Absence of Vasoactive Intestinal Peptide Signaling Significantly Enhances T Cell Survival Following MCMV Infection Via Increased Expression of Bcl-2 and Bcl-XL

Background

The nervous system's influence on immune responses has become increasingly appreciated following the identification of a variety of immunomodulatory products secreted from neurons. One such product, vasoactive intestinal peptide (VIP), has been identified as an anti-inflammatory mediator exerting effects on both the innate and adaptive arms of the immune system. Interference with this pathway through blocking the VIP receptors VPAC1 and VPAC2 has been demonstrated to enhance T cell responses to viral infection as well as cancer. VIP antagonism results in increased numbers of antigen-specific cells in the peripheral blood and a shift in the balance of co-stimulatory and co-inhibitory molecules towards a pro-inflammatory phenotype. The consequences of such effects are a reduction in viral load in a model of MCMV infection and a reduced tumor burden and increased survival in a model of lymphoma.

Methods

In order to better understand the molecular mechanisms underlying the effects of VIP absence or antagonism, we used the MCMV infection model. Male VIP WT and VIP KO mice were infected with 10⁵ pfu MCMV ip and sacrificed at various time points. Spleens were harvested and T cells were FACS sorted based on the expression of activation markers. Expression of pro and anti-apoptotic molecules was assessed via Western blot. Expression of VIP and its receptor VPAC1 was determined using qRT-PCR of extracted mRNA. In vitro T cell proliferation was assessed using CFSE dilution of splenocytes stimulated with CD3/CD28 beads and IL-2.

Results

We first examined levels of VIP and VPAC1 over the course of MCMV infection via qRT-PCR of extracted mRNA. Expression of VIP and VPAC1 was found to change in response to infection with an initial decrease in the levels of VPAC1 followed by an increase back to baseline levels. The pattern of VIP expression was opposite with an initial increase followed by a decrease. In order to gain more mechanistic insight into the effects of VIP signaling in T cells during MCMV infection, activated splenic CD8 T cells from WT mice and mice lacking VIP were FACS sorted at various time points. Activated CD69+CD8+ T cells from KO mice expressed higher levels of the anti-apoptotic protein Bcl-X_L both during the initial response and at the peak by Western blot (Figure 1). Intracellular staining revealed an increased frequency of Bcl-2-expressing CD8 T cells in KO mice at the peak of the response (Figure 1). Proliferation of CFSE-labeled CD8 T cells in response to in vitro CD3/CD28 stimulation was found to be identical in wild type and VIP knockout cells suggesting modified T cell survival rather than proliferation. The negative influence of VIP on T cell survival resulted in significantly higher T cell frequency and a significant increase in the absolute numbers of antigen-specific splenic CD8 T cells in knockout mice (Figure 2). These results provide further support for VIP antagonism as a viable option for th e enhancement of CD8 T cell-mediated immunity.