We recently showed that Flt3L levels are elevated in the synovial fluid of RA patients and that local exposure to the ligand increases the severity of arthritis in mice 
. Furthermore, high serum level of Flt3L is identified as a preclinical marker with high predictive value for developing RA 
. In this study, we explored the role of systemic Flt3L supplementation during the development of antigen-induced arthritis using the mBSA mouse model. This is a well-established arthritis model where morphological changes within the inflamed joint show great similarity to the human disease with presence of immune infiltration in synovial tissue, immune complex deposition in the cartilage and progressive destruction of cartilage and bone erosion. The mBSA model also shows immunological similarity to human RA being highly dependent on CD4+ T cells with subsequent activation of B cells 
. Immunization with mBSA induces a permanent activation of CD4+ T cells with formation of antigen specific T cell clones and production of antigen specific antibodies. One of the advantages of the mBSA model is that susceptibility to mBSA it not dependent on MHCII genetics, which is the case in the collagen induced arthritis model 
. Furthermore, the mBSA model has a moderate disease severity with inflammation being restricted only to the antigen challenged joint, thereby reducing animal suffering. Interestingly, we observed that high levels of Flt3L had immunosuppressive effects. It reduced the severity of arthritis, primarily by reducing cartilage destruction, decreasing IL-6 and TNF-α production and reducing antigen-induced proliferation. Also, Flt3L-treated mice had reduced B cell proliferation and antigen-specific antibody response.
We have previously shown that reducing the number of DCs has a pronounced effect on antigen presentation in the mBSA model 
. Present study shows that the adoptive transfer of the entire DC population in combination with T cells from mBSA-immunized mice to naïve recipients is required for inducing susceptibility to arthritis, whereas transfer of DCs alone is not. This suggests an important antigen presenting cross-talk between DCs and T cells in this arthritis model.
As expected, treatment with Flt3L was associated with an expansion of cDCs and pDCs in the spleen and lymph nodes. Further analysis of cDC subpopulations revealed that Flt3L induced an almost selective expansion of CD8+ cDC, an effect also reported by others 
. Flt3L-treatment was also associated with increased CD11c expression on both CD8+ cDCs and pDCs. This would indicate a potent effect of Flt3L on the expansion of pDCs and CD8+ cDCs. Both CD8+ cDCs and pDCs improve the outcome of experimental arthritis 
, as well as the outcome of allograft rejection and autoimmunity 
. Also, recent reports show a protective effect of Flt3 signaling dependent DCs against atherosclerosis 
. This suggests that Flt3L induces a regulatory and tolerogenic DC phenotype with the potential to control inflammatory and autoimmune conditions.
We found that Flt3L was associated with not only increased frequency of cDCs and pDCs, but also with expansion of Treg cells in the spleen and lymph nodes. Flt3L increases the frequency of Treg cells in lymphoid organs of mice, an effect that is DC dependent and identifies an Flt3L-dependent feedback loop between these cells 
. CD8+ cDCs and pDCs have distinct functions during Treg formation. CD8+ cDCs induce the generation of FoxP3+ Tregs from FoxP3− T cells 
, and produce the anti-inflammatory and Treg inducing cytokine TGF-β 
. Also, pDCs induces expansion of Tregs, a function dependent on MHCII expression 
. We observed correlation between the level of MHCII expression by pDCs and the frequency of Tregs in the spleen. Also, the expression of PD-L1 on pDCs would further support the importance of these cells in Treg homeostasis. DCs have been shown to maintain FoxP3 expression by Tregs 
. Accordingly, the intensity and gene expression of FoxP3 was increased in the spleens of Flt3L-treated mice, despite the less prominent increase of these cells at day 28. It was recently reported that Tregs with increased level of FoxP3 expression have a better suppressive capacity and greater potential to prevent allograft rejection and suppress T cell activation 
. The reduced proliferation after mBSA stimulation in splenocyte cultures from Flt3L-treated mice further supports an increase in potent antigen-specific Tregs. These results indicate that Flt3L induces a DC phenotype, which promotes formation, expansion and maintenance of Tregs, further supporting the immunosuppressive effects of Flt3L observed in our arthritis model.
Polymorphisms in the Blimp-1 gene have now been identified in patients with RA and systemic lupus erythematosus 
. Blimp-1 regulates the function and differentiation of B and T cells and was also recently reported to play a vital role in the development and tolerogenic function of DCs 
. Deletion of Blimp-1 in hematopoietic lineages revealed its important inhibitory function towards CD8− cDCs development 
, and specific deletion of Blimp-1 in DCs results in an autoimmune syndrome 
. We found increased Blimp-1 gene expression in Flt3L-treated mice, which correlated with the gene expression of Flt3 and a decreased frequency of CD4−CD8− cDCs. This supports the notion of a suppressive function for Blimp-1 on the development of the CD4−CD8− cDC subpopulation and could explain the tolerogenic DC profile induced by Flt3L.
Increased levels of Flt3L have been observed in mice lacking Flt3, or after inhibition of Flt3 signaling, possibly as a consequence of reduced DC numbers 
. Increased levels of Flt3L have also been observed in mice after Treg cell depletion 
. Also, humans suffering from a syndrome causing total absence of blood DCs and pDC, show severely reduced numbers of Tregs and significantly increased serum levels of Flt3L 
. This suggests that increased Flt3L levels may be a consequence of reduced numbers of DCs or Tregs. Therefore, the increased levels of Flt3L seen in RA patients may reflect a feedback mechanism that compensates for a deficiency in Flt3-derived DCs and functional Tregs. This is in agreement with our findings showing that Flt3L increases the frequency of cDCs, pDCs and Tregs in the lymphoid organs and controls inflammatory response and severity of arthritis.
The present study supports recent findings on a potential involvement of Flt3L in the pathogenesis of autoimmune disease such as RA. We provide experimental evidence that Flt3L has potent immunoregulatory properties. Flt3L induce formation of DCs, which are important regulators of adaptive immune responses in the mBSA model. In addition, Flt3L facilitates formation of Treg cells and by this mechanism reduces the inflammatory response and subsequent severity of arthritis in mice. We suggest that high systemic levels of Flt3L have the potential to induce formation of regulatory cell types and prevent autoreactivity and autoimmunity.