Non-obese diabetic (NOD) mice develop spontaneous T-cell responses against pancreatic beta-cells,... more Non-obese diabetic (NOD) mice develop spontaneous T-cell responses against pancreatic beta-cells, leading to islet cell destruction and diabetes. Despite high genetic similarity, non-obese resistant (NOR) mice do not develop diabetes. We show here that spleen cells of both NOD and NOR mice respond to the islet cell antigen glutamic acid decarboxylase-65 in IFN-gamma-ELISPOT assays. Moreover, NOR-T cells induce periinsulitis in NOD SCID recipient mice. Thus, a potentially pathogenic islet cell-specific T-cell response arises in NOR and NOD mice alike; the mechanism that prevents the autoimmune progression of self-reactive T cells in NOR mice presumably acts at the level of effector function. Consistent with this hypothesis, CD4+CD25+ cell-depleted spleen cells from NOR mice mediated islet cell destruction and overt diabetes in NOD SCID mice. Therefore, islet cell-specific effector cells in NOR mice appear to be under the control of CD4+CD25+ regulatory T cells, confirming the importance of regulatory cells in the control of autoimmune diabetes.
Treatment-resistant Lyme arthritis is associated with immune reactivity to outer surface protein ... more Treatment-resistant Lyme arthritis is associated with immune reactivity to outer surface protein A (OspA) of Borrelia burgdorferi, the agent of Lyme disease, and the major histocompatibility complex class II allele DRB1*0401. The immunodominant epitope of OspA for T helper cells was identified. A homology search revealed a peptide from human leukocyte function-associated antigen-1 (hLFA-1) as a candidate autoantigen. Individuals with treatment-resistant Lyme arthritis, but not other forms of arthritis, generated responses to OspA, hLFA-1, and their highly related peptide epitopes. Identification of the initiating bacterial antigen and a cross-reactive autoantigen may provide a model for development of autoimmune disease.
It is unknown to what extent the heterogeneity of antigen presenting cells (APC) influences the I... more It is unknown to what extent the heterogeneity of antigen presenting cells (APC) influences the IFN-gamma response of CD4 memory cells. We re-stimulated DO11.10 T cell receptor (TCR)-transgenic cells and wild-type CD4 memory cells with OVA-peptide 323-339 presented on purified dendritic cells (DC), macrophages, and B cells. Using IFN-gamma ELISPOT assays, we measured the number of cytokine producing T cells and the amount of cytokine produced by individual T cells at different time points after antigen encounter. The data showed that, when CD4 cells recognized antigen on DC, the induction of cytokine production was accelerated compared to macrophages and B cells. In contrast, the per-cell cytokine productivity was independent of the type of APC by which the T cells were re-stimulated. Moreover, the peptide concentration required for CD4 cell activation was comparable for the different APC. The data suggest that DC induce cytokine production in memory cells with accelerated activatio...
Validation of Cell-Based Assays in the GLP Setting, 2008
ABSTRACT SummaryELISPOT assays have recently emerged as a primary tool for monitoring antigen-spe... more ABSTRACT SummaryELISPOT assays have recently emerged as a primary tool for monitoring antigen-specific low frequency measurements of cellular immunity, recording two cardinal features of cell-mediated immunity: the clonal sizes and the cytokine effector class. Obtaining information on these parameters is crucial for the comprehensive assessment, among other things, of any immunotherapy. This is particularly useful in the field of pre-clinical/clinical studies and vaccine trials, where high sample numbers from various sites have to be collected and stored before they can be processed in a timely fashion. With relatively little cell material and labor involved, ELISPOT assays would ideally lend themselves for high throughput analysis. While long employed in the academic research setting, the ability to obtain valid ELISPOT data on study samples is beginning to greatly enhance ex vivo immune monitoring capabilities for the pharmaceutical companies. To be used in a regulated environment, however, the assay needs to be validated. This chapter will focus on the standardization and validation of an ELISPOT assay using reference samples.
Experimental allergic encephalomyelitis (EAE) is an animal model of multiple sclerosis. While EAE... more Experimental allergic encephalomyelitis (EAE) is an animal model of multiple sclerosis. While EAE is mediated by the cytokines produced by specific T cells, the cytokine signature of these effector cells is unresolved. We tested CD4 cells from MOG peptide 35-55 immunized C57BL/6 mice for their peptide induced cytokine production on antigen presenting cells of the respective cytokine knockout mice, or wild type mice. IL-4 and IL-6 production was seen on wild type antigen presenting cells, suggesting that IL-4 and IL-6 are not T cell products. In contrast, IFN-gamma, IL-2 and IL-3 were found to be produced by the MOG specific CD4 cells. Understanding the cognate vs. bystander cytokine production in EAE might help dissect the contribution of cytokines to the pathogenesis of the disease.
Gene knock-out and knock-in mice are becoming increasingly indispensable for mechanism-oriented s... more Gene knock-out and knock-in mice are becoming increasingly indispensable for mechanism-oriented studies of EAE. Most gene-modified mice are on the C57BL/6 background, for which presently there are only two EAE models available, the MOG peptide 35-55 and the PLP 178-191 peptide induced disease. However, because MS is not a single pathogenic entity, different EAE models are required to reproduce and study its various features. Here we are introducing MBP-PLP fusion protein (MP4)-induced EAE for C57BL/6 mice. B cell- and CD8+ T cell-dependence, as well as multi-determinant recognition are among the unique features of this demyelinating EAE.
Vaccinations typically rely on immunization with live virus for eliciting protective CD8 T cell i... more Vaccinations typically rely on immunization with live virus for eliciting protective CD8 T cell immunity. There is increasing interest to use subunit vaccination strategies to achieve such responses. Complete Freund's adjuvant (CFA) and unmethylated cytosine-guanine dinucleotide containing DNA are considered some of the most potent adjuvants for eliciting immunity. Whereas a wealth of information is available on how these adjuvants affect CD4 T cell responses, their effects on engaging CD8 T cell immunity are not completely understood. We immunized C57BL/6J mice with the class I restricted peptides Uty or SIINFEKL using these 2 adjuvants and tested for cytokine secretion, proliferation, in vivo cytotoxicity, and delayed-type hypersensitivity (DTH). Our data show that CFA-induced CD8 T cells to proliferate, mediate DTH, and to secrete interferon-gamma, interleukin (IL)-2 and IL-17. Despite these markers of CD8 T cell activation, CFA failed to induce an early cytotoxic CD8 T cell response. In contrast, unmethylated cytosine-guanine dinucleotide containing DNA promoted a vigorous cytolytic response without activating substantial cytokine production, proliferation or DTH. These data have implications for CD8 T cell subunit vaccine design in which cytotoxicity versus DTH plays a key role in host defense.
Non-obese diabetic (NOD) mice develop spontaneous T-cell responses against pancreatic beta-cells,... more Non-obese diabetic (NOD) mice develop spontaneous T-cell responses against pancreatic beta-cells, leading to islet cell destruction and diabetes. Despite high genetic similarity, non-obese resistant (NOR) mice do not develop diabetes. We show here that spleen cells of both NOD and NOR mice respond to the islet cell antigen glutamic acid decarboxylase-65 in IFN-gamma-ELISPOT assays. Moreover, NOR-T cells induce periinsulitis in NOD SCID recipient mice. Thus, a potentially pathogenic islet cell-specific T-cell response arises in NOR and NOD mice alike; the mechanism that prevents the autoimmune progression of self-reactive T cells in NOR mice presumably acts at the level of effector function. Consistent with this hypothesis, CD4+CD25+ cell-depleted spleen cells from NOR mice mediated islet cell destruction and overt diabetes in NOD SCID mice. Therefore, islet cell-specific effector cells in NOR mice appear to be under the control of CD4+CD25+ regulatory T cells, confirming the importance of regulatory cells in the control of autoimmune diabetes.
Treatment-resistant Lyme arthritis is associated with immune reactivity to outer surface protein ... more Treatment-resistant Lyme arthritis is associated with immune reactivity to outer surface protein A (OspA) of Borrelia burgdorferi, the agent of Lyme disease, and the major histocompatibility complex class II allele DRB1*0401. The immunodominant epitope of OspA for T helper cells was identified. A homology search revealed a peptide from human leukocyte function-associated antigen-1 (hLFA-1) as a candidate autoantigen. Individuals with treatment-resistant Lyme arthritis, but not other forms of arthritis, generated responses to OspA, hLFA-1, and their highly related peptide epitopes. Identification of the initiating bacterial antigen and a cross-reactive autoantigen may provide a model for development of autoimmune disease.
It is unknown to what extent the heterogeneity of antigen presenting cells (APC) influences the I... more It is unknown to what extent the heterogeneity of antigen presenting cells (APC) influences the IFN-gamma response of CD4 memory cells. We re-stimulated DO11.10 T cell receptor (TCR)-transgenic cells and wild-type CD4 memory cells with OVA-peptide 323-339 presented on purified dendritic cells (DC), macrophages, and B cells. Using IFN-gamma ELISPOT assays, we measured the number of cytokine producing T cells and the amount of cytokine produced by individual T cells at different time points after antigen encounter. The data showed that, when CD4 cells recognized antigen on DC, the induction of cytokine production was accelerated compared to macrophages and B cells. In contrast, the per-cell cytokine productivity was independent of the type of APC by which the T cells were re-stimulated. Moreover, the peptide concentration required for CD4 cell activation was comparable for the different APC. The data suggest that DC induce cytokine production in memory cells with accelerated activatio...
Validation of Cell-Based Assays in the GLP Setting, 2008
ABSTRACT SummaryELISPOT assays have recently emerged as a primary tool for monitoring antigen-spe... more ABSTRACT SummaryELISPOT assays have recently emerged as a primary tool for monitoring antigen-specific low frequency measurements of cellular immunity, recording two cardinal features of cell-mediated immunity: the clonal sizes and the cytokine effector class. Obtaining information on these parameters is crucial for the comprehensive assessment, among other things, of any immunotherapy. This is particularly useful in the field of pre-clinical/clinical studies and vaccine trials, where high sample numbers from various sites have to be collected and stored before they can be processed in a timely fashion. With relatively little cell material and labor involved, ELISPOT assays would ideally lend themselves for high throughput analysis. While long employed in the academic research setting, the ability to obtain valid ELISPOT data on study samples is beginning to greatly enhance ex vivo immune monitoring capabilities for the pharmaceutical companies. To be used in a regulated environment, however, the assay needs to be validated. This chapter will focus on the standardization and validation of an ELISPOT assay using reference samples.
Experimental allergic encephalomyelitis (EAE) is an animal model of multiple sclerosis. While EAE... more Experimental allergic encephalomyelitis (EAE) is an animal model of multiple sclerosis. While EAE is mediated by the cytokines produced by specific T cells, the cytokine signature of these effector cells is unresolved. We tested CD4 cells from MOG peptide 35-55 immunized C57BL/6 mice for their peptide induced cytokine production on antigen presenting cells of the respective cytokine knockout mice, or wild type mice. IL-4 and IL-6 production was seen on wild type antigen presenting cells, suggesting that IL-4 and IL-6 are not T cell products. In contrast, IFN-gamma, IL-2 and IL-3 were found to be produced by the MOG specific CD4 cells. Understanding the cognate vs. bystander cytokine production in EAE might help dissect the contribution of cytokines to the pathogenesis of the disease.
Gene knock-out and knock-in mice are becoming increasingly indispensable for mechanism-oriented s... more Gene knock-out and knock-in mice are becoming increasingly indispensable for mechanism-oriented studies of EAE. Most gene-modified mice are on the C57BL/6 background, for which presently there are only two EAE models available, the MOG peptide 35-55 and the PLP 178-191 peptide induced disease. However, because MS is not a single pathogenic entity, different EAE models are required to reproduce and study its various features. Here we are introducing MBP-PLP fusion protein (MP4)-induced EAE for C57BL/6 mice. B cell- and CD8+ T cell-dependence, as well as multi-determinant recognition are among the unique features of this demyelinating EAE.
Vaccinations typically rely on immunization with live virus for eliciting protective CD8 T cell i... more Vaccinations typically rely on immunization with live virus for eliciting protective CD8 T cell immunity. There is increasing interest to use subunit vaccination strategies to achieve such responses. Complete Freund's adjuvant (CFA) and unmethylated cytosine-guanine dinucleotide containing DNA are considered some of the most potent adjuvants for eliciting immunity. Whereas a wealth of information is available on how these adjuvants affect CD4 T cell responses, their effects on engaging CD8 T cell immunity are not completely understood. We immunized C57BL/6J mice with the class I restricted peptides Uty or SIINFEKL using these 2 adjuvants and tested for cytokine secretion, proliferation, in vivo cytotoxicity, and delayed-type hypersensitivity (DTH). Our data show that CFA-induced CD8 T cells to proliferate, mediate DTH, and to secrete interferon-gamma, interleukin (IL)-2 and IL-17. Despite these markers of CD8 T cell activation, CFA failed to induce an early cytotoxic CD8 T cell response. In contrast, unmethylated cytosine-guanine dinucleotide containing DNA promoted a vigorous cytolytic response without activating substantial cytokine production, proliferation or DTH. These data have implications for CD8 T cell subunit vaccine design in which cytotoxicity versus DTH plays a key role in host defense.
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