Positive conversion of negative signaling of CTLA4 potentiates antitumor efficacy of adoptive T-cell therapy in murine tumor models.

Cytotoxic T lymphocyte-associated antigen 4 (CTLA4) has been known to be a strong tolerance-inducing inhibitory receptor on T-cell surface. Systemic blocking of CTLA4 function with blocking antibodies has been regarded as an attractive strategy to enhance antitumor immunity. However, this strategy accompanies systemic autoimmune side effects that are sometimes problematic. Therefore, we developed a novel CTLA4 mutant that could be expressed in tumor antigen-specific T cells to enhance antitumor effect without systemic autoimmunity. This mutant, named CTLA4-CD28 chimera, consists of extracellular and transmembrane domains of CTLA4, linked with cytoplasmic CD28 domain. Overexpression of CTLA4-CD28 chimera in T cells delivered stimulatory signals rather than inhibitory signals of CTLA4 and significantly enhanced T-cell reactivity. Although this effect was observed in both CD4 and CD8 T cells, the effect on CD4 T cells was predominant. CTLA4-CD28 chimera gene modification of CD4 T cells significantly enhanced antitumor effect of unmodified CD8 T cells. Nonetheless, the gene modification of CD8 T cells along with CD4 T cells further maximized antitumor effect of T cells in 2 different murine tumor models. Thus, CTLA4-CD28 chimera gene modification of both tumor antigen-specific CD4 and CD8 T cells would be an ideal way of modulating CTLA4 function to enhance tumor-specific T-cell reactivity.

Adenovirus expressing both thymidine kinase and soluble PD1 enhances antitumor immunity by strengthening CD8 T-cell response.

Adenoviruses harboring the herpes simplex virus thymidine kinase (HSVtk) gene under the regulation of a trans-splicing ribozyme targeting human telomerase reverse transcriptase (hTERT-TR) show marked and specific antitumor activity. In addition to inducing tumor cell death by direct cytotoxicity, it is becoming clear that HSVtk also induces antitumor immunity. Programmed death ligand 1 (PD-L1) expressed on tumor cell surfaces mediates tumor-induced immunoresistance by inhibiting PD1-expressing tumor-infiltrating T cells. Here, we explored whether a soluble form of PD1 (sPD1-Ig), which blocks PD-L1, could synergize with TERT-TR-regulated HSVtk to enhance the adenoviral therapeutic efficacy by boosting antitumor immunity. Tumor antigen released by HSVtk-transduced tumors successfully primed tumor antigen-specific CD8 T cells via dendritic cells (DC). Regression of murine tumors was markedly enhanced when sPD1-Ig was incorporated into the adenovirus as compared with a single-module adenovirus expressing only HSVtk. This effect was abolished by CD8 T-cell depletion. Consistent with this, following adoptive transfer of tumor antigen-specific CD8 T cells into tumor-bearing Rag1(-/-) mice, dual-module adenovirus significantly enhanced CD8 T cell-mediated tumor rejection. In addition, secondary tumor challenge at a distal site was completely suppressed in mice treated with a dual-module adenovirus. These results suggest that a dual-targeting strategy to elicit both tumor antigen priming and tumor-induced immunoresistance enhances CD8 T cell-mediated antitumor immunity.

Transglutaminase 2 exacerbates experimental autoimmune encephalomyelitis through positive regulation of encephalitogenic T cell differentiation and inflammation.

The increased activity of transglutaminase 2 (TG2) in various inflammatory and fibrotic conditions results in the development of numerous disease processes. Experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, is an inflammatory and demyelinating disease of the central nervous system and is mediated by many inflammatory cytokines and mediators. We examined the role of TG2 in encephalitogenic CD4(+) T cell responses and EAE development using mice lacking TG2 (TG2(-/-)). TG2(-/-) mice showed decreased disease severity as compared with wild-type (WT) mice. Treatment with cysteamine, a TG2 inhibitor, ameliorated disease severity in WT mice. Exacerbated disability in WT mice resulted from the increased infiltration of cytokine-producing CD4(+) T cells and sustained expression of inflammatory cytokines and mediators in the lesion. The increased number of IL-17- and IFN-γ-producing cells in the spinal cord resulted from peripheral expansion of these cells after immunization with myelin-derived antigen. In vitro differentiation of WT and TG2(-/-) splenocytes revealed that proliferation and activation-induced cell death did not differ, but differentiation into IL-17- or IFN-γ-producing cells was increased in WT mice. Adoptive transfer experiments revealed that pathogenic CD4(+) T cell differentiation and disease progression were caused by both the T cell-intrinsic and -extrinsic effects of TG2. This study is the first to report a pathogenic role for TG2 in the EAE progress and suggests that therapeutic targeting of TG2 may be effective against multiple sclerosis.

Stereoselective synthesis and osteogenic activity of subglutinols A and B.

Since clinically approved immunosuppressive drugs (e.g., cyclosporin A, FK506) possess dose-dependent biphasic effects that cause undesirable side effects on bone structure, including osteopenia, osteoporosis, and increased incidence of bone fractures, considerable effort has been devoted to the identification of immunosuppressive drugs that promote bone formation in a dose-dependent manner. Herein, we report the stereoselective synthesis of subglutinols A and B and present initial biological data showing the significant potential of subglutinol A as an immunosuppressive drug with dose-dependent osteogenic activity. We also show that activating protein 1 (AP-1) family transcription factors could be one of the key regulators for the anabolic activity of subglutinol A. Such drugs with dose-dependent osteogenic activity might help reduce bone-associated side effects and be clinically useful for bone tissue transplantation.

Conservative treatment using a sponge cast for transfer fractures in nursing home patients.

BACKGROUND: Transfer fractures in the lower limbs of bedridden and chair-bound nursing home patients can result from trauma induced by the usual lifting, moving, turning, or transferring maneuvers. Treatment entails immobilization for pain control and position change; however, splints/hard casts increase the risk of pressure sores. Therefore, we evaluated the use of a sponge cast. MATERIALS AND METHODS: Between March 2011 and October 2017, 17 patients with a lower limb transfer fracture due to transferring maneuvers in a nursing home were recruited. We evaluated the improvement in pseudo-motion and divided the patients as having bony union, fibrous union, or remaining pseudo-motion. We also investigated the occurrence of pressure sores due to immobilization up until the final follow-up. RESULTS: Femur fractures occurred in 15 patients and lower leg fractures in two. Six of the 15 femur fractures were periprosthetic (four hip arthroplasty and two knee arthroplasty). Pseudo-motion was improved in 15 of 17 cases, within an average of 17.3 weeks for the improvement (14-23 weeks; bony union: 11 cases and fibrous union: four cases). Pseudo-motion remained in two cases: one periprosthetic fracture around the knee arthroplasty and the other, a femur neck fracture. No pressure sores occurred. CONCLUSIONS: A sponge cast appears to be one of the effective treatment options available for bedridden or chair-bound patients with a lower limb fracture due to its low risk of complications and satisfactory clinical results.

The Versatility of Framework Regions of Chicken VH and VL to Mutations.

To identify the interchangeability of VH and VL framework region (FR) residues, we artificially introduced random mutations at all residue positions in a chicken monoclonal antibody, which has only one functional VH and Vλ gene. When we classified the amino acids into 5 groups by their physicochemical properties, all FR residues could be replaced by another group except L23 (C), H36 (W), H86 (D), H104 (G), and H106 (G). Eighty-two (50.9%), 48 (29.8%), 17 (10.6%), and 9 FR residues (5.6%) could be replaced by 4, 3, 2, and 1 group(s), individually, without significant loss of reactivity. We also confirmed a similar level of versatility with 2 different chicken antibodies. This high level of versatility on FR residues has not been predicted because it has not been observed in the 150 chicken antibodies that we previously generated or in the 1,269 naïve chicken VH sequences publically available. In conclusion, chicken antibody FR residues are highly interchangeable and this property can be applied for improving the physicochemical property of antibody including thermal stability, solubility and viscosity.

CTLA4-CD28 chimera gene modification of T cells enhances the therapeutic efficacy of donor lymphocyte infusion for hematological malignancy.

Donor lymphocyte infusion (DLI) followed by hematopoietic stem cell transplantation has served as an effective prevention/treatment modality against the relapse of some hematologic tumors, such as chronic myeloid leukemia (CML). However, the therapeutic efficacies of DLI for other types of leukemia, including acute lymphocytic leukemia (ALL), have been limited thus far. Therefore, we examined whether increasing the reactivity of donor T cells by gene modification could enhance the therapeutic efficacy of DLI in a murine model of ALL. When a CTLA4-CD28 chimera gene (CTC28) in which the intracellular signaling domain of CTLA4 was replaced with the CD28 signaling domain was introduced into CD4 and CD8 T cells in DLI, the graft-versus-tumor (GVT) effect was significantly increased. This effect was correlated with an increased expansion of donor CD8 T cells in vivo, and the depletion of CD8 T cells abolished this effect. The CD8 T cell expansion and the enhanced GVT effect were dependent on the transduction of both CD4 and CD8 T cells with CTC28, which emphasizes the role of dual modification in this therapeutic effect. The CTC28-transduced T cells that expanded in vivo also exhibited enhanced functionality. Although the potentiation of the GVT effect mediated by the CTC28 gene modification of T cells was accompanied by an increase of graft-versus-host disease (GVHD), the GVHD was not lethal and was mitigated by treatment with IL-10 gene-modified third-party mesenchymal stem cells. Thus, the combined genetic modification of CD4 and CD8 donor T cells with CTC28 could be a promising strategy for enhancing the therapeutic efficacy of DLI.

Enhanced Anti-tumor Reactivity of Cytotoxic T Lymphocytes Expressing PD-1 Decoy.

Programmed death-1 (PD-1) is a strong negative regulator of T lymphocytes in tumor-microenvironment. By engaging PD-1 ligand (PD-L1) on tumor cells, PD-1 on T cell surface inhibits anti-tumor reactivity of tumor-infiltrating T cells. Systemic blockade of PD-1 function using blocking antibodies has shown significant therapeutic efficacy in clinical trials. However, approximately 10 to 15% of treated patients exhibited serious autoimmune responses due to the activation of self-reactive lymphocytes. To achieve selective activation of tumor-specific T cells, we generated T cells expressing a dominant-negative deletion mutant of PD-1 (PD-1 decoy) via retroviral transduction. PD-1 decoy increased IFN-γ secretion of antigen-specific T cells in response to tumor cells expressing the cognate antigen. Adoptive transfer of PD-1 decoy-expressing T cells into tumor-bearing mice potentiated T cell-mediated tumor regression. Thus, T cell-specific blockade of PD-1 could be a useful strategy for enhancing both efficacy and safety of anti-tumor T cell therapy.

Paraplegia following cervical epidural catheterization using loss of resistance technique with air: a case report.

We report a case of paraplegia without neurologic deficit of upper extremities following cervical epidural catheterization using air during the loss of resistance technique. A 41-year-old woman diagnosed with complex regional pain syndrome had upper and lower extremity pain. A thoracic epidural lead was inserted for a trial spinal cord stimulation for treating lower extremity pain and cervical epidural catheterization was performed for treating upper extremity pain. Rapidly progressive paraplegia developed six hours after cervical epidural catheterization. Spine CT revealed air entrapment in multiple thoracic intervertebral foraminal spaces and surrounding epidural space without obvious spinal cord compression before the decompressive operation, which disappeared one day after the decompressive operation. Her paraplegia symptoms were normalized immediately after the operation. The presumed cause of paraplegia was transient interruption of blood supply to the spinal cord through the segmental radiculomedullary arteries feeding the spinal cord at the thoracic level of the intervertebral foramen caused by the air.

Effect-site concentration of remifentanil for smooth inhalational induction with desflurane.

Objective To determine the effect-site concentration (Ce) of remifentanil target-controlled infusion required for a smooth inhalational induction without airway irritation using desflurane in a stepwise incremental manner for 50% of patients (EC50) and 95% of patients (EC95). Methods Patients with an American Society of Anesthesiologists physical status I and II, aged 19-60 years undergoing elective surgery were enrolled in this study. When target Ce of remifentanil was reached, desflurane was inhaled at 4 vol% initially and then it was increased to 8 and 12 vol% at intervals of 30 s. Smooth induction was regarded as an absence of airway irritation signs and excitatory movements. The EC50 and EC95 values for remifentanil were determined using a modified Dixon's up-and-down method as well as an isotonic regression method with a bootstrapping approach. Results The EC50 and EC95 of remifentanil for smooth induction during inhalation of desflurane were 3.40 ng/ml (95% confidence interval [CI] 2.42, 4.38 ng/ml) and 4.31 ng/ml (95% CI 2.15, 5.98 ng/ml), respectively. Conclusion Prior administration of remifentanil could provide smooth inhalational induction with desflurane in a stepwise increment.

Ndrg1 is a T-cell clonal anergy factor negatively regulated by CD28 costimulation and interleukin-2.

Induction of T-cell clonal anergy involves serial activation of transcription factors, including NFAT and Egr2/3. However, downstream effector mechanisms of these transcription factors are not fully understood yet. Here we identify Ndrg1 as an anergy factor induced by Egr2. Ndrg1 is upregulated by anergic signalling and maintained at high levels in resting anergic T cells. Overexpression of Ndrg1 mimics the anergic state and knockout of the gene prevents anergy induction. Interestingly, Ndrg1 is phosphorylated and degraded by CD28 signalling in a proteasome-dependent manner, explaining the costimulation dependence of anergy prevention. Similarly, IL-2 treatment of anergic T cells, under conditions that lead to the reversal of anergy, also induces Ndrg1 phosphorylation and degradation. Finally, older Ndrg1-deficient mice show T-cell hyperresponsiveness and Ndrg1-deficient T cells aggravate inducible autoimmune inflammation. Thus, Ndrg1 contributes to the maintenance of clonal anergy and inhibition of T-cell-mediated inflammation.

Epithelial transglutaminase 2 is needed for T cell interleukin-17 production and subsequent pulmonary inflammation and fibrosis in bleomycin-treated mice.

Pulmonary fibrosis is a potentially life-threatening disease that may be caused by overt or asymptomatic inflammatory responses. However, the precise mechanisms by which tissue injury is translated into inflammation and consequent fibrosis remain to be established. Here, we show that in a lung injury model, bleomycin induced the secretion of IL-6 by epithelial cells in a transglutaminase 2 (TG2)-dependent manner. This response represents a key step in the differentiation of IL-17-producing T cells and subsequent inflammatory amplification in the lung. The essential role of epithelial cells, but not inflammatory cells, TG2 was confirmed in bone marrow chimeras; chimeras made in TG2-deficient recipients showed reduced inflammation and fibrosis, compared with those in wild-type mice, regardless of the bone marrow cell phenotype. Epithelial TG2 thus appears to be a critical inducer of inflammation after noninfectious pulmonary injury. We further demonstrated that fibroblast-derived TG2, acting downstream of transforming growth factor-ß, is also important in the effector phase of fibrogenesis. Therefore, TG2 represents an interesting potential target for therapeutic intervention.

CP-690550, a Janus kinase inhibitor, suppresses CD4+ T-cell-mediated acute graft-versus-host disease by inhibiting the interferon-γ pathway.

BACKGROUND: Acute graft-versus-host disease (GVHD) is a critical obstacle to bone marrow transplantation. Although numerous studies have described immunosuppression protocols to mitigate acute GVHD, the need still exists for a more efficient immunosuppressant with fewer side effects. Here, we evaluated the protective effect of CP-690550, a newly developed Janus kinase inhibitor, in an acute GVHD model. METHODS: CP-690550 was chemically synthesized. Acute GVHD was induced through the transfer of parent B6 (H-2) bone marrow and CD4 T cells into lethally irradiated (B6×bm12)F1 (H-2) mice. RESULTS.: CP-690550 treatments confined to days -3 to 11 of GVHD induction provided full protection against allogeneic, acute GVHD-related lethality and histopathology. An analysis of the initial donor-derived CD4 T-cell responses revealed that the inhibitory effects of CP-690550 were largely related to the suppression of donor CD4 T-cell-mediated interferon (IFN)-γ production. Enhanced inhibition of T helper 1 cell differentiation, rather than the inhibition of allogeneic CD4 T-cell proliferation or T helper 17 cell differentiation, was also confirmed in allogeneic mixed lymphocyte reactions. Because lethality was considerably delayed by the systemic blockade of IFN-γ, the principal protective effect of CP-690550 occurred through the modulation of IFN-γ production. CONCLUSION: The targeting of Janus kinase with a sensitive and specific inhibitor, CP-690550, conferred effective protection from acute GVHD induced by a semiallogeneic major histocompatibility complex class II-disparate combination. Protection from acute GVHD was largely mediated by the inhibition of IFN-γ production.

Total synthesis, assignment of the absolute stereochemistry, and structure-activity relationship studies of subglutinols A and B.

Immunosuppressive drugs are used to prevent rejection of transplanted organs and treat autoimmune diseases. Clinically approved immunosuppressive drugs possess undesirable side effects, including acute neurological toxicity, chronic nephrotoxicity, and osteoporosis. As a result, considerable efforts have been devoted to the identification of immunosuppressive natural products that lack cytotoxicity and undesirable side effects on bone structure. Subglutinols A (1 a) and B (1 b) are diterpene pyrones isolated from Fusarium subglutinans. Compounds 1 a and 1 b are equipotent in the mixed lymphocyte reaction assay and thymocyte proliferation assay (IC(50) = 0.1 microM). Owing to the lack of toxicity, 1 a and 1 b are expected to be promising new immunosuppressive drugs. Herein, we detail our efforts that have culminated in a stereoselective synthesis of 1 a and 1 b from the (S)-(+)-5-methyl-Wieland-Miescher ketone and determined their absolute stereochemistries. We also present initial biological data to show the great potential of 1 a as an immunosuppressive drug with dose-dependent osteogenic activity.

Porcine PD-L1: cloning, characterization, and implications during xenotransplantation.

BACKGROUND: Effective intervention achieved by manipulating cell-mediated xenogeneic immune responses would critically increase the clinical feasibility of xenotransplantation as immediate hyperacute rejections become controllable through genetic modulations of donor organs. Endogenous negative regulatory signals like the programmed death 1 (PD-1)-programmed death ligand 1 (PD-L1) system are candidate targets for the control of cell-mediated xenogeneic immune response. METHODS: A porcine PD-L1 molecule was cloned using RACE (rapid amplification of cDNA ends) technology based on the human PD-L1 sequence. The functional effects of cloned porcine PD-L1 were tested on human CD4(+) T cell activation using porcine PD-L1-transfected bystander cells. Cellular proliferation was monitored by [3H] thymidine incorporation, and human T cell apoptosis was measured by flow cytometry. RESULTS: Porcine PD-L1 (GenBank accession number AY837780) was found to have 73.8% sequence homology with human PD-L1 and to contain two immunoglobulin domains in its extracellular region. Moreover, porcine PD-L1 expressed on Chinese hamster ovary (CHO) cells inhibited human CD4(+) T cell proliferation stimulated with anti-CD3 only or anti-CD3 plus anti-CD28. Percentages of apoptotic activated human T cells increased by over 30% in the presence of porcine PD-L1/CHO cells, and the addition of recombinant human PD-1-Fc fusion proteins during human T cell activation reversed the inhibitory effects of porcine PD-L1. CONCLUSIONS: Cloned porcine PD-L1 showed high sequence homology with human PD-L1 and a similar molecular structure. Moreover, porcine PD-L1 inhibited human CD4(+) T cell activation in human PD-1-dependent manner, and this involved activated T cell apoptosis. The authors suggest that PD-1-PD-L1 might play an important endogenous immune regulatory role during xenogeneic transplantation, and that the effective application of this system would improve transplanted xenogeneic organ survival.

Acquisition of anergic and suppressive activities in transforming growth factor-beta-costimulated CD4+CD25- T cells.

Regulatory T (Tr) cells have been shown to arise in the periphery during induction of peripheral tolerance. However, the mechanism involved remains elusive. In the present study, we investigated the potential role of transforming growth factor (TGF)-beta in the peripheral induction of regulatory phenotypes in the conventional CD4(+)CD25(-) T cells. Upon priming in the presence of TGF-beta, there was greatly enhanced expression of CD25, cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), and the natural Tr cell-specific transcription factor Foxp3 in naive CD4(+)CD25(-) T cells. The CD25(+) cells that emerged later only in the TGF-beta-treated culture failed to express CD69, so distinguishing this population from activated CD25(+) effector cells. The TGF-beta-treated T cells entered an anergic state following restimulation, as judged by enhanced induction of programmed death (PD)-1, as well as impaired responses in terms of proliferation and IL-2 production. Importantly, the TGF-beta-costimulated CD4(+)CD25(-) T cells, prior to conversion to CD25(+) cells, were able to suppress the proliferation of responder T cells via contact-dependent and interleukin-10-independent mechanisms. Taken together, these results demonstrate that the existence of TGF-beta during early phase of priming is sufficient to induce CD4(+)CD25(-) Tr cells with anergic and immunoregulatory activities equivalent to thymus-derived CD4(+)CD25(+) Tr cells, and these cells are programmed to be CD25(+) cells under the prolonged resting conditions. Thus, our findings provide a novel mechanism by which TGF-beta mediates infectious tolerance in the periphery.

Association of reduced CD4 T cell responses specific to varicella zoster virus with high incidence of herpes zoster in patients with systemic lupus erythematosus.

OBJECTIVE: To examine whether the high incidence of herpes zoster in patients with systemic lupus erythematosus (SLE) is associated with the frequency of memory T cells specific to varicella zoster virus (VZV). METHODS: Whole blood samples from 47 subjects [24 patients with SLE, 11 with rheumatoid arthritis (RA) as a disease control, and 12 healthy negative controls] were stimulated with VZV antigen, stained for surface CD4 and CD8 and intracellularly stained for the cytokines interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), interleukin 4 (IL-4), and IL-10, followed by flow cytometry analyses. Correlations of VZV-specific T cell frequencies with the clinical status of patients were analyzed. RESULTS: Percentage of IFN-gamma-positive CD4 T cells was significantly lower in patients with SLE (0.043 +/- 0.009%) than in RA (0.102 +/- 0.019%) and healthy controls (0.126 +/- 0.025%) upon VZV stimulation. A similar pattern was seen in TNF-alpha-positive CD4 T cell responses. These low frequencies of VZV-specific CD4 T cells in patients with SLE were significantly related with disease activity (r = -0.435, p = 0.043). CONCLUSION: These data suggest that the high incidence of herpes zoster in patients with SLE was related to the intrinsic defects in controlling VZV reactivation, and thus VZV-specific CD4 T cell frequency could be another practical risk factor of herpes zoster in patients with SLE.