Molecular characterisation of Ovine CD1

The CD1 molecules are a family of ß2microglobulin- associated glycoproteins with strong structural homology, but weaker sequence homology, to the MHC class I antigens. In contrast to the classical class I antigens, CD1 molecules exhibit restricted tissue expression (cortical thymocytes, dendritic cells, a subset of B cells and some intestinal epithelial cells), and are nonpolymorphic. Five CD1 genes have been identified in humans, two in the mouse and several in other mammalian species (Calabi et al, 1991). CD1 expression has also been detected by immunohistological techniques in the cow, sheep and pig.The MHC class I -like structure of CD1 and the expression on classical antigen presenting cells of the immune system has pointed to a role for CD1 in antigen presentation. Indeed, evidence has been accumulating over the past few years to support this view, with several reports suggesting that CD4 -8- T cells in particular may be able to recognise nonclassical presentational elements including MHC class lb molecules such as TLa and Qa, as well as CD1. Most recently, CD1b molecules on human monocytes have been demonstrated to restrict the response of CD4 -8- T cells to antigens derived from M. tuberculosis (Porcelli et al, 1992).Previous studies on the ovine CD1 family have involved the use of monoclonal antibodies to assess tissue expression and distribution, and biochemical analyses of the ovine CD1 antigens. However, no studies have been carried out to investigate ovine CD1 at the molecular level. Therefore, a human CD1 C a3 probe was used to screen several sheep thymocyte cDNA libraries. The HCD1 B -like clone SCD1 A25 was isolated from a foetal thymocyte library. A homologous probe comprising the a3/TM /CYT domains from this clone was derived by PCR amplification and used to identify a further three ovine clones - SCD1 B -42, SCD1 B -52 and SCD1T10. Three of the four clones are truncated at the 5' end, with sequences beginning towards the end of the al domain or the start of the a2 domain. These 5' truncation events probably reflect poor reverse transcriptase activity during library preparation. The fourth clone, SCD1 B -52, represents a transcript containing a precise a3 deletion. The PCR technique was used to amplify the missing 5' ends from two of the three truncated$equences, thus generating full length coding sequence for two of the four ovine CD1's identified.Comparison of the ovine CD1 sequences amongst themselves has shown them to be 81 -96% identical at the nucleotide level and 79 -90% identical at the amino acid level, suggesting that the four clones represent different gene products rather than allelic variants of CD1. The sheep sequences have also been analysed by comparison to the human, mouse and rabbit coding seqences. Perhaps unexpectedly, given the existence of five different human CD1 genes, all of the ovine CD1 sequences are most homologous to human CD1 B at both the nucleotide and amino acid levels. The sheep CD1 sequences also show a high percentage sequence identity to the cottontail rabbit sequence, which is itself most similar to HCD1 B.Southern blot analysis of genomic DNA digested with a variety of enzymes and probed with the homologous a3 probe has indicated the possible existence of up to seven ovine CD1 genes. Further studies are required to determine which of these genes are expressed and to identify the genes encoding the CD1 molecules recognised by the monoclonal antibodies. The significance and implications of these results are discussed and potential further experiments suggested

Human natural killer cell committed thymocytes and their relation to the T cell lineage.

Recent studies have demonstrated that mature natural killer (NK) cells can be grown from human triple negative (TN; CD3-, CD4-, CD8-) thymocytes, suggesting that a common NK/T cell precursor exists within the thymus that can give rise to both NK cells and T cells under appropriate conditions. In the present study, we have investigated human fetal and postnatal thymus to determine whether NK cells and their precursors exist within this tissue and whether NK cells can be distinguished from T cell progenitors. Based on the surface expression of CD56 (an NK cell-associated antigen) and CD5 (a T cell-associated antigen), three phenotypically distinctive populations of TN thymocytes were identified. CD56+, CD5-; CD56-, CD5-, and CD56-, CD5+. The CD56+, CD5- population of TN thymocytes, although displaying a low cytolytic function against NK sensitive tumor cell targets, were similar in antigenic phenotype to fetal liver NK cells, gave rise to NK cell clones, and were unable to generate T cells in mouse fetal thymic organ cultures (mFTOC). This population of thymocytes represents a relatively mature population of lineage-committed NK cells. The CD56-, CD5- population of TN thymocytes were similar to thymic NK cells in antigenic phenotype and NK cell clonogenic potential. Clones derived from this population of TN thymocytes acquired CD56 surface expression and NK cell cytolytic function. CD56-, CD5- TN thymocytes thus contain a novel population of NK cell-committed precursors. The CD56-, CD5- population of TN thymocytes also contains a small percentage of CD34+ cells, which demonstrate no in vitro clonogenic potential, but possess T cell reconstituting capabilities in mFTOC. The majority of TN thymocytes do not express CD56, but coexpress CD34 and CD5. These CD56-, CD5+, CD34+ cells demonstrate no NK or T cell clonogenic potential, but are extremely efficient in repopulating mFTOC and differentiating into CD3+, CD4+, CD8+ T cells. The results of this investigation have identified NK cells and NK cell precursors in the human thymus and have shown that these cell types are unable to differentiate along the T cell lineage pathway. Thus, while a common NK/T cell progenitor likely exists, once committed to the NK cell lineage these cells no longer have the capacity to develop along the T cell developmental pathway

Genetic Regulation of NKT Cell Function

NKT cells are specialized T cells that play important roles in the host immune response to bacteria and viruses. NKT cells produce a wide variety of cytokines and chemokines after being activated by glycolipids such as α-galactosylceramide (αGalCer). Previous work suggested that the ability of NKT cells to be activated by aGalCer mapped to a genetic region encompassing a gene family (Slam genes) that is known to be important in NKT cell development, but the exact gene in this region which regulates NKT cells is unknown. This study utilizes a panel of C57BL/6 (B6) mice containing different regions of chromosome 1 derived from 129X1/SvJ mice (B6.129 congenics) to identify candidate genes regulating NKT cell function by positionally mapping the genes within this locus. We assessed NKT cell function in B6.129c2 (C2), B6.129c3 (C3), and B6.129c4 (C4) mice, which contain 129 intervals ranging from 0.1-1 megabase pairs (Mbp). To assess NKT cell function, we injected mice with αGalCer, which specifically activates NKT cells. Flow cytometry was utilized to determine NKT cell IL-4, TNF, and IFN-g expression on a per cell basis and ELISA assays were conducted to observe the overall magnitude of the NKT cell response. There was a significant reduction in the TNF, IL-4, and IFNγ production in all congenic mice as compared to B6 controls. These data suggested that the NKT cell response to αGalCer mapped to a 0.1 Mbp region on chromosome 1 (the C3 interval), which excluded Slam genes as potential genes regulating these NKT cell functions. Possible candidate genes of interest in this locus are ApoA2, which encodes a protein involved in lipid transport, and Fcer1g, which encodes a protein that has recently been implicated in the development of different NKT cell subsets

Regulation of CD1 Antigen-presenting Complex Stability

For major histocompatibility complex class I and II molecules, the binding of specific peptide antigens is essential for assembly and trafficking and is at the center of their quality control mechanism. However, the role of lipid antigen binding in stabilization and quality control of CD1 heavy chain (HC).beta(2)-microglobulin (beta(2)m) complexes is unclear. Furthermore, the distinct trafficking and loading routes of CD1 proteins take them from mildly acidic pH in early endososmal compartments (pH 6.0) to markedly acidic pH in lysosomes (pH 5.0) and back to neutral pH of the cell surface (pH 7.4). Here, we present evidence that the stability of each CD1 HC.beta(2)m complex is determined by the distinct pH optima identical to that of the intracellular compartments in which each CD1 isoform resides. Although stable at acidic endosomal pH, complexes are only stable at cell surface pH 7.4 when bound to specific lipid antigens. The proposed model outlines a quality control program that allows lipid exchange at low endosomal pH without dissociation of the CD1 HC.beta(2)m complex and then stabilizes the antigen-loaded complex at neutral pH at the cell surface

A structural perspective of how T cell receptors recognise the CD1 family of lipid antigen-presenting molecules

The CD1 family of antigen-presenting molecules adopt a Major Histocompatibility Complex class I (MHC-I) fold. Whereas MHC molecules present peptides, the CD1 family has evolved to bind self- and foreign-lipids. The CD1 family of antigen-presenting molecules comprises four members, CD1a, CD1b, CD1c, CD1d, that differ in their architecture around the lipid-binding cleft, thereby enabling diverse lipids to be accommodated. These CD1-lipid complexes are recognised by T cell receptors (TCRs) expressed on T cells, either through dual recognition of CD1 and lipid or in a new model whereby the TCR directly contacts CD1, thereby triggering an immune response. Chemical syntheses of lipid antigens, and analogues thereof, have been crucial in understanding the underlying specificity of T cell-mediated lipid immunity. This review will focus on our current understanding of how TCRs interact with CD1-lipid complexes, highlighting how it can be fundamentally different from TCR-MHC-peptide co-recognition

A structural perspective of how T cell receptors recognise the CD1 family of lipid antigen-presenting molecules

The CD1 family of antigen-presenting molecules adopt a Major Histocompatibility Complex class I (MHC-I) fold. Whereas MHC molecules present peptides, the CD1 family has evolved to bind self- and foreign-lipids. The CD1 family of antigen-presenting molecules comprises four members, CD1a, CD1b, CD1c, CD1d, that differ in their architecture around the lipid-binding cleft, thereby enabling diverse lipids to be accommodated. These CD1-lipid complexes are recognised by T cell receptors (TCRs) expressed on T cells, either through dual recognition of CD1 and lipid or in a new model whereby the TCR directly contacts CD1, thereby triggering an immune response. Chemical syntheses of lipid antigens, and analogues thereof, have been crucial in understanding the underlying specificity of T cell-mediated lipid immunity. This review will focus on our current understanding of how TCRs interact with CD1-lipid complexes, highlighting how it can be fundamentally different from TCR-MHC-peptide co-recognition

ER stress in antigen‐presenting cells promotes NKT cell activation through endogenous neutral lipids

CD1d-restricted invariant natural killer T (iNKT) cells constitute a common glycolipid-reactive innate-like T-cell subset with a broad impact on innate and adaptive immunity. While several microbial glycolipids are known to activate iNKT cells, the cellular mechanisms leading to endogenous CD1d-dependent glycolipid responses remain largely unclear. Here, we show that endoplasmic reticulum (ER) stress in APCs is a potent inducer of CD1d-dependent iNKT cell autoreactivity. This pathway relies on the presence of two transducers of the unfolded protein response: inositol-requiring enzyme-1a (IRE1α) and protein kinase R-like ER kinase (PERK). Surprisingly, the neutral but not the polar lipids generated within APCs undergoing ER stress are capable of activating iNKT cells. These data reveal that ER stress is an important mechanism to elicit endogenous CD1d-restricted iNKT cell responses through induction of distinct classes of neutral lipids

Lipid antigen presentation and thymic selection of iNKT cells

The previous introductory part has reviewed the current knowledge on CD1 and lipid immunology. Although the last 20 years witnessed a dramatic expansion of the field, many aspects remain elusive and require additional studies. Little is known about the mechanisms by which stimulatory CD1:lipid antigen complexes are generated and how is the influence of CD1d lipid antigen presentation on thymic selection and peripheral activation of iNKT cells. The aim of this study is to investigate the mechanisms contributing to generation of lipid-specific complexes. In particular, I aim to investigate the impact of LTP residing in different cellular compartments on lipid antigen generation, loading on CD1 molecules, presentation to T cells, positive selection in the thymus as well as stimulation of peripheral CD1-restricted T cells. These studies are divided in three parts: 1. Impact of the absence of the lysosomal LTP NPC2 or of the enzyme β- Galactosidase on lipid antigen presentation and on thymic selection of iNKT cells. Recent studies demonstrate that different lysosomal lipid transfer proteins, like saposins and GM2-A, are implicated in loading of endogenous and exogenous lipid antigens onto CD1d and thus in CD1d-restricted presentation of lipid antigens important for iNKT cells thymic selection [84, 87]. Development of iNKT cells is also completely abolished in mice deficient in saposins [84]. Deficiencies in the lysosomal enzyme Hexb, which is involved in the lipid degradation pathway, or in saposins cause severe imbalances in lipid metabolism. It has never been addressed whether lipid metabolism alteration, and thus accumulation of storage lipids, could contribute to the impairment of lipid antigen presentation and iNKT cell thymic selection. This study has directly assessed the contribution of imbalance in lipid metabolism, consequence of ablation of the LTP NPC2 and of the enzyme β-Galactosidase, on generation of stimulatory CD1d:lipid antigen complexes, on their presentation to iNKT cells, and thus on thymic selection of iNKT cells. 2. Investigation of the functional interaction of CD1e with the other CD1 molecules CD1e is the fifth member of the CD1 family. It is the only member of the CD1 family that does not reach the plasma membrane and is soluble in lysosomes, therefore it might have functions going beyond presentation of lipid antigen to T cells. CD1e binds lipid molecules in the lysosomal compartment and facilitates processing of complex glycolipid antigens, thus participating in lipid editing of CD1b-presented antigens [16]. Nothing is known about the role of CD1e in editing CD1 restricted lipids, either of self or of microbial origin. This study has demonstrated the capacity of CD1e to interact with all lysosomal CD1 molecules and to influence the response of lipid specific-T cells, including that of iNKT cells. Generation of transgenic mice expressing CD1e in professional APC allowed us to study the contribution of CD1e in the formation of antigenic CD1d:lipid complexes and their capacity to activate iNKT cells. 3. Impact of the absence of the peroxisomal and cytoplasmic LTP SCP-2 on lipid antigen presentation and on thymic selection of iNKT cells. All the LTP influencing lipid antigenicity and thus iNKT cell thymic selection that have been investigated so far are localised within the lysosome. The role of LTP residing in other subcellular compartments, like the cytoplasm and the peroxisomes, on presentation of lipid antigens has never been investigated. Furthermore, whether cytoplasmic LTP are involved in generation and intracellular trafficking of endogenous-lipid antigens remains unknown. This study has directly investigated the role of a cytoplasmic/peroxisomal LTP in the generation of the repertoire of selecting lipids, and its impact on thymic selection of iNKT cells