Donor Plasmacytoid Dendritic Cells Synthesis Vasoactive Intestinal Peptide To Regulate Mice Intestinal GVHD

Background:Allogeneic stem cell transplantation(Allo-HSCT)is a curative therapy for high-risk and/or advanced-stage hematologic malignancies,but its use in clinical practice is limited by graft-versus-host disease(GVHD).Vasoactive intestinal peptide(VIP)is an anti-inflammatory neuropeptide with pleiotropic effects that induce differentiation of regulatory dendritic cells,thereby limiting host GVHD.There are preclinical and clinical data available suggesting that bone marrow donor grafts containing higher plasmacytoid dendritic cells(pDC)that can limit GVHD,but the exact mechanism is not yet understood.Purpose:The aim of this study was to investigate the mechanism of VIP-synthesizing bone marrow pDCs in the context of Allo-HSCT to limit murine intestinal GVHD.Methods:The following assays were performed on sorted bone marrow pDC from VIP knockout(VIP-KO)mice on C57BL/6(B6)background,or from wild-type(WT)B6 mice:RT-q PCR and western blot to detect VIP synthesis levels of pDC in mice and humans;Flow cytometry to probe the VIP-KO/WT pDC VIP expression ratio and phenotype;the proliferation rate and polarization effects of WT/VIP-KO pDC on T cells were observed using pDC and T cell co-culture system.Subsequently,a male B6(H2-K~b)→male B10.BR(H2-K~k)or BALB/c(H2-K~d)mouse transplantation model was constructed.After administration of a lethal dose(11 Gy)of radiation,an equal amount(5×10~3)of flow-sorted VIP-KO/WT pDC,bone marrow stem cells,and magnetic bead-sorted T cells were transplanted.The recipient mice were evaluated for survival,clinical GVHD,intestinal pathological GVHD,serum inflammatory factor levels by ELISA,splenic T cell Th1/Th2/Th17/Tregs differentiation status by intracellular staining and flow assay,immune-related gene expression profiles by RNA-seq.Finally,donor T cells from luciferase transgenic(Luc~+)mice or bone marrow pDC were purified and transplanted into BALB/c mice to reveal the early homing pattern of Luc~+pDC and Luc~+T cells in mice.The model was also used to detect the proliferation and movement of T cells in recipient mice receiving VIP-KO pDC or WT pDC.Results:(1)VIP can be synthesized in pDC of fresh or activated mice bone marrow and from human peripheral blood.There were more pDCs in the bone marrow of WT mice than in VIP-KO mice,without difference in the number of pDCs in the spleen.(2)T cells co-cultured with VIP-KO pDC in vitro resulted in more significant CD4~+/CD8~+T cell proliferation,Th1polarization,and more IFN-γ+TNF-α+activation.(3)Successful establishment of reductive mouse acute GVHD models(B6→B10.BR,B6→BALB/c);Donor WT pDC mediated higher survival,lower clinical GVHD,fewer colonic gland necrotic cells,and lower pathological GVHD scores compared to VIP-KO pDC in mouse bone marrow transplantation.(4)After 14 days of Allo-HSCT in mice,significant proliferation of donor pDC and donor T cells was seen in the spleen and gastrointestinal tract.This finding indicates the possibility of both cells migrating to the same organ and interacting with each other in the early post-transplantation period.Donor T cells proliferated significantly more in the lung and skin when co-transplanted with VIP-KO pDC.(5)Mice receiving VIP-KO pDC had higher levels of GM-CSF and inflammatory cytokines in serum compared to WT pDC recipients,while transcriptome analysis of immune-related donor T cells showed higher expression of CCL3/CCL4 gene;a higher percentage of induced Tregs was found in co-transplanted pDC recipients.Conclusions:These results suggest that paracrine VIP production by pDC limits pathogenic T cell inflammation,supporting a novel mechanism by which donor immune cells regulate T cell activation and GVHD in allo-HSCT.