Immune Checkpoint Receptor Signaling Pathways in T Cells
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Immune Checkpoint Receptor Signaling Pathways in T Cells
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Immune Checkpoint Receptor Signaling Pathways in T Cells
The rapid development of research on receptors that negatively regulate lymphocyte function is driven by the success of tumor immunotherapy.
While much research focuses on characterizing these immune checkpoint receptors from a functional perspective, there is relatively less emphasis on studying their signaling mechanisms.
Current research has demonstrated that the extracellular portions of some receptors act as bait receptors for activating ligands, while in most cases, the phosphorylation of tyrosine residues in their cytoplasmic tails drives critical inhibitory signals.
These negative signals are mediated by key signaling transducers, such as tyrosine kinases, inositol phosphatases, and diacylglycerol kinases, allowing them to counteract T-cell receptor (TCR)-mediated activation.
The features of these signaling pathways are crucial for developing new immunotherapies and overcoming the limitations of current immune checkpoint inhibitors.
T-Cell Signaling Pathways
T-cell signaling mechanisms primarily facilitate the rapid expansion, differentiation, and effector response of T lymphocytes when recognizing non-self antigens presented by antigen-presenting cells (APCs). This extraordinary discriminatory ability arises from the integration of activating and inhibitory signals within the cell.
The first activation signal is delivered by the T-cell receptor (TCR), which recognizes antigens bound to the major histocompatibility complex (MHC) on APCs. Activated TCR triggers the assembly of a signalosome, with key components including tyrosine kinases like Lck and Zap70, scaffold proteins such as Lat, SLP76, and Themis, and phospholipase Cγ1 (PLC γ1), as well as tyrosine phosphatases and E3 ubiquitin ligases like SHP1 and Cbl, among others.
The second activation signal for T cells is provided by co-stimulatory receptors like CD28. CD28 enhances TCR-driven tyrosine phosphorylation and interacts with phosphatidylinositol 3-kinase (PI3K) and Grb2, initiating the Akt-mTOR and Ras-MAPK pathways.
The most characteristic inhibitory receptors for lymphocytes are PD-1 and CTLA-4, which represent examples of T-cell inhibitory receptors. Both continuously suppress cytokine secretion and proliferation induced by TCR, as well as glucose uptake and metabolism. The cytoplasmic tails of these inhibitory receptors contain tyrosine-based inhibitory motifs (ITIMs) and switch motifs (ITSMs) with common sequence features. These motifs are phosphorylated by Src family kinases (SFKs) upon receptor activation and play crucial roles in regulating the immune system. ITIMs negatively modulate cell activation by recruiting phosphatases such as protein tyrosine phosphatases 1 and 2 (SHP-1 and SHP-2), as well as inositol 5′-phosphatases 1 and 2 (SHIP-1 and SHIP-2). ITSMs can transmit positive or negative signals by recruiting adapters like signaling lymphocytic activation molecule (SLAM)-associated protein (SAP).
PD-1 Signaling Pathway
Programmed cell death protein 1 (PD-1, CD279) is a transmembrane glycoprotein that belongs to the CD28 receptor superfamily. Unlike CD28, PD-1 exists as a monomer on the cell surface. Structurally, PD-1 consists of extracellular immunoglobulin (Ig)-like variable domains, a transmembrane domain, and a cytoplasmic tail responsible for signal transduction and binding to scaffold molecules. The cytoplasmic tail of PD-1 contains two tyrosine-based motifs, an ITIM (VDY223GEL), and an ITSM (TEY248ATI). Both motifs are phosphorylated by the tyrosine kinase Lck when PD-1 ligands bind to PD-1.
PD-1 is expressed on activated T cells, B cells, natural killer (NK) cells, monocytes, dendritic cells, and cancer cells such as melanoma. PD-1 is activated through its interaction with PD-L1 (B7-H1, CD274) and exhibits higher affinity for PD-L2 (B7-DC, CD273). Both ligands are induced by interferon/cytokines but have distinct expression patterns: PD-L1 is broadly expressed in hematopoietic and non-hematopoietic cells, while PD-L2 is primarily expressed on APCs.
In immune cells, PD-1 signaling relies on tyrosine phosphatase SHP-2. Disruption of the PD-1/SHP-2 signaling axis is partly responsible for the clinical response of PD-1 antibodies in the tumor microenvironment. After ligand binding, SHP-2 is recruited to the phosphorylated ITSM of PD-1. Phosphorylation of ITSM induces a conformational change in SHP-2 towards an active state. Microscopy studies of reconstructed immune synapses show that in the presence of PD-L1, PD-1 and CD28 associate in the central TCR-enriched area. PD-1 recruits SHP-2, promoting the dephosphorylation of CD3ζ and CD28 and negatively affecting TCR signaling strength. PD-1-mediated CD28 dephosphorylation significantly impacts PI3K recruitment at the TCR signalosome, reducing PI3K/AKT pathway activity and its downstream transcriptional targets, such as Bcl-xL. Furthermore, SHP-2 is thought to not only block CD28 co-stimulatory signals but also inhibit TCR-mediated ZAP70 phosphorylation and its association with CD3ζ, which leads to PKCθ and ERK activation, as well as downstream IL-2 production and amplification.
CTLA-4 Signaling Pathway
Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) interacts with the co-stimulatory ligands B7-1 (CD80) and B7-2 (CD86) with higher affinity than CD28 itself. These interactions yield CTLA-4’s inhibitory function by competing with CD28 for ligands, thus reducing the second signal required for full T-cell activation. Additionally, CTLA-4 constitutively internalizes ligands on APCs, decreasing their T-cell activating ability.
Structurally, CTLA-4 shares extensive homology with CD28. Their extracellular portions have immunoglobulin-like V domains, allowing the formation of disulfide-bonded homodimers, while the cytoplasmic tail is phosphorylated by SFKs upon activation. CTLA-4’s cytoplasmic tail is highly conserved and contains two tyrosine substrates (Y201VKM and Y218FIP), which can be activated by Fyn, Lck, and potentially other kinases. These tyrosines are not typical ITSM motifs but play a role in CTLA-4’s inhibitory function by recruiting signaling molecules with SH3 domains.
The internal region of CTLA-4 recruits many effectors similar to those recruited by CD28, such as PI3K and the tyrosine/syrosine phosphatase PP2A. Although CD28 is the typical second signal required for full activation, CTLA-4 also contributes by recruiting SHP-1 or SHP-2 to reduce early TCR signaling events, including ζ chain, Zap70, and LAT phosphorylation, as well as MAPK pathway activity. However, CTLA-4 lacks ITSM motifs for SHP-2 binding, indicating indirect recruitment.
BTLA Signaling Pathway
B and T lymphocyte attenuator (BTLA) is one of several receptors in the tumor necrosis factor receptor (TNFR) superfamily, and it can also act as a ligand, allowing bidirectional signaling. Since both BTLA and herpes virus entry mediator (HVEM, also known as TNFRSF14) are expressed in T cells, heterotypic homodimerization might occur, allowing BTLA to inhibit HVEM-dependent NFκB activation. Conversely, HVEM binding induces BTLA phosphorylation, which suppresses T-cell proliferation and IL-2 production. BTLA is expressed on monocytes, B cells, NK cells, and resting T lymphocytes, and its expression is upregulated on activated T cells, NK cells, and tumor-infiltrating T cells.
BTLA is structurally related to PD-1 and CTLA-4, including extracellular Ig-like V domains, a transmembrane region, and a cytoplasmic tail. Similar to PD-1, BTLA’s tail contains two additional tyrosines (Y226 and Y243) involved in Grb2 binding, an ITIM (IVY257ASL), and an ITSM (TEY282ASI). These four tyrosines are essential for BTLA’s inhibitory function. BTLA preferentially recruits SHP-1 through Y257 and Y282, effectively promoting dephosphorylation of CD28 and CD3ζ. Additionally, BTLA complexes with the B-cell receptor (BCR) signalosome and recruits SHP-1, reducing B-cell proliferation and cytokine secretion, leading to Syk kinase dephosphorylation and reduced activity of PLCγ2 and NF-kB.
TIM-3 Signaling Pathway
T-cell immunoglobulin and mucin-domain-containing protein 3 (TIM-3) belongs to the TIM protein family and is expressed on Th1, CD4+ and CD8+ T cells, NK cells, and dendritic cells. Structurally, TIM-3 contains an N-terminal immunoglobulin V domain involved in ligand interactions, a mucin-like domain, a transmembrane region, and a cytoplasmic tail responsible for tyrosine-dependent signal transduction. TIM-3’s ligands include CEACAM1, HMGB1, and Gal-9.
Intracellular Tim-3 signaling in T cells depends on its cytoplasmic tail, although the specifics remain unclear. A recent proteomics study indicated that Tim-3 co-precipitates with 37 different proteins, 11 of which are dynamically regulated by sodium orthovanadate, including E3 ubiquitin ligase CBL-B, SHP-1, and Grb2. However, these interactions remain controversial with respect to TIM-3’s function.
LAIR-1 Signaling Pathway
Leukocyte-associated immunoglobulin-like receptor 1 (LAIR-1, also known as CD305) is an inhibitory receptor widely expressed on collagen and collagen-like domain proteins, such as complement C1q. Binding of various collagen subtypes to LAIR-1’s extracellular domain inhibits NK cell cytotoxicity and the activation of effector T cells.
LAIR-1’s intracellular region contains two ITIMs (Y251 and Y281), which are phosphorylated upon LAIR-1 activation and are necessary for recruiting SHP-1 and SHP-2. LAIR-1’s binding to collagen inhibits the phosphorylation of key components in the TCR-triggered signaling pathways, such as Lck, Lyn, CD3ζ, Zap70, and MAPK.
LAG-3 Signaling Pathway
Lymphocyte activation gene-3 (LAG-3) is an inhibitory receptor that possesses four Ig-like domains, similar to the CD4 receptor. LAG-3 interacts with MHC II, but with higher affinity than CD4 itself, inhibiting T-cell activation by interfering with the binding of pMHC to CD4.
The cytoplasmic region of LAG-3 consists of approximately 60 amino acids and lacks typical inhibitory motifs. However, it contains several amino acid sequences that are highly conserved among different species, and not shared with other inhibitory co-receptors. These sequences include adjacent membrane-proximal FSAL regions, a central region with KIEELE, 10-15 consecutive glutamic acid repeat sequences, and an EX-repeat in the C-terminal region. LAG-3’s inhibitory function requires intracellular signal transduction via these sequences, transmitting various inhibitory signals.
SLAM Signaling Pathway
The signaling lymphocytic activation molecule (SLAM) family includes receptors primarily expressed on hematopoietic cells, such as SLAM/CD150, CD48, Ly-9/CD229, CD84, 2B4/CD244, NTB-a/Ly108, and CRACC/CD319. These receptors are homophilic (self-binding) but are an exception to 2B4, which recognizes CD48.
Structurally, SLAM family proteins feature extracellular Ig-like V and C domains, followed by a transmembrane region and a cytoplasmic tail with one or more ITSMs. These ITSMs can interact with SH2-containing signaling molecules like SAP and its homologue EAT-2, or inhibitory molecules such as SHP-1, SHP-2, and SHIP-1. When these receptors bind to ligands, SAP activates downstream signaling through the recruitment of SFKs like Fyn and Lck, while also competing with phosphatases like SHP-1, SHP-2, and SHIP-1. Consequently, the balance between these cell-specific effectors determines whether SLAM receptors transmit positive or negative signals to cells, regulating differentiation and effector functions.
In Summary
The role of immune signaling networks in immune checkpoint receptors is an exciting area of research. However, our understanding of the immune checkpoint signaling pathways is still limited, as signal integration is the result of finely regulated and activated signal complexes.
At present, some key signaling molecules of immune checkpoint receptors have drawn attention because they may target common inhibitory mechanisms rather than specific receptors.
The most advanced target is SHP-2, the inhibitor of which has entered multiple phase I clinical trials, while SHIP and DGK inhibitors remain in the preclinical stage. In the future, as research into the immune checkpoint signaling pathway networks advances, we may discover new therapeutic targets.
Immune Checkpoint Receptor Signaling Pathways in T Cells
References:
Immune Checkpoint Receptors Signaling in T Cells. Int J Mol Sci. 2022 Apr; 23(7): 3529.
(source:internet, reference only)
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