Beom K. Choi

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.

Immune Responses in 4-1BB (CD137)-Deficient Mice

The 4-1BB (a TNFR superfamily member) is an inducible costimulatory molecule that can exert regulatory effects on T cells independently of CD28 stimulation. The in vitro expression of 4-1BB (CD137) is induced following activation of T cells with various stimuli, including anti-TCR mAbs, lectins, and a combination of PMA and ionomycin. To delineate further the physiological role of 4-1BB in immunity, mice deficient in this receptor were generated. These mutant mice developed normally, and were viable and fertile. Humoral responses to vesicular stomatitis virus were comparable with those seen in wild-type mice, whereas the IgG2a and IgG3 isotype responses to keyhole limpet hemocyanin were somewhat reduced in the mutant mice. The 4-1BB-deficient mice demonstrated enhanced T cell proliferation in response to mitogens or anti-CD3 even in the environment of reduced ability to secrete growth-supporting cytokines (IL-2 and IL-4). Although T cells from 4-1BB-deficient mice showed enhanced proliferation, the T cell immune responses of these animals, such as cytokine production and CTL activity, were diminished. In addition, 4-1BB deletion appears to play a role in the regulation of myeloid progenitor cell growth, leading to an increase in these precursor cells in peripheral blood, bone marrow, and spleen.

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 (4–1BB) Deficiency Reduces Atherosclerosis in Hyperlipidemic Mice

Background— The tumor necrosis factor receptor superfamily, which includes CD40, LIGHT, and OX40, plays important roles in atherosclerosis. CD137 (4-1BB), a member of the tumor necrosis factor receptor superfamily, has been reported to be expressed in human atherosclerotic lesions. However, limited information is available on the precise role of CD137 in atherosclerosis and the effects of blocking CD137/CD137 ligand signaling on lesion formation. Methods and Results— We generated CD137-deficient apolipoprotein E–knockout mice (ApoE−/−CD137−/−) and LDL-receptor–knockout mice (Ldlr−/−CD137−/−) to investigate the role of CD137 in atherogenesis. The deficiency of CD137 induced a reduction in atherosclerotic plaque lesions in both atherosclerosis mouse models, which was attributed to the downregulation of cytokines such as interferon-&ggr;, monocyte chemoattractant protein-1, and tumor necrosis factor-&agr;. CD137 signaling promoted the production of inflammatory molecules, including monocyte chemoattractant protein-1, interleukin-6, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1, in endothelial cells. Stimulation of CD137 ligand signaling activated monocytes/macrophages and augmented the production of proinflammatory cytokines in atherosclerotic vessels. Conclusions— CD137/CD137 ligand signaling plays multiple roles in the progression of atherosclerosis, and thus, blockade of this pathway is a promising therapeutic target for the disease.

Mechanisms Involved in Synergistic Anticancer Immunity of Anti-4-1BB and Anti-CD4 Therapy

Anti-4-1BB-mediated anticancer effects were potentiated by depletion of CD4+ cells in B16F10 melanoma-bearing C57BL/6 mice. Anti-4-1BB induced the expansion and differentiation of polyclonal tumor-specific CD8+ T cells into IFN-gamma-producing CD11c+CD8+ T cells. The CD4+ cell depletion was responsible for facilitating immune cell infiltration into tumor tissues and removing some regulatory barriers such as T regulatory and indoleamine-2,3-dioxygenase (IDO)+ dendritic cells. Both monoclonal antibodies (mAb) contributed to the efficient induction of MHC class I molecules on the tumor cells in vivo. The effectors that mediated the anti-4-1BB effect were NKG2D+KLRG1+CD11c+CD8+ T cells that accumulated preferentially in the tumor tissues. Blocking NKG2D reduced the therapeutic effect by 20% to 26%, which may indicate that NKG2D contributes partially to tumor killing by the differentiated CD8+ T cells. Our results indicate that the combination of the two mAbs, agonistic anti-4-1BB and depleting anti-CD4, results in enhanced production of efficient tumor-killing CTLs, facilitation of their infiltration, and production of a susceptible tumor microenvironment.

4-1BB functions as a survival factor in dendritic cells.

4-1BB (CD137) is expressed on dendritic cells (DCs) and its biological function has remained largely unresolved. By comparing 4-1BB-intact (4-1BB+/+) and 4-1BB-deficient (4-1BB−/−) DCs, we found that 4-1BB was strongly induced on DCs during the maturation and that DC maturation was normal in the absence of 4-1BB. However, DC survival rate was low in the absence of 4-1BB, which was due to the decreased Bcl-2 and Bcl-xL in 4-1BB−/− DCs compared with 4-1BB+/+ DCs after DC maturation. Consistent with these results, 4-1BB−/− DCs showed an increased turnover rate in steady state and more severely decreased in spleen by injecting LPS compared with 4-1BB+/+ DCs. When OVA-pulsed DCs were adoptively transferred to recipient mice along with OVA-specific CD4+ T cells, 4-1BB−/− DCs did not properly migrate to the T cell zone in lymph nodes and poorly induced proliferation of CD4+ T cells, although both DCs comparably expressed functional CCR7. Eventually, 4-1BB−/− DCs generated a reduced number of OVA-specific memory CD4+ T cells compared with 4-1BB+/+ DCs. To further assess the role of 4-1BB on DC longevity in vivo, 4-1BB+/+ and 4-1BB−/− C57BL/6 were administrated with Propionibacterium acnes that develop liver granuloma by recruiting DCs. Number and size of granuloma were reduced in the absence of 4-1BB, but the inflammatory cytokine level was comparable between the mice, which implied that the granuloma might be reduced due to the decreased longevity of DCs. These results demonstrate that 4-1BB on DCs controls the duration, DC-T interaction, and, therefore, immunogenicity.

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-γ–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 ∼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. [Mol Cancer Ther 2009;8(2):469–78

Combination Therapy with Cisplatin and Anti–4-1BB: Synergistic Anticancer Effects and Amelioration of Cisplatin-Induced Nephrotoxicity

Anti-4-1BB and cisplatin showed synergistic anticancer effects in the CT-26 colon carcinoma model, producing complete regression in >60% of mice with either preventive or therapeutic treatment. The tumor-free mice formed long-lasting CD8(+) T cell-dependent tumor-specific memory. Anti-4-1BB induced rapid repopulation of T and B cells from cisplatin-mediated lymphopenia and differentiation and expansion of IFN-gamma(+)CD11c(+)CD8(+) T cells. Cisplatin facilitated expansion of naïve, effector, and memory CD8(+) T cells; combination therapy produced almost twice as many lymphoid cells as anti-4-1BB alone. Cisplatin increased 4-1BB on antigen-primed T cells and induced 4-1BB de novo on kidney tubular epithelium. Cross-linking of 4-1BB protected the T cells and kidney epithelium from cisplatin-mediated apoptosis by increasing expression of antiapoptotic molecules. Thus, cisplatin-induced 4-1BB provided a mechanism for amelioration of the lymphopenia and nephrotoxicity inherent in cisplatin treatment. We concluded that chemoimmunotherapy with anti-4-1BB and cisplatin is synergistic in tumor killing and prevention of organ-specific toxicity.

Reverse signaling through BAFF differentially regulates the expression of inflammatory mediators and cytoskeletal movements in THP-1 cells

Most members of the tumor‐necrosis factor superfamily have been reported to mediate reverse signaling in T cells, macrophages, and/or dendritic cells. BAFF has been reported to have important functions in B‐cell survival through forward signaling, but the presence of reverse signaling has not been explored. To investigate the possibility of BAFF‐mediated reverse signaling, the expression patterns and functions of BAFF were analyzed in monocytic cell lines including the human macrophage‐like cell line, THP‐1. The expression of BAFF and its receptors was detected in monocytic cell lines, either before or after activation. The stimulation of BAFF induced the expression of matrix metalloproteinase (MMP)‐9, interleukin ‐8, and transforming growth factor‐β‐induced gene product (βig‐h3) and the upregulation of intercellular adhesion molecule‐1 in THP‐1 cells. The activation of mitogen‐activated protein kinase extracellular signal‐regulated kinase1/2 and nuclear factor‐κB was required for these responses. In addition to these stimulatory effects, BAFF‐mediated signaling inhibited processes involving cytoskeletal movement such as phagocytosis and transmigration through blocking the activation of phosphatidylinositol 3‐kinase/AKT and Rac‐1. Furthermore, murine primary macrophage culture such as peritoneal macrophages expressed BAFF and stimulation of it induced the expression of MMP‐9. These observations show that the reverse signaling initiated from BAFF induces the expression of inflammatory mediators while suppressing the cytoskeletal movements associated with phagocytosis and transmigration.