Byoung S. Kwon

4-1BB-mediated immunotherapy of rheumatoid arthritis

Collagen type II–induced arthritis is a CD4+ T-cell–dependent chronic inflammation in susceptible DBA/1 mice and represents an animal model of human rheumatoid arthritis. We found that development of this condition, and even established disease, are inhibited by an agonistic anti-4-1BB monoclonal antibody. Anti-4-1BB suppressed serum antibodies to collagen type II and CD4+ T-cell recall responses to collagen type II. Crosslinking of 4-1BB evoked an antigen-specific, active suppression mechanism that differed from the results of blocking the interaction between 4-1BB and its ligand, 4-1BBL. Anti-4-1BB monoclonal antibodies induced massive, antigen-dependent clonal expansion of CD11c+CD8+ T cells and accumulation of indoleamine 2,3-dioxygenase in CD11b+ monocytes and CD11c+ dendritic cells. Both anti-interferon-γ and 1-methyltryptophan, a pharmacological inhibitor of indoleamine 2,3-dioxygenase, reversed the anti-4-1BB effect. We conclude that the suppression of collagen-induced arthritis was caused by an expansion of new CD11c+CD8+ T cells, and that interferon-γ produced by these cells suppresses antigen-specific CD4+ T cells through an indoleamine 2,3-dioxygenase–dependent mechanism.

4-1BB Promotes the Survival of CD8+ T Lymphocytes by Increasing Expression of Bcl-xL and Bfl-1

4-1BB, a T cell costimulatory receptor, prolongs CD8+ T cell survival. In these studies, 4-1BB stimulation was shown to increase expression of the antiapoptotic genes bcl-xL and bfl-1 via 4-1BB-mediated NF-κB activation. This signaling pathway was specifically inhibited by PDTC and was different from the pathways that enhanced CD8+ T cell proliferation. The results suggest a role for the antiapoptotic activities of Bcl-xL and Bfl-1 proteins in 4-1BB-mediated CD8+ T cell survival in vivo.

4-1BB-dependent inhibition of immunosuppression by activated CD4+CD25+ T cells

4‐1BB (CD137) is a costimulatory molecule involved in the activation and survival of CD4, CD8, and natural killer cells. Although a great deal has been learned as to how 4‐1BB‐mediated signaling governs the immunity of conventional T cells, the functional role of 4‐1BB in the context of CD4+CD25+ regulatory T cell (Tr) activation is largely unknown. Using 4‐1BB‐intact and ‐deficient mice, we investigated the effect of the 4‐1BB/4‐1BB ligand pathway on the suppressive function of Tr cells. Our data indicate that although 4‐1BB is expressed on Tr cells, its contribution to their proliferation is minimal. We also showed that signaling through the 4‐1BB receptor inhibited the suppressive function of Tr cells in vitro and in vivo. It is interesting that anti‐4‐1BB‐mediated but not anti‐GITR‐directed inhibition was more potent when Tr cells were preactivated. Collectively, these data indicate that 4‐1BB signaling is critical in Tr cell immunity.

CD137-Deficient Mice Have Reduced NK/NKT Cell Numbers and Function, Are Resistant to Lipopolysaccharide-Induced Shock Syndromes, and Have Lower IL-4 Responses

CD137, a member of the TNF superfamily, is involved in T cell and NK cell activation and cytokine production. To establish its in vivo role in systems dependent on NK and NKT cells, we studied the response of CD137−/− mice to LPS-induced shock, tumor killing, and their IL-4-controlled Th2 responses. In both high and low dose shock models, all the CD137-deficient mice, but none of the wild-type BALB/c mice, survived. After injection of LPS/2-amino-2-deoxy-d-galactose (D-gal), CD137−/− mice had reduced serum cytokine levels and substantially impaired liver IFN-γ and TNF-α mRNA levels. Phenotypic analysis of mononuclear cells revealed fewer NK and NKT cells in the CD137−/− mice. The knockout mice did not generate a rapid IL-4 response after systemic T cell activation, or effective Ag-specific Th2 responses. In addition, both in vitro and in vivo NK-specific cytolytic activities were reduced. These findings suggest that CD137-directed NK/NKT cells play an important role in the inflammatory response leading to the production of proinflammatory cytokines, LPS-induced septic shock, and tumor killing, as well as IL-4-dependent Th2 responses.

Dual immunoregulatory pathways of 4-1BB signaling

It is perhaps rare to encounter among the various immunologically competent receptor–ligand pairs that a single cell surface determinant unleashes both a hidden suppressive function and costimulation. 4-1BB, an activation-induced tumor necrosis factor receptor family member chiefly viewed as a powerful T-cell costimulatory molecule, is one such example. Accumulated evidence in recent years uncovered an unknown facet of in vivo 4-1BB signaling (i.e., “active suppression”). Although in vitro signaling via 4-1BB is shown to support both CD4+ and CD8+ T-cell responses, the same induces a predominant CD8+ T-cell response suppressing CD4+ T-cell function when applied in vivo. How, when, and why such dual immunoregulatory effect of anti-4-1BB monoclonal antibody (MAB) comes into play is currently the focus of intense research. Existing data, although not complete, uncover several important aspects of in vivo 4-1BB signaling in the amelioration or exacerbation of various immune disorders. Despite minor disagreements, a majority agree that upregulation of interferon (IFN)-γ is critical to anti-4-1BB MAB therapy in addition to immune modulators such as interleukin 2, transforming growth factor β, and indolamine 2,3-dioxygenase5, all of which contribute greatly to the success of anti-4-1BB MAB-based immunotherapy. Anti-4-1BB MAB-mediated expansion of novel CD11c+CD8+ T cells is additional weaponry that appears critical for its in vivo suppressive function. These CD11c+CD8+ T cells express high levels of IFN-γ, become effective killers, and mediate selective suppression of CD4+ T cells. In this review, we discuss the dual nature (costimulatory and suppressive) of 4-1BB-mediated immune regulation, its current status, future direction, and its impact on the immune system, with special reference to its immunotherapy.

New insights into the role of 4-1BB in immune responses: beyond CD8+ T cells

Since the discovery of 4-1BB 15 years ago, the receptor and its ligand (4-1BBL) have remained enigmatic molecules because of their highly regulated pattern of expression. Classically, 4-1BB is known to function as a costimulator for T cells and as a potent survival factor for CD8+ T cells. Recent studies highlight the participation of 4-1BB and its ligand 4-1BBL in a more complex network of immune cell responses and suggest that intervening in the 4-1BB costimulatory pathway might have some potential as a therapeutic approach to immune disorders.

LIGHT enhances the bactericidal activity of human monocytes and neutrophils via HVEM.

Human monocytes and neutrophils play major roles in clearing bacteria from human blood and tissues. We found that the herpes virus entry mediator (HVEM) was highly expressed in monocytes and neutrophils, and its interaction with “homologous to lymphotoxins, shows inducible expression, and competes with herpes simplex virus glycoprotein D for HVEM/tumor necrosis factor (TNF)‐related 2” (LIGHT) enhanced bactericidal activity against Listeria monocytogenes and Staphylococcus aureus. The LIGHT‐HVEM interaction increased levels of phagocytosis, interleukin (IL)‐8, TNF‐α, nitric oxide (NO), and reactive oxygen species (ROS) in monocytes and neutrophils. Anti‐HVEM monoclonal antibody was able to block LIGHT‐induced bactericidal activity, cytokine production (IL‐8 and TNF‐α), and ROS generation. Moreover, inhibition of ROS and NO production blocked LIGHT‐induced bactericidal activity. Our results indicate that the LIGHT/HVEM interaction in monocytes and neutrophils contributes to antibacterial activity.

Glucocorticoid-induced tumor necrosis factor receptor-related protein co-stimulation facilitates tumor regression by inducing IL-9-producing helper T cells

T cell stimulation via glucocorticoid-induced tumor necrosis factor receptor (TNFR)-related protein (GITR) elicits antitumor activity in various tumor models; however, the underlying mechanism of action remains unclear. Here we demonstrate a crucial role for interleukin (IL)-9 in antitumor immunity generated by the GITR agonistic antibody DTA-1. IL-4 receptor knockout (Il4ra−/−) mice, which have reduced expression of IL-9, were resistant to tumor growth inhibition by DTA-1. Notably, neutralization of IL-9 considerably impaired tumor rejection induced by DTA-1. In particular, DTA-1–induced IL-9 promoted tumor-specific cytotoxic T lymphocyte (CTL) responses by enhancing the function of dendritic cells in vivo. Furthermore, GITR signaling enhanced the differentiation of IL-9–producing CD4+ T-helper (TH9) cells in a TNFR-associated factor 6 (TRAF6)- and NF-κB–dependent manner and inhibited the generation of induced regulatory T cells in vitro. Our findings demonstrate that GITR co-stimulation mediates antitumor immunity by promoting TH9 cell differentiation and enhancing CTL responses and thus provide a mechanism of action for GITR agonist–mediated cancer immunotherapies.

Blocking 4-1BB/4-1BB Ligand Interactions Prevents Herpetic Stromal Keratitis

Herpetic stromal keratitis (HSK) is a chronic inflammatory process in corneal stroma that results from recurrent HSV type 1 infection. We used the murine model of HSK to demonstrate the importance of the interaction between an inducible T cell costimulatory receptor, 4-1BB, and its ligand, 4-1BB ligand (4-1BBL), in the development of this disease. In BALB/c mice, HSK ordinarily induced by infection with the RE strain of herpes was prevented by blocking 4-1BB/4-1BBL interaction, either by deleting 4-1BB (in mutant 4-1BB−/− mice) or by introducing mAbs against 4-1BBL. The majority of T cells infiltrating the infected corneas were 4-1BB+ activated effector cells that expressed cell surface markers CD44, CD25, and/or CD62L, as well as chemokine receptors CCR1, CCR2, and CCR5, and a limited number of TCR Vβ chains (Vβ8.1/8.2, Vβ8.3, Vβ10b, and Vβ5.1/5.2, in order of abundance). Analysis of cell surface phenotypes showed that the failure to develop HSK in the 4-1BB−/− mice was associated with a reduced expression of CD62L at the time of T cell migration into the corneal stroma.

Gene expression analyzed by microarrays in HSV-1 latent mouse trigeminal ganglion following heat stress.

An understanding of the cellular genes whose expression is altered during HSV reactivation will enable us to better understand host responses and biochemical pathways involved in the process. Furthermore, this knowledge could allow us to develop gene-targeted inhibitors to prevent viral reactivation. Mice latent with HSV-1 strain McKrae and uninfected control mice were subjected to hyperthermic stress (43°C for 10 min) and their trigeminal ganglia (TG) collected 1 h later. Two additional groups included HSV-1 latently infected and uninfected mice not subjected to hyperthermic stress. Poly A+ mRNA was enriched from total mouse TG RNA and reverse transcribed using MMLV RT. Radioactively labeled cDNAs were analyzed by microarray analysis. A stress/toxicology array of 149 mouse genes on a nylon membrane was used. The labeled cDNAs prepared from latently infected, stressed mice demonstrated 3-fold or greater increases in certain mRNA-early response genes (ERGs) compared to cDNAs from uninfected, stressed control mice. The ERG mRNAs that showed increases included two heat shock proteins (HSP60 and HSP40), a basic transcription factor (BTF T62), a DNA repair enzyme, two kinases [MAP kinase and a stress-induced protein kinase (SADK)], an oxidative stress-induced protein, a manganese superoxide dismutase precursor-2 (SOD-2), and cyclooxygenase 2 (COX-2). The gene expression in unstressed, infected TGs was similar to the gene expression in unstressed, uninfected controls. These results suggest that there is a significant difference in the ERG expression profile in latently infected TGs undergoing stress-induced reactivation compared to uninfected TGs.