In Vivo Induction of Regulatory T Cells Via CTLA-4 Signaling Peptide to Control Autoimmune Encephalomyelitis and Prevent Disease Relapse.

Regulatory T cells play a key role in immune tolerance to self-antigens, thereby preventing autoimmune diseases. However, no drugs targeting Treg cells have been approved for clinical trials yet. Here, a chimeric peptide is generated by conjugation of the cytoplasmic domain of CTLA-4 (ctCTLA-4) with dNP2 for intracellular delivery, dNP2-ctCTLA-4, and evaluated Foxp3 expression during Th0, Th1, Treg, and Th17 differentiation dependent on TGF-ß. The lysine motif of ctCTLA-4, not tyrosine motif, is required for Foxp3 expression for Treg induction and amelioration of experimental autoimmune encephalomyelitis (EAE). Transcriptome analysis reveals that dNP2-ctCTLA-4-treated T cells express Treg transcriptomic patterns with properties of suppressive functions. In addition, the molecular interaction between the lysine motif of ctCTLA-4 and PKC-η is critical for Foxp3 expression. Although both CTLA-4-Ig and dNP2-ctCTLA-4 treatment in vivo ameliorated EAE progression, only dNP2-ctCTLA-4 requires Treg cells for inhibition of disease progression and prevention of relapse. Furthermore, the CTLA-4 signaling peptide is able to induce human Tregs in vitro and in vivo as well as from peripheral blood mononuclear cells (PBMCs) of multiple sclerosis patients. These results collectively suggest that the chimeric CTLA-4 signaling peptide can be used for successful induction of regulatory T cells in vivo to control autoimmune diseases, such as multiple sclerosis.

Intranuclear delivery of the transcription modulation domain of Tbet-improved lupus nephritis in (NZB/NZW) F1 lupus-prone mice.

Excessive expression of Tbet and IFNγ is evidence of systemic lupus erythematosus (SLE) in lupus patients. In this study, the nucleus-transducible form of Transcription Modulation Domain (TMD) of Tbet (ntTbet-TMD), which is a fusion protein between Protein Transduction Domain Hph-1 (Hph-1-PTD) and the TMD of Tbet comprising DNA binding domain and isotype-specific domain, was generated to inhibit Tbet-mediated transcription in the interactomic manner. ntTbet-TMD was effectively delivered into the nucleus of the cells and specifically inhibited Tbet-mediated transcription without influencing the differentiation of other T cell subsets and signaling events for T cell activation. The severity of nephritis was significantly reduced by ntTbet-TMD as effectively as methylprednisolone in lupus-prone mice. The number of Th1, Th2 or Th17 cells and the secretion of their cytokines substantially decreased in the spleen and kidney of lupus-prone mice by ntTbet-TMD treatment. In contrast to methylprednisolone, the marked increase of Treg cells and the secretion of their immunosuppressive cytokine were detected in the spleen of (NZB/NZW) F1 mice treated with ntTbet-TMD. Thus, ntTbet-TMD can improve nephritis in lupus-prone mice by modulating the overall proinflammatory microenvironment and rebalancing T cell subsets, leading to new immune therapeutics for Th1-mediated autoimmune diseases.

Intranuclear interactomic inhibition of NF-κB suppresses LPS-induced severe sepsis.

Suppression of nuclear factor-κB (NF-κB) activation, which is best known as a major regulator of innate and adaptive immune responses, is a potent strategy for the treatment of endotoxic sepsis. To inhibit NF-κB functions, we designed the intra-nuclear transducible form of transcription modulation domain (TMD) of RelA (p65), called nt-p65-TMD, which can be delivered effectively into the nucleus without influencing the cell viability, and work as interactomic inhibitors via disruption of the endogenous p65-mediated transcription complex. nt-p65-TMD effectively inhibited the secretion of pro-inflammatory cytokines, including TNF-α, IL-1ß, or IL-6 from BV2 microglia cells stimulated by lipopolysaccharide (LPS). nt-p65-TMD did not inhibit tyrosine phosphorylation of signaling mediators such as ZAP-70, p38, JNK, or ERK involved in T cell activation, but was capable of suppressing the transcriptional activity of NF-κB without the functional effect on that of NFAT upon T-cell receptor (TCR) stimulation. The transduced nt-p65-TMD in T cell did not affect the expression of CD69, however significantly inhibited the secretion of T cell-specific cytokines such as IL-2, IFN-γ, IL-4, IL-17A, or IL-10. Systemic administration of nt-p65-TMD showed a significant therapeutic effect on LPS-induced sepsis model by inhibiting pro-inflammatory cytokines secretion. Therefore, nt-p65-TMD can be a novel therapeutics for the treatment of various inflammatory diseases, including sepsis, where a transcription factor has a key role in pathogenesis, and further allows us to discover new functions of p65 under normal physiological condition without genetic alteration.

RORγt-specific transcriptional interactomic inhibition suppresses autoimmunity associated with TH17 cells.

The nuclear hormone receptor retinoic acid-related orphan receptor gamma t (RORγt) is a transcription factor (TF) specific to TH17 cells that produce interleukin (IL)-17 and have been implicated in a wide range of autoimmunity. Here, we developed a novel therapeutic strategy to modulate the functions of RORγt using cell-transducible form of transcription modulation domain of RORγt (tRORγt-TMD), which can be delivered effectively into the nucleus of cells and into the central nerve system (CNS). tRORγt-TMD specifically inhibited TH17-related cytokines induced by RORγt, thereby suppressing the differentiation of naïve T cells into TH17, but not into TH1, TH2, or Treg cells. tRORγt-TMD injected into experimental autoimmune encephalomyelitis (EAE) animal model can be delivered effectively in the splenic CD4(+) T cells and spinal cord-infiltrating CD4(+) T cells, and suppress the functions of TH17 cells. The clinical severity and incidence of EAE were ameliorated by tRORγt-TMD in preventive and therapeutic manner, and significant reduction of both infiltrating CD4(+) IL-17(+) T cells and inflammatory cells into the CNS was observed. As a result, the number of spinal cord demyelination was also reduced after tRORγt-TMD treatment. With the same proof of concept, tTbet-TMD specifically blocking TH1 differentiation improved the clinical incidence of rheumatoid arthritis (RA). Therefore, tRORγt-TMD and tTbet-TMD can be novel therapeutic reagents with the natural specificity for the treatment of inflammatory diseases associated with TH17 or TH1. This strategy can be applied to treat various diseases where a specific transcription factor has a key role in pathogenesis.

Alleviation of abnormal synaptic neurotransmitter release by cell-permeable form of the truncated SNAP-25 upon transcutaneous delivery.

Abnormal releases of neurotransmitters result in movement disorders such as dystonia, chorea, tics, blepharospasm and wrinkle formation, and intra-muscular injection of Botulinum neurotoxin is widely used for the treatment of these complications. But it is potentially poisonous and must be intra-muscularly injected with precision by a well-trained physician. For novel therapeutic development with high safety profile and easy skin penetration for these complications, we generated Trans-X, a cell permeable form of the truncated SNAP-25 that is one of the SNARE complex for vesicle exocytosis of neuron. Upon topical administration, Trans-X efficiently penetrated through skin, reached the dermis layer and remained stable. Trans-X, which did not show any ocular or skin allergic sensitivity, can block the pre-synaptic neurotransmitter transport via acting as a competitive inhibitor of SNARE complex formation, and effectively induced muscle paralysis comparable to BOTOX(®) evaluated by measuring compound muscle action potential. Topical treatment of the facial skin with Trans-X in clinical study can prevent the wrinkle formation and improve the skin roughness. Therefore, our study suggests that Trans-X may be a convenient and effective medical and cosmetic treatment for local management of movement disorder without systemic toxicity.

Anti-inflammatory effect of quetiapine on collagen-induced arthritis of mouse.

Quetiapine is an atypical antipsychotic and has also been used in the treatment of depression. Since anti-inflammatory effects of antidepressants are well established, we hypothesized that quetiapine may also exert anti-inflammatory effects. Thus this study was designed to examine the anti-inflammatory effect of quetiapine in murine collagen-induced arthritis. Mice were immunized with collagen type II for the induction of arthritis and treated with quetiapine (10mg/kg) daily for 2weeks. Mice were divided into 3 groups: control, CIA, and CIA+quetiapine treatment. Arthritic index and paw thickness were used to compare severity of arthritis. In additions, radiological and histological assessments were employed. Anti-type II collagen-specific antibody, interleukin-6 (IL-6), interleukin-17 (IL-17), and prostaglandin E(2) (PGE(2)) were evaluated at the end of the treatment period. Both arthritic index and paw thickness were markedly improved in CIA+quetiapine treatment group compared with those in CIA groups (arthritic index; P<0.01, paw thickness; P<0.05). Radiologic assessment revealed decreased cartilage damage and bone erosion in CIA+quetiapine treatment group compared with those in CIA groups. Articular cartilage destruction observed in CIA group was not found in CIA+quetiapine group. The concentrations of anti-type II collagen-specific antibody, IL-6, IL-17, and PGE(2) in CIA+quetiapine group were significantly lower than those in CIA groups (P<0.05). Weight gain which is commonly observed with the treatment of antipsychotics was not observed. Taken together, these results suggest that quetiapine shows anti-inflammatory effects in murine collagen-induced arthritis.

Immunonucleochemistry: a new method for in situ detection of antigens in the nucleus of cells in culture.

The advancement of immunocytochemistry (ICC) allows one to observe detailed spatial distribution of cellular antigens, but, with some limitations. Using conventional ICC, it is difficult to distinguish the nuclear localization from cytoplasm, as two large subcellular compartments overlap on the z-axis. In this study, we have investigated whether in situ immunostaining of 'naked' nuclei could provide an unambiguous method for detection of nuclear antigens. We have designed a protocol that efficiently lyses plasmalemma, while keeping the nuclear envelope intact. The optimal condition for lysing the plasmalemma was 0.5% Nonidet P-40 for 5 min in both neuronal and non-neuronal cultured cells. Using this protocol, we could unambiguously isolate nuclear from cytoplasmic ICC signals. Since the present protocol has been designed for immunostaining of 'naked' nuclei from cultured or isolated cells, we have coined a new term to refer to this procedure as 'immunonucleochemistry' ('INC' for abbreviation).

Immunological factors relating to the antitumor effect of temozolomide chemoimmunotherapy in a murine glioma model.

In this study, we investigated the potential of combined treatment with temozolomide (TMZ) chemotherapy and tumor antigen-pulsed dendritic cells (DCs) and the underlying immunological factors of TMZ chemoimmunotherapy with an intracranial GL26 glioma animal model. The combined treatment enhanced the tumor-specific immune responses and prolonged the survival more effectively than either single therapy in GL26 tumor-bearing animals. Apoptosis was induced in the tumors of the animals by the treatment with TMZ. Calreticulin (CRT) surface exposure was detected by immunofluorescence staining of TMZ-treated GL26 cells. TMZ chemotherapy increased tumor antigen cross-priming from tumor cells, leading to cross-priming of tumor antigen-specific CD4(+) T cells and CD8(+) T cells. This chemotherapy appeared to suppress the frequency of CD4(+) CD25(+) regulatory T cells (Treg). Moreover, this combined therapy resulted in an increase in the tumor infiltration of CD4(+) and CD8(+) T cells. Collectively, the findings of this study provide evidence that the combination of TMZ chemotherapy and treatment with DC-based vaccines leads to the enhancement of antitumor immunity through increased tumor-specific immune responses via the cross-priming of apoptotic tumor cell death mediated by CRT exposure and, in part, the suppression of Treg. Therefore, CRT exposure, regulatory T cells, and cross-priming by TMZ chemotherapy may be immunological factors related to the enhancement of the antitumor effects of chemoimmunotherapy in an experimental brain tumor model.

Enhanced antitumour immunity by combined use of temozolomide and TAT-survivin pulsed dendritic cells in a murine glioma.

Although chemotherapy remains among the best treatment options for most cancers, adjuvant therapies such as dendritic cell (DC)-based immunotherapy have been added to treatment protocols to destroy residual tumour cells. Combination treatment with low-dose temozolomide (TMZ) chemotherapy followed by vaccination with TAT-survivin-pulsed DCs enhanced T-cell responses specific for survivin and improved survival rate, as compared with DC alone or TMZ alone. Moreover, antigen-specific immunity appears to be mediated by CD8(+) T cells, as determined by in vitro T-cell subset depletion. These studies demonstrated that a combination of low-dose TMZ chemotherapy and TAT-based DC immunotherapy may be a novel strategy for safe and effective treatment of malignant gliomas.

Cross-priming by temozolomide enhances antitumor immunity of dendritic cell vaccination in murine brain tumor model.

Although chemotherapy remains among the best treatment options for most cancers, adjuvant therapies such as dendritic cell (DC)-based immunotherapy have been added to treatment protocols to destroy residual tumor cells. IFN-gamma secreting T cells specific for survivin was found after temozolomide (TMZ) treatment in C57BL/6 mice intracranial (i.c.) inoculated with GL26 cells. Furthermore, combination treatment with low-dose TMZ (2.5mg/kg/day, i.p.) chemotherapy followed by vaccination with survivin RNA-transfected DCs (1 x 10(6)cells/mouse, s.c.) enhanced T cells responses specific for survivin and improved survival rate compared with DC vaccination alone or TMZ treatment alone in tumor inoculated mice. However, these enhancements of T cells responses by TMZ treatment were not observed in mice without tumor inoculation. These results suggested that cross-priming by TMZ may enhance antitumor immunity of DC vaccination in murine brain tumor model.

Enhancement of anti-tumor immunity specific to murine glioma by vaccination with tumor cell lysate-pulsed dendritic cells engineered to produce interleukin-12.

AIM: The aim of this study was to develop an immunotherapy specific to a malignant glioma by examining the efficacy of glioma tumor-specific cytotoxic T lymphocytes (CTL) as well as the anti-tumor immunity by vaccination with dendritic cells (DC) engineered to express murine IL-12 using adenovirus-mediated gene transfer and pulsed with a GL26 glioma cell lysate (AdVIL-12/DC+GL26) was investigated. EXPERIMENT1: For measuring CTL activity, splenocytes were harvested from the mice immunized with AdVIL-12/DC+GL26 and restimulated with syngeneic GL26 for 7 days. The frequencies of antigen-specific cytokine-secreting T cell were determined with mIFN-gamma ELISPOT. The cytotoxicity of CTL was assessed in a standard 51Cr-release assay. For the protective study in the subcutaneous tumor model, the mice were vaccinated subcutaneously (s.c) with 1x10(6) AdVIL-12/DC+GL26 in the right flanks on day -21, -14 and -7. On day 7, the mice were challenged with 1x10(6) GL26 tumor cells in the shaved left flank. For a protective study in the intracranial tumor model, the mice were vaccinated with 1x10(6) AdVIL-12/DC+GL26 s.c in the right flanks on days -21, -14 and -7. Fresh 1x10(4) GL26 cells were inoculated into the brain on day 0. To prove a therapeutic benefit in established tumors, subcutaneous or intracranial GL26 tumor-bearing mice were vaccinated s.c with 1x10(6) AdVIL-12/DC+GL26 on day 5, 12 and 19 after tumor cell inoculation. RESULTS: Splenocytes from the mice vaccinated with the AdVIL-12/DC+GL26 showed enhanced induction of tumor-specific CTL and increased numbers of IFN-gamma: secreting T cells by ELISPOT. Moreover, vaccination of AdVIL-12/DC+GL26 enhanced the induction of anti-tumor immunity in both the subcutaneous and intracranial tumor models. CONCLUSIONS: These preclinical model results suggest that DC engineered to express IL-12 and pulsed with a tumor lysate could be used in a possible immunotherapeutic strategy for malignant glioma.