Inhibition of AKT signaling uncouples T cell differentiation from expansion for receptor-engineered adoptive immunotherapy
Christopher A. Klebanoff, Joseph G. Crompton, Anthony J. Leonardi, Tori N. Yamamoto, Smita S. Chandran, Robert L. Eil, Madhusudhanan Sukumar, Suman K. Vodnala, Jinhui Hu, Yun Ji, David Clever, Mary A. Black, Devikala Gurusamy, Michael J. Kruhlak, Ping Jin, David F. Stroncek, Luca Gattinoni, Steven A. Feldman, Nicholas P. Restifo
Christopher A. Klebanoff, Joseph G. Crompton, Anthony J. Leonardi, Tori N. Yamamoto, Smita S. Chandran, Robert L. Eil, Madhusudhanan Sukumar, Suman K. Vodnala, Jinhui Hu, Yun Ji, David Clever, Mary A. Black, Devikala Gurusamy, Michael J. Kruhlak, Ping Jin, David F. Stroncek, Luca Gattinoni, Steven A. Feldman, Nicholas P. Restifo
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Research Article Immunology Oncology

Inhibition of AKT signaling uncouples T cell differentiation from expansion for receptor-engineered adoptive immunotherapy

Abstract

Adoptive immunotherapies using T cells genetically redirected with a chimeric antigen receptor (CAR) or T cell receptor (TCR) are entering mainstream clinical practice. Despite encouraging results, some patients do not respond to current therapies. In part, this phenomenon has been associated with infusion of reduced numbers of early memory T cells. Herein, we report that AKT signaling inhibition is compatible with CAR and TCR retroviral transduction of human T cells while promoting a CD62L-expressing central memory phenotype. Critically, this intervention did not compromise cell yield. Mechanistically, disruption of AKT signaling preserved MAPK activation and promoted the intranuclear localization of FOXO1, a transcriptional regulator of T cell memory. Consequently, AKT signaling inhibition synchronized the transcriptional profile for FOXO1-dependent target genes across multiple donors. Expression of an AKT-resistant FOXO1 mutant phenocopied the influence of AKT signaling inhibition, while addition of AKT signaling inhibition to T cells expressing mutant FOXO1 failed to further augment the frequency of CD62L-expressing cells. Finally, treatment of established B cell acute lymphoblastic leukemia was superior using anti-CD19 CAR–modified T cells transduced and expanded in the presence of an AKT inhibitor compared with conventionally grown T cells. Thus, inhibition of signaling along the PI3K/AKT axis represents a generalizable strategy to generate large numbers of receptor-modified T cells with an early memory phenotype and superior antitumor efficacy.

Authors

Christopher A. Klebanoff, Joseph G. Crompton, Anthony J. Leonardi, Tori N. Yamamoto, Smita S. Chandran, Robert L. Eil, Madhusudhanan Sukumar, Suman K. Vodnala, Jinhui Hu, Yun Ji, David Clever, Mary A. Black, Devikala Gurusamy, Michael J. Kruhlak, Ping Jin, David F. Stroncek, Luca Gattinoni, Steven A. Feldman, Nicholas P. Restifo

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Figure 2

Blockade of AKT signaling minimizes gene expression changes associated with human T cell differentiation and promotes nuclear accumulation of the transcription factor FOXO1.

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Blockade of AKT signaling minimizes gene expression changes associated w...
(A) Unsupervised hierarchical clustering of gene expression from peripheral blood T cells at baseline and 10d after stimulation, retroviral transduction (RV Td) with an anti-CD19 chimeric antigen receptor (CAR), and expansion in the continuous presence of IL-2 (300 IU ml–1) and AKT inhibitor VIII (AKTi; 1 μM) or a vehicle control (Veh). Clustering was based on all significantly expressed genes (1-way ANOVA, FDR-corrected P < 0.05) using a Pearson correlation. T cells were derived from 3 independent donors using an n = 3–4 replicates per donor, per time point, and per condition. (B) Heat map of expression for selected genes that are direct targets of the transcription factor FOXO1 or genes involved in T cell costimulation, effector function, and apoptosis. Red and blue colors indicate relative increased and decreased expression, respectively. Each column represents the average expression of a gene from an individual donor, and each row represents the indicated gene. (C) Robust multichip analysis–normalized (RMA-normalized) intensity of selected FOXO1-target genes and genes associated with T cell differentiation at baseline and 10d after ex vivo expansion in the continuous presence of AKTi or Veh. The bar graphs represent the mean ± SEM of n = 3 overlaid biological replicates per condition from a single donor and are representative of experiments observed with all 3 independently analyzed donors. (D) Representative confocal immunofluorescence images and (E) summary graph quantifying the dose-dependent intranuclear accumulation of FOXO1 4d following T cell activation and expansion in indicated concentrations of AKTi or Veh control. Red, FOXO1; blue, Hoechst; and green, phalloidin. Scale bar: 10 μm. Data shown are representative of 2 independently performed experiments using 2 separate donors. (F) The ratio of gene expression levels for FOXO1 in AKTi- versus Veh-treated cells from n =3 independently evaluated donors. Dashed horizontal line indicates an expression ratio of 1. Data shown in panels A–C and F are from patient-derived T cells, and panels D and E are from healthy donors (HDs). Statistical comparisons in panels C and E were performed using an unpaired 2-tailed Student’s t test corrected for multiple comparisons by a Bonferroni adjustment. ***P < 0.001; **P < 0.01; *P < 0.0167.