Constitutive expression of functional 4-1BB (CD137) ligand on carcinoma cells

Members of the TNF superfamily, including Fas, Fas ligand, and CD40, have been shown to be expressed on tumor cells. In the studies described in this work, we report that another family member, the ligand for 4-1BB (CD137), is expressed on various human carcinoma cell lines, on cells of solid tumors derived from these cell lines, and cells obtained from human tumors. Expression of 4-1BB ligand (4-1BBL) mRNA was detected by both RT-PCR and Northern blot analysis, and expression of 4-1BBL protein was detected by Western blot analysis of whole cell lysates and by FACS analysis of tumor cells and cell lines. Incubation of tumor cells with a 4-1BB-Ig fusion protein led to the production of IL-8 by the cells, demonstrating that the 4-1BBL is functionally active and signals back into the tumor cells. Furthermore, 4-1BBL expressed on the carcinoma cells functioned as a costimulatory molecule for the production of cytokines (most notably IFN-gamma) in cocultures of T cells and tumor cells. These findings suggest that 4-1BBL expressed on carcinoma cells may significantly influence the outcome of a T cell-tumor cell interaction.     

Immune responses in 4-1BB (CD137)-deficient mice

The 4-1BB (a TNFR superfamily member) is an inducible costimulatory molecule that can exert regulatory effects on T cells independently of CD28 stimulation. The in vitro expression of 4-1BB (CD137) is induced following activation of T cells with various stimuli, including anti-TCR mAbs, lectins, and a combination of PMA and ionomycin. To delineate further the physiological role of 4-1BB in immunity, mice deficient in this receptor were generated. These mutant mice developed normally, and were viable and fertile. Humoral responses to vesicular stomatitis virus were comparable with those seen in wild-type mice, whereas the IgG2a and IgG3 isotype responses to keyhole limpet hemocyanin were somewhat reduced in the mutant mice. The 4-1BB-deficient mice demonstrated enhanced T cell proliferation in response to mitogens or anti-CD3 even in the environment of reduced ability to secrete growth-supporting cytokines (IL-2 and IL-4). Although T cells from 4-1BB-deficient mice showed enhanced proliferation, the T cell immune responses of these animals, such as cytokine production and CTL activity, were diminished. In addition, 4-1BB deletion appears to play a role in the regulation of myeloid progenitor cell growth, leading to an increase in these precursor cells in peripheral blood, bone marrow, and spleen.     

Immunity in the absence of CD28 and CD137 (4-1BB) molecules

We report the generation and immune regulation of mice that are deficient in CD28 and 4-1BB (CD137) genes. These mice were viable, fertile and did not display any overt abnormalities and had a normal T cell phenotype in thymus and spleen. Proliferative responses to anti-CD3 and ConA were enhanced in 4-1BB-/- but not in either CD28-/- or double mutant mice, while levels of interleukin-2 were decreased in all mutant mice. Although the 4-1BB-/- mice displayed increased basal levels of most immunoglobulin isotypes tested, the plateau levels of immunoglobulin G2a, immunoglobulin G2b and immunoglobulin A were particularly high compared to wild type controls. The immunoglobulin class switch to T-dependent antigen was normal in 4-1BB-/- mice but was greatly affected in both CD28-/- and 4-1BB-/- CD28-/- mice. Vesicular stomatitis virus-specific cytotoxic T lymphocyte responses and plaque reduction neutralizing ability was differentially reduced in all mutant mice. Contact sensitivity to allergens showed marginal but not significant change in ear thickness in 4-1BB-/- mice, but an ability to mount contact hypersensitivity to the same antigens was greatly curtailed in CD28-/- and double mutant mice.     

Defective T cell priming associated with aging can be rescued by signaling through 4-1BB (CD137)

Aging is associated with an increased susceptibility to infectious agents and correlates with a decreased ability to mount an immune response. It has been postulated that the major defect is related to a reduced capacity of an aged T cell to proliferate and to survive after encounter with Ag. This is similar to the phenotype associated with T cell tolerance in young adults. In this study, we determined whether targeting 4-1BB (CD137), a member of the TNFR family implicated in providing expansion and survival signals to T cells, can rescue defective priming in aged and tolerized animals. Agonist Abs to 4-1BB injected in vivo were capable of preventing CD4 T cell tolerance to soluble peptide in young mice. Moreover, anti-4-1BB rescued defective priming of aged TCR transgenic CD4 T cells responding to peptide Ag in a young host, and as importantly, anti-4-1BB completely restored T cell priming to protein Ag in nontransgenic aged mice. These studies demonstrate that 4-1BB, and potentially other costimulatory members of the TNFR family, are targets for therapies aimed at augmenting weak T cell responses in elderly immunocompromised individuals.     

Differential expression and costimulatory effect of 4-1BB (CD137) and CD28 molecules on cytokine-induced murine CD8(+) Tc1 and Tc2 cells

In this study we report that the relative expression of 4-1BB (CD137) and CD28 molecules can differentially be modulated on CD8(+) T cells by combinations of various cytokines and anti-cytokine antibodies. During allostimulation of naive CD8(+) T cells in the presence of IL-2, IFN-gamma, IL-12, and anti-IL-4, they evolved into IL-2, IFN-gamma-producing Tc1 cells and showed inability to upregulate 4-1BB expression but not CD28. On the other hand, the Tc2 cells, generated in the presence of allogeneic APCs, IL-2, IL-10, IL-4, and anti-IFN-gamma, demonstrated intact and elevated 4-1BB and CD28 molecules. Activation of Tc1 and Tc2 cells with anti-CD3 and plate-bound anti-4-1BB and anti-CD28 mAbs revealed differential proliferative and cytokine secretory patterns. The 4-1BB signaling in the context of anti-CD3 as first signal led to the increased secretion of IL-4 by the Tc2 cells and not by Tc1 cells, while CD28 triggering produced IL-4 from Tc2 and IL-2 and IFN-gamma from Tc1 cells. Flow cytometric analysis of cell surface expression on Tc1 and Tc2 cells strengthened our observation that 4-1BB expression but not CD28 is poorly expressed on Tc1 cells. Both of the polarized CD8(+) T cell subsets exhibited comparable cytotoxic abilities and perforin and granzyme expression. The regeneration of 4-1BB expression is possible on Tc1 cells when back cultured in a Tc2 cytokine environment, but its expression could not be significantly altered on the Tc2 population unless IL-12 was included in the system.     

Activation of c-jun N-terminal kinase by 4-1BB (CD137), a T cell co-stimulatory molecule

It has been widely accepted that T cell activation requires two signals; one from the binding of the antigen/major histocompatibility complex to the T-cell receptor (TCR)/CD3 complex and the other from the interaction between a surface molecule on antigen presenting cells and its receptor on T cells. The second signal is considered as co-stimulatory and the B7/CD28 pair has been well studied as a prototype. Recently 4-1BB (CD137) has been characterized as another co-stimulatory molecule for T cell activation. However, unlike the CD28/B7 pair, 4-1BB and its ligand 4-1BBL constitute a member of the tumor necrosis factor (TNF) receptor/TNF pair superfamily. The signaling mechanism of 4-1BB has not been revealed in detail. To investigate whether 4-1BB takes the signaling pathways analogous to those for TNF receptors, we generated polyclonal antibodies against human 4-1BB and 4-1BBL and established stable transfectants of the receptor and the ligand with a high level of cell surface expression. Over-expression of h4-1BB was found to result in the activation of c-Jun N-terminal kinase (JNK) in the human embryonic kidney cell line 293. In T cells, it has been previously demonstrated that JNK activation requires dual signals such as the ligation of TCR/CD3 complex plus CD28 co-stimulation or PMA plus ionomycin. The JNK activation by 4-1BB in Jurkat T cells was also found to require stimulation of the TCR/CD3 complex, consistent with the notion that 4-1BB functions as a co-stimulatory molecule for T cell activation.     

Heparin is an adhesive ligand for the leukocyte integrin Mac-1 (CD11b/CD1)

Previous studies have demonstrated that the leukocyte integrin Mac-1 adheres to several cell surface and soluble ligands including intercellular adhesion molecule-1, fibrinogen, iC3b, and factor X. However, experiments with Mac-1-expressing transfectants, purified Mac- 1, and mAbs to Mac-1 indicate the existence of additional ligands. In this paper, we demonstrate a direct interaction between Mac-1 and heparan sulfate glycans. Heparin affinity resins immunoprecipitate Mac- 1, and neutrophils and transfectant cells that express Mac-1 bind to heparin and heparan sulfate, but not to other sulfated glycosaminoglycans. Inhibition studies with mAbs and chemically modified forms of heparin suggest the I domain as a recognition site on Mac-1 for heparin, and suggest that either N- or O-sulfation is sufficient for heparin to bind efficiently to Mac-1. Under conditions of continuous flow in which heparins and E-selectin are cosubstrates, neutrophils tether to E-selectin and form firm adhesions through a Mac- 1-heparin interaction.     

4-1BB (CD137), an inducible costimulatory receptor,as a specific target for cancer therapy

Although considerable progress has been made in understanding how tumors evade immune surveillance, measures to counter the same have not kept pace with the advances made in designing effective strategies. 4-1BB (CD137; TNFRS9), an activation-induced costimulatory molecule, is an important regulator of immune responses. Targeting 4-1BB or its natural ligand 4-1BB ligand (4-1BBL) has important implications in many clinical conditions, including cancer. In-depth analysis revealed that 4-1BB-mediated anti-cancer effects are based on its ability to induce activation of cytotoxic T lymphocytes (CTL), and among others, high amounts of IFN-γ. In this review, we will discuss the various aspects of 4-1BB-mediated anti-tumor responses, the basis of such responses, and future directions. [BMB Reports 2014; 47(3): 122-129]     

A signal through 4-1BB ligand inhibits receptor for activation of nuclear factor-kappaB ligand (RANKL)-induced osteoclastogenesis by increasing interferon (IFN)-beta production

We tested whether any intracellular signals are transmitted through 4-1BB/CD137 ligand (4-1BBL), using a 4-1BB-Fc fusion protein and 4-1BB-deficient mice. Immobilized 4-1BB-Fc fusion protein strongly inhibited osteoclastogenesis induced by macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-kappaB ligand (RANKL) derived from bone marrow macrophages (BMM). Incubation of BMM with M-CSF increased 4-1BBL mRNA and surface expression of 4-1BBL protein. Cross-linking 4-1BBL with immobilized 4-1BB-Fc also dramatically reduced the number of tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells (MNC) derived from the BMM from 4-1BB-deficient mice, suggesting that the inhibitory effect of immobilized 4-1BB on osteoclastogenesis is due to a signal through 4-1BBL. Reverse signaling by 4-1BB-Fc increased the level of interferon (IFN)-beta in BMM and neutralization of IFN-beta reversed the inhibitory effect of immobilized 4-1BB-Fc. Inhibition of osteoclastogenesis by immobilized 4-1BB-Fc is, therefore, at least in part, due to elevation of the level of the negative regulator, IFN-beta in BMM.     

4-1BB ligand induces cell division, sustains survival, and enhances effector function of CD4 and CD8 T cells with similar efficacy

A costimulatory member of the TNFR family, 4-1BB, is expressed on activated T cells. Although some reports have suggested that 4-1BB is primarily involved in CD8 T cell activation, in this report we demonstrate that both CD4 and CD8 T cells respond to 4-1BB ligand (4-1BBL) with similar efficacy. CD4 and CD8 TCR transgenic T cells up-regulate 4-1BB, OX40, and CD27 and respond to 4-1BBL-mediated costimulation during a primary response to peptide Ag. 4-1BBL enhanced proliferation, cytokine production, and CTL effector function of TCR transgenic T cells. To compare CD4 vs CD8 responses to 4-1BBL under similar conditions of antigenic stimulation, we performed MLRs with purified CD4 or CD8 responders from CD28(+/+) and CD28(-/-) mice. We found that CD8 T cells produced IL-2 and IFN-gamma in a 4-1BBL-dependent manner, whereas under the same conditions the CD4 T cells produced IL-2 and IL-4. 4-1BBL promoted survival of CD4 and CD8 T cells, particularly at late stages of the MLR. CD4 and CD8 T cells both responded to anti-CD3 plus s4-1BBL with a similar cytokine profile as observed in the MLR. CD4 and CD8 T cells exhibited enhanced proliferation and earlier cell division when stimulated with anti-CD3 plus anti-CD28 compared with anti-CD3 plus 4-1BBL, and both subsets responded comparably to anti-CD3 plus 4-1BBL. These data support the idea that CD28 plays a primary role in initial T cell expansion, whereas 4-1BB/4-1BBL sustains both CD4 and CD8 T cell responses, as well as enhances cell division and T cell effector function.     

Temporal segregation of 4-1BB versus CD28-mediated costimulation: 4-1BB ligand influences T cell numbers late in the primary response and regulates the size of the T cell memory response following influenza infection

In this report, we demonstrate that CD28(-/-) mice are severely impaired in the initial expansion of D(b)/NP366-374-specific CD8 T cells in response to influenza virus infection, whereas 4-1BB ligand (4-1BBL)(-/-) mice show no defect in primary T cell expansion to influenza virus. In contrast, 4-1BBL(-/-) mice show a decrease in D(b)/NP366-374-specific T cells late in the primary response. Upon secondary challenge with influenza virus, 4-1BBL(-/-) mice show a decrease in the number of D(b)/NP366-374-specific T cells compared to wild-type mice such that the level of the CD8 T cell expansion during the in vivo secondary response is reduced to the level of a primary response, with concomitant reduction of CTL effector function. In contrast, Ab responses, as well as secondary CD4 T cell responses, to influenza are unaffected by 4-1BBL deficiency. Thus, CD28 is critical for initial T cell expansion, whereas 4-1BB/4-1BBL signaling affects T cell numbers much later in the response and is essential for the survival and/or responsiveness of the memory CD8 T cell pool.     

Increasing the expression of programmed death ligand 2 (PD-L2) but not 4-1BB ligand in colorectal cancer cells

Molecular Biology Reports - Immune checkpoint (ICP) molecules modulate the immune response by either inducing or preventing T cell activation. Over-expression of some ICPs on malignant cells has...     

Enhanced CD4 T cell responsiveness in the absence of 4-1BB

The 4-1BB (CD137) is a member of the TNFR superfamily, and is expressed on several cell types, including activated T cells. Although 4-1BB ligation by agonistic Ab or 4-1BB ligand-expressing APCs can costimulate T cells, the physiological significance of 4-1BB expression in vivo during T cell responses is still being elucidated. In this study, we have addressed the impact on CD4 T cell priming when 4-1BB is absent after gene targeting. Surprisingly, 4-1BB(-/-) mice generated more enhanced effector CD4 T cell responses to OVA protein in adjuvant, even though Ab responses in 4-1BB(-/-) mice were normal. Using an adoptive transfer system with OT-II TCR transgenic CD4 T cells, we found that 4-1BB(-/-) CD4 cells responding in a 4-1BB-sufficient environment had enhanced cell division compared with wild-type cells and displayed augmented clonal expansion during the primary response. This was not due to a developmental defect as 4-1BB-deficient CD4 cells could respond normally to Ag in vitro. These results demonstrate that the absence of 4-1BB can make CD4 T cells hyperresponsive to protein Ag in vivo, suggesting a new unappreciated negative regulatory role of 4-1BB when expressed on a T cell.     

4-1BB signaling synergizes with programmed death ligand 1 blockade to augment CD8 T cell responses during chronic viral infection

Previous studies have identified the inhibitory role that the programmed death 1 (PD-1) pathway plays during chronic infection. Blockade of this pathway results in rescue of viral-specific CD8 T cells, as well as reduction of viral loads in mice chronically infected with lymphocytic choriomeningitis virus (LCMV). We tested the effect of combining PD ligand 1 (PD-L1) blockade with an agonistic regimen that induces 4-1BB costimulation during chronic LCMV infection. There is a boosting effect in the rescue of LCMV-specific CD8 T cell responses after dual treatment with PD-L1 blockade and 4-1BB agonistic Abs when the amount and timing of 4-1BB costimulation are carefully controlled. When PD-L1-blocking Abs are given together with a single low dose of anti-4-1BB agonistic Abs, there is an enhanced and stable expansion of viral-specific CD8 T cells. Conversely, when blocking Abs to PD-L1 are given with a repetitive high dose of anti-4-1BB, there is an initial synergistic expansion of viral-specific CD8 T cells by day 7, followed by dramatic apoptosis by day 14. Viral control paralleled CD8 T cell kinetics after dual treatment. By day 7 posttreatment, viral titers were lower in both of the combined regimens (compared with PD-L1 blockade alone). However, whereas the high dose of anti-4-1BB plus PD-L1 blockade resulted in rebound of viral titers to original levels, the low dose of anti-4-1BB plus PD-L1 blockade resulted in a stable reduction of viral loads. These findings demonstrate the importance of carefully manipulating the balance between activating and inhibitory signals to enhance T cell responses during chronic infection.     

Analysis of 4-1BB ligand (4-1BBL)-deficient mice and of mice lacking both 4-1BBL and CD28 reveals a role for 4-1BBL in skin allograft rejection and in the cytotoxic T cell response to influenza virus.

4-1BB ligand (4-1BBL) is a member of the TNF family expressed on activated APC. 4-1BBL binds to 4-1BB (CD137) on activated CD4 and CD8 T cells and in conjunction with strong signals through the TCR provides a CD28-independent costimulatory signal leading to high level IL-2 production by primary resting T cells. Here we report the immunological characterization of mice lacking 4-1BBL and of mice lacking both 4-1BBL and CD28. 4-1BBL-/- mice mount neutralizing IgM and IgG responses to vesicular stomatitis virus that are indistinguishable from those of wild-type mice. 4-1BBL-/- mice show unimpaired CTL responses to lymphocytic choriomeningitis virus (LCMV) and exhibit normal skin allograft rejection but have a weaker CTL response to influenza virus than wild-type mice. 4-1BBL-/-CD28-/- mice retain the CTL response to LCMV, respond poorly to influenza virus, and exhibit a delay in skin allograft rejection. In agreement with these in vivo results, allogeneic CTL responses of CD28-/- but not CD28+/+ T cells to 4-1BBL-expressing APC are substantially inhibited by soluble 4-1BB receptor as is the in vitro secondary response of CD28+ T cells to influenza virus peptides. TCR-transgenic CD28-/- LCMV glycoprotein-specific T cells are insensitive to the presence of 4-1BBL when a wild-type peptide is used, but the response to a weak agonist peptide is greatly augmented by the presence of 4-1BBL. These results further substantiate the idea that different immune responses vary in their dependence on costimulation and suggest a role for 4-1BBL in augmenting suboptimal CTL responses in vivo.     

CD8+ T cell dysfunction and increase in murine gammaherpesvirus latent viral burden in the absence of 4-1BB ligand

Studies of costimulatory receptors belonging to the TNFR family have revealed their diverse roles in affecting different stages of the T cell response. The 4-1BB ligand (4-1BBL)/4-1BB pathway has emerged as a receptor-ligand pair that impacts not the initial priming, but later phases of the T cell response, such as sustaining clonal expansion and survival, maintaining memory CD8(+) T cells, and supporting secondary expansion upon Ag challenge. Although the role of this costimulatory pathway in CD8(+) T cell responses to acute viral infections has been well-studied, its role in controlling chronic viral infections in vivo is not known to date. Using the murine gammaherpesvirus-68 (MHV-68) model, we show that 4-1BBL-deficient mice lack control of MHV-68 during latency and show significantly increased latent viral loads. In contrast to acute influenza infection, the numbers of MHV-68-specific memory CD8(+) T cells were maintained during latency. However, the virus-specific CD8(+) T cells showed defects in function, including decreased cytolytic function and impaired secondary expansion. Thus, 4-1BBL deficiency significantly affects the function, but not the number, of virus-specific CD8(+) T cells during gammaherpesvirus latency, and its absence results in an increased viral burden. Our study suggests that the 4-1BB costimulatory pathway plays an important role in controlling chronic viral infections.     

Age-associated thymic atrophy is not associated with a deficiency in the CD44(+)CD25(-)CD3(-)CD4(-)CD8(-) thymocyte population.

Age-associated thymic atrophy has been proposed to be due to changes in both the thymic microenvironment and in the intrinsic properties of the early T cell progenitors, the CD44(+)CD25(-)CD3(-)CD4(-)CD8(-) cells. We have purified these cells from the thymus of both old and young mice and demonstrate no age-associated defect in their ability to differentiate into their progeny in vitro when used to reconstitute fetal thymic organ cultures. We also demonstrate that in the presence of anti-IL-7, CD44(+)CD25(-)CD3(-)CD4(-)CD8(-) cells from young mice show reduced thymocyte development in fetal thymic organ cultures compared with controls. Finally we have shown that old mice treated with IL-7 show improved thymopoiesis compared with control groups. The increased thymopoiesis seen in the old animals occurs in the sequential manner which would be anticipated for an agent working directly on the early stages, including the CD44(+)CD25(-)CD3(-)CD4(-)CD8(-) cells.     

The 1A4 molecule (CD27) is involved in T cell activation.

We developed a new mAb, anti-1A4, which recognizes an epitope on the CD27 molecule distinct from those recognized by several known anti-CD27 mAb. Although it has been suggested that the CD27 molecule is a T cell activation Ag, there was little direct evidence that the structure was involved in the T cell activation process. In this study, we showed that anti-1A4 inhibited anti-CD2, anti-CD3, mitogens, or soluble Ag-induced T cell proliferation as well as PWM-driven B cell IgG synthesis. Interestingly, anti-1A4 inhibited IL-2 secretion without affecting IL-2R expression. In addition, pretreatment of T cells with anti-1A4 inhibited the normally sustained intracellular calcium mobilization seen after triggering of T cells via the CD2 or CD3 pathways. Thus, binding of anti-1A4 to the CD27 molecule appears to induce a negative effect on T cell activation. This may be due to either a direct signal to T cells or the blocking of an interaction between T cells and accessory cells or both. These findings support the notion that the CD27 molecule plays an integral role in the process of T cell activation.     

4-1BB ligand, a member of the TNF family, is important for the generation of antiviral CD8 T cell responses.

4-1BB (CD137) is a costimulatory molecule expressed on activated T cells and interacts with 4-1BB ligand (4-1BBL) on APCs. To investigate the role of 4-1BB costimulation for the development of primary immune responses, 4-1BBL-deficient (4-1BBL-/-) mice were infected with lymphocytic choriomeningitis virus (LCMV). 4-1BBL-/- mice were able to generate CTL and eliminate acute LCMV infection with normal kinetics, but CD8 T cell expansion was 2- to 3-fold lower than in wild-type (+/+) mice. In the same mice, virus-specific CD4 Th and B cell responses were minimally affected, indicating that 4-1BB costimulation preferentially affects CD8 T cell responses. This result contrasts with our earlier work with CD40L-deficient (CD40L-/-) mice, in which the CD8 T cell response was unaffected and the CD4 T cell response was markedly impaired. When both 4-1BBL- and B7-dependent signals were absent, CD8 T cell expansion was further reduced, resulting in lower CTL activity and impairing their ability to clear LCMV. Altogether, these results indicate that T cells have distinct costimulatory requirements: optimal CD8 responses require 4-1BBL-dependent interactions, whereas CD4 responses are minimally affected by 4-1BB costimulation, but require CD40-CD40L and B7-dependent interactions.     

Ligation of 4-1BB (CDw137) regulates graft-versus-host disease, graft-versus-leukemia, and graft rejection in allogeneic bone marrow transplant recipients

4-1BB is expressed on activated CD4(+) and CD8(+) T cells; its ligand, 4-1BB ligand is expressed on APCs. Despite expression on both T cell subpopulations, 4-1BB has been reported to predominantly affect CD8(+) T cell responses. By quantifying graft-vs-host disease alloresponses in vivo, we demonstrate that both CD4(+) and CD8(+) T cell-mediated alloresponses are regulated by 4-1BB/4-1BB ligand interactions to approximately the same extent. 4-1BB receptor-facilitated CD4(+) T cell-mediated alloresponses were partly CD28 independent. In two distinct marrow graft rejection systems, host CD8(+) and CD4(+) T cells each separately contributed to host anti-donor T cell-mediated allograft rejection. alpha 4-1BB mAb increased the graft-vs-leukemia effect of a suboptimal number of donor splenocytes given later post bone marrow transplantation by bolstering allogeneic responses resulting in leukemia elimination. In summary, 4-1BB ligation is a potent regulator of CD4(+) and CD8(+) T cell-mediated allogeneic responses in vivo. Modifying the ligation of 4-1BB represents a new approach to altering the graft-vs-host disease and graft-vs-leukemia effects of allogeneic T cells post bone marrow transplantation.