Background Killer immunoglobulin-like receptors (KIRs) are a family of regulatory cell surface molecules expressed on natural killer (NK) cells and in subsets of memory T cells.^1,2 Interaction of KIR2DL2 with its HLA-C1 ligands leads to inhibition of NK cell activation and decoupling of T cell effector function by inhibiting actin cytoskeleton rearrangement and modifying T cell transcriptional profile.^3-5 Here we assess the effect of KIR2DL2 on CAR-T cell effector function both in vitro and in vivo and propose KIR2DL2 as an immune checkpoint. We have developed a strategy for genomic ablation of KIR2DL2 during T cell manufacturing as a method for enhancement of adoptive cell immunotherapy (ACT).

Methods We generated prostate stem cell antigen (PSCA)-CAR-T cells, alone or in combination with KIR2DL2, using a bicistronic retroviral vector. We tested KIR2DL2 in vitro inhibitory role in presence or absence of HLA-C1 using real time cytotoxicity assays at different effector:target ratios (2.5:1, 1:1, 0.5:1, 0.25:1). We evaluated IFN-γ production by ELISA. To assess the inhibitory role of KIR2DL2 in vitro we used a mouse model bearing HLA-C1⁺ or HLA-C1^- subcutaneous xenografts of human pancreatic adenocarcinoma (HPAC) cells, treated with 5x10⁶ KIR2DL2⁺ or KIR2DL2^- CAR-T cells. We used GFP-transduced T cells as a negative control. CRISPR/Cas9 genome editing was used for KIR2DL2 ablation in CAR-T cells.

Results In vitro, KIR2DL2 impaired CAR-T cell responses in an HLA-C1-dependent manner. KIR2DL2⁺ PSCA-CAR-T cells were significantly less cytotoxic in presence of HLA-C1 and secreted less IFN-γ than their KIR2DL2^- counterparts. In vivo, KIR2DL2⁺ PSCA-CAR-T cells injected in NSG mice harboring PSCA⁺/HLA-C1⁺ pancreatic tumors were not able to eliminate tumors in the presence of HLA-C1. In contrast, KIR2DL2⁺ CAR-T cells transferred to mice harboring PSCA⁺/HLA-C1^-/- tumor cells performed as well as their KIR2DL2^- counterparts. We designed guide RNAs targeting KIR2DL2 and developed a strategy for KIR2DL2 abrogation during adoptively transferred CAR-T cell manufacturing.

Conclusions We evaluated for the first time the biological and molecular function of KIR2DL2 within adoptively transferred T cells. Engagement of KIR2DL2 with its HLA-C1 ligand(s) impaired CAR-T cell effector function both in vitro and in vivo, as CAR⁺/KIR2DL2⁺ cells were less cytotoxic and secreted less IFN-γ than their CAR⁺/KIR2DL2^- counterparts. KIR2DL2 abrogation in CAR-T cells may enhance ACT by limiting its inhibitory signaling. Current efforts are focused on evaluating the impact of KIR2DL2 ablation on the therapeutic effect of CAR- and TCR-T cells.

Acknowledgements This work has been supported in part by the Flow Cytometry and Comparative Medicine Core Facilities at Moffitt Cancer, a National Cancer Institute (NCI) designated Comprehensive Cancer Center (P30 CA076292); and by a donation by the Steinman Family Foundation.

References

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2. Bjorkstrom, N.K., et al., CD8 T cells express randomly selected KIRs with distinct specificities compared with NK cells. Blood, 2012; 120(17): p. 3455-65.

3. Burshtyn, D.N., et al., Recruitment of tyrosine phosphatase HCP by the killer cell inhibitor receptor. Immunity, 1996; 4(1): p. 77-85.

4. Olcese, L., et al., Human and mouse killer-cell inhibitory receptors recruit PTP1C and PTP1D protein tyrosine phosphatases. J Immunol, 1996; 156(12): p. 4531-4.

5. Stebbins, C.C., et al., Vav1 dephosphorylation by the tyrosine phosphatase SHP-1 as a mechanism for inhibition of cellular cytotoxicity. Mol Cell Biol, 2003; 23(17): p. 6291-9.

Ethics Approval This study was approved by the Institutional Animal Care and Use Committee (IACUC); approval number R ISO00010727.