Immune Regulatory Activity Research Articles

Novel SiRNA Delivery Technology Promises Hope of New Approach for a Treatment of Rejection in Organ Transplantation - SiRNATargeting Co-Stimulatory Signal

Introduction: Silencing of gene expression by siRNAs has the potential to rapidly become a powerful therapeutics development platform. However, the efficacious siRNA delivery to target organs and cells with effective endosomal escape is still a developing area. SPG is a fungal cell surface ligand for the mammalian cell surface receptor dectin-1 (b-glucan receptor) present on the surface of antigen presenting cells (for response to fungal infections) which internalize the b-glucan after binding. We have devised a delivery complex comprised of schizophyllan (SPG) duplexed with a chimeric siRNA-poly-dA molecule (siRNA/SPG) targeting dectin-1 expressing cells. While targeting the CD40 mRNA, we report successful delivery of poly-dA linked siRNA complexed with SPG into mice to show the suppression of co-stimulatory activity and to explore therapeutic applications. Materials and methods: Poly-dA linked siRNA was chemically synthesized and complexed with SPG using our proprietary approach. We have performed confocal microscopic observation, FACS analysis, mixed lymphocyte reaction (MLR) to prove the delivery and bioactivity of siCD40/SPG both in vitro and in vivo. Results: We observed co-localization of siCD40/SPG and TRBP2 (core subunit of RLC) by using a confocal microscope in splenocyte and dRaw cells (dectin-1 over-expressing RAW264.7 cells). Using Alexa modified siCD40, we observed its uptake into dRaw cells when added complexed with SPG in the culture medium, while not by naked siCD40. In dRaw cells, the siCD40/SPG knocked down the CD40 gene expression in dose dependent manner. Also, we found that the siCD40/SPG maintained RNAi activity for at least 36 hours. Utilizing in vitro MLR and ex-vivo MLR of Balb/c (stimulator) and B6 (responder) combination, the siCD40/SPG successfully suppressed 60% of cellular proliferation (see fig.). In ex-vivo MLR, we obtained same results either when simulator or responder mouse was pre-treated with siCD40/SPG. The siCD40/SPG demonstrated bioactivity in the splenocytes strongly suggesting that the siCD40/SPG traffics to the cytoplasm through dectin-1 mediated internalization. The experimental conditions determined from these studies were successfully used to obtain long term graft survival in mouse MHC fully mismatched allogeneic heart transplantation (presented in a separate publication in this Congress).Figure: [Result of Ex-vivo MLR]Conclusion: We conclude: siRNA/SPG binds RLC in complex form, indicating that the poly-dA linked siRNA is protected from RNase attack by SPG and it traffics to the cytoplasm. Subsequently, the siCD40/SPG causes mRNA degradation. siRNA/SPG is internalized through Dectin-1 mediated pathway, into splenocytes in sufficient amount and traffics to cytoplasm. siRNA for CD40 (siCD40) showed sufficient immune-regulatory activity both in vitro and in vivo. Our results indicate a potential application of our siRNA delivery technology for organ transplantation.

Lactobacillus plantarumNRIC1832 Enhances IL-10 Production from CD4+T Cellsin Vitro

The anti-inflammatory effect of lactic acid bacteria (LAB) has been reported in several models for autoimmune diseases. It was considered in those studies that IL-10 induced by LAB might have been involved in such anti-inflammatory activity. We therefore examined the IL-10-inducing activity of LAB in detail by using an in vitro culture system of DO11.10 splenocytes. Most strains of LAB tested in this study increased IL-10 production. A further study using one of the tested strains with potent immune-regulatory activity, Lactobacillus plantarum NRIC1832, showed that the enhanced IL-10 was mainly produced by T cells. However, this enhancement required several types of cells other than T cells. NRIC1832 enhanced IL-10 production after short-term exposure to T cells, but this effect diminished after long-term exposure, indicating that the enhancement of IL-10 production by NRIC1832 was temporary, in contrast to the enhancement of IFN-γ production which was still apparent after long-term exposure.

An exploratory study to investigate the immunomodulatory activity of BMS-936558 in patients with metastatic clear-cell renal cell carcinoma.

TPS2613 Background: With the enhanced understanding of the immune response to a tumor, new immunotherapeutic agents may expand treatment options for metastatic renal cell carcinoma (mRCC). The programmed death-1 (PD-1) receptor is upregulated by activated lymphocytes, and upon binding to its ligands, inhibits lymphocyte function. In a phase 1 study of the anti-PD-1 monoclonal antibody BMS-936558, encouraging antitumor activity has been observed in patients with previously treated mRCC. Responses occurred in extensive mRCC and were durable in many patients. Here we describe an exploratory study to investigate the immune-regulatory activity of various doses of BMS-936558 in patients with metastatic clear-cell RCC. Methods: Eighty patients with metastatic clear-cell RCC will be enrolled in this open-label, parallel group, randomized study. Patients who have received prior treatment with ≥3 therapies, including ≥1 anti-angiogenic therapy, will be enrolled in Arms 1-3 (~20 patients per arm); 20 treatment-naïve patients will be enrolled in Arm 4. BMS-936558 will be administered every 3 weeks until disease progression or unacceptable toxicity at 0.3 mg/kg (Arm 1), 2 mg/kg (Arm 2), and 10 mg/kg (Arms 3 and 4). Patients in the 0.3-mg/kg group may escalate to 2 mg/kg if not responding. Tumor assessments will occur every 6 weeks for the first 12 months then every 12 weeks for the remainder of the study. The primary objective is to evaluate the effects of BMS-936558 on the frequency of circulating immune cell subsets, levels of soluble factors associated with immune responses, and tumor infiltrating lymphocyte populations (based on pre-treatment and week 5 paired biopsies). Secondary objectives are to assess the safety, tolerability, antitumor activity, and immunogenicity of BMS-936558. Exploratory objectives include the association of clinical activity and safety with selected biomarkers in peripheral blood and tumor microenvironment and the characterization of the pharmacokinetics of BMS-936558. As of February 1, 2012, 30 patients were enrolled.

Abstract 556: L-Tag-driven systemic immune regulatory profile as a potential signature for polyomavirus BK involvement in prostate cancer

Abstract Background: Polyomavirus BK (BKV) large tumor antigen (L-Tag) contributes to oncogenesis by regulating crucial pathways of human cell cycle when non-permissive cells are infected. Therefore, L-Tag has been identified as an important target of immune surveillance in L-Tag expressing tumors. The role of L-Tag as a target of tumor immune surveillance in prostate cancer (PCa) patients has not been investigated so far. In this study we thus analyzed the immunological profile exerted by L-Tag peptide-specific induction in newly diagnosed PCa patients by means of BKV L-Tag serology and local molecular testing, and compared it to age-matched benign prostatic hyperplasia (BPH) patients. Methods: We enrolled 60 newly diagnosed PCa patients and 50 age-matched BPH. Detection of BKV L-Tag DNA was carried out in random punches from tumor areas of surgically excised specimens. BKV L-Tag seroprevalence was determined by enzyme immunoassay (EIA). Systemic immune-regulatory activity was tested upon BKV L-Tag peptide-pool induction and analyzed using cytokine gene expressions (qrt-PCR), antigen-specific regulatory T cell (Treg) characterization (flow cytometry) and immune-regulatory protein release (multiplex fluorescent bead immunoassay). Data were correlated to 5-year follow-up clinical information. Five-year follow-up after surgery was completed on 48/60 PCa patients. Biochemical recurrence (BR) with early censoring was defined as the first PSA ≤ 0.2ng/ml. Results: In peripheral blood mononuclear cells (PBMCs) from PCa patients with evidence of biochemical recurrence (BR) and BKV L-Tag positive lesions, BKV L-Tag peptide-pool, but not the Epstein Barr/Influenza virus/Cytomegalovirus (CEF) peptide-pool, induced a particular gene profile, which was characterized by high expression of IL-10 and TGFβ-1 and a low expression of IFN-γ and TNF-α genes. This signature was confirmed by protein secretion data and correlated with levels of anti-BKV L-Tag IgG activity. Furthermore, in these PCa patients the L-Tag peptide pool significantly boosted IL-10 secreting CD4+CD25+(high)/FoxP3+(high)/CD103+ T cells exerting suppressive functions on autologous effectors (CD4+, CD25− and CD8+ T cell fractions) by reducing cell proliferation and IFN-γ production. Conclusion: Significant associations between immune regulatory activity exerted by BKV L-Tag induction, BKV L-Tag DNA positive tumor lesions and IgG activity against L-Tag were observed in PCa patients. In detail, BR+ PCa patients shared features such as functional BKV L-Tag specific immune regulatory profile with marked BKV L-Tag positive lesion and strong L-Tag-specific IgG activity, thereby hinting at a potential BKV involvement in prostate cancer progression. Our findings are thus consistent with a systemic BKV L-Tag-related tolerogenic environment governing prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 556. doi:1538-7445.AM2012-556

The Tight Relationship Between Osteoclasts and the Immune System

Osteoimmunology is an interdisciplinary field addressing the interplay between the skeletal and the immune system. A substantial body of evidence demonstrated the existence of two-way regulatory mechanisms that affect both systems, placing them in much closer association to each other than one could ever predict. Inflammatory diseases have long been known to induce alterations in bone metabolism, and inflammatory cytokines play prominent roles in the control of bone resorption, representing communication pathways bridging the two systems. Osteoclasts are particularly linked to the immune cells because they belong to the monocyte/macrophage family, have tight relationships with B and T cells, and differentiate in response to RANKL which is also produced by lymphocytes and regulates lymphopoiesis. Osteoclasts are negatively regulated by cytokines and other factors known for their anti-inflammatory and immune regulatory activity. Finally, they express immune co-receptor typical of immune cells which are indispensable for their differentiation, thus leading to the hypothesis that osteoclasts are immune cells themselves. The underlying principle why an immune cell is required to resorb bone has not yet been elucidated. Data from early literature suggest that the bone matrix could trigger an innate immune response activating giant cells that could destroy large bone areas because of their unique property of resorbing bone extracellularly. Bone resorption could though be prevented by the physical barrier made by osteoblasts and lining cells, whose retraction would be required to give access to osteoclasts when specific pathways signal their precursors to differentiate and mature osteoclasts to reach the uncovered bone surface.

Myeloid-Derived Suppressor Cells Protect Islet Transplants by B7-H1 Mediated Enhancement of T Regulatory Cells

Side effects of lifetime immunosuppression for cell transplants often outweigh the benefits; therefore, induction of transplant tolerance is needed. We have shown that cotransplantation with myeloid-derived suppressor cells (MDSC) effectively protect islet allografts from rejection without requirement of immunosuppression. This study was to investigate the underlying mechanisms. MDSC were generated by addition of hepatic stellate cells from various stain mice into dendritic cell (DC) culture. The quality of MDSC was monitored by phenotype and function analyses. MDSC mixed with islet allografts were transplanted into diabetic recipients. T-cell response was analyzed after transplantation by using flow and histochemical analyses, and was compared with islet alone and islet/DC transplant groups. B7-H1 knockout mice were used to determine the role of B7-H1 on MDSC in regulation of T-cell response. Cotransplantation with MDSC (not DC) effectively protected islet allografts without requirement of immunosuppression. This is associated with attenuation of CD8 T cells in the grafts and marked expansion of regulatory T (Treg) cells, which contributed to MDSC-induced T-cell hyporesponsiveness. Antigen-specific Treg cells were prone to accumulate in lymphoid organs close to the grafts. Both in vitro and in vivo data demonstrated that B7-H1 was absolutely required for MDSC to exert immune regulatory activity and induction of Treg cells. The described approach holds great clinical application potential and may overcome the limitation of requiring chronic administration of immunosuppression in cell transplants. Understanding the underlying mechanisms will facilitate the development of this novel therapeutic strategy.

Diverse ability of maternal immune stimulation to reduce birth defects in mice exposed to teratogens: a review.

Stimulating the maternal immune system before or during pregnancy can dramatically improve morphologic outcome in mice that have been exposed to teratogens. For example, maternal immune stimulation in mice reduced craniofacial and palate defects, heart defects, digit and limb defects, tail malformations and neural tube defects caused by diverse teratogens that included chemical agents, hyperthermia, X-rays and diabetes mellitus. Several different procedures of immune stimulation were effective and included footpad injection with Freund's Complete Adjuvant, intraperitoneal (IP) injection with inert particles or attenuated Bacillus Calmette-Guerin, intrauterine injection with allogenic or xenogenic lymphocytes, or intravascular, intrauterine or IP injection with immunomodulatory cytokines. Limited information is available regarding mechanisms by which such immune stimulation reduces fetal dysmorphogenesis; however, cytokines of maternal origin have been suggested as effector molecules that act on the placenta or fetus to improve development. These collective data raise novel questions about the possibility of unrecognized maternal immune system regulatory activity in normal fetal development. This manuscript reviews the literature showing maternal immune protection against morphologic birth defects. Potential operating mechanisms are discussed, and the possibility is considered that a suppressed maternal immune system may negatively impact fetal development.

C17 Prevents Inflammatory Arthritis and Associated Joint Destruction in Mice

C17 was first described about ten years ago as a gene expressed in CD34+ cells. A more recent study has suggested a role for C17 in chondrogenesis and development of cartilage. However, based on sequence analysis, we believe that C17 has homology to IL-2 and hence we present the hypothesis that C17 is a cytokine possessing immune-regulatory properties. We provide evidence that C17 is a secreted protein preferentially expressed in chondrocytes, hence in cartilage-rich tissues. Systemic expression of C17 in vivo reduces disease in a collagen antibody-induced arthritis model in mice (CAIA). Joint protection is evident by delayed disease onset, minimal edema, bone protection and absence of diverse histological features of disease. Expression of genes typically associated with acute joint inflammation and erosion of cartilage or bone is blunted in the presence of C17. Consistent with the observed reduction in bone erosion, we demonstrate reduced levels of RANKL in the paws and sera of mice over-expressing C17. Administration of C17 at the peak of disease, however, had no effect on disease progression, indicating that C17's immune-regulatory activity must be most prominent prior to or at the onset of severe joint inflammation. Based on this data we propose C17 as a cytokine that s contributes to immune homeostasis systemically or in a tissue-specific manner in the joint.

Modulation of chicken macrophage effector function by T H1/T H2 cytokines

Regulation of macrophage activity by T H1/2 cytokines is important to maintain the balance of immunity to provide adequate protective immunity while avoiding excessive inflammation. IFN-γ and IL-4 are the hallmark T H1 and T H2 cytokines, respectively. In avian species, information concerning regulation of macrophage activity by T H1/2 cytokines is limited. Here, we investigated the regulatory function of chicken T H1 cytokines IFN-γ, IL-18 and T H2 cytokines IL-4, IL-10 on the HD11 macrophage cell line. Chicken IFN-γ stimulated nitric oxide (NO) synthesis in HD11 cells and primed the cells to produce significantly greater amounts of NO when exposed to microbial agonists, lipopolysaccharide, lipoteichoic acid, peptidoglycan, CpG-ODN, and poly I:C. In contrast, chicken IL-4 exhibited bi-directional immune regulatory activity: it activated macrophage NO synthesis in the absence of inflammatory agonists, but inhibited NO production by macrophages in response to microbial agonists. Both IFN-γ and IL-4, however, enhanced oxidative burst activity of the HD11 cells when exposed to Salmonella enteritidis. IL-18 and IL-10 did not affect NO production nor oxidative burst in HD11 cells. Phagocytosis and bacterial killing by the HD11 cells were not affected by the treatments of these cytokines. Infection of HD11 cells with S. enteritidis was shown to completely abolish NO production regardless of IFN-γ treatment. This study has demonstrated that IFN-γ and IL-4 are important T H1 and T H2 cytokines that regulate macrophage function in chickens.

The Alpha-Melanocyte Stimulating Hormone Induces Conversion of Effector T Cells into Treg Cells

The neuropeptide alpha-melanocyte stimulating hormone (α-MSH) has an important role in modulating immunity and homeostasis. The production of IFN-γ by effector T cells is suppressed by α-MSH, while TGF-β production is promoted in the same cells. Such α-MSH-treated T cells have immune regulatory activity and suppress hypersensitivity, autoimmune diseases, and graft rejection. Previous characterizations of the α-MSH-induced Treg cells showed that the cells are CD4+ T cells expressing the same levels of CD25 as effector T cells. Therefore, we further analyzed the α-MSH-induced Treg cells for expression of effector and regulatory T-cell markers. Also, we examined the potential for α-MSH-induced Treg cells to be from the effector T-cell population. We found that the α-MSH-induced Treg cells are CD25+ CD4+ T cells that share similar surface markers as effector T cells, except that they express on their surface LAP. Also, the α-MSH treatment augments FoxP3 message in the effector T cells, and α-MSH induction of regulatory activity was limited to the effector CD25+ T-cell population. Therefore, α-MSH converts effector T cells into Treg cells, which suppress immunity targeting specific antigens and tissues.

Multiple functions of human T cells generated by experimental malaria challenge

Protective immunity generated following malaria infection may be comprised of Ab or T cells against malaria Ag of different stages; however, the short-lived immunity that is observed suggests deficiency in immune memory or regulatory activity. In this study, cellular immune responses were investigated in individuals receiving Plasmodium falciparum sporozoite challenge by the natural (mosquito bite) route as part of a malaria vaccine efficacy trial. Parasitemia, monitored by blood film microscopy and PCR, was subsequently cleared with drugs. All individuals demonstrated stable IFN-gamma, IL-2 and IL-4 ex vivo ELISPOT effector responses against P. falciparum-infected RBC (iRBC) Ag, 28 and 90 days after challenge. However, infected RBC-specific central memory responses, as measured by IFN-gamma cultured ELISPOT, were low and unstable over time, despite CD4(+) T cells being highly proliferative by CFSE dilution, and showed an inverse relationship to parasite density. In support of the observation of poor memory, co-culture experiments showed reduced responses to common recall Ag, indicating malaria-specific regulatory activity. This activity could not be accounted for by the expression of IL-10, TGF-beta, FOXP3 or CTLA-4, but proliferating T cells expressed high levels of CD95, indicating a pro-apoptotic phenotype. Lastly, there was an inverse relationship between FOXP3 expression, when measured 10 days after challenge, and ex vivo IFN-gamma measured more than 100 days later. This study shows that malaria infection elicits specific Th1 and Th2 effector cells, but concomitant weak central memory and regulatory activity, which may help to explain the short-lived immunity observed.

Immunostimulatory Activity of Specific CpG Oligonucleotides from Bifidobacterium longum Genome on RAW 264.7 Macrophage Cells

Bacterial CpG motifs have attracted considerable interests because of their immunomodulatory activities. Since Bifidobacterium is an important probiotic bacteria with characteristically high G+C content of 60 to 67%, investigation on the immune regulatory activity of CpG oligodeoxynucleotides (ODNs) from Bifidobacterium is warranted. The ODNs containing repeated TCG motifs were designed and synthesized based upon B. longum genomic sequence. Four 21-mer ODNs (ODNBL1∼4) and three 25-mer ODNs (CpG ODN1∼2, ODNBL5) induced macrophages to secrete cytokines such as IL-6, IL-12p40 and TNF-α. In particular, ODNBL5 induced macrophages to secret high amount of IL-6 and IL-12 compared with positive control PE2080 which was reported as a strong immune activating CpG ODN. This indicated that the specific functional sequences in the genome of B. longum could activate murine macrophage RAW 264.7 possibly acting as an immunostimulatory modulator for immune response.

Heat Shock Protein 90 Inhibitor BIIB021 (CNF2024) Depletes NF-κB and Sensitizes Hodgkin's Lymphoma Cells for Natural Killer Cell–Mediated Cytotoxicity

In Hodgkin's lymphoma, constitutive activation of NF-kappaB promotes tumor cell survival and proliferation. The molecular chaperone heat shock protein 90 (HSP90) has immune regulatory activity and supports the activation of NF-kappaB in Hodgkin's lymphoma cells. We analyzed the effect of HSP90 inhibition on viability and NF-kappaB activity in Hodgkin's lymphoma cells and the consequences for their recognition and killing through natural killer (NK) cells. The novel orally administrable HSP90 inhibitor BIIB021 (CNF2024) inhibited Hodgkin's lymphoma cell viability at low nanomolar concentrations in synergy with doxorubicin and gemcitabine. Annexin V/7-aminoactinomycin D binding assay revealed that BIIB021 selectively induced cell death in Hodgkin's lymphoma cells but not in lymphocytes from healthy individuals. We observed that BIIB021 inhibited the constitutive activity of NF-kappaB and this was independent of IkappaB mutations. Furthermore, we analyzed the effect of HSP90 inhibition on NK cell-mediated cytotoxicity. BIIB021 induced the expression of ligands for the activating NK cell receptor NKG2D on Hodgkin's lymphoma cells resulting in an increased susceptibility to NK cell-mediated killing. In a xenograft model of Hodgkin's lymphoma, HSP90 inhibition significantly delayed tumor growth. HSP90 inhibition has direct antitumor activity in Hodgkin's lymphoma in vitro and in vivo. Moreover, HSP90 inhibition may sensitize Hodgkin's lymphoma cells for NK cell-mediated killing via up-regulation of ligands engaging activating NK cell receptors.

Mechanistic insights into immunomodulation by hepatic stellate cells in mice: A critical role of interferon-γ signaling

The liver is considered to be an immune-privileged organ that favors the induction of tolerance. The underlying mechanisms are not completely understood. Interestingly, liver transplants are spontaneously accepted in several animal models, but hepatocyte transplants are acutely rejected, suggesting that liver nonparenchymal cells may effectively protect the parenchymal cells from immune attack. We have shown the profound T cell inhibitory activity of hepatic stellate cells (HSCs). Thus, cotransplantation with HSCs effectively protects islet allografts from rejection in mice. In this study, using T cell receptor transgenic and gene knockout approaches, we provided definitive evidence that HSCs protected cotransplanted islet allografts by exerting comprehensive inhibitory effects on T cells, including apoptotic death in graft-infiltrating antigen-specific effector T cells and marked expansion of CD4(+) Forkhead box protein (Foxp)3(+) T regulatory (Treg) cells. All these effects required an intact interferon-gamma (IFN-gamma) signaling in HSCs, demonstrated by using HSCs isolated from IFN-gamma receptor 1 knockout mice. B7-H1 expression on HSCs, a product molecule of IFN-gamma signaling, was responsible for induction of T cells apoptosis, but had no effect on expansion of Treg cells, suggesting that undetermined effector molecules produced by IFN-gamma signaling is involved in this process. Upon inflammatory stimulation, specific organ stromal cells (such as HSCs in the liver) demonstrate potent immune regulatory activity. Understanding of the mechanisms involved may lead to development of novel strategies for clinical applications in transplantation and autoimmune diseases.

Select procyanidins prime the γδ T cell by modulating transcriptome degradation (134.88)

Abstract Tannins from many plant-derived traditional medicines demonstrate immune regulatory activity. Recently, we reported that condensed tannins (procyanidins) isolated from a select number of traditional medicines act as γδ T cell agonists. γδ T cells play an important role in mucosal immunity by regulating the mucosal immune response and maintaining epithelial health. Procyanidins induce a priming state on the γδ T cell, thus improving its ability to quickly respond to secondary stimuli. This priming state is characterized, in part, by an increased half-life of select transcripts in the γδ T cell relevant to innate immunity. This increase in transcript half-life could enable the cell to more quickly produce appropriate immune responses to secondary stimuli. Previously we reported that oligomeric procyanidin fractions, but not monomeric procyanidins, act as γδ T cell agonists. We have further defined the activity of different sizes of procyanidins. The tetrameric and pentameric procyanidins from apple peel have greater specificity for γδ T cells, whereas larger oligomers demonstrate less specificity. Our results suggest procyanidins with specific oligomeric structures prime γδ T cells by stabilizing transcripts with critical functions in innate immunity. Funded by: NIH/ NIAID Contract HHSN266200400009/N01-AI40009 and NCCAM P01 AT004986-01

Heat shock protein-containing exosomes in mid-trimester amniotic fluids

Exosomes are multivesicular bodies formed by inverse membrane budding into the lumen of an endocytic compartment. Fusion with the plasma membrane leads to their release into the external milieu. The incorporation of heat shock proteins into exosomes has been associated with immune regulatory activity. We have examined whether heat shock protein-containing exosomes are present in mid-trimester amniotic fluid. Exosomes were isolated from mid-trimester amniotic fluids by sequential low-speed and high-speed centrifugation followed by sucrose density gradient centrifugation. Biochemical characterization included floatation pattern in sucrose gradients, acetylcholinesterase (AChE) activity and Western blot analysis for exosome-containing proteins. Exosomes were present in each of 23 amniotic fluids tested. They banded at a density of 1.17 g/ml in sucrose gradients, were positive for AChE activity and contained tubulin, the inducible 72 kDa heat shock protein, Hsp72 and the constitutively expressed heat shock protein, Hsc73; they were negative for calnexin. Exosome concentrations correlated positively with the number of pregnancies. Heat shock protein-containing exosomes are constituents of mid-trimester amniotic fluids and may contribute to immune regulation within the amniotic cavity.

Foxp3+CD25+ T Regulatory Cells Stimulate IFN-γ-Independent CD152-Mediated Activation of Tryptophan Catabolism That Provides Dendritic Cells with Immune Regulatory Activity in Mice Unresponsive to Staphylococcal Enterotoxin B

Mice made unresponsive by repeated injection of staphylococcal enterotoxin B (SEB) contained SEB-specific CD25(+)CD4(+)TCRBV8(+) T cells that were able to transfer their state of unresponsiveness to primary-stimulated T cells. About one-half of these cells stably up-regulated the expression of CD152. We undertook the present study to determine whether CD152(high) cells seen in this system were T regulatory cells responsible for suppression or whether they represented SEB-activated CD4(+) T effector cells. Our results show that, among SEB-specific TCRBV8(+) T cells isolated from unresponsive mice, all CD152(high)CD25(+)CD4(+) T cells expressed Foxp3, the NF required for differentiation and function of natural T regulatory cells. Moreover, suppression by CD25(+)CD4(+)TCRBV8(+) T cells was fully inhibited by anti-CD152 Abs. Following stimulation by soluble CD152-Ig, dendritic cells (DC) isolated from unresponsive mice strongly increased the expression and the function of indoleamine-2,3-dioxygenase (IDO), the enzyme responsible for the catabolism of tryptophan. This capacity to activate IDO was independent of IFN-gamma production by DC because CD152-Ig stimulation of DC isolated from SEB-treated IFN-gamma-deficient animals activated IDO expression and function. Finally, adding 1-methyl-tryptophan, an inhibitor of tryptophan catabolism, increased substantially the capacity of DC from unresponsive animals to stimulate primary T cell response toward SEB. Thus, we conclude that IFN-gamma-independent CD152-mediated activation of tryptophan catabolism by Foxp3(+)CD25(+) T regulatory cells provides DC with immune regulatory activity in mice unresponsive to SEB.

In vivo immune modulatory activity of hepatic stellate cells in mice

Accumulating data suggest that hepatic tolerance, initially demonstrated by spontaneous acceptance of liver allografts in many species, results from an immune regulatory activity occurring in the liver. However, the responsible cellular and molecular components have not been completely understood. We have recently described profound T cell inhibitory activity of hepatic stellate cells (HSCs) in vitro. In this study, we demonstrate in vivo evidence of immune modulatory activity of HSCs in mice using an islet transplantation model. Co-transplanted HSCs effectively protected islet allografts from rejection, forming a multi-layered capsule, which reduced allograft immunocyte infiltrates by enhancement of apoptotic death. The immune modulation by HSCs appeared to be a local effect, and regulated by inducible expression of B7-H1, an inhibitory molecule of B7 family. This may reflect an intrinsic mechanism of immune inhibition mediated by liver-derived tissue cells. In conclusion, these results may lead to better understanding of liver immunobiology and development of new strategies for treatment of liver diseases.

Immune regulatory activity of liver‐derived dendritic cells generated in vivo

Hepatic tolerance is demonstrated by spontaneous acceptance of liver allografts in mice. Hepatic dendritic cells (DC) play a crucial role in determining immunity or tolerance. In this study, we adopted an approach to transfect gene(s) into the mouse liver by tail-vein injection of plasmid-carrying genes. Transfection with GM-CSF expanded liver CD11c+ myeloid DC (LMDC), while liver B220+CD11c- lymphoid DC (LLDC) were expanded after transfection of IL-3 and CD40L. Flow analysis revealed that these liver DC subsets were phenotypically mature following overnight culture. However, in contrast to LMDC, LLDC induced hyporesponsiveness in allogeneic T-cells, with suppressed secretion of both IL-2 and IFN-gamma, and prolonged cardiac allograft survival. This immune regulatory DC population in the liver may play a role in modulating T-cell immunity in the liver.

Reduced Cord Blood Immune Effector‐Cell Responsiveness Mediated by CD4+Cells Induced in Utero as a Consequence of PlacentalPlasmodium falciparumInfection

To determine mechanisms of neonatal parasite antigen (Ag)-specific immune suppression associated with placental Plasmodium falciparum infection, we isolated cord blood mononuclear cells (CBMCs) from Gabonese neonates born to mothers with differing histories of P. falciparum infection and performed ex vivo and in vitro studies to evaluate immune regulatory activity. We found increased ex vivo percentages of CD4(+)CD25(hi) and CD4(+)CD25(+)CTLA-4(+) cells and increased interleukin (IL)-10 responses to parasite Ag in vitro in CBMCs from neonates born to mothers with placental P. falciparum infection at delivery. Depleting CBMCs of CD4(+)CD25(+) cells before cell culture led to the abrogation of parasite Ag-specific IL-10 responses, to enhanced interferon- gamma responses, and to enhanced expression of CD25 on CD8(+) T cells and of major histocompatibility complex class I and II on monocytes. These data demonstrate that parasite Ag-specific CD4(+) regulatory cells are generated in utero as a consequence of placental P. falciparum infection.