Abstract A21: Adenosine is a rheostat for radiation therapy-induced immune activation
Authors: Wennerberg Erik , Kawashima Noriko , Demaria Sandra 
Published: October 2015

Abstract
Background: Administration of anti-CTLA-4 antibodies has shown clinical benefit in a small percentage of cancer patients. However, most patients are unresponsive to anti-CTLA-4 antibody treatment, due to immunosuppressive mechanisms and/or the absence of sufficient spontaneous anti-tumor T cells that can be activated by CTLA-4 blockade. We have previously shown that radiotherapy (RT) induces immunogenic tumor cell death and priming of anti-tumor T cells making tumors susceptible to anti-CTLA-4 treatment. RT also triggers the release of ATP in the tumor microenvironment, which has proinflammatory effects. However, ATP is rapidly converted into adenosine, a pleiotropic immunosuppressive mediator, by ectonucleotidases CD39 and CD73 expressed on cancer cells and tumor stromal cells. We hypothesized that adenosine generation in the tumor microenvironment acts as a rheostat modulating RT ability to prime effective anti-tumor immunity. To test this hypothesis we used a mouse tumor model where we have previously characterized two RT regimens that were efficient (8Gy x 3) or inefficient (20 Gy) in inducing anti-tumor immunity in combination with anti-CTLA-4 antibodies.
Method: BALB/c mice were inoculated s.c. with 1 x 105 TSA cells, a poorly immunogenic breast cancer cell line on day 0. Anti-CD73 mAb TY/23 (200 µg) was administered i.p. every 4 days starting on day 11. RT was given locally to the tumor as a single 20 Gy dose on day 12 or in three doses of 8 Gy each given on day 12, 13 and 14. At day 19, tumors were harvested and infiltrating immune cells were isolated for analysis of DC and T cell phenotype by flow cytometry. In another experiment, mice were randomly assigned (n=5/group) to be treated with: (1) Isotype control mAb (2) TY/23 (3) TY/23 + anti-CTLA-4 mAb 9H10 (4) RT 20 Gy (5) RT 20 Gy + TY/23 (6) RT 20 Gy + 9H10 (6) RT 20 Gy + TY/23+ 9H10. RT was given on day 12 and mAbs were administered i.p. on day 11, 14, 17 and 20 after tumor inoculation. Mice were monitored for tumor growth.
Results: In tumors that had received 8GyX3 RT there was a four-fold increase in CD70 expression (MFI 436 ± 72 in 8GyX3 vs. 110 ± 30 in control, p&lt;0.05) on CD8α+ dendritic cells (DCs), which are responsible for tumor-antigen cross-presentation. In addition, CD8+ T cells showed a two-fold increase in PD-1 expression (MFI 550 ± 22 in 8GyX3 vs. 271 ± 35 in control, p&lt;0.01) and 1.6-fold increase in CD69 expression (MFI 351 ± 95 in 8GyX3 vs. 225 ± 40 in control), indicating increased activation of both DC and T cells in tumors treated with 8GyX3 RT. In contrast, there was no evidence of activation of DC and T cells infiltrating tumors treated with 20Gy RT. However, when 20Gy RT was given in combination with TY/23 to block adenosine generation, DC and T cells showed an increase in expression of activation markers comparable to what was observed in tumors treated with 8GyX3. Interestingly, the frequency of intratumoral CD4+CD25+FoxP3+ regulatory T cells (Treg) was increased in mice receiving TY/23 + 8GyX3 (62% increase, p&lt;0.05) compared to TY/23 alone, suggesting that the inflammation induced by RT may trigger Treg recruitment to the tumor when it is not controlled by adenosine. TY/23 and 9H10 given alone or in combination did not have any effect on tumor growth. However, they improved tumor inhibition obtained with 20 Gy RT (Two-way ANOVA: p=0.08 for RT+anti-CD73 and p&lt;0.05 for RT+9H10 vs. RT alone). Moreover, blockade of both CD73 and CTLA-4 in combination with 20Gy RT further improved tumor control resulting in complete tumor regression in 2/5 mice (p&lt;0.01 for RT+TY/23+9H10 vs. RT alone).
Conclusions: Data indicate that the ability of RT to promote immune activation is dependent on the radiation regimen employed and is regulated by adenosine generation by the ectonucleotidase CD73. Importantly, data suggest that blocking CD73 can improve the ability of RT to synergize with immunotherapy.
Citation Format: Erik Wennerberg, Noriko Kawashima, Sandra Demaria. Adenosine is a rheostat for radiation therapy-induced immune activation. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A21.