Identification of self-lipids presented by CD1c and CD1d proteins

The CD1 family consists of five proteins that are related to the peptide-presenting MHC class I family. T cells can recognize the presentation of both foreign and self-derived lipids on four CD1 family members. The identities of the self-lipids capable of stimulating autoreactive T cell responses remain elusive or controversial. Here, we employed mass spectrometry to analyze the lipid content of highly purified CD1c and CD1d protein samples. We report the identification of 11 novel self-lipids presented by CD1c and nine by CD1d. Rigorous controls provide strong evidence that the identified lipids were specifically loaded into the lipid-binding site of the CD1 molecules. The diverse but distinct population of lipids identified from each CD1 family member implies each present a different subset of self-lipids, and the enrichment of particular motifs indicates that the lipids that are presented by CD1 family members could be predicted. Finally, our results imply the CD1 system surveys the endoplasmic reticulum, Golgi apparatus, and/or secretory compartments, in addition to its well characterized surveillance of the endocytic and lysosomal compartments.     

Effect of HIF1α on Foxp3 expression in CD4+CD25− T lymphocytes

The aim of the present study was to investigate the effect of HIF1α on Foxp3 expression in CD4⁺CD25^? T lymphocytes. CD4⁺CD25^? T lymphocytes were sorted from PBMC using a CD4⁺CD25⁺ regulatory T cell isolation kit. Lentivirus containing lentiviral vector that overexpressed HIF1α (HIF‐lenti) and those containing empty expression vector (control‐lenti) were produced. Meanwhile, lentivirus that contained lentiviral vector that suppressed HIF1α expression (siHIF‐lenti) and those containing control vector (sicontrol‐lenti) were also generated. The sorted CD4⁺CD25^? T lymphocytes were infected with HIF‐lenti, control‐lenti, siHIF‐lenti, and sicontrol‐lenti, respectively. Approximately 72 hr after transduction, real‐time PCR and Western blot were carried out to analyze the RNA and protein expression level of HIF1α and Foxp3. CD4⁺CD25^? T lymphocytes cultured under 21% O₂, 5% CO₂ (normoxia) and 1% O₂, 5% CO₂ (hypoxia) were used as control. Our results showed that overexpression of HIF1α increased both mRNA and protein expression of Foxp3 and, meanwhile, suppression of HIF1α expression by RNAi could reverse high Foxp3 expression in CD4⁺CD25^? T lymphocytes caused by hypoxic culture. These results suggested that hypoxia could stimulate Foxp3 expression by increasing HIF1α expression in CD4⁺ T lymphocytes which may promote CD4⁺ T lymphocytes to convert to Treg.

     

The bovine CD1 family contains group 1 CD1 proteins, but no functional CD1d

The CD1 family of proteins presents lipid Ags to T cells. Human CD1a, CD1b, and CD1c have been shown in humans to present mycobacterial lipid Ags. Cattle, like humans, are a natural host of several mycobacterial pathogens. In this study, we describe the CD1 family of genes in cattle (Bos taurus) and provide evidence that B. taurus expresses CD1a, CD1e, and multiple CD1b molecules, but no CD1c and CD1d molecules. In mice and humans, CD1d is known to present Ag to NKT cells, a T cell lineage that is characterized by a limited TCR repertoire, capable of rapidly secreting large amounts of IFN-gamma and IL-4. In cattle, two CD1D pseudogenes were found and no intact CD1D genes. Consistent with this, we found complete lack of reactivity to a potent, cross-reactive Ag for NKT cells in mice and humans, alpha-galactosylceramide. Our data suggest the absence of NKT cells in cattle. It remains open whether other cells with the NKT-like phenotype and functions are present in this species. With its functional CD1A and CD1B genes, B. taurus is well equipped to present Ags to CD1-restricted T cells other than NKT cells. Cattle can be used as a model to study group 1 CD1-restricted T cell immunity, including its role in the defense against mycobacterial infections that occur naturally in this species.     

Tissue-specific segregation of CD1d-dependent and CD1d-independent NK T cells.

NKT cells, defined as T cells expressing the NK cell marker NK1.1, are involved in tumor rejection and regulation of autoimmunity via the production of cytokines. We show in this study that two types of NKT cells can be defined on the basis of their reactivity to the monomorphic MHC class I-like molecule CD1d. One type of NKT cell is positively selected by CD1d and expresses a biased TCR repertoire together with a phenotype found on activated T cells. A second type of NKT cell, in contrast, develops in the absence of CD1d, and expresses a diverse TCR repertoire and a phenotype found on naive T cells and NK cells. Importantly, the two types of NKT cells segregate in distinct tissues. Whereas thymus and liver contain primarily CD1d-dependent NKT cells, spleen and bone marrow are enriched in CD1d-independent NKT cells. Collectively, our data suggest that recognition of tissue-specific ligands by the TCR controls localization and activation of NKT cells.     

CD1 and CD1-restricted T cells in infections with intracellular bacteria

Glycolipid-specific, CD1a-, b- and c-dependent cytotoxic T cells have recently been shown to be involved in the host response against tuberculosis. These CD1 molecules 'sample' mycobacterial glycolipids from different intracellular sites in the infected cell. Additionally, upon microbial encounter, CD1d-dependent natural killer T cells promptly produce cytokines and perform regulatory activities. Here, we discuss the intracellular localization of CD1 molecules and mycobacterial lipids and the role of CD1-mediated T-cell responses in mycobacterial infections.     

Glycosyl-phosphatidylinositol reanchoring unmasks distinct antigen-presenting pathways for CD1b and CD1c

Human CD1 proteins present lipid and glycolipid Ags to T cells. Cellular trafficking patterns of CD1 proteins may determine the ability of differing isoforms of CD1 to acquire, bind, and present these Ags to T cells. To test this hypothesis, glycosyl-phosphatidylinositol (GPI)-modified variants of CD1b and CD1c were engineered by chimerization with a GPI modification signal sequence derived from decay-accelerating factor (DAF). GPI reanchoring was confirmed by demonstrating the phosphatidylinositol-specific phospholipase C sensitivity of the CD1b. DAF and CD1c. DAF fusion proteins expressed on transfectant cell surfaces. Using cytotoxicity and cytokine release assays as functional readouts, we demonstrated that CD1c. DAF is as efficient as native CD1c in presenting mycobacterial Ags to the human CD1c-restricted T cell line CD8-1. In contrast, CD1b. DAF, although also capable of presenting Ag (in this case to the CD1b-restricted T cell line LDN5), was less efficient than its native CD1b counterpart. The data support the idea that CD1c. DAF maintains the capacity to access CD1c Ag-loading compartment(s), whereas CD1b. DAF is diverted by its GPI anchor away from the optimal CD1b Ag-loading compartment(s). This constitutes the first GPI reanchoring of CD1 proteins and provides evidence that CD1b and CD1c have nonoverlapping Ag-presenting pathways, suggesting that these two Ag-presenting molecules may have distinct roles in lipid Ag presentation.     

Amino-terminal sequencing of sheep CD1 antigens and identification of a sheep CD1D gene

 The anti-CD1 monoclonal antibodies IAH-CC14 and SBU-T6 were used to immunopurify CD1 antigens from sheep thymocytes. The amino-terminal sequence of IAH-CC14 yielded 13 amino acids, and 29 amino acids were obtained from the SBU-T6 antigen. The sequence of the IAH-CC14 antigen was 100% identical to the predicted sequence of the sheep CD1B clone, SCD1B-42. The 29 amino acid sequence of the SBU-T6 antigen did not match identically with the derived amino acid sequence of any of the previously reported sheep CD1 genes but had closest similarity to the derived sequence of human CD1E. Degenerate polymerase chain reaction primers based on this sequence identified a group 2 sheep CD1 gene. The predicted amino acid sequence of this gene shows that it is not identical to the SBU-T6 peptide, indicating that a different, CD1D-like gene was cloned. Received: 22 June 1998 / Revised: 16 September 1998     

FOXO1 constrains activation and regulates senescence in CD8 T cells

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GOT1 regulates CD8+ effector and memory T cell generation

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CD1 expression and CD1-restricted T cell activity in normal and tumour-bearing human liver

CD1d-restricted natural killer T (NKT) cells expressing invariant Vα14Jα18 T cell receptor α-chains are abundant in murine liver and are implicated in the control of malignancy, infection and autoimmunity. Invariant NKT cells have potent anti-metastatic effects in mice and phase I clinical trials involving their homologues in humans are ongoing. However, invariant NKT cells are less abundant in human liver (∼0.5% of hepatic T cells) than in murine liver (up to 50%) and it is not known if other hepatic T cells are CD1-restricted. We have examined expression of CD1a, CD1b, CD1c and CD1d mRNA and protein in human liver and evaluated the reactivity of mononuclear cells (MNC) from histologically normal and tumour-bearing human liver specimens against these CD1 isoforms. Messenger RNA for all CD1 isotypes was detectable in all liver samples. CD1c and CD1d were expressed at the protein level by hepatic MNC. CD1d, only, was detectable at the cell surface, but CD1c and CD1d were found at an intracellular location in significant numbers of liver MNC. CD1b was not expressed by MNC from healthy livers but was detectable within MNC in all tumour samples tested. Hepatic T cells exhibited reactivity against C1R cells expressing transfected CD1c and CD1d, but neither CD1a nor CD1b. These cells secreted interferon-γ (IFN-γ) but not interleukin-4 (IL-4) upon stimulation. In contrast, similar numbers of peripheral T cells released 13- and 16-fold less IFN-γ in response to CD1c and CD1d, respectively. CD1c and CD1d expression and T cell reactivity were not altered in tumour-bearing liver specimens compared to histologically normal livers. These data suggest that, in addition to invariant CD1d-restricted NKT cells, autoreactive T cells that recognise CD1c and CD1d and release inflammatory cytokines are abundant in human liver.     

CD1d‐mediated lipid presentation by CD11c+ cells regulates intestinal homeostasis

Intestinal homeostasis relies on a continuous dialogue between the commensal bacteria and the immune system. Natural killer T (NKT) cells, which recognize CD1d‐restricted microbial lipids and self‐lipids, contribute to the regulation of mucosal immunity, yet the mechanisms underlying their functions remain poorly understood. Here, we demonstrate that NKT cells respond to intestinal lipids and CD11c⁺ cells (including dendritic cells (DCs) and macrophages) are essential to mediate lipid presentation within the gut ultimately controlling intestinal NKT cell homeostasis and activation. Conversely, CD1d and NKT cells participate in the control of the intestinal bacteria composition and compartmentalization, in the regulation of the IgA repertoire and in the induction of regulatory T cells within the gut. These changes in intestinal homeostasis require CD1d expression on DC/macrophage populations as mice with conditional deletion of CD1d on CD11c⁺ cells exhibit dysbiosis and altered immune homeostasis. These results unveil the importance of CD11c⁺ cells in controlling lipid‐dependent immunity in the intestinal compartment and reveal an NKT cell–DC crosstalk as a key mechanism for the regulation of gut homeostasis.     

Contrasting effect of transforming growth factor type beta 1 (TGF-β1) on proliferation and interleukin-2 receptor expression in activated and rapidly cycling immature (CD3−CD4−CD8−) thymocytes

Transforming growth factor β (TGF-β) is a cytokine with immunoregulatory properties that acts negatively on T lymphocyte proliferation. However, with the EL 4–6.1 variant of the murine thymoma EL 4 activated with phorbol ester and/or interleukin-1 (IL-1), we recently found that it up-regulates interleukin-2-receptor (IL-2R) expression. Since EL 4–6.1 cells share phenotypic and functional characteristics with the immature thymic subset lacking CD4 and CD8 accessory molecules (DN), we investigated the effect of TGF-β1 on the IL-2R 55kD α chain expression and proliferation of activated DN cells and especially in DN cells that do not express CD3. We observed that TGF-β1 was able to increase both the percentage of CD3^?DN cells expressing IL-2Rα chains and the expression of IL-2Rα chain in these cells. This stimulatory effect of TGF-β1 was distal from early transduction events. In addition, TGF-β1 was found to modulate CD3^?DN cell proliferation. During differentiation in the thymus, CD3^?DN cells transiently express the IL-2Rα chain of the IL-2R and these IL-2R⁺ CD3^?DN cells are preprogrammed to down-regulate the IL-2Rα chain and up-regulate the CD4 and CD8 accessory molecule. We thus also tested the effect of TGF-β1 on IL-2Rα chain expression in these in vitro differentiating CD3^?DN cells. We found that TGF-β1 neither significantly affected IL-2R expression nor changed CD4 or CD8 expression. Hence, in CD3^?DN cells, the effect of TGF-β1 on IL-2R expression seems to be restricted to proliferating cells. © 1993 Wiley-Liss, Inc.     

Bin1 and CD2AP polarise the endocytic generation of beta‐amyloid

The mechanisms driving pathological beta‐amyloid (Aβ) generation in late‐onset Alzheimer's disease (AD) are unclear. Two late‐onset AD risk factors, Bin1 and CD2AP, are regulators of endocytic trafficking, but it is unclear how their endocytic function regulates Aβ generation in neurons. We identify a novel neuron‐specific polarisation of Aβ generation controlled by Bin1 and CD2AP. We discover that Bin1 and CD2AP control Aβ generation in axonal and dendritic early endosomes, respectively. Both Bin1 loss of function and CD2AP loss of function raise Aβ generation by increasing APP and BACE1 convergence in early endosomes, however via distinct sorting events. When Bin1 levels are reduced, BACE1 is trapped in tubules of early endosomes and fails to recycle in axons. When CD2AP levels are reduced, APP is trapped at the limiting membrane of early endosomes and fails to be sorted for degradation in dendrites. Hence, Bin1 and CD2AP keep APP and BACE1 apart in early endosomes by distinct mechanisms in axon and dendrites. Individuals carrying variants of either factor would slowly accumulate Aβ in neurons increasing the risk for late‐onset AD.     

Prominin‐1 (CD133) modulates the architecture and dynamics of microvilli

Prominin‐1 is a cell surface biomarker that allows the identification of stem and cancer stem cells from different organs. It is also expressed in several differentiated epithelial and non‐epithelial cells. Irrespective of the cell type, prominin‐1 is associated with plasma membrane protrusions. Here, we investigate its impact on the architecture of membrane protrusions using microvilli of Madin‐Darby canine kidney cells as the main model. Our high‐resolution analysis revealed that upon the overexpression of prominin‐1 the number of microvilli and clusters of them increased. Microvilli with branched and/or knob‐like morphologies were observed and stimulated by mutations in the ganglioside‐binding site of prominin‐1. The altered phenotypes were caused by the interaction of prominin‐1 with phosphoinositide 3‐kinase and Arp2/3 complex. Mutation of tyrosine 828 of prominin‐1 impaired its phosphorylation and thereby inhibited the aforementioned interactions abolishing altered microvilli. This suggests that the interplay of prominin‐1‐ganglioside membrane complexes, phosphoinositide 3‐kinase and cytoskeleton components regulates microvillar architecture. Lastly, the expression of prominin‐1 and its mutants modified the structure of filopodia emerging from fibroblast‐like cells and silencing human prominin‐1 in primary hematopoietic stem cells resulted in the loss of uropod‐associated microvilli. Altogether, these findings strengthen the role of prominin‐1 as an organizer of cellular protrusions.      

CD1a and CD1c activate intrathyroidal T cells during Graves' disease and Hashimoto's thyroiditis

Molecular studies have shown that CD1 proteins present self and foreign lipid Ags to T cells, but the possible roles of CD1 in human autoimmune diseases in vivo are not known, especially for the group 1 CD1 isoforms (CD1a, CD1b, and CD1c). To investigate the hypothesis that CD1-restricted T cells might be activated and home to target tissues involved in Hashimoto's thyroiditis and Graves' disease, we performed ex vivo analysis of lymphocytes from peripheral blood and autoinflammatory lesions of thyroid tissue. Immunofluorescence analysis identified two types of CD1-expressing APCs in inflamed thyroid tissues. CD1a, CD1b, and CD1c were expressed on CD83+ dendritic cells, and CD1c was expressed on an abundant population of CD20+ IgD+ CD23- CD38- B cells that selectively localized to the mantle zone of lymphoid follicles within the thyroid gland. CD1c-restricted, glycolipid-specific T cells could not be detected in the peripheral blood, but were present in polyclonal lymphocyte populations isolated from affected thyroid glands. In addition, polyclonal thyroid-derived lymphocytes and short-term T cell lines were found to recognize and lyse targets in a CD1a- or CD1c-dependent manner. The targeting of CD1-restricted T cells and large numbers of CD1-expressing APCs to the thyroid gland during the early stages of autoimmune thyroiditis suggests a possible effector function of CD1-restricted T cells in tissue destruction and point to a new model of organ-specific autoimmune disease involving lipid Ag presentation.     

TL1A Increases Expression of CD25, LFA-1, CD134 and CD154, and Induces IL-22 and GM-CSF Production from Effector CD4 T-Cells

Elevated levels of the cytokine TL1A is associated with several autoimmune diseases e.g. rheumatoid arthritis and inflammatory bowel disease. However, the exact role of TL1A remains elusive. In this study, we investigated the function of TL1A in a pro-inflammatory setting. We show that TL1A together with IL-12, IL-15 and IL-18 increases expression of the co-stimulatory molecules CD154 (CD40 ligand) and CD134 (OX40) on previously activated CD4⁺ T cells. This indicates that TL1A functions as a co-stimulatory molecule, decreasing the activation threshold of T-cells. We have previously shown that TL1A co-stimulation strongly induces IL-6 in human healthy leukocytes. Interestingly, the cytokine-activated effector T-cells did not produce IL-6 in response to TL1A, indicating distinct effects of TL1A on different cell populations. We further show that this co-stimulation increases the expression of CD25 (IL-2Rα) and CD11a (α-chain of LFA-1) on CD4 T-cells, likely governing increased IL-2/IL-15 sensitivity and cell-cell contact. Along with this, TL1A co-stimulation caused a specific induction of IL-22 and GM-CSF from the activated T-cells. These results substantially contribute to the explanation of TL1A''s role in inflammation. Our results suggest that TL1A should be considered as a target for immunotherapeutic treatment of rheumatoid arthritis and inflammatory bowel disease.