completely block the in vitro B cell activation mediated by Lat(Y136F) CD4(+) T cells. The role of CD80-CD86 in T-B cooperation in vivo remained elusive due to the upstream implication of these costimulatory molecules in the expansion of Lat(Y136F) CD4(+) T cells. Together, our data suggest that CD80 and CD86 costimulators play a key role in the polyclonal B cell activation mediated by Lat(Y136F) CD4(+) T cells even though additional costimulatory molecules or cytokines are likely to be required in this process.