DOI: 10.5061/dryad.4mw6m90g8
title: Data from: DGKα/ζ inhibition lowers the TCR affinity threshold and potentiates anti-tumor immunity

description: Checkpoint blockade immunotherapies expand neoantigen- or virus-specific T
 cells, and poor responsiveness to immunotherapy is associated with lower
 mutational burden in tumors of non-viral origin. Although mouse models
 demonstrate that lower affinity T cells recognizing self-antigens can
 contribute to tumor control if sufficiently activated, therapeutic options
 for enhancing T cell priming are limited. Diacylglycerol kinases (DGKs)
 attenuate DAG signaling by converting DAG to phosphatidic acid, thereby
 suppressing pathways downstream of TCR signaling. Using a novel dual DGK
 alpha and zeta inhibitor (DGKi), tumor-specific CD8 T cells with different
 affinities (TRP1high and TRP1low), and a series of altered peptide
 ligands, we demonstrate that inhibition of DGKα/ζ can lower the signaling
 threshold for T cell priming. TRP1high and TRP1low CD8 T cells produced
 more IL-2, IFNγ, and other effector cytokines in the presence of cognate
 antigen and DGKi. Effector TRP1high- and TRP1low-mediated cytolysis of
 tumor cells with low antigen load was MHC-restricted, mediated by IFNγ,
 and augmented by DGKi. Adoptive T cell transfer into mice bearing
 pancreatic or melanoma tumors synergized with single-agent DGKi or DGKi
 and αPD1, with increased expansion of low-affinity T cells and increased
 cytokine production observed in tumor infiltrates of treated mice.
 Collectively, our findings highlight DGKα/ζ as therapeutic targets for
 augmenting tumor-specific CD8 T cell function.
description: Methods used are described in detail in the manuscript.
description: # Data from: DGKα/ζ inhibition lowers the TCR affinity threshold and
 potentiates anti-tumor immunity Data for Kureshi et al Raw Quantitative
 values for all the figure data sets. Each sheet is labeled with the
 corresponding figures and corresponding panels are labeled in bold font.
 Figure 1. Inhibition of DGKα/ζ in antigen-specific CD8 T cells enhances
 proliferation and cytokine production in a dose-dependent manner. Panel A
 & B. Trp1high CD8 T cells were activated with αCD3/28 antibodies for
 5,15,30, and 60 minutes and western blot was conducted for phosphorylated
 and total ERK1/2 levels. Band intensity values from imageJ are provided in
 the Excel sheet. Panel C. Trp1high Cd8 T cells were activated with B cells
 pulsed with 0, 50, and 500pg/mL peptide at denoted concentrations of DGK
 inhibitor. Proliferation indices are provided in the Excel sheet and was
 calculated from CTV staining. Fold change in proliferation was calculated
 in reference to vehicle-treated group for each peptide concentration.
 Panel D. IFNg and IL-2 ELISA were conducted from supernatants from priming
 assay in panel C. Protein levels from ELISA are provided. Panel E & F.
 The same experiments were conducted as panel C and D but with Trp1low CD8
 T cells. Panel G. Trp1high CD8 T cells were activated with B cells pulsed
 with Trp1 peptide variants. Proliferation index was similarly calculated
 from CTV staining and provided in the excel sheet. Fold change in
 proliferation was calculated in reference to vehicle-treated group for
 each respective peptide variant. Panel H. IFNg and IL-2 ELISA values from
 supernatants from panel G priming assay. Panel I & J. The same
 experiments were conducted as panel G and H, but with Trp1low CD8 T cells.
 Figure 2. DGKα/ζ inhibition in CD8 T cells enhances killing of pancreatic
 cancer cells Panel C. Effector Trp1high Cd8 T cells cocultured with B16
 zsgreen, C2VTrp1, and C2 zsgreen cells. Celigo imaging cytometer measure
 zsgreen confluency. Percent cytotoxicity values were calculated from
 zsgreen confluency and are provided. Panel D. Supernatants from the
 cytotoxicity assay in panel C were measured for IFNg, IL-2, TNFa protein
 levels. Data from ELISA is provided in the excel sheet. Panel E & F.
 Same cytotoxicity assay as panel C & D but with effector Trp1low CD8 T
 cells. Panel G & H. Same cytotoxicity assay as panel C & D but
 with effector OT-I CD8 T cells and the addition of B16-OVA tumor cells.
 Figure 3. DGKi-mediated cytotoxicity by CD8 T cells occurs even at very
 low peptide MHC densities Panel A. MFI values for PD-L1 and MHC-I
 (H2Kb/Db) surface expression on C2VTrp1 WT and b2m-/- cells in the
 presence or absence of IFNg. Panel B. Effector Trp1high CD8 T cells were
 cocultured with C2VTrp1 WT or b2m-/- with or without IFNg pretreatment.
 Percent cytotoxicity values are provided. Panel C. Effector Trp1low
 cytotoxicity was measured just like in panel B. Panel D and F. IFNg and
 TNFa protein levels from Panel B cytotoxicity experiment. Panel E and G.
 IFNg and TNFa protein levels from Panel C cytotoxicity experiment. Panel
 I. Effector Trp1high T cells cocultured with C2VTrp1 cells and a
 caspase-3,7 dye. Percent of tumor cells positive for caspase-3,7 activity
 was measured by a celigo imaging cytometer and is provided. Figure 4.
 DGKi-dependent cytotoxicity is mediated by IFN-γ but not perforin Panel B.
 Effector Trp1high WT, IFNg-/-, or Prf1-/- CD8 T cells were cocultured with
 C2VTrp1 tumor cells and percent cytotoxicity values are provided. Panel C
 and D. IFNg and TNFa protein levels are provided and were measured from
 supernatants from panel B's cytotoxicity assay. Figure 5. Timing of
 DGKi treatment affects TRP1high cytotoxicity of C2VTrp1 cells Panel B.
 Trp1high Cd8 T cells were treated with DGKi at three phases - T cell
 activation, expansion/differentiation, and coculture with C2VTrp1 tumor
 cells. Cytotoxicity values are provided after 18 and 48 hours of
 coculture. Panel C and D. IFNg and TNFa protein levels are provided and
 were measured from supernatants collected from panel B's cytotoxicity
 assay after 48 hours of coculture. Panel E. Effector Trp1high CD8 T cells
 were cocultured with C2VTrp1 tumor cells at different T cell:tumor cell
 ratios. Cytotoxicity values after 48 hours of coculture are provided.
 Panel F. Supernatant from Panel E assay were measured for IFNg levels.
 Figure 6. DGKi synergizes with αPD-1 therapy to control melanoma and
 pancreatic tumor growth in an antigen-dependent manner Panel B and C.
 Contralateral tumors - C2VTrp1 and C2 WT. Tumor volume measurements are
 provided. Panel E. Tumor volume measurements for B16 tumor growth with
 Trp1high and Trp1low CD8 T cells adoptively transferred into mice one day
 prior to tumor inoculation. Panel F. Survival Curve from Panel E. 1
 denotes endpoint for a mouse (this is how graphpad prism requires input of
 survival data). Figure 7. DGKi increases proliferation and cytokine
 production in antigen-specific T cells in mice bearing C2VTrp1 tumors
 Panel D. Proliferation indices are provided. Panel E. Percentage of
 Trp1high and Trp1low Cd8 T cells out of total number of lymph node cells
 is provided. Panel G. Tumor weights of C2VTrp1 tumors 18 days after
 inoculation. Panel H. Ratio of the percentage of Trp1high Cd8 T cells to
 Trp1low CD8 T cells Panel I. Percentage of Trp1high and Trp1low CD8 T
 cells from total cells in the tumor. Panel J-L. Intracellular cytokine
 staining and the percentage of Trp1high cells that are IFNg, IL-2, or TNFa
 positive out of all Trp1high and low CD8 T cells. Panel M-O. Fraction of
 Trp1low CD8 T cells that IFNg, IL-2, or TNFa positive out of all Trp1high
 and low CD8 T cells. Figure S3. IL-2 augments DGKi-mediated proliferation
 Panel A. Trp1high CD8 T cells activated for 72 hours in the presence or
 absence of peptide and/or 100U/mL IL-2. Proliferation indices are
 provided. Panel C. Same as panel A, but with Trp1low Cd8 T cells. Figure
 S4. DGKi upregulates cytokine transcript and protein expression during T
 cell priming. Panel A. Proliferation index from Trp1high T cells activated
 with 500pg/ml native Trp1 peptide. Panel B. qPCR fold-change data for
 IFNg, IL-2, and TNFa transcript levels were measured from Trp1high T cells
 activated at 24, 48, and 72 hours. Panel C. ELISA data measuring protein
 levels of aforementioned three cytokines. Figure S5. DGKi upregulates Myc
 expression in Trp1high CD8 T cells in a temporal manner. Panel A. Trp1high
 Myc-GFP CD8 T cells were activated and proliferation indices are provided
 from CTV staining. Panel B. Percentage of CD8 T cells that are myc-gfp+
 Panel C. Myc-GFP MFI Panel D. Myc expression (MFI) for every division for
 vehicle-treated Trp1high Cd8 T cells Panel E. Myc expression (MFI) for
 every division for DGKi-treated Trp1high Cd8 T cells Figure S6. Tumor
 growth is unaffected by DGKi in the absence of antigen-specific T cells.
 Panel A and B. B16 and C2VTrp1 confluency over time (24h and 48h time
 points) in the presence of DGKi but in the absence of antigen-specific
 Trp1 CD8 T cells. Panel C and D. Trp1high percent cytotoxicity values when
 Trp1high Cd8 T cells were cocultured with B16 or C2VTrp1 tumor cells and
 indicated DGKi concentrations. Figure S7. DGKi enhances CD8 T cell
 transcript levels and protein expression during coculture with tumor
 cells. Panel A. Percent cytotoxicity data when Trp1high CD8 T cells were
 cocultured with C2VTrp1 tumor cells in the presence or absence of DGKi.
 Panel B. qPCR fold-change data for IL-2, IFNg, and TNFa transcript levels
 from panel A assay. Panel C. ELISA protein levels for IL-2, IFNg, and TNFa
 from panel A assay. Figure S8. DGKi and aPD-1 in combination decreases CD8
 T cell PD-1 expression and increases tumor cell PD-L1 expression. Panel B.
 Percentage of PD-L1+ cells out of zsgreen+ cells for the four treatment
 conditions. Panel C. PDL1 MFI from zsgreen+ cells Panel D. Percentage of
 CD8 T cells that are PD-1+ Panel E. PD-1 MFI from PD1+CD8+ T cells.
 Figures S11. Enhanced cytokine production by antigen-specific T cells in
 DGKi treated mice was not found in the spleen. Panel A. Percentage of
 adoptively transferred CD8 T cells found in the spleen from mice bearing
 C2VTrp1 tumors Panel B. The ratio of TRP1low to TRP1high cells found in
 the spleen. Panel C and D. Frequency of (C) TRP1high or (D) TRP1low
 producing IL2, IFNγ, and TNFα, was determined via intracellular cytokine
 staining.