Robert S. Mittler

Intracellular Trafficking of CTLA-4 and Focal Localization Towards Sites of TCR Engagement

T lymphocyte receptor CTLA-4 binds costimulatory molecules CD80 (B7-1) and CD86 (B7-2) with high avidity and negatively regulates T cell activation. CTLA-4 functions at the cell surface, yet is primarily localized in intracellular vesicles. Here, we demonstrate cycling of CTLA-4 between intracellular stores and the cell surface. Intracellular vesicles containing CTLA-4 overlapped with endocytic compartment(s) and with perforin-containing secretory granules. Cell surface expression of CTLA-4 was rapidly increased by raising intracellular calcium levels. During T cell activation, intracellular and cell surface CTLA-4 became focused towards sites of TCR activation. Cycling and directional control of CTLA-4 expression may regulate its functional interaction with APCs bearing peptide-MHC complexes of appropriate specificity and avidity.

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)-γ in the tumor-draining lymph node, consistent with a crucial requirement for CD8+ T cells and IFN-γ 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.*

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.

Amplification of tumor immunity by gene transfer of the co-stimulatory 4-1BB ligand: synergy with the CD28 co-stimulatory pathway

We have explored the role of an activation‐induced T cell molecule, 4‐1BB (CDw137), in the amplification of tumor immunity by retrovirus‐mediated transduction of the 4‐1BB ligand (4‐1BBL) into tumor cells. Mice inoculated with P815 tumor cells expressing 4‐1BBL developed a strong cytotoxic T lymphocyte (CTL) response and long‐term immunity against wild‐type tumor. The optimal effect of 4‐1BBL in CTL stimulation required B7‐CD28 interaction since blockade of this interaction by antibodies down‐regulated the expression of 4‐1BB on T cells and decreased CTL activity. Furthermore, co‐expression of 4‐1BBL and B7‐1 in the poorly immunogenic AG104A sarcoma enhanced the induction of effector CTL and the rejection of the wild‐type tumor while neither 4‐1BBL nor B7‐1 single transfectants were effective, suggesting a synergistic effect between the 4‐1BB and the CD28 co‐stimulatory pathways. Our results underscore the importance of the 4‐1BB T cell stimulation pathway in the amplification of an antitumor immune response.

CD137 costimulatory T cell receptor engagement reverses acute disease in lupus-prone NZB × NZW F1 mice

Systemic lupus erythematosus (SLE) is a CD4(+) T cell-dependent, immune complex-mediated, autoimmune disease that primarily affects women of childbearing age. Generation of high-titer affinity-matured IgG autoantibodies, specific for double-stranded DNA and other nuclear antigens, coincides with disease progression. Current forms of treatment of SLE including glucocorticosteroids are often inadequate and induce severe side effects. Immunological approaches for treating SLE in mice using anti-CD4 mAbs or CTLA4-Ig and anti-CD154 mAbs have proven to be effective. However, like steroid treatment, these regimens induce global immunosuppression, and their withdrawal allows for disease progression. In this report we show that lupus-prone NZB x NZW F(1) mice given three injections of anti-CD137 (4-1BB) mAbs between 26 and 35 weeks of age reversed acute disease, blocked chronic disease, and extended the mices lifespan from 10 months to more than 2 years. Autoantibody production in recipients was rapidly suppressed without inducing immunosuppression. Successful treatment could be traced to the fact that NZB x NZW F(1) mice, regardless of their age or disease status, could not maintain pathogenic IgG autoantibody production in the absence of continuous CD4(+) T cell help. Our data support the hypothesis that CD137-mediated signaling anergized CD4(+) T cells during priming at the DC interface.

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-γ staining, and IFN-γ 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.

Cutting Edge: Deficiency in the E3 Ubiquitin Ligase Cbl-b Results in a Multifunctional Defect in T Cell TGF-β Sensitivity In Vitro and In Vivo

Mice deficient in the E3 ubiquitin ligase Cbl-b have CD28-independent T cells and develop autoimmunity. We previously reported that Cbl-b−/− CD4+CD25− T effector cells are resistant in vitro to the antiproliferative effects of CD4+CD25+ regulatory T cells and TGF-β. We have now asked whether the resistance noted in Cbl-b−/− T cells is restricted solely to TGF-β’s antiproliferative effects, whether the TGF-β resistance has in vivo relevance, and whether a defect can be identified in the TGF-β signaling pathway. We now demonstrate the following: 1) in vitro, Cbl-b deficiency prevents the TGF-β-mediated induction of Foxp3+ functional regulatory T cells; 2) in vivo, Cbl-b−/− mice show a significantly enhanced response to a tumor that is strictly TGF-β regulated; and 3) Cbl-b−/− T effector cells have defective TGF-β-mediated Smad2 phosphorylation. These studies are the first to document that the E3 ubiquitin ligase Cbl-b plays an integral role in T cell TGF-β signaling, and that its absence results in multifunctional TGF-β-related defects that have important disease-related implications.

Cytokine-Mediated Disruption of Lymphocyte Trafficking, Hemopoiesis, and Induction of Lymphopenia, Anemia, and Thrombocytopenia in Anti-CD137-Treated Mice

CD137-mediated signals costimulate T cells and protect them from activation-induced apoptosis; they induce curative antitumor immunity and enhance antiviral immune responses in mice. In contrast, anti-CD137 agonistic mAbs can suppress T-dependent humoral immunity and reverse the course of established autoimmune disease. These results have provided a rationale for assessing the therapeutic potential of CD137 ligands in human clinical trials. In this study, we report that a single 200-μg injection of anti-CD137 given to otherwise naive BALB/c or C57BL/6 mice led to the development of a series of immunological anomalies. These included splenomegaly, lymphadenopathy, hepatomegaly, multifocal hepatitis, anemia, altered trafficking of B cells and CD8 T cells, loss of NK cells, and a 10-fold increase in bone marrow (BM) cells bearing the phenotype of hemopoietic stem cells. These events were dependent on CD8 T cells, TNF-α, IFN-γ, and type I IFNs. BM cells up-regulated Fas, and there was a significant increase in the number of CD8+ T cells that correlated with a loss of CD19+ and Ab-secreting cells in the BM. TCR Vαβ usage was random and polyclonal among liver-infiltrating CD8 T cells, and multifocal CD8+ T cell infiltrates were resolved upon termination of anti-CD137 treatment. Anti-CD137-treated mice developed lymphopenia, thrombocytopenia, and anemia, and had lowered levels of hemoglobin and increased numbers of reticulocytes.

In Vivo Triggering Through 4-1BB Enables Th-Independent Priming of CTL in the Presence of an Intact CD28 Costimulatory Pathway

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

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.