Cluster Of Differentiation 1 Research Articles

Features of the CD1 gene family in rodents and the uniqueness of the immune system of naked mole-rat

Cluster of Differentiation 1 (CD1) proteins are widely expressed throughout jawed vertebrates and present lipid antigens to specific CD1-restricted T lymphocytes. CD1 molecules play an important role in immune defense with the presence or absence of particular CD1 proteins frequently associated with the functional characteristics of the immune system. Here, we show the evolution of CD1 proteins in the Rodentia family and the diversity among its members. Based on the analysis of CD1 protein-coding regions in rodent genomes and the reconstruction of protein structures, we found that Heterocephalus glaber represents a unique member of the suborder Hystricomorpha with significant changes in protein sequences and structures of the CD1 family. Multiple lines of evidence point to the absence of CD1d and CD1e and probably a dysfunctional CD1b protein in Heterocephalus glaber. In addition, the impact of CD1d loss on the CD1d/Natural killer T (NKT) cell axis in the naked mole-rat and its potential implications for immune system function are discussed in detail.

Functional and biological implications of clonotypic diversity among human donor-unrestricted T cells.

Tcells express a T-cell receptor (TCR) heterodimer that is the product of germline rearrangement and junctional editing resulting in immense clonotypic diversity. The generation of diverse TCR repertoires enables the recognition of pathogen-derived peptide antigens presented by polymorphic major histocompatibility complex (MHC) molecules. However, Tcells also recognize nonpeptide antigens through nearly monomorphic antigen-presenting systems, such as cluster of differentiation 1 (CD1), MHC-related protein 1 (MR1) and butyrophilins (BTNs). This potential for shared immune responses across genetically diverse populations led to their designation as donor-unrestricted Tcells (DURTs). As might be expected, some CD1-, MR1- and BTN-restricted Tcells express a TCR that is conserved across unrelated individuals. However, several recent studies have reported unexpected diversity among DURT TCRs, and increasing evidence suggests that this diversity has functional consequences. Recent reports also challenge the dogma that immune cells are either innate or adaptive and suggest that DURT TCRs may act in both capacities. Here, we review this evidence and propose an expanded view of the role for clonotypic diversity among DURTs in humans, including new perspectives on how DURT TCRs may integrate their adaptive and innate immune functions.

A study on etiology of incontinence in double knockout mouse model

Stress urinary incontinence is of concern in both pediatric and adult population. Double mutant GLI family zinc finger Gli2+/-; Gli3Δ699/+ murine model of stress incontinence has been recently developed as a reliable model which does not require surgical manipulation to create incontinence and is shown to survive to adulthood. The aim of this study was to establish the etiology of incontinence in the double mutant Gli2+/-; Gli3Δ699/+ mice. We used 13 cluster of differentiation 1 (CD-1) mice (7-9 weeks) for demonstration of histology of the bladder and urethra. There were 3 Wild Gli2+/- females, 2 Wild Gli2+/- males, 4 Gli2+/-;Gli3Δ699/+ females and 4 Gli2+/-;Gli3Δ699/+ males. The Wild Gli2+/- mice served as the control group and Gli2+/-;Gli3Δ699/+ mice served as the test group. Additionally, eight 16.5 days mice (2 each of Wild Gli2+/- females, Wild Gli2+/- males, double knockout (DKO) Gli2+/-;Gli3Δ699/+ females and Gli2+/-;Gli3Δ699/+ males) were used to assess the histology of the spinal cord. The gross appearance of bladder and urethra was studied using ink injection assays. Immunohistochemistry was done for smooth muscle actin and cytokeratin. Gross and histologic appearance confirmed the previously reported widening of bladder outlet and hypoplasia of smooth muscles in female urethra and also established them in the male urethra of Gli2+/-;Gli3Δ699/+ mice compared to Gli2+/- mice. The double knockout mice were smaller than the Gli2 mice (5.2 vs 6.1cm, p=0.002). Immunohistochemistry demonstrated epithelial hyperplasia and smooth muscle hypoplasia. Additionally, there was prostatic hypoplasia in the Gli2+/-;Gli3Δ699/+ male mice. The spinal cord length for body size appeared comparable between the Gli2+/- and Gli2+/-;Gli3Δ699/+ mice but histological evaluation revealed abnormal development of the caudal end of the vertebral body with premature termination of the spinal cord (Figure). The histological changes in the bladder neck and urethra were consistent to those previously reported. While previous report described the findings in female mice only, we confirmed that these findings are also present in males as well as prostatic hypoplasia, a possible additional factor leading to stress incontinence. The most important finding in the present study however, was the detection of premature termination of spinal cord in the DKO Gli2+/-; Gli3Δ699/+ mice which has not been reported previously and is likely a major contributor to incontinence in this model. The incontinence in male as well as female Gli2+/-; Gli3Δ699/+ mice is due to both myogenic and neurogenic involvement. These double knockout mice are a valuable model of stress incontinence related to neurogenic bladder due to low outlet resistance.

Offspring from oocytes grown in frozen-thawed ovarian tissues transplanted to male and female bodies

Abstract Objective: There are few reports of live births from heterotopic transplantation of frozen-thawed ovarian tissue. The purpose of this study is to assess the follicular development in the frozen-thawed ovarian tissues following heterotopic transplantation in both female and male bodies. Methods: Cluster of differentiation 1 (CD1) mice (6-8 weeks) were used in this study as ovarian tissue donors and foster mothers for embryo transfer. Sperm from CD1 male mice were used for insemination by intracytoplasmic sperm injection (ICSI). Nude severe combined immunodeficiency mice (8 weeks) were employed as recipients of ovarian tissue transplantation. The frozen-thawed ovarian tissues were transplanted to 4 sites on each mouse, female and male, subcutaneously. After 3 months, both female and male mice were injected with 5.0 IU gonadotropins intraperitoneally. Post 48 hours of injection, the mouse was killed for ovarian transplant collection. Only fully grown oocytes with contacted cumulus cells (cumulus-oocyte complexes) were then selected for maturation in vitro. In vitro matured oocytes were inseminated with fresh sperm by ICSI, and the developed blastocysts were frozen using the vitrification method and stored until embryo transfer. After thawing, the thawed blastocysts were incubated for at least 2 hours before the transfer. The foster mice mothers mated with vasectomised male 3 days previously. Live birth was monitored at 19 days after transfer, and the resulted offspring was raised for fertility test. Results: The relatively high recovery rates of the transplanted ovarian tissues were collected in both frozen-thawed and fresh ovarian tissue transplants from both female and male bodies. The fully grown immature oocytes became mature in vitro and the fertilized zygotes developed to blastocyst stage. There are no differences between frozen-thawed and fresh ovarian transplants in term of oocyte quality and embryo development to blastocyst rates. Nineteen-day post-transfer, 3 foster mothers from the frozen-thawed ovarian tissue transplant group delivered 13 pups and the 4 foster mothers of the fresh ovarian tissue transplant group delivered 12 live pups. The produced offspring were normal in appearance and grew healthy and fertile. Conclusions: Our results attest that the follicles can survive and develop in the frozen-thawed ovarian tissues following the subcutaneous transplant to adult male mouse's body regardless of basal endocrinal environment. Those fully grown oocytes can produce healthy and fertile offspring which will provide the possibility for further mechanistic understanding of endocrinology of folliculogenesis.

Current Understanding of the Roles of CD1a-Restricted T Cells in the Immune System.

Cluster of differentiation 1 (CD1) is a family of cell-surface glycoproteins that present lipid antigens to T cells. Humans have five CD1 isoforms. CD1a is distinguished by the small volume of its antigen-binding groove and its stunted A′ pocket, its high and exclusive expression on Langerhans cells, and its localization in the early endosomal and recycling intracellular trafficking compartments. Its ligands originate from self or foreign sources. There are three modes by which the T-cell receptors of CD1a-restricted T cells interact with the CD1a:lipid complex: they bind to both the CD1a surface and the antigen or to only CD1a itself, which activates the T cell, or they are unable to bind because of bulky motifs protruding from the antigen-binding groove, which might inhibit autoreactive T-cell activation. Recently, several studies have shown that by producing TH2 or TH17 cytokines, CD1a-restricted T cells contribute to inflammatory skin disorders, including atopic dermatitis, psoriasis, allergic contact dermatitis, and wasp/bee venom allergy. They may also participate in other diseases, including pulmonary disorders and cancer, because CD1a-expressing dendritic cells are also located in non-skin tissues. In this mini-review, we discuss the current knowledge regarding the biology of CD1a-reactive T cells and their potential roles in disease.

Novel Molecular Insights into Human Lipid-Mediated T Cell Immunity.

T cells represent a critical arm of our immune defense against pathogens. Over the past two decades, considerable inroads have been made in understanding the fundamental principles underpinning the molecular presentation of peptide-based antigens by the Major Histocompatibility Complex molecules (MHC-I and II), and their molecular recognition by specialized subsets of T cells. However, some T cells can recognize lipid-based antigens presented by MHC-I-like molecules that belong to the Cluster of Differentiation 1 (CD1) family. Here, we will review the advances that have been made in the last five years to understand the molecular mechanisms orchestrating the presentation of novel endogenous and exogenous lipid-based antigens by the CD1 glycoproteins and their recognition by specific populations of CD1-reactive T cells.

Diversification of CD1 Molecules Shapes Lipid Antigen Selectivity.

Molecular studies of host–pathogen evolution have largely focused on the consequences of variation at protein–protein interaction surfaces. The potential for other microbe-associated macromolecules to promote arms race dynamics with host factors remains unclear. The cluster of differentiation 1 (CD1) family of vertebrate cell surface receptors plays a crucial role in adaptive immunity through binding and presentation of lipid antigens to T-cells. Although CD1 proteins present a variety of endogenous and microbial lipids to various T-cell types, they are less diverse within vertebrate populations than the related major histocompatibility complex (MHC) molecules. We discovered that CD1 genes exhibit a high level of divergence between simian primate species, altering predicted lipid-binding properties and T-cell receptor interactions. These findings suggest that lipid–protein conflicts have shaped CD1 genetic variation during primate evolution. Consistent with this hypothesis, multiple primate CD1 family proteins exhibit signatures of repeated positive selection at surfaces impacting antigen presentation, binding pocket morphology, and T-cell receptor accessibility. Using a molecular modeling approach, we observe that interspecies variation as well as single mutations at rapidly-evolving sites in CD1a drastically alter predicted lipid binding and structural features of the T-cell recognition surface. We further show that alterations in both endogenous and microbial lipid-binding affinities influence the ability of CD1a to undergo antigen swapping required for T-cell activation. Together these findings establish lipid–protein interactions as a critical force of host–pathogen conflict and inform potential strategies for lipid-based vaccine development.

Harnessing Unconventional T Cells for Immunotherapy of Tuberculosis.

Even if the incidence of tuberculosis (TB) has been decreasing over the last years, the number of patients with TB is increasing worldwide. The emergence of multidrug-resistant and extensively drug-resistant TB is making control of TB more difficult. Mycobacterium bovis bacillus Calmette–Guérin vaccine fails to prevent pulmonary TB in adults, and there is an urgent need for a vaccine that is also effective in patients with human immunodeficiency virus (HIV) coinfection. Therefore, TB control may benefit on novel therapeutic options beyond antimicrobial treatment. Host-directed immunotherapies could offer therapeutic strategies for patients with drug-resistant TB or with HIV and TB coinfection. In the last years, the use of donor lymphocytes after hematopoietic stem cell transplantation has emerged as a new strategy in the cure of hematologic malignancies in order to induce graft-versus leukemia and graft-versus-infection effects. Moreover, adoptive therapy has proven to be effective in controlling cytomegalovirus and Epstein-Barr virus reactivation in immunocompromised patients with ex vivo expanded viral antigen-specific T cells. Unconventional T cells are a heterogeneous group of T lymphocytes with limited diversity. One of their characteristics is that antigen recognition is not restricted by the classical major histocompatibility complex (MHC). They include CD1 (cluster of differentiation 1)–restricted T cells, MHC-related protein-1–restricted mucosal-associated invariant T (MAIT) cells, MHC class Ib–reactive T cells, and γδ T cells. Because these T cells are genotype-independent, they are also termed “donor unrestricted” T cells. The combined features of low donor diversity and the lack of genetic restriction make these cells suitable candidates for T cell–based immunotherapy of TB.

Hypersensitivities following allergen antigen recognition by unconventional T cells.

Conventional T cells recognise protein-derived antigens in the context of major histocompatibility complex (MHC) class Ia and class II molecules and provide anti-microbial and anti-tumour immunity. Conventional T cells have also been implicated in type IV (also termed delayed-type or T cell-mediated) hypersensitivity reactions in response to protein-derived allergen antigens. In addition to conventional T cells, subsets of unconventional T cells exist, which recognise non-protein antigens in the context of monomorphic MHC class I-like molecules. These include T cells that are restricted to the cluster of differentiation 1 (CD1) family members, known as CD1-restricted T cells, and mucosal-associated invariant T cells (MAIT cells) that are restricted to the MHC-related protein 1 (MR1). Compared with conventional T cells, much less is known about the immune functions of unconventional T cells and their role in hypersensitivities. Here, we review allergen antigen presentation by MHC-I-like molecules, their recognition by unconventional T cells, and the potential role of unconventional T cells in hypersensitivities. We also speculate on possible scenarios of allergen antigen presentation by MHC-I-like molecules to unconventional T cells, the hallmarks of such responses, and the expected frequencies of hypersensitivities within the human population.

Lipid-Reactive T Cells in Immunological Disorders of the Lung.

Regulation of T cell-mediated immunity in the lungs is critical for prevention of immune-related lung disorders and for host protection from pathogens. While the prevalent view of pulmonary T cell responses is based on peptide recognition by antigen receptors, called T cell receptors (TCR), on the T cell surface in the context of classical major histocompatibility complex (MHC) molecules, novel pathways involving the presentation of lipid antigens by cluster of differentiation 1 (CD1) molecules to lipid-reactive T cells are emerging as key players in pulmonary immune system. Whereas, genetic conservation of group II CD1 (CD1d) in mouse and human genomes facilitated numerous in vivo studies of CD1d-restricted invariant natural killer T (iNKT) cells in lung diseases, the recent development of human CD1-transgenic mice has made it possible to examine the physiological roles of group I CD1 (CD1a-c) molecules in lung immunity. Here, we discuss current understanding of the biology of CD1-reactive T cells with a specific focus on their roles in several pulmonary disorders.

CD1d-dependent natural killer T cells attenuate angiotensin II-induced cardiac remodelling via IL-10 signalling in mice.

CD1d is a member of the cluster of differentiation 1 (CD1) family of glycoproteins expressed on the surface of various antigen-presenting cells, which is recognized by natural killer T (NKT) cells. CD1d-dependent NKT cells play an important role in immune-mediated diseases; but the role of these cells in regulating cardiac remodelling remains unknown. Cardiac remodelling was induced by angiotensin (Ang) II infusion for 2 weeks. Ang II-induced increase in hypertension, cardiac performance, hypertrophy and fibrosis, inflammatory response, and activation of the NF-kB and TGF-β1/Smad2/3 pathways was significantly aggravated in CD1d knockout (CD1dko) mice compared with wild-type (WT) mice, but these effects were markedly abrogated in WT mice treated with α-galactosylceramide (αGC), a specific activator of NKT cells. Adoptive transfer of CD1dko bone marrow cells to WT mice further confirmed the deleterious effect of CD1dko. Moreover, IL-10 expression was significantly decreased in CD1dko hearts but increased in αGC-treated mice. Co-culture experiments revealed that CD1dko dendritic cells significantly reduced IL-10 mRNA expression from NKT cells. Administration of recombinant murine IL-10 to CD1dko mice improved hypertension, cardiac performance, and adverse cardiac remodelling induced by Ang II, and its cardioprotective effect was possibly associated with activation of STAT3, and inhibition of the TGF-β1 and NF-kB pathways. These findings revealed a previously undefined role for CD1d-dependent NKT cells in Ang II-induced cardiac remodelling, hence activation of NKT cells may be a novel therapeutic target for hypertensive cardiac disease.

CD1-Restricted T Cells During Persistent Virus Infections: "Sympathy for the Devil".

Some of the clinically most important viruses persist in the human host after acute infection. In this situation, the host immune system and the viral pathogen attempt to establish an equilibrium. At best, overt disease is avoided. This attempt may fail, however, resulting in eventual loss of viral control or inadequate immune regulation. Consequently, direct virus-induced tissue damage or immunopathology may occur. The cluster of differentiation 1 (CD1) family of non-classical major histocompatibility complex class I molecules are known to present hydrophobic, primarily lipid antigens. There is ample evidence that both CD1-dependent and CD1-independent mechanisms activate CD1-restricted T cells during persistent virus infections. Sophisticated viral mechanisms subvert these immune responses and help the pathogens to avoid clearance from the host organism. CD1-restricted T cells are not only crucial for the antiviral host defense but may also contribute to tissue damage. This review highlights the two edged role of CD1-restricted T cells in persistent virus infections and summarizes the viral immune evasion mechanisms that target these fascinating immune cells.

A Comparative Study of Human Saposins.

Saposins are small proteins implicated in trafficking and loading of lipids onto Cluster of Differentiation 1 (CD1) receptor proteins that in turn present lipid antigens to T cells and a variety of T-cell receptors, thus playing a crucial role in innate and adaptive immune responses in humans. Despite their low sequence identity, the four types of human saposins share a similar folding pattern consisting of four helices linked by three conserved disulfide bridges. However, their lipid-binding abilities as well as their activities in extracting, transporting and loading onto CD1 molecules a variety of sphingo- and phospholipids in biological membranes display two striking characteristics: a strong pH-dependence and a structural change between a compact, closed conformation and an open conformation. In this work, we present a comparative computational study of structural, electrostatic, and dynamic features of human saposins based upon their available experimental structures. By means of structural alignments, surface analyses, calculation of pH-dependent protonation states, Poisson-Boltzmann electrostatic potentials, and molecular dynamics simulations at three pH values representative of biological media where saposins fulfill their function, our results shed light into their intrinsic features. The similarities and differences in this class of proteins depend on tiny variations of local structural details that allow saposins to be key players in triggering responses in the human immune system.

β2-microglobulin gene duplication in cetartiodactyla remains intact only in pigs and possibly confers selective advantage to the species.

Several β2-microglobulin (B2M) -bound protein complexes undertake key roles in various immune system pathways, including the neonatal Fc receptor (FcRn), cluster of differentiation 1 (CD1) protein, non-classical major histocompatibility complex (MHC), and well-known MHC class I molecules. Therefore, the duplication of B2M may lead to an increase in the biological competence of organisms to the environment. Based on the pig genome assembly SSC10.2, a segmental duplication of ~45.5 kb, encoding the entire B2M protein, was identified in pig chromosome 1. Through experimental validation, we confirmed the functional duplication of the B2M gene with a completely identical coding sequence between two copies in pigs. Considering the importance of B2M in the immune system, we performed the phylogenetic analysis of B2M duplication in ten mammalian species, confirming the presence of B2M duplication in cetartioldactyls, like cattle, sheep, goats, pigs and whales, but non-cetartiodactyl species, like mice, cats, dogs, horses, and humans. The density of long interspersed nuclear element (LINE) at the edges of duplicated blocks (39 to 66%) was found to be 2 to 3-fold higher than the average (20.12%) of the pig genome, suggesting its role in the duplication event. The B2M mRNA expression level in pigs was 12.71 and 7.57 times (2-ΔΔCt values) higher than humans and mice, respectively. However, we were unable to experimentally demonstrate the difference in the level of B2M protein because species specific anti-B2M antibodies are not available. We reported, for the first time, the functional duplication of the B2M gene in animals. The identification of partially remaining duplicated B2M sequences in the genomes of only cetartiodactyls indicates that the event was lineage specific. B2M duplication could be beneficial to the immune system of pigs by increasing the availability of MHC class I light chain protein, B2M, to complex with the proteins encoded by the relatively large number of MHC class I heavy chain genes in pigs. Further studies are necessary to address the biological meaning of increased expression of B2M.

Inhibition of endocytic lipid antigen presentation by common lipophilic environmental pollutants

Environmental pollutants as non-heritable factors are now recognized as triggers for multiple human inflammatory diseases involving T cells. We postulated that lipid antigen presentation mediated by cluster of differentiation 1 (CD1) proteins for T cell activation is susceptible to lipophilic environmental pollutants. To test this notion, we determined whether the common lipophilic pollutants benzo[a]pyrene and diesel exhaust particles impact on the activation of lipid-specific T cells. Our results demonstrated that the expression of CD1a and CD1d proteins, and the activation of CD1a- and CD1d-restricted T cells were sensitively inhibited by benzo[a]pyrene even at the low concentrations detectable in exposed human populations. Similarly, diesel exhaust particles showed a marginal inhibitory effect. Using transcriptomic profiling, we discovered that the gene expression for regulating endocytic and lipid metabolic pathways was perturbed by benzo[a]pyrene. Imaging flow cytometry also showed that CD1a and CD1d proteins were retained in early and late endosomal compartments, respectively, supporting an impaired endocytic lipid antigen presentation for T cell activation upon benzo[a]pyrene exposure. This work conceptually demonstrates that lipid antigen presentation for T cell activation is inhibited by lipophilic pollutants through profound interference with gene expression and endocytic function, likely further disrupting regulatory cytokine secretion and ultimately exacerbating inflammatory diseases.

Lipophilic air pollutants inhibit endocytic lipid antigen presentation

Abstract Air pollutants as non-heritable factors are recognized as triggers for various human inflammatory diseases, in which T cells are central in producing cytokines and determining disease outcomes. We postulated that lipid antigen presentation mediated by cluster of differentiation 1 (CD1) proteins for T cell activation is susceptible to lipophilic air pollutants. To test this notion, we determined whether common lipophilic pollutants benzo[a]pyrene and diesel exhaust particles impact on the activation of lipid-specific T cells. Our results demonstrated that the expression of CD1a and CD1d proteins, and the activation of CD1a- and CD1d-restricted T cells were sensitively inhibited by benzo[a]pyrene even at low concentrations detectable in exposed human populations. Similarly, diesel exhaust particles showed marginal inhibitory effect. Using transcriptomic profiling, we discovered that gene expression for regulating endocytic and lipid metabolic pathways was perturbed by benzo[a]pyrene. Imaging flow cytometry also showed that CD1a and CD1d proteins were respectively retained in early and late endosomal compartments, supporting an impaired endocytic lipid antigen presentation for T cell activation upon benzo[a]pyrene exposure. This work conceptually demonstrates that lipid antigen presentation for T cell activation is inhibited by lipophilic pollutants through profound interference with gene expression and endocytic function, likely further disrupting regulatory cytokine secretion and ultimately exacerbating inflammatory diseases.

Immunological study of expression of CD1 in preimplantation and midgestation mouse embryo cells and placental trophoblasts and the role of decidual natural killer T cells at the feto–maternal interface

Background Expression of major histocompatibility complex molecules in the preimplantation embryo is important for control of development and immune recognition of the embryo by the mother. Four types of major histocompatibility complex class I molecules that have been identified are as follows: class Ia, class Ib, class Ic, and class Id. Expression of class Ic and class Id antigens in preimplantation embryos has not yet been fully evaluated. Cluster of differentiation 1 (CD1) molecule is a member of class Id antigen. Localization and regulation of CD1 expression remain unclear. The aim of this study was to detect expression of the CD1 molecule in a preimplantation mouse embryo at two-cell, four-cell, eight-cell, and blastocyst stages and in the embryo cells and placental trophoblasts at 10 days of gestation. Detection of invariant natural killer T (iNKT) cells in the decidua at the site of attachment of the placenta was also investigated. Materials and methods Mice of the C57BL/6 genetic background were used. Forty-eight female and 16 male mice of 10–12 weeks of age were divided into two groups. Group 1 included 24 female and eight male mice. Eight breeding colonies were constructed in which each cage contained one male and three female mice. The pregnant female mice were killed at 36, 48, 60, and 80 h of appearance of the vaginal plug to collect two-cell, four-cell, eight-cell, and blastocyst stages of embryos, respectively. The embryos were then processed for immunohistochemistry. Group 2 animals included 24 female and eight male mice. Eight breeding colonies were constructed in which each cage contained one male and three female mice. The pregnant female mice were killed at 10 days of gestation when embryo cells, placental cells, and decidual cells were isolated and processed for culturing and immunohistochemistry. Results This study showed no expression of CD1 molecules in any of the preimplantation mouse embryo stages till the blastocyst stage, but were detected on the cell surface and inside the cytoplasm of embryo cells and placental trophoblasts at 10 days of gestation. A high proportion of iNKT cells was also detected in the decidua at the site of attachment of the placenta. Conclusion The CD1 molecule is not actively involved in the regulation of early development of embryo till the blastocyst stage, and the true biological role of CD1 in preimplantation development remains to be determined. However, it may play a role in the placenta and in embryo development during midgestation by preventing lysis of fetal and placental cells through an interaction with decidual iNKT cells at the feto–maternal interface.

Synthesis of Dideoxymycobactin Antigens Presented by CD1a Reveals T Cell Fine Specificity for Natural Lipopeptide Structures

Mycobacterium tuberculosis survival in cells requires mycobactin siderophores. Recently, the search for lipid antigens presented by the CD1a antigen-presenting protein led to the discovery of a mycobactin-like compound, dideoxymycobactin (DDM). Here we synthesize DDMs using solution phase and solid phase peptide synthesis chemistry. Comparison of synthetic standards to natural mycobacterial mycobactins by nuclear magnetic resonance and mass spectrometry allowed identification of an unexpected alpha-methyl serine unit in natural DDM. This finding further distinguishes these pre-siderophores as foreign compounds distinct from conventional peptides, and we provide evidence that this chemical variation influences the T cell response. One synthetic DDM recapitulated natural structures and potently stimulated T cells, making it suitable for patient studies of CD1a in infectious disease. DDM analogs differing in the stereochemistry of their butyrate or oxazoline moieties were not recognized by human T cells. Therefore, we conclude that T cells show precise specificity for both arms of the peptide, which are predicted to lie at the CD1a-T cell receptor interface.

Endoplasmic reticulum stress, hepatocyte CD1d and NKT cell abnormalities in murine fatty livers

The liver regulates lipid homeostasis and is enriched with natural killer T (NKT) cells that respond to lipid antigens. Optimal maturation and activation of NKT cells requires their interaction with lipid antigens that are presented by cluster of differentiation-1 (CD-1) molecules on antigen-presenting cells. Hepatocytes express CD1d and present lipid antigens to NKT cells. Depletion and dysregulation of hepatic NKT cells occurs in mice with fatty livers. Herein, we assess whether reduced CD1d content on steatotic hepatocytes contributes to fatty liver-associated NKT cell abnormalities. We show that despite expressing normal levels of CD1d mRNA, fatty hepatocytes from ob/ob mice have significantly less CD1d on their plasma membranes than normal hepatocytes. This has functional significance because ob/ob hepatocytes are less able to activate CD1d-restricted T-cell responses in vitro, and CD1d-reactive NKT cells are reduced in ob/ob livers. Events in the endoplasmic reticulum (ER) normally regulate CD1d trafficking to plasma membranes. Hepatic steatosis has been associated with ER stress. To determine if ER stress reduces CD-1 accumulation on hepatocytes, we evaluated hepatic ER stress in ob/ob mice and treated cultured hepatocytes and lean mice with tunicamycin to induce ER stress. Lipid accumulation and ER stress occurred in the livers of both ob/ob and tunicamycin-treated mice. Tunicamycin caused dose-dependent decreases in hepatocyte CD1d, inhibited hepatocyte activation of CD1d-restricted T-cell responses, depleted liver populations of CD1d-reactive NKT cells and promoted Th-1 polarization of hepatic cytokine production. In conclusion, ER stress-related decreases in hepatocyte CD1d contribute to NKT cell dysregulation in fatty livers.

An N-Linked Glycan Modulates the Interaction between the CD1d Heavy Chain and β2-Microglobulin

Human CD1d molecules consist of a transmembrane CD1 (cluster of differentiation 1) heavy chain in association with beta(2)-microglobulin (beta(2)m). Assembly occurs in the endoplasmic reticulum (ER) and involves the initial glycan-dependent association of the free heavy chain with calreticulin and calnexin and the thiol oxidoreductase ERp57. Folding and disulfide bond formation within the heavy chain occurs prior to beta(2)m binding. There are four N-linked glycans on the CD1d heavy chain, and we mutated them individually to ascertain their importance for the assembly and function of CD1d-beta(2)m heterodimers. None of the four were indispensable for assembly or the ability to bind alpha-galactosyl ceramide and to present it to human NKT cells. Nor were any required for the CD1d molecule to bind and present alpha-galactosyl ceramide after lysosomal processing of a precursor lipid, galactosyl-(alpha1-2)-galactosyl ceramide. However, one glycan, glycan 2 at Asn-42, proved to be of particular importance for the stability of the CD1d-beta(2)m heterodimer. A mutant CD1d heavy chain lacking glycan 2 assembled with beta(2)m and transported from the ER more rapidly than wild-type CD1d and dissociated more readily from beta(2)m upon exposure to detergents. A mutant expressing only glycan 1 dissociated completely from beta(2)m upon exposure to the detergent Triton X-100, whereas a mutant expressing only glycan 2 at Asn-42 was more stable. In addition, glycan 2 was not processed efficiently to the complex form in mature wild-type CD1d molecules. Modeling the glycans on the published structure indicated that glycan 2 interacts significantly with both the CD1d heavy chain and beta(2)m, which may explain these unusual properties.