Phosphatidylserine binding directly regulates TIM-3 function

Abstract

Co-signaling receptors for the T cell receptor (TCR) are important therapeutic targets, with blockade of co-inhibitory receptors such as PD-1 now central in immuno-oncology. Advancing additional therapeutic immune modulation approaches requires understanding ligand regulation of other co-signaling receptors. One poorly understood potential therapeutic target is TIM-3 (T cell immunoglobulin and mucin domain containing-3). Which of TIM-3's several proposed regulatory ligands is/are relevant for signaling is unclear, and different studies have reported TIM-3 as a co-inhibitory or co-stimulatory receptor in T cells. Here, we show that TIM-3 promotes NF-κB signaling and IL-2 secretion following TCR stimulation in Jurkat cells, and that this activity is regulated by binding to phosphatidylserine (PS). TIM-3 signaling is stimulated by PS exposed constitutively in cultured Jurkat cells, and can be blocked by mutating the PS-binding site or by occluding this site with an antibody. We also find that TIM-3 signaling alters CD28 phosphorylation. Our findings clarify the importance of PS as a functional TIM-3 ligand, and may inform the future exploitation of TIM-3 as a therapeutic target.

Switch receptor T3/28 improves long-term persistence and antitumor efficacy of CAR-T cells

“… 56 As the most recently discovered ligand, CEACAM1, which is highly expressed by some tumor cells, plays a role in dampening T cell responses. 57 58 As a functional TIM-3 ligand, 59 PtdSer is an essential component of bilayer cell membranes and is normally present in the inner leaflet. However, PtdSer is present in multiple viable tumor cells, and its exposure is significantly increased on the surface of tumor cells or tumor cell-derived microvesicles in the tumor microenvironment, which have intrinsic immunosuppressive properties and facilitate tumor growth and metastasis.…”

Immunotherapy for Colorectal Cancer: Mechanisms and Predictive Biomarkers

“…Co-inhibition of TIM-3 and CEACAM1 results in improved elimination of CRC tumors in mice [ 74 ]; however, no clinical trials are currently investigating this combination for CRC. PtdSer is released when cells undergo apoptosis and directly regulates TIM-3 function [ 75 ]. HMGB1 binds DNA released from dying cells and mediates cytokine production and activation of an innate immune reaction that is inhibited upon TIM-3-HMGB1 binding [ 14 ].…”

Immune Checkpoint Receptors Signaling in T Cells

“…TIM-3 presents a membrane distal Ig-like V domain involved in ligand interactions, a membrane proximal mucin domain, and a transmembrane domain connected to an intracellular cytoplasmic tail involved in phosphotyrosine-dependent signaling. TIM-3 is a promiscuous receptor suggested to bind heterologous ligands, such as phosphatidylserine, from apoptotic cells [ 69 ], carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1, [ 70 ]), the alarmin protein high mobility group B1 (HMGB1), and the carbohydrate receptor galectin-9 [ 71 ]. Tim-3 is actively recruited to the IS, where it associates with other transmembrane proteins, including the CD45 and CD148 phosphatases, which may perturb IS stability [ 72 , 73 ].…”

“…A recent proteomic study indicated a coprecipitation of Tim-3 with 37 proteins, of which 11 are dynamically regulated by pervanadate, including the E3 ubiquitin ligase CBL-B, SHP-1, and Grb2 [ 72 ]. Precedent works have added PI3Kα, Lck, interleukin inducible T cell kinase (ITK), and the adaptor HLA-B-Associated Transcript 3 (Bat3) to the list of cytoplasmatic-phosphotyrosine-dependent TIM-3 interactors [ 69 , 74 , 75 , 76 ]. However, the functional output of these interactions and the physiological Tim-3 agonists in T cells is debatable as it is hard to determine whether Tim-3 is resting or engaged by some ligand in the experimental system used and whether the antibodies binding to it are agonists or competing with endogenous ligands [ 77 ].…”

“…However, the functional output of these interactions and the physiological Tim-3 agonists in T cells is debatable as it is hard to determine whether Tim-3 is resting or engaged by some ligand in the experimental system used and whether the antibodies binding to it are agonists or competing with endogenous ligands [ 77 ]. In some studies, unbound or phosphatidylserine-stimulated Tim-3 enhanced TCR signaling [ 69 , 74 ], while in other reports, unbound or galectin-9/CEACAM1 triggered Tim-3 suppression of TCR signaling [ 75 , 76 , 78 ]. A current working model implies a permissive Tim-3 bound to Bat3 recruiting Lck and costimulating T cells in opposition to a Tim-3 phosphorylated on tyrosines 256 and 263 by ITK switching from Bat3 to Fyn.…”