Regulation of CD8 T-cell signaling, metabolism, and cytotoxic activity by extracellular lysophosphatidic acid

Abstract

Lysophosphatidic acid (LPA) is an endogenous bioactive lipid that is produced extracellularly and signals to cells via cognate LPA receptors, which are G-protein coupled receptors (GPCRs). Mature lymphocytes in mice and humans express three LPA receptors, LPA₂ , LPA_5, and LPA₆ , and work from our group has determined that LPA₅ signaling by T lymphocytes inhibits specific antigen-receptor signaling pathways that ultimately impair lymphocyte activation, proliferation, and function. In this review, we discuss previous and ongoing work characterizing the ability of an LPA-LPA₅ axis to serve as a peripheral immunological tolerance mechanism that restrains adaptive immunity but is subverted during settings of chronic inflammation. Specifically, LPA-LPA₅ signaling is found to regulate effector cytotoxic CD8 T cells by (at least) two mechanisms: (i) regulating the actin-microtubule cytoskeleton in a manner that impairs immunological synapse formation between an effector CD8 T cell and antigen-specific target cell, thus directly impairing cytotoxic activity, and (ii) shifting T-cell metabolism to depend on fatty-acid oxidation for mitochondrial respiration and reducing metabolic efficiency. The in vivo outcome of LPA₅ inhibitory activity impairs CD8 T-cell killing and tumor immunity in mouse models providing impetus to consider LPA₅ antagonism for the treatment of malignancies and chronic infections.

Keywords: T cells; cytotoxic; cytotoxicity; lipid mediators; signal transduction cancer.

Figure 1.

Model of autocrine/paracrine LPA action. Autotaxin (ATX) is secreted by select cells and binds to integrins on the cell surface and hydrolyzes LPC to produce LPA. LPA then signals via nearby LPA G-protein-coupled receptors on the same or nearby cells.

Figure 2.

LPA signals via LPA₅ to impair IP₃R activity and resulting in reduced intracellular calcium stores release and inhibited lymphocyte antigen receptor signaling. Top: The presence of LPA at the time of BCR- and TCR-induced signaling suppresses intracellular calcium stores release for follicular (FO) and marginal zone (MZ) B cells (left) and CD4 and CD8 T cells (right). Bottom: Schematic illustrating TCR- and BCR-proximal kinase and PLCg activity is intact in the presence of LPA₅ signaling while IP₃R activity is depressed resulting in reduced antigen receptor-induced cytosolic calcium levels.

Figure 3.

LPA alters the regulation of the cytoskeleton during CD8 T cell:target cell IS formation in manner feasibly accounting for the resulting impairment in TCR signaling and killing activity. Top: Immunofluorescence microscopy of primary cytotoxic CD8 T cells (T; cell on right) shortly after immune synapse formation with antigen-specific target B cell (TC: cell on left). IS were established in the presence of vehicle or with wild type cells (left 3 images) or in the presence of LPA (OTP) or with mDia1^−/− T cells (right 3 images). Staining: DAPI (blue) F-actin (green), perforin (red) and IP₃R1, mDia1, IFNγ (yellow in top 4 rows). Bottom left: Schematic of an IS between an antigen-specific CD8 T cell:target cell showing IP₃R, RhoA mDia1, polymerized actin and stable microtubules positioned at the IS where cytotoxic granules and cytokines are transported to be secreted. Bottom right: Presence of LPA results in inefficient IP₃R localization to the IS and altered localization of RhoA and F-actin and perturbed microtubule detyrosination that impairs TCR signaling (see text), perforin and cytokine targeted release.

Figure 4.

Lpar5^−/− tumor-specific CD8 T cells provide better control of B16 lung tumors than wild type CD8 T cells. A) Representative hematoxylin & eosin (H&E) histology images of day 20 B16.cOVA tumors after i.v. transfer together with either wild type (left) or Lpar5^−/− (right) OT-I CD8 T cells. Scale bars=100 μm. Right: Quantification of B16.cOva tumor size after transfer of B6 (filled bar) or Lpar5^−/− (open bar) OT-I CD8 T cells. B) Number of B6 (filled bar) or Lpar5^−/− (open bar) OT-I CD8 T cells found in lung tumors. Student’s t test *p < 0.05 and **p < 0.005.

Figure 5.

Schematic of induction of ENPP2 expression and subsequent ATX production by a transformed cell leading to local LPA production. LPA then signals via LPA_1–3 expressed by malignant cells to promote autocrine cell proliferation and tumorigenesis (left) or LPA₅ expressed by tumor-specific CD8 T cells to suppress tumor immunity.

Figure 6.

LPA promotes an increase in basal T cell respiration, maximal respiratory capacity, proton leak and transiently elevates ATP production. Naïve (A) and effector (B) OT-I CD8 T cells were cultured in vitro with media (red) or 1 μM LPA for 30 minutes (green), 2 hrs (blue) or 4 hrs (teal) and oxygen consumption rate (OCR; left graphs) and extracellular acidification rate (ECAR; right graphs) measured. Assay was performed with injections of oligomycin (oligo), (4-(trifluoromethoxy) phenyl) carbonohydrazonoyl dicyanide (FCCP), antimycin A (ant), and rotenone (rot) at 18-minute intervals in media supplemented with 25 mM glucose. Data are n=6 technical replicates.

Figure 7.

Lysophosphatidic acid as a potential prognostic marker in melanoma. A) Data analysis from The Cancer Genome Atlas (TCGA) on progression-free survival. Data was from pan-cancer data from all solid tumors in cBioPortal from the complete curated non-redundant studies (as of June 18, 2021). Cohorts were stratified based on genomic status of amplification of ENNP2, MYC, or wild type for both genes. B) Relative abundance of LPA 16:0 in stage IV melanoma responder patients (blue; complete and partial response) and non-responder patients (red; stable disease and progressive disease) measured both pre- and post-therapy treatment. Unpaired Student’s t-test where p<0.05.