Radiotherapy (RT) is commonly employed to treat solid tumors. Immune checkpoint blockade of programmed cell death protein 1 (PD-1) and CTLA-4 improves survival in RT patients, yet many fail to respond to combination therapy. Natural killer group 2 (NKG2) family receptors, particularly inhibitory NKG2A and activating NKG2D, have emerged as promising therapeutic targets to improve antitumor T cell responses; thus, we examined how these receptors and their ligands (Qa-1b and retinoic acid early inducible 1 [Rae-1], respectively) regulate the RT response in C57BL/6 mice bearing syngeneic B16F10 melanoma and MC38 colorectal adenocarcinoma tumors. RT (15 Gy) transiently reduced B16F10 tumor burden, whereas MC38 tumors exhibited durable response to RT. Intratumoral NK and CD8 T cells expressed NKG2A and NKG2D in both models, which was unaltered by RT. In vitro/in vivo RT increased tumor/stromal cell Qa-1b and Rae-1 expression in both models, especially B16F10 tumors, but IFN-γ stimulation induced both Qa-1b and Rae-1 only in B16F10 tumors. NKG2A/Qa-1b inhibition alone did not improve RT response in either model, but combined RT and NKG2A/PD-1 blockade improved survival in the B16F10 model. Depletion experiments indicate that the triple therapy efficacy is CD8 T cell–dependent with negligible NK cell contribution. RNA sequencing of CD8 T cells from triple therapy–treated B16F10 tumors showed increased proliferative capacity compared with RT and PD-1 blockade alone. Our work demonstrates that RT modulates NKG2A ligand expression, which inhibits RT-induced T cell responses in tumors that fail to respond to combined RT and PD-1 blockade. These results provide a rationale for combining NKG2A blockade with immune checkpoint blockade therapies and RT to improve clinical response.

More than half of patients with solid tumor malignancies undergo treatment with radiotherapy (RT). In addition to causing direct tumor cell death, RT results in release of tumor antigen and damage associated molecular patterns that elicit a CD8 T cell and IFN-g dependent anti-tumor immune response. To further bolster this response, RT has been combined with immune checkpoint inhibitors such as anti-PD-1 and anti-CTLA-4. Despite a modest increase in survival when these therapies are used alone or together in conjunction with RT, many patients fail to respond to treatment, suggesting other mechanisms of immune suppression exist in the tumor microenvironment (TME). Using syngeneic B16 F10 melanoma and C38 colorectal adenocarcinoma models, we have studied the role of the inhibitory receptor NKG2A. We have observed that NKG2A is expressed only on tumor infiltrating lymphocytes (TILs) and expression of this receptor does not change with RT. Furthermore, RT increases intratumoral expression of the ligand for NKG2A, Qa-1b, as does IFN-g stimulation in vitro. Blockade of NKG2A alone through use of B16 F10 cells lacking Qa-1b expression or blocking antibodies did not significantly increase survival of mice treated with RT. Further analysis revealed that among CD8 TILs, only a minority of cells express NKG2A alone, with most TILs co-expressing NKG2A and PD-1 or PD-1 alone. Thus, we combined anti-NKG2A and anti-PD-1 blockade and observed increased survival in mice treated with RT, whereas either therapy alone was ineffective. These results suggest that NKG2A blockade could be combined with RT and existing immunotherapies clinically to improve patient response. Supported by awards T32AI007285 from the NIAID and R01CA028332 from the NCI.

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