IL-28B/IFN-��3 Drives Granzyme B Loading and Significantly Increases CTL Killing Activity in Macaques

Type III/λ interferons (IFNs) were discovered less than a decade ago and are still in the process of being characterized. Although previous studies have focused on the function of IFN-λ3 (also known as interleukin (IL)-28B) in a small animal model, it is unknown whether these functions would translate to a larger, more relevant model. Thus in the present study, we have used DNA vaccination as a method of studying the influence of IFN-λ3 on adaptive immune responses in rhesus macaques. Results of our study show for the first time that IFN-λ3 has significant influence on antigen-specific CD8⁺ T-cell function, especially in regards to cytotoxicity. Peripheral CD8⁺ T cells from animals that were administered IFN-λ3 showed substantially increased cytotoxic responses as gauged by CD107a and granzyme B coexpression as well as perforin release. Moreover, CD8⁺ T cells isolated from the mesenteric lymph nodes (MLN) of animals receiving IFN-λ3 loaded significant amounts of granzyme B upon extended antigenic stimulation and induced significantly more granzyme B-mediated cell death of peptide pulsed targets. These data suggest that IFN-λ3 is a potent effector of the immune system with special emphasis on CD8⁺ T-cell killing functions which warrants further study as a possible immunoadjuvant.     

CD28 Costimulation Impairs the Efficacy of a Redirected T-cell Antitumor Attack in the Presence of Regulatory T cells Which Can Be Overcome by Preventing Lck Activation

Adoptive T-cell transfer showed promising efficacy in recent trials raising interest in T cells with redirected specificity against tumors. T cells were engineered with a chimeric antigen receptor (CAR) with predefined binding and CD3ζ signaling to initiate T-cell activation. CD28 costimulation provided by a CD28-CD3ζ signaling CAR moreover improved T cell activation and persistence; however, it failed to meet the expectations with respect to mounting attacks against solid tumors infiltrated with regulatory T (Treg) cells. We revealed that a CD28 CAR–redirected T-cell attack is accompanied by higher numbers of Treg cells infiltrating the tumor and is less efficient against cancer cells in presence of Treg cells than a CD3ζ CAR T-cell attack. Deletion of the lck binding moiety in the CD28 CAR endodomain, however, improved redirected anti-tumor activity in presence of Treg cells without impairing interferon-γ (IFN-γ) secretion, proliferation, and cytolysis. CD28 modification abrogated interleukin-2 (IL-2) induction upon CAR engagement which in turn is no longer available to sustain Treg cell persistence. CARs with the modified CD28 endodomain thereby expedite the implementation of adoptive T-cell therapy in patients with a variety of cancer types that are heavily infiltrated by Treg cells.     

T helper 1 cells and interferon gamma regulate allergic airway inflammation and mucus production.

CD4 T helper (Th) type 1 and Th2 cells have been identified in the airways of asthmatic patients. Th2 cells are believed to contribute to pathogenesis of the disease, but the role of Th1 cells is not well defined. In a mouse model, we previously reported that transferred T cell receptor–transgenic Th2 cells activated in the respiratory tract led to airway inflammation with many of the pathologic features of asthma, including airway eosinophilia and mucus production. Th1 cells caused inflammation with none of the pathology associated with asthma. In this report, we investigate the role of Th1 cells in regulating airway inflammation. When Th1 and Th2 cells are transferred together into recipient mice, there is a marked reduction in airway eosinophilia and mucus staining. To address the precise role of Th1 cells, we asked (i), Are Th2-induced responses inhibited by interferon (IFN)-γ? and (ii) Can Th1 cells induce eosinophilia and mucus in the absence of IFN-γ? In IFN-γ receptor^−/− recipient mice exposed to inhaled antigen, the inhibitory effects of Th1 cells on both airway eosinophilia and mucus production were abolished. In the absence of IFN-γ receptor signaling, Th1 cells induced mucus but not eosinophilia. Thus, we have identified new regulatory pathways for mucus production; mucus can be induced by Th2 and non-Th2 inflammatory responses in the lung, both of which are inhibited by IFN-γ. The blockade of eosinophilia and mucus production by IFN-γ likely occurs through different inhibitory pathways that are activated downstream of Th2 cytokine secretion and require IFN-γ signaling in tissue of recipient mice.     

Genetic-induced Variations in the GAD65 T-cell Repertoire Governs Efficacy of Anti-CD3/GAD65 Combination Therapy in New-onset Type 1 Diabetes

To enhance efficacy of forthcoming type 1 diabetes (T1D) clinical trials, combination therapies (CTs) are envisaged. In this study, we showed that efficacy of a CT, using anti-CD3 antibody and glutamic acid decarboxylase of 65 kd (GAD65)-expressing plasmid, to reverse new-onset T1D was dependent upon the genetic background. Synergism between both treatments was only observed in the RIP-LCMV-GP but not in the nonobese diabetic (NOD) or RIP-LCMV-NOD models. Efficacy was associated with an expansion of bystander suppressor regulatory T cells (Tregs) recognizing the C-terminal region of GAD65 and secreting interleukin-10 (IL-10), transforming growth factor-β (TGF-β), and interferon-γ (IFN-γ). In addition, we found that frequency and epitope specificity of GAD65-reactive CD4⁺ T cells during antigen priming at diabetes onset and Tregs detected after CT correlated. Consequently, NOD mice harbored significantly lower levels of GAD65-reactive CD4⁺ T cells than RIP-LCMV-GP before and after treatment. Our results demonstrate that antigen-specific T cells available at treatment may differ between various major histocompatibility complex (MHC) and genetic backgrounds. These cells play a major role in shaping T-cell responses following antigen-specific immune intervention and determine whether a beneficial Tregs response is generated. Our findings hold important implications to understand and predict the success of antigen-based clinical trials, where responsiveness to immunotherapy might vary from patient to patient.     

Therapeutic Immunity by Adoptive Tumor-primed CD4+ T-cell Transfer in Combination With In Vivo GITR Ligation

Tumor-primed CD4⁺ T cells from splenocytes of tumor-rejection mice in combination with in vivo glucocorticoid-induced tumor necrosis factor receptor (GITR) ligation (the combination therapy) elicited effective host CD8⁺ T cell–dependent therapeutic immunity against a murine breast tumor. GITR ligation in vitro enhanced tumor-primed CD4⁺ T-cell activity and partially abrogated regulatory T cells (Treg) suppressor function. Dendritic cells (DCs) from tumor-draining lymph nodes (TDLNs) of tumor-bearing mice treated by the combination therapy stimulated Ag-specific T cells and produced interleukin (IL)-12 ex vivo. Whereas tumor-primed CD4⁺ T cells or in vivo GITR ligation alone induced a tumor-specific interferon (IFN)-γ-producing cellular response, the combination therapy enhanced and sustained it. Furthermore, the combination therapy in vivo attenuated Treg''s ability to suppress IL-12 production by DCs and IFN-γ production by effectors ex vivo. Importantly, tumor-primed CD4⁺ CD25⁻ T cells from splenocytes of untreated tumor-bearing mice in combination with in vivo GITR ligation also elicited an effective therapeutic effect in this model. These data suggest that the combination therapy may improve DC function, accentuate tumor-specific T-cell responses, and attenuate Treg suppressor function, thereby eliciting effective therapeutic immunity.     

Bispecific T-cells Expressing Polyclonal Repertoire of Endogenous γδ T-cell Receptors and Introduced CD19-specific Chimeric Antigen Receptor

Even though other γδ T-cell subsets exhibit antitumor activity, adoptive transfer of γδ Tcells is currently limited to one subset (expressing Vγ9Vδ2 T-cell receptor (TCR)) due to dependence on aminobisphosphonates as the only clinically appealing reagent for propagating γδ T cells. Therefore, we developed an approach to propagate polyclonal γδ T cells and rendered them bispecific through expression of a CD19-specific chimeric antigen receptor (CAR). Peripheral blood mononuclear cells (PBMC) were electroporated with Sleeping Beauty (SB) transposon and transposase to enforce expression of CAR in multiple γδ T-cell subsets. CAR⁺γδ T cells were expanded on CD19⁺ artificial antigen-presenting cells (aAPC), which resulted in >10⁹ CAR⁺γδ T cells from <10⁶ total cells. Digital multiplex assay detected TCR mRNA coding for Vδ1, Vδ2, and Vδ3 with Vγ2, Vγ7, Vγ8, Vγ9, and Vγ10 alleles. Polyclonal CAR⁺γδ T cells were functional when TCRγδ and CAR were stimulated and displayed enhanced killing of CD19⁺ tumor cell lines compared with CAR^negγδ T cells. CD19⁺ leukemia xenografts in mice were reduced with CAR⁺γδ T cells compared with control mice. Since CAR, SB, and aAPC have been adapted for human application, clinical trials can now focus on the therapeutic potential of polyclonal γδ T cells.     

IFN-γ-induced iNOS Expression in Mouse Regulatory Macrophages Prolongs Allograft Survival in Fully Immunocompetent Recipients

Mouse monocytes exposed to macrophage colony-stimulating factor (M-CSF) and interferon-γ (IFN-γ) were driven to a novel suppressor phenotype. These regulatory macrophages (M regs) expressed markers distinguishing them from M0-, M1-, and M2-polarized macrophages and monocyte-derived dendritic cells (DCs). M regs completely suppressed polyclonal T cell proliferation through an inducible nitric oxide synthase (iNOS)-dependent mechanism. Additionally, M regs eliminated cocultured T cells in an allospecific fashion. In a heterotopic heart transplant model, a single intravenous administration of 5 × 10⁶ donor-strain M regs before transplantation significantly prolonged allograft survival in fully immunocompetent recipients using both the stringent C3H-to-BALB/c (32.6 ± 4.5 versus 8.7 ± 0.2 days) and B6-to-BALB/c (31.1 ± 12 versus 9.7 ± 0.4 days) strain combinations. Nos2-deficient M regs did not prolong allograft survival, proving that M reg function in vivo is iNOS-dependent and mediated by living cells. M regs were detectable for at least 2 weeks postinfusion in allogeneic recipients. In their origin, development, phenotypic relationship with other in vitro-derived macrophages and functions, there are solid grounds to assert a near-equivalence of mouse and human M regs. It is concluded that mouse M regs represent a novel, phenotypically distinct subset of suppressor macrophages. Clinical applications of M reg therapy as an adjunct immunosuppressive therapy are currently being investigated within The ONE Study.     

Nanogel-based delivery of mycophenolic acid ameliorates systemic lupus erythematosus in mice

The ability to selectively inactivate immune cells with immunosuppressants is a much sought-after modality for the treatment of systemic lupus erythematosus and autoimmunity in general. Here, we designed and tested a novel nanogel drug delivery vehicle for the immunosuppressant mycophenolic acid (MPA). Treatment with MPA-loaded nanogels increased the median survival time (MST) of lupus-prone NZB/W F1 mice by 3 months with prophylactic use (MST was 50 weeks versus 38 weeks without treatment), and by 2 months when administered after the development of severe renal damage (MST after proteinuria onset was 12.5 weeks versus 4 weeks without treatment). Equivalent and greater doses of MPA administered in buffer were not efficacious. Nanogels had enhanced biodistribution to organs and association with immune cells. CD4-targeted nanogels yielded similar therapeutic results compared with nontargeted formulations, with protection from glomerulonephritis and decreases in IFN-γ–positive CD4 T cells. DCs that internalized nanogels helped mediate immunosuppression, as they had reduced production of inflammatory cytokines such as IFN-γ and IL-12. Our results demonstrate efficacy of nanogel-based lupus therapy and implicate a mechanism by which immunosuppression is enhanced, in part, by the targeting of antigen-presenting cells.     

Intravaginal immunization with HPV vectors induces tissue-resident CD8+ T cell responses

The induction of persistent intraepithelial CD8⁺ T cell responses may be key to the development of vaccines against mucosally transmitted pathogens, particularly for sexually transmitted diseases. Here we investigated CD8⁺ T cell responses in the female mouse cervicovaginal mucosa after intravaginal immunization with human papillomavirus vectors (HPV pseudoviruses) that transiently expressed a model antigen, respiratory syncytial virus (RSV) M/M2, in cervicovaginal keratinocytes. An HPV intravaginal prime/boost with different HPV serotypes induced 10-fold more cervicovaginal antigen-specific CD8⁺ T cells than priming alone. Antigen-specific T cell numbers decreased only 2-fold after 6 months. Most genital antigen-specific CD8⁺ T cells were intra- or subepithelial, expressed α_E-integrin CD103, produced IFN-γ and TNF-α, and displayed in vivo cytotoxicity. Using a sphingosine-1-phosphate analog (FTY720), we found that the primed CD8⁺ T cells proliferated in the cervicovaginal mucosa upon HPV intravaginal boost. Intravaginal HPV prime/boost reduced cervicovaginal viral titers 1,000-fold after intravaginal challenge with vaccinia virus expressing the CD8 epitope M2. In contrast, intramuscular prime/boost with an adenovirus type 5 vector induced a higher level of systemic CD8⁺ T cells but failed to induce intraepithelial CD103⁺CD8⁺ T cells or protect against recombinant vaccinia vaginal challenge. Thus, HPV vectors are attractive gene-delivery platforms for inducing durable intraepithelial cervicovaginal CD8⁺ T cell responses by promoting local proliferation and retention of primed antigen-specific CD8⁺ T cells.     

Protective effect of theaflavins on neuron against 6-hydroxydopamine-induced apoptosis in SH-SY5Y cells

Theaflavins, the oxidation products of tea polyphenols are important biologically active components of black tea. 6-hydroxydopamine is a pro-parkinsonian neurotoxin. Theaflavins could inhibit the auto-oxidation of 6-hydroxydopamine in a dose-dependent manner from 0.5 µg/ml to 25 µg/ml. Here we investigated the protective effect of theaflavins on 6-hydroxydopamine induced SH-SY5Y cells against apoptosis (within this concentration range). It was found that pretreating SH-SY5Y cells with 0.5 µg/ml of theaflavins prevented 6-hydroxydopamine-induced loss of cell viability, condensed nuclear morphology, attenuated 6-hydroxydopamine-induced apoptosis, decrease of mitochondrial membrane potential and the increase of intracellular nitric oxide levels. Our results indicated that theaflavins had protective effect against 6-hydroxydopamine induced apoptosis at low concentrations, possibly through inhibition of reactive oxygen species and nitric oxide production.     

PPAR-γ对人胰腺癌BxPc-3细胞增殖的影响

目的 通过促进人胰腺癌BxPc-3细胞中过氧化物酶体增殖物激活受体-γ（PPAR-γ）的表达,分析其对BxPc-3细胞增殖的影响。方法 培养BxPc-3细胞,分别给予终浓度为5、10、20、40 μmol/L的PPAR-γ 激动剂罗格列酮处理细胞,蛋白免疫印迹法检测PPAR-γ蛋白的表达,四甲基偶氮唑蓝（MTT）法观察细胞增殖活性。结果 BxPc-3细胞经5、10 μmol/L罗格列酮处理后其PPAR-γ表达与对照组比较差异均无统计学意义（P均> 0.05）,经20、40 μmol/L罗格列酮处理后PPAR-γ表达升高,与对照组比较差异有统计学意义（P均< 0.05）,且40 μmol/L罗格列酮组PPAR-γ表达较20 μmol/L罗格列酮组升高（P < 0.05）;BxPc-3细胞给予浓度为5、10 μmol/L罗格列酮处理后BxPc-3细胞增殖活性分别为0.60±0.07、0.57±0.06,与对照组的0.62±0.09比较差异均无统计学意义（P均> 0.05）;经40 μmol/L罗格列酮处理后BxPc-3细胞增殖活性为0.30±0.02,低于20 μmol/L罗格列酮的0.45±0.03,且2组BxPc-3细胞增殖活性均低于对照组、5 μmol/L罗格列酮组和10 μmol/L罗格列酮组（P均< 0.05）。结论 促进PPAR-γ表达可以有效抑制人胰腺癌BxPc-3细胞增殖。     

Scavenger receptors target glycolipids for natural killer T cell activation

NKT cells are innate-like T cells with powerful regulatory functions that are a promising target for immunotherapy. The efficacy of glycolipids, such as the prototypic NKT cell antagonist α-galactosylceramide (αGalCer), is currently being evaluated in clinical trials, but little is known about factors that target lipid antigens for CD1d presentation and NKT cell activation in vivo. Lipid uptake via the LDL receptor (LDLR) has been shown for digalactosylceramide; however, whether this pathway contributes to CD1d presentation of other important NKT cell agonists remains unclear. We therefore investigated receptor-mediated uptake pathways for CD1d presentation using a panel of structurally diverse lipid antigens. We found that uptake via scavenger receptors was essential for the CD1d presentation of αGalCer and Sphingomonas glycolipids. Moreover, in vivo NKT cell responses, i.e., cytokine production, proliferation, and NKT cell help for adaptive CD4⁺ and CD8⁺ T cells, required the uptake of αGalCer via scavenger receptor A. Importantly, our data indicate that structural characteristics of glycolipids determine their receptor binding and direct individual lipids toward different uptake pathways. These results reveal an important contribution of scavenger receptors in the selection of lipids for CD1d presentation and identify structural motifs that may prove useful for therapeutic NKT cell vaccination.     

Anti-invasive activity of α-tocopherol against hepatoma cells in culture via protein kinase C inhibition

Effects of α-, β-, γ- and δ-tocopherols on the proliferation and invasion of AH109A hepatoma cells and their modes of action were investigated. Four tocopherols inhibited the invasion as well as the proliferation of AH109A cells. Their inhibitory effects were more prominent on the invasion than on the proliferation. At 1 µM, α-tocopherol showed most potent anti-invasive activity without any influence on the proliferation. We have previously demonstrated that reactive oxygen species increase the invasion of AH109A cells. α-Tocopherol suppressed the reactive oxygen species-induced invasion but failed to suppress the reactive oxygen species-induced rises in intracellular peroxide level. GF 109203X, a protein kinase C inhibitor, decreased the invasive activity of AH109A cells. In contrast, phorbol-12-myristate-13-acetate, a protein kinase C activator, increased the invasive capacity of AH109A cells. α-Tocopherol suppressed the phorbol-12-myristate-13-acetate-induced increase in the invasion, and canceled the phorbol-12-myristate-13-acetate-induced rises in protein kinase C activity and phosphorylation of extracellular signal-regulated kinase. These results suggest that tocopherols, especially α-tocopherol, possess inhibitory effect more strongly on the invasion of AH109A cells than on the proliferation. They also suggest that the anti-invasive activity of α-tocopherol is raised through suppression of PKC/ERK signaling.