Post-Transplant Administration of Flt3 Ligand Enhances Innate Cellular-Mediated Immune Responses to Toll-Like Receptor Agonists in Syngeneic Bone Marrow Transplant Mice.

Innate cellular-mediated immunity (iCMI) responses are initiated through activation of pattern recognition receptors like Toll-like receptors (TLRs) by pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharide (LPS). FL (Flt3 ligand) is a hematopoietic growth factor that promotes the expansion of dendritic cells (DCs) and natural killer (NK) cells, key immunomodulatory effector cells involved in host defense. We have previously shown that reconstituted splenocyte iCMI following murine syngeneic BMT occurs in distinct phases in which donor-derived, innate effector cell recovery correlates with inducible cytokine production. Therefore, we studied whether post-transplant administration of FL could enhance reconstitution of innate effector cells and their cytokine responses to established TLR agonists. After split-fraction radiation with 12 Gy 137 Cs, C57BL/6 recipient mice were tail-vein injected with 5x10 6 unselected BM cells from congeneic donor mice. Transplant mice were injected intraperitoneally (IP) with either 10μg FL/100ml 0.1%MSA/PBS (FL) or 100ml 0.1%MSA/PBS alone (Sham) for 10 consecutive days starting the first day after transplant (D1). Non-transplant C57BL/6 mice served as controls and were similarly treated with IP injections. At 2 (D12) and 10 days (D22) post-FL and sham therapy, splenocytes were harvested and pooled per group and were characterized phenotypically by flow cytometry and functionally via ex vivo cell-culture stimulation with established PAMP and cell-specific agonists. Unselected splenocytes were cultured in triplicate in the following stimulation conditions: media only; Staphylococcus aureus consorbin (SAC, TLR-2); polyinosine-polycytidylic acid (polyI:C, TLR-3); Salmonella enteriditis lipopolysaccharide (LPS, TLR-4); anti-CD40 alone; anti-CD40 plus recombinant IL-4 (rIL-4); combination anti-CD40, rIL-4, and anti-IL-10; R848 (TLR7/8); unmethylated CpG oligodeoxynucleotide 1826 (CpG, TLR-9); and lastly, combination rIL-12 and rIL-18. 24h cell-culture supernatants were assessed for cytokine levels using standard ELISA. Three main observations were noted. First, FL therapy increased total numbers of splenocyte innate effector cells in both control and D12 transplants by 6.2- and 3.2-fold, respectively. Of effector cell expansion, DC increases were the most dramatic (16.4 and 9.7-fold for control and D12 transplants, respectively). Specifically, numbers of reconstituted lymphoid, myeloid and plasmacytoid DCs were 17-, 7- and 5-fold higher, respectively, in FL than sham D12 transplants. Reconstituted NK cells had only modest increases (2-fold) after FL therapy. Second, levels of TLR-2 and TLR-9 induction of IL-12p70 and IFN-γ were 3 to 5-fold higher and DC-specific p70 induction was 5-fold higher in FL than sham D12 mice. Finally, FL augmentation of cellularity and cytokine responses was not long-lasting, as post-FL and sham D22 transplant mice had similar levels of innate effector cells and inducible cytokines. Together, these results show that post-transplant FL immunotherapy transiently enhances reconstituted iCMI and cytokine responses to PAMP agonists. Therapy targeting iCMI response could potentially be used to improve anti-microbial host defense during absent or incomplete adaptive immune recovery.