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The ABCD1 protein, one of the four ATP-binding cassette (ABC) proteins in subfamily D, is located on the peroxisomal membrane and is involved in the transport of very long chain fatty acid (VLCFA)-CoA into peroxisomes. Its mutation causes X-linked adrenoleukodystophy (X-ALD): an inborn error of peroxisomal β-oxidation of VLCFA. Whether ABCD1 transports VLCFA-CoA as a CoA ester or free fatty acid is controversial. Recently, Comatose (CTS), a plant homologue of human ABCD1, has been shown to possess acyl-CoA thioesterase (ACOT) activity, and it is suggested that this activity is required for transport of acyl-CoA into peroxisomes. However, the precise transport mechanism is unknown. Here, we expressed human His-tagged ABCD1 in methylotrophic yeast, and characterized its ACOT activity and transport mechanism. The expressed ABCD1 possessed both ATPase and ACOT activities. The ACOT activity of ABCD1 was inhibited by p -chloromercuribenzoic acid ( p CMB), a cysteine-reactive compound. Furthermore, we performed a transport assay with ABCD1-containing liposomes using 7-nitro-2–1,3-benzoxadiazol-4-yl (NBD)-labeled acyl-CoA as the substrate. The results showed that the fatty acid produced from VLCFA-CoA by ABCD1 is transported into liposomes and that ACOT activity is essential during this transport process. We propose a detailed mechanism of VLCFA-CoA transport by ABCD1.

Tumor necrosis factor receptor-associated factors (TRAFs) are a family of adaptor proteins that transmit signals from immunoregulatory receptors—such as TNF receptors, Toll-like receptors, and interleukin receptors—to coordinate immune and inflammatory responses. Among them, TRAF5 is highly expressed in lymphocytes and implicated in obesity-associated inflammation, but its role in secondary lymphoid organs during chronic low-grade inflammation remains unclear. We examined splenic B and T cell phenotypes in wild-type (WT) and Traf5-deficient (KO) mice fed a high-fat diet (HFD). Although lymphocyte composition was broadly comparable, KO mice showed reduced spontaneous immunoglobulin G2c (IgG2c) production ex vivo—about 1.5-fold lower than WT. Notably, despite elevated TNF-α and CD40 ligand (CD40L) expression in HFD-fed KO splenocytes, IgG2c production remained diminished—about 1.9-fold lower than WT—upon soluble CD40L stimulation, indicating impaired CD40-mediated class-switch recombination (CSR). Consistently, B cells from KO mice on a normal diet exhibited reduced activation-induced cytidine deaminase (AID) expression—about 4.4-fold lower than WT—after CD40L stimulation, and decreased IgG2c secretion—about 6.6-fold lower—upon CD40L and IFN-γ co-stimulation in vitro. Collectively, these findings suggest that TRAF5 is involved in CD40-dependent CSR in B cells under inflammatory conditions and may contribute to sustaining adaptive immune responses during obesity-associated chronic inflammation.

There is growing evidence that tumor necrosis factor (TNF) receptor-associated factors (TRAFs) bind to unconventional membrane-bound receptors in many cell types and control their key signaling activity, in both positive and negative ways. TRAFs function in a variety of biological processes in health and disease, and dysregulation of TRAF expression or activity often leads to a patho-physiological outcome. We have identified a novel attribute of TRAF2 and TRAF5 in interleukin-6 (IL-6) receptor signaling in CD4 T cells. TRAF2 and TRAF5 are highly expressed by naïve CD4 T cells and constitutively bind to the signal-transducing receptor common chain gp130 via the C-terminal TRAF domain. The binding between TRAF and gp130 limits the early signaling activity of the IL-6 receptor complex by preventing proximal interaction of Janus kinases (JAKs) associated with gp130. In this reason, TRAF2 and TRAF5 in naïve CD4 T cells negatively regulate IL-6-mediated activation of signal transducer and activator of transcription 3 (STAT3) that is required for the development of IL-17-secreting CD4 T 17 cells. Indeed, -knockdown in differentiating CD4 T cells strongly promotes T 17 development. donor CD4 T cells exacerbate the development of neuroinflammation in experimental autoimmune encephalomyelitis (EAE) in wild-type recipient mice. In this review, we summarize the current understanding of the role for TRAF2 and TRAF5 in the regulation of IL-6-driven differentiation of pro-inflammatory CD4 T cells, especially focusing on the molecular mechanism by which TRAF2 and TRAF5 inhibit the JAK-STAT pathway that is initiated in the IL-6 receptor signaling complex.

BackgroundThe Toll-like receptor signaling pathway contributes to the regulation of intestinal homeostasis through interactions with commensal bacteria. Although the transcriptional regulator IκB-ζ can be induced by Toll-like receptor signaling, its role in intestinal homeostasis is still unclear.AimsTo investigate the role of IκB-ζ in gut homeostasis.MethodsDSS-administration induced colitis in control and IκB-ζ-deficient mice. The level of immunoglobulins in feces was detected by ELISA. The immunological population in lamina propria (LP) was analyzed by FACS.ResultsIκB-ζ-deficient mice showed severe inflammatory diseases with DSS administration in the gut. The level of IgM in the feces after DSS administration was less in IκB-ζ-deficient mice compared to control mice. Upon administration of DSS, IκB-ζ-deficient mice showed exaggerated intestinal inflammation (more IFN-g-producing CD4+ T cells in LP), and antibiotic treatment canceled this inflammatory phenotype.ConclusionIκB-ζ plays a crucial role in maintaining homeostasis in the gut.

OX40, a member of the tumor necrosis factor (TNF) receptor superfamily, is expressed on the surface of activated T cells. Upon interaction with its cognate ligand, OX40L, OX40 transmits costimulatory signals to antigen-primed T cells, promoting their activation, differentiation, and survival — processes essential for the establishment of adaptive immunity. Although the OX40-OX40L interaction has been extensively studied in the context of disease treatment, developing a substitute for the naturally expressed membrane-bound OX40L, particularly a multimerized OX40L trimers, that effectively regulates OX40-driven T cell responses remains a significant challenge. In this study, we successfully engineered soluble OX40L-fusion proteins capable of robustly activating OX40 on T cells. This was achieved by incorporating functional multimerization domains into the TNF homology domain of OX40L. These OX40L proteins bound to OX40, subsequently activated NF-κB signaling, and induced cytokine production by T cells in vitro . In vivo , mice treated with one of the OX40L-fusion proteins — comprising a single-chain OX40L trimer linked to the C-terminus of the human IgG1 Fc domain, forming a dimer of trimers — exhibited significantly enhanced clonal expansion of antigen-specific CD4 + T cells during the primary phase of the immune response. A comparable antibody-fusion single-chain TNF protein incorporating 4-1BBL, CD70 (CD27L), or GITRL in place of OX40L elicited similar in vivo T cell responses. Thus, we propose that optimizing the multimerization of OX40L proteins through innovative design strategies may facilitate the development of more effective agonists for targeted immunotherapies.

NF-κB activation is essential for T-cell responses, and costimulatory molecules in the TNF receptor (TNFR) superfamily are viewed as a major source of this signal. Although the TNFR family recruits TNFR-associated factor (TRAF) molecules leading to IKKα/β/γ activation, it is not clear whether simple binding of TRAFs explains why they are such strong activators of NF-κB and so important for T-cell immunity. We now show that one TNFR family member, OX40 (CD134), after ligation by OX40L, assembles a unique complex that not only contains TRAF2, RIP, and IKKα/β/γ but also CARMA1, MALT1, BCL10, and PKCO, molecules previously shown to regulate NF-KB activation through the T-cell receptor (TCR). The OX40 signalosome is formed in membrane microdomains irrespective of TCR engagement, and strongly promotes NF-κB activation only if CARMA1 and PKC6 are recruited. This NF-κB signal allows effector/memory T cells to survive when antigen is no longer available. Thus, by recruiting TCR-related intracellular molecules into the TRAF2 complex, OX40 provides the T cell with a high level of NF-κB activity needed for longevity.

T cell helper type 2 (Th2) differentiation is driven by a source of IL-4 receptor (IL-4R) that mobilizes IL-4R signaling pathways and the transcription factor GATA-3. Naïve CD4 cells can secrete IL-4 independently of IL-4R signals, but how this secretion is regulated is not understood. Here we demonstrate that costimulation through the tumor necrosis factor receptor family molecule OX40, in synergy with CD28, is essential for high levels of nuclear factor of activated T cells c1 to accumulate in the nucleus of a recently activated naive T cell. This action is not dependent on either IL-4R or IL-2R signals and results in OX40 controlling initial naïve T cell IL-4 transcription. OX40 signals subsequently enhance nuclear GATA-3 accumulation through an IL-4R-dependent action, leading to Th2 differentiation. These data show that, in the absence of an exogenous source of IL-4, OX40 provides a critical synergistic and temporal signal with other noncytokine receptors to modulate nuclear factor of activated T cells cl and to promote optimal Th2 generation.

The costimulatory signal regulated by the members of the tumor necrosis factor receptor (TNFR) superfamily expressed by T cells plays essential roles for T cell responses and has emerged as a promising target for cancer immunotherapy. However, it is unclear how the difference in TNFR costimulation contributes to T cell responses. In this study, to clarify the functional significance of four different TNFRs, OX40, 4-1BB, CD27 and GITR, we prepared corresponding single-chain TNF ligand proteins (scTNFLs) connected to IgG Fc domain with beneficial characteristics, i.e., Fc−scOX40L, Fc−sc4-1BBL, Fc−scCD27L (CD70) and Fc−scGITRL. Without intentional cross-linking, these soluble Fc−scTNFL proteins bound to corresponding TNFRs induced NF-kB signaling and promoted proliferative and cytokine responses in CD4+ and CD8+ T cells with different dose-dependencies in vitro. Mice injected with one of the Fc−scTNFL proteins displayed significantly augmented delayed-type hypersensitivity responses, showing in vivo activity. The results demonstrate that each individual Fc−scTNFL protein provides a critical costimulatory signal and exhibits quantitatively distinct activity toward T cells. Our findings provide important insights into the TNFR costimulation that would be valuable for investigators conducting basic research in cancer immunology and also have implications for T cell-mediated immune regulation by designer TNFL proteins.

The TNF receptor 4-1BB functions as a costimulatory molecule in T cells. Croft and colleagues show that binding of 4-1BB to its ligand regulates the production of dendritic cells by inhibiting myelopoiesis. The costimulatory molecule 4-1BB and its ligand 4-1BBL can control adaptive immunity, but here we show that their interaction also suppressed myelopoiesis. We found that 4-1BBL was expressed on hematopoietic stem cells, differentiating common myeloid progenitors and granulocyte-macrophage progenitors, and 4-1BB was inducible on activated myeloid progenitors. Steady-state numbers of granulocyte-macrophage progenitors, myeloid-lineage cells and mature dendritic cells were higher in 4-1BB- and 4-1BBL-deficient mice, indicative of a negative function, and we confirmed that result with bone marrow chimeras and in vitro , where the absence of interactions between 4-1BB and 4-1BBL led to enhanced differentiation into dendritic cell lineages. The regulatory activity was mediated by 4-1BBL, with binding by 4-1BB inhibiting differentiation of myeloid progenitors. Thus, 4-1BB and 4-1BBL have a previously unknown function in limiting myelopoiesis and the development of dendritic cells.

X-linked severe combined immunodeficiency (SCID-X1) is an inherited genetic immunodeficiency associated with mutations in the common cytokine receptor γ chain (γc) gene, and characterized by a complete defect of T and natural killer (NK) cells. Gene therapy for SCID-X1 using conventional retroviral (RV) vectors carrying the γc gene results in the successful reconstitution of T cell immunity. However, the high incidence of vector-mediated T cell leukemia, caused by vector insertion near or within cancer-related genes has been a serious problem. In this study, we established a gene therapy model of mouse SCID-X1 using a modified foamy virus (FV) vector expressing human γc. Analysis of vector integration in a human T cell line demonstrated that the FV vector integration sites were significantly less likely to be located within or near transcriptional start sites than RV vector integration sites. To evaluate the therapeutic efficacy, bone marrow cells from γc-knockout (γc-KO) mice were infected with the FV vector and transplanted into γc-KO mice. Transplantation of the FV-treated cells resulted in the successful reconstitution of functionally active T and B cells. These data suggest that FV vectors can be effective and may be safer than conventional RV vectors for gene therapy for SCID-X1.