Oncogene-induced TIM-3 ligand expression dictates susceptibility to anti–TIM-3 therapy in mice
Nana Talvard-Balland, et al.
Nana Talvard-Balland, et al.
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Research Article

Oncogene-induced TIM-3 ligand expression dictates susceptibility to anti–TIM-3 therapy in mice

Abstract

Leukemia relapse is a major cause of death after allogeneic hematopoietic cell transplantation (allo-HCT). We tested the potential of targeting T cell (Tc) immunoglobulin and mucin-containing molecule 3 (TIM-3) for improving graft-versus-leukemia (GVL) effects. We observed differential expression of TIM-3 ligands when hematopoietic stem cells overexpressed certain oncogenic-driver mutations. Anti–TIM-3 Ab treatment improved survival of mice bearing leukemia with oncogene-induced TIM-3 ligand expression. Conversely, leukemia cells with low ligand expression were anti–TIM-3 treatment resistant. In vitro, TIM-3 blockade or genetic deletion in CD8+ Tc enhanced Tc activation, proliferation, and IFN-γ production while enhancing GVL effects, preventing Tc exhaustion, and improving Tc cytotoxicity and glycolysis in vivo. Conversely, TIM-3 deletion in myeloid cells did not affect allogeneic Tc proliferation and activation in vitro, suggesting that anti–TIM-3 treatment–mediated GVL effects are Tc induced. In contrast to anti–programmed cell death protein 1 (anti–PD-1) and anti–cytotoxic T lymphocyte–associated protein 4 (anti–CTLA-4) treatment, anti–TIM-3-treatment did not enhance acute graft-versus-host disease (aGVHD). TIM-3 and its ligands were frequently expressed in acute myeloid leukemia (AML) cells of patients with post–allo-HCT relapse. We decipher the connections between oncogenic mutations found in AML and TIM-3 ligand expression and identify anti–TIM-3 treatment as a strategy for enhancing GVL effects via metabolic and transcriptional Tc reprogramming without exacerbation of aGVHD. Our findings support clinical testing of anti–TIM-3 Ab in patients with AML relapse after allo-HCT.

Authors

Nana Talvard-Balland, Lukas M. Braun, Karen O. Dixon, Melissa Zwick, Helena Engel, Alina Hartmann, Sandra Duquesne, Livius Penter, Geoffroy Andrieux, Lukas Rindlisbacher, Andrea Acerbis, Jule Ehmann, Christoph Köllerer, Michela Ansuinelli, Andres Rettig, Kevin Moschallski, Petya Apostolova, Tilman Brummer, Anna L. Illert, Markus A. Schramm, Yurong Cheng, Anna Köttgen, Justus Duyster, Hans D. Menssen, Jerome Ritz, Bruce R. Blazar, Melanie Boerries, Annette Schmitt-Gräff, Nurefsan Sariipek, Peter Van Galen, Joerg M. Buescher, Nina Cabezas-Wallscheid, Heike L. Pahl, Erika L. Pearce, Robert J. Soiffer, Catherine J. Wu, Luca Vago, Burkhard Becher, Natalie Köhler, Tobias Wertheimer, Vijay K. Kuchroo, Robert Zeiser

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Figure 2

Anti–TIM-3 Ab treatment after allo-HCT reduces Tc exhaustion, increases glycolytic capacity of Tc, and induces changes in myeloid subsets.

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Anti–TIM-3 Ab treatment after allo-HCT reduces Tc exhaustion, increases ...
(A and B) BALB/c recipient mice were injected with WEHI-3B cells, 5 × 106 allogeneic BM, and Tc and treated with isotype (n = 12) or anti–TIM-3 Ab (n = 13). The proportion of TIM-3+PD-1+ cells within CD4+ (A) or CD8+ Tc (B) in the indicated organ was determined by FC at day 23. Data are represented as mean ± SEM, and P values were calculated using Mann-Whitney U test. (C–N) C57BL/6 recipient mice were injected with FLT3-ITD MLL-PTD cells, 5 × 106 allogeneic BM, and Tc and treated with isotype Ab or anti–TIM-3 Ab. Donor Tc were isolated from the spleen at day 23 after allo-HCT. (C) Analysis using high-resolution spectral FC allows UMAP visualization of the immune landscape. (D and E) Scaled expression of 25 phenotypic or functional markers using the FlowSOM algorithm among 6 CD4+ or 4 CD8+ Tc subsets. Log fold change of isotype Ab (n = 6) compared with anti–TIM-3 Ab (n = 8) treatment is shown (blue color depicts higher expression in isotype, red color higher expression in anti–TIM-3). Differentially expressed proteins with P < 0.05 tested with moderated t test of limma are presented. (F and G) Glycolytic capacity of Tc subsets at day 23 assessed by FC-based single-cell metabolism. Data are represented as mean ± SEM of n = 9 biological replicates for each condition. P values were calculated using Mann-Whitney U test. (H) Volcano plot of 1,249 metabolic features from nontargeted LC-MS analysis. Features that were identified as members of the KEGG module “glycolysis” are highlighted. Results show n = 5 isotype and n = 8 anti–TIM-3-treated mice. DHAP, dihydroxyacetonphosphate; PGA, phosphoglyceric acid; PEP, phosphoenolpyruvate; G6P, glucose-6-phosphate; F6P, fructose-6-phosphate; FBP, fructose-bisphosphate. (I) Analysis using high-resolution spectral FC allows UMAP visualization of immune cells. (J) Proportion of each cell subset among total cells in the respective condition. Proportion of leukemia blasts (K), neutrophils (L), cDC1s (M), and macrophages (N) among total cells (%). P values were calculated using unpaired Student’s t test.