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643 Vasoactive intestinal peptide as a novel immune checkpoint molecule in activated T cells
  1. Sruthi Ravindranathan,
  2. Tenzin Passang Fnu and
  3. Edmund Waller
  1. Emory University, Atlanta, GA, United States


Background Only a fraction of cancer patients responds to current antibody-based immune checkpoint inhibitors.1 Our lab has identified vasoactive intestinal peptide-receptor (VIP-R) signaling as a targetable immune checkpoint pathway in cancer. VIP is a small neuropeptide with known immunosuppressive effects on T cells, in particular, CD4+ T cells.2–5 However, little is known about VIP-R signaling in CD8+ T cells. To define mechanisms by which VIP limits T cell activation and function, we studied the regulation of VIP and VIP receptors (VIP-R) in T cells following their activation in vitro and in mouse models of cancer.

Methods T cells from healthy human donors and murine splenocytes were activated using anti-CD3 coated plates. Western blots measured intracellular pre-pro-VIP, along with its cognate receptors; VPAC1 and VPAC2. Purified cultures of CD4+ and CD8+ T cells were used to interrogate the protein expression on specific T cell subsets. Activation and chemokine receptor expression was assessed by flow cytometry to evaluate T cell response to VIP-R antagonists in vitro and in tumor-bearing mice engrafted with pancreatic cancer cell lines.

Results Both murine and human T cells upregulate pre-pro-VIP following TCR stimulation with similar kinetics of VIP receptors between species. VIP expression is upregulated in vivo following treatment of tumor-bearing mice with anti-PD1 MoAb. VIP expression is temporally correlated with the upregulation of other co-inhibitory molecules. VPAC1 expression modestly increased in activated T cells while VPAC2 expression decreased. A non-canonical high molecular weight (HMW) form of VPAC2-related protein robustly and transiently increase in activated T cells. Expression of HMW form of VPAC2 is only detected in activated CD4+ T cells. Of note, activated CD4+ but not CD8+ T cells upregulate pre-pro-VIP. Pharmacological inhibition of VIP-R signaling significantly increased CD69+, OX40+, Lag3+, and PD1+ expression in CD4+ subsets compared to activated T cells without VIP-R antagonists (p < 0.05). In contrast, CD8+ T cells upregulate VPAC1 but not VPAC2 receptor following activation. VIP-R antagonist treatment of activated CD8+ T cells significantly decreased CXCR4+ expression (p < 0.05). CXCR3 and CXCR5 expression were not affected by VIP-R antagonist treatment.

Conclusions VIP-R signaling is a novel immune autocrine and paracrine checkpoint pathway in activated CD4+ T cells. Activated CD4+ and CD8+ T cells demonstrate different kinetics of VPAC1 and VPAC2 expression, suggesting different immune-regulatory responses to VIP-R antagonists. Understanding VIP-R signaling induced during T cell activation can lead to specific drugs that target VIP-R pathways to enhance cancer immunotherapy.

Acknowledgements We thank healthy volunteers for blood samples. The authors also thank the shared resources at Emory University, namely, Emory Flow Cytometry Core (EFCC) and Integrated Cellular Imaging Core (ICI) and Yerkes Nonhuman Primate Genomics Core that provided services or instruments at subsidized cost to conduct some of the reported experiments. This work was supported in part by Katz Foundation funding, Georgia Research Alliance, and Emory School of Medicine Dean's Imagine, Innovate and Impact (I3) venture award to Edmund K. Waller.


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  2. Wang HY, Jiang XM, Ganea D. The Neuropeptides VIP and PACAP Inhibit IL-2 Transcription by Decreasing c-Jun and Increasing JunB Expression in T Cells. J Neuroimmunol 2000;104(1):68–78.

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  4. Anderson P, Gonzalez-Rey E. Vasoactive intestinal peptide induces cell cycle arrest and regulatory functions in human T cells at multiple levels. Mol Cell Biol 2010.

  5. Delgado M, Ganea D. Vasoactive intestinal peptide: a neuropeptide with pleiotropic immune functions. Amino Acids. NIH Public Access July 2013, 25–39.

Ethics Approval De-identified blood samples from consented healthy volunteers (IRB 00046063) were obtained with approval from Institutional Review Boards.

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