Diacylglycerol lipase-b (DAGLb) hydrolyzes arachidonic acid (AA)-esterified diacylglycerols to produce 2-arachidonoylglycerol (2-AG) and downstream prostanoids that mediate inflammatory responses of macrophages. Here, we utilized DAGL-tailored activity-based protein profiling and genetic disruption models to discover that DAGLb regulates inflammatory lipid and protein signaling pathways in primary dendritic cells (DCs). DCs serve as an important link between innate and adaptive immune pathways by relaying innate signals and antigen to drive T cell clonal expansion and prime antigen-specific immunity. We discovered that disruption of DAGLb in DCs lowers cellular 2-AG and AA that is accompanied by reductions in lipopolysaccharide (LPS) stimulated tumor necrosis factor a secretion. Cell-based vaccination studies revealed that DC maturation ex vivo and immunogenicity in vivo was surprisingly unaffected by DAGLb inactivation. Collectively, we identify DAGLb pathways as a means for attenuating DC inflammatory signaling while sparing critical adaptive immune functions and further expand the utility of targeting lipid pathways for immunomodulation.

We previously demonstrated that CD27 co-stimulation during a primary CD8+ T cell response was critical for the expression of IL-7Rα on acute effector CD8+ T cells, providing an essential element in the generation of CD8+ T cell memory to infectious pathogens. IL-7 plays a critical role in the generation and maintenance of memory CD8+ T cells, and IL-7Rα has been regarded as a functional marker of long-lived memory precursor effector cells. While IL-7Rα is down-regulated acutely upon TCR stimulation, the regulation of the emergence of IL-7Rα expressing cells around the peak of primary CD8+ responses is less clear. Re-expression could be a default outcome after withdrawal of TCR stimulation. Alternatively, specific stimuli actively antagonize the down-regulation or promote the recovery of IL-7Rα in Ag-activated CD8+ T cells. By utilizing agonistic mAb and transgenic models, here we show: 1) CD27 stimulation acts directly on CD8+ T cells to enhance IL-7Rα-expressing effectors; 2) CD27 stimulation neither alleviates the down-regulation of IL-7Rα upon TCR signaling nor promotes the expansion/survival of IL-7Rα-expressing effectors, but facilitates IL-7Rα re-expression; 3) CD27 stimulation regulates Il7ra mRNA abundance but not protein distribution. Importantly, CD27 stimulation promotes not only IL-7Rα, but also the common γ chain of the receptor and the downstream signaling mediated by pSTAT5. Our results demonstrate a previously unappreciated role of CD27 stimulation as a positive regulator of IL-7Rα during CD8 T cell responses, provide insights into the mechanistic basis by which CD27 stimulation influences CD8+ T cell memory differentiation, and highlight the potential of targeting CD27-CD70 axis to enhance IL-7 signaling for antiviral/antitumor immunotherapy.

Melanoma develops the ability to evade immune recognition through multiple mechanisms, despite being highly immunogenic. This is demonstrated by the effective and additive response checkpoint blockade therapies have had during combination clinical trials in melanoma [1]. While the results of these trials have been promising, large portions of patients do not respond to treatment [1,2]. Additionally, many responders take months to show a response using standard criteria. In order to monitor patient response and understand the limitations of immunotherapy, we performed phage display to discover peptides targeted to tumor infiltrating lymphocytes (TILs) in melanoma tumors. To improve phage library characterization, we developed a method to deep sequence phage with the Ilumina MiSeq platform. Phage screens using PhD7 library (NEB) performed on HUVECS and on TILs, naïve and effector T cells, and B cells harvested from mice. The phage libraries generated from these screens were deep sequenced using the Illumina MiSeq system. The sequencing data was analyzed with an efficient, custom MATLAB script, which arranges sequences by frequency determining peptide frequencies for each library. Peptide frequency was normalized to a reference library generated by amplifying an unscreened phage library. T cell screens were compared with B cell and endothelial cell screens in sequence frequency matrices. Top TIL targeting sequences were cloned into phage, and then fluorescently labeled along with insert-less control phage. Labeled phage were incubated with TILs, or effector T cells and analyzed for specificity using flow cytometry. Four phage clones were identified with at least four fold increased binding for TILs over effector T cells isolated from the spleen. Interestingly, only subsets of the TILs were bound by the targeted phage. We have identified and validated peptides that demonstrate specificity for CD8 TILs. These peptide sequences represent candidates for development into companion diagnostic imaging agents for use with checkpoint therapies. We are currently evaluating these peptides in an in vivo tumor model to further validate their specificity and to determine their biodistributions. Peptides that validate in vivo could be developed into useful imaging agents. After peptide sequences have been validated in vivo, we will determine the phage binding partner using a phage based pulldown. The peptide binding partners discovered through this approach will provide insight into the subset of TILs bound by the phage. [1] Wolchok, J. D. et al., N Engl J Med 2013; 369:122-133. [2] Drake, CG. et al., Clin Cancer Res November 15, 201117. Citation Format: Dustin Bauknight, Andrew Buckner, Lindsey Brinton, Timothy Bullock, Kimberly Kelly. T cell targeted peptides for monitoring immune response in melanoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4147.