Takanori So

The significance of OX40 and OX40L to T‐cell biology and immune disease

Summary:u2002 OX40 (CD134) and its binding partner, OX40L (CD252), are members of the tumor necrosis factor receptor/tumor necrosis factor superfamily and are expressed on activated CD4+ and CD8+ T cells as well as on a number of other lymphoid and non‐lymphoid cells. Costimulatory signals from OX40 to a conventional T cell promote division and survival, augmenting the clonal expansion of effector and memory populations as they are being generated to antigen. OX40 additionally suppresses the differentiation and activity of T‐regulatory cells, further amplifying this process. OX40 and OX40L also regulate cytokine production from T cells, antigen‐presenting cells, natural killer cells, and natural killer T cells, and modulate cytokine receptor signaling. In line with these important modulatory functions, OX40–OX40L interactions have been found to play a central role in the development of multiple inflammatory and autoimmune diseases, making them attractive candidates for intervention in the clinic. Conversely, stimulating OX40 has shown it to be a candidate for therapeutic immunization strategies for cancer and infectious disease. This review provides a broad overview of the biology of OX40 including the intracellular signals from OX40 that impact many aspects of immune function and have promoted OX40 as one of the most prominent costimulatory molecules known to control T cells.

The kinases aurora B and mTOR regulate the G1–S cell cycle progression of T lymphocytes

CD28-deficient T cells arrest at the G1–S transition of the cell cycle. Here we show that this is controlled by the kinase aurora B, which exists in a complex with survivin and mammalian target of rapamycin (mTOR). Expression of aurora B in Cd28−/− T cells augmented phosphorylation of mTOR substrates, expression of cyclin A, hyperphosphorylation of retinoblastoma protein and activation of cyclin-dependent kinases 1 and 2 and promoted cell cycle progression. Interleukin 2 enhanced aurora B activity, and inactive aurora B prevented interleukin 2–induced proliferation. Moreover, expression of aurora B restored Cd28−/− T cell proliferation and promoted inflammation in vivo. These data identify aurora B, along with survivin and mTOR, as a regulator of the G1–S checkpoint in T cells.

Immune regulation and control of regulatory T cells by OX40 and 4-1BB.

The TNFR family members OX40 (CD134) and 4-1BB (CD137) have been found to play major roles as costimulatory receptors for both CD4 and CD8 T cells. In particular, in many situations, they can control proliferation, survival, and cytokine production, and hence are thought to dictate accumulation of protective T cells during anti-viral and anti-tumor responses and pathogenic T cells during autoimmune reactions. As opposed to simply controlling the activity of naïve, effector, and memory T cells, recent data have suggested that both molecules are also instrumental in controlling the generation and activity of so-called regulatory or suppressor T cells (Treg), perhaps in both positive and negative manners. Part of the action on Treg might function to further promote protective or pathogenic T cells, but alternate activities of OX40 and 4-1BB on Treg are also being described that suggest that there might be control by these molecules at multiple levels that will alter the biological outcome when these receptors are ligated. This review specifically focuses on recent studies of regulatory T cells, and regulatory or suppressive activity, that are modulated by OX40 or 4-1BB.

Tumor Necrosis Factor/Tumor Necrosis Factor Receptor Family Members That Positively Regulate Immunity

The interactions between members of the tumor necrosis factor (TNF) family and their specific receptors (TNFRs) are influential in controlling cell division, life, and death. Recent evidence suggests that these interactions control the functionality and longevity of many types of cells involved in immune responses. In particular, it has become evident that certain interactions support the clonal expansion and survival of T-cells, B-cells, and dendritic cells and thus are essential for establishing a robust immune response. This review describes select TNF/TNFR family members that principally support activation and survival and prevent excessive cell death of T-cells (OX40L/OX40, 4-1BBL/4-1BB, CD30L/CD30, LIGHT/HVEM, CD70/CD27, and GITRL/GITR), B-cells (BAFF/BAFFR), and dendritic cells (RANKL/RANK). Expression of these ligands and receptors on the cell surface is highly regulated, and communication via them occurs during contact between T-cells and dendritic cells and between T-cells and B-cells.The functional dynamic between these TNF/TNFR members is slowly being unraveled, including whether these molecules act together or sequentially or control different type of immune responses. This review summarizes aspects of these TNF/TNFR interactions that are potentially important to immune responses.

Identification of regulatory functions for 4-1BB and 4-1BBL in myelopoiesis and the development of dendritic cells

The costimulatory molecule 4-1BB and its ligand 4-1BBL can control adaptive immunity, but here we show that their interaction also suppressed myelopoiesis. We found that 4-1BBL was expressed on hematopoietic stem cells, differentiating common myeloid progenitors and granulocyte-macrophage progenitors, and 4-1BB was inducible on activated myeloid progenitors. Steady-state numbers of granulocyte-macrophage progenitors, myeloid-lineage cells and mature dendritic cells were higher in 4-1BB- and 4-1BBL-deficient mice, indicative of a negative function, and we confirmed that result with bone marrow chimeras and in vitro, where the absence of interactions between 4-1BB and 4-1BBL led to enhanced differentiation into dendritic cell lineages. The regulatory activity was mediated by 4-1BBL, with binding by 4-1BB inhibiting differentiation of myeloid progenitors. Thus, 4-1BB and 4-1BBL have a previously unknown function in limiting myelopoiesis and the development of dendritic cells.

ACTIVATION OF NF–κB1 BY OX40 CONTRIBUTES TO ANTIGEN-DRIVEN T CELL EXPANSION AND SURVIVAL

The costimulatory molecule OX40 (CD134) is required in many instances for effective T cell-mediated immunity, controlling proliferation, and survival of T cells after encountering specific Ag. We previously found that the functional targets of OX40 are survivin and aurora B that regulate proliferation and Bcl-2 antiapoptotic family members that regulate survival. However, the intracellular pathways from OX40 that mediate these effects are unclear. In this study, we show that OX40 signaling can target the canonical NF-κB (NF-κB1) pathway in peripheral Ag-responding CD4 T cells. Phosphorylation of IκBα, nuclear translocation of NF-κB1/p50 and RelA, and NF-κB1 activity, are impaired in OX40-deficient T cells. Retroviral transduction of active IκB kinase that constitutively activates NF-κB1 rescues the poor expansion and survival of OX40-deficient T cells, directly correlating with increased expression and activity of survivin, aurora B, and Bcl-2 family members. Moreover, active IκB kinase expression alone is sufficient to restore the defective expansion and survival of OX40-deficient T cells in vivo when responding to Ag. Thus, OX40 signals regulate T cell number and viability through the NF-κB1 pathway that controls expression and activity of intracellular targets for proliferation and survival.

Antagonism of Airway Tolerance by Endotoxin/Lipopolysaccharide through Promoting OX40L and Suppressing Antigen-Specific Foxp3+ T Regulatory Cells

Respiratory exposure to allergens can lead to airway tolerance. Factors that antagonize tolerance mechanisms in the lung might result in susceptibility to diseases such as asthma. We show that inhalation of endotoxin/LPS with Ag prevented airway tolerance and abolished protection from T cell-driven asthmatic lung inflammation. Under conditions leading to tolerance, adaptive Ag-specific CD4+Foxp3+ T regulatory cells (Treg) were generated following exposure to intranasal Ag and outnumbered IL-4- and IFN-γ-producing CD4 T cells by 100:1 or greater. Inhaled LPS altered the ratio of Treg to IL-4+ or IFN-γ+ T cells by concomitantly suppressing Treg generation and promoting effector T cell generation. LPS induced OX40L expression on dendritic cells and B cells that resulted in a synergistic activity between TLR4 and OX40 signals, leading to production of IL-4, IFN-γ, and IL-6, which blocked Treg development. Furthermore, inhibiting OX40/OX40L interactions prevented LPS from suppressing tolerance, and resulted in the generation of greater numbers of adaptive Treg. Thus, cooperation between TLR4 and OX40 controls susceptibility to developing airway disease via modulating the balance between adaptive Treg and IL-4+ or IFN-γ+ T cells. Targeting OX40L then has the potential to improve the efficacy of Ag immunotherapy to promote tolerance.

OX40 Complexes with Phosphoinositide 3-Kinase and Protein Kinase B (PKB) To Augment TCR-Dependent PKB Signaling

T lymphocyte activation requires signal 1 from the TCR and signal 2 from costimulatory receptors. For long-lasting immunity, growth and survival signals imparted through the Akt/protein kinase B (PKB) pathway in activated or effector T cells are important, and these can be strongly influenced by signaling from OX40 (CD134), a member of the TNFR superfamily. In the absence of OX40, T cells do not expand efficiently to Ag, and memory formation is impaired. How most costimulatory receptors integrate their signals with those from Ag through the TCR is not clear, including whether OX40 directly recruits PKB or molecules that regulate PKB. We show that OX40 after ligation by OX40L assembled a signaling complex that contained the adapter TNFR-associated factor 2 as well as PKB and its upstream activator phosphoinositide 3-kinase (PI3K). Recruitment of PKB and PI3K were dependent on TNFR-associated factor 2 and on translocation of OX40 into detergent-insoluble membrane lipid microdomains but independent of TCR engagement. However, OX40 only resulted in strong phosphorylation and functional activation of the PI3K–PKB pathway when Ag was recognized. Therefore, OX40 primarily functions to augment PKB signaling in T cells by enhancing the amount of PI3K and PKB available to the TCR. This highlights a quantitative role of this TNFR family second signal to supplement signal 1.

Inducible CD4+LAP+Foxp3− Regulatory T Cells Suppress Allergic Inflammation

Regulatory T cells (Tregs) play a critical role in the maintenance of airway tolerance. We report that inhaled soluble Ag induces adaptive Foxp3+ Tregs, as well as a regulatory population of CD4+ T cells in the lungs and lung-draining lymph nodes that express latency-associated peptide (LAP) on their cell surface but do not express Foxp3. Blocking the cytokine IL-10 or TGF-β prevented the generation of LAP+ Tregs and Foxp3+ Tregs in vivo, and the LAP+ Tregs could also be generated concomitantly with Foxp3+ Tregs in vitro by culturing naive CD4+ T cells with Ag and exogenous TGF-β. The LAP+ Tregs strongly suppressed naive CD4+ T cell proliferation, and transfer of sorted OVA-specific LAP+ Tregs in vivo inhibited allergic eosinophilia and Th2 cytokine expression in the lung, either when present at the time of Th2 sensitization or when injected after Th2 cells were formed. Furthermore, inflammatory innate stimuli from house dust mite extract, nucleotide-binding oligomerization domain containing 2 ligand, and LPS, which are sufficient for blocking airway tolerance, strongly decreased the induction of LAP+ Tregs. Taken together, we concluded that inducible Ag-specific LAP+ Tregs can suppress asthmatic lung inflammation and constitute a mediator of airway tolerance together with Foxp3+ Tregs.

TNF Receptor-Associated Factor 5 Limits the Induction of Th2 Immune Responses

The TNF receptor-associated factor (TRAF) family of molecules acts as adapter proteins for signaling pathways initiated by several members of the TNF receptor (TNFR) superfamily. TRAF5−/− animals are viable and have normal development of the immune system despite interacting with several TNFR family members. A clear role for TRAF5 has yet to emerge. OX40 (CD134) interacts with TRAF5, suggesting that this pathway could be involved in regulating T cell differentiation into Th1 or Th2 cells. In tissue culture, OX40 stimulation of TRAF5−/− T cells resulted in a pronounced Th2 phenotype with elevated levels of IL-4 and IL-5. Similarly, in vivo immunization with protein in adjuvant in the presence of an agonist anti-OX40 Ab resulted in enhanced Th2 development in TRAF5−/− mice. Additionally, lung inflammation induced by T cells, which is critically controlled by OX40, was more pronounced in TRAF5−/− mice, characterized by higher levels of Th2 cytokines. These results suggest that TRAF5 can limit the induction of Th2 responses, and that TRAF5 can play a role in modulating responses driven by OX40 costimulation.