Zinc and immunity

Nutritional deficiency of zinc is widespread throughout the developing countries and a conditioned deficiency of zinc is known to occur in many diseased states. Zinc is known to play an important role in the immune system and zinc deficient subjects may experience increased susceptibility to a variety of pathogens. We have studied the effects of a mild deficiency of zinc on T cells in an experimental model of human zinc deficiency. We showed that T cell functions were affected adversely even when the deficiency of zinc was mild in humans. Characteristically during zinc deficiency, the serum thymulin activity (a thymic hormone) was decreased which was restored following zinc supplementation. Our studies also showed that zinc deficiency caused an imbalance between TH1 and TH2 functions. The production of IFN-g, IL-2, TNF-a (products of TH1 cells) were decreased, whereas the production of IL-4, IL-6 and IL-10 (products of TH2) were not affected during zinc deficiency. T cell subpopulation studies revealed that the CD4+ CD45RA+ to CD4+ CD45RO+ ratio was decreased as a result of zinc deficiency, suggesting that zinc may be required for the regeneration of new CD4+ T cells. We further documented that zinc deficiency decreased NK cell lytic activity and caused a decrease in the percentage of CD8+ CD73+ T cells which are known to be predominantly precursors of cytotoxic T cells. In a suitable cell culture model our studies revealed that the gene expression of a DNA synthesizing enzyme TK was affected adversely which resulted in delayed cell cycle and decreased cell growth. The above immunological consequences of zinc deficiency may be responsible for decreased cell mediated immune functions in zinc deficient subjects.     

Cross-presentation of Listeria-derived CD8 T cell epitopes requires unstable bacterial translation products

Presentation of bacteria-derived CD8 T cell epitopes by dendritic cells (DC) requires either their direct infection or that DC acquire and cross-present Ags from other infected cells. We found that cross-presentation of Listeria monocytogenes-derived CD8 T cell epitopes was much stronger than direct Ag presentation by infected murine DC. Cross-presentation of Listeria-derived CD8 T cell epitopes showed unique physiological requirements. It was dependent upon the delivery of unstable bacterial translation products by infected, but still viable, Ag donor cells. Cross-presentation was enhanced both when unstable translation products in infected Ag donor cells were protected from proteasomal degradation and when the production of misfolded bacterial proteins was increased. The requirement of unstable translation products for cross-presentation may represent a novel pathway that functions to focus the CD8 T cell response toward epitopes derived from newly synthesized proteins.     

Alloreactive murine CD8+ T cell clones secrete the Th1 pattern of cytokines

A large panel of CD8+ mouse T cell clones expressed the cytokine synthesis pattern characteristic of Th1 clones. CD8+ clones synthesized IFN-gamma and lymphotoxin at levels similar to Th1 clones, whereas IL-2 was synthesized by only 50% of the clones and at significantly lower levels compared to Th1 clones. CD8+ clones also produced substantial amounts of granulocyte/macrophage-CSF, TY5, P500, and TNF-alpha which are expressed preferentially by Th1 clones and at lower levels by Th2 clones. The level of IL-3 produced by CD8+ clones was approximately 10% of that produced by Th1 and Th2 clones. Some CD8+ clones expressed low levels of the Th2-preferential product preproenkelphalin. None of the CD8+ clones expressed detectable levels of the Th2-specific products IL-4, IL-5, and P600, and the great majority did not express IL-6. The cytokine profile of CD8+ clones is representative of that secreted by activated normal CD8+ splenocytes, which includes IFN-gamma, low levels of IL-2 and IL-3 but no IL-4 or IL-5. Inasmuch as many Th1/Th2 functions are cytokine mediated, the striking similarity of the Th1 and CD8+ cytokine secretion patterns helps to explain why these two cell types share certain functions such as DTH, and also suggests that further common functions may be discovered in the future.     

Signaling properties of CD38 in the mouse immune system: enzyme-dependent and -independent roles in immunity

The 5th international CD38 meeting, held in Torino, Italy, spanned a range of topics from the role of CD38 as a signaling receptor in lymphocytic tumors to the importance of CD38-derived metabolites in NAD(+) metabolism, calcium signaling, and immune function. This meeting was particularly exciting as data were presented demonstrating that collaborative experiments between enzymologists, biochemists, cell biologists, immunologists, and clinicians have started to unravel the secrets of CD38 biology. It is now clear that all of the products of the CD38 enzyme reaction regulate calcium signal transduction in cell types as diverse as sea urchin oocytes and mammalian lymphocytes. It is also apparent that CD38 plays important immunomodulatory role(s), however there is still much debate on how CD38 mediates its immunoregulatory functions and whether the enzymatic products generated by CD38 are important for immunity. The data presented at this meeting have begun to resolve some of these controversies. First, CD38 regulates the function of leukocytes by enzyme-dependent and enzyme-independent mechanisms. Second, CD38 regulates inflammatory responses by modulating the activity of the responding leukocytes and by altering the activity of non-hematopoietic cells in the inflamed tissue. Finally, crosstalk between CD38 and other NAD(+) utilizing enzymes such as ART2, SIRT1, and PARP-1 impacts NAD(+) homeostasis, inflammation, and immunity. Thus, immunity is regulated by CD38 in multiple and unexpected ways and the new research challenge will be to determine whether we can exploit the complex biology of CD38 to therapeutically regulate the immune system.     

NK cell triggering by the human costimulatory molecules CD80 and CD86.

NK cell-mediated effector functions are regulated by a delicate balance between positive and negative signals. Receptors transmitting negative signals upon engagement with target cell MHC class I molecules have been characterized in detail in recent years. In contrast, less information is available about receptor-ligand interactions involved in the transmission of positive or "triggering" signals to NK cells. Recently, it has been described that murine NK cells are triggered by the costimulatory molecules CD80, CD86, and CD40. Using NK cell lines derived from PBMC as effectors, we demonstrate that the human CD80 and CD86 gene products can function as triggering molecules for NK cell-mediated cytotoxicity. Expression of human CD80 or CD86 molecules in murine B16.F1 melanoma cells rendered these significantly more susceptible to lysis by human NK cell lines. Blocking of the transfected gene products with specific mAb reduced lysis levels to that of nontransfected control cell lines. Triggering of human NK cells by CD80 and CD86 appeared to be independent of CD28 and CTLA-4, at least as determined by the reagents used in the present study, because the expression of these molecules could not be detected on the NK cell lines by either flow cytometry or in redirected lysis assays. Thus, human NK cells may use receptors other than CD28 and CTLA-4 in their interactions with CD80 and CD86 molecules. Alternatively, interactions may involve variants of CD28 (and possibly CTLA-4) that are not recognized by certain anti-CD28 mAb.     

Tissue expression of human Toll-like receptors and differential regulation of Toll-like receptor mRNAs in leukocytes in response to microbes, their products, and cytokines.

Members of the Toll-like receptor (TLR) family mediate dorsoventral patterning and cellular adhesion in insects as well as immune responses to microbial products in both insects and mammals. TLRs are characterized by extracellular leucine-rich repeat domains and an intracellular signaling domain that shares homology with cytoplasmic sequences of the mammalian IL-1 receptor and plant disease resistance genes. Ten human TLRs have been cloned as well as RP105, a protein similar to TLR4 but lacking the intracellular signaling domain. However, only five TLRs have described functions as receptors for bacterial products (e.g., LPS, lipoproteins). To identify potential sites of action, we used quantitative real-time RT-PCR to examine systematically the expression of mRNAs encoding all known human TLRs, RP105, and several other proteins important in TLR functions (e.g., MD-1, MD-2, CD14, MyD88). Most tissues tested expressed at least one TLR, and several expressed all (spleen, peripheral blood leukocytes). Analysis of TLR expression in fractionated primary human leukocytes (CD4(+), CD8(+), CD19(+), monocytes, and granulocytes) indicates that professional phagocytes express the greatest variety of TLR mRNAs although several TLRs appear more restricted to B cells, suggesting additional roles for TLRs in adaptive immunity. Monocyte-like THP-1 cells regulate TLR mRNA levels in response to a variety of stimuli including phorbol esters, LPS, bacterial lipoproteins, live bacteria, and cytokines. Furthermore, addition of Escherichia coli to human blood ex vivo caused distinct changes in TLR expression, suggesting that important roles exist for these receptors in the establishment and resolution of infections and inflammation.     

Isolation and expression of cDNA encoding the murine homologues of CD1.

The cDNA encoding the murine CD1.1 and CD1.2 gene products were isolated and their complete nucleotide sequence was determined. The nucleotide sequence and genomic organization of these molecules were similar to human CD1. The sequences in the alpha 1- alpha 3 domains were almost identical to previously reported genomic clones from a different strain, indicating limited polymorphism among these molecules. The predicted amino acid sequence in the transmembrane region and in the cytoplasmic tail was identical for CD1.1 and CD1.2. The two cDNA were also homologous in the 5' untranslated region but diverged in the 3' untranslated region. In contrast to human CD1, which is expressed at high levels in thymus, the expression of CD1 message in murine thymus was not detected in either thymus leukemia Ag positive or negative strains. Cell expressing murine CD1.1 were generated after transfer of the CD1.1 cDNA into murine cell lines. Immunoprecipitation with a rat anti-mouse CD1.1 mAb showed that the transfected CD1 was expressed on the cell surface as a beta 2-microglobulin-linked heterodimer. These results demonstrate that the murine and human CD1 genes, although encoding homologous transmembrane glycoproteins, are expressed in distinct tissues and may serve different functions.     

Human CD1d molecules are resistant to human cytomegalovirus US2- and US11-mediated degradation

Natural killer T (NKT) cells may play a crucial role in controlling viral infection by bridging the innate and adaptive immune systems. These cells are activated by lipids presented by CD1d molecules, which are structurally homologous to major histocompatibility complex class I (MHC-I) molecules. Although human cytomegalovirus (HCMV) can avoid T cell recognition by down-regulating MHC-I-mediated antigen presentation, it remains unknown whether it can also interfere with CD1d-mediated lipid presentation. Here, we show that CD1d is resistant to rapid degradation induced by the HCMV gene products US2 and US11, which cause dislocation of MHC-I molecules from the endoplasmic reticulum (ER) to the cytosol for destruction by proteasomes. The resistance of CD1d to US11 is mainly due to the short cytosolic tail of CD1d; a hybrid CD1d protein, whose cytosolic tail was replaced with that of HLA-A2.1, was efficiently degraded by US11. Finally, we found that HCMV infection did not significantly influence the cell surface expression of CD1d. Thus, these results suggest that antigen presentation by CD1d is largely unaffected by the multiple immune-modulating functions of HCMV.     

A unique mechanism for innate cytokine promotion of T cell responses to viral infections

The kinetics of CD8 T cell IFN-gamma responses as they occur in situ are defined here during lymphocytic choriomeningitis virus (LCMV) infections, and a unique mechanism for the innate cytokines IFN-alphabeta and IL-18 in promoting these responses is defined. Infections of mice with Armstrong or WE strains of LCMV induced an unexpectedly early day 4 IFN-gamma response detectable in serum samples and spleen and liver homogenates. Production of IFN-gamma was MHC class I/CD8 dependent, but did not require IL-12, NK cells, TCR-gammadelta T cells, MHC class II, or CD4 T cells. Peak response required specific Ag recognition, as administration of antagonist peptide partially impaired day 4 IFN-gamma induction, and viral peptide stimulation enhanced CD8 T cell IFN-gamma expression in culture. The IFN-gamma response was associated with IL-18 and IFN-alphabeta expression. Furthermore, both factors augmented peptide-driven IFN-gamma production in culture, and mice lacking IL-18 or IFN-alphabeta functions had reduced day 4 IFN-gamma. Collectively, these results demonstrate that during viral infections, there is a dramatic in vivo CD8 T cell response preceding maximal expansion of these cells, and that the mechanism supporting this response is dependent on endogenous innate cytokines. Because stimulation by microbial products is linked to innate cytokine expression, the studies also suggest a pathway for precisely limiting T cell functions to times of need.     

Double-negative (CD4- CD8-) T cells with an alpha/beta T cell receptor. Non-MHC-restricted cytolytic activity and lymphokine production

T cell lines with a novel phenotype (CD3+ TCR-alpha/beta+ CD4- CD8-) were developed from the peripheral blood of a patient with a combined immunodeficiency and tissue injury resembling graft-vs-host disease. One of these IL-2-dependent T cell lines demonstrated non-MHC-restricted cytolytic function against tumor targets, syngeneic and allogeneic fibroblasts, and PHA blasts from allogeneic donors. The other cell line only became cytotoxic in the presence of lectin or anti-CD3 antibody. The two cell lines also differed in their expression of the T-200 gene products CD45RO (gp180) and CD45RA (gp220). Both cell lines produced tumor necrosis factor-alpha and -beta and IFN-gamma activity when activated with mitogens or PMA and IL-1. The in vitro functions of these T-cell lines suggest a potential role for alpha/beta double-negative T lymphocytes in tissue injury resembling graft-vs-host disease.     

CD1d function is regulated by microsomal triglyceride transfer protein

CD1d is a major histocompatibility complex (MHC) class I-related molecule that functions in glycolipid antigen presentation to distinct subsets of T cells that express natural killer receptors and an invariant T-cell receptor-alpha chain (invariant NKT cells). The acquisition of glycolipid antigens by CD1d occurs, in part, in endosomes through the function of resident lipid transfer proteins, namely saposins. Here we show that microsomal triglyceride transfer protein (MTP), a protein that resides in the endoplasmic reticulum of hepatocytes and intestinal epithelial cells (IECs) and is essential for lipidation of apolipoprotein B, associates with CD1d in hepatocytes. Hepatocytes from animals in which Mttp (the gene encoding MTP) has been conditionally deleted, and IECs in which Mttp gene products have been silenced, are unable to activate invariant NKT cells. Conditional deletion of the Mttp gene in hepatocytes is associated with a redistribution of CD1d expression, and Mttp-deleted mice are resistant to immunopathologies associated with invariant NKT cell-mediated hepatitis and colitis. These studies indicate that the CD1d-regulating function of MTP in the endoplasmic reticulum is complementary to that of the saposins in endosomes in vivo.     

A second subunit of CD8 is expressed in human T cells.

The CD8 glycoprotein plays important functions in T cell development and in T cell activation. In rodents, CD8 is a heterodimer, consisting of an alpha-chain (Lyt2) and a beta-chain (Lyt3). In humans, only the alpha-chain has been detected, and it has been thought that CD8 consists of homodimers of this protein. We have isolated functional cDNA clones encoding human CD8 beta, and show that the CD8 beta protein is expressed on the surface of CD8+ human T cells. cDNA clones encoding multiple forms of the human CD8 beta-chain have been isolated and characterized. These structural variants, which are likely to arise by alternative splicing, differ in the sequences encoding the cytoplasmic domain, which can consist of 19, 30, or 52 amino acids. One of the cDNAs lacks nucleotide sequences corresponding to a hydrophobic transmembrane domain, and may encode a secreted CD8 beta protein. The protein product of the human CD8 beta gene can be detected by a recently described anti-CD8 monoclonal antibody, 597. Expression of the epitope recognized by this antibody requires co-expression of the CD8 alpha and CD8 beta gene products. About 90% of human CD8 alpha positive thymocytes and peripheral blood lymphocytes express CD8 beta at the cell surface. Expression of the CD8 beta chain is thus conserved between human and rodents, and the variant CD8 beta polypeptides may have distinct roles in T cell function and development.     

Silibinin Inhibits HIV-1 Infection by Reducing Cellular Activation and Proliferation

Purified silymarin-derived natural products from the milk thistle plant (Silybum marianum) block hepatitis C virus (HCV) infection and inhibit T cell proliferation in vitro. An intravenous formulation of silibinin (SIL), a major component of silymarin, displays anti-HCV effects in humans and also inhibits T-cell proliferation in vitro. We show that SIL inhibited replication of HIV-1 in TZM-bl cells, PBMCs, and CEM cells in vitro. SIL suppression of HIV-1 coincided with dose-dependent reductions in actively proliferating CD19+, CD4+, and CD8+ cells, resulting in fewer CD4+ T cells expressing the HIV-1 co-receptors CXCR4 and CCR5. SIL inhibition of T-cell growth was not due to cytotoxicity measured by cell cycle arrest, apoptosis, or necrosis. SIL also blocked induction of the activation markers CD38, HLA-DR, Ki67, and CCR5 on CD4+ T cells. The data suggest that SIL attenuated cellular functions involved in T-cell activation, proliferation, and HIV-1 infection. Silymarin-derived compounds provide cytoprotection by suppressing virus infection, immune activation, and inflammation, and as such may be relevant for both HIV mono-infected and HIV/HCV co-infected subjects.     

Role for CD14, TLR2, and TLR4 in bacterial product-induced anorexia

The cell surface component CD14 and the toll-like receptors 2 and 4 (TLR2 and TLR4) are important in mediating the immune responses to bacterial products in mammals. Using mice genetically deficient in CD14, TLR2, or TLR4, we studied the role of these molecules in the anorectic effects of LPS and muramyl dipeptide (MDP). CD14 or TLR2 knockout (KO) and TLR4-deficient (TLR4-DEF) mice as well as corresponding wild-type (WT) colittermates were injected intraperitoneally at dark onset with LPS (2 microg/mouse), MDP (10 mg/kg), interleukin-1 beta (IL-1 beta, 150 ng/mouse), or vehicle, and food intake was recorded. LPS and MDP reduced food intake in WT mice of all genotypes tested. The anorectic effect of LPS was attenuated (P < 0.04) in CD14-KO and TLR4-DEF mice but not in TLR2-KO (P > 0.05). The anorectic effect of MDP was blunted in CD14-KO and TLR2-KO (P < 0.02) mice but not in TLR4-DEF mice. IL-1 beta reduced food intake similarly in all genotypes tested. These results indicate that CD14 is involved in mediating the anorectic effects of both LPS and MDP. Furthermore, TLR4 and TLR2 are specifically involved in mediating the anorectic effects of LPS and MDP, respectively. The results are consistent with the hypothesis that TLR4 functions as the true LPS receptor and that TLR2 is involved in recognition of gram-positive bacterial products.     

Tissue-specific expression of the human CD19 gene in transgenic mice inhibits antigen-independent B-lymphocyte development.

CD19 is a B-cell-specific member of the immunoglobulin superfamily expressed from early pre-B-cell development until plasma cell differentiation. In vitro studies demonstrate that the CD19 signal transduction molecule can serve as a costimulatory molecule for activation through other B-lymphocyte cell surface molecules. However, much remains to be known regarding how CD19 functions in vivo and whether CD19 has different roles at particular stages of B-cell differentiation. Therefore, transgenic mice overexpressing the human CD19 (hCD19) gene were generated to determine whether this transgene would be expressed in a B-lineage-specific fashion and to dissect the in vivo role of CD19 in B-cell development and activation. Expression of the human transgene product was specifically restricted to all B-lineage cells and appeared early in development as occurs with hCD19. In addition, expression of hCD19 severely impaired the development of immature B cells in the bone marrow, with dramatically fewer B cells found in the spleen, peripheral circulation, and peritoneal cavity. The level of hCD19 expressed on the cell surface correlated directly with the severity of the defect in different transgenic lines. These results demonstrate that the hCD19 gene is expressed in a lineage-specific fashion in mice, indicating that the hCD19 gene may be useful for mediating B-lineage-specific expression of other transgene products. In addition, these results indicate an important role for the lineage-specific CD19 molecule during early B-cell development before antigen-dependent activation.     

CD66b regulates adhesion and activation of human eosinophils

Eosinophils and their products are likely important in the pathophysiology of allergic diseases, such as bronchial asthma, and in host immunity to parasitic organisms. However, the mechanisms for proinflammatory mediator release by eosinophils are poorly understood. CD66b (CEACAM8, CGM6, NCA-95) is a single chain, GPI-anchored, highly glycosylated protein belonging to the carcinoembryonic Ag supergene family. CD66b is an activation marker for human granulocytes; however, its biological functions are largely unknown in eosinophils. We found that CD66b is highly expressed on the surface of human peripheral blood eosinophils isolated from healthy individuals. Engagement of CD66b, but not CD66a, by mAb or a natural ligand, galectin-3, activated a Src kinase family molecule, hemopoietic cell kinase (Hck), and induced cellular adhesion, superoxide production, and degranulation of eosinophils. CD66b molecules were localized in lipid rafts, and disruption of lipid rafts or removal of the GPI anchor inhibited the adhesion and activation of eosinophils. Importantly, CD66b was constitutively and physically associated with a beta2 integrin, CD11b, and cross-linking of CD66b induced a striking clustering of CD11b molecules. Thus, CD66b molecules are involved in regulating adhesion and activation of eosinophils, possibly through their localization in lipid rafts and interaction with other cell surface molecules, such as CD11b. Binding of exogenous or endogenous carbohydrate ligands(s) to CD66b may be important in the release of proinflammatory mediators by human eosinophils.     

Differential Expression of CD96 Surface Molecule Represents CD8+ T Cells with Dissimilar Effector Function during HIV-1 Infection

During HIV-1 infection, immune dysregulation and aberrant lymphocyte functions are well-established characteristics. Cell surface molecules are important for immunological functions and changes in expression can affect lymphocyte effector functions, thereby contributing to pathogenesis and disease progression. In this study we have focused on CD96, a member of the IgG superfamily receptors that have generated increasing recent interest due to their adhesive and co-stimulatory functions in addition to immunoregulatory capacity. CD96 is expressed by both T and NK cells. Although the function of CD96 is not completely elucidated, it has been shown to have adhesive functions and enhance cytotoxicity. Interestingly, CD96 may also have inhibitory functions due to its immunoreceptor tyrosine-based inhibitory motif (ITIM). The clinical significance of CD96 is still comparatively limited although it has been associated with chronic Hepatitis B infection and disease progression. CD96 has not previously been studied in the context of HIV-1 infection, but due to its potential importance in immune regulation and relevance to chronic disease, we examined CD96 expression in relation to HIV-1 pathogenesis. In a cross-sectional analysis, we investigated the CD8⁺ T cell expression of CD96 in cohorts of untreated HIV-1 infected adults with high viral loads (non-controllers) and low viral loads (“elite” controllers). We demonstrated that elite controllers have significantly higher CD96 mean fluorescence intensity on CD8⁺ T cells compared to HIV-1 non-controllers and CD96 expression was positively associated with CD4⁺ T cell counts. Functional assessment showed that CD8⁺ T cells lacking CD96 expression represented a population that produced both perforin and IFN-γ following stimulation. Furthermore, CD96 expression on CD8⁺ T cells was decreased in presence of lipopolysaccharide in vitro. Overall, these findings indicate that down-regulation of CD96 is an important aspect of HIV-1 pathogenesis and differential expression is related to cell effector functions and HIV-1 disease course.     

The moonlighting enzyme CD13: old and new functions to target

Aminopeptidase N (CD13) is a widely expressed ectoenzyme with functions that do not always depend on its enzymatic activity: an aspect that has been overlooked. Numerous CD13-targeting tools have been developed in the last few years. Several of them are already undergoing clinical trials, and there are promising reports on the effectiveness of others in animal models of disease. However, their efficacy might be obscured by their effects on unrecognized functions of CD13, resulting in unexpected complications. The purpose of this review is (i) to discuss the various functions ascribed to CD13 and the possible mechanisms behind them and (ii) to consider some of the questions that need to be answered to achieve a better understanding of the biological relevance of these functions, a more precise interpretation of the results obtained after their manipulation and a more rational design of CD13-targeting agents.