Interleukin 12 suppresses autoantibody production by reversing helper T-cell phenotype in hepatitis B e antigen transgenic mice.

Helper T (Th) cells are classified as Th1 or Th2 cells by virtue of cytokine secretion and function as mediators of cellular or humoral immunity, respectively. Cytokines also regulate the differentiation of Th cells. For example, interleukin (IL)-12 promotes Th1 and suppresses Th2 cell development, suggesting that IL-12 may be useful therapeutically in Th2-mediated autoimmune and allergic disorders. Therefore, the effect of systemic IL-12 treatment on in vivo autoantibody synthesis in hepatitis B e antigen (HBeAg)-expressing transgenic mice, which is dependent on self-reactive Th2 cells, was examined. Low-dose IL-12 significantly inhibited autoantibody production by shifting the Th2-mediated response toward Th1 predominance. Additionally, previous studies suggest that a predominance of HBeAg-specific Th2-type cells may contribute to chronicity in hepatitis B virus infection. Therefore, IL-12 may also prove beneficial in modulating the HBeAg-specific Th response to favor viral clearance in chronic hepatitis B virus infection.

Regulation of the polarization of T cells toward antigen-presenting cells by Ras-related GTPase CDC42.

The mechanisms by which cells rapidly polarize in the direction of external signals are not understood. Helper T cells, when contacted by an antigen-presenting cell, polarize their cytoskeletons toward the antigen-presenting cell within minutes. Here we show that, in T cells, the mammalian Ras-related GTPase CDC42 (the homologue of yeast CDC42, a protein involved in budding polarity) can regulate the polarization of both actin and microtubules toward antigen-presenting cells but is not involved in other T-cell signaling processes such as those which culminate in interleukin 2 production. Although T-cell polarization appears dispensable for signaling leading to interleukin 2 production, polarization may direct lymphokine secretion towards the correct antigen-presenting cell in a crowded cellular environment. Inhibitor experiments suggest that phosphatidylinositol 3-kinase is required for cytoskeletal polarization but that calcineurin activity, known to be important for other aspects of signaling, is not. Apparent conservation of CDC42 function between yeast and T cells suggests that this GTPase is a general regulator of cytoskeletal polarity in many cell types.

Switch from a type 2 to a type 1 T helper cell response and cure of established Leishmania major infection in mice is induced by combined therapy with interleukin 12 and Pentostam.

Successful treatment in allergic, autoimmune, and infectious diseases often requires altering the nature of a detrimental immune response mediated by a particular CD4+ T helper (Th) cell subset. While several factors contribute to the development of CD4+ Th1 and Th2 cells, the requirements for switching an established response are not understood. Here we use infection with Leishmania major as a model to investigate those requirements. We report that treatment with interleukin 12 (IL-12), in combination with the antimony-based leishmanicidal drug Pentostam, induces healing in L. major-infected mice and that healing is associated with a switch from a Th2 to a Th1 response. The data suggest that decreasing antigen levels may be required for IL-12 to inhibit a Th2 response and enhance a Th1 response. These observations are important for treatment of nonhealing forms of human leishmaniasis and also demonstrate that in a chronic infectious disease an inappropriate Th2 response can be switched to an effective Th1 response.

IgE receptor-positive non-B/non-T cells dominate the production of interleukin 4 and interleukin 6 in immunized mice.

The phenotype and antigenic specificity of cells secreting interleukin (IL) 4, IL-6, and interferon gamma was studied in mice during primary and secondary immune responses. T lymphocytes were the major source of interferon gamma, whereas non-B/non-T cells were the dominant source of IL-4 and IL-6 in the spleens of immunized animals. Cytokine-secreting non-B/non-T cells expressed surface receptors for IgE and/or IgG types II/III. Exposing these cells to antigen-specific IgE or IgG in vivo (or in vitro) "armed" them to release IL-4 and IL-6 upon subsequent antigenic challenge. These findings suggest that non-B/non-T cells may represent the "natural immunity" analogue of CD4+ T helper type 2 cells and participate in a positive feedback loop involved in the perpetuation of T helper type 2 cell responses.

Functional memory CD8+ T cells can be generated in vivo without evident T help

Synthetic cytotoxic T cell (CTL) epitope peptides provide an effective and safe means of vaccination against cancers and viruses, as these peptides can induce specific CD8+ effector T cells in vivo. However, the effector CD8+ T cells induced by the minimal CTL epitope peptides do not last past about 3 weeks after the induction and no functional memory CD8+ T cells are generated. It is held that simultaneous induction of CD4+ T cells by incorporating peptides containing T-helper epitopes in the vaccine at the time of primary vaccination are necessary for the induction of long-lived functional memory CD8+ T cells. We now report that, surprisingly, incorporation of medium length (>20 AA) peptides devoid of detectable T-helper epitopes in a minimal CTL epitope-based vaccine can also induce long-lasting! functional rumour antigen specific memory CD8+ T cells that are capable of promoting protection against tumour challenge. This observation may have implications for the formulation of therapeutic anti-cancer and anti-virus peptide vaccines where a strong induction of CD4 T help would be undesirable. (C) 2004 Elsevier Ltd. All rights reserved.

Molecular Insights of CD4+ T Cell Differentiation, Effector Formation and Helper Function

CD4+ T cells play a crucial in the adaptive immune system. They function as the central hub to orchestrate the rest of immunity: CD4+ T cells are essential governing machinery in antibacterial and antiviral responses by facilitating B cell affinity maturation and coordinating the innate and adaptive immune systems to boost the overall immune outcome; on the contrary, hyperactivation of the inflammatory lineages of CD4+ T cells, as well as the impairments of suppressive CD4+ regulatory T cells, are the etiology of various autoimmunity and inflammatory diseases. The broad role of CD4+ T cells in both physiological and pathological contexts prompted me to explore the modulation of CD4+ T cells on the molecular level.

microRNAs (miRNAs) are small RNA molecules capable of regulating gene expression post-transcriptionally. miRNAs have been shown to exert substantial regulatory effects on CD4+ T cell activation, differentiation and helper function. Specifically, my lab has previously established the function of the miR-17-92 cluster in Th1 differentiation and anti-tumor responses. Here, I further analyzed the role of this miRNA cluster in Th17 differentiation, specifically, in the context of autoimmune diseases. Using both gain- and loss-of-function approaches, I demonstrated that miRNAs in miR-17-92, specifically, miR-17 and miR-19b in this cluster, is a crucial promoter of Th17 differentiation. Consequently, loss of miR-17-92 expression in T cells mitigated the progression of experimental autoimmune encephalomyelitis and T cell-induced colitis. In combination with my previous data, the molecular dissection of this cluster establishes that miR-19b and miR-17 play a comprehensive role in promoting multiple aspects of inflammatory T cell responses, which underscore them as potential targets for oligonucleotide-based therapy in treating autoimmune diseases.

To systematically study miRNA regulation in effector CD4+ T cells, I devised a large-scale miRNAome profiling to track in vivo miRNA changes in antigen-specific CD4+ T cells activated by Listeria challenge. From this screening, I identified that miR-23a expression tightly correlates with CD4+ effector expansion. Ectopic expression and genetic deletion strategies validated that miR-23a was required for antigen-stimulated effector CD4+ T cell survival in vitro and in vivo. I further determined that miR-23a targets Ppif, a gatekeeper of mitochondrial reactive oxygen species (ROS) release that protects CD4+ T cells from necrosis. Necrosis is a type of cell death that provokes inflammation, and it is prominently triggered by ROS release and its consequent oxidative stress. My finding that miR-23a curbs ROS-mediated necrosis highlights the essential role of this miRNA in maintaining immune homeostasis.

A key feature of miRNAs is their ability to modulate different biological aspects in different cell populations. Previously, my lab found that miR-23a potently suppresses CD8+ T cell cytotoxicity by restricting BLIMP1 expression. Since BLIMP1 has been found to inhibit T follicular helper (Tfh) differentiation by antagonizing the master transcription factor BCL6, I investigated whether miR-23a is also involved in Tfh differentiation. However, I found that miR-23a does not target BLIMP1 in CD4+ T cells and loss of miR-23a even fostered Tfh differentiation. This data indicate that miR-23a may target other pathways in CD4+ T cells regarding the Tfh differentiation pathway.

Although the lineage identity and regulatory networks for Tfh cells have been defined, the differentiation path of Tfh cells remains elusive. Two models have been proposed to explain the differentiation process of Tfh cells: in the parallel differentiation model, the Tfh lineage is segregated from other effector lineages at the early stage of antigen activation; alternatively, the sequential differentiation model suggests that naïve CD4+ T cells first differentiate into various effector lineages, then further program into Tfh cells. To address this question, I developed a novel in vitro co-culture system that employed antigen-specific CD4+ T cells, naïve B cells presenting cognate T cell antigen and BAFF-producing feeder cells to mimic germinal center. Using this system, I were able to robustly generate GC-like B cells. Notably, well-differentiated Th1 or Th2 effector cells also quickly acquired Tfh phenotype and function during in vitro co-culture, which suggested a sequential differentiation path for Tfh cells. To examine this path in vivo, under conditions of classical Th1- or Th2-type immunizations, I employed a TCRβ repertoire sequencing technique to track the clonotype origin of Tfh cells. Under both Th1- and Th2- immunization conditions, I observed profound repertoire overlaps between the Teff and Tfh populations, which strongly supports the proposed sequential differentiation model. Therefore, my studies establish a new platform to conveniently study Tfh-GC B cell interactions and provide insights into Tfh differentiation processes.

IL-34- and M-CSF-induced macrophages switch memory T cells into Th17 cells via membrane IL-1α: Immunomodulation

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Expression of Potato virus Y cytoplasmic inclusion protein in tobacco results in disorganization of parenchyma cells, distortion of epidermal cells, and induces mitochondrial and chloroplast abnormalities, formation of membrane whorls and atypical lipid accumulation

Infection of plant cells by potyviruses induces the formation of cytoplasmic inclusions ranging in size from 200 to 1000 nm. To determine if the ability to form these ordered, insoluble structures is intrinsic to the potyviral cytoplasmic inclusion protein, we have expressed the cytoplasmic inclusion protein from Potato virus Y in tobacco under the control of the chrysanthemum ribulose-1,5-bisphosphate carboxylase small subunit promoter, a highly active, green tissue promoter. No cytoplasmic inclusions were observed in the leaves of transgenic tobacco using transmission electron microscopy, despite being able to clearly visualize these inclusions in Potato virus Y infected tobacco leaves under the same conditions. However, we did observe a wide range of tissue and sub-cellular abnormalities associated with the expression of the Potato virus Y cytoplasmic inclusion protein. These changes included the disruption of normal cell morphology and organization in leaves, mitochondrial and chloroplast internal reorganization, and the formation of atypical lipid accumulations. Despite these significant structural changes, however, transgenic tobacco plants were viable and the results are discussed in the context of potyviral cytoplasmic inclusion protein function.

Multilineage differentiation potential of bone and cartilage cells derived from explant culture

To date, mesenchymal stem cells (MSCs) from various tissues have been reported, but the yield and differentiation potential of different tissue-derived MSCs is still not clear. This study was undertaken in an attempt to investigate the multilineage stem cell potential of bone and cartilage explant cultures in comparison with bone marrow derived mesenchymal stem cells (BMSCs). The results showed that the surface antigen expression of tissue-derived cells was consistent with that of mesenchymal stem cells, such as lacking the haematopoietic and common leukocyte markers (CD34, CD45) while expressing markers related to adhesion (CD29, CD166) and stem cells (CD90, CD105). The tissue-derived cells were able to differentiate into osteoblast, chondrocyte and adipocyte lineage pathways when stimulated in the appropriate differentiating conditions. However, compared with BMSCs, tissue-derived cells showed less capacity for multilineage differentiation when the level of differentiation was assessed in monolayer culture by analysing the expression of tissue-specific genes by reverse transcription polymerase chain reaction (RT-PCR) and histology. In high density pellet cultures, tissue-derived cells were able to differentiate into chondrocytes, expressing chondrocyte markers such as proteoglycans, type II collagen and aggrecan. Taken together, these results indicate that cells derived from tissue explant cultures reserved certain degree of differentiation properties of MSCs in vitro.

Stem cell regulatory gene expression in human adult dental pulp and periodontal ligament cells undergoing odontogenic/osteogenic differentiation

Introduction During development and regeneration, odontogenesis and osteogenesis are initiated by a cascade of signals driven by several master regulatory genes. Methods In this study, we investigated the differential expression of 84 stem cell–related genes in dental pulp cells (DPCs) and periodontal ligament cells (PDLCs) undergoing odontogenic/osteogenic differentiation. Results Our results showed that, although there was considerable overlap, certain genes had more differential expression in PDLCs than in DPCs. CCND2, DLL1, and MME were the major upregulated genes in both PDLCs and DPCs, whereas KRT15 was the only gene significantly downregulated in PDLCs and DPCs in both odontogenic and osteogenic differentiation. Interestingly, a large number of regulatory genes in odontogenic and osteogenic differentiation interact or crosstalk via Notch, Wnt, transforming growth factor β (TGF-β)/bone morphogenic protein (BMP), and cadherin signaling pathways, such as the regulation of APC, DLL1, CCND2, BMP2, and CDH1. Using a rat dental pulp and periodontal defect model, the expression and distribution of both BMP2 and CDH1 have been verified for their spatial localization in dental pulp and periodontal tissue regeneration. Conclusions This study has generated an overview of stem cell–related gene expression in DPCs and PDLCs during odontogenic/osteogenic differentiation and revealed that these genes may interact through the Notch, Wnt, TGF-β/BMP, and cadherin signalling pathways to play a crucial role in determining the fate of dental derived cell and dental tissue regeneration. These findings provided a new insight into the molecular mechanisms of the dental tissue mineralization and regeneration

Amniotic fluid stem cells produce robust mineral deposits on biodegradable scaffolds

Insufficient availability of osteogenic cells limits bone regeneration through cell-based therapies. This study investigated the potential of amniotic fluid–derived stem (AFS) cells to synthesize mineralized extracellular matrix within porous medical-grade poly-e-caprolactone (mPCL) scaffolds. The AFS cells were initially differentiated in two-dimensional (2D) culture to determine appropriate osteogenic culture conditions and verify physiologic mineral production by the AFS cells. The AFS cells were then cultured on 3D mPCL scaffolds (6-mm diameter9-mm height) and analyzed for their ability to differentiate to osteoblastic cells in this environment. The amount and distribution of mineralized matrix production was quantified throughout the mPCL scaffold using nondestructive micro computed tomography (microCT) analysis and confirmed through biochemical assays. Sterile microCT scanning provided longitudinal analysis of long-term cultured mPCL constructs to determine the rate and distribution of mineral matrix within the scaffolds. The AFS cells deposited mineralized matrix throughout the mPCL scaffolds and remained viable after 15 weeks of 3D culture. The effect of predifferentiation of the AFS cells on the subsequent bone formation in vivo was determined in a rat subcutaneous model. Cells that were pre-differentiated for 28 days in vitro produced seven times more mineralized matrix when implanted subcutaneously in vivo. This study demonstrated the potential of AFS cells to produce 3D mineralized bioengineered constructs in vitro and in vivo and suggests that AFS cells may be an effective cell source for functional repair of large bone defects

Cellular senescence and longevity of osteophyte-derived mesenchymal stem cells compared to patient-matched bone marrow stromal cells

This study aimed to determine the cellular aging of osteophyte-derived mesenchymal cells (oMSCs) in comparison to patient-matched bone marrow stromal cells (bMSCs). Extensive expansion of the cell cultures was performed and early and late passage cells (passages 4 and 9, respectively) were used to study signs of cellular aging, telomere length, telomerase activity, and cell-cycle-related gene expression. Our results showed that cellular aging was more prominent in bMSCs than in oMSCs, and that oMSCs had longer telomere length in late passages compared with bMSCs, although there was no significant difference in telomere lengths in the early passages in either cell type. Telomerase activity was detectable only in early passage oMSCs and not in bMSCs. In osteophyte tissues telomerase-positive cells were found to be located perivascularly and were Stro-1 positive. Fifteen cell-cycle regulator genes were investigated and only three genes (APC, CCND2, and BMP2) were differentially expressed between bMSC and oMSC. Our results indicate that oMSCs retain a level of telomerase activity in vitro, which may account for the relatively greater longevity of these cells, compared with bMSCs, by preventing replicative senescence. J. Cell. Biochem. 108: 839-850, 2009. (c) 2009 Wiley-Liss, Inc.