OX40 Regulates Both Innate and Adaptive Immunity and Promotes Nonalcoholic Steatohepatitis.

Both innate and adaptive immune cells are involved in the pathogenesis of nonalcoholic steatohepatitis (NASH), but the crosstalk between innate and adaptive immunity is largely unknown. Here we show that compared with WT mice, OX40-/- mice exhibit decreased liver fat accumulation, lobular inflammation, and focal necrosis after feeding with diets that induce NASH. Mechanistically, OX40 deficiency suppresses Th1 and Th17 differentiation, and OX40 deficiency in T cells inhibits monocyte migration, antigen presentation, and M1 polarization. Soluble OX40 stimulation alone upregulates antigen presentation, chemokine receptor expression, and proinflammatory cytokine secretion by liver monocytes. Furthermore, plasma soluble OX40 levels are positively associated with NASH in humans, suggesting clinical relevance of the findings. In conclusion, we show a mechanism for T cell regulation of innate immune cells. OX40 is a key regulator of both intrahepatic innate and adaptive immunity, generates two-way signals, and promotes both proinflammatory monocyte and macrophage and T cell function, resulting in NASH development.

PKC-ѳ is dispensable for OX40L-induced TCR-independent Treg proliferation but contributes by enabling IL-2 production from effector T-cells.

We have previously shown that OX40L/OX40 interaction is critical for TCR-independent selective proliferation of Foxp3+ Tregs, but not Foxp3- effector T-cells (Teff), when CD4+ T-cells are co-cultured with GM-CSF derived bone marrow dendritic cells (G-BMDCs). Events downstream of OX40L/OX40 interaction in Tregs responsible for this novel mechanism are not understood. Earlier, OX40L/OX40 interaction has been shown to stimulate CD4+ T-cells through the formation of a signalosome involving TRAF2/PKC-Ѳ leading to NF-kB activation. In this study, using CD4+ T-cells from WT and OX40-/- mice we first established that OX40 mediated activation of NF-kB was critical for this Treg proliferation. Although CD4+ T-cells from PKC-Ѳ-/- mice were also defective in G-BMDC induced Treg proliferation ex vivo, this defect could be readily corrected by adding exogenous IL-2 to the co-cultures. Furthermore, by treating WT, OX40-/-, and PKC-Ѳ-/- mice with soluble OX40L we established that OX40L/OX40 interaction was required and sufficient to induce Treg proliferation in vivo independent of PKC-Ѳ status. Although PKC-Ѳ is dispensable for TCR-independent Treg proliferation per se, it is essential for optimum IL-2 production by Teff cells. Finally, our findings suggest that OX40L binding to OX40 likely results in recruitment of TRAF1 for downstream signalling.

Regulation of A1 by OX40 contributes to CD8(+) T cell survival and anti-tumor activity.

The TNFR family member OX40 (CD134) is critical for optimal clonal expansion and survival of T cells. However, the intracellular targets of OX40 in CD8 T cells are not fully understood. Here we show that A1, a Bcl-2 family protein, is regulated by OX40 in effector CD8 T cells. In contrast to wild-type T cells, OX40-deficient CD8 T cells failed to maintain A1 expression driven by antigen. Conversely, enforced OX40 stimulation promoted A1 expression. In both situations, the expression of A1 directly correlated with CD8 T cell survival. In addition, exogenous expression of A1 in OX40-deficient CD8 T cells reversed their survival defect in vitro and in vivo. Moreover, forced expression of A1 in CD8 T cells from OX40-deficient mice restored the ability of these T cells to suppress tumor growth in a murine model. These results indicate that OX40 signals regulate CD8 T cell survival at least in part through maintaining expression of the anti-apoptotic molecule A1, and provide new insight into the mechanism by which OX40 may impact anti-tumor immunity.

Inherited human OX40 deficiency underlying classic Kaposi sarcoma of childhood.

Kaposi sarcoma (KS), a human herpes virus 8 (HHV-8; also called KSHV)-induced endothelial tumor, develops only in a small fraction of individuals infected with HHV-8. We hypothesized that inborn errors of immunity to HHV-8 might underlie the exceedingly rare development of classic KS in childhood. We report here autosomal recessive OX40 deficiency in an otherwise healthy adult with childhood-onset classic KS. OX40 is a co-stimulatory receptor expressed on activated T cells. Its ligand, OX40L, is expressed on various cell types, including endothelial cells. We found OX40L was abundantly expressed in KS lesions. The mutant OX40 protein was poorly expressed on the cell surface and failed to bind OX40L, resulting in complete functional OX40 deficiency. The patient had a low proportion of effector memory CD4(+) T cells in the peripheral blood, consistent with impaired CD4(+) T cell responses to recall antigens in vitro. The proportion of effector memory CD8(+) T cells was less diminished. The proportion of circulating memory B cells was low, but the antibody response in vivo was intact, including the response to a vaccine boost. Together, these findings suggest that human OX40 is necessary for robust CD4(+) T cell memory and confers apparently selective protective immunity against HHV-8 infection in endothelial cells.

Transgenic expression of survivin compensates for OX40-deficiency in driving Th2 development and allergic inflammation.

Survivin, an inhibitor of apoptosis family molecule, has been proposed as a crucial intermediate in the signaling pathways leading to T-cell development, proliferation, and expansion. However, the importance of survivin to T-cell-driven inflammatory responses has not been demonstrated. Here, we show that survivin transgenic mice exhibit an increased antigen-driven Th2 lung inflammation and that constitutive expression of survivin reversed the defective lung inflammation even in the absence of OX40 costimulation. We found that OX40-deficient mice were compromised in generating Th2 cells, airway eosinophilia, and IgE responses. In contrast, OX40-deficient/survivin transgenic mice generated normal Th2 responses and exhibited strong lung inflammation. These results suggest that OX40 costimulation crucially engages survivin during antigen-mediated Th2 responses. These findings also promote the notion that OX40 costimulation regulates allergic responses or lung inflammation by targeting survivin thereby enhancing T-cell proliferation and resulting in more differentiated Th2 cells in the allergic inflammatory response.

New insights on OX40 in the control of T cell immunity and immune tolerance in vivo.

OX40 is a T cell costimulatory molecule that belongs to the TNFR superfamily. In the absence of immune activation, OX40 is selectively expressed by Foxp3(+) regulatory T cells (Tregs), but not by resting conventional T cells. The exact role of OX40 in Treg homeostasis and function remains incompletely defined. In this study, we demonstrate that OX40 engagement in vivo in naive mice induces initial expansion of Foxp3(+) Tregs, but the expanded Tregs have poor suppressive function and exhibit features of exhaustion. We also show that OX40 enables the activation of the Akt and Stat5 pathways in Tregs, resulting in transient proliferation of Tregs and reduced levels of Foxp3 expression. This creates a state of relative IL-2 deficiency in naive mice that further impacts Tregs. This exhausted Treg phenotype can be prevented by exogenous IL-2, as both OX40 and IL-2 agonists drive further expansion of Tregs in vivo. Importantly, Tregs expanded by both OX40 and IL-2 agonists are potent suppressor cells, and in a heart transplant model, they promote long-term allograft survival. Our data reveal a novel role for OX40 in promoting immune tolerance and may have important clinical implications.

Targeting OX40 promotes lung-resident memory CD8 T cell populations that protect against respiratory poxvirus infection.

One goal of vaccination is to promote development of mucosal effector cells that can immediately respond to peripheral infection. This is especially important for protection against viruses that enter the host through the respiratory tract. We show that targeting the OX40 costimulatory receptor (CD134) strongly promotes mucosal memory in the CD8 T cell compartment. Systemic injection of an agonist antibody to OX40 strongly enhanced development of polyfunctional effector CD8 T cells that were induced after intraperitoneal infection with a highly virulent strain of vaccinia virus. These cells were located in lymphoid organs and also the lung, and importantly, long-term memory CD8 T cells were maintained in the lung over 1 year. Anti-OX40 also boosted memory development when mice were vaccinated subcutaneously with viral peptide. These CD8 T cells were sufficient to provide protection from lethal respiratory infection with live vaccinia virus independent of CD4 T cells and antibody. Again, the CD8 T cell populations that were induced after secondary infection displayed polyfunctionality and were maintained in the lung for over a year. These data suggest that agonists to the OX40 costimulatory receptor represent potential candidates for incorporation into vaccines for respiratory viruses.

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.

The TNFR family members OX40 and CD27 link viral virulence to protective T cell vaccines in mice.

Induction of CD8+ T cell immunity is a key characteristic of an effective vaccine. For safety reasons, human vaccination strategies largely use attenuated nonreplicating or weakly replicating poxvirus-based vectors, but these often elicit poor CD8+ T cell immunity and might not result in optimal protection. Recent studies have suggested that virulence is directly linked to immunogenicity, but the molecular mechanisms underlying optimal CD8+ T cell responses remain to be defined. Here, using natural and recombinant vaccinia virus (VACV) strains, we have shown in mice that VACV strains of differing virulence induce distinct levels of T cell memory because of the differential use of TNF receptor (TNFR) family costimulatory receptors. With strongly replicating (i.e., virulent) VACV, the TNFR family costimulatory receptors OX40 (also known as CD134) and CD27 were engaged and promoted the generation of high numbers of memory CD8+ T cells, which protected against a lethal virus challenge in the absence of other mechanisms, including antibody and help from CD4+ T cells. In contrast, weakly replicating (i.e., low-virulence) VACV strains were poor at eliciting protective CD8+ T cell memory, as only the Ig family costimulatory receptor CD28 was engaged, and not OX40 or CD27. Our results suggest that the virulence of a virus dictates costimulatory receptor usage to determine the level of protective CD8+ T cell immunity.

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-gamma-producing CD4 T cells by 100:1 or greater. Inhaled LPS altered the ratio of Treg to IL-4(+) or IFN-gamma(+) 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-gamma, 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-gamma(+) T cells. Targeting OX40L then has the potential to improve the efficacy of Ag immunotherapy to promote tolerance.

OX40 costimulatory signals potentiate the memory commitment of effector CD8+ T cells.

A T cell costimulatory molecule, OX40, contributes to T cell expansion, survival, and cytokine production. Although several roles for OX40 in CD8(+) T cell responses to tumors and viral infection have been shown, the precise function of these signals in the generation of memory CD8(+) T cells remains to be elucidated. To address this, we examined the generation and maintenance of memory CD8(+) T cells during infection with Listeria monocytogenes in the presence and absence of OX40 signaling. We used the expression of killer cell lectin-like receptor G1 (KLRG1), a recently reported marker, to distinguish between short-lived effector and memory precursor effector T cells (MPECs). Although OX40 was dispensable for the generation of effector T cells in general, the lack of OX40 signals significantly reduced the number and proportion of KLRG1(low) MPECs, and, subsequently, markedly impaired the generation of memory CD8(+) T cells. Moreover, memory T cells that were generated in the absence of OX40 signals in a host animal did not show self-renewal in a second host, suggesting that OX40 is important for the maintenance of memory T cells. Additional experiments making use of an inhibitory mAb against the OX40 ligand demonstrated that OX40 signals are essential during priming, not only for the survival of KLRG1(low) MPECs, but also for their self-renewing ability, both of which contribute to the homeostasis of memory CD8(+) T cells.

Critical synergy of CD30 and OX40 signals in CD4 T cell homeostasis and Th1 immunity to Salmonella.

CD30 and OX40 (CD134) are members of the TNFR superfamily expressed on activated CD4 T cells, and mice deficient in both these molecules harbor a striking defect in the capacity to mount CD4 T cell-dependent memory Ab responses. This article shows that these mice also fail to control Salmonella infection because both CD30 and OX40 signals are required for the survival but not commitment of CD4 Th1 cells. These signals are also needed for the survival of CD4 T cells activated in a lymphopenic environment. Finally, Salmonella and lymphopenia are shown to act synergistically in selectively depleting CD4 T cells deficient in OX40 and CD30. Collectively these findings identify a novel mechanism by which Th1 responses are sustained.

Differential requirements for OX40 signals on generation of effector and central memory CD4+ T cells.

Memory T cells can be divided into effector memory (T(EM)) and central memory (T(CM)) subsets based on their effector function and homing characteristics. Although previous studies have demonstrated that TCR and cytokine signals mediate the generation of the two memory subsets of CD8(+) T cells, the mechanisms for generation of the CD4(+) T(EM) and T(CM) cell subsets are unknown. We found that OX40-deficient mice showed a marked reduction in the number of CD4(+) T(EM) cells, whereas the number of CD4(+) T(CM) cells was normal. Adoptive transfer experiments using Ag-specific CD4(+) T cells revealed that OX40 signals during the priming phase were indispensable for the optimal generation of the CD4(+) T(EM), but not the CD4(+) T(CM) population. In a different transfer experiment with in vitro established CD4(+)CD44(high)CD62L(low) (T(EM) precursor) and CD4(+)CD44(high)CD62L(high) (T(CM) precursor) subpopulations, OX40-KO T(EM) precursor cells could not survive in the recipient mice, whereas wild-type T(EM) precursor cells differentiated into both T(EM) and T(CM) cells. In contrast, T(CM) precursor cells mainly produced T(CM) cells regardless of OX40 signals, implying the dispensability of OX40 for generation of T(CM) cells. Nevertheless, survival of OX40-KO T(EM) cells was partially rescued in lymphopenic mice. During in vitro recall responses, the OX40-KO T(EM) cells that were generated in lymphopenic recipient mice showed impaired cytokine production, suggesting an essential role for OX40 not only on generation but also on effector function of CD4(+) T(EM) cells. Collectively, the present results indicate differential requirements for OX40 signals on generation of CD4(+) T(EM) and T(CM) cells.

OX40 controls functionally different T cell subsets and their resistance to depletion therapy.

T cell depletion is a widely used approach in clinical transplantation. However, not all T cells are equally sensitive to depletion therapies and a significant fraction of T cells persists even after aggressive treatment. The functional attributes of such T cells and the mechanisms responsible for their resistance to depletion are poorly studied. In the present study, we showed that CD4(+) T cells that are resistant to polyclonal anti-lymphocyte serum (ALS) mediated depletion exhibit phenotypic features of memory cells and uniformly express OX40 on the cell surface. Studies using the foxp3gfp knockin mice revealed that the remaining CD4(+)OX40(+) cells consist of Foxp3(+) Tregs and Foxp3(-) T effector/memory cells. The ALS-resistant CD4(+)OX40(+) cells failed to mediate skin allograft rejection upon adoptive transferring into congenic Rag(-/-) mice, but removal of Foxp3(+) Tregs from the OX40(+) cells resulted in prompt skin allograft rejection. Importantly, OX40 is critical to survival of both Foxp3(+) Tregs and T effector/memory cells. However, OX40 exhibits opposing effects on the functional status of Foxp3(+) Tregs and T effector/memory cells, as stimulation of OX40 on T effector cells induced amplified cell proliferation but stimulation of OX40 on the Foxp3(+) Tregs impaired their suppressor functions. Our study demonstrates that OX40 is a critical molecule in regulating survival and functions of depletion-resistant T cells; and these findings may have important clinical implications.

OX40 costimulation promotes persistence of cytomegalovirus-specific CD8 T Cells: A CD4-dependent mechanism.

The mechanisms that regulate CMV-specific T cell responses in vivo are poorly understood. During murine CMV infection of B6 mice, primary responses in the spleen are dominated by CD8 T cells reactive with antigenic epitopes in M45, M57, and m139 murine CMV gene products. However, during the later persistent phase of infection, CD8 T cell responses to epitopes in m139 and M38 viral gene products predominate. The basis for this shift in CD8 T populations is unknown. In this study, we demonstrate that OX40, a TNFR superfamily member, specifically regulates the accumulation of CD8 T cells reactive with the persistent-phase epitopes. Defective CD8 T cell responses in OX40(-/-) mice were replicated in MHC class II(-/-) mice implying that CD4 T cells in part controlled the differentiation of the CD8 T cell clones responsive to these epitopes during persistent infection. Furthermore, treatment of infected mice with an agonist OX40 Ab induced expansion of protective primary virus-specific CD8 T cells independent of CD4 T cell help, but CD4 T cells were crucial for anti-OX40 to promote CD8 T cells reactive to the persistent dominant epitopes. Collectively, these results indicate manipulation of OX40 may be useful in improving cellular immunotherapy regimes for treatment of persistent virus infections.