ABSTRACT
Innate lymphoid cells (ILCs) are tissue-resident lymphoid cells that reside mostly at barrier surfaces and participate in the initial response against pathogens. They are classified into different types based on effector programs that are based on cytokine production and transcription factor expression. They all derive from the common lymphoid precursor, but the molecular mechanisms regulating ILC subset development is not well understood. Experiments using Id2 knockout mice have previously shown that E protein activity inhibition is an absolute requirement for the development of all ILC subsets. In this study, we use a genetic approach to demonstrate that small increases in E protein activity during ILC development selectively inhibit type 2 ILC development. Type 1 ILCs are mostly unperturbed, and type 3 ILC show only a minor inhibition. This effect is first evident at the ILC2 progenitor stage and is ILC intrinsic. Therefore, our results demonstrate that modulation of E protein activity can bias cell fate decisions in developing ILCs.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Immunity, Innate/immunology , Natural Killer T-Cells/immunology , Transcription Factor 4/metabolism , Animals , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Differentiation/immunology , Female , Gene Expression Regulation/immunology , Gene Knock-In Techniques , Lymphoid Progenitor Cells/metabolism , Male , Mice , Mice, TransgenicABSTRACT
Transduction of lymphoid progenitors with retroviral or lentiviral vectors is a powerful experimental strategy to tease out the role of a gene or pathway in T cell development via gain-of-function or loss-of-function strategies. Here we discuss different approaches to use this powerful technology, and present some protocols that we use to transduce murine HSCs, thymocytes, and lymphoid cell lines with these viral vectors.
Subject(s)
Genetic Vectors/genetics , Precursor Cells, T-Lymphoid/metabolism , Retroviridae/genetics , T-Lymphocytes/metabolism , Transduction, Genetic , Animals , Cell Line , Humans , MiceABSTRACT
E protein transcription factors and their natural inhibitors, Id proteins, play critical and complex roles during lymphoid development. In this article, we report that partial maintenance of E protein activity during positive selection results in a change in the cell fate determination of developing iNKT cells, with a block in the development of iNKT1 cells and a parallel increase in the iNKT2 and iNKT17 subsets. Because the expression levels of the transcription factors that drive these alternative functional fates (GATA-3, RORγT, T-bet, and Runx-3) are not altered, our results suggest that E protein activity controls a novel checkpoint that regulates the number of iNKT precursors that choose each fate.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Cell Differentiation/immunology , Lymphocyte Activation/immunology , Natural Killer T-Cells/metabolism , Animals , Cell Proliferation , Cells, Cultured , Core Binding Factor Alpha 3 Subunit/biosynthesis , Interferon-gamma/biosynthesis , Interleukin-2 Receptor beta Subunit/biosynthesis , Lymphocyte Subsets/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , RNA, Untranslated/genetics , Signal Transduction/immunology , T-Box Domain Proteins/biosynthesisABSTRACT
iNKT cells derive from CD4(+)CD8(+) DP thymocytes, and are selected by thymocyte-thymocyte interactions through signals from their invariant Vα14-Jα18 TCR and from the costimulatory molecules SLAMF1 and SLAMF6. Genetic studies have demonstrated the contribution of different signaling pathways to this process. Surprisingly, current models imply that the Ras/MAPK pathway, one of the critical mediators of conventional αß T cell positive selection, is not necessary for iNKT cell development. Using mice defective at different levels of this pathway our results refute this paradigm, and demonstrate that Ras, and its downstream effectors Egr-1 and Egr-2 are required for positive selection of iNKT cells. Interestingly our results also show that there are differences in the contributions of several of these molecules to the development of iNKT and conventional αß T cells.
Subject(s)
MAP Kinase Signaling System , Natural Killer T-Cells/cytology , Natural Killer T-Cells/enzymology , ras Proteins/metabolism , Animals , Antigens, CD/metabolism , Antigens, CD1d/metabolism , Early Growth Response Protein 1/metabolism , Early Growth Response Protein 2/metabolism , Integrases/metabolism , Mice , Mice, Knockout , Proto-Oncogene Proteins c-bcl-2/metabolism , Receptors, Cell Surface/metabolism , Signaling Lymphocytic Activation Molecule Family , Signaling Lymphocytic Activation Molecule Family Member 1 , Thymus Gland/cytologyABSTRACT
GATA-3 is necessary for the development of MHC class II-restricted CD4 T cells, and its expression is increased during positive selection of these cells. TCR signals drive this upregulation, but the signaling pathways that control this process are not well understood. Using genetic and pharmacological approaches, we show that GATA-3 upregulation during thymocyte-positive selection is the result of additive inputs from the Ras/MAPK and calcineurin pathways. This upregulation requires the presence of the transcription factor c-Myb. Furthermore, we show that TH-POK can also upregulate GATA-3 in double-positive thymocytes, suggesting the existence of a positive feedback loop that contributes to lock in the initial commitment to the CD4 lineage during differentiation.
Subject(s)
CD4-Positive T-Lymphocytes/cytology , CD4-Positive T-Lymphocytes/immunology , Cell Differentiation/immunology , Cell Lineage/immunology , GATA3 Transcription Factor/metabolism , Animals , CD4-Positive T-Lymphocytes/metabolism , Calcineurin/physiology , Cell Differentiation/genetics , Cell Lineage/genetics , DNA-Binding Proteins/physiology , GATA3 Transcription Factor/biosynthesis , GATA3 Transcription Factor/genetics , Gene Expression Regulation/immunology , Gene Knock-In Techniques , HEK293 Cells , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Proto-Oncogene Proteins c-myb/physiology , Signal Transduction/genetics , Signal Transduction/immunology , Transcription Factors/physiology , ras Proteins/physiologyABSTRACT
Type I invariant NKT cells (iNKT cells) are a subset of alphabeta T cells characterized by the expression of an invariant alpha-chain variable region 14-alpha-chain joining region 18 (V(alpha)14J(alpha)18) T cell antigen receptor (TCR) alpha-chain. The iNKT cells derive from CD4(+)CD8(+) double-positive (DP) thymocytes, and their generation requires a long half-life of DP thymocytes to allow V(alpha)14-J(alpha)18 rearrangements, expression of glycolipid-loaded CD1d on DP thymocytes, and signaling through the signaling-activation molecule SLAM-adaptor SAP pathway. Here we show that the transcription factor c-Myb has a central role in priming DP thymocytes to enter the iNKT lineage by simultaneously regulating CD1d expression, the half-life of DP cells and expression of SLAMF1, SLAMF6 and SAP.
Subject(s)
Antigens, CD1d/metabolism , Natural Killer T-Cells/metabolism , Precursor Cells, T-Lymphoid/metabolism , Proto-Oncogene Proteins c-myb/metabolism , bcl-X Protein/metabolism , Animals , Antigens, CD/genetics , Antigens, CD/metabolism , Antigens, CD1d/genetics , Antigens, CD1d/immunology , Bone Marrow Transplantation , CD4 Antigens/biosynthesis , CD8 Antigens/biosynthesis , Cell Differentiation/genetics , Cell Differentiation/immunology , Cell Lineage/genetics , Cell Lineage/immunology , Cell Survival/genetics , Cell Survival/immunology , GATA3 Transcription Factor/genetics , Gene Rearrangement, T-Lymphocyte/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Natural Killer T-Cells/cytology , Natural Killer T-Cells/immunology , Precursor Cells, T-Lymphoid/cytology , Precursor Cells, T-Lymphoid/immunology , Proto-Oncogene Proteins c-myb/genetics , Proto-Oncogene Proteins c-myb/immunology , Radiation Chimera , Receptors, Cell Surface/genetics , Receptors, Cell Surface/metabolism , Saposins/genetics , Saposins/metabolism , Signal Transduction/genetics , Signal Transduction/immunology , Signaling Lymphocytic Activation Molecule Family , Signaling Lymphocytic Activation Molecule Family Member 1 , Thymus Gland/cytology , bcl-X Protein/genetics , bcl-X Protein/immunologyABSTRACT
During inflammation, elevated granulocyte macrophage-colony-stimulating factor (GM-CSF) directs the development of new dendritic cells (DCs). This pathway is influenced by environmental factors, and we previously showed that physiologic levels of estradiol, acting through estrogen receptor alpha (ERalpha), promote the GM-CSF-mediated differentiation of a CD11b(+) DC subset from myeloid progenitors (MPs). We now have identified interferon regulatory factor 4 (IRF4), a transcription factor induced by GM-CSF and critical for CD11b(+) DC development in vivo, as a target of ERalpha signaling during this process. In MPs, ERalpha potentiates and sustains GM-CSF induction of IRF4. Furthermore, retroviral delivery of the Irf4 cDNA to undifferentiated ERalpha(-/-) bone marrow cells restored the development of the estradiol/ERalpha-dependent DC population, indicating that an elevated amount of IRF4 protein substitutes for ERalpha signaling. Thus at an early stage in the MP response to GM-CSF, ERalpha signaling induces an elevated amount of IRF4, which leads to a developmental program underlying CD11b(+) DC differentiation.
