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Marina Rode von Essen,
Martin Kongsbak,
Trine Bøegh Levring,
Ann Kathrine Hansen,
Lasse Boding,
Jens Peter Holst Lauritsen,
Anders Woetmann,
Gottfried Baier,
Niels Odum, Charlotte Menné Bonefeld,
Carsten Geisler
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ABSTRACT: PKCθ plays a central role in TCR-induced IL-2 production and T-cell proliferation. The aim of the present study was to analyze how PKCθ is regulated in human T cells during T-cell activation and differentiation. We show that PKCθ is found in a high-molecular disulfide-linked complex in naïve T cells, and that PKCθ most likely is inactive in this form. In parallel with the accumulation of the major redox regulators glutathione and thioredoxin, PKCθ is gradually reduced to the 82 kDa active form during T-cell activation. We demonstrate that PKCθ is recruited to the plasma membrane in the disulfide-linked form in naïve T cells, and that activation of PKCθ is redox-dependent and requires de novo synthesis of glutathione. This is the first study that shows that the activity of PKCθ is regulated by the intracellular redox state, and that PKCθ is recruited to the plasma membrane in an inactive form in naïve T cells. Our observations underscore the existence of major differences in TCR signalling in naïve versus primed T cells.
European Journal of Immunology 02/2013; · 5.10 Impact Factor
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ABSTRACT: Because naïve T cells are unable to import cystine due to the absence of cystine transporters, it has been suggested that T cell activation is dependent on cysteine generated by antigen presenting cells. The aim of this study was to determine at which phases during T cell activation exogenous cystine/cysteine is required and how T cells meet this requirement. We found that early activation of T cells is independent of exogenous cystine/cysteine, whereas T cell proliferation is strictly dependent of uptake of exogenous cystine/cysteine. Naïve T cells express no or very low levels of both cystine and cysteine transporters. However, we found that these transporters become strongly up-regulated during T cell activation and provide activated T cells with the required amount of cystine/cysteine needed for T cell proliferation. Thus, T cells are equipped with mechanisms that allow T cell activation and proliferation independently of cysteine generated by antigen presenting cells.
Scientific Reports 01/2012; 2:266.
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ABSTRACT: Perfumes are complex mixtures composed of many fragrance ingredients, many of which are known to be only weak allergens when tested individually. It is therefore surprising that fragrance contact allergy is one of the most common forms of contact allergy.
To investigate whether mixing different fragrance allergens leads to increased sensitization potency, and to examine the difference in the challenge response to one chemical in mice sensitized either with the mixture of allergens or with only the relevant allergen.
CBA mice were sensitized with three different concentrations of three fragrance allergens alone or as a mixture. The sensitization and elicitation responses were measured by ear thickness plus infiltration of B and T cells and T cell proliferation in the draining lymph nodes.
We found a dose-dependent sensitization response for each of the allergens. An increased response was seen when the allergens were mixed. A stronger challenge response to cinnamal was seen in mice sensitized with the allergen mixture than in mice sensitized with cinnamal alone.
Our findings suggest that mixtures of allergens increase the primary response that potentiates the generation of memory T cells in response to the specific allergen. Thus, allergen mixtures enhance both induction and elicitation of contact allergy.
Contact Dermatitis 07/2011; 65(6):336-42. · 3.51 Impact Factor
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Lasse Boding,
Martin Weiss Nielsen, Charlotte Menné Bonefeld,
Marina Rode von Essen,
Bodil Lisbeth Nielsen,
Jens Peter H Lauritsen,
Ann Kathrine Hansen,
Morten Milek Nielsen,
Martin Kongsbak,
Maria Rubin,
Marie Torp Vennegaard,
Niels Odum,
Carsten Geisler
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ABSTRACT: T cell receptor (TCR) structure and function have been thoroughly studied for decades. Production and analyses of knock-out and knock-in mice with mutations in the CD3 chains have contributed significantly to these studies. The generation of such gene-modified mice relies on the availability of suitable embryonic stem (ES) cell lines. Traditionally, ES cell lines from the 129 mouse strains have been used followed by backcrossing to the C57BL/6 strain. In the present study, we demonstrate the existence of polymorphisms in the CD3 genes from mice of the 129 and C57BL/6 strains. These polymorphisms result in amino acid substitutions in the ectodomains of both the CD3delta and CD3epsilon chains in 129 mice compared to C57BL/6 mice. The amino acid substitutions do not change the stoichiometry or surface expression level of the TCR complex in 129 T cells but cause reduced anti-CD3 antibody binding to 129 T cells. Further, when stimulated with mitogenic anti-CD3 antibodies, T cells from the 129 strains show reduced expression of the activation marker CD69, Ca(2+) flux, IL-2 production and proliferative responses compared to C57BL/6 T cells. These findings demonstrate that polymorphisms of the CD3delta and epsilon ectodomains exist in mice, and that some of these polymorphisms lead to amino acid substitutions which cause structural changes and affect anti-CD3 antibody binding. Thus, functional T cell studies should be interpreted with caution when anti-CD3 antibodies are used for stimulation of T cells derived from gene-modified mice originating from 129 ES cell lines.
Molecular Immunology 09/2010; 47(15):2450-7. · 2.90 Impact Factor
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ABSTRACT: TCR and cytokine receptor signaling play key roles in the complex homeostatic mechanisms that maintain a relative stable number of T cells throughout life. Despite the homeostatic mechanisms, a slow decline in naive T cells is typically observed with age. The CD3gamma di-leucine-based motif controls TCR down-regulation and plays a central role in fine-tuning TCR expression and signaling in T cells. In this study, we show that the age-associated decline of naive T cells is strongly accelerated in CD3gammaLLAA knock-in mice homozygous for a double leucine to alanine mutation in the CD3gamma di-leucine-based motif, whereas the number of memory T cells is unaffected by the mutation. This results in premature T cell population senescence with a severe dominance of memory T cells and very few naive T cells in middle-aged to old CD3gamma mutant mice. The reduced number of naive T cells in CD3gamma mutant mice was caused by the combination of reduced thymic output, decreased T cell apoptosis, and increased transition of naive T cells to memory T cells. Experiments with bone marrow chimeric mice confirmed that the CD3gammaLLAA mutation exerted a T cell intrinsic effect on T cell homeostasis that resulted in an increased transition of CD3gammaLLAA naive T cells to memory T cells and a survival advantage of CD3gammaLLAA T cells compared with wild-type T cells. The experimental observations were further supported by mathematical modeling of T cell homeostasis. Our study thus identifies an important role of CD3gamma-mediated TCR down-regulation in T cell homeostasis.
The Journal of Immunology 10/2009; 183(8):4994-5005. · 5.79 Impact Factor
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Charlotte Menné Bonefeld,
Mariëlle Haks,
Bodil Nielsen,
Marina von Essen,
Lasse Boding,
Ann Kathrine Hansen,
Jeppe Madura Larsen,
Niels Odum,
Paul Krimpenfort,
Ada Kruisbeek,
Jan Pravsgaard Christensen,
Allan Randrup Thomsen,
Carsten Geisler
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ABSTRACT: The CD3gamma di-leucine-based motif plays a central role in TCR down-regulation. However, little is understood about the role of the CD3gamma di-leucine-based motif in physiological T cell responses. In this study, we show that the expansion in numbers of virus-specific CD8(+) T cells is impaired in mice with a mutated CD3gamma di-leucine-based motif. The CD3gamma mutation did not impair early TCR signaling, nor did it compromise recruitment or proliferation of virus-specific T cells, but it increased the apoptosis rate of the activated T cells by increasing down-regulation of the antiapoptotic molecule Bcl-2. This resulted in a 2-fold reduction in the clonal expansion of virus-specific CD8(+) T cells during the acute phase of vesicular stomatitis virus and lymphocytic choriomeningitis virus infections. These results identify an important role of CD3gamma-mediated TCR down-regulation in virus-specific CD8(+) T cell responses.
The Journal of Immunology 01/2009; 181(11):7786-99. · 5.79 Impact Factor
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ABSTRACT: IL-17-producing T(H) (T(H)17) cells are key mediators of chronic inflammation in mice. Recent studies have implicated T(H)17-mediated inflammation in the pathogenesis of human autoimmune diseases; however, the involvement of T(H)17 cells in allergic disorders remains largely elusive.
To investigate T(H)17-mediated inflammation in human beings with allergic contact dermatitis; in particular, the innate response of keratinocytes to contact allergen, the induction of allergen-specific T(H)17 cells, and the presence of T(H)17-related effector cells in inflamed skin.
Human keratinocytes were stimulated with nickel in vitro followed by measurements of IL-23 and IL-12 production by quantitative PCR and ELISA. Allergen-specific memory T cells from the blood of individuals with nickel allergy and healthy controls were identified and characterized by using a short-term ex vivo assay. Nickel patch test lesions and normal skin were analyzed for the expression of T(H)17-related cells and molecules by using immunohistochemistry.
Keratinocytes were found to produce IL-23, but no detectable IL-12, in a response to nickel stimulation. Memory T cells isolated from peripheral blood of individuals with nickel allergy, but not healthy controls, contained T(H)17 and T(H)1 cells proliferating in response to nickel-pulsed DCs. Inflamed skin of nickel-challenged allergic individuals contained infiltrating neutrophils and cells expressing IL-17, IL-22, CCR6, and IL-22R.
Our results demonstrate the involvement of T(H)17-mediated immunopathology in human allergic contact dermatitis, including both innate and adaptive immune responses to contact allergens.
The Journal of allergy and clinical immunology 12/2008; 123(2):486-92. · 9.17 Impact Factor
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ABSTRACT: The different role of various immunological effector cells in contact hypersensitivity (CHS) is receiving increased attention. During the past decade, the involvement of different cell types in CHS has been investigated by the use of antibody-induced depletion of specific subtypes of immunological cells and by studying knockout mice lacking one or more of these immunological cell populations.
To develop a method for studying the collective cellular dynamics of immune cells in the draining lymph nodes during CHS in intact animals.
Mice were sensitized and/or challenged with 2,4-dinitrofluorobenzene or oxazolone. Using multi-parameter flow cytometry we determined the proliferation, activation state, and absolute number of helper T cells, cytotoxic T cells, B cells, and natural killer cells in the draining lymph nodes.
The presented method can be applied to evaluate the effect of different contact allergens on various cell populations of the immune system.
Our study support recent findings that several cell types seem to be involved in CHS.
Contact Dermatitis 12/2007; 57(5):300-8. · 3.51 Impact Factor
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ABSTRACT: The T-cell receptor (TCR) is a multimeric receptor composed of the Ti alpha beta heterodimer and the noncovalently associated CD3 gamma delta epsilon and zeta(2) chains. All of the TCR chains are required for efficient cell surface expression of the TCR. Previous studies on chimeric molecules containing the di-leucine-based endocytosis motif of the TCR subunit CD3 gamma have indicated that the zeta chain can mask this motif. In this study, we show that successive truncations of the cytoplasmic tail of zeta led to reduced surface expression levels of completely assembled TCR complexes. The reduced TCR expression levels were caused by an increase in the TCR endocytic rate constant in combination with an unaffected exocytic rate constant. Furthermore, the TCR degradation rate constant was increased in cells with truncated zeta. Introduction of a CD3 gamma chain with a disrupted di-leucine-based endocytosis motif partially restored TCR expression in cells with truncated zeta chains, indicating that the zeta chain masks the endocytosis motif in CD3 gamma and thereby stabilizes TCR cell surface expression.
Traffic 10/2004; 5(9):672-84. · 4.92 Impact Factor
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ABSTRACT: Modulation of TCR expression levels is a central event during T cell development and activation, and it probably plays an important role in adjusting T cell responsiveness. Conflicting data have been published on down-regulation and degradation rates of the individual TCR subunits, and several divergent models for TCR down-regulation and degradation have been suggested. The aims of this study were to determine the rate constants for constitutive and ligand-induced TCR degradation and to determine whether the TCR subunits segregate or are processed as an intact unit during TCR down-regulation and degradation. We found that the TCR subunits in nonstimulated Jurkat cells were degraded with rate constants of approximately 0.0011 min(-1), resulting in a half-life of approximately 10.5 h. Triggering of the TCR by anti-TCR Abs resulted in a 3-fold increase in the degradation rate constants to approximately 0.0033 min(-1), resulting in a half-life of approximately 3.5 h. The subunits of the TCR complex were down-regulated from the cell surface and degraded with identical kinetics, and most likely remained associated during the passage throughout the endocytic pathway from the cell surface to the lysosomes. Similar results were obtained in studies of primary human Vbeta8+ T cells stimulated with superantigen. Based on these results, the simplest model for TCR internalization, sorting, and degradation is proposed.
The Journal of Immunology 08/2004; 173(1):384-93. · 5.79 Impact Factor
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ABSTRACT: The T-cell receptor (TCR) is a multimeric receptor composed of the Tiαβ heterodimer and the noncovalently associated CD3γδε and ζ2 chains. All of the TCR chains are required for efficient cell surface expression of the TCR. Previous studies on chimeric molecules containing the di-leucine-based endocytosis motif of the TCR subunit CD3γ have indicated that the ζ chain can mask this motif. In this study, we show that successive truncations of the cytoplasmic tail of ζ led to reduced surface expression levels of completely assembled TCR complexes. The reduced TCR expression levels were caused by an increase in the TCR endocytic rate constant in combination with an unaffected exocytic rate constant. Furthermore, the TCR degradation rate constant was increased in cells with truncated ζ. Introduction of a CD3γ chain with a disrupted di-leucine-based endocytosis motif partially restored TCR expression in cells with truncated ζ chains, indicating that the ζ chain masks the endocytosis motif in CD3γ and thereby stabilizes TCR cell surface expression.
Traffic 07/2004; 5(9):672 - 684. · 4.92 Impact Factor
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ABSTRACT: One of the earliest events following TCR triggering is TCR down-regulation. However, the mechanisms behind TCR down-regulation are still not fully known. Some studies have suggested that only directly triggered TCR are internalized, whereas others studies have indicated that, in addition to triggered receptors, nonengaged TCR are also internalized (comodulated). In this study, we used transfected T cells expressing two different TCR to analyze whether comodulation took place. We show that TCR triggering by anti-TCR mAb and peptide-MHC complexes clearly induced internalization of nonengaged TCR. By using a panel of mAb against the Ti beta chain, we demonstrate that the comodulation kinetics depended on the affinity of the ligand. Thus, high-affinity mAb (K(D) = 2.3 nM) induced a rapid but reversible comodulation, whereas low-affinity mAb (K(D) = 6200 nM) induced a slower but more permanent type of comodulation. Like internalization of engaged TCR, comodulation was dependent on protein tyrosine kinase activity. Finally, we found that in contrast to internalization of engaged TCR, comodulation was highly dependent on protein kinase C activity and the CD3 gamma di-leucine-based motif. Based on these observations, a physiological role of comodulation is proposed and the plausibility of the TCR serial triggering model is discussed.
The Journal of Immunology 10/2003; 171(6):3003-9. · 5.79 Impact Factor
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Lasse Boding,
Martin Weiss Nielsen, Charlotte Menné Bonefeld,
Marina Rode von Essen,
Bodil Lisbeth Nielsen,
Jens Peter H. Lauritsen,
Ann Kathrine Hansen,
Morten Milek Nielsen,
Martin Kongsbak,
Maria Rubin,
Marie Torp Vennegaard,
Niels Ødum,
Carsten Geisler
[show abstract]
[hide abstract]
ABSTRACT: T cell receptor (TCR) structure and function have been thoroughly studied for decades. Production and analyses of knock-out and knock-in mice with mutations in the CD3 chains have contributed significantly to these studies. The generation of such gene-modified mice relies on the availability of suitable embryonic stem (ES) cell lines. Traditionally, ES cell lines from the 129 mouse strains have been used followed by backcrossing to the C57BL/6 strain. In the present study, we demonstrate the existence of polymorphisms in the CD3 genes from mice of the 129 and C57BL/6 strains. These polymorphisms result in amino acid substitutions in the ectodomains of both the CD3δ and CD3ɛ chains in 129 mice compared to C57BL/6 mice. The amino acid substitutions do not change the stoichiometry or surface expression level of the TCR complex in 129 T cells but cause reduced anti-CD3 antibody binding to 129 T cells. Further, when stimulated with mitogenic anti-CD3 antibodies, T cells from the 129 strains show reduced expression of the activation marker CD69, Ca2+ flux, IL-2 production and proliferative responses compared to C57BL/6 T cells. These findings demonstrate that polymorphisms of the CD3δ and ɛ ectodomains exist in mice, and that some of these polymorphisms lead to amino acid substitutions which cause structural changes and affect anti-CD3 antibody binding. Thus, functional T cell studies should be interpreted with caution when anti-CD3 antibodies are used for stimulation of T cells derived from gene-modified mice originating from 129 ES cell lines.
Molecular Immunology.
