Chang Hee Kim

4-1BB Triggering Ameliorates Experimental Autoimmune Encephalomyelitis by Modulating the Balance between Th17 and Regulatory T Cells

Agonistic anti–4-1BB Ab is known to ameliorate experimental autoimmune encephalomyelitis. 4-1BB triggering typically leads to the expansion of CD8+ T cells, which produce abundant IFN-γ, and this in turn results in IDO-dependent suppression of autoimmune responses. However, because neutralization of IFN-γ or depletion of CD8+ T cell only partially abrogates the effect of 4-1BB triggering, we sought to identify an additional mechanism of 4-1BB–triggered suppression of autoimmune responses using IFN-γ- or IFN-γR–deficient mice. 4-1BB triggering inhibited the generation of Th17 cells that is responsible for experimental autoimmune encephalomyelitis induction and progression, and increased Foxp3+CD4+ regulatory T (Treg) cells, particularly among CD4+ T cells. This was not due to a direct effect of 4-1BB signaling on CD4+ T cell differentiation: 4-1BB signaling not only reduced Th17 cells and increased Treg cells in wild-type mice, which could be due to IFN-γ production by the CD8+ T cells, but also did so in IFN-γ–deficient mice, in that case by downregulating IL-6 production. These results show that although secondary suppressive mechanisms evoked by 4-1BB triggering are usually masked by the strong effects of IFN-γ, 4-1BB signaling seems to modulate autoimmune responses by a number of mechanisms, and modulation of the Th17 versus Treg cell balance is one of those mechanisms.

Origins and functional basis of regulatory CD11c+CD8+ T cells

Previously, we showed that CD11c defines a novel subset of CD8+ T cells whose in vivo activity is therapeutic for arthritis; however, the mechanisms directing their development, identity of their precursors, and basis of their effector function remain unknown. Here, we show that the novel subset develops from CD11csurface−CD8+ T cells and undergoes robust expansion in an antigen‐ and 4‐1BB (CD137)‐dependent manner. CD11c+CD8+ T cells exist in naïve mice (<3%) with limited suppressive activity. Once activated, they suppress CD4+ T cells in vivo and in vitro. Suppression of CD4+ by CD11c+CD8+ T cells is related to an increase in IDO activity induced in competent cells via the general control non‐derepressible‐2 pathway. CD11c+CD8+ T cells are refractory to the effect of IDO but constrict in a novel 1‐methyl D,L‐tryptophan‐dependent mechanism resulting in reversal of their suppressive effects. Thus, our data uncover, for the first time, the origin, development, and basis of the suppressive function of this novel CD11c+CD8+ T‐cell subpopulation that has many signature features of Treg.

Authentic GITR Signaling Fails To Induce Tumor Regression unless Foxp3+ Regulatory T Cells Are Depleted

The glucocorticoid-induced TNFR family–related protein (GITR, TNFRSF18, CD357) is expressed on effector and regulatory T (Treg) cells. Previous studies demonstrated that GITR triggering by anti-GITR mAb enhanced T and B cell–mediated immune responses. GITR-deficient T cells, however, also proliferate more than normal T cells, and this effect is unexplained. Because the activities of mAbs are controlled by their Fc regions, the true effect of GITR signaling needs to be determined by examining its interaction with authentic ligand. Therefore, we generated a pentamerized form of the GITRL extracellular domain (pGITRL) for ligation to GITR and compared its effect on T cells with that of anti-GITR mAb. The pGITRL was more effective than anti-GITR mAb in enhancing the proliferation of effector and regulatory cells in vitro and in vivo. Nonetheless, the growth of MC38 adenocarcinoma cells in vivo was only suppressed for initial 15 d by pGITRL, whereas it was suppressed indefinitely by anti-GITR mAb. Detailed analysis revealed that pGITRL induced extensive proliferation of Foxp3+CD4+ Treg cells and led to the accumulation of activated Treg cells in tumor tissue and draining lymph nodes. Because GITR signaling could not neutralize the suppressive activity of activated Treg cells, pGITRL seems to lose its adjuvant effect when sufficient activated Treg cells have accumulated in the lymph nodes and tumor tissue. Indeed, the antitumor effects of pGITRL were markedly enhanced by depleting CD4+ cells. These results suggest that GITR signaling has stimulatory effects on effector T cells and inhibitory effects through Treg cells.

Peripheral 4-1BB Signaling Negatively Regulates NK Cell Development through IFN-γ

Stimulation of 4-1BB (CD137) was shown to produce strong anticancer effects in vivo. In contrast, 4-1BB–deficient (4-1BB−/−) B6 mice are remarkably resistant to tumor growth. We set out to determine the mechanisms involved in these seemingly contradictory observations. We found that the therapeutic effects of 4-1BB triggering were mainly dependent on CD8+ T cells and partially on NK cells, whereas CD8+ T and NK cells were equally needed to suppress tumor growth in 4-1BB−/− mice. Cellular analysis showed that the frequency and number of NK cells in the spleen and bone marrow were decreased by 4-1BB triggering but were increased in the absence of 4-1BB signaling in tumor-challenged mice. The 4-1BB–mediated downregulation of NK cell development was primarily dependent on IFN-γ, which was produced by peripheral CD8+ T and NK cells. The suppression of NK cell development by 4-1BB–mediated IFN-γ production occurred in the bone marrow. As 4-1BB signaling increased in the periphery, more CD8+ T cells but fewer NK cells contributed to the antitumor immunity. As 4-1BB signaling decreased, more NK cells participated in the antitumor immunity. We conclude that 4-1BB signaling results in a shift of the dominant type of immune cell in antitumor immunity from the innate NK cell to the adaptive CD8+ T cell and that the level of IFN-γ is critical for this 4-1BB–mediated shift.

4-1BB Signaling Activates the T Cell Factor 1 Effector/β-Catenin Pathway with Delayed Kinetics via ERK Signaling and Delayed PI3K/AKT Activation to Promote the Proliferation of CD8+ T Cells

4-1BB (CD137), an inducible costimulatory molecule, strongly enhances the proliferation and effector function of CD8+ T cells. Since the serine/threonine kinase, glycogen synthase kinase-3 (GSK-3), is involved in a variety of signaling pathways of cellular proliferation, migration, immune responses, and apoptosis, we examined whether 4-1BB signaling activates GSK-3/β-catenin signaling and downstream transcription factors to enhance the proliferation of CD8+ T cells. 4-1BB signaling induces rapid activation of ERK and IκB degradation, and shows delayed activation of AKT at 24 h post 4-1BB stimulation on anti-CD3 activated T cells. ERK and AKT signals were required for sustained β-catenin levels by inactivating GSK-3, which was also observed with delayed kinetics after 4-1BB stimulation. As a transcriptional partner of β-catenin, 4-1BB signaling decreased levels of FOXO1 and increased levels of stimulatory TCF1 in CD8+ T cells at 2–3 days but not at early time points after 4-1BB engagement. The enhanced proliferation of CD8+ T cells due to 4-1BB signaling was completely abolished by treatment with the TCF1/β-catenin inhibitor quercetin. These results show that 4-1BB signaling enhances the proliferation of activated CD8+ T cells by activating the TCF1/β-catenin axis via the PI3K/AKT/ERK pathway. As effects of 4-1BB on AKT, FOXO1, β-catenin and GSK-3β showed delayed kinetics it is likely that an intervening molecule induced by 4-1BB and ERK signaling in activated T cells is responsible for these effects. These effects were observed on CD8+ but not on CD4+ T cells. Moreover, 4-1BB appeared to be unique among several TNFRs tested in inducing increase in stimulatory over inhibitory TCF-1.

PDCA Expression by B Lymphocytes Reveals Important Functional Attributes

We have demonstrated in this study the existence of a PDCA-expressing functional B cell population (PDCA+ B lymphocytes), which differentiates from activated conventional B (PDCA−IgM+) lymphocytes. Stimulation with anti-μ, LPS, CpG oligodeoxynucleotide, HSV-1, or CTLA-4 Ig activates the PDCA+ B lymphocytes, leading to cell division and induction of type I IFNs and IDO. Notably, the PDCA+ B lymphocytes are capable of Ag-specific Ab production and Ig class switching, which is corroborated by transfer experiments in B- and PDCA+ B lymphocyte-deficient μMT mice. Importantly, in lupus-prone MRL-Faslpr mice, PDCA+ B lymphocytes remain the principal source of autoantibodies. The PDCA+ B lymphocytes have phenotypes with plasmacytoid dendritic cells, but are a distinct cell population in that they develop from C-kit+B220+ pro-B precursors. Thus, our data suggest that not all PDCA+ cells are dendritic cell-derived plasmacytoid dendritic cells and that a significant majority is the PDCA+ B lymphocyte population having distinct phenotype and function.

4-1BB-based isolation and expansion of CD8+ T cells specific for self-tumor and non-self-tumor antigens for adoptive T-cell therapy.

Adoptive T-cell therapy is a promising approach to the immunotherapy of cancer, but for it to be a general cancer therapy a simple and standardized procedure for producing tumor-specific CD8+ T cells is needed. On the basis of a unique property of 4-1BB (CD137), the selective expression on activated T cells, we have developed a simple and practical protocol to produce antigen-specific CD8+ T cells from peripheral blood mononuclear cells. We have proved the feasibility of this procedure by isolating and expanding cytomegalovirus-specific CD8+ T cells, and applied the procedure to produce Epstein-Barr virus (EBV)-specific CD8+ T cells. By using this procedure, we could readily produce 109–1010 antigen-specific CD8+ T cells from 30 to 50 mL of blood in about 4 weeks. Moreover, our protocol allowed us to produce, from solid cancer patients, CD8+ T cells that were specific for self/tumor antigens such as human telomerase reverse transcriptase (hTERT). It is interesting to note that, we were unable to amplify hTERT-specific CD8+ T cells from healthy donors. Our protocol can be readily translated into cGMP-compliant production and is currently being used to produce EBV-specific CD8+ T cells for phase I clinical trial. We believe that our method will provide a practical and effective option for adoptive T-cell therapy in the clinic.

Regulation of Mouse 4-1BB Expression: Multiple Promoter Usages and a Splice Variant

The expression of 4-1BB has been known to be dependent on T cell activation. Recent studies have, however, revealed that 4-1BB expression is not restricted to T cells. We sought to determine the molecular basis for the differential gene expression. Here we report the expression pattern of two mouse 4-1BB transcripts, type I and type II. Whereas the type I transcript was specifically expressed on immune organ as previously reported, the type II transcript was ubiquitously expressed in tissues and various cell lines. However, both type I and type II transcript were highly induced on activated T cells. Primer extension assay of the two 4-1BB transcripts suggested that mouse 4-1BB had more than two transcripts. Using luciferase assay we have identified three promoter regions (PI, PII and PIII), which located on upstream region of second exon 1, first exon 1, and exon 2, respectively. In particular, the type I transcript was preferentially induced when naïve T cells are stimulated by anti-CD3 monoclonal antibody (mAb) since NF-κB specifically binds to the putative NF-κB element of PI. We have also shown that a splice variant, in which the transmembrane domain was deleted, could inhibit 4-1BB signaling. The splicing variant was highly induced by TCR stimulation. Our results reveal 4-1BB also has a negative regulation system through soluble 4-1BB produced from a splice variant induced under activation conditions.

Exposure of a distinct PDCA-1+ (CD317) B cell population to agonistic anti-4-1BB (CD137) inhibits T and B cell responses both in vitro and in vivo.

4-1BB (CD137) is an important T cell activating molecule. Here we report that it also promotes development of a distinct B cell subpopulation co-expressing PDCA-1. 4-1BB is expressed constitutively, and its expression is increased when PDCA-1+ B cells are activated. We found that despite a high level of surface expression of 4-1BB on PDCA-1+ B cells, treatment of these cells with agonistic anti-4-1BB mAb stimulated the expression of only a few activation markers (B7-2, MHC II, PD-L2), cytokines (IL-12p40/p70), and chemokines (MCP-1, RANTES), as well as sTNFR1, and the immunosuppressive enzyme, IDO. Although the PDCA-1+ B cells stimulated by anti-4-1BB expressed MHC II at high levels and took up antigens efficiently, Ig class switching was inhibited when they were pulsed with T-independent (TI) or T-dependent (TD) Ags and adoptively transferred into syngeneic recipients. Furthermore, when anti-4-1BB-treated PDCA-1+ B cells were pulsed with OVA peptide and combined with Vα2+CD4+ T cells, Ag-specific cell division was inhibited both in vitro and in vivo. Our findings suggest that the 4-1BB signal transforms PDCA-1+ B cells into propagators of negative immune regulation, and establish an important role for 4-1BB in PDCA-1+ B cell development and function.

Unified immune modulation by 4-1BB triggering leads to diverse effects on disease progression in vivo.

4-1BB (CD137) is a powerful T-cell costimulatory molecule in the treatment of virus infections and tumors, but recent studies have also uncovered regulatory functions of 4-1BB signaling. Since 4-1BB triggering suppresses autoimmunity by accumulating indoleamine 2,3-dioxygenase (IDO) in dendritic cells (DCs) in an interferon (IFN)-γ-dependent manner, we asked whether similar molecular and cellular changes were induced by 4-1BB triggering in virus-infected mice. 4-1BB triggering increased IFN-γ and IDO, and suppressed CD4(+) T cells, in C57BL/6 mice infected with the type 1 KOS strain of Herpes simplex virus (HSV-1), as it does in an autoimmune disease model. Detailed analysis of the CD4(+) T suppression showed that freshly activated CD62L(high) T cells underwent apoptosis in the early phase of suppression, and CD62L(low) effector/memory T cells in the later phase. Although 4-1BB triggering resulted in similar cellular changes - increased CD8(+) T and decreased CD4(+) T cells, it had different effects on mortality in mice infected with HSV-1 RE, influenza, and Japanese encephalitis virus (JEV); it increased mortality in influenza-infected mice but decreased it in JEV-infected mice. Since the dominant type of immune cell generated to protect the host was different for each virus - CD4(+) T cells and neutrophils in HSV-1 RE infection, both CD4(+) T and CD8(+) T cells in influenza infection, and a crucial role for B cells in JEV infection, 4-1BB triggering resulted in different therapeutic outcomes. We conclude that the therapeutic outcome of 4-1BB triggering is determined by whether the protective immunity generated against the virus was beneficially altered by the 4-1BB triggering.