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 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.     

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.     

4-1BB costimulation promotes human T cell adhesion to fibronectin

CD28 and 4-1BB (CD137) are costimulatory molecules for T cells. In this study we investigated the role of 4-1BB in T cell adhesion to fibronectin (FN). Unlike CD28, 4-1BB is present in only a small subset of T cells prepared from fresh human peripheral blood mononuclear cells, but was induced after prolonged TCR/CD28 activation in vitro. 4-1BB-expressing T cells were characteristically unique in their strong responsiveness to FN. Anti-4-1BB cross-linking synergized CD28 costimulation by lowering the threshold of CD3 signal required for CD28-mediated maximal proliferative response. In addition to increasing proliferative responses, 4-1BB promoted T cell adhesion to FN in the presence of CD28 costimulation. 4-1BB-mediated cell adhesion to FN was blocked by anti-beta1 integrin, suggesting that 4-1BB mediates beta1 integrin activation. The role of 4-1BB in inducing CD4(+) T cell adhesion to FN was confirmed by showing that the human leukemic CD4(+) T cell line, Jurkat, when transfected with cDNA encoding 4-1BB, became adherent to FN with anti-4-1BB stimulation. Taken together, our results suggest that 4-1BB-promoted T cell adhesion to extracellular matrix proteins is an important postactivation process for T cell migration.     

Cutting edge: a crucial role for B7-CD28 in transmitting T help from APC to CTL

Although APC activation via CD40-CD40L signaling plays a critical role in enabling CD4(+) T cells to provide the "help" necessary for cross-priming of naive CTL, it is unclear how this makes the APC competent for priming. We have investigated the roles of B7-1/B7-2 and their receptors [corrected] CD28/CTLA-4 in cross-priming of CD4-dependent CTL in vivo. We find that both CD28 and B7-1/B7-2 are required for CD40-activated APC to cross-prime CTL, and that priming by CD40-activated APC was prevented by blockade of CD28. Conversely, augmenting CD28 signals with an agonistic Ab bypassed the requirement for CD4(+) T help or CD40 activation. Interestingly, blockade of the negative regulatory B7 receptor CTLA-4 failed to prime CTL in the absence of T help. These results support a model in which activation-induced up-regulation of B7 molecules on APC leads to increased CD28 signaling and a commitment to cross-priming of CD4-dependent CTL.     

The role of intercellular adhesion molecule-1/LFA-1 interactions in the generation of tumor-specific CD8+ T cell responses

The activation of naive CD4+ T cells requires both TCR engagement and a second costimulatory signal mediated by the interaction of CD28 with CD80/CD86 expressed on professional APC. However, the situation for naive CD8+ T cells is less clear. Although evidence indicates that induction of CD8+ T cell responses is also dependent on professional APC, the ability of some tumors, which do not express CD80/CD86, to induce CTL suggests that other pathways of costimulation exist for the activation of CD8+ T cells. We examined the ability of tumor cells expressing different levels of a tumor-specific Ag to directly prime CD8+ T cells. We demonstrate that CD8+ T cells are directly activated by tumor cells in a CD80/CD86-CD28 independent manner. In this system, costimulation requires ICAM-1/LFA-1 interaction. This results in the generation of CTL capable of inhibiting tumor growth in vivo, and maintaining long-term survival.     

Engagement of 4-1BB inhibits the development of experimental allergic conjunctivitis in mice

The 4-1BB receptor acts as a costimulator in CD8(+) T cell activation. Agonistic stimulation through this molecule by treatment with anti-4-1BB Abs has been demonstrated to inhibit various experimentally induced diseases in animals. However, the effect of anti-4-1BB Abs on experimental allergic diseases has not been reported. We investigated the effect of anti-4-1BB Abs on the development and progression of experimental allergic conjunctivitis in mice. To examine the effects of Abs during the induction or effector phase, actively immunized mice or passively immunized mice by splenocyte transfer were treated with agonistic anti-4-1BB Abs, blocking anti-4-1BB ligand Abs, or normal rat IgG. Eosinophil infiltration into the conjunctiva was significantly reduced in wild-type mice by the anti-4-1BB Ab treatment during either induction or effector phase. Th2 cytokine production by splenocytes and total serum IgE were significantly reduced by the anti-4-1BB Ab treatment, while IFN-gamma production was increased. The anti-4-1BB Ab treatment induced a relative increase of CD8-positive cell numbers in the spleens. Moreover, inhibition of eosinophil infiltration by the treatment with anti-4-1BB Abs was also noted in actively immunized IFN-gamma knockout mice. Taken altogether, in vivo treatment with agonistic anti-4-1BB Abs in either induction or effector phase inhibits the development of experimental allergic conjunctivitis, and this inhibition is likely to be mediated by suppression of Th2 immune responses rather than up-regulation of IFN-gamma.     

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.     

Co-Stimulation through 4-1BB/CD137 Improves the Expansion and Function of CD8+ Melanoma Tumor-Infiltrating Lymphocytes for Adoptive T-Cell Therapy

Adoptive T-cell therapy (ACT) using tumor-infiltrating lymphocytes (TIL) can induce tumor regression in up to 50% or more of patients with unresectable metastatic melanoma. However, current methods to expand melanoma TIL, especially the “rapid expansion protocol” (REP) were not designed to enhance the generation of optimal effector-memory CD8⁺ T cells for infusion. One approach to this problem is to manipulate specific co-stimulatory signaling pathways to enhance CD8⁺ effector-memory T-cell expansion. In this study, we determined the effects of activating the TNF-R family member 4-1BB/CD137, specifically induced in activated CD8⁺ T cells, on the yield, phenotype, and functional activity of expanded CD8⁺ T cells during the REP. We found that CD8⁺ TIL up-regulate 4-1BB expression early during the REP after initial TCR stimulation, but neither the PBMC feeder cells in the REP or the activated TIL expressed 4-1BB ligand. However, addition of an exogenous agonistic anti-4-1BB IgG₄ (BMS 663513) to the REP significantly enhanced the frequency and total yield of CD8⁺ T cells as well as their maintenance of CD28 and increased their anti-tumor CTL activity. Gene expression analysis found an increase in bcl-2 and survivin expression induced by 4-1BB that was associated with an enhanced survival capability of CD8⁺ post-REP TIL when re-cultured in the absence or presence of cytokines. Our findings suggest that adding an agonistic anti-4-1BB antibody during the time of TIL REP initiation produces a CD8⁺ T cell population capable of improved effector function and survival. This may greatly improve TIL persistence and anti-tumor activity in vivo after adoptive transfer into patients.     

4-1BB is superior to CD28 costimulation for generating CD8+ cytotoxic lymphocytes for adoptive immunotherapy

Artificial APCs (aAPCs) genetically modified to express selective costimulatory molecules provide a reproducible, cost-effective, and convenient method for polyclonal and Ag-specific expansion of human T cells for adoptive immunotherapy. Among the variety of aAPCs that have been studied, acellular beads expressing anti-CD3/anti-CD28 efficiently expand CD4+ cells, but not CD8+ T cells. Cell-based aAPCs can effectively expand cytolytic CD8+ cells, but optimal costimulatory signals have not been defined. 4-1BB, a costimulatory molecule expressed by a minority of resting CD8+ T cells, is transiently up-regulated by all CD8+ T cells following activation. We compared expansion of human cytolytic CD8+ T cells using cell-based aAPCs providing costimulation via 4-1BB vs CD28. Whereas anti-CD3/anti-CD28 aAPCs mostly expand naive cells, anti-CD3/4-1BBL aAPCs preferentially expand memory cells, resulting in superior enrichment of Ag-reactive T cells which recognize previously primed Ags and efficient expansion of electronically sorted CD8+ populations reactive toward viral or self-Ags. Using HLA-A2-Fc fusion proteins linked to 4-1BBL aAPCs, 3-log expansion of Ag-specific CD8+ CTL was induced over 14 days, whereas similar Ag-specific CD8+ T cell expansion did not occur using HLA-A2-Fc/anti-CD28 aAPCs. Furthermore, when compared with cytolytic T cells expanded using CD28 costimulation, CTL expanded using 4-1BB costimulation mediate enhanced cytolytic capacity due, in part, to NKG2D up-regulation. These results demonstrate that 4-1BB costimulation is essential for expanding memory CD8+ T cells ex vivo and is superior to CD28 costimulation for generating Ag-specific products for adoptive cell therapy.     

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.     

Administration of agonistic anti-4-1BB monoclonal antibody leads to the amelioration of experimental autoimmune encephalomyelitis.

4-1BB, a member of the TNFR superfamily, is a costimulatory receptor primarily expressed on activated T cells. It has been shown that the administration of agonistic anti-4-1BB Abs enhances tumor immunity and allogenic immune responses. Paradoxically, we found that the administration of an agonistic anti-4-1BB mAb (2A) dramatically reduced the incidence and severity of experimental autoimmune encephalomyelitis (EAE). Adoptive transfer of T cells from such treated mice failed to induce EAE, whereas anti-4-1BB treatment following adoptive transfer of encephalitogenic T cells did not prevent EAE pathogenesis. These results suggest that anti-4-1BB treatment during the induction phase inhibits autoreactive T cell immune responses rather than preventing T cell trafficking into the CNS. This was substantiated by the observations that draining lymph node cells from anti-4-1BB-treated mice failed to respond to Ag stimulation in vitro. In addition, we found that such treatment initially promotes the activation and proliferation of Ag-specific CD4+ T cells but subsequently increases their probability of undergoing activation-induced cell death, thereby inhibiting effector T cell responses. More importantly, 2A treatment also inhibits the relapse of EAE in a clinically relevant murine model of multiple sclerosis. This study indicates that the agonistic Ab against 4-1BB can potentially be used as a novel immunotherapeutic agent for treating autoimmune diseases.     

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.     

Immunity to melanoma mediated by 4-1BB is associated with enhanced activity of tumour-infiltrating lymphocytes

4-1BB costimulates T cells to carry out effector functions such as eradication of established tumours. 4-1BB (CD137) is a member of the TNF receptor family, and its triggering by either 4-1BB ligand or antibody ligation induces T-cell activation and growth. We analysed tumour-infiltrating lymphocytes (TIL) in the experimental B16F10 melanoma model to determine the mechanisms involved in 4-1BB-mediated tumour suppression. 4-1BB(+/+) mice survived longer than 4-1BB(-/-) mice, and survival was further prolonged by triggering 4-1BB with an agonistic mAb. The number of metastatic B16F10 colonies in the lung was much greater in 4-1BB(-/-) mice than in their 4-1BB(+/+) littermates. Administration of agonistic anti-4-1BB mAb increased the number of TIL in the tumour masses in the lungs of 4-1BB(+/+) mice. The numbers of CD4(+) T, CD8(+) T and CD11b(+) TIL increased in these mice. Anti-4-1BB mAb induced not only CD8(+) 4-1BB(+) T cells but also a CD8(+) IFN-gamma(+) T-cell population. B16F10 cells from the lungs of anti-4-1BB-treated mice showed enhanced expression of MHC class Iota and IotaIota antigens compared with the same cells from control IgG-treated mice. Thus, the increase in number of CD8(+) T cells and enhanced MHC Iota and IotaIota expression in B16F10 cells that result from augmented IFN-gamma production in response to anti-4-1BB mAb may lead to suppression of tumour growth and metastasis.     

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.     

4-1BB enhances proliferation of beryllium-specific T cells in the lung of subjects with chronic beryllium disease

In contrast to naive T cells, reactivation of memory cells is less dependent on CD28-mediated costimulation. We have shown that circulating beryllium-specific CD4(+) T cells from chronic beryllium disease patients remain CD28-dependent, while those present in the lung no longer require CD28 for T cell activation. In the present study, we analyzed whether other costimulatory molecules are essential for beryllium-induced T cell function in the lung. Enhanced proliferation of a beryllium-responsive, HLA-DP2-restricted T cell line was seen after the induction of 4-1BB ligand expression on the surface of HLA-DP2-expressing fibroblasts. Following beryllium exposure, CD4(+) T cells from blood and bronchoalveolar lavage of chronic beryllium disease patients up-regulate 4-1BB expression, and the majority of beryllium-responsive, IFN-gamma-producing CD4(+) T cells in blood coexpress CD28 and 4-1BB. Conversely, a significant fraction of IFN-gamma-producing bronchoalveolar lavage (BAL) T cells express 4-1BB in the absence of CD28. In contrast to blood, inhibition of the 4-1BB ligand-4-1BB interaction partially blocked beryllium-induced proliferation of BAL CD4(+) T cells, and a lack of 4-1BB expression on BAL T cells was associated with increased beryllium-induced cell death. Taken together, these findings suggest an important role of 4-1BB in the costimulation of beryllium-responsive CD4(+) T cells in the target organ.     

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.     

Fas ligand costimulates the in vivo proliferation of CD8+ T cells

Fas ligand (FasL/CD95L/APO-1L) is one of a growing number of TNF family members whose triggering costimulates maximal proliferation of activated T cells. In this study we show that maximal Ag-dependent accumulation of transferred TCR-transgenic CD8(+) T cells requires Fas (CD95/APO-1) expression by the adoptive hosts. Additionally, adoptively transferred FasL(+) CD8(+) T cells demonstrate a 2-fold advantage in Ag-driven expansion over their FasL(-)counterparts. This study illustrates the in vivo role of TCR-dependent FasL costimulation in the Ag-specific proliferation of both heterogeneous and homogeneous populations of primary CD8(+) T cells and long-term CTL lines. Thus, cross-linking FasL on naive and Ag-experienced CD8(+) T cells whose Ag-specific TCRs are engaged is required to drive maximal cellular proliferation in vivo.     

Activation of dendritic cells that cross-present tumor-derived antigen licenses CD8+ CTL to cause tumor eradication

The fate of naive CD8(+) T cells is determined by the environment in which they encounter MHC class I presented peptide Ags. The manner in which tumor Ags are presented is a longstanding matter of debate. Ag presentation might be mediated by tumor cells in tumor draining lymph nodes or via cross-presentation by professional APC. Either pathway is insufficient to elicit protective antitumor immunity. We now demonstrate using a syngeneic mouse tumor model, expressing an Ag derived from the early region 1A of human adenovirus type 5, that the inadequate nature of the antitumor CTL response is not due to direct Ag presentation by the tumor cells, but results from presentation of tumor-derived Ag by nonactivated CD11c(+) APC. Although this event results in division of naive CTL in tumor draining lymph nodes, it does not establish a productive immune response. Treatment of tumor-bearing mice with dendritic cell-stimulating agonistic anti-CD40 mAb resulted in systemic efflux of CTL with robust effector function capable to eradicate established tumors. For efficacy of anti-CD40 treatment, CD40 ligation of host APC is required because adoptive transfer of CD40-proficient tumor-specific TCR transgenic CTL into CD40-deficient tumor-bearing mice did not lead to productive antitumor immunity after CD40 triggering in vivo. CpG and detoxified LPS (MPL) acted similarly as agonistic anti-CD40 mAb with respect to CD8(+) CTL efflux and tumor eradication. Together these results indicate that dendritic cells, depending on their activation state, orchestrate the outcome of CTL-mediated immunity against tumors, leading either to an ineffective immune response or potent antitumor immunity.     

Cutting edge: 4-1BB is a bona fide CD8 T cell survival signal

After recognition of Ag/MHC and ligation of a costimulatory molecule, resting T cells will clonally expand and then delete to very low levels. Previously, it was shown that deletion can be prevented by coinjection of cytokines or proinflammatory agents such as adjuvants. Here, we demonstrate that ligation of 4-1BB blocks deletion of superantigen-activated T cells in the absence of adjuvant or additional cytokine treatment. Nearly 10 times as many staphylococcal enterotoxin A-specific T cells were detected in the spleens of mice injected 21 days previously with staphylococcal enterotoxin A and an agonist anti-4-1BB Ab compared with mice given staphylococcal enterotoxin A and a control IgG. Even though both CD4- and CD8-activated T cells expressed 4-1BB, a higher proportion of CD8 T cells were rescued compared CD4 T cells. These data suggest that although 4-1BB provides costimulation, it may also promote long-term T cell survival.