Comparative analysis of dendritic cell density and total number in commonly transplanted organs: morphometric estimation in normal mice

Dendritic cells (DC) are considered to be the major cell type responsible for induction of primary immune responses. While they have been shown to play a critical role in eliciting allosensitization via the direct pathway, there is evidence that maturational and/or activational heterogeneity between DC in different donor organs may be crucial to allograft outcome. Despite such an important perceived role for DC, no accurate estimates of their number in commonly transplanted organs have been reported. Therefore, leukocytes and DC were visualized and enumerated in cryostat sections of normal mouse (C57BL/10, B10.BR, C3H) liver, heart, kidney and pancreas by immunohistochemistry (CD45 and MHC class II staining, respectively). Total immunopositive cell number and MHC class II+ cell density (C57BL/10 mice only) were estimated using established morphometric techniques - the fractionator and disector principles, respectively. Liver contained considerably more leukocytes (similar to 5-20 x 10(6)) and DC (similar to 1-3 x 10(6)) than the other organs examined (pancreas: similar to 0.6 x 10(6) and similar to 0.35 x 10(6): heart: similar to 0.8 x 10(6) and similar to 0.4 x 10(6); kidney similar to 1.2 x 10(6) and 0.65 x 10(6), respectively). In liver, DC comprised a lower proportion of all leukocytes (similar to 15-25%) than in the other parenchymal organs examined (similar to 40-60%). Comparatively, DC density in C57BL/10 mice was heart > kidney > pancreas much greater than liver (similar to 6.6 x 10(6), 5 x 10(6), 4.5 x 10(6) and 1.1 x 10(6) cells/cm(3), respectively). When compared to previously published data on allograft survival, the results indicate that the absolute number of MHC class II+ DC present in a donor organ is a poor predictor of graft outcome. Survival of solid organ allografts is more closely related to the density of the donor DC network within the graft. (C) 2000 Elsevier Science B.V. All rights reserved.

Heavy chain ferritin activates regulatory T cells by induction of changes in dendritic cells

Heavy chain ferritin (H-ferritin) Is a component of the Iron-binding protein, ferritin. We have previously shown that H-ferritin Inhibits anti-CD3-stimulated lymphocyte proliferation and that this was due to Increased production of Interleukin-10 (IL-10). In the present study we have shown that Induction of IL-10 production was due to effects of H-ferritin on adherent antigen-presenting cells (APCs) In blood and monocyte-derived dendritic cells (MoDCs). IL-10 was produced by a subpopulation of CD4 T cells, which expressed the CD25 component of the IL-2 receptor and the CTLA-4 receptor characteristic of regulatory T cells. The changes Induced In MoDCs were compared with those Induced by CD40L and their significance tested by Inhibition with monoclonal antibodies. These studies Indicated that H-ferritin Induced relatively greater expression of CD86 and B7-H1 on MoDCs and that monoclonal antibodies against their receptors, CTLA-4 and programmed death receptor-1 (PD-1), Inhibited IL-10 production from the regulatory T cells. H-ferritin did not appear to Induce direct production of the cytokines IL-2, IL-4, IL-6, IL-10, IL-12, or Interferon-gamma from the DCs. These results are consistent with the thesis that H-ferritin Induces B7-H1 and CD86 (B7-2) on APCs, which In turn Induce IL-10 production from regulatory T cells. This is possibly one mechanism by which melanoma cells may Induce changes In APCs In the vicinity of the tumor and result in suppression of Immune responses by induction of regulatory T cells. (C) 2002 by The American Society of Hematology.

Immunobiology of liver dendritic cells

Dendritic cells (DC) are rare, bone marrow-derived antigen-presenting cells that play a critical role in the induction and regulation of immune reactivity. In this article, we review the identification and characterization of liver DC, their ontogenic development, in vivo mobilization and population dynamics. In addition, we discuss the functions of DC isolated from liver tissue or celiac lymph, or propagated in vitro from liver-resident haemopoietic stem/progenitor cells. Evidence concerning the role of DC in viral hepatitis. liver tumours, autoimmune liver diseases, granulomatous inflammation and the outcome of liver transplantation is also discussed.

Association of increased levels of heavy-chain ferritin with increased CD4(+) CD25(+) regulatory T-Cell levels in patients with melanoma

We have shown previously that melanoma cells in culture release heavy-chain ferritin (H-Ferritin) into supernatants and that this is responsible for the suppression of responses of peripheral blood lymphocytes stimulated by anti-CD3. These effects were mediated by activation of regulatory T cells to produce interleukin (IL)-10. In the present study, we examined whether a similar relation might exist between levels of H-Ferritin and activation of regulatory T cells in patients with melanoma. Ferritin levels were evaluated by ELISA and regulatory T-cell numbers were assessed by three-color flow cytometry to identify CD4(+) CD25(+) CD69(-) T cells. CD69 positive cells were excluded to avoid inclusion of normal activated CD4, CD25 expressing T cells. Measurements of H- and light-chain (L)-Ferritin by ELISA revealed that H- but not L-Ferritin was elevated in the circulation of melanoma patients. In addition, these studies revealed a marked increase in the number of CD4+ CD25+ CD69- T cells in such patients, compared with age-matched controls. The ratio of H-Ferritin:L-Ferritin correlated with the levels of regulatory T cells consistent with a causal relation between unbound H-Ferritin levels and the activation of regulatory T cells. H-Ferritin or regulatory T cells did not, however, correlate with the stage of the melanoma. These results provide evidence for the importance of H-Ferritin in the induction of regulatory T cells in patients with melanoma and provide additional insight into the suppression of immune responses in such patients.

Overcoming original antigenic sin to generate effective immune responses in antigen primed host

Original antigenic sin is failure to mount effective immunity to virus variants in a previously virus infected host. We have previously shown that prior immunity to a virus capsid protein inhibits induction from naive CD8 T cells of an IFN-g response to a MHC class I restricted epitope linked to the capsid protein, following immunisation with a capsid expressing the class I restricted epitope. The inhibition is independent of pre-existing antibody to the viral capsid, and the inhibition is observed in animal lacking B cells. CD8 restricted viral capsid specific T cell responses are also not required, but the inhibition is not observed in IL10 knockout mice. We now demonstrate that capsid antigen primed CD4+ T cells secrete IL10 in response to capsid antigen presented by DC, and deviate CD8 cells specific for the linked MHC Class I restricted epitope from IFN-g production to IL-5 production. Neutralizing IL10, either in vitro or in vivo, restores induction following immunisation of an antigen specific IFN-g response to an MHC Class I restricted epitope. This finding demonstrates a strategy for overcoming bias towards a Tc2 response to MHC Class I epitopes upon immunisation of a host already primed to antigen, facilitating immunotherapy for chronic viral infection or cancer

B, NK and NKT cells contribute to suppression of immunity by dendritic cells

Among the population of antigen presenting cells, dendritic cells (DCs) are considered the sentinels of the immune system. Besides activating naı¨ ve T cells, DC can directly activate naı¨ ve and memory B cells and are also able to regulate effectors of innate immunity such as NK cells and NKT cells. Increasing evidence indicates that DCs are not only decisive for T cell priming, but are also key players to maintain self-tolerance in vivo. Previous results in our lab have shown that DCs treated with a pharmacological NFkB inhibitor (BAY11–7082) confer suppression to a previously immune response. This suppression was IL-10 dependent and results from the induction of Ag specific CD4+ regulatory T cells. To elucidate the mechanism of suppression induced by administration of Bay treated DC, we used a model of infectious tolerance transfer from DC treated mice to primed recipient mice. Our results show that both CD4 + splenic cells and non T cells from animals injected with Bay treated DC, but not from untreated DC, were capable of transferring the suppression. Moreover, sorted B cells and NK cells could transfer antigenspecific infectious tolerance after administration of Bay treated DC. In addition, this suppressive effect could not be seen either in mice depleted of NK cells nor in NKT deficient mice. These observations highlight the role of several immune cells in the maintenance of tolerance, and impact on the design of immunotherapeutic suppression of autoimmune diseases in which NKT cells are deficient or defective, such as diabetes and lupus.

Immune response to vaccination with DNA-hsp65 in a phase I clinical trial with head and neck cancer patients

DNA-hsp65, a DNA vaccine encoding the 65-kDa heat-shock protein of Mycobacterium leprae (Hsp65) is capable of inducing the reduction of established tumors in mouse models. We conducted a phase I clinical trial of DNA-hsp65 in patients with advanced head and neck carcinoma. In this article, we report on the vaccine`s potential to induce immune responses to Hsp65 and to its human homologue, Hsp60, in these patients. Twenty-one patients with unresectable squamous cell carcinoma of the head and neck received three doses of 150, 400 or 600 mu g naked DNA-hsp65 plasmid by ultrasound-guided intratumoral injection. Vaccination did not increase levels of circulating anti-hsp65 IgG or IgM antibody, or lead to detectable Hsp65-specific cell proliferation or interferon-gamma (IFN-gamma) production by blood mononuclear cells. Frequency of antigen-induced IL-10-producing cells increased after vaccination in 4 of 13 patients analyzed. Five patients showed disease stability or regression following immunization; however, we were unable to detect significant differences between these patients and those with disease progression using these parameters. There was also no increase in antibody or IFN-gamma responses to human Hsp60 in these patients. Our results suggest that although DNA-hsp65 was able to induce some degree of immunostimulation with no evidence of pathological autoimmunity, we were unable to differentiate between patients with different clinical outcomes based on the parameters measured. Future studies should focus on characterizing more reliable correlations between immune response parameters and clinical outcome that may be used as predictors of vaccine success in immunosuppressed individuals. Cancer Gene Therapy (2009) 16, 598-608; doi:10.1038/cgt.2009.9; published online 6 February 2009

IL-1 beta breaks tolerance through expansion of CD25(+) effector T cells

IL-1 is a key proinflammatory driver of several autoimmune diseases including juvenile inflammatory arthritis, diseases with mutations in the NALP/cryopyrin complex and Crohn's disease, and is genetically or clinically associated with many others. IL-1 is a pleiotropic proinflammatory cytokine; however the mechanisms by which increased IL-1 signaling promotes autoreactive T cell activity are not clear. Here we show that autoimmune-prone NOD and IL-1 receptor antagonist-deficient C57BL/6 mice both produce high levels of IL-1, which drives autoreactive effector cell expansion. IL-1 beta drives proliferation and cytokine production by CD4(+)CD25(+)FoxP3(-) effector/memory T cells, attenuates CD4(+)CD25(+)FoxP3(+) regulatory T cell function, and allows escape of CD4(+)CD25(-) autoreactive effectors from suppression. Thus, inflammation or constitutive overexpression of IL-1 beta in a genetically predisposed host can promote autoreactive effector T cell expansion and function, which attenuates the ability of regulatory T cells to maintain tolerance to self.

Induction of Indoleamine 2,3-Dioxygenase by Gene Delivery in Allogeneic Islets Prolongs Allograft Survival

Indoleamine 2,3-dioxygenase (IDO), an enzyme that plays a critical role in fetomaternal tolerance, exerts immunoregulatory functions suppressing T-cell responses. The aims of this study were to promote IDO expression in rat islets using a nonviral gene transfer approach, and to analyze the effect of the in vivo induction of IDO in a model of allogeneic islet transplantation. The IDO cDNA was isolated from rat placenta, subcloned into a plasmid and transfected into rat islets using Lipofectamine. The efficiency of transfection was confirmed by qRT-PCR and functional analysis. The in vivo effect of IDO expression was analyzed in streptozotocin-induced diabetic Lewis rats transplanted with allogeneic islets under the renal capsule. Transplantation of IDO-allogeneic islets reversed diabetes and maintained metabolic control, in contrast to transplantation of allogeneic nontransfected islets, which failed shortly after transplantation in all animals. Graft survival of allograft islets transfected with IDO transplanted without any immunosuppression was superior to that observed in diabetic rats receiving nontransfected islets. These data demonstrated that IDO expression induced in islets by lipofection improved metabolic control of streptozotocin-diabetic rats and prolonged allograft survival.

Endotoxin tolerance: Selective alterations in gene expression and protection against lymphocyte death

Extensive lymphocyte apoptosis may be an important cause of immune suppression in sepsis. Here we investigated the effect of LPS tolerance on lymphocyte apoptosis in an experimental model of polymicrobial infection. Tolerance was induced by the injection of lipopolysaccharide (1.0 mg/kg/subcutaneously) once a day for 5 days. Macroarray analysis of mRNA isolated from T-(CD4) lymphocytes was used to identify genes that are differentially expressed during LPS tolerance. In addition, assessment of the expression of apoptosis-associated lymphocyte gene products and apoptotic events was performed on the 8th day; 6 h after the terminal challenge with polymicrobial infection or high-dose LPS administration. Survival studies with polymicrobial infection were also conducted. LPS tolerance induced a broad reprogramming of cell death pathways, including a suppression of receptor-mediated and mitochondrial apoptotic pathways, inflammatory caspases, alternate apoptotic pathways, as well as reduced expression of genes involved in necrosis. These alterations led to a marked resistance of lymphocytes against cell death during the subsequent period of sepsis. In addition, LPS tolerance produced an increased differentiation of T-lymphocytes to T(H)1 and T(H)2, with a T(H)1 differentiation predominance. Thus, in the current study we provide an evidence for a marked reprogramming of gene expression of multiple cell death pathways during LPS tolerance. These alterations may play a significant role in the observed protection of the animals from a subsequent lethal polymicrobial sepsis challenge. (C) 2009 Elsevier GmbH. All rights reserved.

E7 oncoprotein of human papillomavirus type 16 expressed constitutively in the epidermis has no effect on E7-specific B- or Th-repertoires or on the immune response induced or sustained after immunization with E7 protein

A line of FVB (H-2(q)) mice transgenic for the E6/E7 open reading frames of Human Papillomavirus type 16 driven from the alpha-A crystallin promoter expresses E7 mRNA in lens and skin epithelium. E7 protein is detectable in adult skin, coinciding with the development or inflammatory skin disease, which progresses to papillomata and squamous carcinomata in some mice. By examining the outcome of parenteral immunization with E7 protein, we sought to determine whether endogenous expression of E7 in skin had induced a preexisting immune outcome, i.e., specific immunity or tolerance, or whether the mice remain naive (''ignorant'') to E7. Our data show that the antibody response to defined E7 B-epitopes, the proliferative response to Th epitopes, and the delayed-type hypersensitivity (DTH) response to whole E7 did not differ between groups or young and old E6/E7 transgenic mice (likely having different degrees of lifetime exposure to E7 protein) or between E6/E7-transgenic and nontransgenic parental strain control mice. Although an E7-specific CTL response could not be induced in the H-2(q) background of these mice, incorporation of a D-b allele into the genome allowed comparison of D-b-restricted CTL responses in E6/E7 transgenic and nontransgenic mice. Experiments indicated that the E7-immunization-induced CTL response did not differ significantly between E6/E7 transgenic and nontransgenic mice. We interpret these results to indicate that in spite of expression of E7 protein in adult skin, E6/E7 transgenic mice remain immunologically naive (ignorant) of E7 epitopes presented by immunization. (C) 1997 Academic Press.

Immunization of neonatal mice with LAMP/p55 HIV gag DNA elicits robust immune responses that last to adulthood

Successful T cell priming in early postnatal life that can generate effective long-lasting responses until adulthood is critical in HIV vaccination strategies because it prevents early sexual initiation and breastfeeding transmission of HIV. A chimeric DNA vaccine encoding p55 HIV gag associated with lysosome-associated membrane protein 1 (LAMP-1; which drives the antigen to the MIIC compartment), has been used to enhance cellular and humoral antigen-specific responses in adult mice and macaques. Herein, we investigated LAMP-1/gag vaccine immunogenicity in the neonatal period in mice and its ability to generate long-lasting effects. Neonatal vaccination with chimeric LAMP/gag generated stronger Gag-specific immune responses, as measured by the breadth of the Gag peptide-specific IFN-gamma, proliferative responsiveness, cytokine production and antibody production, all of which revealed activation of CD4+ T cells as well as the generation of a more robust CTL response compared to gag vaccine alone. To induce long-lived T and B cell memory responses, it was necessary to immunize neonates with the chimeric IAMP/gag DNA vaccine. The LAMP/gag DNA vaccine strategy could be particularly useful for generating an anti-HIV immune response in the early postnatal period capable of inducing long-term immunological memory. (C) 2010 Elsevier Inc. All rights reserved.

Differential monocyte STAT6 activation and CD4(+)CD25(+)Foxp3(+) T cells in kidney operational tolerance transplanted individuals

In organ transplantation, the immunosuppression withdrawal leads, in most cases, to rejection. Nonetheless, a special group of patients maintain stable graft function after complete withdrawal of immunosuppression, achieving a state called ""operational tolerance."" The study of such patients may be important to understand the mechanisms involved in human transplantation tolerance. We compared the profile of CD4(+)CD25(+)Foxp3(+) T cells and the signaling pathways IL-6/STAT3 (signal transducers and activators of transcription) and IL-4/STAT6 in peripheral blood mononuclear cells of four kidney transplant groups: (i) operational tolerance (OT), (ii) chronic allograft nephropathy (CR), (iii) stable graft function under standard immunosuppression (Sta), (iv) stable graft function under low immunosuppression, and (v) healthy individuals. Both CR and Sta displayed lower numbers and percentages of CD4(+)CD25(+)Foxp3(+) T cells compared with all other groups (p < 0.05). The OT patients displayed a reduced activation of the IL-4/STAT6 pathway in monocytes, compared with all other groups (p < 0.05). The lower numbers of CD4(+)CD25(+)Foxp3(+) T cells observed in CR individuals may be a feature of chronic allograft nephropathy. The differential OT signaling profile, with reduced phosphorylation of STAT6, in monocytes` region, suggests that some altered function of STAT6 signaling may be important for the operational tolerance state. Crown copyright (C) 2010 Published by Elsevier Inc. on behalf of American Society for Histocompatibility and Immunogenetics. All rights reserved.

Oral tolerance attenuates airway inflammation and remodeling in a model of chronic pulmonary allergic inflammation

We investigated the effects of oral tolerance (OT) in controlling inflammatory response, hyperresponsiveness and airway remodeling in guinea pigs (GP) with chronic allergic inflammation. Animals received seven inhalations of ovalbumin (1-5 mg/mL-OVA group) or normal saline (NS group). OT was induced by offering ad libitum ovalbumin 2% in sterile drinking water starting with the 1st ovalbumin inhalation (OT1 group) or after the 4th (OT2 group). The induction of OT in sensitized animals decreased the elastance of respiratory system (Ers) response after both antigen and methacholine challenges, peribronchial edema formation, eosinophilic airway infiltration, eosinophilopoiesis, and airways collagen and elastic fiber content compared to OVA group (P < 0.05). The number of mononuclear cells and resistance of respiratory system (Rrs) responses after antigen and methacholine challenges were decreased only in OT2 group compared to OVA group (P < 0.05). Concluding, our results show that inducing OT attenuates airway remodeling as well as eosinophilic inflammation and respiratory system mechanics. (C) 2008 Elsevier B.V. All rights reserved.

Immunization with pVAXhsp65 Decreases Inflammation and Modulates Immune Response in Experimental Encephalomyelitis

Background: A DNA vaccine (pVAXhsp65) containing the gene of a heat-shock protein (hsp65) from Mycobacterium leprae showed high immunogenicity and protective efficacy against tuberculosis in BALB/c mice. A possible deleterious effect related to autoimmunity needed to be tested because hsp65 is highly homologous to the correspondent mammalian protein. In this investigation we tested the effect of a previous immunization with DNAhsp65 in the development of experimental autoimmune encephalomyelitis (EAE), a rat model of multiple sclerosis. Methods: Female Lewis rats were immunized with 3 pVAXhsp65 doses by intramuscular route. Fifteen days after the last DNA dose the animals were evaluated for specific immunity or submitted to induction of EAE. Animals were evaluated daily for weight loss and clinical score, and euthanized during the recovery phase to assess the immune response and inflammatory infiltration at the central nervous system. Results: Immunization with pVAXhsp65 induced a specific immune response characterized by production of IgG(2b) anti-hsp65 antibodies and IFN-gamma secretion. Previous immunization with pVAXhsp65 did not change EAE clinical manifestations (weight and clinical score). However, the vaccine clearly decreased brain and lumbar spinal cord inflammation. In addition, it downmodulated IFN-gamma and IL-10 production by peripheral lymphoid organs. Conclusion: Our data demonstrated that this vaccine does not trigger a deleterious effect on EAE development and also points to a potential protective effect. Copyright (C) 2010 S. Karger AG, Basel