Oclacitinib,a Janus Kinase Inhibitor,Reduces the Frequency of IL-4- and IL-10-, but Not IFN-γ-, Producing Murine CD4+ and CD8+ T Cells and Counteracts the Induction of Type 1 Regulatory T Cells

The purpose of the present study was to broaden the knowledge and understanding of the effects of oclacitinib (OCL), a Janus kinase inhibitor, on T cells in the context of both the immune mechanisms underlying anti-inflammatory and anti-allergic properties of the drug and its safety. The results indicate that beneficial effects of OCL in the treatment of skin allergic diseases may be partially mediated by the inhibition of IL-4 production in CD4⁺ and CD8⁺ T cells. To a certain extent, the antiproliferative effect of OCL on CD8⁺ T cells may also contribute to its therapeutic effect. The study found that OCL does not affect the proliferation of CD4⁺ T cells or the number of IFN-γ- and IL-17-producing CD4⁺ and CD8⁺ T cells. Moreover, OCL was found to counteract the induction of type 1 regulatory T (Tr1) cells and to act as a strong inhibitor of IL-10 production in both CD4⁺ and CD8⁺ T cells. Thus, these results indicate that beneficial effects of OCL in the treatment of skin allergic diseases are not mediated through: (a) the abolishment of IFN-γ and IL-17-production in CD4⁺ and CD8⁺ T cells; (b) generation of Tr1 cells; (c) inhibition of CD4⁺ T cell proliferation; (d) induction of IL-10 production in CD4⁺ T cells. The results of this study strongly suggest that, with respect to the evaluated parameters, OCL exerts a suppressive effect on Th2- but not Th1-mediated immunity.     

Selective addition of CXCR3+CCR4-CD4+ Th1 cells enhances generation of cytotoxic T cells by dendritic cells in vitro

Increasing importance is being given to the stimulation of Th1 response in cancer immunotherapy because its presence can shift the direction of adaptive immune responses toward protective immunity. Based on chemokine receptor expression, CXCR3⁺CCR4^-CD4⁺ T cells as Th1-type cells were investigated its capacity in monocyte-derived dendritic cell (DC) maturation and polarization, and induction of antigen specific cytotoxic T lymphocytes (CTL) in vitro. The levels of IL-4, IL-5 and IL-10 were decreased to the basal level compared with high production of IFN-γ, TNF-α, and IL-2 in CXCR3⁺CCR4^-CD4⁺ T cells stimulated with anti-CD3 and anti-CD28 antibodies. Co-incubation of activated CD4⁺ or CXCR3⁺CCR4^-CD4⁺ T cells with DC (CD4⁺/DC or CXCR3⁺CD4⁺/DC, respectively) particularly up-regulated IL-12 and CD80 expression compared with DC matured with TNF-α and LPS (mDC). Although there was no significant difference between the effects of the CXCR3⁺CCR4^-CD4⁺ and CD4⁺ T cells on DC phenotype expression, CXCR3⁺CD4⁺/DC in CTL culture were able to expand number of CD8⁺ T cells and increased frequencies of IFN-γ secreting cells and overall cytolytic activity against tumor antigen WT-1. These results demonstrated that the selective addition of CXCR3⁺CCR4^-CD4⁺ T cells to CTL cultures could enhance the induction of CTLs by DC in vitro, and implicated on a novel strategy for adoptive T cell therapy.     

Expression of 4-1BB and 4-1BBL in thymocytes during thymus regeneration

4-1BB, a member of the tumor necrosis factor receptor (TNFR) superfamily, is a major costimulatory receptor that is rapidly expressed on the surface of CD4⁺ and CD8⁺ T cells after antigen- or mitogen-induced activation. The interaction of 4-1BB with 4-1BBL regulates immunity and promotes the survival and expansion of activated T cells. In this study, the expression of 4-1BB and 4-1BBL was examined during regeneration of the murine thymus following acute cyclophosphamide-induced involution. Four-color flow cytometry showed that 4-1BB and 4-1BBL were present in the normal thymus and were preferentially expressed in the regenerating thymus, mainly in CD4⁺CD8⁺ double-positive (DP) thymocytes. Furthermore, the CD4^loCD8^lo, CD4⁺CD8^lo and CD4^loCD8⁺ thymocyte subsets, representing stages of thymocyte differentiation intermediate between DP and single-positive (SP) thymocytes, also expressed 4-1BB and 4-1BBL during thymus regeneration but to a lesser degree. Interestingly, the 4-1BB and 4-1BBL positive cells among the CD4⁺CD8⁺ DP thymocytes present during thymus regeneration were TCR^hi and CD69⁺ unlike the corresponding controls. Moreover, the 4-1BB and 4-1BBL positive cells among the intermediate subsets present during thymus regeneration also exhibited TCR^hi/int and CD69^+/int phenotypes, indicating that 4-1BB and 4-1BBL are predominantly expressed by the positively selected population of the CD4⁺CD8⁺ DP and the intermediate thymocytes during thymus regeneration. RT-PCR and Western blot analyses confirmed the presence and elevated levels of 4-1BB and 4-1BBL mRNA and protein in thymocytes during thymus regeneration. We also found that the interaction of 4-1BB with 4-1BBL promoted thymocyte adhesion to thymic epithelial cells. Our results suggest that 4-1BB and 4-1BBL participate in T lymphopoiesis associated with positive selection during recovery from acute thymic involution.     

Genetic controls of Th17 cell differentiation and plasticity

CD4⁺ T lymphocytes play a major role in regulation of adaptive immunity. Upon activation, naïve T cells differentiate into different functional subsets. In addition to the classical Th1 and Th2 cells, several novel effector T cell subsets have been recently identified, including Th17 cells. There has been rapid progress in characterizing the development and function of Th17 cells. Here I summarize and discuss on the genetic controls of their differentiation and emerging evidence on their plasticity. This information may benefit understanding and treating immune diseases.     

Combination of anti-angiogenic therapy and immune checkpoint blockade normalizes vascular-immune crosstalk to potentiate cancer immunity

Cancer immunotherapy with immune checkpoint inhibitors (ICIs) has revolutionized the treatment of advanced cancers. However, the tumor microenvironment (TME) functions as a formidable barrier that severely impairs the efficacy of ICIs. While the crosstalk between tumor vessels and immune cells determines the nature of anti-tumor immunity, it is skewed toward a destructive cycle in growing tumors. First, the disorganized tumor vessels hinder CD8⁺ T cell trafficking into the TME, disable effector functions, and even kill T cells. Moreover, VEGF, the key driver of angiogenesis, interferes with the maturation of dendritic cells, thereby suppressing T cell priming, and VEGF also induces TOX-mediated exhaustion of CD8⁺ T cells. Meanwhile, a variety of innate and adaptive immune cells contribute to the malformation of tumor vessels. Protumoral M2-like macrophages as well as T_H2 and Treg cells secrete pro-angiogenic factors that accelerate uncontrolled angiogenesis and promote vascular immaturity. While CD8⁺ T and CD4⁺ T_H1 cells suppress angiogenesis and induce vascular maturation by secreting IFN-γ, they are unable to infiltrate the TME due to malformed tumor vessels. These findings led to preclinical studies that demonstrated that simultaneous targeting of tumor vessels and immunity is a viable strategy to normalize aberrant vascular-immune crosstalk and potentiate cancer immunotherapy. Furthermore, this combination strategy has been evidently demonstrated through recent pivotal clinical trials, granted approval from FDA, and is now being used in patients with kidney, liver, lung, or uterine cancer. Overall, combining anti-angiogenic therapy and ICI is a valid therapeutic strategy that can enhance cancer immunity and will further expand the landscape of cancer treatment.Subject terms: Cancer immunotherapy, Cancer microenvironment, Tumour angiogenesis, Tumour immunology, Targeted therapies     

ZIP8 exacerbates collagen-induced arthritis by increasing pathogenic T cell responses

Zinc is a trace element that is essential for immune responses. Therefore, changes in cellular zinc levels in specific immune cells may influence inflammatory autoimmune diseases, such as rheumatoid arthritis (RA). However, the regulation of zinc mobilization in immune cells and its role in the pathogenesis of RA are not fully understood. Thus, we investigated the roles of zinc transporters in RA pathogenesis. We demonstrated that ZIP8 was specifically upregulated in CD4⁺ T cells that infiltrated the inflamed joint and that ZIP8 deficiency in CD4⁺ T cells abrogated collagen-induced arthritis. ZIP8 deficiency dramatically affected zinc influx in effector T cells and profoundly reduced T cell receptor (TCR)-mediated signaling, including NF-κB and MAPK signaling, which are pathways that are involved in T helper (Th) 17 cell differentiation. Taken together, our findings suggest that ZIP8 depletion in CD4⁺ T cells attenuates TCR signaling due to insufficient cellular zinc, thereby reducing the function of effector CD4⁺ T cells, including Th17 cells. Our results also suggest that targeting ZIP8 may be a useful strategy to inhibit RA development and pathogenesis.Subject terms: Autoimmunity, Immunological disorders     

Bombyx batryticatus Protein-Rich Extract Induces Maturation of Dendritic Cells and Th1 Polarization: A Potential Immunological Adjuvant for Cancer Vaccine

Bombyx batryticatus, a protein-rich edible insect, is widely used as a traditional medicine in China. Several pharmacological studies have reported the anticancer activity of B. batryticatus extracts; however, the capacity of B. batryticatus extracts as immune potentiators for increasing the efficacy of cancer immunotherapy is still unverified. In the present study, we investigated the immunomodulatory role of B. batryticatus protein-rich extract (BBPE) in bone marrow-derived dendritic cells (BMDCs) and DC vaccine-immunized mice. BBPE-treated BMDCs displayed characteristics of mature immune status, including high expression of surface molecules (CD80, CD86, major histocompatibility complex (MHC)-I, and MHC-II), increased production of proinflammatory cytokines (tumor necrosis factor-α and interleukin-12p70), enhanced antigen-presenting ability, and reduced endocytosis. BBPE-treated BMDCs promoted naive CD4⁺ and CD8⁺ T-cell proliferation and activation. Furthermore, BBPE/ovalbumin (OVA)-pulsed DC-immunized mice showed a stronger OVA-specific multifunctional T-cell response in CD4⁺ and CD8⁺ T cells and a stronger Th1 antibody response than mice receiving differently treated DCs, which showed the enhanced protective effect against tumor growth in E.G7 tumor-bearing mice. Our data demonstrate that BBPE can be a novel immune potentiator for a DC-based vaccine in anticancer therapy.     

Characterization of Endogenous Protoporphyrin IX Induced by δ-Aminolevulinic Acid in Resting and Activated Peripheral Blood Lymphocytes by Four-Color Flow Cytometry

Lymphocytes treated with δ-aminolevulinic acid (ALA) can accumulate the photoactive, fluorescent heme precursor, protoporphyrin IX (PpIX). With visible light illumination, PpIX can be used in photodynamic therapy (ALA-PDT) to kill or functionally alter cells. The aim of this study was to characterize the effects of ALA and ALA-PDT on resting and activated human peripheral blood T lymphocytes. Accumulation of PpIX depends inversely on the rate of its iron-dependent conversion into heme. Activated, replicating lymphocytes have low intracellular iron levels, with corresponding increases in the transferrin receptor (CD71). Thus, we expected activated lymphocytes would preferentially accumulate PpIX. Using four-color flow cytometry, we examined ALA-induced PpIX levels in T-cell subsets of resting and activated human peripheral blood mononuclear cells and the relationship between CD71 and PpIX. Peripheral blood mononuclear cells stimulated by phytohemagglutinin (PHA) were simultaneously phenotyped for PpIX, CD71 and the T-cell markers CD3 and CD4 or CDS. In activated cells treated with 0-6mM ALA for 4 h, PpIX fluorescence was maximal at 1 mM ALA. On a single cell basis, there was a strong correlation between PpIX ac-cumulation and CD71 expression. The ALA-treated, PHA-stimulated, CD71⁺ lymphocytes had an eight-fold greater mean PpIX fluorescence than nonactivated, CD71- cells. Approximately 87% of the CD4* and 85% of the CD8⁺ T cells accumulated PpIX. The PpIX levels of CDS⁺ cells were about 5% greater than CD4⁺ cells. In addition, mixed lymphocyte reaction-stimulated cells treated with ALA accumulated more PpIX than controls. Thus, activated cells preferentially accumulate endogenous PpIX when exogenous ALA is administered. Cytotoxicity studies showed that the majority of the activated cells following ALA-PDT were killed but resting cells were spared. Also, in examining activation markers by flow cytometry the number of cells that were positive for activation markers CD38 or CD71 dramatically decreased after ALA and light treatment in activated populations. The data suggest a role for ALA-PDT as an immunomodulator or photocytotoxic agent targeting activated lymphocytes.     

Analyses of the TCR repertoire of MHC class II-restricted innate CD4+ T cells

Analysis of the T-cell receptor (TCR) repertoire of innate CD4⁺ T cells selected by major histocompatibility complex (MHC) class II-dependent thymocyte–thymocyte (T-T) interaction (T-T CD4⁺ T cells) is essential for predicting the characteristics of the antigens that bind to these T cells and for distinguishing T-T CD4⁺ T cells from other types of innate T cells. Using the TCR^mini Tg mouse model, we show that the repertoire of TCRα chains in T-T CD4⁺ T cells was extremely diverse, in contrast to the repertoires previously described for other types of innate T cells. The TCRα chain sequences significantly overlapped between T-T CD4⁺ T cells and conventional CD4⁺ T cells in the thymus and spleen. However, the diversity of the TCRα repertoire of T-T CD4⁺ T cells seemed to be restricted compared with that of conventional CD4⁺ T cells. Interestingly, the frequency of the parental OT-II TCRα chains was significantly reduced in the process of T-T interaction. This diverse and shifted repertoire in T-T CD4⁺ T cells has biological relevance in terms of defense against diverse pathogens and a possible regulatory role during peripheral T-T interaction.     

Blockade of NF-κB Translocation and of RANKL/RANK Interaction Decreases the Frequency of Th2 and Th17 Cells Capable of IL-4 and IL-17 Production,Respectively, in a Mouse Model of Allergic Asthma

The main purpose of this study was to investigate whether the blockade of the interaction between the receptor activator of nuclear factor-κB (NF-ĸB) ligand (RANKL) and its receptor RANK as well as the blockade of NF-κB inhibitor kinase (IKK) and of NF-κB translocation have the potential to suppress the pathogenesis of allergic asthma by inhibition and/or enhancement of the production by CD4⁺ and CD8⁺ T cells of important cytokines promoting (i.e., IL-4 and IL-17) and/or inhibiting (i.e., IL-10 and TGF-β), respectively, the development of allergic asthma. Studies using ovalbumin(OVA)-immunized mice have demonstrated that all the tested therapeutic strategies prevented the OVA-induced increase in the absolute number of IL-4- and IL-17-producing CD4⁺ T cells (i.e., Th2 and Th17 cells, respectively) indirectly, i.e., through the inhibition of the clonal expansion of these cells in the mediastinal lymph nodes. Additionally, the blockade of NF-κB translocation and RANKL/RANK interaction, but not IKK, prevented the OVA-induced increase in the percentage of IL-4-, IL-10- and IL-17-producing CD4⁺ T cells. These latter results strongly suggest that both therapeutic strategies can directly decrease IL-4 and IL-17 production by Th2 and Th17 cells, respectively. This action may constitute an important mechanism underlying the anti-asthmatic effect induced by the blockade of NF-κB translocation and of RANKL/RANK interaction. Thus, in this context, both these therapeutic strategies seem to have an advantage over the blockade of IKK. None of the tested therapeutic strategies increased both the absolute number and frequency of IL-10- and TGF-β-producing Treg cells, and hence they lacked the potential to inhibit the development of the disease via this mechanism.     

Regulatory T-cell vaccination independent of auto-antigen

To date, efforts to treat autoimmune diseases have primarily focused on the disease symptoms rather than on the cause of the disease. In large part, this is attributed to not knowing the responsible auto-antigens (auto-Ags) for driving the self-reactivity coupled with the poor success of treating autoimmune diseases using oral tolerance methods. Nonetheless, if tolerogenic approaches or methods that stimulate regulatory T (T_reg) cells can be devised, these could subdue autoimmune diseases. To forward such efforts, our approach with colonization factor antigen I (CFA/I) fimbriae is to establish bystander immunity to ultimately drive the development of auto-Ag-specific T_reg cells. Using an attenuated Salmonella vaccine expressing CFA/I fimbriae, fimbriae-specific T_reg cells were induced without compromising the vaccine''s capacity to protect against travelers'' diarrhea or salmonellosis. By adapting the vaccine''s anti-inflammatory properties, it was found that it could also dampen experimental inflammatory diseases resembling multiple sclerosis (MS) and rheumatoid arthritis. Because of this bystander effect, disease-specific T_reg cells are eventually induced to resolve disease. Interestingly, this same vaccine could elicit the required T_reg cell subset for each disease. For MS-like disease, conventional CD25⁺ T_reg cells are stimulated, but for arthritis CD39⁺ T_reg cells are induced instead. This review article will examine the potential of treating autoimmune diseases without having previous knowledge of the auto-Ag using an innocuous antigen to stimulate T_reg cells via the production of transforming growth factor-β and interleukin-10.     

The presence of CD8+ invariant NKT cells in mice

Invariant natural killer T (iNKT) cells develop in the thymus upon recognition of CD1d expressed on developing thymocytes. Although CD4 and CD8 coreceptors are not directly involved in the interaction between CD1d and the T cell receptors (TCRs) of iNKT cells, a conspicuous lack of CD8⁺ iNKT cells in mice raised the question of whether CD8⁺ iNKT cells are excluded due to negative selection during their thymic development, or if there is no lineage commitment for the development of murine CD8⁺ iNKT cells. To address this question, we analyzed iNKT cell-specific TCR Vα14⁺ transgenic mice, where the Vα14 transgene forces the generation of iNKT cells. This allows detailed study of the iNKT cell repertoire. We were able to identify CD8⁺ iNKT cells which respond to the NKT cell-specific glycolipid ligand α-galactosylceramide. Unlike conventional iNKT cells, CD8⁺ iNKT cells produce predominantly IFN-γ but not IL-4 upon antigen stimulation. We also confirmed the presence of CD8⁺ iNKT cells in wild type mice. Our results suggest that CD8⁺ NKT cells do exist in mice, although their population size is quite small. Their T_h1-skewed phenotype might explain why the population size of this subtype needs to be controlled tightly.     

TGF-��-treated antigen presenting cells suppress collagen-induced arthritis through the promotion of Th2 responses

Collagen-induced arthritis (CIA) is mediated by self-reactive CD4⁺ T cells that produce inflammatory cytokines. TGF-β₂-treated tolerogenic antigen-presenting cells (Tol-APCs) are known to induce tolerance in various autoimmune diseases. In this study, we investigated whether collagen-specific Tol-APCs could induce suppression of CIA. We observed that Tol-APCs could suppress the development and severity of CIA and delay the onset of CIA. Treatment of Tol-APCs reduced the number of IFN-γ- and IL-17-producing CD4⁺ T cells and increased IL-4- and IL-5-producing CD4⁺ T cells upon collagen antigen stimulation in vitro. The suppression of CIA conferred by Tol-APCs correlated with their ability to selectively induce IL-10 production. We also observed that treatment of Tol-APCs inhibited not only cellular immune responses but also humoral immune responses in the process of CIA. Our results suggest that in vitro-generated Tol-APCs have potential therapeutic value for the treatment of rheumatoid arthritis as well as other autoimmune diseases.     

Stimulation of In Vivo Antitumor Immunity with Hollow Mesoporous Silica Nanospheres

The use of appropriate adjuvants that support the generation of robust and long‐lasting antitumor immune responses is crucial for tumor immunotherapy owing to the immunosuppressive environment of the growing tumor. However, the most commonly used adjuvant, aluminum hydroxide, is ineffective for generating such immune responses and therefore not suitable for cancer immunotherapy. It is now shown that plain hollow mesoporous silica nanospheres markedly improve the antitumor immunity, the Th1 and Th2 immunity, and the CD4⁺ and CD8⁺ effector memory T cell population in bone marrow in vivo and may thus be used as immunoadjuvants to treat cancer in humans.     

TARGETING OF PHOTOTOXIC DRUGS TO ANTIGEN-SPECIFIC T LYMPHOCYTES in vitro USING ANTIGEN-PRESENTING CELL MEMBRANES

We have used the complex of antigen with class II major histocompatibility proteins (la) in membrane-bound form to target a phototoxic compound to antigen-specific T cell hybridomas in vitro. The iodoacetamidyl ester of phototoxic pyrene was bound covalently to antigen-presenting cells (APC), and protein antigens were added to the cells for processing, presentation and targeting of the drug to three different T hybridomas specific for myelin basic protein (MBP), ovalbumin (OVA) and keyhole limpet hemocyanin (KLH). The B hybridoma LS102.9 was used as APC to present MBP, KLH and either a tryptic digest of OVA or the synthetic peptide OVA323–339to these T cells. A transformed B lymphoma, which expresses trinitrophenol (TNP)-specific surface IgM, A20-HL, was used to present TNP conjugates of KLH and OVA to T cells. Either the antigen-bearing intact APC or Ia⁺ membranes shed spontaneously from them were used as drug carriers to target pyrene to the T cells. In the dark, or in the absence of pyrene, both the intact APC or the shed membranes stimulated interleukin-2 (IL-2) production by the T cells in an antigen-specific way. After UVA (320–400nm) irradiation, both forms of these drug carriers had an antigen-specific toxic effect on the T hybridoma cells with receptors for the antigen that they carried. Both spontaneous T cell proliferation and antigen-induced IL-2 production were inhibited. The shed membranes had a more antigen-specific toxic effect than the intact APC, which tend to settle out with the T cells in the microtiter plates, possibly causing nonspecific contact. These results indicate that the antigen-la complex in membrane-bound form can be used to antigen-target cytotoxic drugs to antigen-specific T cells. The Ia⁺ membranes shed from APC may be useful to target drugs to antigen-specific clones of T cells in vivo.     

Magnetic bead based immunoassay for enumeration of CD4 T lymphocytes on a microfluidic device

Human immunodeficiency virus (HIV) diagnostics are urgently needed in resource-scarce settings. Monitoring of HIV-infected patients requires accurate counting of CD4⁺ T lymphocytes. However, the current methods for enumeration of CD4⁺ T lymphocytes are of high cost, technically complex and time-consuming. In this paper, we developed a simple, rapid and inexpensive one-step immunomagnetic method for separating and counting CD4⁺ T lymphocytes on microfluidic devices with enlarged reaction chambers. CD4⁺ T lymphocytes were successfully separated and captured from the cell suspension obtained from mouse thymus. CD4 counts were determined under an optical microscope in a rapid and simple format. In order to acquire the maximum efficiency of cell capture, relative parameters were investigated, including section area of the reaction chamber and injection flow rate of the cell suspension. The enlarged reaction chamber with two symmetrical cone-shaped ends was helpful for cell capture, and the maximum capability of captured CD4⁺ T lymphocytes was about 700 cells μL⁻¹. Our investigations avoided the complex sample pre-treatment, and the entire analysis time was significantly reduced to 15 min. This CD4 counting microdevice had the potential to reduce the cost for HIV diagnosis in resource-limited settings.