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210 Regulation of TIM-3 by phosphatidylserine
  1. Courtney Smith,
  2. Alice Li,
  3. Nithya Krishnamurthy and
  4. Mark Lemmon
  1. Yale University, West Haven, CT, USA


Background Immune checkpoint blockade has proven effective in targeting exhausted T-cells to reactivate the immune system against cancer. However, the majority of patients fail to respond to currently available therapies, which primarily target PD-1. Thus, a key challenge for checkpoint blockade therapy is to identify and understand new therapeutic targets. Another immune checkpoint receptor is TIM-3, which – like PD-1 – is expressed on exhausted T-cells in the tumor microenvironment.1, 2 TIM-3 belongs to a family of phosphatidylserine (PS) receptors, including TIM-1 and TIM-4, which have well-documented roles in the engulfment of apoptotic cells by phagocytes.3 However, the role of PS in regulating TIM-3 function is less clear. We therefore investigated how TIM-3 modulates T-cell signaling and how PS influences TIM-3 activity, with the ultimate goal of improving the translation of candidate TIM-3 therapies to the clinic.

Methods Surface plasmon resonance (SPR) was used to quantify the interaction between human TIM-3 and PS. A Jurkat T-cell model was used to investigate the role of TIM-3 in T-cell receptor (TCR) signaling and to determine the role of PS in regulating TIM-3 function.

Results TIM-3 bound PS-containing membranes with low micromolar affinity in vitro. In the Jurkat cell model system, high – but not low – surface levels of TIM-3 promoted T-cell signaling, suggesting a threshold of receptor expression needed to modulate T-cell signaling, similar to what has recently been reported for PD-1.4 However, chimeric receptors that maintained the TIM-3 cytoplasmic tail but were unable to bind PS failed to enhance T-cell signaling like the full-length TIM-3 receptor. Cells expressing mutant TIM-3, which displayed reduced PS binding as quantified by SPR, also displayed reduced T-cell signaling compared to cells expressing wild-type TIM-3. Importantly, treatment of TIM-3-expressing cells with a functional TIM-3 antibody that blocks PS binding also reduced T-cell signaling compared with untreated TIM-3-expressing cells.

Conclusions Our results support a role for PS as a ligand capable of modulating TIM-3 activity. Using chimeric receptors, TIM-3 mutants, changes in receptor expression, and a functional TIM-3 antibody, we show that preventing the interaction between TIM-3 and PS blocks TIM-3 activity. These data suggest that blocking the PS-TIM-3 interaction is a key mechanism for functional antibodies targeting TIM-3. Ultimately, this work supports the development and use of clinical antibodies that block the interaction of TIM-3 with PS and provides new mechanistic insight into how TIM-3 modulates TCR signaling.

Acknowledgements This work was supported by the PhRMA Foundation Pre-Doctoral Fellowship in Pharmacology/Toxicology.


  1. Fourcade J, Sun Z, Benallaoua M, et al. Upregulation of Tim-3 and PD-1 expression is associated with tumor antigen-specific CD8+ T cell dysfunction in melanoma patients. J Exp Med. 2010;207(10):2175–2186.

  2. Zhou Q, Munger ME, Veenstra RG, et al. Coexpression of Tim-3 and PD-1 identifies a CD8+ T-cell exhaustion phenotype in mice with disseminated acute myelogenous leukemia. Blood. 2011;117(17):4501–4510.

  3. Kobayashi N, Karisola P, Peña-Cruz V, et al. TIM-1 and TIM-4 glycoproteins bind phosphatidylserine and mediate uptake of apoptotic cells. Immunity. 2007;27(6):927–940.

  4. Hui E, Cheung J, Zhu J, et al. T cell costimulatory receptor CD28 is a primary target for PD-1-mediated inhibition. Science. 2017;355(6332):1428–1433.

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