Pharmacological inhibition of VIP signaling enhances antiviral immunity and improves survival in murine cytomegalovirus-infected allogeneic bone marrow transplant recipients

Abstract

Cytomegalovirus (CMV) infection following allogeneic bone marrow transplant (allo-BMT) is controlled by donor-derived cellular immunity. Vasoactive intestinal peptide (VIP) suppresses Th1 immunity. We hypothesized that blocking VIP-signaling would enhance anti-CMV immunity in murine recipients of allo-BMT. Recipients were transplanted with bone marrow (BM) and T-cells from major histocompatibility complex (MHC)-mismatched VIP-knockout (KO) or wild-type donors, and treated with 7 daily subcutaneous injections of VIPhyb (peptidic VIP-antagonist) or phosphate-buffered saline (PBS). Genetic and pharmacological blockade of VIP-signaling protected allo-BMT recipients from lethal murine CMV (mCMV) infection, improving survival without increasing graft-versus-host disease. Mice treated with VIPhyb or transplanted with VIP-KO allografts had significantly lower viral loads, increased numbers of mCMV-M45-peptide-MHC-tetramer(+) CD8(+) T-cells, with lower PD-1 expression, and enhanced primary and secondary cellular immune responses after mCMV infection than did PBS-treated mice. These results demonstrate that administration of a VIP antagonist after allo-BMT is a promising safely therapeutic approach to enhance antiviral cellular immunity.

Figure 1

Allo-BMT recipients treated with VIP antagonist or transplanted with VIP-KO donors had improved survival following mCMV infection. B10BR and CB6 F1 mice were transplanted with grafts from either VIP-KO or WT C57BL/6 mice. Transplant recipients were treated with 7 daily s.c. injections of 10 μg of VIPhyb or PBS starting the day prior to mCMV infection on day 8 or on day 35 posttransplant. Mice were examined daily for survival and clinical signs of GVHD. Peripheral blood was collected and tetramer⁺ CD8⁺ T-cells analyzed by flow cytometry. Viral loads in the liver from mice subjected to necropsy at predetermined time points were measured by plaque assay as previously described.^,, (A-C) Survival of B6→B10BR recipients of 5 × 10⁶ TCD-BM alone or TCD-BM plus 0.1 × 10⁶, 0.3 × 10⁶, or 1 × 10⁶ splenic T-cells, treated with VIPhyb (A) or PBS (B, C). Mice were infected with 1 × 10⁵ mCMV PFU on day 35 posttransplant. Data are pooled from 2 replicate experiments with 12 total mice per group. (D-F) GVHD clinical scores for the B6→B10BR groups transplanted in panels A-C.^- (G-I) tetramer⁺ CD8⁺ T-cells in the B6→B10BR groups transplanted in panels A-C. (J) Survival of B6→CB6 F1 recipients of 5 × 10⁶ TCD-BM plus 3 × 10⁶ splenocytes following infection with 5 × 10³ mCMV PFU on day 8 posttransplantation. Data are from 3 independent experiments with a total of n = 22 mice for each group. (K) GVHD clinical scores in mCMV-infected B6→ CB6 F1 transplant recipients in panel J (SD were +/− 18% of mean values at respective time-points and omitted). (L) tetramer⁺ CD8⁺ T-cells in the B6→CB6 F1 groups transplanted in panel J. (M) Survival of B6→CB6 F1 recipients of 5 × 10⁶ TCD-BM plus 3 × 10⁶ splenocytes following 2 × 10⁴ mCMV PFU infection on day 35 posttransplant. Data are pooled from 3 independent experiments; n = 36 for both the WT group and VIP-KO group, and n = 30 for the VIPhyb group. (N) GVHD clinical scores of B6→CB6 F1 transplanted mice from M (SD were +/− 20% of mean values at respective time-points and omitted). (O) Viral load in the liver of B6→CB6 F1 recipients of 5 × 10⁶ TCD-BM plus 3 × 10⁶ splenocytes following infection with 2 × 10⁴ mCMV PFU on day 35 posttransplant. Data in panel O are mean values ± standard error of the mean from 2 replicate experiments with 10 mice per time point. **P < .01 and ***P < .001 signify significant differences between VIP-KO or VIPhyb-treated group and PBS-treated WT group. NS, not significant.

Figure 2

Allo-BMT recipients treated with VIP antagonist or transplanted with VIP-KO donors had enhanced primary and secondary antigen-specific cellular immune responses to Lm-MCMV vaccine and mCMV infection. B10BR mice were transplanted with 5 × 10⁶ TCD-BM plus 0.3 × 10⁶ T-cells as described in Figure 1. Twenty-eight days posttransplant, mice were vaccinated with 1 × 10⁶ colony-forming units Lm-MCMV or PBS by i.p. injection and then infected with 1 × 10⁵ mCMV PFU on day +42 posttransplant. (A–B) Survival of transplant recipients treated on day 28 with either PBS (left) or VIPhyb (right). (C-D) Content of tetramer⁺ CD8⁺ T-cells in the blood of mice from panels A and B, respectively. (E-J) Percentage tetramer⁺ CD8⁺ T-cells expressing PD-1 or CD69. (K-P) Percentage of total CD8⁺ T-cells expressing CD69 or PD-1. Data are mean values ± standard deviation from 2 replicate experiments with 10 mice per time point. *P < .5, **P < .01, and ***P < .001 signify significant differences comparing mCMV-peptide MHC-tetramer⁺ CD8⁺ T-cell levels between VIP-KO or VIPhyb group and WT group.