Abstract 5616: Characterization of a spectrum of DTR models for conditional depletion of specific immune cell lineages
Authors: Jin Ying , Zhou Jiahua , Li Fengge , Sun Ruilin , An Annie Xiaoyu , Li Henry Q. X. , Ouyang Davy Xuesong 
Published: September 2020

Abstract
Introduction: Cancer immunotherapy often works through more than one mechanism of action (MOA) mediated by multiple immune cell subsets. We have demonstrated that the antitumor effects of anti-PD-1 treatment, while predominately driven by CD8+ cytotoxic T cells (CTL), is also led by CD4+ effector T cells in some tumors. For anti-CTLA-4 treatment, it is still under debate whether efficacy is attributed to intrinsic CD8+ T cell activation, Treg depletion, or other extrinsic regulations. Other immune modulators have MOAs with even more potential to impact treatment outcomes, and may differ significantly in different tumor microenvironments. It is critical to dissect the MOA of specific treatments by clarifying their role through different immune cell lineages, which may guide on rational approaches for monotherapy as well as combination strategies.
Methods: We have developed a series of immune lineage-DTR (diphtheria toxin receptor) transgenic mouse models, in which specific immune cell subsets can be easily depleted from the whole mouse following diphtheria toxin (DT) administration. These transgenic mice were engineered to express DTR-EGFP downstream of specific lineage markers, i.e. CD8a, CD4, FoxP3, Ncr-1, CD19, or CD11c. Therefore, expression of DTR and EGFP were both regulated in the same manner as endogenous lineage markers without disrupting their own expression. We have confirmed EGFP expression on specific immune cell lineages by FACS analysis. To test the depletion efficiency by DT in the mice, we engrafted MC38 tumors into DTR mice or naïve mice, treated the mice with DT, and monitored the depletion of specific immune cell lineages over time in blood, spleen, and tumor infiltrating lymphocytes (TILs).
Results: Analysis of depletion efficiency showed that DT administration led to complete depletion of the targeted immune cell populations in blood by Day 2 to Day 6 post DT administration, while repopulation was observed from Day 13 for some lines (e.g. Ncr-1 and CD19). The proportion of the targeted immune cell subset in tumor and spleen remained lower than the naïve mice by study termination on Day 20. The DTR transgene did not affect MC38 tumor growth and the tumor take rate in these mice was 100%. MC38 tumor growth was significantly slower when CD4 and FoxP3 cell populations were depleted. In contrast, CD8 depletion led to a much faster tumor growth rate, whereas NK or B cell depletion appeared not to impact tumor growth.
Conclusions: Taken together, immune cell specific DTR models provide important tools for examining the MOA of various cancer immunotherapies. They also benchmark the maximal tumor growth inhibition that can be achieved by manipulating specific immune cell lineages in specific syngeneic tumors.
Citation Format: Ying Jin, Jiahua Zhou, Fengge Li, Ruilin Sun, Annie Xiaoyu An, Henry Q. X. Li, Davy Xuesong Ouyang. Characterization of a spectrum of DTR models for conditional depletion of specific immune cell lineages [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5616.