Immunity to glycolipid antigens in microbial infections.

T cells recognize ligands of different chemical structures. Recently, it has become clear that also self glycosphingolipids and bacterial lipoglycans may act as T cell stimulatory ligands. This type of antigen recognition is restricted by the non-polymorphic CD1 molecules, which have a structure resembling that of classical MHC molecules. Glycolipids insert their hydrophobic lipid tails in two pockets below the antigen-binding groove and position their hydrophilic heads on the external part of CD1 molecules. TCR interacts with these carbohydrates and discriminates their structural variations. Glycolipid-specific T cells may provide protection during bacterial and parasite infection probably with different mechanisms: by secreting pro-inflammatory lymphokines, by the direct killing of infected target cells, and by helping specific B cells in Ig production. Lipoglycans represent excellent candidates for new anti-microbial vaccines due to their wide distribution in the microbial world and their structural composition which does not change and thus cannot give rise to escape mutants. Moreover, these vaccines might induce anti-microbial protective T cell responses in the whole population due to the non-polymorphic nature of CD1 presenting molecules.     

A method for production of recombinant mCD1d protein in insect cells

CD1 proteins constitute a third class of antigen-presenting molecules. They are cell surface glycoproteins, expressed as approximately 50-kDa glycosylated heavy chains that are noncovalently associated with beta2-microglobulin. They bind lipids rather than peptides. Although their structure confirms the similarity of CD1 proteins to MHC class I and class II antigen presenting molecules, the mCD1d groove is relatively narrow, deep, and highly hydrophobic and it has two binding pockets instead of the several shallow pockets described for the classical MHC-encoded antigen-presenting molecules. Based upon their amino acid sequences, such a hydrobphobic groove provides an ideal environment for the binding of lipid antigens. The Natural Killer T (NKT) cells use their TCR to recognize glycolipids bound to or presented by CD1d. T cells reactive to lipids presented by CD1 have been involved in the protection against autoimmune and infectious diseases and in tumor rejection. Thus, the ability to identify, purify , and track the response of CD1-reactive NKT cell is of great importance . The generation of tetramers of alpha Galactosyl ceramide (a-Galcer) with CD1d has significant insight into the biology of NKT cells. Tetramers constructed from other CD1 molecules have also been generated and these new reagents have greatly expanded the knowledge of the functions of lipid-reactive T cells, with potential use in monitoring the response to lipid-based vaccines and in the diagnosis of autoimmune diseases and other treatments.     

CD1-mediated antigen presentation of glycosphingolipids

CD1 proteins are distinguished by their ability to present lipid antigens to T cells. Group II CD1 or CD1d molecules are recognized by the specialized NK T-cell subset, and this reactivity can be greatly augmented by alpha-galactosylceramide, a glycosphingolipid derived from a marine sponge. Human CD1b, which is only distantly related to the CD1d molecules, can present mammalian glycosphingolipids (gangliosides) to autoreactive T-cell clones derived from multiple sclerosis patients. Thus, CD1 responsive and glycosphingolipid-reactive cells may play an important immune regulatory role, in addition to their well-characterized role in the response to microbial lipids.     

Anatomy of CD1-lipid antigen complexes

CD1 proteins bind lipids to form antigen complexes that contact T-cell receptors and activate T cells. Recent crystal structures of CD1 proteins show that their antigen-binding grooves are composed of up to four pockets (A', C', F' and T') and two antigen portals (C' and F'). Although certain structural features are conserved among CD1 proteins, the grooves of CD1a, CD1b and CD1d differ in the number, shape and connectivity of their antigen-binding pockets. Here, we outline how the portals and pockets of CD1 antigen-binding grooves influence ligand specificity and facilitate the presentation of a surprisingly diverse set of antigenic lipids, glycolipids, lipopeptides and even small, non-lipidic molecules.     

The A' and F' pockets of human CD1b are both required for optimal presentation of lipid antigens to T cells

CD1 proteins are unique in their ability to present lipid Ags to T cells. Human CD1b shares significant amino acid homology with mouse CD1d1, which contains an unusual putative Ag-binding groove formed by two large hydrophobic pockets, A' and F'. We investigated the function of the amino acid residues that line the A' and F' pockets of CD1b by engineering 36 alanine-substitution mutants and analyzing their ability to present mycobacterial glycolipid Ags. Two lipid Ags presented by CD1b were studied, a naturally occurring glucose monomycolate (GMM) isolated from mycobacteria, which contains two long alkyl chains (C54-C62 and C22-C24) and synthetic GMM (sGMM), which includes two short alkyl chains (C18 and C14). We identified eight residues in both the A' and F' pockets that were involved in the presentation of both GMM and sGMM to T cells. Interestingly, four additional residues located in the distal portion of the A' pocket were required for the optimal presentation of GMM, but not sGMM. Conversely, nine residues located between the center of the groove and the F' pocket were necessary for the optimal presentation of sGMM, but not GMM. These data indicate that both the A' and F' pockets of human CD1b are required for the presentation of lipid Ags to T cells.     

Diverse endogenous antigens for mouse NKT cells: self-antigens that are not glycosphingolipids

NKT cells with an invariant Ag receptor (iNKT cells) represent a highly conserved and unique subset of T lymphocytes having properties of innate and adaptive immune cells. They have been reported to regulate a variety of immune responses, including the response to cancers and the development of autoimmunity. The development and activation of iNKT cells is dependent on self-Ags presented by the CD1d Ag-presenting molecule. It is widely believed that these self-Ags are glycosphingolipids (GSLs), molecules that contain ceramide as the lipid backbone. In this study, we used a variety of methods to show that mammalian Ags for mouse iNKT cells need not be GSLs, including the use of cell lines deficient in GSL biosynthesis and an inhibitor of GSL biosynthesis. Presentation of these Ags required the expression of CD1d molecules that could traffic to late endosomes, the site where self-Ag is acquired. Extracts of APCs contain a self-Ag that could stimulate iNKT cells when added to plates coated with soluble, rCD1d molecules. The Ag(s) in these extracts are resistant to sphingolipid-specific hydrolase digestion, consistent with the results using live APCs. Lyosphosphatidylcholine, a potential self-Ag that activated human iNKT cell lines, did not activate mouse iNKT cell hybridomas. Our data indicate that there may be more than one type of self-Ag for iNKT cells, that the self-Ags comparing mouse and human may not be conserved, and that the search to identify these molecules should not be confined to GSLs.     

Dendritic Cell Immunoreceptor Is a New Target for Anti-AIDS Drug Development: Identification of DCIR/HIV-1 Inhibitors

The HIV-1 pandemic continues to expand while no effective vaccine or cure is yet available. Existing therapies have managed to limit mortality and control viral proliferation, but are associated with side effects, do not cure the disease and are subject to development of resistance. Finding new therapeutic targets and drugs is therefore crucial. We have previously shown that the dendritic cell immunoreceptor (DCIR), a C-type lectin receptor expressed on dendritic cells (DCs), acts as an attachment factor for HIV-1 to DCs and contributes to HIV-1 transmission to CD4⁺ T lymphocytes (CD4TL). Directly involved in HIV-1 infection, DCIR is expressed in apoptotic or infected CD4TL and promotes trans-infection to bystander cells. Here we report the 3D modelling of the extracellular domain of DCIR. Based on this structure, two surface accessible pockets containing the carbohydrate recognition domain and the EPS binding motif, respectively, were targeted for screening of chemicals that will disrupt normal interaction with HIV-1 particle. Preliminary screening using Raji-CD4-DCIR cells allowed identification of two inhibitors that decreased HIV-1 attachment and propagation. The impact of these inhibitors on infection of DCs and CD4TL was evaluated as well. The results of this study thus identify novel molecules capable of blocking HIV-1 transmission by DCs and CD4TL.     

Molecular recognition of human CD1b antigen complexes: evidence for a common pattern of interaction with alpha beta TCRs

Ag-specific T cell recognition is mediated through direct interaction of clonotypic TCRs with complexes formed between Ag-presenting molecules and their bound ligands. Although characterized in substantial detail for class I and class II MHC encoded molecules, the molecular interactions responsible for TCR recognition of the CD1 lipid and glycolipid Ag-presenting molecules are not yet well understood. Using a panel of epitope-specific Abs and site-specific mutants of the CD1b molecule, we showed that TCR interactions occur on the membrane distal aspects of the CD1b molecule over the alpha1 and alpha2 domain helices. The location of residues on CD1b important for this interaction suggested that TCRs bind in a diagonal orientation relative to the longitudinal axes of the alpha helices. The data point to a model in which TCR interaction extends over the opening of the putative Ag-binding groove, making multiple direct contacts with both alpha helices and bound Ag. Although reminiscent of TCR interaction with MHC class I, our data also pointed to significant differences between the TCR interactions with CD1 and MHC encoded Ag-presenting molecules, indicating that Ag receptor binding must be modified to accommodate the unique molecular structure of the CD1b molecule and the unusual Ags it presents.     

Lowering glycosphingolipid levels in CD4+ T cells attenuates T cell receptor signaling, cytokine production, and differentiation to the Th17 lineage

Lipid rafts reportedly have a role in coalescing key signaling molecules into the immunological synapse during T cell activation, thereby modulating T cell receptor (TCR) signaling activity. Recent findings suggest that a correlation may exist between increased levels of glycosphingolipids (GSLs) in the lipid rafts of T cells and a heightened response of those T cells toward activation. Here, we show that lowering the levels of GSLs in CD4(+) T cells using a potent inhibitor of glucosylceramide synthase (Genz-122346) led to a moderation of the T cell response toward activation. TCR proximal signaling events, such as phosphorylation of Lck, Zap70 and LAT, as well as early Ca(2+) mobilization, were attenuated by treatment with Genz-122346. Concomitant with these events were significant reductions in IL-2 production and T cell proliferation. Similar findings were obtained with CD4(+) T cells isolated from transgenic mice genetically deficient in GM3 synthase activity. Interestingly, lowering the GSL levels in CD4(+) T cells by either pharmacological inhibition or disruption of the gene for GM3 synthase also specifically inhibited the differentiation of T cells to the Th(17) lineage but not to other Th subsets in vitro. Taken together with the recently reported effects of Raftlin deficiency on Th(17) differentiation, these results strongly suggest that altering the GSL composition of lipid rafts modulates TCR signaling activity and affects Th(17) differentiation.     

Dual role of the CD44 molecule in T cell adhesion and activation

Studies of T cell adhesion and activation reveal two new functions of the CD44 molecule, a molecule now recognized to be identical to three molecules of functional interest: Pgp-1, Hermes, and extracellular matrix receptor type III (ECMRIII). By screening for mAb which inhibit T cell adhesion to E, we have identified a functionally unique CD44-specific mAb, NIH44-1, which partially inhibits T cell rosetting by binding to CD44 on the E. NIH44-1, which immunoprecipitates a protein of 85 to 110 kDa with broad tissue distribution, was determined to be specific for CD44 based on comparison of its tissue distribution with multiple CD44-specific reference mAb and sequential immunoprecipitation with such mAb. Anticipating a role for many adhesion molecules in signal transduction, we studied the effect of CD44 mAb on T cell activation and observed that CD44 mAb dramatically augments T cell proliferation induced by CD3- and CD2-receptor-mediated activation. The augmentation of the response to immobilized CD3 mAb by exhaustively monocyte-depleted T cells indicates that augmentation can be mediated by binding to the T cell. Thus, our studies demonstrate specific new roles for CD44 in T cell adhesion and activation. Furthermore, we suggest that: 1) CD44 has a role in adhesion of cells of multiple lineages; and 2) CD44 may participate in adhesion not (only) by functioning as an adhesion receptor but rather by serving as an anchorage site for other adhesion molecules.     

Glycolipid Sensing and Innate Immunity in Paracoccidioidomycosis

Distinct glycolipid profiles are described in microorganisms, which have been shown to modulate the innate immune system. We tested the hypothesis that glycosphingolipids from Paracoccidioides brasiliensis have immunomodulatory properties on monocytes and dendritic cells of two groups of healthy individuals, one cured of paracoccidioidomycosis in the past (CUR-I) and the other nonexposed to P. brasiliensis (HNE-I). Two classes of glycosphingolipids purified from yeast cells were evaluated: a neutral glycosphingolipid, monohexosylceramide (CMH), and acidic glycosylinositolphosphorylceramides (GIPCs). Both glycosphingolipids affected the functioning of innate immunity cells, interfering with the antigen presenting process: P. brasiliensis yeast cells phagocytosis, IL-10 secretion, and costimulatory molecules and recognition receptors expression by monocytes were altered, while dendritic cell antigen presentation to autologous T cells was markedly down-modulated as shown by reduced T-cell proliferative responses. The mechanisms by which CMH and GIPCs exert their effects differ since the target cells did not always respond similarly to the challenge with the glycosphingolipids. Moreover, CUR-I and HNE-I presented different responses to the glycosphingolipids. Differences not only in the glycosphingolipid structure (such as the polar head group or the ceramide moiety), but also in the innate immunity properties of CUR-I and HNE-I, may underlie these differences and contribute to individual’s susceptibility or resistance to develop paracoccidioidomycosis.     

Differential costimulatory effects of adhesion molecules B7, ICAM-1, LFA-3, and VCAM-1 on resting and antigen-primed CD4+ T lymphocytes.

Optimal proliferation of T cells although initiated via ligation of the CD3/TCR complex requires additional stimulation resulting from adhesive interactions between costimulatory receptors (R) on T cells and their counter-R on APC. At least four distinct adhesion molecules (counter-R) present on APC, B7, ICAM-1 (CD54), LFA-3 (CD58), and VCAM-1 have been individually shown to costimulate T cell activation. Because some of these molecules may be expressed simultaneously on APC, it has been difficult to examine relative contributions of individual counter-R during the induction of T cell proliferation. We have produced soluble IgC gamma 1 fusion chimeras (receptor globulins or Rg) of B7, ICAM-1, LFA-3, and VCAM-1 and compared their relative abilities to costimulate proliferation of resting or Ag-primed CD4+ T cells. When co-immobilized with mAb directed at TCR alpha beta or CD3 but not CD2 or CD28, each Rg induced proliferation of both resting and Ag-primed CD4+ cells. In contrast, similarly co-immobilized CD7 Rg or ELAM-1 Rg were ineffective. Resting CD4+ T cells produced more IL-2, expressed significantly higher levels of IL-2R alpha, and proliferated more efficiently when costimulated with either ICAM-1 Rg or VCAM-1 Rg than with B7 Rg or LFA-3 Rg. CD4+ CD45RO+ memory T cells proliferated more vigorously in response to the costimulation by each of the four Rg than CD4+ CD45RA+ naive T cells. In contrast with the behavior of resting CD4+ T cells, proliferation of Ag-preactivated CD4+ T cells was most efficient when costimulated by B7 Rg. The costimulatory effect of LFA-3 Rg on Ag-primed CD4+ T cells was weaker than that of B7 Rg but was significantly greater than that of either ICAM-1 Rg or VCAM-1 Rg. These results suggest that resting and Ag-primed CD4+ T cells preferentially respond by proliferation to different costimulatory counter-R. ICAM-1 and VCAM-1 may be involved in the initiation of proliferation of Ag-responsive T cells, and B7 and LFA-3 may facilitate sustained proliferation of Ag-primed T cells. The cumulative costimulation by the above counter-R may facilitate optimal expression of various regulatory and effector functions of T cells.     

Costimulation of T cell receptor-triggered IL-2 production by Jurkat T cells via fibroblast growth factor receptor 1 upon its engagement by CD56

Recent studies have demonstrated that neural cell adhesion molecule (NCAM) is involved in multiple adhesive interactions with several different classes of ligands on the cell surface and in the extracellular matrix. One of these ligands is fibroblast growth factor receptor (FGFR) that is expressed on neural cells. While it is known that CD56 is a molecular isoform of NCAM expressed on human NK cells and a subset of T cells, it remains poorly characterized, with its ligand unidentified. Therefore, we were prompted to examine if CD56 molecules on NK cells interact with FGFR expressed on T cells. We demonstrate that ligation of FGFR1 beta on J.C2-14 Jurkat T cells by CD56 on fixed NK-92 cells costimulates TCR/CD3-triggered IL-2 production. CD56-binding mAbs inhibited the costimulatory effect of NK-92 cells in 50-75%. Flow cytometric analysis and cell adhesion assays showed that FGFR1 beta/Fc and FGFR2 beta/Fc chimeric proteins bind to NK-92 cells. The binding of FGFR1 beta/Fc protein to CD56 molecules was verified by immunoprecipitation of CD56 with anti-CD56 mAb followed by Western blotting with FGFR1 beta/Fc. These findings suggest that ligation of FGFR1 by CD56 may contribute to the interaction between NK cells and T cells that we have postulated in our previous studies.     

Staphylococcal exotoxin superantigens induce human immunodeficiency virus type 1 expression in naturally infected CD4+ T cells.

A high proportion of Staphylococcus aureus strains of human origin produce one or more exotoxins. In vivo, these toxins may give rise to a variety of clinical syndromes. In vitro, staphylococcal exotoxins have been shown to bind both to human leukocyte antigen (HLA) class II molecules on antigen-presenting cells and to the T-cell receptors on large fractions of T cells. The result of this interaction may be proliferation of the T cells, T-cell anergy, or apoptosis, depending on several factors, including the state of the responding cells and the presence of accessory molecules. Using naturally infected peripheral blood mononuclear cells depleted of CD8+ T cells, we have shown that staphylococcal exotoxins are powerful inducers of human immunodeficiency virus type 1 expression and that they induce expression at low concentrations and with greater efficiency than other T-cell mitogens. Human immunodeficiency virus type 1 was produced entirely by CD4+ T cells in this model; monocytes were expendable both as a source of virus and as a source of HLA class II molecules as long as other cells expressing HLA class II molecules were present. The results suggest that infection by S. aureus may be a cofactor in the immunopathogenesis of AIDS.     

Induction of HLA class II molecules on human T cells: relationship to immunoregulation and the pathogenesis of AIDS.

Human T cells express HLA class II molecules upon activation. The factors that regulate the induction of expression of these molecules are for the most part unknown. Here we report preliminary results indicating that tumor necrosis factor-alpha (TNF-alpha) regulates the induction of cell-surface HLA-DR, DO, and DP molecules in human T cells stimulated with PHA. In contrast, recombinant interferon-gamma (rIFN-gamma), recombinant interleukin-1 alpha (rIL-1 alpha), or rIL-4 appear to have no effect on class II expression. The role of class II molecules on activated T cells is discussed in relationship to immunoregulation and the progression of HIV infection. Three non-mutually exclusive hypotheses are discussed. In the first hypothesis, we consider the role of these class II molecules in antigen presentation of endogenously synthesized HIV envelope by CD4+ cells. The second is a clonal inactivation of virus-specific helper T cells that might occur as a consequence of a direct T cell to T cell interaction and a bypass of the "accessory signal" normally delivered by antigen-presenting cells such as macrophages. The third is a molecular mimicry between HIV envelope proteins and HLA class II molecules, which may lead to the development of autoimmunity against CD4+ T-cell-expressing class II molecules.     

Bromelain treatment of human T cells removes CD44, CD45RA, E2/MIC2, CD6, CD7, CD8, and Leu 8/LAM1 surface molecules and markedly enhances CD2-mediated T cell activation.

Treatment of T cells with the cysteine protease bromelain has been widely used to enhance the binding of human T cells to human E (autologous E rosettes) and has been shown to remove surface T cell CD44 molecules. Ligand binding to CD44 has been shown to markedly augment T cell activation. To study the activation potential of bromelain-treated CD44 T cells, we have compared the proliferation of sham- and bromelain-treated normal human PBMC to mitogenic CD2 mAb. We found that bromelain not only removed T cell CD44, but also removed the CD45RA isoform of CD45 as well as E2/MIC2, CD6, CD7, CD8, and Leu 8/LAM1 molecules. T cell proliferation in response to CD2 mAb was increased 325% in bromelain-treated PBMC compared to sham-treated PBMC (p < 0.005). Reciprocal treatment experiments using purified T cells and monocytes demonstrated that the enhancement of T cell CD2 activation by bromelain occurred only when T cells were treated with bromelain and was accompanied by increased adhesion of T cells to monocytes. These data demonstrate that expression of portions of the extracellular domains of the CD44, CD45RA, E2/MIC2, CD6, CD7, CD8, and Leu 8/LAM1 surface molecules are not required for CD2 activation of human T cells. Rather, the removal of these surface molecules by bromelain is associated with enhanced T cell-monocyte aggregation and enhanced CD2-mediated T cell activation. Taken together with data that CD44, E2/MIC2, CD6, and CD7 mAb inhibit CD2/lymphocyte function-associated Ag-3-mediated cellular interactions and also augment CD2-mediated triggering of T cells, these data suggest that members of the bromelain-sensitive group of surface molecules may comprise a set of CD2-associated adhesion ligands that acts in concert to modulate human T cell activation.     

T-cell activation induced by anti-CD3 × anti-B-cell lymphoma monoclonal antibody is enhanced by pretreatment of lymphoma cells with soluble CD40 ligand

 T cells play a key role in the control of abnormal B cell proliferation. Factors that play a role in inadequate T cell responses include absence of expression of costimulatory and adhesion molecules by the malignant B cells and lack of cytotoxic T cells specific for tumor-associated antigens. A number of approaches have been used to enhance T cell response against malignant B cells. Agents such as soluble CD40 ligand can enhance expression of costimulatory molecules by the malignant B cells and improve their ability to activate T cells. Anti-CD3-based bispecific antibodies can retarget T cells toward the tumor cells irrespective of T cell specificity. We used the V 38C13 murine lymphoma model to assess whether the combination of soluble CD40 ligand and anti-CD3-based bispecific antibody can enhance T cell activation induced by malignant B cells more effectively than either approach alone. Expression of CD80, CD86, and ICAM-1 on lymphoma cells was up-regulated by soluble CD40 ligand. Syngeneic T cells were activated more extensively by lymphoma cells when the lymphoma cells were pre-treated with soluble CD40 ligand. Bispecific-antibody induced T cell activation was more extensive when lymphoma cells pretreated with soluble CD40 ligand were present. The combination of soluble CD40 ligand plus bispecific antibody enhanced the median survival of mice compared to mice treated with bispecific anibody alone. We conclude that pretreatment of tumor cells with agents capable of inducing costimulatory molecule expression, such as soluble CD40 ligand can enhance the ability of malignant B cells to activate T cells. This effect is enhanced by the addition of bispecific antibody. The combination of enhanced expression of costimulatory molecules and retargeting of T cells by bispecific antibody may allow for a more effective T-cell-based immunotherapy. Accepted: 14 October 1997     

Th17 augmentation in OTII TCR plus T cell-selective type 1 sphingosine 1-phosphate receptor double transgenic mice

Sphingosine 1-phosphate (S1P) in blood and lymph controls lymphoid traffic and tissue migration of T cells through signals from the type 1 S1PR (S1P(1)), but less is known of effects of the S1P-S1P(1) axis on nonmigration functions of T cells. CD4 T cells from a double transgenic (DTG) mouse express OTII TCRs specific for OVA peptide 323-339 (OVA) and a high level of transgenic S1P(1), resistant to suppression by T cell activation. OVA-activated DTG CD4 T cells respond as expected to S1P by chemotactic migration and reduction in secretion of IFN-gamma. In addition, DTG CD4 T cells stimulated by OVA secrete a mean of 2.5-fold more IL-17 than those from OTII single transgenic mice with concomitantly higher levels of mRNA encoding IL-17 by real-time PCR and of CD4 T cells with intracellular IL-17 detected by ELISPOT assays. OVA challenge of s.c. air pockets elicited influx of more OTII TCR-positive T cells producing a higher level of IL-17 in DTG mice than OTII control mice. Augmentation of the number and activity of Th17 cells by the S1P-S1P(1) axis may thus enhance host defense against microbes and in other settings increase host susceptibility to autoimmune diseases.