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Biomedical subjects

S A Porcelli

Publications and source records attributed to S A Porcelli.

At least 19 recordsLinked to original sources
PubMed2004

Mycobacterium tuberculosis lipomannan induces apoptosis and interleukin-12 production in macrophages.

The mycobacterial cell wall component lipoarabinomannan (LAM) has been described as a virulence factor of Mycobacterium tuberculosis, and modification of the terminal arabinan residues of this compound with mannose caps (producing mannosyl-capped LAM [ManLAM]) in M. tuberculosis or with phosphoinositol caps (producing phosphoinositol-capped LAM [PILAM]) in Mycobacterium smegmatis has been implicated in various functions associated with these lipoglycans. A structure-function analysis was performed by using LAMs and their biosynthetic precursor lipomannans (LMs) isolated from different mycobacterial species on the basis of their capacity to induce the production of interleukin-12 (IL-12) and/or apoptosis of macrophage cell lines. Independent of the mycobacterial species, ManLAMs did not induce IL-12 gene expression or apoptosis of macrophages, whereas PILAMs induced IL-12 secretion and apoptosis. Interestingly, uncapped LAM purified from Mycobacterium chelonae did not induce IL-12 secretion or apoptosis. Furthermore, LMs, independent of their mycobacterial origins, were potent inducers of IL-12 and apoptosis. The precursor of LM, phosphatidyl-myo-inositol dimannoside, had no activity, suggesting that the mannan core of LM was required for the activity of LM. The specific interaction of LM with Toll-like receptor 2 (TLR-2) but not with TLR-4 suggested that these responses were mediated via the TLR-2 signaling pathway. Our experiments revealed an important immunostimulatory activity of the biosynthetic LAM precursor LM. The ratio of LAM to LM in the cell wall of mycobacteria may be an important determinant of virulence, and enzymes that modify LM could provide targets for development of antituberculosis drugs and for derivation of attenuated strains of M. tuberculosis.

Animals↗
PubMed2002

Intracellular trafficking pathway of newly synthesized CD1b molecules.

The intracellular trafficking of major histocompatibility complex (MHC) class I and class II molecules has evolved to support their function in peptide antigen presentation optimally. We have analyzed the intracellular trafficking of newly synthesized human CD1b, a lipid antigen-presenting molecule, to understand how this relates to its antigen-presenting function. Nascent CD1b was transported rapidly to the cell surface after leaving the Golgi, and then entered the endocytic system by internalization via AP-2-dependent sorting at the plasma membrane. A second sorting event, possibly involving AP-3 complexes, led to prominent accumulation of CD1b in MHC class II compartments (MIICs). Functional studies demonstrated the importance of nascent CD1b for the efficient presentation of a foreign lipid antigen. Therefore, the intracellular trafficking of nascent CD1b via the cell surface to reach MIICs may allow the efficient sampling of lipid antigens present in endocytic compartments.

Adaptor Protein Complex 3↗
PubMed2002

The CD1 family and T cell recognition of lipid antigens.

For many years it was thought that T lymphocytes recognized only peptide antigens presented by MHC class I or class II molecules. Recently, it has become clear that a wide variety of lipids and glycolipids are also targets of the T cell response. This novel form of cell-mediated immune recognition is mediated by a family of lipid binding and presenting molecules known as CD1. The CD1 proteins represent a small to moderate sized family of beta2-microglobulin-associated transmembrane proteins that are distantly related to MHC class I and class II molecules. They are conserved in most or all mammals, and control the development and function of T cell populations that participate in innate and adaptive immune responses through the recognition of self and foreign lipid antigens. Here we review the current state of our understanding of the structure and function of CD1 proteins, and the role of CD1-restricted T cell responses in the immune system.

Animals↗
PubMed2001

Distinct cytokine profiles of neonatal natural killer T cells after expansion with subsets of dendritic cells.

Natural killer T (NKT) cells are a highly conserved subset of T cells that have been shown to play a critical role in suppressing T helper cell type 1-mediated autoimmune diseases and graft versus host disease in an interleukin (IL)-4-dependent manner. Thus, it is important to understand how the development of IL-4- versus interferon (IFN)-gamma-producing NKT cells is regulated. Here, we show that NKT cells from adult blood and those from cord blood undergo massive expansion in cell numbers (500-70,000-fold) during a 4-wk culture with IL-2, IL-7, phytohemagglutinin, anti-CD3, and anti-CD28 mAbs. Unlike adult NKT cells that preferentially produce both IL-4 and IFN-gamma, neonatal NKT cells preferentially produce IL-4 after polyclonal activation. Addition of type 2 dendritic cells (DC2) enhances the development of neonatal NKT cells into IL-4(+)IFN-gamma(-) NKT2 cells, whereas addition of type 1 dendritic cells (DC1) induces polarization towards IL-4(-)IFN-gamma(+) NKT1 cells. Adult NKT cells display limited plasticity for polarization induced by DC1 or DC2. Thus, newly generated NKT cells may possess the potent ability to develop into IL-4(+)IFN-gamma(-) NKT2 cells in response to appropriate stimuli and may thereafter acquire the tendency to produce both IL-4 and IFN-gamma.

Adult↗
PubMed2001

Potent expansion of human natural killer T cells using alpha-galactosylceramide (KRN7000)-loaded monocyte-derived dendritic cells, cultured in the presence of IL-7 and IL-15.

Natural killer T (NKT) cells have an extremely restricted T-cell receptor repertoire, in man consisting of a Valpha24 chain preferentially paired with a Vbeta11 chain, and play crucial roles in various immune responses. Characterization of circulating Valpha24(+)Vbeta11(+)-T cells is hampered by their low frequencies. The alpha-galactosylceramide KRN7000 was reported to be presented by CD1d to NKT cells. Since dendritic cells (DC) are potent antigen presenting cells, and have been shown to express CD1d, we analyzed whether these cells could efficiently mediate expansion of Valpha24(+)Vbeta11(+)-T cells. During a 7-day co-culture of peripheral blood mononuclear cells and KRN7000-loaded mature monocyte derived DC (moDC) in the presence of interleukin-7 (IL-7) and IL-15, we observed up to 76-fold expansion of Valpha24(+)Vbeta11(+)-T cells. The expanded Valpha24(+)Vbeta11(+)-T cells expressed the cytotoxic molecule granzyme B, showed negligible expression of Fas ligand and could be induced to express high levels of interferon-gamma, while retaining the capacity to produce IL-4. B cells, expressing CD1d, could also present KRN7000, but Valpha24(+)Vbeta11(+)-T cell expansion was only observed in the presence of IL-7 and/or IL-15. Considering the low frequency of circulating Valpha24(+)Vbeta11(+)-T cells, the present method for expansion of Valpha24(+)Vbeta11(+)-T cells using KRN7000-loaded mature moDC will be of value for the further characterization of this unique T cell subset.

Adult↗
PubMed2001

CD1 trafficking: invariant chain gives a new twist to the tale.

The CD1 family of MHC class I-related proteins present foreign and self-lipid antigens for specific recognition by T cells. Based on previous experience with MHC class I and II molecules, it seems likely that a thorough knowledge of the intracellular trafficking and localization of CD1 proteins will be essential to fully understand their functions in antigen presentation and immune responses. Two studies in this issue of Immunity take a detailed look at factors affecting the localization of mouse CD1 proteins to the endocytic system of antigen-presenting cells. Their results provide intriguing evidence for the involvement of two critical components of the MHC class II endosomal processing pathway, cathepsin S and the invariant chain, in the normal functioning of CD1.

Amino Acid Motifs↗
PubMed2001

Influence of Mycobacterium bovis bacillus Calmette Guérin on in vitro induction of CD1 molecules in human adherent mononuclear cells.

Nonpeptide antigens (including glycolipids of microbial origin) can be presented to T cells by CD1 molecules expressed on monocyte-derived dendritic cells. These HLA unrestricted responses appear to play a role in host immunity against Mycobacterium tuberculosis and other pathogenic bacteria. It is known that vaccination with Mycobacterium bovis bacillus Calmette-Guérin (BCG) has limited efficacy in many clinical settings, although the reasons for its inadequacy remain unclear. Here we have investigated the influence of BCG on the induction of CD1b on human monocytes by granulocyte-macrophage colony-stimulating factor (GM-CSF), which is believed to be the principal inducer of this antigen-presenting molecule. Although BCG alone led to a slight induction of CD1b expression, this agent reduced markedly the ability of GM-CSF to induce high levels of CD1b that were typically observed in uninfected cells. Inhibition of CD1b expression in BCG-infected monocytes was apparent at both the mRNA transcript and CD1b protein levels. Down-regulation of CD1b expression by BCG was mediated, at least in part, by one or more soluble factors and could not be reversed with high concentrations of GM-CSF or a variety of other cytokines. The present results suggest that BCG could diminish the efficiency of CD1-restricted T-cell responses against nonpeptide mycobacterial antigens by reducing CD1 expression on antigen-presenting cells. These findings have potential implications for understanding the nature of the immune response elicited by BCG in humans and suggest potential strategies that could be important for the development of better vaccines for the prevention of tuberculosis.

Antigen Presentation↗
PubMed2000

Human double-negative T cells in systemic lupus erythematosus provide help for IgG and are restricted by CD1c.

To understand the mechanism of T cell help for IgG production in systemic lupus erythematosus (SLE) we investigated the response of CD4- and CD8-negative (double-negative (DN)) T cells because 1) DN T cells are present at unusually high frequency in patients with SLE and can induce pathogenic autoantibodies; 2) the DN T cell repertoire includes cells restricted by CD1 Ag-presenting molecules; and 3) CD1c is expressed on a population of circulating B cells. We derived DN T cell lines from SLE patients and healthy individuals. In the presence of CD1(+) APCs, DN T cell lines from SLE patients produced both IL-4 and IFN-gamma, whereas DN T cells from healthy donors produced IFN-gamma, but no IL-4. In general, cells from patients with highly active disease produced high levels of IFN-gamma; cells from those with little activity produced high IL-4. Coculture of CD1c-directly reactive T cells from healthy donors with CD1c(+) B cells elicited IgM Abs, but little or no IgG. In contrast, CD1c-directly reactive T cells from SLE patients induced isotype switching, with a striking increase in IgG production. Neutralizing Abs to CD1c inhibited the ability of DN T cells to induce IgG production from CD1c(+) B cells, further indicating that CD1c mediated the T and B cell interaction. IgG production was also inhibited by neutralizing Abs to IL-4, correlating with the cytokine pattern of DN T cells derived from these patients. The data suggest that CD1c-restricted T cells from SLE patients can provide help to CD1c(+) B cells for IgG production and could therefore promote pathogenic autoantibody responses in SLE.

Adolescent↗
PubMed2000

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.

Antibodies, Blocking↗
PubMed2000

CD1b-mediated T cell recognition of a glycolipid antigen generated from mycobacterial lipid and host carbohydrate during infection.

T cells recognize microbial glycolipids presented by CD1 proteins, but there is no information regarding the generation of natural glycolipid antigens within infected tissues. Therefore, we determined the molecular basis of CD1b-restricted T cell recognition of mycobacterial glycosylated mycolates, including those produced during tissue infection in vivo. Transfection of the T cell receptor (TCR) alpha and beta chains from a glucose monomycolate (GMM)-specific T cell line reconstituted GMM recognition in TCR-deficient T lymphoblastoma cells. This TCR-mediated response was highly specific for natural mycobacterial glucose-6-O-(2R, 3R) monomycolate, including the precise structure of the glucose moiety, the stereochemistry of the mycolate lipid, and the linkage between the carbohydrate and the lipid. Mycobacterial production of antigenic GMM absolutely required a nonmycobacterial source of glucose that could be supplied by adding glucose to media at concentrations found in mammalian tissues or by infecting tissue in vivo. These results indicate that mycobacteria synthesized antigenic GMM by coupling mycobacterial mycolates to host-derived glucose. Specific T cell recognition of an epitope formed by interaction of host and pathogen biosynthetic pathways provides a mechanism for immune response to those pathogenic mycobacteria that have productively infected tissues, as distinguished from ubiquitous, but innocuous, environmental mycobacteria.

Animals↗
PubMed2000

Overexpression of CD1d by keratinocytes in psoriasis and CD1d-dependent IFN-gamma production by NK-T cells.

The MHC class I-like protein CD1d is a nonpolymorphic molecule that plays a central role in development and activation of a subset of T cells that coexpress receptors used by NK cells (NK-T cells). Recently, T cells bearing NK receptors were identified in acute and chronic lesions of psoriasis. To determine whether NK-T cells could interact with epidermal cells, we examined the pattern of expression of CD1d in normal skin, psoriasis, and related skin disorders, using a panel of CD1d-specific mAbs. CD1d was expressed by keratinocytes in normal skin, although expression was at a relatively low level and was generally confined to upper level keratinocytes immediately beneath the lipid-rich stratum corneum. In contrast, there was overexpression of CD1d in chronic, active psoriatic plaques. CD1d could be rapidly induced on keratinocytes in normal skin by physical trauma that disrupted barrier function or by application of a potent contact-sensitizing agent. Keratinocytes displayed enhanced CD1d following exposure to IFN-gamma. Combining CD1d-positive keratinocytes with human NK-T cell clones resulted in clustering of NK-T cells, and while no significant proliferation ensued, NK-T cells became activated to produce large amounts of IFN-gamma. We conclude that CD1d can be expressed in a functionally active form by keratinocytes and is up-regulated in psoriasis and other inflammatory dermatoses. The ability of IFN-gamma to enhance keratinocyte CD1d expression and the subsequent ability of CD1d-positive keratinocytes to activate NK-T cells to produce IFN-gamma, could provide a mechanism that contributes to the pathogenesis of psoriasis and other skin disorders.

Adult↗
PubMed2000

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

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

Antigen Presentation↗
PubMed2000

Alpha-glycosylceramides enhance the antitumor cytotoxicity of hepatic lymphocytes obtained from cancer patients by activating CD3-CD56+ NK cells in vitro.

Alpha-glycosylceramides, such as alpha-galactosylceramide and alpha-glucosylceramide, induce antitumor immunity in various murine cancer models. In the murine hepatic metastasis model, V alpha 14 TCR+NK1.1+ T cells, which accumulate preferentially in the liver, are considered to play a key role in the induction of antitumor immunity by alpha-glycosylceramides. We recently reported that V alpha 24 TCR+ NKT cells, the human homologues of murine V alpha 14 TCR+NK1.1+ cells, are rarely seen among freshly isolated human hepatic lymphocytes. Therefore, it is important to examine whether alpha-glycosylceramides also enhance the antitumor cytotoxicity of human hepatic lymphocytes, as they have been shown to do in murine systems, to determine the usefulness of alpha-glycosylceramides in cancer immunotherapy in humans. Here, we show that alpha-glycosylceramides greatly enhance the cytotoxicity of human hepatic lymphocytes obtained from cancer patients against the tumor cell lines, K562 and Colo201, in vitro. The direct effector cells of the elicited cytotoxicity were CD3-CD56+ NK cells. Even though V alpha 24 TCR+NKT cells proliferated remarkably in response to alpha-glycosylceramides, they did not contribute directly to the cytotoxicity. Our observations strongly suggest the potential usefulness of alpha-glycosylceramides for immunotherapy of liver cancer in humans based on their ability to activate CD3-CD56+ NK cells in the liver.

Adjuvants, Immunologic↗
PubMed2000

Human CD1b and CD1c isoforms survey different intracellular compartments for the presentation of microbial lipid antigens.

CD1b and CD1c are antigen-presenting molecules that mediate recognition of bacterial lipids by T cells, but it is currently not known whether these two molecules are redundant or are specialized to perform different immunological functions. Here, we show that the distribution of CD1c in human dendritic cells was characterized by a high ratio of cell surface to intracellular molecules, whereas CD1b showed a reciprocal pattern of distribution. In contrast to the accumulation of CD1b in lysosomal major histocompatibility complex class II compartments, intracellular CD1c molecules accumulated in other endocytic compartments, most likely early and late endosomes. Deletion of the cytoplasmic tail of CD1c, containing a tyrosine-based internalization motif, abolished most of its intracellular localization. Functional studies using T cells specific for defined lipid antigens revealed that in contrast to CD1b-mediated antigen presentation, antigen presentation by CD1c was resistant to drugs inhibiting endosomal acidification and was independent of endosomal localization of CD1c. Taken together, these results support the hypothesis that CD1b and CD1c are specialized to survey the lipid content of different intracellular compartments.

Antigen Presentation↗
PubMed2000

Analysis of human V alpha 24+ CD4+ NKT cells activated by alpha-glycosylceramide-pulsed monocyte-derived dendritic cells.

Human V alpha 24+ NKT cells with an invariant TCR (V alpha 24-J alpha Q) have been shown to be specifically activated by synthetic glycolipids such as alpha-galactosylceramide and alpha-glucosylceramide in a CD1d-restricted and V alpha 24 TCR-mediated manner. We recently characterized V alpha 24+ CD4- CD8- double negative (DN) NKT cells using alpha-galactosylceramide-pulsed monocyte-derived dendritic cells. Here, we compare V alpha 24+ CD4+ NKT cells with human V alpha 24+ DN NKT cells from the same donor using alpha-galactosylceramide-pulsed monocyte-derived dendritic cells. Human V alpha 24+ CD4+ NKT cells were phenotypically and functionally similar to the human V alpha 24+ DN NKT cells characterized previously. Both of them use V alpha 24-J alpha Q-V beta 11 TCR and express CD161 (NKR-P1A), but not the other NK receptors tested so far. They also produce cytokines such as IL-4 and IFN-gamma, and, in regard to IL-4 production, V alpha 24+ CD4+ NKT cells produce more IL-4 than V alpha 24+ DN NKT cells. The cells exhibit marked cytotoxic activity against the U937 tumor cell line, but not against the NK target cell line, K562. Although at least some of the factors responsible for the stimulation of V alpha 24+ NKT cells have been clarified, little is known regarding the killing phase of these cells. Here we show that the cytotoxic activity of V alpha 24+ NKT cells against U937 cells is mediated mainly through the perforin pathway and that ICAM-1/LFA-1 as well as CD44/hyaluronic acid interactions are important for the effector phase of V alpha 24+ NKT cell-mediated cytotoxicity against U937 cells.

Antigens, CD1↗
PubMed2000

Evidence for human CD4+ T cells in the CD1-restricted repertoire: derivation of mycobacteria-reactive T cells from leprosy lesions.

Both the CD4-CD8- (double negative) and CD4-CD8+ T cell lineages have been shown to contain T cells which recognize microbial lipid and glycolipid Ags in the context of human CD1 molecules. To determine whether T cells expressing the CD4 coreceptor could recognize Ag in the context of CD1, we derived CD4+ T cell lines from the lesions of leprosy patients. We identified three CD4+ Mycobacterium leprae-reactive, CD1-restricted T cell lines: two CD1b restricted and one CD1c restricted. These T cell lines recognize mycobacterial Ags, one of which has not been previously described for CD1-restricted T cells. The response of CD4+ CD1-restricted T cells, unlike MHC class II-restricted T cells, was not inhibited by anti-CD4 mAb, suggesting that the CD4 coreceptor does not impact positive or negative selection of CD1-restricted T cells. The CD4+ CD1-restricted T cell lines produced IFN-gamma and GM-CSF, the Th1 pattern of cytokines required for cell-mediated immunity against intracellular pathogens, but no detectable IL-4. The existence of CD4+ CD1-restricted T cells that produce a Th1 cytokine pattern suggests a contributory role in immunity to mycobacterial infection.

Antigen Presentation↗
PubMed2000

CD1c-mediated T-cell recognition of isoprenoid glycolipids in Mycobacterium tuberculosis infection.

The discovery of the CD1 antigen presentation pathway has expanded the spectrum of T-cell antigens to include lipids, but the range of natural lipid antigens and functions of CD1-restricted T cells in vivo remain poorly understood. Here we show that the T-cell antigen receptor and the CD1c protein mediate recognition of an evolutionarily conserved family of isoprenoid glycolipids whose members include essential components of protein glycosylation and cell-wall synthesis pathways. A CD1c-restricted, mycobacteria-specific T-cell line recognized two previously unknown mycobacterial hexosyl-1-phosphoisoprenoids and structurally related mannosyl-beta1-phosphodolichols. Responses to mannosyl-beta1-phosphodolichols were common among CD1c-restricted T-cell lines and peripheral blood T lymphocytes of human subjects recently infected with M. tuberculosis, but were not seen in naive control subjects. These results define a new class of broadly distributed lipid antigens presented by the CD1 system during infection in vivo and suggest an immune mechanism for recognition of senescent or transformed cells that are known to have altered dolichol lipids.

Adult↗