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.     

4-1BB costimulation is required for protective anti-viral immunity after peptide vaccination

Peptide vaccination induces T cell activation and cytotoxic T cell development. In an effort to understand what factors can improve immune responses to peptide vaccination, the role of 4-1BB (CD137) costimulation was examined, since 4-1BB has been shown to promote T cell responses in other systems. 4-1BBL-deficient (-/-) and wild-type (+/+) mice were immunized with a lipidated lymphocytic choriomeningitis virus (LCMV) peptide NP396-404. Analysis of peptide-specific responses early after immunization by CTL assay, intracellular IFN-gamma staining, and IFN-gamma enzyme-linked immunospot assay (ELISPOT) indicated that CD8 T cell responses were reduced 3- to 10-fold in the absence of 4-1BB costimulation. Moreover, when agonistic anti-4-1BB Ab was given, CD8 T cell responses in 4-1BBL-/- mice were augmented to levels similar to those in 4-1BBL+/+ mice. Two months after immunization, 4-1BBL+/+ mice still had epitope-specific cells and were protected against viral challenge, demonstrating that peptide vaccination can induce long-term protection. In fact, 70% of CD8 T cells were specific for the immunizing peptide after viral challenge, demonstrating that strong, epitope-specific CD8 T cell responses are generated after peptide vaccination. In contrast, peptide-immunized 4-1BBL-/- mice had fewer epitope-specific cells and were impaired in their ability to resolve the infection. These results show that immunization with a single LCMV peptide provides long term protection against LCMV infection and point to costimulatory molecules such as 4-1BB as important components for generating protective immunity after vaccination.     

IL-15-dependent induction of 4-1BB promotes antigen-independent CD8 memory T cell survival

Mice lacking CD137L (4-1BBL) show normal primary expansion and contraction of the CD8+ T cell response to influenza virus, but exhibit a defect in Ag-specific CD8+ T cell numbers at 3-6 wk postinfection. Previous results showed that the decrease in CD8+ T cell numbers in this model is not due to a programming defect during primary expansion. Thus, it appears that 4-1BB/4-1BBL interactions control the number of surviving CD8+ effector memory cells, late in the primary response. In this report, we asked how 4-1BB on T cells could play a role after Ag has apparently been cleared from the host. We show that IL-15, a cytokine involved in regulation of CD8+ memory T cell survival, induces the expression of 4-1BB on CD8+CD44(high) memory phenotype T cells, but not on CD4+ T cells. The Ag-independent induction of 4-1BB by IL-15 was dependent on MAPK p38 and ERK activation. Transfer of in vitro-generated OT-I CD8+ memory T cells into unimmunized wild-type or 4-1BBL-deficient hosts revealed a 2- to 3-fold survival advantage when 4-1BBL was present, recapitulating the effect seen in the endogenous response to influenza in mice. Decreases in the overall number of memory CD8+ T cells were also observed in the bone marrow of unmanipulated 4-1BBL-deficient mice. These data suggest a model whereby 4-1BB expression on memory CD8+ T cells, perhaps due to encounter with IL-15 in the bone marrow, allows 4-1BB/4-1BBL interactions to maintain memory CD8 T cell survival in the absence of Ag.     

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.     

4-1BB and OX40 act independently to facilitate robust CD8 and CD4 recall responses

Mice deficient in OX40 or 4-1BB costimulatory pathways show defects in T cell recall responses, with predominant effects on CD4 vs CD8 T cells, respectively. However, OX40L can also stimulate CD8 T cells and 4-1BBL can influence CD4 T cells, raising the possibility of redundancy between the two TNFR family costimulators. To test this possibility, we generated mice deficient in both 4-1BBL and OX40L. In an adoptive transfer model, CD4 T cells expressed 4-1BB and OX40 sequentially in response to immunization, with little or no overlap in the timing of their expression. Under the same conditions, CD8 T cells expressed 4-1BB, but no detectable OX40. Thus, in vivo expression of 4-1BB and OX40 can be temporally and spatially segregated. In the absence of OX40L, there were decreased CD4 T cells late in the primary response and no detectable secondary expansion of adoptively transferred CD4 T cells under conditions in which primary expansion was unaffected. The 4-1BBL had a minor effect on the primary response of CD4 T cells in this model, but showed larger effects on the secondary response, although 4-1BBL(-/-) mice show less impairment in CD4 secondary responses than OX40L(-/-) mice. The 4-1BBL(-/-) and double knockout mice were similarly impaired in the CD8 T cell response, whereas OX40L(-/-) and double knockout mice were similarly impaired in the CD4 T cell response to both protein Ag and influenza virus. Thus, 4-1BB and OX40 act independently and nonredundantly to facilitate robust CD4 and CD8 recall responses.     

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.     

A switch in costimulation from CD28 to 4-1BB during primary versus secondary CD8 T cell response to influenza in vivo

4-1BBL(-/-) mice exhibit normal primary CD8 T cell responses to influenza virus, but show decreased CD8 T cell numbers late in the primary response as well as decreased secondary responses. In contrast, CD28(-/-) mice are defective in initial CD8 T cell expansion. Using agonistic anti-4-1BB Ab to replace the CD28 or 4-1BB signal, we examined the timing of the required signals for CD28 vs 4-1BB costimulation. A single dose of agonistic anti-4-1BB Ab added only during priming restores the secondary CD8 T cell response in CD28(-/-) mice. Once the T cell numbers in the primary response reach a minimum threshold, a full secondary response is achieved even in the absence of CD28. In contrast, anti-4-1BB added during priming fails to correct the defective secondary response in 4-1BBL(-/-) mice, whereas addition of anti-4-1BB during challenge fully restores this response. Thus, there is a switch in costimulatory requirement from CD28 to 4-1BB during primary vs recall responses. Adoptive transfer studies show that T cells primed in 4-1BBL(-/-) or wild-type mice are equally capable of re-expansion when rechallenged in wild-type mice. These studies rule out a model in which signals delivered through 4-1BB during priming program the T cells to give a full recall response and suggest that 4-1BB-4-1BBL interactions take place at later stages in the immune response. The results indicate that anti-4-1BB or 4-1BBL therapy will be most effective during the boost phase of a prime-boost vaccination strategy.     

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.     

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.     

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.     

4-1BB ligand-mediated costimulation of human T cells induces CD4 and CD8 T cell expansion, cytokine production, and the development of cytolytic effector function

4-1BB (CD137) is a costimulatory member of the TNFR family expressed on activated T cells. Its ligand, 4-1BBL, is expressed on activated APC. In the mouse, CD8 T cells are preferentially activated by agonistic anti-murine 4-1BB Abs. However, murine 4-1BBL can stimulate both CD4 and CD8 T cells. To date, there are only limited data on the effects of 4-1BBL on human T cell responses. To further understand the role of 4-1BBL in human T cell responses, we compared human CD4 and CD8 T cell responses to transfected human 4-1BBL plus TCR-mediated stimulation. Both human CD4 and CD8 T cells responded to 4-1BBL. The presence of 4-1BBL on the APC led to increased expansion, cytokine production, and the development of cytolytic effector function by human T cells. In unfractionated T cell cultures, CD4 and CD8 T cells could expand to a similar extent in response to signals through the TCR and 4-1BB, as measured by CFSE labeling and by quantitating T cell numbers in the cultures. In contrast to the results with total T cells, isolated CD8 T cells produced less IL-2 and expanded to a lesser extent than isolated CD4 T cells responding to 4-1BBL. Thus, 4-1BBL is most effective when both CD4 and CD8 T cells are included in the cultures. CD28 and 4-1BB were found to synergize in the induction of IL-2 by human T cells, and CTLA-Ig partially blocked 4-1BBL-dependent IL-2 production. However, a portion of the 4-1BBL-mediated effects were independent of CD28-B7 interaction.     

A novel adenovirus expressing human 4-1BB ligand enhances antitumor immunity

4-1BB ligand (4-1BBL), a member of the tumor necrosis factor (TNF) superfamily, interacts with 4-1BB (CDw137) expressed on activated T cells and delivers a costimulatory signal for T cell activation and growth. Various studies have demonstrated a role for murine 4-1BB in immune function, but relatively few investigations of human 4-1BB have been conducted. Here we report on the construction of a recombinant E1/E3-deleted adenovirus encoding human 4-1BBL (Ad4-1BBL) and its stimulation of antitumor immunity. Ad4-1BBL was able to efficiently infect several human adenocarcinoma cell lines and induce 4-1BBL expression on the cell surface within 24 h, this enhancing the antitumor activity not only of lymphokine-activated killer cells with a T cell phenotype (T-LAK) but also naive peripheral blood mononuclear cells (PBMC). This antitumor activity with T-LAK cells was further enhanced by addition of bispecific antibody (BsAb; anti-MUC1xanti-CD3). Cocultivation of Ad4-1BBL-infected tumor cells with either T-LAK cells or PBMC resulted in significant elevation of interferon-gamma (IFN-gamma), interleukin-2 (IL-2), and granulocyte-macrophage colony-stimulating factor (GM-CSF) production. Furthermore, remarkable tumor growth inhibition was observed in cholangiocarcinoma-grafted severe combined immunodeficient (SCID) mice to which Ad4-1BBL and T-LAK cells were administered when tumor size exceeded 5 mm in diameter. These results provide strong evidence in support of the efficacy of adenovirally delivered 4-1BBL for genetic immunotherapy of cancer.     

MCP-1 derived from stromal keratocyte induces corneal infiltration of CD4+ T cells in herpetic stromal keratitis

Herpetic stromal keratitis (HSK) is an inflammatory disorder induced by HSV-1 infection and characterized by T cell-dependent destruction of corneal tissues. It is not known what triggers CD4(+) T cell migration into the stroma of HSV-1-infected corneas. The keratocyte is a fibroblast-like cell that can function as an antigen-presenting cell in the mouse cornea by expressing MHC class II and costimulatory molecules after HSV-1 infection. We hypothesized that chemokines produced by stromal keratocytes are involved in CD4(+) T cell infiltration into the cornea. We found that keratocytes produce several cytokines and chemokines, including MCP-1, RANTES, and T cell activation (TCA)-3. HSV-1 infection increased the production of MCP-1 and RANTES by keratocytes, and these acted as chemoattractants for HSV-1-primed CD4(+) T cells expressing CCR2 and CCR5. Expression of MCP-1 in the corneal stroma was confirmed in vivo. Finally, when HSV-1-primed CD4(+) T cells were adoptively transferred into wild type and MCP-1-deficient mice that had been sublethally irradiated to minimize chemokine production from immune cells, infiltration of CD4(+) T cells was markedly reduced in the MCP-1-deficient mice, suggesting that it is the MCP-1 from HSV-1-infected keratocytes that attracts CD4(+) T cells into the cornea.     

Infection-induced up-regulation of the costimulatory molecule 4-1BB in osteoblastic cells and its inhibitory effect on M-CSF/RANKL-induced in vitro osteoclastogenesis

Bacterial infection sometimes impairs bone metabolism. In this study, we infected the osteoblastic cell line MC3T3-E1 with Mycobacterium bovis bacillus Calmette-Guérin (BCG) and identified genes that were up-regulated in the BCG-infected cells by the suppression subtractive hybridization method. A gene encoding 4-1BB (CD137), a member of the tumor necrosis factor-alpha receptor family, was found to be one of the up-regulated genes. Up-regulation of 4-1BB was also observed by infection with Escherichia coli, Salmonella typhimurium, and Staphylococcus aureus, and by treatment with lipopolysaccharides and heat-killed BCG. Bone marrow cells and the macrophage-like cell lines J774 and RAW264.7 were found to express 4-1BB ligand (4-1BBL). Recombinant 4-1BB (r4-1BB) that was immobilized on culture plates strongly inhibited macrophage colony stimulating factor (M-CSF)/receptor activator of nuclear factor-kappaB ligand (RANKL)-induced in vitro osteoclast formation from bone marrow cells. Anti-4-1BBL antibody also inhibited osteoclast formation to a lesser extent, indicating involvement of reverse signaling through 4-1BBL during inhibition of osteoclast formation. A casein kinase I (CKI) inhibitor markedly suppressed the inhibitory effect of r4-1BB on M-CSF/RANKL-induced osteoclast formation, suggesting that CKI might be involved in 4-1BB/4-1BBL reverse signaling. r4-1BB showed no effects on M-CSF- or RANKL-induced phosphorylation of I-kappaB, ERK1/2, p38, or JNK, whereas RANKL-induced phosphorylation of Akt, a downstream target of phosphatidylinositol 3-kinase (PI3K), was completely abolished by r4-1BB, suggesting that 4-1BB/4-1BBL reverse signaling may interfere with PI3K/Akt pathway. r4-1BB also abolished RANKL-mediated induction of nuclear factor of activated T cells-2. This study may elucidate a novel role of 4-1BB in cell metabolism, especially osteoclastogenesis.     

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.     

Cooperation between 4-1BB and ICOS in the immune response to influenza virus revealed by studies of CD28/ICOS-deficient mice

CD28, ICOS, and 4-1BB each play distinct roles in the CD8 T cell response to influenza virus. CD28-/- mice are severely impaired in primary CD8 T cell expansion and fail to mount a secondary response to influenza. Influenza-specific CD8 T cells expand normally in ICOS-/- mice, with only a small and transient defect late in the primary response and an unimpaired secondary response. Conversely, 4-1BB/4-1BBL interaction is dispensable for the primary CD8 T cell response to influenza, but maintains CD8 T cell survival and controls the size of the secondary response. Previous results showed that a single dose of agonistic anti-4-1BB Ab at priming allowed partial restoration of primary CD8 T cell expansion and full recovery of the secondary CD8 T cell responses to influenza in CD28-/- mice. In this study we show that anti-4-1BB fails to correct the CD8 T cell defect in CD28-/-ICOS-/- mice, suggesting that ICOS partially compensates for CD28 in this model. In support of this hypothesis, we found that anti-4-1BB enhances ICOS expression on both T cell subsets and that anti-4-1BB and anti-ICOS can synergistically activate CD4 and CD8 T cells. Furthermore, ICOS and 4-1BB can cooperate to directly stimulate isolated CD28-/- CD8 T cells. These results reveal a novel interaction between the ICOS and 4-1BB costimulatory pathways as well as unexpected redundancy between CD28 and ICOS in primary CD8 T cell expansion. These findings have implications for costimulation of human T cell responses in diseases such as AIDS or rheumatoid arthritis, in which CD28- T cells accumulate.     

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.     

Ex vivo expansion of CD4+CD25+FoxP3+ T regulatory cells based on synergy between IL-2 and 4-1BB signaling

Naturally occurring CD4(+)CD25(+)FoxP3(+) T regulatory (Treg) cells require three distinct signals transduced via TCR, CD28, and IL-2R for their development and maintenance. These requirements served as the basis for several recently developed ex vivo expansion protocols that relied on the use of solid support-bound Abs to CD3 and CD28 in the presence of high dose IL-2. We report in this study that Treg cells up-regulate the expression of inducible costimulatory receptor 4-1BB in response to IL-2, and stimulation using this receptor via a novel form of 4-1BB ligand (4-1BBL) fused to a modified form of core streptavidin (SA-4-1BBL) was effective in expanding these cells up to 110-fold within 3 wk. Expanded cells up-regulated CD25, 4-1BB, and membranous TGF-beta, suppressed T cell proliferation, and prevented the rejection of allogeneic islets upon adoptive transfer into graft recipients. Importantly, SA-4-1BBL rendered CD4(+)CD25(-) T effector cells refractive to suppression by Treg cells. This dual function of signaling via 4-1BB, vis-à-vis Treg cell expansion and licensing T effector cells resistant to Treg cell suppression, as well as the up-regulation of 4-1BB by IL-2 may serve as important regulatory mechanisms for immune homeostasis following antigenic challenge. Stimulation using a soluble form of SA-4-1BBL represents a novel approach to expand Treg cells with potential therapeutic applications in autoimmunity and transplantation.     

4-1BB-mediated amelioration of experimental autoimmune uveoretinitis is caused by indoleamine 2,3-dioxygenase-dependent mechanisms

Interphotoreceptor retinoid binding protein (IRBP)-induced experimental autoimmune uveoretinitis (EAU) is a CD4+ T cell-mediated autoimmune disease. Development of EAU is inhibited by treatment with an agonistic anti-4-1BB mAb. Even established EAU was alleviated by anti-4-1BB mAb. However, inhibition of 4-1BB/4-1BB ligand (4-1BBL) interaction does not suppress the development of EAU. It appears that cross-linking of 4-1BB evokes an active antigen-specific suppression mechanism rather than merely blocking 4-1BB/4-1BBL interaction. We found that administration of anti-4-1BB mAb induced massive clonal expansion of CD11c+CD8+ T cells that produced IFN-gamma, resulting in accumulation of a high level of indoleamine 2,3-dioxygenase (IDO) in CD11c+ dendritic cells. 4-1BB-mediated suppression of EAU was reversed by the pharmacological IDO inhibitor, 1-methyl-tryptophan (1-MT). These studies demonstrate that suppression of EAU results from antigen-driven, 4-1BB-mediated expansion of novel CD11c+CD8+ T cells that suppress antigen-specific CD4+ T cells via an IDO-dependent mechanism.     

Progressive depletion of peripheral B lymphocytes in 4-1BB (CD137) ligand/I-Ealpha)-transgenic mice

Interaction of 4-1BB (CD137) and its ligand (4-1BBL) is thought to positively regulate cell-mediated and humoral immune responses. We have prepared transgenic mouse strains that express 4-1BBL cDNA under the control of MHC class II I-Ealpha promoter. The 4-1BBL-transgenic mice show progressive splenomegaly and selective depletion of B220(+) B cells accompanied with low levels of circulating IgG and defective humoral responses to Ag challenge. In addition, splenocytes from the transgenic mice fail to provide stimulation for allogeneic T cells in both lymphoproliferative and CTL responses in vitro, whereas their T cells remain functionally normal. Our results reveal unexpected functions of 4-1BBL in the regulation of humoral immune responses and Ag presentation.