CD200 Immune-Checkpoint Peptide Elicits an Anti-glioma Response Through the DAP10 Signaling Pathway

Abstract

Numerous therapies aimed at driving an effective anti-glioma response have been employed over the last decade; nevertheless, survival outcomes for patients remain dismal. This may be due to the expression of immune-checkpoint ligands such as PD-L1 by glioblastoma (GBM) cells which interact with their respective receptors on tumor-infiltrating effector T cells curtailing the activation of anti-GBM CD8⁺ T cell-mediated responses. Therefore, a combinatorial regimen to abolish immunosuppression would provide a powerful therapeutic approach against GBM. We developed a peptide ligand (CD200AR-L) that binds an uncharacterized CD200 immune-checkpoint activation receptor (CD200AR). We sought to test the hypothesis that CD200AR-L/CD200AR binding signals via he DAP10&12 pathways through in vitro studies by analyzing transcription, protein, and phosphorylation, and in vivo loss of function studies using inhibitors to select signaling molecules. We report that CD200AR-L/CD200AR binding induces an initial activation of the DAP10&12 pathways followed by a decrease in activity within 30 min, followed by reactivation via a positive feedback loop. Further in vivo studies using DAP10&12KO mice revealed that DAP10, but not DAP12, is required for tumor control. When we combined CD200AR-L with an immune-stimulatory gene therapy, in an intracranial GBM model in vivo, we observed increased median survival, and long-term survivors. These studies are the first to characterize the signaling pathway used by the CD200AR, demonstrating a novel strategy for modulating immune checkpoints for immunotherapy currently being analyzed in a phase I adult trial.

Keywords: CD200AR; GBM; Immune checkpoints; Immunotherapy; Phase 1.

Fig. 1

CD200AR-L modeling. A Cartoon representation of the mouse CD200R1 (red) and CD200 (green) crystal structure (PDB: 4BFI) with the highlighted sequence location of CD200AR-L (yellow). Association between CD200 and CD200R1 involves the 2 N-domains which includes CD200AR-L residues 32–44 region that accounts for 42% of the CD200R1 binding site. B Sequence conservation of CD200AR-L across the 26 available mammalian CD200 sequences from NCBI. C Multiple sequence alignment of mammalian CD200 sequences from NCBI. The highly conserved CD200AR-L sequence motif (boxed) is highlighted. D Electrostatic potential surface of the binding site highlighting the electrostatic complementarity between CD200AR-L and CD200R4 residues

Fig. 2

CD200AR-L upregulated DAP10 and DAP12 pathways. Preliminary nanostring analysis suggest that the CD200AR-L activated the DAP10 and DAP 12 pathways. To validate these results, CD11b cells isolated from wild-type mice were pulsed with the CD200AR-L, cells were harvested at various times analyzed for A transcription levels and B, C Western analysis for protein levels. In separate experiments, D-H cells were harvested at 6 and 18 h; select signaling molecules were analyzed for transcription levels. I To profile phosphorylation, cells were pulsed for 5 min and analyzed by Fe-IMAC enrichment and LC-MS/MS to derive a Sigmoidal plot of quantitative changes (log2) between CD200AR-L treatment and control. Error bars represent standard deviation, *p < 0.05, **p < 0.005, ***p < 0.0005 by t test

Fig. 3

Signaling molecule inhibitors reduce the CD200AR-L activity. CD11b cells isolated from wild-type mice were pulsed with the CD200AR-L ± various signaling molecule inhibitor for 6 h and analyzed for transcription levels of A p38MAPK, B ERK1/2, C Jak 1, and D Jak 2. CD11b cells isolated from wild-type mice were pulsed with the CD200AR-L ± various signaling molecule inhibitor and incubated for 48 h. Supernatants were analyzed for E TNF-alpha production by bead array. CD200AR-L in combination with gene therapy (GT) results in increased efficacy. F Timeline of treatment of the combined CD200AR-L + GT survival study. 2 × 10⁴ GL261 cells were implanted at day 0. G Kaplan–Meier GT. All mice (5/5) treated with the combination CD200AR-L + GT exhibits 100% long-term survival with no signs of residual tumor. Error bars represent standard deviation. Survival was analyzed by log-rank; Mantel–Cox test, graphs by t test; *p < 0.05, **p < 0.001, *p < 0.05, **p < 0.005, ***p < 0.0005

Fig. 4

Knocking out the DAP10 and DAP12 pathways inhibits ability the of CD200AR-L to activate CD11b cells. A CD11b cells isolated from wildtype, DAP 10, and DAP12 mice were pulsed with the CD200AR-L and incubated for 48 h. Supernatants were analyzed for TNF-alpha production by bead array. B Wildtype, C DAP12KO, and D DAP10K0 tumor-bearing mice were vaccinated with tumor lysate or CD200AR-L + tumor lysate and monitored for tumor growth and E survival. Error bars represent standard deviation, *p < 0.05, **p < 0.005, ***p < 0.0005 by t test or 2-way ANOVA

Fig. 5

CD200AR-L modulates CD200 checkpoint receptor expression. CD11b cells isolated from wild-type mice were pulsed with the CD200AR-L; cells were harvested at various times analyzed for transcription levels for the A inhibitory receptor, CD200R1, and the B activation receptors, CD200AR2, C CD200AR3, and D CD200AR4 expression levels. CD11b cells isolated from wild-type mice were pulsed with the CD200AR-L; cells were E analyzed by flow cytometry for the inhibitory CD200R1 expression. F In a separate experiment, CD11b cells isolated from wildtype mice were pulsed with the CD200AR-L + OVA, washed, and added to T-cells. CD3⁺CD200R1⁺ cells were analyzed by flow cytometry. G Wildtype and DAP10KO mice were vaccinated with CD200AR-L for 3 consecutive days over the inguinal lymph nodes. Mice were sacrificed on day 4; lymphocytes isolated from inguinal lymph nodes were analyzed for CD11b⁺CD200R1⁺ cells were analyzed by flow cytometry. MFIs were normalized to no pulsed controls. Error bars represent standard deviation, *p < 0.05, **p < 0.005, ***p < 0.0005 by t test

Fig. 6

Model of the CD200AR-L signaling pathway. CD200AR-L binds to the CD200AR complex activating the DAP10 pathway and associated signaling molecules (PLCy, PI3K, Grb3, Slp76, Vav1, Rac, Pak1, Mek, ERK, and C Jun) DAP12 pathway and associated signaling molecules (SKY, SLP76, SHC, GRB2, and Ras) and Jak1/2 molecules