Background Adoptive T cell transfer therapies, including chimeric antigen receptor (CAR)-T, have been successful in treating circulating B cell malignancies. However, efficient intratumoral infiltration of adoptively transferred cells remains a significant barrier to effectively treating solid tumors. Identification of T cell-intrinsic mechanisms promoting infiltration into tumors could lead to novel approaches that would enhance existing therapies and support development of new therapeutic interventions. We recently performed an in vivo, genome-wide screen to identify T cell-intrinsic regulators of endogenous T cell intratumoral accumulation into solid tumors using the Sleeping Beauty (SB) transposon system.1 The most strongly selected mutation was overexpression of a truncated AP-2 associated kinase 1 (Aak1). We hypothesize that overexpression of truncated AAK1 (AAK1DN125) in adoptively transferred T cells would increase intratumoral accumulation into solid tumors.
Aak1 kinase activity potentiates clathrin assembly by phosphorylating the µ2 subunit of AP2 during initiation of clathrin-mediated endocytosis.2 Aak1 may support T cell migration by regulating the chemokine receptor, CXCR3, which undergoes clathrin-mediated endocytosis upon interacting with chemokines CXCL9/10. Supporting this, in vitro assays show that OT-1 cells treated with Aak1 inhibitor increased migration toward CXCL10.1 Here, we test whether overexpression of AAK1DN125 produces the same effect on promoting T cell migration toward CXCL10.
Methods In vitro Transwell migration assays were performed with primary T cells overexpressing full-length AAK1 or AAK1DN125 and CXCR3 ligands CXCL9 and CXCL10. To measure impact of AAK1DN125 on tumor infiltration, we adoptively transferred polyclonally activated T cells overexpressing either AAK1DN125 or empty vector into EG7 tumor bearing mice. Immune infiltration one week later was measured using flow cytometry. To further characterize the relationship between Aak1 and Cxcr3, colocalization studies were performed using confocal microscopy, with further investigation ongoing through co-immunoprecipitation studies.
Results In vitro assays show increased migration of T cells overexpressing AAK1DN125 toward CXCR3 ligands compared to empty vector or full-length AAK1. In vivo migration of adoptively transferred T cells into tumors, but not spleen or lymph nodes, was significantly increased when the T cells overexpressed AAK1DN125 . Further, AAK1DN125 expression improves selective T cell migration in vitro toward chemokine receptor CXCR3 ligands, but not CCR2, CCR5, or CCR7 ligands, suggesting there may be specificity for inflamed tissues.
Conclusions Our data strongly support developing AAK1DN125 as a strategy to increase adoptively transferred T cell homing to the tumor microenvironment and expand the efficacy of adoptive T cell transfer therapies beyond circulating malignancies.
Rogers LM, Wang Z, Mott SL, Dupuy AJ, Weiner GJ. A Genetic Screen to Identify Gain- and Loss-of-Function Modifications that Enhance T-cell Infiltration into Tumors. Cancer Immunol Res. 2020;8:1206–1214.
Conner SD, Schmid SL. Identification of an adaptor-associated kinase, AAK1, as a regulator of clathrin-mediated endocytosis. J Cell Biol. 2002;156:921–929.
Ethics Approval The Mayo Clinic Animal Care and Use Committee (IACUC) approved all uses in this study. Mice were purchased from Jackson Laboratories (JAX stock #000664 and #003831) and were housed in specific pathogen-free facility at Mayo Clinic.
This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See http://creativecommons.org/licenses/by-nc/4.0/.
Statistics from Altmetric.com
If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.