ABSTRACT
Self-reactive T cell clones that escape negative selection are either deleted or rendered functionally unresponsive (anergic), thus preventing them from propagating host tissue damage. By using an in vivo model, we investigated molecular mechanisms for T cell tolerance, finding that despite a characteristic inability to generate effector cytokine proteins, self-reactive T cells express large amounts of cytokine mRNAs. This disconnect between cytokine message and protein was not observed in T cells mounting productive responses to foreign antigens but, instead, was seen only in those responding to self, where the block in protein translation was shown to involve conserved AU-rich elements within cytokine 3'UTRs. These studies reveal that translation of abundant cytokine mRNAs is limited in self-reactive T cells and, thus, identify posttranscriptional silencing of antigen-driven gene expression as a key mechanism underlying the anergic phenotype of self-reactive T cells.
Subject(s)
Autoantigens/immunology , CD4-Positive T-Lymphocytes/metabolism , Cytokines/metabolism , RNA, Messenger/metabolism , RNA-Induced Silencing Complex/metabolism , 3' Untranslated Regions/genetics , Adoptive Transfer , Animals , Autoimmunity , CD4-Positive T-Lymphocytes/cytology , CD4-Positive T-Lymphocytes/immunology , Clonal Anergy , Cytokines/genetics , Cytokines/immunology , Immune Tolerance , Mice , Mice, Transgenic , Protein Biosynthesis/immunology , RNA, Messenger/genetics , RNA, Messenger/immunology , RNA-Induced Silencing Complex/immunology , Response Elements/geneticsABSTRACT
BALB/c IL-2-deficient (IL-2-KO) mice develop systemic autoimmunity, dying within 3 to 5 wk from complications of autoimmune hemolytic anemia. Disease in these mice is Th1 mediated, and IFN-γ production is required for early autoimmunity. In this study, we show that dendritic cells (DCs) are required for optimal IFN-γ production by T cells in the IL-2-KO mouse. Disease is marked by DC accumulation, activation, and elevated production of Th1-inducing cytokines. IL-2-KO DCs induce heightened proliferation and cytokine production by naive T cells compared with wild-type DCs. The depletion of either conventional or plasmacytoid DCs significantly prolongs the survival of IL-2-KO mice, demonstrating that DCs contribute to the progression of autoimmunity. Elimination of Th1-inducing cytokine signals (type 1 IFN and IL-12) reduces RBC-specific Ab production and augments survival, indicating that cytokines derived from both plasmacytoid DCs and conventional DCs contribute to disease severity. DC activation likely precedes T cell activation because DCs are functionally activated even in an environment lacking overt T cell activation. These data indicate that both conventional and plasmacytoid DCs are critical regulators in the development of this systemic Ab-mediated autoimmune disease, in large part through the production of IL-12 and type 1 IFNs.
Subject(s)
Anemia, Hemolytic, Autoimmune/immunology , Autoimmunity/immunology , Dendritic Cells/immunology , Adoptive Transfer , Anemia, Hemolytic, Autoimmune/metabolism , Animals , Autoantibodies/immunology , Disease Models, Animal , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Interferon Type I/biosynthesis , Interleukin-12/biosynthesis , Interleukin-2 , Lymphocyte Activation/immunology , Mice , Mice, Inbred BALB C , Mice, Knockout , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , T-Lymphocytes/immunologyABSTRACT
T cell-APC interactions are essential for the initiation of effector responses against foreign and self-antigens, but the role of these interactions in generating different populations of effector T cells in vivo remains unclear. Using a model of CD4(+) T cell responses to a systemic self-antigen without adjuvants or infection, we demonstrate that activation of APCs augments Th17 responses much more than Th1 responses. Recognition of systemic Ag induces tolerance in self-reactive CD4(+) T cells, but induction of CD40 signaling, even under tolerogenic conditions, results in a strong, Ag-specific IL-17 response without large numbers of IFN-γ-producing cells. Transfer of the same CD4(+) T cells into lymphopenic recipients expressing the self-antigen results in uncontrolled production of IL-17, IFN-γ, and systemic inflammation. If the Ag-specific T cells lack CD40L, production of IL-17 but not IFN-γ is decreased, and the survival time of recipient mice is significantly increased. In addition, transient blockade of the initial MHC class II-dependent T cell-APC interaction results in a greater reduction of IL-17 than of IFN-γ production. These data suggest that Th17 differentiation is more sensitive to T cell interactions with APCs than is the Th1 response, and interrupting this interaction, specifically the CD40 pathway, may be key to controlling Th17-mediated autoimmunity.
Subject(s)
Autoantigens/immunology , CD4-Positive T-Lymphocytes/immunology , Th1 Cells/immunology , Th17 Cells/immunology , Animals , Antigen-Presenting Cells/immunology , Antigen-Presenting Cells/metabolism , CD4-Positive T-Lymphocytes/metabolism , CD40 Antigens/immunology , CD40 Antigens/metabolism , CD40 Ligand/genetics , CD40 Ligand/immunology , CD40 Ligand/metabolism , Cell Communication/immunology , Cell Differentiation/immunology , Cells, Cultured , Dendritic Cells/immunology , Dendritic Cells/metabolism , Female , Flow Cytometry , Interferon-gamma/immunology , Interferon-gamma/metabolism , Interleukin-17/immunology , Interleukin-17/metabolism , Interleukin-6/immunology , Interleukin-6/metabolism , Lymphocyte Activation/immunology , Male , Mice , Mice, Inbred BALB C , Mice, Knockout , Mice, Transgenic , Ovalbumin/genetics , Ovalbumin/immunology , Th1 Cells/metabolism , Th17 Cells/metabolismABSTRACT
The early events that determine the decision between lymphocyte tolerance and activation are not well-understood. Using a model of systemic self-antigen recognition by CD4(+) T cells, we show, using single-cell biochemical analyses, that tolerance is characterized by transient signaling events downstream of T-cell receptor engagement in the mammalian target of rapamycin (mTOR) and NF-κB pathways. Parallel studies done by live cell imaging show that the key difference between tolerance and activation is the duration of the T cell-antigen presenting cell (APC) interaction, as revealed by stable T-cell immobilization on antigen encounter. Brief T cell-APC interactions result in tolerance, and prolonged interactions are associated with activation and the development of effector cells. These studies show that the duration of T cell-APC interactions and magnitude of associated TCR-mediated signaling are key determinants of lymphocyte tolerance vs. activation.
Subject(s)
Immune Tolerance , Lymphocyte Activation , Receptors, Antigen, T-Cell/metabolism , Signal Transduction , Animals , Antigen-Presenting Cells/immunology , Flow Cytometry , Lymphopenia/immunology , Mice , Mice, Inbred BALB CABSTRACT
Patients with Wiskott-Aldrich syndrome (WAS) have numerous immune cell deficiencies, but it remains unclear how abnormalities in individual cell types contribute to the pathologies of WAS. In T cells, the WAS protein (WASp) regulates actin polymerization and transcription, and plays a role in the dynamics of the immunologic synapse. To examine how these events influence CD4 function, we isolated the WASp deficiency to CD4(+) T cells by adoptive transfer into wild-type mice to study T-cell priming and effector function. WAS(-/-) CD4(+) T cells mediated protective T-helper 1 (Th1) responses to Leishmania major in vivo, but were unable to support Th2 immunity to Nippostrongylus brasiliensis or L major. Mechanistically, WASp was not required for Th2 programming but was required for Th2 effector function. WAS(-/-) CD4(+) T cells up-regulated IL-4 and GATA3 mRNA and secreted IL-4 protein during Th2 differentiation. In contrast, cytokine transcription was uncoupled from protein production in WAS(-/-) Th2-primed effectors. WAS(-/-) Th2s failed to produce IL-4 protein on restimulation despite elevated IL-4/GATA3 mRNA. Moreover, dominant-negative WASp expression in WT effector T cells blocked IL-4 production, but had no effect on IFNgamma. Thus WASp plays a selective, posttranscriptional role in Th2 effector function.
Subject(s)
Th2 Cells/immunology , Wiskott-Aldrich Syndrome Protein/immunology , Animals , GATA3 Transcription Factor/biosynthesis , GATA3 Transcription Factor/genetics , GATA3 Transcription Factor/immunology , Humans , Interferon-gamma/genetics , Interferon-gamma/immunology , Interferon-gamma/metabolism , Interleukin-4/biosynthesis , Interleukin-4/genetics , Interleukin-4/immunology , Leishmania major/immunology , Leishmaniasis, Cutaneous/genetics , Leishmaniasis, Cutaneous/immunology , Leishmaniasis, Cutaneous/metabolism , Mice , Mice, Inbred BALB C , Mice, Knockout , Nippostrongylus/immunology , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , RNA, Messenger/immunology , Strongylida Infections/genetics , Strongylida Infections/immunology , Strongylida Infections/metabolism , Th1 Cells/immunology , Th2 Cells/metabolism , Transcription, Genetic/genetics , Transcription, Genetic/immunology , Up-Regulation/genetics , Up-Regulation/immunology , Wiskott-Aldrich Syndrome/genetics , Wiskott-Aldrich Syndrome/immunology , Wiskott-Aldrich Syndrome/metabolism , Wiskott-Aldrich Syndrome Protein/genetics , Wiskott-Aldrich Syndrome Protein/metabolismABSTRACT
The possibility that effector T cells can be converted into forkhead box P3(+) regulatory T cells (Tregs) has potential therapeutic implications. To analyze the relationship between Th1 effectors and Tregs, we have used a model of systemic autoimmunity in which both effector and Tregs arise from a single population specific for a transgene-encoded systemic protein. In vitro, the presence of IFN-gamma inhibits Treg generation during activation. Using IFN-gamma reporter mice, we demonstrate that IFN-gamma-producing cells tend not to develop into Tregs, and Th1 priming of T cells prior to cell transfer limits the number of forkhead box P3(+) T cells generated in vivo. Moreover, transfer of IFN-gamma(-/-) or STAT1(-/-) T cells resulted in an increase in the number of Tregs. These data support a role for Th1 effector molecules and transcription factors in the control of peripheral Treg generation and demonstrates the limited plasticity of Th1 populations.
Subject(s)
T-Lymphocyte Subsets/cytology , T-Lymphocytes, Regulatory/cytology , Th1 Cells/cytology , Adoptive Transfer , Animals , Cell Differentiation/immunology , Flow Cytometry , Forkhead Transcription Factors , Interferon-gamma/immunology , Lymphocyte Activation/immunology , Mice , Mice, Transgenic , T-Lymphocyte Subsets/immunology , T-Lymphocytes, Regulatory/immunology , Th1 Cells/immunologyABSTRACT
Compartmentalization of immunity ensures tight regulation of T cell activation in the LN and precise effector T cell delivery to inflamed sites. Herein we show that the tissue-specific accumulation of effector T cells can be subverted by a pathogen at the infection site. Using the Leishmania major mouse model of dermal infection, we observed a restricted chemokine profile at the infection site, i.e., the expression of Th2 cell-attracting CCL7 but not of Th1 cell-attracting chemokines. Consistent with these chemokine expression data, recruitment of cytokine-producing T cells to the infection site was also selective. Both IL-4- and IFN-gamma-producing effector T cells homed to inflamed OVA/CFA-immunized dermis, but only IL-4-producing cells homed to L. major-infected dermis. The narrowing of the cytokine repertoire at the site of infection with L. major was driven, in part, by pathogen-induced CCL7. Inflammatory signals failed to disrupt the early restrictive L. major infection site, which suggests that L. major dominantly modifies the local milieu. We have highlighted an emerging principle in pathogen-host interactions: that the cytokine repertoire at the infection site and the LN draining the infection site can be different because of the ability of the pathogen to modify the chemokine profile at the infection site. Thus, pathogens may edit the LN cytokine repertoire through differential recruitment of cytokine-producing cells.
Subject(s)
Chemokines/metabolism , Cytokines/metabolism , Host-Pathogen Interactions/immunology , Leishmania major , Leishmaniasis, Cutaneous/immunology , Animals , Disease Models, Animal , Lymph Nodes/immunology , Lymph Nodes/parasitology , Mice , Mice, Inbred Strains , Th1 Cells/immunology , Th2 Cells/immunologyABSTRACT
Regulation of the magnitude and quality of immune responses is dependent on the integration of multiple signals which typically operate through positive and negative feedback loops. Cytokines that promote or limit T cell expansion and differentiation are often both present in the complex lymphoid environment where antigen-initiated T cell responses take place. The nature and strength of the cytokine signal received by the responding cell, as well as by surrounding regulatory cells, will determine the extent of clonal expansion and the progression towards effector and memory cell differentiation. The mechanisms that determine how much cytokine is produced and how cytokine activities are controlled by receptor expression and intracellular regulators of signaling are not fully understood. Here we discuss the opposing functions of two members of the common receptor gamma chain (γc) cytokines, IL-2 and IL-7 in the generation and regulation of immune responses in vivo.
Subject(s)
Immunity/immunology , Interleukin-2/immunology , Interleukin-7/immunology , Animals , Humans , Immunologic Memory/immunology , Signal Transduction/immunology , Skin/immunologyABSTRACT
Specifications (specs) grading is a grading system in which mastery of specific educational outcomes is the basis for the final grade a student earns in the course. Implementation of the types of assessments used for specs grading has shown to be beneficial for student learning and motivation compared to traditional grading systems. We designed a specs grading strategy in an undergraduate Cell Biology course, creating 20 individual learning outcomes (LOs). The grade earned in lecture depended on the number of LOs the student mastered. If students were unable to master the content on their initial attempt, they could earn retakes for each LO assessment by completing an assignment associated with the information covered in that LO. A student's final class grade was dependent on the number of LOs mastered combined with the grade earned on their final exam. Here, we present how specifications grading was implemented in Cell Biology, differences in overall grade distribution between grading systems, improved performance on content-related assessment questions in sections using specifications grading, and more-positive attitudes for sections using specifications grading than for traditionally graded sections.
ABSTRACT
Interleukin 4 (IL-4) plays a central role in the orchestration of Type 2 immunity. During T cell activation in the lymph node, IL-4 promotes Th2 differentiation and inhibits Th1 generation. In the inflamed tissue, IL-4 signals promote innate and adaptive Type-2 immune recruitment and effector function, positively amplifying the local Th2 response. In this study, we identify an additional negative regulatory role for IL-4 in limiting the recruitment of Th1 cells to inflamed tissues. To test IL-4 effects on inflammation subsequent to Th2 differentiation, we transiently blocked IL-4 during ongoing dermal inflammation (using anti-IL-4 mAb) and analyzed changes in gene expression. Neutralization of IL-4 led to the upregulation of a number of genes linked to Th1 trafficking, including CXCR3 chemokines, CCL5 and CCR5 and an associated increase in IFNγ, Tbet and TNFα genes. These gene expression changes correlated with increased numbers of IFNγ-producing CD4+ T cells in the inflamed dermis. Moreover, using an adoptive transfer approach to directly test the role of IL-4 in T cell trafficking to the inflamed tissues, we found IL-4 neutralization led to an early increase in Th1 cell recruitment to the inflamed dermis. These data support a model whereby IL-4 dampens Th1-chemokines at the site of inflammation limiting Th1 recruitment. To determine biological significance, we infected mice with Leishmania major, as pathogen clearance is highly dependent on IFNγ-producing CD4+ T cells at the infection site. Short-term IL-4 blockade in established L. major infection led to a significant increase in the number of IFNγ-producing CD4+ T cells in the infected ear dermis, with no change in the draining LN. Increased lymphocyte influx into the infected tissue correlated with a significant decrease in parasite number. Thus, independent of IL-4's role in the generation of immune effectors, IL-4 attenuates lymphocyte recruitment to the inflamed/infected dermis and limits pathogen clearance.
Subject(s)
Cell Movement/immunology , Chemokines/immunology , Inflammation/immunology , Interleukin-4/immunology , Th1 Cells/immunology , Animals , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , Cell Movement/genetics , Cells, Cultured , Chemokines/genetics , Chemokines/metabolism , Cluster Analysis , Dermis/immunology , Dermis/metabolism , Dermis/parasitology , Enzyme-Linked Immunosorbent Assay , Host-Parasite Interactions/immunology , Inflammation/genetics , Inflammation/metabolism , Interferon-gamma/genetics , Interferon-gamma/immunology , Interferon-gamma/metabolism , Interleukin-4/genetics , Interleukin-4/metabolism , Leishmania major/immunology , Leishmania major/physiology , Leishmaniasis, Cutaneous/immunology , Leishmaniasis, Cutaneous/parasitology , Mice , Mice, Inbred BALB C , Oligonucleotide Array Sequence Analysis , Th1 Cells/metabolism , Th2 Cells/immunology , Th2 Cells/metabolism , Transcriptome/immunologyABSTRACT
B cell tolerance to self-antigen is critical to preventing antibody-mediated autoimmunity. Previous work using B cell antigen receptor transgenic animals suggested that self-antigen-specific B cells are either deleted from the repertoire, enter a state of diminished function termed anergy, or are ignorant to the presence of self-antigen. These mechanisms have not been assessed in a normal polyclonal repertoire because of an inability to detect rare antigen-specific B cells. Using a novel detection and enrichment strategy to assess polyclonal self-antigen-specific B cells, we find no evidence of deletion or anergy of cells specific for antigen not bound to membrane, and tolerance to these types of antigens appears to be largely maintained by the absence of T cell help. In contrast, a combination of deleting cells expressing receptors with high affinity for antigen with anergy of the undeleted lower affinity cells maintains tolerance to ubiquitous membrane-bound self-antigens.
Subject(s)
Autoantigens/immunology , B-Lymphocytes/immunology , Clonal Anergy/immunology , Clonal Deletion/immunology , Adoptive Transfer , Animals , Arthritis/immunology , Arthritis/metabolism , Autoantigens/metabolism , B-Lymphocytes/metabolism , Cell Membrane/immunology , Cell Membrane/metabolism , Clone Cells/immunology , Clone Cells/metabolism , Female , Flow Cytometry , Glycosylphosphatidylinositols/chemistry , Glycosylphosphatidylinositols/immunology , Glycosylphosphatidylinositols/metabolism , Lymphocyte Count , Male , Mice , Mice, 129 Strain , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Inbred NOD , Mice, Knockout , Ovalbumin/chemistry , Ovalbumin/immunology , Ovalbumin/metabolism , Protein Multimerization , Receptors, Antigen, B-Cell/immunology , Receptors, Antigen, B-Cell/metabolismABSTRACT
The TCR signals for the release of CD4 effector function are poorly understood. Itk plays an essential role in Th2, but not Th1, responses. However, when Itk is required during Th2 development is unclear. We followed the fate of Itk-deficient T cells during Th2 development in vitro and in vivo using an IL-4/GFP reporter. Surprisingly, a similar frequency of itk(-/-) CD4(+) cells differentiated and committed to the Th2 lineage as wild-type cells. However, Itk-deficient Th2 cells failed to exert effector function upon TCR triggering. Loss of function was marked by defective transcriptional enhancement of Th2 cytokines and GATA3. IL-4 production in itk(-/-) Th2s could be rescued by the expression of kinase-active Itk. Thus, Itk is necessary for the release, but not gain, of Th2 function. We suggest that the liberation of effector function is tightly controlled through qualitative changes in TCR signals, facilitating postdifferentiation regulation of cytokine responses.