Vasoactive Intestinal Peptide Synthesis in Transplant Recipients Critically Regulate Donor T Cell Alloreactivity and Graft-Versus-Host Disease

Introduction: Vasoactive Intestinal peptide (VIP) is a neuropeptide expressed by neural and lymphoid cells with pleiotropic effects on brain, immune, pulmonary and GI systems. The potent immunomodulatory activity of VIP make it an attractive therapeutic target to suppresses immune responses in conditions of deleterious inflammation. In allogeneic bone marrow transplantation (allo-BMT), graft-versus-host disease (GvHD) is mediated by activated alloreactive T cells that also upregulate a variety of co-inhibitory pathway molecules, including VIP expression on T cells. It is unclear whether VIP production by host cells in the transplant recipient influence the severity of GvHD and donor T cell activation. We tested the hypothesis that lack of VIP production by recipient cells would enhance donor T cell activation and increase GvHD in allo-BMT recipients. Methods and Results: We examined the effect of endogenous VIP signaling in host tissues in modulating GvHD in a murine allo-BMT model, in which C57BL/6 wildtype (WT) and VIP-knock-out (VIP-KO) mice received 11Gy irradiation on day -1 and were transplanted i.v. on day 0 with 5x10⁶ T cell depleted (TCD) bone marrow (BM) plus 0, 1x10⁶ or 3x10⁶ splenocytes (SP) from MHC mis-matched B10.BR donors. All three groups of WT recipients had better survival than VIP-KO recipients. VIP-KO mice transplanted with TCD BM alone had 33.3% survival at day 75 compared with 73.3% survival of WT recipients. Chimerism studies on day 20 post-BMT showed equivalent levels of donor chimerism within each group that received the same dose of donor SP. WT and VIP-KO recipients received BM only showed significantly lower donor chimerism compared to either WT or VIP-KO recipients received additional SP (p<0.001), indicating increased mortality in recipients transplanted with BM only was primarily due to early graft rejection. The addition of donor SP to the graft resulted in significantly more GvHD-related mortality in VIP-KO mice compared with WT recipients, with 80% vs 100% day 75 survival among recipients of 1x10⁶ SP (p=NS) and 0% vs 40% survival (p<0.01) among recipients of 3x10⁶ donor SP. WT and VIP-KO mice treated with lethal doses of irradiation without allo-BMT showed no survival difference, suggesting VIP-KO mice are not intrinsically more susceptible to irradiation. To determine how the absence of VIP synthesis by VIP-KO host cells modulates immune effector mechanisms that contributes to increased GvHD, we analyzed donor-derived lymphocytes in the spleen of transplant recipients received 5M TCD BM plus 3 × 10⁶ SP from WT B10BR donors following a time course, with flow cytometry. WT recipients had significantly increased levels of Lag3+ CD4 (p<0.001) on D6, PD1+ CD8 (p<0.01) on D13, Tim3+ CD4 (p<0.05) on D19, and increased levels of Tim3+ CD4 (p<0.001), PD1+ CD4 (p<0.05), and PD1+ CD8 (p<0.001) on D25 post allo-BMT compared to VIP-KO recipients. In addition, VIP-KO recipients had significantly higher expression of selected Th1 and Th17 cytokines. We observed significantly higher levels of IFN-γ+ CD4 (p<0.001) and IL2+ CD4 (p<0.01) on D4, significantly higher levels of TNF-α+ CD4 (p<0.05) and TNF-α+ CD8 (p<0.001) on D19, and significantly increased TNF-α+ CD8 (p<0.05) on D25 post allo-BMT in VIP-KO compared to WT recipients. Consistent with the significant elevation of IL17 in CD4 and CD8 (p<0.001) donor T cells of VIP-KO mice on D19, increased transcriptional factor RORγt in VIP-KO mice were detected in both CD4 (p<0.0001) and CD8 (p<0.0001) donor T cells. Transplant experiments using radiation chimeric recipients in which either the hematopoietic or non-hematopoietic compartment lacked expression of VIP further demonstrated that VIP production in non-hematopoietic cells is the key factor in limiting the GvHD activity of donor T cells (Fig 1). Finally, immunofluorescent imaging of transgenic mice in which GFP is regulated by the VIP promoter showed VIP production is highly expressed in efferent neurons innervating the lungs, and that host expression of VIP in lung tissues continues for at least 15 days post-transplant (Fig 2). Conclusion: These data suggest local production of VIP in the lung may be a key factor in the control of alloreactive donor T cells and subsequent GVHD in epithelial target organs. Furthermore, local production of VIP by host neurons surrounding lung alveoli suggests a novel pathway for pharmacological modulation of allo-reactive T cells and control of GVHD.

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