Dendritic Cell Homeostasis Is Maintained by Non-Hematopoietic and T Cell-Produced Flt3-Ligand In Steady-State and During Immune Responses.

Abstract 1547

Lymphoid tissue dendritic cells (DCs) have a short life-span of a few days and need to be continuously replenished from hematopoietic stem and progenitor cells. Flt3-Ligand (Flt3L) plays non-redundant role in development of DCs (McKenna. H.J. et al., Blood; 2000). Previously we found that Flk2 (fetal liver kinase-2), the cognate receptor for Flt3L is expressed on early dendritic cell progenitors and Flt3L-Flk2 signalling efficiently supports DC development from early progenitors to steady-state DCs in mice and men (Karsunky, H. et al., J Exp Med; 2003; Chicha L. et al. J Exp Med; 2004). Flk2 is also expressed on mature steady-state lymphoid organ DCs; however its function on mature cells remains to be determined. Flt3L is expressed in almost all the tissues in both mice and men (Hannum, C. et al., Nature; 1994) and this cytokine is critical in the maintenance of DC/T regulatory (Treg) cell homeostasis (Darrase-Jéze. G et al., J Exp Med; 2009; Swee LK et al., Blood; 2009; Manz MG, Blood 2009). However, the precise cellular source of Flt3L and the regulation of production in steady-state and immune responses in vivo is not well understood.

Genetic ablation of the Flk2 receptor lead to 10-fold elevated Flt3L levels in the serum of mice. To evaluate if hematopoietic or non-hematopoietic cells are the main consumers of Flt3L in vivo, we generated bone marrow chimeras by transplanting wild type (WT) or Flt3L^-/- c-Kit⁺ hematopoietic stem and progenitor cells into lethally irradiated Flk2^-/- mice. This demonstrated that hematopietic progenitors and DCs expressing Flk2 receptor are the main consumers of Flt3L in vivo. Previously we showed that in vivo Flk2 tyrosine kinase inhibition and consecutive DC reduction lead to 10fold elevated levels of serum Flt3L (Tussiwand. R. et al., J Immunol; 2005). By using CD11c DTR mice (Zaft, T. et al., J Immunol; 2005) in which diphtheria toxin (DT) receptor is cloned under the CD11c promoter and treatment of mice with DT lead to selective depletion of DCs we here show that ablation Flk2 expressing DCs lead to immediate, about 4-fold elevated serum Flt3L levels in mice. However, we observed no change in mRNA expression of Flt3L, which strongly indicates that Flk2 expressed on DCs is acting as “scavenger” for Flt3L.

We then studied sources of Flt3L in vivo. To this end we generated bone marrow chimeras by transplanting WT c-Kit⁺ hematopoietic stem and progenitor cells in to lethally irradiated Flt3L^-/- hosts and vice versa (WT to Fllt3L^-/-, Flt3L^-/- to WT), and found that in vivo DC homeostasis can be achieved by non-hematopoietic and to lesser extend by hematopoietic cell produced Flt3L. Furhtermore, we found that compared to other hematopoietic cells Flt3L mRNA is highly expressed in lymphocytes (T and B cells) and in lymphoid tissues like thymus, spleen and lymph nodes. We thus used bone marrow c-Kit⁺ hematopoietic stem and progenitor cells from mice that lack T and B cells (Rag1^-/-) or that lack T cells (CD3ε^-/-) as donors to transplant lethally conditioned Flt3L^-/- mice, and found that Flt3L produced by T and B cells is necessary to support DC development in non hematopoietic Flt3L deficient mice.

Using BrdU incorporation we evaluated the functional relevance of Flt3L produced by T cells in an ongoing immune response. Experiments revealed that in lymph nodes with proliferating T cells producing Flt3L a higher percent of BrdU⁺ DCs, i.e. DCs derived from proliferating progenitors were detected. This indicates that Flt3L produced by T cells in an ongoing immune response helps in faster regeneration of DCs from DC committed progenitors. Earlier it has been shown that Treg ablation in Foxp3-DTR mice lead to expansion of DCs in lymph nodes and spleen through Flk2 mediated pathway (Liu, K. et al., Science; 2009); however, the source of Flt3L remained unknown. Here we provide evidence that Treg ablation leads to activation and proliferation of CD4⁺ T cells that in turn release Flt3L to enhance DC development.

These key observations provide insight into the regulation of DC homeostasis and function via tailored adaptation of the Flt3L cytokine milieu by non-hematopoietic and T cells during steady state and during adaptive immune responses.

Supported by the Swiss National Science Foundation (310000-116637) and the European Commission FP6 Network of Excellence initiative (LSHB-CT-2004-512074 DC-THERA)