Identification of regulatory functions for 4-1BB and 4-1BBL in myelopoiesis and the development of dendritic cells.

The costimulatory molecule 4-1BB and its ligand 4-1BBL can control adaptive immunity, but here we show that their interaction also suppressed myelopoiesis. We found that 4-1BBL was expressed on hematopoietic stem cells, differentiating common myeloid progenitors and granulocyte-macrophage progenitors, and 4-1BB was inducible on activated myeloid progenitors. Steady-state numbers of granulocyte-macrophage progenitors, myeloid-lineage cells and mature dendritic cells were higher in 4-1BB- and 4-1BBL-deficient mice, indicative of a negative function, and we confirmed that result with bone marrow chimeras and in vitro, where the absence of interactions between 4-1BB and 4-1BBL led to enhanced differentiation into dendritic cell lineages. The regulatory activity was mediated by 4-1BBL, with binding by 4-1BB inhibiting differentiation of myeloid progenitors. Thus, 4-1BB and 4-1BBL have a previously unknown function in limiting myelopoiesis and the development of dendritic cells.

Targeting CD137 enhances vaccine-elicited anti-respiratory syncytial virus CD8+ T cell responses in aged mice.

Respiratory syncytial virus (RSV) causes significant morbidity and mortality in children and the elderly. No vaccines for RSV are in use. Because of immunosenescence, the immunologic requirements for a successful RSV vaccine in the elderly might differ from a RSV vaccine for young children. Using an aged mouse model of RSV pathogenesis, we found that aged mice had impaired Ag-specific CD8(+) T cell responses and delayed RSV clearance compared with young mice. To study vaccine-elicited RSV-specific CD8(+) T cells in aged mice, we used a peptide vaccine approach. TriVax is a commixture of a peptide representing immunodominant RSV CD8(+) T cell epitope M282-90, a TLR agonist (polyinosinic-polycytidylic acid), and a costimulatory anti-CD40 Ab. TriVax vaccination generated robust, polyfunctional, and protective CD8(+) T cell responses in young BALB/c mice, but not in 18-mo-old (aged) BALB/c mice. We hypothesized that treatment of aged mice with agonistic anti-CD137 (41BB) mAb will partially restore T cell responses and TriVax efficacy in aged mice. We immunized 18-mo-old BALB/c mice twice with TriVax + anti-41BB mAb or TriVax + isotype control Ab. Coadministration of anti-41BB mAb with TriVax enhanced RSV-specific CD8(+) T cell responses and TriVax efficacy in challenge experiments. Triggering the 41BB costimulatory pathway may be a strategy for enhancing T cell responses to vaccines in the elderly.

TLR2 signaling in tubular epithelial cells regulates NK cell recruitment in kidney ischemia-reperfusion injury.

Damage-associated molecular patterns released from damaged kidney cells initiate postischemic inflammation, an essential step in the progression of kidney ischemia-reperfusion injury (IRI). However, the mechanism that coordinates this highly specific process in ischemic kidneys remains to be clarified. Previously, we demonstrated that CD137 from NK cells specifically stimulates CD137 ligand (CD137L) on tubular epithelial cells (TECs) such that TECs produced the high CXCR2 chemokine levels required for neutrophil chemotaxis. We report in the present study that endogenous TLR2 ligands released from ischemic TECs induce CCR5 chemokine expression, which is critical to promoting NK cell recruitment. By implanting CD137L(-/-) TECs into the kidney capsule of TLR2(-/-) mice, we further showed that TLR2-mediated NK cell recruitment is an uncoupled event that can occur independently of CD137L signaling in TECs, which is responsible for recruiting neutrophils. Therefore, our findings identify TECs as both a target for kidney damage and also as a master regulator that actively modulates stepwise signaling, leading to the initiation and amplification of acute sterile inflammation that inflicts kidney IRI. Being clinically important, the signaling pathway of innate receptors in epithelial cells may therefore be a good target to block acute sterile inflammation resulting from tissue damage, including kidney IRI.

Reverse signaling through the costimulatory ligand CD137L in epithelial cells is essential for natural killer cell-mediated acute tissue inflammation.

Renal ischemia-reperfusion injury (IRI) after kidney transplantation is a major cause of delayed graft function. Even though IRI is recognized as a highly coordinated and specific process, the pathways and mechanisms through which the innate response is activated are poorly understood. In this study, we used a mouse model of acute kidney IRI to examine whether the interactions of costimulatory receptor CD137 and its ligand (CD137L) are involved in the early phase of acute kidney inflammation caused by IRI. We report here that the specific expressions of CD137 on natural killer cells and of CD137L on tubular epithelial cells (TECs) are required for acute kidney IRI. Reverse signaling through CD137L in TECs results in their production of the chemokine (C-X-C motif) receptor 2 ligands CXCL1 and CXCL2 and the subsequent induction of neutrophil recruitment, resulting in a cascade of proinflammatory events during kidney IRI. Our findings identify an innate pathogenic pathway for renal IRI involving the natural killer cell-TEC-neutrophil axis, whereby CD137-CD137L interactions provide the causal contribution of epithelial cell dysregulation to renal IRI. The CD137L reverse signaling pathway in epithelial cells therefore may represent a good target for blocking the initial stage of inflammatory diseases, including renal IRI.

The B10 Idd9.3 locus mediates accumulation of functionally superior CD137(+) regulatory T cells in the nonobese diabetic type 1 diabetes model.

CD137 is a T cell costimulatory molecule encoded by the prime candidate gene (designated Tnfrsf9) in NOD.B10 Idd9.3 congenic mice protected from type 1 diabetes (T1D). NOD T cells show decreased CD137-mediated T cell signaling compared with NOD.B10 Idd9.3 T cells, but it has been unclear how this decreased CD137 T cell signaling could mediate susceptibility to T1D. We and others have shown that a subset of regulatory T cells (Tregs) constitutively expresses CD137 (whereas effector T cells do not, and only express CD137 briefly after activation). In this study, we show that the B10 Idd9.3 region intrinsically contributes to accumulation of CD137(+) Tregs with age. NOD.B10 Idd9.3 mice showed significantly increased percentages and numbers of CD137(+) peripheral Tregs compared with NOD mice. Moreover, Tregs expressing the B10 Idd9.3 region preferentially accumulated in mixed bone marrow chimeric mice reconstituted with allotypically marked NOD and NOD.B10 Idd9.3 bone marrow. We demonstrate a possible significance of increased numbers of CD137(+) Tregs by showing functional superiority of FACS-purified CD137(+) Tregs in vitro compared with CD137(-) Tregs in T cell-suppression assays. Increased functional suppression was also associated with increased production of the alternatively spliced CD137 isoform, soluble CD137, which has been shown to suppress T cell proliferation. We show for the first time, to our knowledge, that CD137(+) Tregs are the primary cellular source of soluble CD137. NOD.B10 Idd9.3 mice showed significantly increased serum soluble CD137 compared with NOD mice with age, consistent with their increased numbers of CD137(+) Tregs with age. These studies demonstrate the importance of CD137(+) Tregs in T1D and offer a new hypothesis for how the NOD Idd9.3 region could act to increase T1D susceptibility.

Recombinant soluble CD137 prevents type one diabetes in nonobese diabetic mice.

Nonobese diabetic (NOD) mice are genetically programmed to spontaneously develop type one diabetes (T1D). Multiple Insulin dependent diabetes (Idd) genetic loci have been identified but their functional effects are mostly poorly understood. TnfsfR9, expressing the protein product CD137, is a strong candidate gene in the Idd9.3 locus, and NOD.B10 Idd9.3 mice are significantly protected from type one diabetes (T1D). We previously showed that nonobese diabetic (NOD) mice have a deficiency in the numbers of CD137(pos) T regulatory cells, that CD137(pos) Tregs are the source of soluble CD137 (sCD137), and that NOD mice have low serum levels of sCD137. To test the hypothesis that correcting low levels of sCD137 could affect the disease, we constructed a lentiviral vector producing recombinant sCD137; this physiologic sCD137 is glycosylated and exists primarily as a dimer. NOD mice treated with the recombinant sCD137 are protected from developing T1D. Insulitis is significantly decreased, but not eliminated in the sCD137 treated mice, however insulin producing pancreatic beta cells are preserved despite residual insulitis. To begin to understand the protective immune mechanisms of sCD137, we tested sCD137 in vitro. It was previously suggested that sCD137 simply blocked the interaction between CD137 (on T cells) and CD137 ligand (on antigen presenting cells (APCs)). Here however, we use an APC independent assay and demonstrate that sCD137 can actively suppress highly purified CD4 T cells in a CD137L dependent fashion. These results support the hypothesis that sCD137 acts in a negative feedback loop to actively suppress over-zealous immune responses, and that it can be used clinically to suppress autoimmunity. sCD137 is an important Treg derived natural immunosuppressive molecule that regulates effector T cells to avert diabetes in vivo.

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.

CD134 plus CD137 dual costimulation induces Eomesodermin in CD4 T cells to program cytotoxic Th1 differentiation.

Cytotoxic CD4 Th1 cells are emerging as a therapeutically useful T cell lineage that can effectively target tumors, but until now the pathways that govern their differentiation have been poorly understood. We demonstrate that CD134 (OX40) costimulation programs naive self- and virus-reactive CD4 T cells to undergo in vivo differentiation into cytotoxic Th1 effectors. CD137 (4-1BB) costimulation maximized clonal expansion, and IL-2 was necessary for cytotoxic Th1 differentiation. Importantly, the T-box transcription factor Eomesodermin was critical for inducing the cytotoxic marker granzyme B. CD134 plus CD137 dual costimulation also imprinted a cytotoxic phenotype on bystanding CD4 T cells. Thus, to our knowledge, the current study identifies for the first time a specific costimulatory pathway and an intracellular mechanism relying on Eomesodermin that induces both Ag-specific and bystander cytotoxic CD4 Th1 cells. This mechanism might be therapeutically useful because CD134 plus CD137 dual costimulation induced CD4 T cell-dependent tumoricidal function in a mouse melanoma model.

Eradication of established tumors in mice by a combination antibody-based therapy.

Tumor-cell apoptosis is the basis of many cancer therapies, and tumor-specific T cells are the principal effectors of successful antitumor immunotherapies. Here we show that induction of tumor-cell apoptosis by an agonistic monoclonal antibody to DR5, the apoptosis-inducing receptor for TNF-related apoptosis-inducing ligand (TRAIL), combined with T-cell activation by agonistic monoclonal antibodies to the costimulatory molecules CD40 and CD137, potently and rapidly stimulated tumor-specific effector CD8+ T cells capable of eradicating preestablished tumors. Primary fibrosarcomas initiated with the carcinogen 3-methylcholanthrene (MCA), multiorgan metastases and a primary tumor containing as many as 90% tumor cells resistant to DR5-specific monoclonal antibody were rejected without apparent toxicity or induction of autoimmunity. This combination therapy of three monoclonal antibodies (trimAb) rapidly induced tumor-specific CD8+ T cells producing interferon (IFN)-gamma in the tumor-draining lymph node, consistent with a crucial requirement for CD8+ T cells and IFN-gamma in the tumor rejection process. These results in mice indicate that a rational monoclonal antibody-based therapy that both causes tumor-cell apoptosis through DR5 and activates T cells may be an effective strategy for cancer immunotherapy in humans.

Host CD25+CD4+Foxp3+ regulatory T cells primed by anti-CD137 mAbs inhibit graft-versus-host disease.

CD25(+)CD4(+)Foxp3(+) regulatory T cells (Tregs) play a pivotal role in the maintenance of self-tolerance and regulation of immune responses. Previous studies have demonstrated that CD137 signals can promote proliferation and survival of Tregs in vitro. Here, we show that in vivo CD137-induced expansion of Tregs in naive mice was dependent upon IL-2 secreted by memory T cells. Tregs primed by anti-CD137 mAbs had a higher immunosuppressive capacity. Preconditioning with anti-CD137 mAbs significantly inhibited graft-versus-host disease (GVHD) in the C57BL/6 → (C57BL/6 × DBA/2) F1 acute GVHD model. In this disease model, a high proportion of host Tregs remained long-term in the recipient spleen, whereas donor hematopoietic cells replaced other host bone marrow-derived cells. Transient depletion of Tregs before transfer of donor cells completely abrogated the inhibitory effect of anti-CD137 mAbs on GVHD. In addition, adoptive transfer of anti-CD137-primed Tregs ameliorated GVHD. Our results demonstrate that it is possible to enhance the survival and/or the immunosuppressive activity of host Tregs in nonmyeloablative GVHD, and that 1 way of accomplishing this is through the prophylactic use of anti-CD137 mAbs in nonmyeloablative GVHD.

Lineage-specific T-cell reconstitution following in vivo CD4+ and CD8+ lymphocyte depletion in nonhuman primates.

Many features of T-cell homeostasis in primates are still unclear, thus limiting our understanding of AIDS pathogenesis, in which T-cell homeostasis is lost. Here, we performed experiments of in vivo CD4(+) or CD8(+) lymphocyte depletion in 2 nonhuman primate species, rhesus macaques (RMs) and sooty mangabeys (SMs). Whereas RMs develop AIDS after infection with simian immunodeficiency virus (SIV), SIV-infected SMs are typically AIDS-resistant. We found that, in both species, most CD4(+) or CD8(+) T cells in blood and lymph nodes were depleted after treatment with their respective antibodies. These CD4(+) and CD8(+) lymphocyte depletions were followed by a largely lineage-specific CD4(+) and CD8(+) T-cell proliferation, involving mainly memory T cells, which correlated with interleukin-7 plasma levels. Interestingly, SMs showed a faster repopulation of naive CD4(+) T cells than RMs. In addition, in both species CD8(+) T-cell repopulation was faster than that of CD4(+) T cells, with CD8(+) T cells reconstituting a normal pool within 60 days and CD4(+) T cells remaining below baseline levels up to day 180 after depletion. While this study revealed subtle differences in CD4(+) T-cell repopulation in an AIDS-sensitive versus an AIDS-resistant species, such differences may have particular relevance in the presence of active SIV repli cation, where CD4(+) T-cell destruction is chronic.

Dendritic cells and Stat3 are essential for CD137-induced CD8 T cell activation-induced cell death.

Agonistic anti-CD137 mAbs either positively or negatively regulate T cell function. When administered at the beginning of lymphocytic choriomeningitis virus Armstrong infection anti-CD137 induced immunosuppression and T cell deletion, and in the case of influenza infection led to increased mortality. In contrast, 72 h delay in anti-CD137 treatment led to an enhanced virus-specific CD8 T cell response and rapid viral clearance. Virus-specific CD8 T cells in anti-CD137-injected mice rapidly upregulate Fas expression, and although necessary, was insufficient to induce CD8 T cell deletion. Strikingly, CD137 signaling in T cells was found to be insufficient to induce suppression or deletion. Rather, immunosuppression and T cell deletion was only observed if CD137 signals were provided to T cells and dendritic cells (DCs). In vitro CD137 crosslinking in DCs led to phosphorylation of Stat3, and importantly, anti-CD137 treatment of lymphocytic choriomeningitis virus Armstrong infected Stat3 conditional knock-out mice induced neither immune suppression or T cell deletion. Taken together, these data suggest that CD137 signaling in DCs can regulate CD8 T cell survival through a Stat3 and Fas-mediated pathway.

Immunity to pre-1950 H1N1 influenza viruses confers cross-protection against the pandemic swine-origin 2009 A (H1N1) influenza virus.

The 2009 H1N1 influenza virus outbreak is the first pandemic of the twenty-first century. Epidemiological data reveal that of all the people afflicted with H1N1 virus, <5% are over 51 y of age. Interestingly, in the uninfected population, 33% of those >60 y old have pre-existing neutralizing Abs against the 2009 H1N1 virus. This finding suggests that influenza strains that circulated 50-60 y ago might provide cross-protection against the swine-origin 2009 H1N1 influenza virus. To test this, we determined the ability of representative H1N1 influenza viruses that circulated in the human population from 1930 to 2000, to induce cross-reactivity to and cross-protection against the pandemic swine-origin H1N1 virus, A/California/04/09. We show that exposure of mice to the 1947 virus, A/FM/1/47, or the 1934 virus, A/PR/8/34, induced robust cross-protective immune responses and these mice were protected against a lethal challenge with mouse-adapted A/California/04/09 H1N1 virus. Conversely, we observed that mice exposed to the 2009 H1N1 virus were protected against a lethal challenge with mouse-adapted 1947 or 1934 H1N1 viruses. In addition, exposure to the 2009 H1N1 virus induced broad cross-reactivity against H1N1 as well as H3N2 influenza viruses. Finally, we show that vaccination with the older H1N1 viruses, particularly A/FM/1/47, confers protective immunity against the 2009 pandemic H1N1 virus. Taken together, our data provide an explanation for the decreased susceptibility of the elderly to the 2009 H1N1 outbreak and demonstrate that vaccination with the pre-1950 influenza strains can cross-protect against the pandemic swine-origin 2009 H1N1 influenza virus.

Multivalent 4-1BB binding aptamers costimulate CD8+ T cells and inhibit tumor growth in mice.

4-1BB is a major costimulatory receptor that promotes the survival and expansion of activated T cells. Administration of agonistic anti-4-1BB Abs has been previously shown to enhance tumor immunity in mice. Abs are cell-based products posing significant cost, manufacturing, and regulatory challenges. Aptamers are oligonucleotide-based ligands that exhibit specificity and avidity comparable to, or exceeding, that of Abs. To date, various aptamers have been shown to inhibit the function of their cognate target. Here, we have described the development of an aptamer that binds 4-1BB expressed on the surface of activated mouse T cells and shown that multivalent configurations of the aptamer costimulated T cell activation in vitro and mediated tumor rejection in mice. Because aptamers can be chemically synthesized, manufacturing and the regulatory approval process should be substantially simpler and less costly than for Abs. Agonistic aptamers could therefore represent a superior alternative to Abs for the therapeutic manipulation of the immune system.

Bone marrow-based homeostatic proliferation of mature T cells in nonhuman primates: implications for AIDS pathogenesis.

Bone marrow (BM) is the key hematopoietic organ in mammals and is involved in the homeostatic proliferation of memory CD8(+) T cells. Here we expanded on our previous observation that BM is a preferential site for T-cell proliferation in simian immunodeficiency virus (SIV)-infected sooty mangabeys (SMs) that do not progress to AIDS despite high viremia. We found high levels of mature T-cell proliferation, involving both naive and memory cells, in healthy SMs and rhesus macaques (RMs). In addition, we observed in both species that lineage-specific, BM-based T-cell proliferation follows antibody-mediated in vivo CD4(+) or CD8(+) T-cell depletion, thus indicating a role for the BM in maintaining T-cell homeostasis under depleting circumstances. We also observed that, in SIV-infected SMs, but not RMs, the level of proliferation of BM-based CD4(+) T cells is higher than that of circulating CD4(+) T cells. Interestingly, limited BM-based CD4(+) T-cell proliferation was found in SIV-infected SMs with low CD4(+) T-cell counts, suggesting a regenerative failure in these animals. Collectively, these results indicate that BM is involved in maintaining T-cell homeostasis in primates and suggest a role for BM-based CD4(+) T-cell proliferation in determining the benign nature of natural SIV infection of SMs.

Hypercostimulation through 4-1BB distorts homeostasis of immune cells.

The deleterious side effects associated with a recent clinical trial with anti-CD28 superagonist Abs have questioned the use of reagents to costimulatory molecules in human therapy. We now show that sustained signaling from an agonist Ab to 4-1BB, a member of the TNFR superfamily, results in detrimental effects on immune cell homeostasis. Repeated anti-4-1BB treatment during the reconstitution of hematopoietic cells in irradiated mice engrafted with bone marrow, or in mice infected with vaccinia virus, induced abnormal apoptosis of premature and immature B cells in the bone marrow, and led to peripheral B cell depletion. Inhibition of B cell development was indirect and due to costimulation of CD8 T cells and dependent on IFN-gamma. Moreover, anti-4-1BB also suppressed the development of NK and NKT cells, but in this case independently of T cells and IFN-gamma. The altered NK cell homeostasis resulted from activation-induced cell death triggered by anti-4-1BB. These results show that hypercostimulation elicits strong T cell immunity, but it can simultaneously distort immune homeostasis, suggesting that careful attention to activity, dose, and periodicity of treatment will be needed in any immunotherapeutic strategy with agonist Abs to costimulatory molecules.

Immune suppression or enhancement by CD137 T cell costimulation during acute viral infection is time dependent.

CD137 is expressed on activated T cells and ligands to this costimulatory molecule have clinical potential for amplifying CD8 T cell immunity to tumors and viruses, while suppressing CD4 autoimmune T cell responses. To understand the basis for this dichotomy in T cell function, CD4 and CD8 antiviral immunity was measured in lymphocytic choriomeningitis virus (LCMV) Armstrong- or A/PR8/34 influenza-infected mice injected with anti-CD137 mAbs. We found that the timing of administration of anti-CD137 mAbs profoundly altered the nature of the antiviral immune response during acute infection. Antiviral immunity progressed normally for the first 72 hours when the mAb was administered early in infection before undergoing complete collapse by day 8 postinfection. Anti-CD137-injected LCMV-infected mice became tolerant to, and persistently infected with, LCMV Armstrong. Elevated levels of IL-10 early in the response was key to the loss of CD4(+) T cells, whereas CD8(+) T cell deletion was dependent on a prolonged TNF-alpha response, IL-10, and upregulation of Fas. Blocking IL-10 function rescued CD4 antiviral immunity but not CD8(+) T cell deletion. Anti-CD137 treatment given beyond 72 hours after infection significantly enhanced antiviral immunity. Mice treated with anti-CD137 mAb 1 day before infection with A/PR8/34 virus experienced 80% mortality compared with 40% mortality of controls. When treatment was delayed until day 1 postinfection, 100% of the infected mice survived. These data show that anti-CD137 mAbs can induce T cell activation-induced cell death or enhance antiviral immunity depending on the timing of treatment, which may be important for vaccine development.

Self-antigen prevents CD8 T cell effector differentiation by CD134 and CD137 dual costimulation.

We compared how CD4 vs CD8 cells attain the capacity to express the effector cytokine IFN-gamma under both immunogenic and tolerogenic conditions. Although the Ifng gene locus was epigenetically repressed in naive Ag-inexperienced CD4 cells, it had already undergone partial remodeling toward a transcriptionally competent configuration in naive CD8 cells. After TCR stimulation, CD8 cells fully remodeled the Ifng locus and gained the capacity to express high levels of IFN-gamma more rapidly than CD4 cells. Enforced dual costimulation through OX40 and 4-1BB redirected CD8 cells encountering soluble exogenous peptide to expand and differentiate into IFN-gamma and TNF-alpha double-producing effectors rather than becoming tolerant. Despite this and the stronger tendency of CD8 compared with CD4 cells to differentiate into IFN-gamma-expressing effectors, when parenchymal self-Ag was the source of tolerizing Ag, enforced dual costimulation selectively boosted expansion but did not push effector differentiation in CD8 cells while both expansion and effector differentiation were dramatically boosted in CD4 cells. Notably, enforced dual costimulation was able to push effector differentiation in CD8 cells encountering cognate parenchymal self-Ag when CD4 cells were simultaneously engaged. Thus, the ability of enforced OX40 plus 4-1BB dual costimulation to redirect CD8 cells to undergo effector differentiation was unexpectedly influenced by the source of tolerizing Ag and help was selectively required to facilitate CD8 cell effector differentiation when the tolerizing Ag derived from self.

The IKK-neutralizing compound Bay11 kills supereffector CD8 T cells by altering caspase-dependent activation-induced cell death.

Antigen with dual costimulation through CD137 and CD134 induces powerful CD8 T cell responses. These effector T cells are endowed with an intrinsic survival program resulting in their accumulation in vivo, but the signaling components required for survival are unknown. We tested a cadre of pathway inhibitors and found one preclinical compound, Bay11-7082 (Bay11), which prevented survival. Even the gammac cytokine family members IL-2, -4, -7, and -15 could not block death, nor could pretreatment with IL-7. We found that dual costimulation caused loading of phosphorylated IkappaBalpha (p-IkappaBalpha) and high basal levels of NF-kappaB activity in the effector CD8 T cells. Bay11 trumped both events by reducing the presence of p-IkappaBalpha and ensuing NF-kappaB activity. Not all pathways were impacted to this degree, however, as mitogen-mediated ERK phosphorylation was evident during NF-kappaB inhibition. Nonetheless, Bay11 blocked TCR-stimulated cytokine synthesis by rapidly accentuating activation-induced cell death through elicitation of a caspase-independent pathway. Thus, in effector CD8 T cells, Bay11 forces a dominant caspase-independent death signal that cannot be overcome by an intrinsic survival program nor by survival-inducing cytokines. Therefore, Bay11 may be a useful tool to deliberately kill death-resistant effector T cells for therapeutic benefit.

Mechanisms involved in synergistic anticancer effects of anti-4-1BB and cyclophosphamide therapy.

Chemotherapy can precondition for immunotherapy by creating an environment for homeostatic lymphoproliferation and eliminating some of the suppressive immune networks. We found that combination therapy with anti-4-1BB and cyclophosphamide (CTX) produced synergistic anticancer effects in the poorly immunogenic B16 melanoma model in mice. The antitumor effect of the combination therapy depended mainly on CD8(+) T cells, the 4-1BB-dependent expansion and differentiation of which into IFN-gamma-producing CD11c(+)CD8(+) T cells was enhanced by CTX. Anti-4-1BB induced a rapid repopulation of T and B cells from CTX-mediated lymphopenia. Anti-4-1BB protected naïve T cells from CTX and promoted proliferation of memory/effector and memory T cells. The combination treatment produced approximately 60- and 2.2-fold more CTLs per tumor-associated antigen compared with CTX or anti-4-1BB alone, respectively. This indicates that anti-4-1BB promoted a preferential expansion of tumor-specific CD8(+) T cells among the repopulated lymphocytes following CTX-mediated lymphopenia. CTX treatment enhanced 4-1BB expression on CD4 and CD8 T cells, and CTX alone or in combination with anti-4-1BB effectively suppressed peripheral regulatory T cells. Our results indicate that anti-4-1BB and CTX can be practical partners in cancer therapy because CTX creates an environment in which anti-4-1BB actively promotes the differentiation and expansion of tumor-specific CTLs.