Yun Hee Cho

TL1A Is a TNF-like Ligand for DR3 and TR6/DcR3 and Functions as a T Cell Costimulator

DR3 is a death domain-containing receptor that is upregulated during T cell activation and whose overexpression induces apoptosis and NF-kappaB activation in cell lines. Here we show that an endothelial cell-derived TNF-like factor, TL1A, is a ligand for DR3 and decoy receptor TR6/DcR3 and that its expression is inducible by TNF and IL-1alpha. TL1A induces NF-kappaB activation and apoptosis in DR3-expressing cell lines, while TR6-Fc protein antagonizes these signaling events. Interestingly, in T cells, TL1A acts as a costimulator that increases IL-2 responsiveness and secretion of proinflammatory cytokines both in vitro and in vivo. Our data suggest that interaction of TL1A with DR3 promotes T cell expansion during an immune response, whereas TR6 has an opposing effect.

Tumor Necrosis Factor (TNF) Receptor Superfamily Member TACI Is a High Affinity Receptor for TNF Family Members APRIL and BLyS

An expression cloning approach was employed to identify the receptor for B-lymphocyte stimulator (BLyS) and identified the tumor necrosis factor receptor superfamily member TACI as a BLyS-binding protein. Expression of TACI in HEK293T cells confers on the cells the ability to bind BLyS with subnanomolar affinity. Furthermore, a TACI-Fc fusion protein recognizes both the cleaved, soluble form of BLyS as well as the membrane BLyS present on the cell surface of a recombinant cell line. TACI mRNA is found predominantly in B-cells and correlates with BLyS binding in a panel of B-cell lines. We also demonstrate that TACI interacts with nanomolar affinity with the BLyS-related tumor necrosis factor homologue APRIL for which no clear in vivo role has been described. BLyS and APRIL are capable of signaling through TACI to mediate NF-κB responses in HEK293 cells. We conclude that TACI is a receptor for BLyS and APRIL and discuss the implications for B-cell biology.

BLyS inhibition eliminates primary B cells but leaves natural and acquired humoral immunity intact

We have used an inhibiting antibody to determine whether preimmune versus antigen-experienced B cells differ in their requisites for BLyS, a cytokine that controls differentiation and survival. Whereas in vivo BLyS inhibition profoundly reduced naïve B cell numbers and primary immune responses, it had a markedly smaller effect on memory B cells and long-lived plasma cells, as well as secondary immune responses. There was heterogeneity within the memory pools, because IgM-bearing memory cells were sensitive to BLyS depletion whereas IgG-bearing memory cells were not, although both were more resistant than naïve cells. There was also heterogeneity within B1 pools, as splenic but not peritoneal B1 cells were diminished by anti-BLyS treatment, yet the number of natural antibody-secreting cells remained constant. Together, these findings show that memory B cells and natural antibody-secreting cells are BLyS-independent and suggest that these pools can be separately manipulated.

Modulation of T-cell responses to alloantigens by TR6/DcR3

TR6 (DcR3) is a new member of the TNF receptor (TNFR) family that lacks a transmembrane domain in its sequence, indicating that it is a secreted molecule. TR6 can bind to FasL and prevent FasL-induced apoptosis; it can also associate with LIGHT, another TNF family member. The role of TR6 in immune responses was investigated in this study. According to flow cytometry, recombinant human TR6-Fc binds to human LIGHT expressed on 293 cells or on activated human T cells and competes with the LIGHT receptor TR2 for the binding to LIGHT on these cells. Human TR6 could cross-react with mouse LIGHT in immunoprecipitation. TR6-Fc also downregulates cytotoxic T lymphocyte activity in vitro and graft-versus-host responses in mice. Moreover, TR6-Fc modulates lymphokine production by alloantigen-stimulated mouse T cells. TR6-Fc ameliorated rejection response to mouse heart allograft. These results indicate that TR6 can dampen T-cell responses to alloantigens. Such regulatory effects of TR6 probably occur via interference with interaction between pairs of related TNF and TNFR family members, LIGHT/TR2 being one of the possible candidate pairs.

Regulatory Effect of IFN-κ, A Novel Type I IFN, On Cytokine Production by Cells of the Innate Immune System

IFN-κ is a recently identified type I IFN that exhibits both structural and functional homology with the other type I IFN subclasses. In this study, we have investigated the effect of IFN-κ on cells of the innate immune system by comparing cytokine release following treatment of human cells with either IFN-κ or two recombinant IFN subtypes, IFN-β and IFN-α2a. Although IFN-α2a failed to stimulate monocyte cytokine secretion, IFN-κ, like IFN-β, induced the release of several cytokines from both monocytes and dendritic cells, without the requirement of a costimulatory signal. IFN-κ was particularly effective in inhibiting inducible IL-12 release from monocytes. Unlike IFN-β, IFN-κ did not induce release of IFN-γ by PBL. Expression of the IFN-κ mRNA was observed in resting dendritic cells and monocytes, and it was up-regulated by IFN-γ stimulation in monocytes, while IFN-β mRNA was minimally detectable under the same conditions. Monocyte and dendritic cell expression of IFN-κ was also confirmed in vivo in chronic lesions of psoriasis vulgaris and atopic dermatitis. Finally, biosensor-based binding kinetic analysis revealed that IFN-κ, like IFN-β, binds strongly to heparin (Kd: 2.1 nM), suggesting that the cytokine can be retained close to the local site of production. The pattern of cytokines induced by IFN-κ in monocytes, coupled with the unique induction of IFN-κ mRNA by IFN-γ, indicates a potential role for IFN-κ in the regulation of immune cell functions.

Abstract 3861: Fusion toxin BLyS-gelonin inhibits growth of B-NHL cell lines in vitro and in vivo

B lymphocyte stimulator (BLyS) is a member of the TNF superfamily of cytokines. BLyS is expressed by cells of myeloid origin and is one of many soluble factors that regulate human B-cell activation. The biological activity of BLyS is mediated by three cell surface receptors: B-cell-activating factor-receptor (BAFF-R/BR3), transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) and B-cell maturation antigen (BCMA). These receptors are expressed by normal and malignant B-cells at various stages of development. To target malignant B cells expressing BLyS receptors, the recombinant plant-derived toxin gelonin was fused to the N-terminus of BLyS to generate the fusion toxin BLyS-gel. Gelonin is an N-glycosidase that removes a critical adenine from eukaryotic 28S rRNA, disrupting ribosome function and inhibiting protein synthesis. Importantly, gelonin is not toxic to cells unless coupled with a targeting moiety that can enter the cell. BLyS-gel was used to treat a panel of nearly 50 malignant non-Hodgkin lymphoma (NHL) cell lines expressing BLyS receptors. NHL subtypes mantle cell lymphoma (MCL), diffuse large B cell lymphoma (DLBCL) and B cell precursor-acute lymphocytic leukemia (BCP-ALL) were preferentially sensitive to BLyS-gel mediated cytotoxicity, with EC50 values in the low picomolar range. BLyS-gel cytotoxicity was mediated primarily by BR3 and TACI in sensitive cell lines; however, cell surface expression of these BLyS receptors did not necessarily confer sensitivity to BLyS-gel. The internalization of BLyS-gel was confirmed in both sensitive and resistant cell lines, indicating resistance was not due to a lack of internalization. As expected, BLyS-gel treatment inhibited protein synthesis in sensitive, but not resistant cell lines. In most cell lines, cell death seemed to be mediated by the “ribotoxic stress response.” This response involves activation of p38 MAPK and JNK/SAPK, and BLyS-gel mediated cytotoxicity was inhibited by the p38/JNK inhibitor SB203580. Finally, BLyS-gel treatment significantly prolonged survival in three distinct disseminated xenograft models (BCP-ALL, DLBCL & MCL) in SCID mice, and BLyS-gel was shown to specifically localize to sites of disease. Together, these findings suggest BLyS has potential as a targeting ligand for the therapeutic delivery of toxins and/or drugs directly to malignant B cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3861. doi:1538-7445.AM2012-3861