ABSTRACT
T lymphocytes must regulate nutrient uptake to meet the metabolic demands of an immune response. Here we show that the intracellular supply of large neutral amino acids (LNAAs) in T cells was regulated by pathogens and the T cell antigen receptor (TCR). T cells responded to antigen by upregulating expression of many amino-acid transporters, but a single System L ('leucine-preferring system') transporter, Slc7a5, mediated uptake of LNAAs in activated T cells. Slc7a5-null T cells were unable to metabolically reprogram in response to antigen and did not undergo clonal expansion or effector differentiation. The metabolic catastrophe caused by loss of Slc7a5 reflected the requirement for sustained uptake of the LNAA leucine for activation of the serine-threonine kinase complex mTORC1 and for expression of the transcription factor c-Myc. Control of expression of the System L transporter by pathogens is thus a critical metabolic checkpoint for T cells.
Subject(s)
Amino Acids, Neutral/metabolism , Large Neutral Amino Acid-Transporter 1/metabolism , T-Lymphocytes, Cytotoxic/immunology , Animals , Cell Differentiation/genetics , Cell Proliferation , Cytotoxicity, Immunologic , Interferon-gamma/metabolism , Interleukin-2/metabolism , Large Neutral Amino Acid-Transporter 1/genetics , Mice , Mice, Inbred C57BL , Mice, Mutant Strains , Protein Transport , Receptors, Antigen, T-Cell, alpha-beta/metabolism , Up-RegulationABSTRACT
T follicular helper (Tfh) cells are essential in the induction of high-affinity, class-switched antibodies. The differentiation of Tfh cells is a multi-step process that depends upon the co-receptor ICOS and the activation of phosphoinositide-3 kinase leading to the expression of key Tfh cell genes. We report that ICOS signaling inactivates the transcription factor FOXO1, and a Foxo1 genetic deletion allowed for generation of Tfh cells with reduced dependence on ICOS ligand. Conversely, enforced nuclear localization of FOXO1 inhibited Tfh cell development even though ICOS was overexpressed. FOXO1 regulated Tfh cell differentiation through a broad program of gene expression exemplified by its negative regulation of Bcl6. Final differentiation to germinal center Tfh cells (GC-Tfh) was instead FOXO1 dependent as the Foxo1(-/-) GC-Tfh cell population was substantially reduced. We propose that ICOS signaling transiently inactivates FOXO1 to initiate a Tfh cell contingency that is completed in a FOXO1-dependent manner.
Subject(s)
Cell Differentiation/immunology , DNA-Binding Proteins/biosynthesis , Forkhead Transcription Factors/genetics , Inducible T-Cell Co-Stimulator Protein/immunology , T-Lymphocytes, Helper-Inducer/cytology , Animals , Enzyme Activation , Forkhead Box Protein O1 , Forkhead Transcription Factors/immunology , Gene Expression Regulation , Lymphocyte Activation/immunology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-bcl-6 , Signal Transduction , T-Lymphocytes, Helper-Inducer/immunologyABSTRACT
Mammalian PKD (protein kinase D) isoforms have been implicated in the regulation of diverse biological processes in response to diacylglycerol and PKC (protein kinase C) signalling. To compare the functions of PKD1 and PKD2 in vivo, we generated mice deficient in either PKD1 or PKD2 enzymatic activity, via homozygous expression of PKD1(S744A/S748A) or PKD2(S707A/S711A) 'knockin' alleles. We also examined PKD2-deficient mice generated using 'gene-trap' technology. We demonstrate that, unlike PKD1, PKD2 catalytic activity is dispensable for normal embryogenesis. We also show that PKD2 is the major PKD isoform expressed in lymphoid tissues, but that PKD2 catalytic activity is not essential for the development of mature peripheral T- and B-lymphocytes. PKD2 catalytic activity is, however, required for efficient antigen receptor-induced cytokine production in T-lymphocytes and for optimal T-cell-dependent antibody responses in vivo. Our results reveal a key in vivo role for PKD2 in regulating the function of mature peripheral lymphocytes during adaptive immune responses. They also confirm the functional importance of PKC-mediated serine phosphorylation of the PKD catalytic domain for PKD activation and downstream signalling and reveal that different PKD family members have unique and non-redundant roles in vivo.
Subject(s)
Lymphocytes/metabolism , Lymphoid Tissue/metabolism , Protein Kinase C/metabolism , Protein Kinases/metabolism , Animals , Blotting, Western , CD4-Positive T-Lymphocytes/cytology , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Cell Proliferation , Cells, Cultured , Cytokines/immunology , Cytokines/metabolism , Embryonic Development/genetics , Female , Flow Cytometry , Gene Expression Profiling , Gene Expression Regulation, Developmental , Lymphocytes/cytology , Lymphocytes/immunology , Lymphoid Tissue/cytology , Lymphoid Tissue/immunology , Male , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Knockout , Phosphorylation , Protein Kinase C/genetics , Protein Kinase D2 , Protein Kinases/genetics , Receptors, Antigen, T-Cell/immunology , Receptors, Antigen, T-Cell/metabolismABSTRACT
The genetic manipulation of donor cells before nuclear transfer (NT) enables prior selection for transgene integration. However, selection for genetically modified cells using antibiotic drugs often results in mixed populations, resulting in a mixture of transgenic and nontransgenic donor cells for NT. In this study, we attempted to develop efficient strategies for the generation of human bile salt-stimulated lipase (BSSL) transgenic cows. Preimplantation screening by either biopsy or green fluorescent protein (GFP) expression was used to detect NT-derived BSSL transgenic embryos to ensure that the calf born would be transgenic. We compared the development rates of NT-derived embryos from G418- and GFP-selected donor cells. There were no significant differences (P < 0.001) in cleavage rate (67.2% vs. 60.0%) and blastocyst formation rate (44.9% vs. 41.2%). We also compared the pregnancy rates of the G418/biopsy and GFP preimplantation screened NT-derived blastocysts. The Day 40 pregnancy rate of the G418/biopsy group (40%) was lower than that of the GFP group (57%), but the calf birth rate of the G418/biopsy group (40%) was higher than that of the GFP group (21%). Healthy BSSL transgenic calves were born after both screening processes. This is the first report of biopsy-screened cloned transgenic animals. The results suggest that both selection methods are useful for detecting transgenic NT embryos without negatively affecting their development into viable transgenic offspring.