OX40 and OX40L: Mastering Immune Activation and Regulation

What are OX40 and OX40L respectively?

OX40, also known as CD134 or TNFRSF4, is a protein within the immune system categorized under the tumor necrosis factor receptor superfamily (TNFRSF). It is predominantly expressed on the surface of T cells, a subset of lymphocytes, and serves as a pivotal regulator in immune response modulation. OX40 holds a crucial role in governing the activation and expansion of T cells, contributing significantly to their activation, differentiation, and long-term survival.

OX40L, or OX40 Ligand (also known as CD252), is a membrane-bound protein belonging to the tumor necrosis factor superfamily (TNFSF). The interaction between OX40L and its receptor, OX40 (CD134), plays a vital regulatory role within the immune system. This interaction profoundly influences the activation, proliferation, and functional capabilities of T cells, marking its significance in orchestrating immune responses.

Insights into the Structure and Expression of OX40 and OX40L

OX40 (also referred to as ACT35, CD134, or TNFRSF4) is a type 1 transmembrane protein belonging to the TNFR superfamily. Comprising 249 amino acids, it contains a 49-amino acid cytoplasmic tail and a 186-amino acid extracellular region [1-2]. Initially identified on activated rat CD4+ T cells in 1987, OX40's presence was subsequently confirmed in mice and humans [3-5]. While predominantly expressed on activated CD4+ and CD8+ T cells, OX40 exhibits comparatively low expression levels on NK cells and NKT cells [6].

OX40L (also known as CD252, TNFSF4, CD134L, or gp34), serving as OX40's ligand, is a type II glycoprotein. It features a 23-amino acid cytoplasmic tail and a 133-amino acid extracellular domain. As a member of the TNF superfamily, OX40L exists in a trimeric form. Initially identified as the gp34 protein on human T-cell leukemia virus-transformed cells in 1985, subsequent research revealed its primary expression on antigen-presenting cells like B cells and dendritic cells [7]. Similar to other members of the TNF family, OX40 signals are transduced through TNF receptor-related factors (TRAFs). In vivo, OX40 signals are transmitted to T cells via TRAF2 and TRAF5, while in vitro, this involves TRAF1, TRAF3, and TRAF5 [9].

The expression of both OX40 and OX40L is tightly regulated by various factors. OX40 expression on T cells is induced by signals such as TCR, CD28/CD80, and CD40/CD40L, peaking at 48-72 hours post T cell activation [10]. TCR signaling initiates OX40 expression on a range of cells, with CD28 and other cytokines further enhancing its expression on activated T cells. Research also highlights that IL-2, IL-4, and TNF can amplify or prolong OX40 expression. Notably, in an animal model of nonalcoholic steatohepatitis, Sun et al. discovered that IL-2, TNF-α, and IFN-γ were highly expressed in liver tissues. However, only exogenous IL-2 stimulation led to an upregulation of OX40 expression on CD4+ T cells [11]. Meanwhile, CD40 signaling and inflammatory signals transmitted via Toll-like receptors prompt OX40L expression on antigen-presenting cells. Additional factors like IL-18, IFN-γ, thymic stromal lymphopoietin (TSLP), and prostaglandin E2 contribute to the promotion of OX40L expression [12-13]. Inflammatory contexts lead to increased OX40L expression on APCs, ensuring activated OX40+ T cells receive OX40 signals from neighboring cells.

Fig.1 Human cells expressing OX40 and/or OX40L.[14]

Functions of OX40 and OX40L

As a pair of imperative costimulatory molecules, OX40 and OX40L jointly orchestrate T cell activation, particularly during the later phases of immune responses. Their functions prove pivotal in bolstering the effectiveness of effector T cells, sustaining their survival, and curbing apoptosis. Studies conducted on OX40-deficient mice highlighted a marked reduction in the percentage of antigen-specific T cells. Moreover, antiapoptotic molecules such as Bcl-xL and Bcl-2 demonstrated evident downregulation in OX40-/- T cells and CD28-/- T cells after antigen stimulation. Remarkably, when CD28-/- T cells were subjected to anti-OX40 agonist antibody stimulation, the expression of Bcl-xL and Bcl-2 witnessed an increase, consequently inhibiting T cell apoptosis [15]. Notably, OX40-deficient T cells normally underwent proliferation and differentiation into effector T cells within 2-3 days of TCR signaling activation. However, their survival rate considerably diminished after 12-13 days of activation, suggesting that while OX40 signaling might not be imperative during the early stages of T cell activation, it may play a crucial role in promoting T cell proliferation and sustaining their survival during later phases [16].

The OX40/OX40L signaling holds a critical role in the differentiation of various T helper (Th) cells. It tends to foster the differentiation of naive CD4+ T cells into Th2 cells, while promoting Th1 differentiation under the influence of antigens or IL-12. Furthermore, OX40 has been recognized for its involvement in Th9 cell differentiation through the nonclassical NF–κB pathway by activating tumor necrosis factor receptor-associated factor 6 (TRAF6) [17]. The nature of Th cell differentiation prompted by this signaling might vary based on the specific environmental context. Typically, OX40 expression undergoes downregulation post the effector phase of the primary T cell response, but it promptly resurfaces in memory T cells following secondary exposure to the same antigen. This resurgence subsequently triggers the activation and recruitment of memory effector T cells, underscoring the indispensability of the OX40/OX40L interaction during the recall response. Notably, OX40 is also constitutively expressed on mouse regulatory T (Treg) cells [18]. Evidence highlights the essential role of OX40 signaling in dampening Treg cell function. Researchers have reported that engagement of the OX40/OX40L axis results in Foxp3 downregulation in Tregs, leading to decreased Treg-mediated suppression of effector T cell proliferation. OX40's upregulation of BATF3 and BATF contributes to a closed chromatin configuration that represses Foxp3 expression in a Sirt1/7-dependent manner. Nevertheless, contrasting reports suggest that OX40 agonists can both enhance Treg cell proliferation and inhibit their function. Gavin MA et al. noted a reduction in the number of Treg cells in the spleen of OX40-deficient mice, while the thymus of OX40L-overexpressing mice exhibited an increase in Treg cell numbers, hinting at an interference of aberrant OX40/OX40L signaling with Treg cell development [19].

Beyond its paramount role in T cells, OX40/OX40L signaling exerts a significant influence on the differentiation and maturation of dendritic cells (DCs). In human immature DCs, OX40L expression remains absent; however, sCD40L stimulation rapidly induces OX40L expression. This interaction elevates the expression of CD80, CD86, CD54, and CD40 on mononuclear-derived DCs in the reversible phase and concurrently amplifies the secretion of IL-4, IL-6, IL-12, TNF-α, and IL-1β by 4- to 35-fold. This observation underscores the role of the OX40L reverse signal in bolstering DC maturation. Additionally, B cells, recognized as OX40L-expressing antigen-presenting cells, assume a crucial role in germinal center formation [20]. Cross-linking of OX40L on stimulated B cells not only significantly augments the proliferative response but also advances immunoglobulin secretion [21]. Research has revealed that CD134L engagement on human B cells enhances the IgG production rate per cell, rather than increasing the number of plasma cells [22]. Hence, the bidirectional OX40/OX40L signal profoundly impacts not only T cells but also assumes pivotal roles in the differentiation and maturation of antigen-presenting cells, especially DCs and B cells.

Fig.2 OX40/OX40L signaling in Tfh differentiation and function.[23]

Signaling Pathways Initiated by OX40 in Tfh Cells

In the realm of T cells, OX40 initiates two primary signal transduction pathways, each with distinct characteristics. These pathways include the antigen-independent NF-κB pathway and the antigen-dependent PI3K-Akt pathway.

When OX40L binds, it triggers the trimerization of OX40 monomers and the recruitment of TRAF2, 3, and 5. Among these adaptors, TRAF2 emerges as a key player capable of not only activating the NF-κB signal but also facilitating PI3K recruitment. The NF-κB pathway entails the engagement of OX40 on activated or effector T cells by OX40L. This interaction assembles a signalosome encompassing the TRAF-RIP-IKKα/β/γ complex and the PCARMA1–BCL10–MALT1–PKCθ complex[24]. This intricate signalosome orchestrates NF-κB activation independently of antigen/TCR engagement. Operating through the TRAF-RIP-IKKα/β/γ complex, phosphorylation and subsequent degradation of IκBα occur, ultimately activating NF-κB1. Consequently, p50 and RelA translocate into the nucleus, offering crucial survival cues to T cells even in the absence of antigens. Notably, the CARMA1–MALT1–BCL10–PKCθ complex, conjoined with OX40 at the immune synapse, assumes a pivotal role in sustaining protracted NF-κB activity. This complex is particularly vital in promoting the survival of effector T cells during the later phases of T cell responses, post-antigen clearance. In an additional facet, OX40 incites the phosphorylation of IKKα and activation of NIK, thereby initiating the noncanonical NF-κB2 pathway[25].

Conversely, the PI3K-Akt pathway comes into play upon OX40L binding. This interaction leads to the formation of a signaling complex involving TRAF2, PKB, and its upstream activator, PI3K[26]. Notably, this complex triggers robust phosphorylation and functional activation of the PI3K-Akt pathway solely in the presence of antigen presentation. Consequently, OX40 effectively heightens TCR signaling via the PI3K-Akt pathway. Furthermore, OX40 collaborates synergistically with TCR, facilitating Ca2+ influx and the nuclear accumulation of NFATc1 and NFATc2.

Fig.3 OX40 signaling pathways mediating Tfh differentiation.[23]

OX40 and OX40L Protein

Recombinant Human OX40 Protein (C-6His)

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Synonym : Tumor necrosis factor receptor superfamily member 4; TNFRSF4; OX40; CD134; Txgp1

Recombinant Human OX40L Protein (N-6His)

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Synonym : Tumor necrosis factor ligand superfamily member 4; Glycoprotein Gp34; OX40 ligand; OX40L; TAX transcriptionally-activated glycoprotein 1; TNFSF4; CD252; TXGP1

References:

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