Induced Human Regulatory T Cells Express the Glucagon-like Peptide-1 Receptor.

The glucagon-like peptide-1 receptor (GLP-1R) plays a key role in metabolism and is an important therapeutic target in diabetes and obesity. Recent studies in experimental animals have shown that certain subsets of T cells express functional GLP-1R, indicating an immune regulatory role of GLP-1. In contrast, less is known about the expression and function of the GLP-1R in human T cells. Here, we provide evidence that activated human T cells express GLP-1R. The expressed GLP-1R was functional, as stimulation with a GLP-1R agonist triggered an increase in intracellular cAMP, which was abrogated by a GLP-1R antagonist. Analysis of CD4+ T cells activated under T helper (Th) 1, Th2, Th17 and regulatory T (Treg) cell differentiation conditions indicated that GLP-1R expression was most pronounced in induced Treg (iTreg) cells. Through multimodal single-cell CITE- and TCR-sequencing, we detected GLP-1R expression in 29-34% of the FoxP3+CD25+CD127- iTreg cells. GLP-1R+ cells showed no difference in their TCR-gene usage nor CDR3 lengths. Finally, we demonstrated the presence of GLP-1R+CD4+ T cells in skin from patients with allergic contact dermatitis. Taken together, the present data demonstrate that T cell activation triggers the expression of functional GLP-1R in human CD4+ T cells. Given the high induction of GLP-1R in human iTreg cells, we hypothesize that GLP-1R+ iTreg cells play a key role in the anti-inflammatory effects ascribed to GLP-1R agonists in humans.

Impaired Vitamin D Signaling in T Cells From a Family With Hereditary Vitamin D Resistant Rickets.

The active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), mediates its immunomodulatory effects by binding to the vitamin D receptor (VDR). Here, we describe a new point mutation in the DNA-binding domain of the VDR and its consequences for 1,25(OH)2D3 signaling in T cells from heterozygous and homozygous carriers of the mutation. The mutation did not affect the overall structure or the ability of the VDR to bind 1,25(OH)2D3 and the retinoid X receptor. However, the subcellular localization of the VDR was strongly affected and the transcriptional activity was abolished by the mutation. In heterozygous carriers of the mutation, 1,25(OH)2D3-induced gene regulation was reduced by ~ 50% indicating that the expression level of wild-type VDR determines 1,25(OH)2D3 responsiveness in T cells. We show that vitamin D-mediated suppression of vitamin A-induced gene regulation depends on an intact ability of the VDR to bind DNA. Furthermore, we demonstrate that vitamin A inhibits 1,25(OH)2D3-induced translocation of the VDR to the nucleus and 1,25(OH)2D3-induced up-regulation of CYP24A1. Taken together, this study unravels novel aspects of vitamin D signaling and function of the VDR in human T cells.

Tumor necrosis factor induces rapid down-regulation of TXNIP in human T cells.

In addition to antigen-driven signals, T cells need co-stimulatory signals for robust activation. Several receptors, including members of the tumor necrosis factor receptor superfamily (TNFRSF), can deliver co-stimulatory signals to T cells. Thioredoxin interacting protein (TXNIP) is an important inhibitor of glucose uptake and cell proliferation, but it is unknown how TXNIP is regulated in T cells. The aim of this study was to determine expression levels and regulation of TXNIP in human T cells. We found that naïve T cells express high levels of TXNIP and that treatment of blood samples with TNF results in rapid down-regulation of TXNIP in the T cells. TNF-induced TXNIP down-regulation correlated with increased glucose uptake. Furthermore, we found that density gradient centrifugation (DGC) induced down-regulation of TXNIP. We demonstrate that DGC induced TNF production that paralleled the TXNIP down-regulation. Treatment of blood with toll-like receptor (TLR) ligands induced TNF production and TXNIP down-regulation, suggesting that damage-associated molecular patterns (DAMPs), such as endogenous TLR ligands, released during DGC play a role in DGC-induced TXNIP down-regulation. Finally, we demonstrate that TNF-induced TXNIP down-regulation is dependent on caspase activity and is caused by caspase-mediated cleavage of TXNIP.

Vitamin D Counteracts Mycobacterium tuberculosis-Induced Cathelicidin Downregulation in Dendritic Cells and Allows Th1 Differentiation and IFNγ Secretion.

Tuberculosis (TB) presents a serious health problem with approximately one-third of the world's population infected with Mycobacterium tuberculosis in a latent state. Experience from the pre-antibiotic era and more recent clinical studies have established a beneficial role of sunlight and vitamin D in patients with TB. At the same time, experimental data have shown that Th1 cells through production of IFNγ are crucial for cathelicidin release by macrophages, bacterial killing, and containment of M. tuberculosis in granulomas. Paradoxically, vitamin D has repeatedly been ascribed an immune-suppressive function inhibiting Th1 differentiation and production of IFNγ in T cells. The aim of this study was to investigate this apparent paradox. We studied naïve human CD4+ T cells activated either with CD3 and CD28 antibodies or with allogeneic dendritic cells (DC) stimulated with heat-killed M. tuberculosis (HKMT) or purified toll-like receptor (TLR) ligands. We show that vitamin D does not block differentiation of human CD4+ T cells to Th1 cells and that interleukin (IL)-12 partially counteracts vitamin D-mediated inhibition of IFNγ production promoting production of equal amounts of IFNγ in Th1 cells in the presence of vitamin D as in T cells activated in the absence of vitamin D and IL-12. Furthermore, we show that HKMT and TLR2 ligands strongly downregulate cathelicidin expression in DC and that vitamin D counteracts this by upregulating cathelicidin expression. In conclusion, we demonstrate that vitamin D counteracts M. tuberculosis-induced cathelicidin downregulation and allows Th1 differentiation and IFNγ secretion.

Human CD4+ T cells require exogenous cystine for glutathione and DNA synthesis.

Adaptive immune responses require activation and expansion of antigen-specific T cells. Whereas early T cell activation is independent of exogenous cystine (Cys2), T cell proliferation is dependent of Cys2. However, the exact roles of Cys2 in T cell proliferation still need to be determined. The aim of this study was to elucidate why activated human T cells require exogenous Cys2 in order to proliferate. We activated purified naïve human CD4+ T cells and found that glutathione (GSH) levels and DNA synthesis were dependent on Cys2 and increased in parallel with increasing concentrations of Cys2. Vice-versa, the GSH synthesis inhibitor L-buthionine-sulfoximine (BSO) and inhibition of Cys2 uptake with glutamate inhibited GSH and DNA synthesis in parallel. We further found that thioredoxin (Trx) can partly substitute for GSH during DNA synthesis. Finally, we show that GSH or Trx is required for the activity of ribonucleotide reductase (RNR), the enzyme responsible for generation of the deoxyribonucleotide DNA building blocks. In conclusion, we show that activated human T cells require exogenous Cys2 to proliferate and that this is partly explained by the fact that Cys2 is required for production of GSH, which in turn is required for optimal RNR-mediated deoxyribonucleotide synthesis and DNA replication.

Fine-tuning of T-cell development by the CD3γ di-leucine-based TCR-sorting motif.

The CD3γ di-leucine-based (diL) receptor-sorting motif plays a central role in TCR down-regulation and in clonal expansion of virus-specific T cells. However, the role of the CD3γ diL motif in T-cell development is not known. In this study, we show that protein kinase C-induced TCR down-regulation is abolished in thymocytes from CD3γLLAA mice with a mutated CD3γ diL motif, and that CD3γLLAA mice have reduced numbers of thymocytes compared with aged-matched wild-type mice. We found that early thymocyte development at the ß-selection checkpoint is impaired resulting in reduced numbers of double negative (DN) 4 cells in CD3γLLAA mice. This was not caused by reduced proliferation but most probably by increased down-regulation of the antiapoptotic molecule Bcl-2 causing enhanced apoptosis during the transition from the DN3 to the DN4 stage. In contrast, proliferation of immature CD8 single positive (ISP) thymocytes was increased resulting in normal numbers of ISP in CD3γLLAA mice. Despite the normal numbers of ISP, CD3γLLAA mice had reduced numbers of double positive and SP thymocytes indicating that the CD3γ diL motif also affected later stages of T-cell development. In accordance, we found that positive and negative selection, differentiation toward CD4 and CD8 SP T cells and the development of nonconventional T cells were affected in CD3γLLAA mice. In conclusion, our study identifies an important role of the CD3γ diL motif in T-cell development most probably mediated by its fine-tuning of pre-TCR and TCR expression, down-regulation and signaling.