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Previous studies in experimental autoimmune encephalomyelitis (EAE) models have shown that some probiotic bacteria beneficially impact the development of this experimental disease. Here, we tested the therapeutic effect of two commercial multispecies probiotics-Lactibiane iki and Vivomixx-on the clinical outcome of established EAE. Lactibiane iki improves EAE clinical outcome in a dose-dependent manner and decreases central nervous system (CNS) demyelination and inflammation. This clinical improvement is related to the inhibition of pro-inflammatory and the stimulation of immunoregulatory mechanisms in the periphery. Moreover, both probiotics modulate the number and phenotype of dendritic cells (DCs). Specifically, Lactibiane iki promotes an immature, tolerogenic phenotype of DCs that can directly induce immune tolerance in the periphery, while Vivomixx decreases the percentage of DCs expressing co-stimulatory molecules. Finally, gut microbiome analysis reveals an altered microbiome composition related to clinical condition and disease progression. This is the first preclinical assay that demonstrates that a commercial probiotic performs a beneficial and dose-dependent effect in EAE mice and one of the few that demonstrates a therapeutic effect once the experimental disease is established. Because this probiotic is already available for clinical trials, further studies are being planned to explore its therapeutic potential in multiple sclerosis patients

The absence of alloantibodies is a feature of transplantation tolerance. Although the lack of T cell help has been evoked to explain this absence, herein we provide evidence for B cell-intrinsic tolerance mechanisms. Using a murine model of heart tolerance, we showed that alloreactive B cells were not deleted but rapidly lost their ability to differentiate into germinal center B cells and secrete donor-specific antibodies. We inferred that tolerant alloreactive B cells retained their ability to sense alloantigen because they continued to drive T cell maturation into CXCR5+PD-1+ T follicular helper cells. Unexpectedly, dysfunctional alloreactive B cells acquired the ability to inhibit antibody production by new naive B cells in an antigen-specific manner. Thus, tolerant alloreactive B cells contribute to transplantation tolerance by foregoing germinal center responses while retaining their ability to function as antigen-presenting cells and by actively suppressing de novo alloreactive B cell responses.

Previous studies in experimental autoimmune encephalomyelitis (EAE) models have shown that some probiotic bacteria beneficially impact the development of this experimental disease. Here, we tested the therapeutic effect of two commercial multispecies probiotics—Lactibiane iki and Vivomixx—on the clinical outcome of established EAE. Lactibiane iki improves EAE clinical outcome in a dose-dependent manner and decreases central nervous system (CNS) demyelination and inflammation. This clinical improvement is related to the inhibition of pro-inflammatory and the stimulation of immunoregulatory mechanisms in the periphery. Moreover, both probiotics modulate the number and phenotype of dendritic cells (DCs). Specifically, Lactibiane iki promotes an immature, tolerogenic phenotype of DCs that can directly induce immune tolerance in the periphery, while Vivomixx decreases the percentage of DCs expressing co-stimulatory molecules. Finally, gut microbiome analysis reveals an altered microbiome composition related to clinical condition and disease progression. This is the first preclinical assay that demonstrates that a commercial probiotic performs a beneficial and dose-dependent effect in EAE mice and one of the few that demonstrates a therapeutic effect once the experimental disease is established. Because this probiotic is already available for clinical trials, further studies are being planned to explore its therapeutic potential in multiple sclerosis patients.

The central nervous system (CNS) is considered to be immune privileged, owing in part to the absence of major histocompatibility (MHC) class II+ cells in the healthy brain parenchyma. However, systemic inflammation can activate microglia to express MHC class II, suggesting that systemic inflammation may be sufficient to mature microglia into functional antigen presenting cells (APCs). We examined the effects of systemic lipopolysaccharide (LPS)-induced inflammation on the phenotype and function of putative APCs within the mouse brain parenchyma, as well as its supporting tissues-the choroid plexus and meninges. Microglia isolated from different regions of the brain demonstrated significant heterogeneity in their ability to present antigen to naïve OT-II CD4+ T cells following exposure to systemic LPS. Olfactory bulb microglia (but not cortical microglia) intimately interacted with T cells in vivo and stimulated T cell proliferation in vitro, albeit in the absence of co-stimulation. In contrast, myeloid cells within the choroid plexus and meninges were immunogenic and upregulated the co-stimulatory molecule CD80 following systemic inflammation. Dural APCs, which clustered around LYVE-1+ lymphatics, were more efficient at stimulating naïve T cell proliferation than choroid plexus APCs, suggesting that the dura may be an under-appreciated site for immune interactions. This study has highlighted the functional diversity of myeloid cells within the sub-compartments of the CNS and its supporting tissues. Furthermore, these findings demonstrate that systemic inflammation can mature selected microglia populations and choroid plexus/meningeal myeloid cells into functional APCs, which may contribute to the pathogenesis of neuroinflammation and neurodegenerative diseases.

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Microbiology and Immunology, 2018., Influenza A viruses continue to pose a threat to human health worldwide and virusspecific CD4 T cells are key to protective immunity. The goal of the experiments within this thesis was to provide novel insight into elements within CD4 T cell responses that influence CD4 T cell specificity and function. Using a combination of novel reporter viruses, intravascular labeling in vivo, multi-parameter flow cytometry, and cytokine ELISpot procedures in two mammalian models, this work examined various stages of the developing immune response and the relationship between antigen acquisition, CD4 T cell fate choice, trafficking and compartmentalization within the inflamed lung, antigen specificity, and cytokine functionality. We found that pandemic 2009 H1N1 virus infection resulted in a broad pattern of viral antigen acquisition in the lung. Complex subpopulations of MHC class II-positive cells from both the hematopoietic and non-hematopoietic lineages were identified, with dynamic shifts over time. Our results demonstrated that antigen access occurs by uptake of exogenous antigen as well as infection. Finally, our results confirmed that many different cell types displayed peptide:MHC class II complexes in situ and possessed the capacity to stimulate influenza-specific CD4 T cells. These results correlated with differences in infection status, cell lineage, and MHC class II expression. These data suggest that there are many different types of antigen presenting cells in the lung that may impose additional checkpoints of CD4 T cell functionality and regulation. At the peak of the immune response, we found that lung CD4 T cells compartmentalized to vasculature and tissue. Antigen-experienced CD4 T cells were enriched in the tissue and expressed higher levels of molecules involved in trafficking to sites of pulmonary infection, damage, and repair. Surprisingly, we found similar representation in the fine specificity, immunodominance hierarchy, and polyfunctional potential of ef

Bioactive peptides secreted by probiotic Bifidobacterium longum (peptide B7) and opportunistic pathogen Bacteroides fragilis (peptide B12) modulate the intestinal cytokine milieu in health. Here, we characterized their capacity to modulate both the mucosal cytokine production and the phenotype of circulating antigen presenting cells (APCs) in active inflammatory bowel disease (IBD). The IBD mucosa produced higher levels of pro-inflammatory cytokines referred to healthy controls (HCs). Peptides B7 and B12, however, did not ameliorate the mucosal cytokine milieu in IBD. Human circulating APCs (B-cells, monocytes, plasmacytoid dendritic cells (pDCs), and conventional dendritic cells (cDCs)) were characterized by flow cytometry in presence/absence of the peptides. Circulating B-cells, monocytes, and cDCs from IBD patients were more activated than those from HCs. Peptide B7, but not B12, decreased CCR2 expression on all APC subsets from HC, but not IBD patients. Moreover, both peptides tend to further increase their pro-inflammatory profile in IBD. In summary, IBD patients display mucosal and circulating APC pro-inflammatory properties. Peptide B7 immunomodulatory capacity elicited over circulating APCs from HC, but not IBD patients, suggests the presence of disrupted modulatory mechanisms for this peptide in IBD. Future studies should address the effect of bacteria-derived immunomodulatory peptides in non-inflamed (quiescent) IBD patients.

It is well understood that antigen-presenting cells (APC) within tumors typically do not maintain cytotoxic T cell (CTL) function, despite engaging them. Across multiple mouse tumor models and human biopsies, we have delineated the intratumoral and lymph node trafficking dendritic-cell (DC) populations as distinct from macrophage subsets. Within these, CD103+ dendritic cells (DCs) are extremely sparse and yet remarkably capable CTL stimulators. These are uniquely dependent upon Irf8, Zbtb46, and Batf3 transcription factors and generated by GM-CSF and Flt3L cytokines. Regressing tumors have higher proportions of these cells, T cell-dependent immune clearance relies upon them, and abundance of their transcripts in human tumors predict clinical outcome. Even further, this population of stimulatory CD103+ DCs is required for the trafficking of tumoral antigen to the lymph node and the generation of anti-tumor T cell responses. As tumoral load of these DCs is predictive of immunotherapeutic clinical benefit, this cell type presents many opportunities for novel prognostic, diagnostic, and therapeutic approaches across multiple cancer types.Current cancer immunotherapies are predicated on enhancing the ability of host or introduced T cells to reject tumors. However, efficient CTL function requires frequent re-priming and abundant tumor macrophages, which capture CTL at the tumor margin, either fail to achieve this and/or actively inhibit T cell responses. Here, we show that the abundant macrophages in tumors have a functional opposite, in the form of antigen-presenting CD103+ DC. These cells efficiently cross-present tumor antigens locally and in the lymph node and are differentially distributed within the tumor microenvironment compared to tolerizing APC. We describe how intratumoral CD103+ DC are uniquely targetable, how their abundance is required for T cell therapy in mice, and how their transcript abundance predicts outcome in human cancers.

It is well understood that antigen-presenting cells (APC) within tumors typically do not maintain cytotoxic T cell (CTL) function, despite engaging them. Across multiple mouse tumor models and human biopsies, we have delineated the intratumoral and lymph node trafficking dendritic-cell (DC) populations as distinct from macrophage subsets. Within these, CD103+ dendritic cells (DCs) are extremely sparse and yet remarkably capable CTL stimulators. These are uniquely dependent upon Irf8, Zbtb46, and Batf3 transcription factors and generated by GM-CSF and Flt3L cytokines. Regressing tumors have higher proportions of these cells, T cell-dependent immune clearance relies upon them, and abundance of their transcripts in human tumors predict clinical outcome. Even further, this population of stimulatory CD103+ DCs is required for the trafficking of tumoral antigen to the lymph node and the generation of anti-tumor T cell responses. As tumoral load of these DCs is predictive of immunotherapeutic clinical benefit, this cell type presents many opportunities for novel prognostic, diagnostic, and therapeutic approaches across multiple cancer types.Current cancer immunotherapies are predicated on enhancing the ability of host or introduced T cells to reject tumors. However, efficient CTL function requires frequent re-priming and abundant tumor macrophages, which capture CTL at the tumor margin, either fail to achieve this and/or actively inhibit T cell responses. Here, we show that the abundant macrophages in tumors have a functional opposite, in the form of antigen-presenting CD103+ DC. These cells efficiently cross-present tumor antigens locally and in the lymph node and are differentially distributed within the tumor microenvironment compared to tolerizing APC. We describe how intratumoral CD103+ DC are uniquely targetable, how their abundance is required for T cell therapy in mice, and how their transcript abundance predicts outcome in human cancers.

Background: Aging is associated with low-grade neuroinflammation that includes basal increases in proinflammatory cytokines and expression of inflammatory markers on microglia. Exercise can reduce neuroinflammation following infection in aged animals, but whether exercise modulates basal changes in microglia activation is unknown. Therefore, we evaluated changes in basal microglia activation in cells isolated from the hippocampus and remaining brain following running-wheel access. Methods: Adult (4 months) and aged (22 months) male and female BALB/c mice were housed with or without running wheels for 10 weeks. Microglia were isolated from the hippocampus or remaining brain. Flow cytometry was used to determine microglia (CD11b+ and CD45low) that co-labeled with CD86, CD206, and MHC II. Results: Aged mice showed a greater proportion of CD86 and MHC II positive microglia. In aged females, access to a running wheel decreased proportion of CD86+ and MHC II+ microglia in the hippocampus whereas aged males in the running group showed a decrease in the proportion of CD86+ microglia in the brain and an increase in the proportion of MHC II+ microglia in hippocampus and brain. Conclusion: Overall, these data indicate that running-wheel access modulates microglia activation, but these effects vary by age, sex, and brain region.

Antigen presenting cells are able to capture, process and present antigens in order to develop an effective immune response. The role of these cells during infectious diseases is crucial to control the disease. Thus, the study of these cells after the infection with Porcine Reproductive and Respiratory Syndrome Virus gives us useful information on how to control this disease., Las células presentadoras de antígeno son aquellas células encargadas de capturar, procesar y presentar antígenos con la finalidad de lograr una respuesta inmune efectiva por parte del organismo. Su papel, como centinelas, es crucial durante el transcurso de diversas enfermedades infecciosas. El estudio de estas células tras la infección con el virus del Síndrome Reproductivo y Respiratorio Porcino nos da información para abordar nuevas estrategias de control frente a esta enfermedad.

Antigen presenting cells are able to capture, process and present antigens in order to develop an effective immune response. The role of these cells during infectious diseases is crucial to control the disease. Thus, the study of these cells after the infection with Porcine Reproductive and Respiratory Syndrome Virus gives us useful information on how to control this disease., Las células presentadoras de antígeno son aquellas células encargadas de capturar, procesar y presentar antígenos con la finalidad de lograr una respuesta inmune efectiva por parte del organismo. Su papel, como centinelas, es crucial durante el transcurso de diversas enfermedades infecciosas. El estudio de estas células tras la infección con el virus del Síndrome Reproductivo y Respiratorio Porcino nos da información para abordar nuevas estrategias de control frente a esta enfermedad.

Dendritic cells [DCs] are potent antigen presenting cells [APC], which plays a vital role in immune system by detecting and capturing pathogens in the body. DCs perform a pivotal role in induction of T cell response. Regulation of immune response can be achieved by specific antigen [Ag] delivery to DCs. A delivery system that can efficiently target and present Ags to DCs for the purpose of anti-tumour activity is currently a topic of significant research interest. DCs are receiving attention due to their key role in anti cancer host response and due to their adjuvanic property in tumour vaccines. Role of toll like receptors [TLR] in innate immune system and their part in eventual stimulation of adaptive immunity is exploited to develop vaccines. TLR agonists in conjugation with vaccines are shown to increase therapeutic efficacy in some cases. TLRs also play a vital role in protecting the cornea from invading pathogens. Due to adverse effects in the treatment of ocular inflammations, cancer and in viral infections, an alternate approach such as the use of TLRs will solve the inquisitive question regarding side effects. The intended delivery is attained by the use of nanoparticles which in turn leads to prolonged half-life in the body. Co-delivery of Ags, TLRs and immunomodulators using nanoparticles has been demonstrated to elicit potent cellular immune responses and are currently under development of clinically applicable immunisations and vaccines.

Delivery of antigens to antigen presenting cell surface receptors represents a promising strategy to improve immune response to weak immunogenic antigens. We have analyzed the potential of porcine sialoadhesin (Sn) and CD163 as antigen targeting receptors using mouse Igs as surrogate antigens. Sn and CD163 are two endocytic receptors mainly expressed on macrophages located in antigen-sampling zones of secondary lymphoid organs. MAbs to CD163 induced in vitro PBMC proliferation at concentrations 50-80 fold lower than the control mAb when using, as responder cells, cells from pigs immunized with mouse serum IgGs. To evaluate in vivo targeting, pigs were immunized s.c. with anti-Sn, anti-CD163 or control mAbs, and the immune response induced to mouse Ig was analyzed. Two weeks after the first immunization, pigs receiving either anti-Sn or anti-CD163 mAbs started to show higher anti-mouse-IgG serum titres than controls. Boosting 6 weeks later, further increased the anti-IgG titres up to 15-60 fold those of controls. In addition, differences in the relative predominance of IgG1 or IgG2 subclasses in the response depending on Sn or CD163 targeting were observed. Peripheral blood mononuclear cells from pigs immunized with anti-Sn mAb showed a higher proliferative response to mouse IgG than cells from pigs immunized with control mAb. These results show that targeting antigen to Sn or CD163 can enhance the immune response in pigs. © 2011 Elsevier Ltd. All rights reserved.

Development of a gene delivery system to transfer the gene of interest selectively and efficiently into targeted cells is essential for achievement of sufficient therapeutic effects by gene therapy. Here, we succeeded in developing the gene transfection method using ultrasound (US)-responsive and mannose-modified gene carriers, named Man-PEG(2000) bubble lipoplexes. Compared with the conventional lipofection method using mannose-modified carriers, this transfection method using Man-PEG(2000) bubble lipoplexes and US exposure enabled approximately 500-800-fold higher gene expressions in the antigen presenting cells (APCs) selectively in vivo. This enhanced gene expression was contributed by the improvement of delivering efficiency of nucleic acids to the targeted organs, and by the increase of introducing efficiency of nucleic acids into the cytoplasm followed by US exposure. Moreover, high anti-tumor effects were demonstrated by applying this method to DNA vaccine therapy using ovalbumin (OVA)-expressing plasmid DNA (pDNA). This US-responsive and cell-specific gene delivery system can be widely applied to medical treatments such as vaccine therapy and anti-inflammation therapy, which its targeted cells are APCs, and our findings may help in establishing innovative methods for in-vivo gene delivery to overcome the poor introducing efficiency of carriers into cytoplasm which the major obstacle associated with gene delivery by non-viral carriers.

A diverse cadre of weapons of mass destruction was utilized by the group to examine pathogenesis and methods of intervention. It was determined that oxidative stress is significant component of the pathogenesis of biological and chemical weapons. Antioxidants had an ameliorate effect on both chemical and biological agents, and has the potential to be used as an ancillary treatment. Antigen presenting cells may be useful for detecting subclinical infectious disease. Dendritic cells are able to differentiate between different types of pathogens thus generating a specific signature for the pathogen.

Immunological synapses (IS) are emerging as highly organized 3D structures -formed by surface and cytoplasmic signalling and cytoskeletal molecules - that assemble at the zone of contact between a T cell and an antigen presenting cell (APC). The IS control functions that allow APC and T cells modulate the immune response.

Adenovirus (Ad)-mediated transduction of dendritic cells (DCs) is inefficient because of the lack of the primary Ad receptor CAR. CD40 is a surface marker expressed by DCs that plays a crucial role in their maturation and subsequent stimulation of T cells. DC infection with Ad targeted to the CD40 results in increased gene transfer. Cells transduced with CD40-targeted Ad5-SV40-TAg vector showed increased expression of transgene and expression of co-stimulatory molecules at 48 hours post-infection compared to cells transduced with untargeted Ad5-SV40-TAg vector. We demonstrated that CD40-targeted gene transfer promotes DC maturation with induction of a complex signaling cascade accompanied by characteristic changes in cyto-kine production. These results demonstrate that DCs can be successfully transduced using a CD40 targeted adenoviral vector and that transduced DCs show activation., The original document contains color images.

Autoimmune diseases develop when the immune system fails to distinguish self from non-self or when the immune system is hypersensitive to endogenous or exogenous danger signals, or when a tissue erroneously sends a danger signal to the immune system. The education of the immune system to distinguish self from non-self is mainly carried out in the thymus and gives rise to central tolerance, whereas the ability to sense a danger or a healthy tissue constitutes peripheral tolerance. In these studies we have investigated the peripheral tolerance mechanisms controlled by the autoimmune regulator (Aire) gene in Aire deficient mice, an animal model of the monogenic disease autoimmune polyendocrine syndrome type I (APS I). Aire-/- mice displayed increased numbers of myeloid-derived antigen-presenting cells (APCs) in the spleen, lymph nodes and peritoneum as well as more blood monocytes and metallophilic macrophages in the spleen. Monocytes were also increased in the blood of APS I patients. Monocyte precursors displayed an accelerated development in the bone marrow of Aire-/- mice, and Aire-/- APCs had an altered phenotype that caused an increased immune response in several different contexts. Aire-/- splenic and lymph node dendritic cells had an increased ability to activate naive T cells, partly as a result of an upregulated expression of the costimulatory molecule VCAM-1. In Aire-/- mice increased activity of the metallophilic macrophages in the splenic marginal zone seems to be responsible both for the activated phenotype of marginal zone B cells and for the frequent development of marginal zone lymphoma with aging. In a TCR transgenic model Aire deficiency caused an increased superantigen-mediated TCR revision in the spleen, perhaps as a result of the altered phenotype of APCs in the spleen. Finally, Aire was shown to influence autoimmune disease development by a macrophage-dependent mechanism in diabetes induced with multiple low dose streptozotocin injections. These, Autoimmune diseases develop when the immune system fails to distinguish self from non-self or when the immune system is hypersensitive to endogenous or exogenous danger signals, or when a tissue erroneously sends a danger signal to the immune system. The education of the immune system to distinguish self from non-self is mainly carried out in the thymus and gives rise to central tolerance, whereas the ability to sense a danger or a healthy tissue constitutes peripheral tolerance. In these studies we have investigated the peripheral tolerance mechanisms controlled by the autoimmune regulator (Aire) gene in Aire deficient mice, an animal model of the monogenic disease autoimmune polyendocrine syndrome type I (APS I). Aire-/- mice displayed increased numbers of myeloid-derived antigen-presenting cells (APCs) in the spleen, lymph nodes and peritoneum as well as more blood monocytes and metallophilic macrophages in the spleen. Monocytes were also increased in the blood of APS I patients. Monocyte precursors displayed an accelerated development in the bone marrow of Aire-/- mice, and Aire-/- APCs had an altered phenotype that caused an increased immune response in several different contexts. Aire-/- splenic and lymph node dendritic cells had an increased ability to activate naive T cells, partly as a result of an upregulated expression of the costimulatory molecule VCAM-1. In Aire-/- mice increased activity of the metallophilic macrophages in the splenic marginal zone seems to be responsible both for the activated phenotype of marginal zone B cells and for the frequent development of marginal zone lymphoma with aging. In a TCR transgenic model Aire deficiency caused an increased superantigen-mediated TCR revision in the spleen, perhaps as a result of the altered phenotype of APCs in the spleen. Finally, Aire was shown to influence autoimmune disease development by a macrophage-dependent mechanism in diabetes induced with multiple low dose streptozotocin injections. These

Autoimmune diseases develop when the immune system fails to distinguish self from non-self or when the immune system is hypersensitive to endogenous or exogenous danger signals, or when a tissue erroneously sends a danger signal to the immune system. The education of the immune system to distinguish self from non-self is mainly carried out in the thymus and gives rise to central tolerance, whereas the ability to sense a danger or a healthy tissue constitutes peripheral tolerance. In these studies we have investigated the peripheral tolerance mechanisms controlled by the autoimmune regulator (Aire) gene in Aire deficient mice, an animal model of the monogenic disease autoimmune polyendocrine syndrome type I (APS I). Aire-/- mice displayed increased numbers of myeloid-derived antigen-presenting cells (APCs) in the spleen, lymph nodes and peritoneum as well as more blood monocytes and metallophilic macrophages in the spleen. Monocytes were also increased in the blood of APS I patients. Monocyte precursors displayed an accelerated development in the bone marrow of Aire-/- mice, and Aire-/- APCs had an altered phenotype that caused an increased immune response in several different contexts. Aire-/- splenic and lymph node dendritic cells had an increased ability to activate naive T cells, partly as a result of an upregulated expression of the costimulatory molecule VCAM-1. In Aire-/- mice increased activity of the metallophilic macrophages in the splenic marginal zone seems to be responsible both for the activated phenotype of marginal zone B cells and for the frequent development of marginal zone lymphoma with aging. In a TCR transgenic model Aire deficiency caused an increased superantigen-mediated TCR revision in the spleen, perhaps as a result of the altered phenotype of APCs in the spleen. Finally, Aire was shown to influence autoimmune disease development by a macrophage-dependent mechanism in diabetes induced with multiple low dose streptozotocin injections. These, Autoimmune diseases develop when the immune system fails to distinguish self from non-self or when the immune system is hypersensitive to endogenous or exogenous danger signals, or when a tissue erroneously sends a danger signal to the immune system. The education of the immune system to distinguish self from non-self is mainly carried out in the thymus and gives rise to central tolerance, whereas the ability to sense a danger or a healthy tissue constitutes peripheral tolerance. In these studies we have investigated the peripheral tolerance mechanisms controlled by the autoimmune regulator (Aire) gene in Aire deficient mice, an animal model of the monogenic disease autoimmune polyendocrine syndrome type I (APS I). Aire-/- mice displayed increased numbers of myeloid-derived antigen-presenting cells (APCs) in the spleen, lymph nodes and peritoneum as well as more blood monocytes and metallophilic macrophages in the spleen. Monocytes were also increased in the blood of APS I patients. Monocyte precursors displayed an accelerated development in the bone marrow of Aire-/- mice, and Aire-/- APCs had an altered phenotype that caused an increased immune response in several different contexts. Aire-/- splenic and lymph node dendritic cells had an increased ability to activate naive T cells, partly as a result of an upregulated expression of the costimulatory molecule VCAM-1. In Aire-/- mice increased activity of the metallophilic macrophages in the splenic marginal zone seems to be responsible both for the activated phenotype of marginal zone B cells and for the frequent development of marginal zone lymphoma with aging. In a TCR transgenic model Aire deficiency caused an increased superantigen-mediated TCR revision in the spleen, perhaps as a result of the altered phenotype of APCs in the spleen. Finally, Aire was shown to influence autoimmune disease development by a macrophage-dependent mechanism in diabetes induced with multiple low dose streptozotocin injections. These

Autoimmune diseases develop when the immune system fails to distinguish self from non-self or when the immune system is hypersensitive to endogenous or exogenous danger signals, or when a tissue erroneously sends a danger signal to the immune system. The education of the immune system to distinguish self from non-self is mainly carried out in the thymus and gives rise to central tolerance, whereas the ability to sense a danger or a healthy tissue constitutes peripheral tolerance. In these studies we have investigated the peripheral tolerance mechanisms controlled by the autoimmune regulator (Aire) gene in Aire deficient mice, an animal model of the monogenic disease autoimmune polyendocrine syndrome type I (APS I). Aire-/- mice displayed increased numbers of myeloid-derived antigen-presenting cells (APCs) in the spleen, lymph nodes and peritoneum as well as more blood monocytes and metallophilic macrophages in the spleen. Monocytes were also increased in the blood of APS I patients. Monocyte precursors displayed an accelerated development in the bone marrow of Aire-/- mice, and Aire-/- APCs had an altered phenotype that caused an increased immune response in several different contexts. Aire-/- splenic and lymph node dendritic cells had an increased ability to activate naive T cells, partly as a result of an upregulated expression of the costimulatory molecule VCAM-1. In Aire-/- mice increased activity of the metallophilic macrophages in the splenic marginal zone seems to be responsible both for the activated phenotype of marginal zone B cells and for the frequent development of marginal zone lymphoma with aging. In a TCR transgenic model Aire deficiency caused an increased superantigen-mediated TCR revision in the spleen, perhaps as a result of the altered phenotype of APCs in the spleen. Finally, Aire was shown to influence autoimmune disease development by a macrophage-dependent mechanism in diabetes induced with multiple low dose streptozotocin injections. These, Autoimmune diseases develop when the immune system fails to distinguish self from non-self or when the immune system is hypersensitive to endogenous or exogenous danger signals, or when a tissue erroneously sends a danger signal to the immune system. The education of the immune system to distinguish self from non-self is mainly carried out in the thymus and gives rise to central tolerance, whereas the ability to sense a danger or a healthy tissue constitutes peripheral tolerance. In these studies we have investigated the peripheral tolerance mechanisms controlled by the autoimmune regulator (Aire) gene in Aire deficient mice, an animal model of the monogenic disease autoimmune polyendocrine syndrome type I (APS I). Aire-/- mice displayed increased numbers of myeloid-derived antigen-presenting cells (APCs) in the spleen, lymph nodes and peritoneum as well as more blood monocytes and metallophilic macrophages in the spleen. Monocytes were also increased in the blood of APS I patients. Monocyte precursors displayed an accelerated development in the bone marrow of Aire-/- mice, and Aire-/- APCs had an altered phenotype that caused an increased immune response in several different contexts. Aire-/- splenic and lymph node dendritic cells had an increased ability to activate naive T cells, partly as a result of an upregulated expression of the costimulatory molecule VCAM-1. In Aire-/- mice increased activity of the metallophilic macrophages in the splenic marginal zone seems to be responsible both for the activated phenotype of marginal zone B cells and for the frequent development of marginal zone lymphoma with aging. In a TCR transgenic model Aire deficiency caused an increased superantigen-mediated TCR revision in the spleen, perhaps as a result of the altered phenotype of APCs in the spleen. Finally, Aire was shown to influence autoimmune disease development by a macrophage-dependent mechanism in diabetes induced with multiple low dose streptozotocin injections. These

Autoimmune diseases develop when the immune system fails to distinguish self from non-self or when the immune system is hypersensitive to endogenous or exogenous danger signals, or when a tissue erroneously sends a danger signal to the immune system. The education of the immune system to distinguish self from non-self is mainly carried out in the thymus and gives rise to central tolerance, whereas the ability to sense a danger or a healthy tissue constitutes peripheral tolerance. In these studies we have investigated the peripheral tolerance mechanisms controlled by the autoimmune regulator (Aire) gene in Aire deficient mice, an animal model of the monogenic disease autoimmune polyendocrine syndrome type I (APS I). Aire-/- mice displayed increased numbers of myeloid-derived antigen-presenting cells (APCs) in the spleen, lymph nodes and peritoneum as well as more blood monocytes and metallophilic macrophages in the spleen. Monocytes were also increased in the blood of APS I patients. Monocyte precursors displayed an accelerated development in the bone marrow of Aire-/- mice, and Aire-/- APCs had an altered phenotype that caused an increased immune response in several different contexts. Aire-/- splenic and lymph node dendritic cells had an increased ability to activate naive T cells, partly as a result of an upregulated expression of the costimulatory molecule VCAM-1. In Aire-/- mice increased activity of the metallophilic macrophages in the splenic marginal zone seems to be responsible both for the activated phenotype of marginal zone B cells and for the frequent development of marginal zone lymphoma with aging. In a TCR transgenic model Aire deficiency caused an increased superantigen-mediated TCR revision in the spleen, perhaps as a result of the altered phenotype of APCs in the spleen. Finally, Aire was shown to influence autoimmune disease development by a macrophage-dependent mechanism in diabetes induced with multiple low dose streptozotocin injections. These, Autoimmune diseases develop when the immune system fails to distinguish self from non-self or when the immune system is hypersensitive to endogenous or exogenous danger signals, or when a tissue erroneously sends a danger signal to the immune system. The education of the immune system to distinguish self from non-self is mainly carried out in the thymus and gives rise to central tolerance, whereas the ability to sense a danger or a healthy tissue constitutes peripheral tolerance. In these studies we have investigated the peripheral tolerance mechanisms controlled by the autoimmune regulator (Aire) gene in Aire deficient mice, an animal model of the monogenic disease autoimmune polyendocrine syndrome type I (APS I). Aire-/- mice displayed increased numbers of myeloid-derived antigen-presenting cells (APCs) in the spleen, lymph nodes and peritoneum as well as more blood monocytes and metallophilic macrophages in the spleen. Monocytes were also increased in the blood of APS I patients. Monocyte precursors displayed an accelerated development in the bone marrow of Aire-/- mice, and Aire-/- APCs had an altered phenotype that caused an increased immune response in several different contexts. Aire-/- splenic and lymph node dendritic cells had an increased ability to activate naive T cells, partly as a result of an upregulated expression of the costimulatory molecule VCAM-1. In Aire-/- mice increased activity of the metallophilic macrophages in the splenic marginal zone seems to be responsible both for the activated phenotype of marginal zone B cells and for the frequent development of marginal zone lymphoma with aging. In a TCR transgenic model Aire deficiency caused an increased superantigen-mediated TCR revision in the spleen, perhaps as a result of the altered phenotype of APCs in the spleen. Finally, Aire was shown to influence autoimmune disease development by a macrophage-dependent mechanism in diabetes induced with multiple low dose streptozotocin injections. These

This group has completed the goal set for Task 1 ("To examine the immune responses to a model tumor antigen selectively expressed in DCs differentiated in vivo from transduced hematopoietic progenitor cells"). They have: 1) made lentiviral vectors expressing a model tumor antigen influenza hemagglutinin (HA); 2) developed a method to efficiently transduce mouse bone marrow progenitor cells that reconstitute immune and blood systems in irradiated and transplanted mice; 3) demonstrated that the transplanted and transduced cells can persistently present the HA model tumor antigen and stimulate T cell responses; 4) devised a tripartite strategies to mount powerful and specific attack on HA-expressing experimental tumors; and 5) demonstrated the efficacy to eradicate an aggressive pre-established tumor expressing the HA antigen. This study provides a proof-of-principle in immune competent animals models that transplantation of antigen gene transduced hematopoietic progenitor cells adds a new component to achieve potent and sustained antigen-specific immunotherapy of aggressive tumors. Key discoveries by this group and their Hopkins colleagues were published in a leading biomedical research journal Nature Medicine (10: 882)., The original document contains color images. All DTIC reproductions will be in black and white.

The development of vaccine approaches for breast cancer has the potential to provide an adjuvant therapy with low toxicity for patients at risk for disease recurrence. Polynucleotide vaccines have several advantages compared to traditional vaccines including the ability to elicit antigen-specific T cells, inherent immunogenicity, ability to modify the encoded antigen, and an excellent safety profile. However, clinical efficacy has been disappointing and strategies to enhance the potency of polynucleotide vaccines are needed. Adenoviral vectors also show promise for cancer vaccine approaches, but do not efficiently deliver genes to antigen-presenting cells such as dendritic cells (DCs). The authors are investigating novel vaccine strategies for breast cancer. The target tumor antigen is carcinoembryonic antigen (CEA), which is highly expressed in most breast tumors. DCs show promise for cancer immunotherapy due to their critical role in mediating immune response. Development of an optimal DC transduction protocol for tumor antigen presentation would represent a significant advancement in DC-based vaccination strategies. The authors are investigating methods of DC transduction and antigen modification that will elicit the most potent anti-tumor immune response. These studies have employed a nontransgenic mouse model of adenocarcinoma as well as a more stringent transgenic model. Appendices present three abstracts and two manuscripts of studies funded through this grant. The manuscripts are as follows: A DNA Vaccine Encoding Genetic Fusions of Carcinoembryonic Antigen (CEA) and Granulocyte/Macrophage Colony-Stimulating Factor (GM-CSF), by Jose Lima et al.; Polynucleotide Immunization for Cancer Therapy, by Theresa V. Strong; and CD4O is Expressed on Ovarian Cancer Cells and Can Be Utilized for Targeting Adenoviruses, by Tanja Hakkarainen et al. (1 table, 2 figures, 5 refs.), The original document contains color images. All DTIC reproductions will be in black and white.

Manipulations capable of repealing host tolerance to induce T cell-mediated prostate tissue-specific responses are of central importance to immunotherapeutic approaches to prostate cancer treatment. Hence in the current proposal, we test whether androgen ablation (by castration) can induce T cell responses targeting murine prostate epithelial and tumor cells. To date, we have found that castration of TRAMP mice results in prostate and tumor infiltration by antigen presenting cells (APC's) as well as CD4+ and CD8+ T cells. These studies complete our original Specific Aim 1 and suggest that castration can prime host responses amenable to immunotherapeutic potentiation. Further studies proposed in our original Specific Aims 2 and 3 will address the potential of androgen ablation to facilitate/potentiate novel immunotherapies, including manipulations that license APC's or prevent T cell downregulation, for prostate cancer treatment.

Dendritic cells (DCs) are potent antigen presenting cells reported to undergo irreversible functional 'maturation' in response to inflammatory signals such as TNF-alpha. The current paradigm holds that this DC maturation event is required for full functional capacity and represents terminal differentiation of this cell type, culminating in apoptotic cell death. This provides a possible mechanism for avoiding dysregulated immunostimulatory activity, but imposes constraints on the capacity of DCs to influence subsequent immune responses and to participate in immunological memory. We report that the cell surface and functional effects induced by TNF-alpha are reversible and reinducible. These effects are accompanied by a concordant modulation of cytokine mRNA expression that includes the induction of proinflammatory factors (IL-15, IL-12, LT-alpha, LT-beta, TNF-alpha, RANTES) which is coincident with the down-regulation of counter-regulatory cytokines (IL-10, TGF-beta1, TGF-beta2, IL-1 RA, MCP-1). The resultant net effect is a dendritic cell activation state characterized by a transient proinflammatory posture. These results demonstrate that 1) human DCs do not undergo terminal 'maturation' in response to TNF-alpha, 2) DC phenotypes are more pleiotropic than previously thought, and 3) DCs are potential immunoregulatory effector cells with implications for control of immune responses in both in vivo and in vitro systems.

Rearrangement of the antigen-specific lymphocyte receptor genes generates repertoire of T and B cells that can attack any emerging pathogens as well as host tissue itself. This horror autotoxicus has to be removed by mechanisms collectively called self-tolerance. Recent advances in immunological science revealed that there are multiple mechanisms of tolerance both in central and peripheral organs. However, it has become more and more apparent that cellular mechanisms that induce tolerance (inactivation) and activation of lymphocytes are quite similar. Possible mechanisms by which immune system distinguishes self-structure from pathogens will be discussed., Acta medica Nagasakiensia. 1999, 44(1-2), p.1-10

Dendritic cells (DCs) are potent antigen presenting cells reported to undergo irreversible functional 'maturation' in response to inflammatory signals such as TNF-alpha. The current paradigm holds that this DC maturation event is required for full functional capacity and represents terminal differentiation of this cell type, culminating in apoptotic cell death. This provides a possible mechanism for avoiding dysregulated immunostimulatory activity, but imposes constraints on the capacity of DCs to influence subsequent immune responses and to participate in immunological memory. We report that the cell surface and functional effects induced by TNF-alpha are reversible and reinducible. These effects are accompanied by a concordant modulation of cytokine mRNA expression that includes the induction of proinflammatory factors (IL-15, IL-12, LT-alpha, LT-beta, TNF-alpha, RANTES) which is coincident with the down-regulation of counter-regulatory cytokines (IL-10, TGF-beta1, TGF-beta2, IL-1 RA, MCP-1). The resultant net effect is a dendritic cell activation state characterized by a transient proinflammatory posture. These results demonstrate that 1) human DCs do not undergo terminal 'maturation' in response to TNF-alpha, 2) DC phenotypes are more pleiotropic than previously thought, and 3) DCs are potential immunoregulatory effector cells with implications for control of immune responses in both in vivo and in vitro systems.

[no abstract]

Several lines of evidence support an etiological role of iodine for the initiation and perpetuation of autoimmune thyroid disease. However, varying relapse rates after increased iodine supplementation have been reported for Graves' disease. Furthermore the effects of iodine on the intensity of human autoimmune thyroiditis have previously only been investigated by indirect parameters and actions of iodine on thyroid function and a possible enhancement of the intrathyroidal autoimmune process in Graves' disease are difficult to separate in previous studies. Moreover lymphocytic thyroiditis in animal models has always been induced by considerably higher iodine doses as those used in in vivo studies. Therefore we investigated the effect of low and high iodine concentrations on the intensity of the intrathyroidal autoimmune process in Graves' disease. The intensity of intrathyroidal infiltration by lymphocytes, memory T cells, plasma cells and antigen presenting cells was determined by quantitative immunohistologic methods in 38 Graves' disease patients. 12 patients received additional preoperative iodine (group II) and 26 were treated with thiourelene antithyroid drugs only (group I). Urinary and intrathyroidal iodine concentrations were determined by a modified cer arsenit method in both groups. Application of high iodine doses in group II induced a significant increase of kappa and lambda positive plasma cells and interdigitating reticulum cells. This was not observed for activated T cells. There was no correlation between the extent of intrathyroidal infiltration by activated T cells, plasma cells and antigen presenting cells, and intrathyoidal or urinary iodine or intrathyoidal iodine concentrations in group I. High iodine doses therefore aggravate the intrathyoidal autoimmune process in Graves' disease by inducing an accumulation of immunocompetent cells involved in antigen presentation and antibody production. Howerver variations of iodine concentrations within th, SCOPUS: ar.j, info:eu-repo/semantics/published

Several lines of evidence support an etiological role of iodine for the initiation and perpetuation of autoimmune thyroid disease. However, varying relapse rates after increased iodine supplementation have been reported for Graves' disease. Furthermore the effects of iodine on the intensity of human autoimmune thyroiditis have previously only been investigated by indirect parameters and actions of iodine on thyroid function and a possible enhancement of the intrathyroidal autoimmune process in Graves' disease are difficult to separate in previous studies. Moreover lymphocytic thyroiditis in animal models has always been induced by considerably higher iodine doses as those used in in vivo studies. Therefore we investigated the effect of low and high iodine concentrations on the intensity of the intrathyroidal autoimmune process in Graves' disease. The intensity of intrathyroidal infiltration by lymphocytes, memory T cells, plasma cells and antigen presenting cells was determined by quantitative immunohistologic methods in 38 Graves' disease patients. 12 patients received additional preoperative iodine (group II) and 26 were treated with thiourelene antithyroid drugs only (group I). Urinary and intrathyroidal iodine concentrations were determined by a modified cer arsenit method in both groups. Application of high iodine doses in group II induced a significant increase of kappa and lambda positive plasma cells and interdigitating reticulum cells. This was not observed for activated T cells. There was no correlation between the extent of intrathyroidal infiltration by activated T cells, plasma cells and antigen presenting cells, and intrathyoidal or urinary iodine or intrathyoidal iodine concentrations in group I. High iodine doses therefore aggravate the intrathyoidal autoimmune process in Graves' disease by inducing an accumulation of immunocompetent cells involved in antigen presentation and antibody production. Howerver variations of iodine concentrations within th, SCOPUS: ar.j, info:eu-repo/semantics/published

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Microbiology and Immunology, 2021., Often megakaryocytes (Mks) are described as bone marrow (BM) resident cells that produce platelets. These Mks are known to be critical cellular mediators of thrombosis, but recently a new function for them has emerged as an immune regulatory cell. Lung Mks were first noted over 100 years ago, but it was only recently demonstrated that lung Mks have a more inflammatory gene expression profile compared to BM Mks. Using single cell RNA-sequencing and imaging flow cytometry we demonstrate that lung Mks express genes and functions that are similar to professional antigen presenting cells (APCs). To do this raw sequencing was processed and aligned to the Mus musculus genome assembly (mm10) using Cell Ranger software (v3, 10x Genomics), the quality control and analysis were done on Seurat. After creating an isolation method, using a negative selection kit with antibodies for CD3e, Ter-119, B220, and CD11c, for primary megakaryocytes, we were able to find that lung Mks express constitutively higher immune molecules than BM Mks. In a collaboration with Mark Looney at University of San Francisco we preformed lung intravital imaging using DQ-OVA, which is a full-length ovalbumin, that was given oropharyngeal (OP) with LPS to Pf4-Cre Rosa26-LSL-tdTomato mice (Mks and platelets are seen in red). These data showed us that lung Mks have the ability to process full-length antigen. With the use of a GFP E. Coli expressing OVA we were able to show that lung Mks are able to process intact, live pathogens in vivo and in vitro. Additionally, both BM and Lung Mks are able to present antigen to CD4+ T cells. Using a mouse deficient in Mks and platelets and a mouse made in-house with a selective reduction in MHC-II on Mks, we were able to determine that Mks were important for CD4 T cell activation during a lung bacterial response. These data demonstrate that lung Mks have several different immune modulatory roles, including activating CD4+ T cells at least in part in an antigen and MHC II