Summary T lymphocytes regulate nutrient uptake to meet the metabolic demands of immune activation. The present study shows that the intracellular supply of large neutral amino acids (LNAAs) in T cells is regulated by pathogen and the T cell antigen receptor (TCR). A single System L transporter, Slc7a5, mediated LNAA uptake in activated T cells. Slc7a5-null T cells could not metabolically reprogram in response to antigen and failed clonal expansion and effector differentiation. The metabolic catastrophe caused by Slc7a5 loss reflects the requirement for sustained uptake of the LNAA leucine for activation of mammalian target of rapamycin complex 1 (mTORC1) and for expression of c-myc. Pathogen control of System L transporters is thus a critical metabolic checkpoint for T cells.

Reactive oxygen species have been implicated both in the ageing process and in degenerative diseases, including arthritis and cancer. Bacteria adapt to the lethal effects of oxidants such as hydrogen peroxide by inducing the expression of protective stress genes. Analogous responses have been identified in human cells. For example, haem oxygenase is a major stress protein in human cells treated with oxidants, and reactive oxygen intermediates activate NF-kappa B, a transcriptional regulator of genes involved in inflammatory and acute-phase responses. We report here the isolation and characterization of a novel complementary DNA (CL100) corresponding to a messenger RNA that is highly inducible by oxidative stress and heat shock in human skin cells. The cDNA contains an open reading frame specifying a protein of M(r) 39.3K with the structural features of a non-receptor-type protein-tyrosine phosphatase and which has significant amino-acid sequence similarity to a Tyr/Ser-protein phosphatase encoded by the late gene H1 of vaccinia virus. The purified protein encoded by the CL100 open reading frame expressed in bacteria has intrinsic phosphatase activity. Given the relationship between the levels of protein-tyrosine phosphorylation, receptor activity, cellular proliferation and cell-cycle control, the induction of this gene may play an important regulatory role in the human cellular response to environmental stress.

SummaryT 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.

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 PKD1S744A/S748A or PKD2S707A/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.

This study explores the regulation and importance of System L amino acid transport in a murine model of T-cell acute lymphoblastic leukemia (T-ALL) caused by deletion of phosphatase and tensin homolog deleted on chromosome 10 (PTEN). There has been a strong focus on glucose transport in leukemias but the present data show that primary T-ALL cells have increased transport of multiple nutrients. Specifically, increased leucine transport in T-ALL fuels mammalian target of rapamycin complex 1 (mTORC1) activity which then sustains expression of hypoxia inducible factor-1α (HIF1α) and c-Myc; drivers of glucose metabolism in T cells. A key finding is that PTEN deletion and phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) accumulation is insufficient to initiate leucine uptake, mTORC1 activity, HIF1α or c-Myc expression in T cells and hence cannot drive T-ALL metabolic reprogramming. Instead, a key regulator for leucine transport in T-ALL is identified as NOTCH. Mass spectrometry based proteomics identifies SLC7A5 as the predominant amino acid transporter in primary PTEN−/− T-ALL cells. Importantly, expression of SLC7A5 is critical for the malignant transformation induced by PTEN deletion. These data reveal the importance of regulated amino acid transport for T-cell malignancies, highlighting how a single amino acid transporter can have a key role.