4-1BB Triggering Research Articles

According to Hepatitis C Virus (HCV) Infection Stage, Interleukin-7 Plus 4-1BB Triggering Alone or Combined with PD-1 Blockade Increases TRAF1low HCV-Specific CD8+ Cell Reactivity.

Hepatitis C virus (HCV)-specific CD8+ T cells suffer a progressive exhaustion during persistent infection (PI) with HCV. This process could involve the positive immune checkpoint 4-1BB/4-1BBL through the loss of its signal transducer, TRAF1. To address this issue, peripheral HCV-specific CD8+ T cells (pentamer-positive [pentamer+]/CD8+ T cells) from patients with PI and resolved infection (RI) after treatment were studied. The duration of HCV infection and the liver fibrosis progression rate inversely correlated with the likelihood of detection of peripheral pentamer+/CD8+ cells. In PI, pentamer+/CD8+ cells had impaired antigen-specific reactivity that worsened when these cells were not detectable ex vivo Short/midduration PI was characterized by detectable peripheral PD-1+ CD127low TRAF1low cells. After triggering of T cell receptors (TCR), the TRAF1 level positively correlated with the levels of CD127, Mcl-1, and CD107a expression and proliferation intensity but negatively with PD-1 expression, linking TRAF1low to exhaustion. In vitro treatment with interleukin-7 (IL-7) upregulated TRAF1 expression, while treatment with transforming growth factor-β1 (TGF-β1) did the opposite, suggesting that the IL-7/TGF-β1 balance, besides TCR stimulation, could be involved in TRAF1 regulation. In fact, the serum TGF-β1 concentration was higher in patients with PI than in patients with RI, and it negatively correlated with TRAF1 expression. In line with IL-7 increasing the level of TRAF1 expression, IL-7 plus 4-1BBL treatment in vitro enhanced T cell reactivity in patients with short/midduration infection. However, in patients with long-lasting PI, anti-PD-L1, in addition to the combination of IL-7 and 4-1BBL, was necessary to reestablish T cell proliferation in individuals with slowly progressing liver fibrosis (slow fibrosers) but had no effect in rapid fibrosers. In conclusion, a peripheral hyporeactive TRAF1low HCV-specific CD8+ T cell response, restorable by IL-7 plus 4-1BBL treatment, characterizes short/midduration PI. In long-lasting disease, HCV-specific CD8+ T cells are rarely detectable ex vivo, but treatment with IL-7, 4-1BBL, and anti-PD-L1 recovers their reactivity in vitro in slow fibrosers.IMPORTANCE Hepatitis C virus (HCV) infects 71 million people worldwide. Two-thirds develop a chronic disease that can lead to cirrhosis and hepatocellular carcinoma. Direct-acting antivirals clear the infection, but there are still patients who relapse. In these cases, additional immunotherapy could play a vital role. A successful anti-HCV immune response depends on virus-specific CD8+ T cells. During chronic infection, these cells are functionally impaired, which could be due to the failure of costimulation. This study describes exhausted specific T cells, characterized by low levels of expression of the signal transducer TRAF1 of the positive costimulatory pathway 4-1BB/4-1BBL. IL-7 upregulated TRAF1 expression and improved T cell reactivity in patients with short/midduration disease, while in patients with long-lasting infection, it was also necessary to block the negative PD-1/PD-L1 checkpoint. When the results are taken together, this work supports novel ways of restoring the specific CD8+ T cell response, shedding light on the importance of TRAF1 signaling. This could be a promising target for future immunotherapy.

4-1BB Signaling Enhances Primary and Secondary Population Expansion of CD8+ T Cells by Maximizing Autocrine IL-2/IL-2 Receptor Signaling.

4-1BB (CD137), a member of the tumor necrosis factor receptor superfamily (TNFRSF), is primarily expressed on activated T cells and is known to enhance proliferation of T cells, prevent activation-induced cell death, and promote memory formation of CD8+ T cells. In particular, it is well acknowledged that 4-1BB triggering preferentially enhances the expansion of CD8+ T cells rather than CD4+ T cells, but the underlying mechanism remains unclear. Here we found that 4-1BB triggering markedly increased IL-2Rα (CD25) and IL-2 expressions of CD8+ T cells but minimally for CD4+ T cells. Proliferation of CD8+ T cells was moderately enhanced by direct 4-1BB triggering in the absence of signaling through IL-2Rα/IL-2 interactions, but further promoted in the presence of IL-2Rα/IL-2 interactions. Among the TNFRSF members including OX40, GITR, CD30, and CD27, 4-1BB was superior in the ability to induce IL-2Rα expression on CD8+ T cells. When the primary and secondary expansions of CD8+ T cells in vivo were examined by adoptively transferring OVA-specific CD8+ T cells along with the treatment with agonistic anti-4-1BB and/or antagonistic anti-CD25 F(ab’)2 mAb, 4-1BB triggering enhanced both primary and secondary expansion of CD8+ T cells in vivo, and the 4-1BB effects were moderately suppressed in primary expansion while completely abolished in secondary expansion of OVA-specific CD8+ T cells by blocking IL-2Rα. These results suggest that 4-1BB-mediated increases of IL-2Rα and IL-2 prolong the effects of transient TCR- and 4-1BB-mediated signaling in CD8+ T cells, and that 4-1BB triggering preferentially enhances the expansion of CD8+ T cells through the amplification of autocrine IL-2/IL-2R signaling loop.

4-1BB Signaling Breaks the Tolerance of Maternal CD8+ T Cells That Are Reactive with Alloantigens

4-1BB (CD137, TNFRSF9), a member of the activation-induced tumor necrosis factor receptor family, is a powerful T-cell costimulatory molecule. It generally enhances CD8+ T responses and even breaks the tolerance of CD8+ T cells in an antigen-specific manner. In the present study we found that it was expressed in the placentas of pregnant mice and that its expression coincided with that of the immunesuppressive enzyme indoleamine 2,3-dioxygenase (IDO). Therefore, we investigated whether 4-1BB signaling is involved in fetal rejection using agonistic anti-4-1BB mAb and 4-1BB-deficient mice. Treatment with agonistic anti-4-1BB mAb markedly increased the rate of rejection of allogeneic but not syngeneic fetuses, and this was primarily dependent on CD8+ T cells. Complement component 3 (C3) seemed to be the effector molecule because 4-1BB triggering resulting in accumulation of C3 in the placenta, and this accumulation was also reversed by anti-CD8 mAb treatment. These findings demonstrate that 4-1BB triggering breaks the tolerance of CD8+ T cells to alloantigens in the placenta. Moreover, triggering 4-1BB protected the pregnant mice from Listeria monocytogenes (LM) infection, but led to rejection of semi-allogeneic fetuses. Therefore, given the cross-recognition of alloantigen by pathogen-reactive CD8+ T cells, the true function of 4-1BB may be to reverse the hypo-responsiveness of pathogen-reactive CD8+ T cells in the placenta in cases of infection, even if that risks losing the fetus.

4-1BB Triggering Ameliorates Experimental Autoimmune Encephalomyelitis by Modulating the Balance between Th17 and Regulatory T Cells

Agonistic anti-4-1BB Ab is known to ameliorate experimental autoimmune encephalomyelitis. 4-1BB triggering typically leads to the expansion of CD8(+) T cells, which produce abundant IFN-γ, and this in turn results in IDO-dependent suppression of autoimmune responses. However, because neutralization of IFN-γ or depletion of CD8(+) T cell only partially abrogates the effect of 4-1BB triggering, we sought to identify an additional mechanism of 4-1BB-triggered suppression of autoimmune responses using IFN-γ- or IFN-γR-deficient mice. 4-1BB triggering inhibited the generation of Th17 cells that is responsible for experimental autoimmune encephalomyelitis induction and progression, and increased Foxp3(+)CD4(+) regulatory T (Treg) cells, particularly among CD4(+) T cells. This was not due to a direct effect of 4-1BB signaling on CD4(+) T cell differentiation: 4-1BB signaling not only reduced Th17 cells and increased Treg cells in wild-type mice, which could be due to IFN-γ production by the CD8(+) T cells, but also did so in IFN-γ-deficient mice, in that case by downregulating IL-6 production. These results show that although secondary suppressive mechanisms evoked by 4-1BB triggering are usually masked by the strong effects of IFN-γ, 4-1BB signaling seems to modulate autoimmune responses by a number of mechanisms, and modulation of the Th17 versus Treg cell balance is one of those mechanisms.

Unified immune modulation by 4-1BB triggering leads to diverse effects on disease progression in vivo

4-1BB (CD137) is a powerful T-cell costimulatory molecule in the treatment of virus infections and tumors, but recent studies have also uncovered regulatory functions of 4-1BB signaling. Since 4-1BB triggering suppresses autoimmunity by accumulating indoleamine 2,3-dioxygenase (IDO) in dendritic cells (DCs) in an interferon (IFN)-γ-dependent manner, we asked whether similar molecular and cellular changes were induced by 4-1BB triggering in virus-infected mice. 4-1BB triggering increased IFN-γ and IDO, and suppressed CD4 + T cells, in C57BL/6 mice infected with the type 1 KOS strain of Herpes simplex virus (HSV-1), as it does in an autoimmune disease model. Detailed analysis of the CD4 + T suppression showed that freshly activated CD62L high T cells underwent apoptosis in the early phase of suppression, and CD62L low effector/memory T cells in the later phase. Although 4-1BB triggering resulted in similar cellular changes – increased CD8 + T and decreased CD4 + T cells, it had different effects on mortality in mice infected with HSV-1 RE, influenza, and Japanese encephalitis virus (JEV); it increased mortality in influenza-infected mice but decreased it in JEV-infected mice. Since the dominant type of immune cell generated to protect the host was different for each virus – CD4 + T cells and neutrophils in HSV-1 RE infection, both CD4 + T and CD8 + T cells in influenza infection, and a crucial role for B cells in JEV infection, 4-1BB triggering resulted in different therapeutic outcomes. We conclude that the therapeutic outcome of 4-1BB triggering is determined by whether the protective immunity generated against the virus was beneficially altered by the 4-1BB triggering.

Peripheral 4-1BB Signaling Negatively Regulates NK Cell Development through IFN-γ

Stimulation of 4-1BB (CD137) was shown to produce strong anticancer effects in vivo. In contrast, 4-1BB-deficient (4-1BB(-/-)) B6 mice are remarkably resistant to tumor growth. We set out to determine the mechanisms involved in these seemingly contradictory observations. We found that the therapeutic effects of 4-1BB triggering were mainly dependent on CD8(+) T cells and partially on NK cells, whereas CD8(+) T and NK cells were equally needed to suppress tumor growth in 4-1BB(-/-) mice. Cellular analysis showed that the frequency and number of NK cells in the spleen and bone marrow were decreased by 4-1BB triggering but were increased in the absence of 4-1BB signaling in tumor-challenged mice. The 4-1BB-mediated downregulation of NK cell development was primarily dependent on IFN-gamma, which was produced by peripheral CD8(+) T and NK cells. The suppression of NK cell development by 4-1BB-mediated IFN-gamma production occurred in the bone marrow. As 4-1BB signaling increased in the periphery, more CD8(+) T cells but fewer NK cells contributed to the antitumor immunity. As 4-1BB signaling decreased, more NK cells participated in the antitumor immunity. We conclude that 4-1BB signaling results in a shift of the dominant type of immune cell in antitumor immunity from the innate NK cell to the adaptive CD8(+) T cell and that the level of IFN-gamma is critical for this 4-1BB-mediated shift.

IFN-γ-indoleamine-2,3 dioxygenase acts as a major suppressive factor in 4-1BB-mediated immune suppression in vivo

It has been reported that 4-1BB triggering in vivo selectively suppressed the recall response of staphylococcal enterotoxin A (SEA)-specific CD4(+) T cells, in which CD8(+) T-derived TGF-beta was involved. Here, we have examined an alternative mechanism for the 4-1BB-mediated CD4(+) T suppression, as the neutralization of TGF-beta is only effective in rescuing the SEA-specific recall response at high cellular concentrations. We show that this selective suppression of CD4(+) T cells by 4-1BB triggering in vivo is mediated mainly by induction of indoleamine 2,3-dioxygenase (IDO) in an IFN-gamma-dependent manner. SEA-specific CD4(+) T responses were suppressed partly by TGF-beta-expressing CD8(+) T cells, particularly CD11c(+)CD8(+) T cells, but strongly inhibited by dendritic cells (DCs) expressing IDO. IFN-gamma that increased IDO in DCs was produced primarily from CD11c(+)CD8(+) T cells, which were expanded selectively by 4-1BB stimulation. CD4(+), CD8(+), and plasmacytoid DCs exerted a similar suppressive activity toward the SEA-specific CD4(+) T cells. Neutralization of IFN-gamma or IDO activity in vivo largely reversed the 4-1BB-mediated CD4(+) T suppression. Collectively, these data indicate that 4-1BB-dependent suppression of SEA-specific CD4(+) T responses was mediated mainly by IFN-gamma-dependent IDO induction and partially by TGF-beta.

4-1BB triggers IL-13 production from T cells to limit the polarized, Th1-mediated inflammation

4-1BB (CD137) triggering typically induces Th1 response by increasing IFN-gamma from T cells upon TCR ligation. We found recently that 4-1BB costimulation increased the expression of IL-13 from CD4(+) T cells, as well as CD8(+) T cells. The enhanced IL-13 expression by agonistic anti-4-1BB treatment was mediated via MAPK1/2, PI-3K, JNK, mammalian target of rapamycin, NF-AT, and NF-kappaB signaling pathways. The signaling for IL-13 induction was similar to that of IFN-gamma production by anti-4-1BB treatment in T cells. When the anti-4-1BB-mediated IL-13 expression was tested in an in vivo viral infection model such as HSV-1 and vesicular stomatitis virus, 4-1BB stimulation enhanced IL-13 expression of CD4(+) T, rather than CD8(+) T cells. Although IL-13 was enhanced by anti-4-1BB treatment, the increased IL-13 did not significantly alter the anti-4-1BB-induced Th1 polarization of T cells--increase of T-bet and decrease of GATA-3. Nevertheless, anti-4-1BB treatment polarized T cells excessively in the absence of IL-13 and even became detrimental to the mice by causing liver inflammation. Therefore, we concluded that IL-13 was coinduced following 4-1BB triggering to maintain the Th1/2 balance of immune response.

4-1BB costimulation enhances HSV-1-specific CD8 + T cell responses by the induction of CD11c +CD8 + T cells

Since 4-1BB plays a predominant role in CD8 + T cell responses, we investigated the effects of 4-1BB triggering on the primary and memory CD8 + T responses to HSV-1 infection. 4-1BB was detected on 10–15% of CD4 + and CD8 + T cells following the infection. 4-1BB-positive T cells were in the proliferative mode and showed the enhanced expression of anti-apoptotic proteins. Agonistic anti-4-1BB treatment exerted preferential expansion of CD8 + T cells and gB/H-2K b-positive CD8 + T cells, and enhanced cytotoxicity against HSV-1 that was mainly mediated by CD11c +CD8 + T cells. CD11c +CD8 + T cells were re-expanded following re-challenge with HSV-1 at post-infection day 50, indicating that CD11c +CD8 + phenotype was maintained in memory CD8 + T cell pool. Our studies demonstrated that 4-1BB stimulation enhanced both primary and memory anti-HSV-1 CD8 + T cell responses, which was mediated by a massive expansion of antigen-specific CD11c +CD8 + T cells.

Stimulation with 4-1BB (CD137) inhibits chronic graft-versus-host disease by inducing activation-induced cell death of donor CD4+ T cells

Abstract4-1BB, a member of the tumor necrosis factor (TNF) receptor superfamily, is a costimulator for activated T cells. Previous studies have established that treatment with agonistic anti–4-BB monoclonal antibody (3H3) is effective in reversing the progression of spontaneous systemic lupus erythematosus. Its therapeutic effect is mediated by suppression of autoantibody production. In this report, we show that a single injection of 3H3 blocks chronic graft-versus-host disease (cGVHD) in the parent-into-F1 model. In particular, donor CD4+ T cells are rapidly eliminated from host spleens by activation-induced cell death after 4-1BB triggering. Since donor CD4+ T cells are required for the development of cGVHD, and 3H3-mediated inhibition of autoantibody production occurs without donor CD8+ T cells, 3H3 blocks cGVHD by preventing alloreactive donor CD4+ T cells from activating host B cells. Importantly, 3H3 treatment can reverse the progression of advanced cGVHD. Our findings indicate that agonistic anti–4-1BB monoclonal antibody has potential as an immunotherapeutic agent for preventing and treating cGVHD.

Effector CD8 T cells possess suppressor function after 4-1BB and Toll-like receptor triggering.

To better understand how innate and adaptive immune responses interact with each other, we combined 4-1BB T cell costimulation with specific adjuvants to determine whether these treatments would influence specific T cell expansion and function in vivo. In the presence of 4-1BB ligation and Toll-like receptor 3 (TLR)3 and/or TLR4 triggering, CD8 T cell clonal expansion and survival was augmented profoundly. Specific T cells primed in vivo with TLR ligands responded normally to in vitro recall stimulus, but, surprisingly, copriming with 4-1BB costimulation significantly impaired the recall response even though many more specific effector T cells were rescued in vivo. Here, we demonstrate that the rescued CD8 T cells suppressed CD4 T cell proliferation via a type beta transforming growth factor-dependent mechanism. Thus, 4-1BB and TLR ligands induce survival of specific effector CD8 T cells with suppressive recall potential, which may explain the dual role that 4-1BB activation plays in mediating tumor clearance and prevention of autoimmune disease.

4-1BB co-stimulation enhances human CD8(+) T cell priming by augmenting the proliferation and survival of effector CD8(+) T cells.

Interactions between 4-1BB and its ligand, 4-1BBL, enhance CD8(+) T cell-mediated antiviral and antitumor immunity in vivo. However, mechanisms regulating the priming of CD8(+) T cell responses by 4-1BB remain unclear, particularly in humans. The 4-1BB receptor was undetectable on naive or resting human CD8(+) T cells and induced in vitro by TCR triggering. Naive cord blood cells were therefore primed in vitro against peptides or cellular antigens and then co-stimulated with 4-1BBL or agonistic antibodies. Co-stimulation enhanced effector function such as IFN-gamma production and cytotoxicity by augmenting numbers of antigen-specific and effector CD8(+) T cells. OKT3 responses also showed reduced cell death and revealed that the proliferation of CD8(+) T cells required two independently regulated events. One, the induction of IL-2 production, could be directly triggered by 4-1BB engagement on CD8(+) T cells in the absence of accessory cells. The other, expression of CD25, was induced with variable efficacy by accessory cells. Thus, suboptimal accessory cells and 4-1BB co-stimulation combined their effects to enhance IL-2 production and proliferation. Reduced apoptosis observed after co-stimulation in the presence of accessory cells correlated with increased levels of Bcl-X(L) in CD8(+) T cells, while Bcl-2 expression remained unchanged. Altogether, 4-1BB enhanced expansion, survival and effector functions of newly primed CD8(+) T cells, acting in part directly on these cells. As 4-1BB triggering could be protracted from the TCR signal, 4-1BB agonists may function through these mechanisms to enhance or rescue suboptimal immune responses.

In vivo triggering through 4-1BB enables Th-independent priming of CTL in the presence of an intact CD28 costimulatory pathway.

Triggering of 4-1BB, a member of the TNFR family, through in vivo administration of agonistic anti-4-1BB Ab delivers a powerful costimulatory signal to CTL. We found this signal to effectively replace the need for CD4(+) T cell help in the cross-priming of tumor-specific CTL immunity. Furthermore, 4-1BB Ab can convert an otherwise tolerogenic peptide vaccine into a formulation capable of efficient CTL priming. Initial activation of naive CTL can occur in the absence of 4-1BB costimulation, but this signal permits increased survival of Ag-stimulated CTL. Because naive CTL do not express 4-1BB at their surface, susceptibility to 4-1BB triggering depends on prior up-regulation of this receptor. We show that this requires both stimulation of the TCR and CD28-dependent costimulation. Accordingly, blockade of the CD28-costimulatory pathway abrogates the capacity of agonistic anti-4-1BB Ab to trigger Th-independent CTL immunity. In conclusion, our data reveal that the 4-1BB-mediated survival signal is positioned downstream of Ag-specific TCR triggering and CD28-dependent costimulation of naive CTL. The powerful effects of 4-1BB triggering on the induction, amplification, and persistence of CTL responses provide a novel strategy for increasing the potency of vaccines against cancers.