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Steven A Porcelli

Publications and source records attributed to Steven A Porcelli.

At least 19 recordsLinked to original sources
PubMed2006

Characterization of the protective T-cell response generated in CD4-deficient mice by a live attenuated Mycobacterium tuberculosis vaccine.

The global epidemic of tuberculosis, fuelled by acquired immune-deficiency syndrome, necessitates the development of a safe and effective vaccine. We have constructed a DeltaRD1DeltapanCD mutant of Mycobacterium tuberculosis (mc(2)6030) that undergoes limited replication and is severely attenuated in immunocompromised mice, yet induces significant protection against tuberculosis in wild-type mice and even in mice that completely lack CD4(+) T cells as a result of targeted disruption of their CD4 genes (CD4(-/-) mice). Ex vivo studies of T cells from mc(2)6030-immunized mice showed that these immune cells responded to protein antigens of M. tuberculosis in a major histocompatibility complex (MHC) class II-restricted manner. Antibody depletion experiments showed that antituberculosis protective responses in the lung were not diminished by removal of CD8(+), T-cell receptor gammadelta (TCR-gammadelta(+)) and NK1.1(+) T cells from vaccinated CD4(-/-) mice before challenge, implying that the observed recall and immune effector functions resulting from vaccination of CD4(-/-) mice with mc(2)6030 were attributable to a population of CD4(-) CD8(-) (double-negative) TCR-alphabeta(+), TCR-gammadelta(-), NK1.1(-) T cells. Transfer of highly enriched double-negative TCR-alphabeta(+) T cells from mc(2)6030-immunized CD4(-/-) mice into naive CD4(-/-) mice resulted in significant protection against an aerosol tuberculosis challenge. Enriched pulmonary double-negative T cells transcribed significantly more interferon-gamma and interleukin-2 mRNA than double-negative T cells from naive mice after a tuberculous challenge. These results confirmed previous findings on the potential for a subset of MHC class II-restricted T cells to develop and function without expression of CD4 and suggest novel vaccination strategies to assist in the control of tuberculosis in human immunodeficiency virus-infected humans who have chronic depletion of their CD4(+) T cells.

Adoptive Transfer↗
PubMed2006

An N-linked glycan modulates the interaction between the CD1d heavy chain and beta 2-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.

Amino Acid Sequence↗
PubMed2006

Pivotal advance: alpha-galactosylceramide induces protection against lipopolysaccharide-induced shock.

alpha-galactosylceramide, a natural killer T cell ligand, and its synthetic homolog, KRN7000, consistently influence IFN-gamma and TNF-alpha release, both mediators of LPS-induced shock. To modify the course of endotoxin shock, we injected KRN7000 at different time points of experimental systemic Shwartzman reaction. Mice treated with KRN7000 survived when it was injected within 2 h before and after LPS challenge. Mice survival was associated with low levels of T helper 1 (Th1) cytokines, such as IFN-gamma and TNF-alpha. By contrast, protection from endotoxin shock was associated with an increase of T helper 2 (Th2) cytokines, like IL-4 and IL-10. A role of Th2 cytokines in counteracting LPS-induced shock was supported by experiments in which the protection against Shwartzman reaction by KRN7000 was abrogated by in vivo coadministration of anti-Th2 cytokines antibodies. In addition, cytofluorimetric analysis showed that surviving animals have higher percentages of NKT-IL-10-positive cells and lower percentages of NKT-IFN-gamma and macrophages/TNF-alpha-stained cells than nonprotected mice. Taken together, our data demonstrate that KRN7000 treatment given at times near LPS challenge is protective for endotoxin shock inhibiting IFN-gamma and TNF-alpha release. Moreover, KRN7000-mediated protection occurs through an increased production of IL-4 and IL-10, which are mainly secreted by NKT cells. Since IFN-gamma release by NKT requires a longer TCR stimulation than that required for Th2 cytokines production, we demonstrate that timing of KRN7000 in vivo exposure affect the pattern of cytokines expression protecting animals by endotoxin shock.

Animals↗
PubMed2006

Expression of CD1d molecules by human schwann cells and potential interactions with immunoregulatory invariant NK T cells.

CD1d-restricted NKT cells expressing invariant TCR alpha-chains (iNKT cells) produce both proinflammatory and anti-inflammatory cytokines rapidly upon activation, and are believed to play an important role in both host defense and immunoregulation. To address the potential implications of iNKT cell responses for infectious or inflammatory diseases of the nervous system, we investigated the expression of CD1d in human peripheral nerve. We found that CD1d was expressed on the surface of Schwann cells in situ and on primary or immortalized Schwann cell lines in culture. Schwann cells activated iNKT cells in a CD1d-dependent manner in the presence of alpha-galactosylceramide. Surprisingly, the cytokine production of iNKT cells stimulated by alpha-galactosylceramide presented by CD1d+ Schwann cells showed a predominance of Th2-associated cytokines such as IL-5 and IL-13 with a marked deficiency of proinflammatory Th1 cytokines such as IFN-gamma or TNF-alpha. Our findings suggest a mechanism by which iNKT cells may restrain inflammatory responses in peripheral nerves, and raise the possibility that the expression of CD1d by Schwann cells could be relevant in the pathogenesis of infectious and inflammatory diseases of the peripheral nervous system.

Antigens, CD1↗
PubMed2006

CD38 is required for the peripheral survival of immunotolerogenic CD4+ invariant NK T cells in nonobese diabetic mice.

T cell-mediated autoimmune type-1 diabetes (T1D) in NOD mice partly results from this strain's numerical and functional defects in invariant NK T (iNKT) cells. T1D is inhibited in NOD mice treated with the iNKT cell superagonist alpha-galactosylceramide through a process involving enhanced accumulation of immunotolerogenic dendritic cells in pancreatic lymph nodes. Conversely, T1D is accelerated in NOD mice lacking CD38 molecules that play a role in dendritic cell migration to inflamed tissues. Unlike in standard NOD mice, alpha-galactosylceramide pretreatment did not protect the CD38-deficient stock from T1D induced by an adoptively transferred pancreatic beta cell-autoreactive CD8 T cell clone (AI4). We found that in the absence of CD38, ADP-ribosyltransferase 2 preferentially activates apoptotic deletion of peripheral iNKT cells, especially the CD4+ subset. Therefore, this study documents a previously unrecognized role for CD38 in maintaining survival of an iNKT cell subset that preferentially contributes to the maintenance of immunological tolerance.

ADP Ribose Transferases↗
PubMed2006

CD1 expression and CD1-restricted T cell activity in normal and tumour-bearing human liver.

CD1d-restricted natural killer T (NKT) cells expressing invariant Valpha14Jalpha18 T cell receptor alpha-chains are abundant in murine liver and are implicated in the control of malignancy, infection and autoimmunity. Invariant NKT cells have potent anti-metastatic effects in mice and phase I clinical trials involving their homologues in humans are ongoing. However, invariant NKT cells are less abundant in human liver ( approximately 0.5% of hepatic T cells) than in murine liver (up to 50%) and it is not known if other hepatic T cells are CD1-restricted. We have examined expression of CD1a, CD1b, CD1c and CD1d mRNA and protein in human liver and evaluated the reactivity of mononuclear cells (MNC) from histologically normal and tumour-bearing human liver specimens against these CD1 isoforms. Messenger RNA for all CD1 isotypes was detectable in all liver samples. CD1c and CD1d were expressed at the protein level by hepatic MNC. CD1d, only, was detectable at the cell surface, but CD1c and CD1d were found at an intracellular location in significant numbers of liver MNC. CD1b was not expressed by MNC from healthy livers but was detectable within MNC in all tumour samples tested. Hepatic T cells exhibited reactivity against C1R cells expressing transfected CD1c and CD1d, but neither CD1a nor CD1b. These cells secreted interferon-gamma (IFN-gamma) but not interleukin-4 (IL-4) upon stimulation. In contrast, similar numbers of peripheral T cells released 13- and 16-fold less IFN-gamma in response to CD1c and CD1d, respectively. CD1c and CD1d expression and T cell reactivity were not altered in tumour-bearing liver specimens compared to histologically normal livers. These data suggest that, in addition to invariant CD1d-restricted NKT cells, autoreactive T cells that recognise CD1c and CD1d and release inflammatory cytokines are abundant in human liver.

Antigens, CD1↗
PubMed2006

Mycobacterium tuberculosis DeltaRD1 DeltapanCD: a safe and limited replicating mutant strain that protects immunocompetent and immunocompromised mice against experimental tuberculosis.

The global epidemic of tuberculosis (TB), fueled by the growing HIV pandemic, warrants the development of a safe and effective vaccine against TB. We report the construction and characterization of an unlinked double deletion mutant of Mycobacterium tuberculosis H37Rv that deletes both the primary attenuating mutation of BCG (DeltaRD1) and two genes required for the synthesis of pantothenate (DeltapanCD). The M. tuberculosis DeltaRD1 DeltapanCD (mc(2)6030) mutant undergoes limited replication in mice, and yet is both significantly safer than BCG in immunocompromised mice and also safe in guinea pigs. Additionally, the mc(2)6030 strain does not reactivate in a mouse chemo-immunosuppression model. Importantly, long-lived protective immune responses following immunization with the mc(2)6030 strain prolong the survival of wild type mice, and CD4-deficient mice against an aerosol challenge with virulent M. tuberculosis. Given its overall safety and effectiveness, the mc(2)6030 live attenuated strain should be considered as a human vaccine candidate for protecting both healthy and HIV-infected individuals against TB.

Animals↗
PubMed2006

The bovine CD1 family contains group 1 CD1 proteins, but no functional CD1d.

The CD1 family of proteins presents lipid Ags to T cells. Human CD1a, CD1b, and CD1c have been shown in humans to present mycobacterial lipid Ags. Cattle, like humans, are a natural host of several mycobacterial pathogens. In this study, we describe the CD1 family of genes in cattle (Bos taurus) and provide evidence that B. taurus expresses CD1a, CD1e, and multiple CD1b molecules, but no CD1c and CD1d molecules. In mice and humans, CD1d is known to present Ag to NKT cells, a T cell lineage that is characterized by a limited TCR repertoire, capable of rapidly secreting large amounts of IFN-gamma and IL-4. In cattle, two CD1D pseudogenes were found and no intact CD1D genes. Consistent with this, we found complete lack of reactivity to a potent, cross-reactive Ag for NKT cells in mice and humans, alpha-galactosylceramide. Our data suggest the absence of NKT cells in cattle. It remains open whether other cells with the NKT-like phenotype and functions are present in this species. With its functional CD1A and CD1B genes, B. taurus is well equipped to present Ags to CD1-restricted T cells other than NKT cells. Cattle can be used as a model to study group 1 CD1-restricted T cell immunity, including its role in the defense against mycobacterial infections that occur naturally in this species.

Animals↗
PubMed2006

Ceramide-dependent regulation of human epidermal keratinocyte CD1d expression during terminal differentiation.

Human keratinocytes (KC), when cultured under conditions to remain undifferentiated or to terminally differentiate, changed their cellular distribution of CD1d. As studied by confocal microscopy, undifferentiated KC had a pool of cytoplasmic CD1d, whereas after terminal differentiation, this molecule localized in the cell membrane, which recapitulates CD1d expression in vivo. A comparison of undifferentiated and differentiated cultured KC did not reveal any differences in the association with beta(2)-microglobulin, invariant chain of class II MHC, or patterns of glycosylation, suggesting that these biochemical properties are not regulating the cellular distribution of CD1d. Time-course studies of CD1d gene expression indicated that KC slowly increased gene expression with CaCl(2)-induced terminal differentiation. Increased CD1d gene expression was dependent on ceramide synthesis, because fumonisin B1, a ceramide synthetase inhibitor, blocked the increase in CD1d gene expression during terminal differentiation. Similarly, exogenous ceramide or the ceramidase inhibitor, B13, induced CD1d gene expression by undifferentiated, but not terminally differentiated, KC. A protein kinase C-zeta (PKC-zeta) inhibitor (a pseudosubstrate oligopeptide), but not a PKC-alphabeta inhibitor, significantly decreased CD1d gene expression by undifferentiated or ceramide-stimulated cultured, undifferentiated KC. As expected, downstream signaling events of PKC-zeta (JNK phosphorylation and NF-kappaBeta accumulation in the nucleus) were also attenuated. The calcineurin phosphatase inhibitor cyclosporine A, which blocks KC terminal differentiation, also blocked CD1d gene expression by cultured KC. In conclusion, this novel function of cellular ceramides extends the importance of this class of biologically active lipids beyond that of terminal differentiation and barrier function in normal human skin.

Antigens, CD1↗
PubMed2005

Synthesis and evaluation of sphinganine analogues of KRN7000 and OCH.

[structures: see text] The phytosphingosine-containing alpha-galactosylceramides (alpha-GalCers), KRN7000 and OCH, have been shown to activate NKT cells via interaction with CD1d, a member of the CD1 family of antigen presenting proteins. Evidence from KRN7000 stimulation of NKT cells suggests that alpha-GalCers may have applications in the treatment or prevention of a range of viral, bacterial, and autoimmune conditions. Moreover, OCH, a truncated analogue of KRN7000, appears to induce a T(H)2 bias, which could have implications for the treatment of autoimmune and inflammatory conditions. We have prepared the direct sphinganine-containing analogues of KRN7000 and OCH, 1 and 2, and found them to be comparable in activity to the parent compounds in inducing the release of IL-2, IL-4, and IFNgamma. In addition, compound 2 leads to a cytokine bias similar to that seen with OCH. This is significant because sphinganines are more easily accessed than phytosphingosines, which should facilitate SAR studies.

Animals↗
PubMed2005

Anthrax lethal toxin-mediated killing of human and murine dendritic cells impairs the adaptive immune response.

Many pathogens have acquired strategies to combat the immune response. Bacillus anthracis interferes with host defenses by releasing anthrax lethal toxin (LT), which inactivates mitogen-activated protein kinase pathways, rendering dendritic cells (DCs) and T lymphocytes nonresponsive to immune stimulation. However, these cell types are considered resistant to killing by LT. Here we show that LT kills primary human DCs in vitro, and murine DCs in vitro and in vivo. Kinetics of LT-mediated killing of murine DCs, as well as cell death pathways induced, were dependent upon genetic background: LT triggered rapid necrosis in BALB/c-derived DCs, and slow apoptosis in C57BL/6-derived DCs. This is consistent with rapid and slow killing of LT-injected BALB/c and C57BL/6 mice, respectively. We present evidence that anthrax LT impairs adaptive immunity by specifically targeting DCs. This may represent an immune-evasion strategy of the bacterium, and contribute to anthrax disease progression. We also established that genetic background determines whether apoptosis or necrosis is induced by LT. Finally, killing of C57BL/6-derived DCs by LT mirrors that of human DCs, suggesting that C57BL/6 DCs represent a better model system for human anthrax than the prototypical BALB/c macrophages.

Journal Article↗
PubMed2005

NKT cell-dependent leukemia eradication following stem cell mobilization with potent G-CSF analogs.

NKT cells have pivotal roles in immune regulation and tumor immunosurveillance. We report that the G-CSF and FMS-like tyrosine kinase 3 ligand (Flt-3L) chimeric cytokine, progenipoietin-1, markedly expands the splenic and hepatic NKT cell population and enhances functional responses to alpha-galactosylceramide. In a murine model of allogeneic stem cell transplantation, donor NKT cells promoted host DC activation and enhanced perforin-restricted CD8+ T cell cytotoxicity against host-type antigens. Following leukemic challenge, donor treatment with progenipoietin-1 significantly improved overall survival when compared with G-CSF or control, attributable to reduced graft-versus-host disease mortality and paradoxical augmentation of graft-versus-leukemia (GVL) effects. Enhanced cellular cytotoxicity was dependent on donor NKT cells, and leukemia clearance was profoundly impaired in recipients of NKT cell-deficient grafts. Enhanced cytotoxicity and GVL effects were not associated with Flt-3L signaling or effects on DCs but were reproduced by prolonged G-CSF receptor engagement with pegylated G-CSF. Thus, modified G-CSF signaling during stem cell mobilization augments NKT cell-dependent CD8+ cytotoxicity, effectively separating graft-versus-host disease and GVL and greatly expanding the potential applicability of allogeneic stem cell transplantation for the therapy of malignant disease.

Animals↗
PubMed2005

The diverse functions of CD1d-restricted NKT cells and their potential for immunotherapy.

CD1d-restricted NKT cells have been identified as an important component of the immune system that have the capacity both to augment beneficial host immunity and to prevent harmful autoimmunity. These cells have the ability to produce a wide variety of cytokines, including both proinflammatory and antiinflammatory cytokines that can have multiple different effects on the outcome of immune reactions. The discovery that these T cells are activated by specific recognition of glycolipids in the glycosylceramide family has led to new approaches to manipulate the pleiotropic functions of these cells. Here, we review the multiple activities that have been attributed to NKT cells in a variety of different disease models, and the current state of our understanding of the mechanisms that control the functional outcome of NKT cell activation.

Adoptive Transfer↗
PubMed2005

Expansion and hyperactivity of CD1d-restricted NKT cells during the progression of systemic lupus erythematosus in (New Zealand Black x New Zealand White)F1 mice.

CD1d-restricted NKT cells expressing invariant TCR alpha-chain rearrangements (iNKT cells) have been reported to be deficient in humans with a variety of autoimmune syndromes and in certain strains of autoimmune mice. In addition, injection of mice with alpha-galactosylceramide, a specific glycolipid agonist of iNKT cells, activates these T cells and ameliorates autoimmunity in several different disease models. Thus, deficiency and reduced function in iNKT cells are considered to be risk factors for the development of such diseases. In this study we report that the development of systemic lupus erythematosus in (New Zealand Black (NZB) x New Zealand White (NZW))F(1) mice was paradoxically associated with an expansion and activation of iNKT cells. Although young (NZB x NZW)F(1) mice had normal levels of iNKT cells, these expanded with age and became phenotypically and functionally hyperactive. Activation of iNKT cells in (NZB x NZW)F(1) mice in vivo or in vitro with alpha-galactosylceramide indicated that the immunoregulatory role of iNKT cells varied over time, revealing a marked increase in their potential to contribute to production of IFN-gamma with advancing age and disease progression. This evolution of iNKT cell function during the progression of autoimmunity may have important implications for the mechanism of disease in this model of systemic lupus erythematosus and for the development of therapies using iNKT cell agonists.

Animals↗
PubMed2005

Human CD1-restricted T cell recognition of lipids from pollens.

Plant pollens are an important source of environmental antigens that stimulate allergic responses. In addition to acting as vehicles for foreign protein antigens, they contain lipids that incorporate saturated and unsaturated fatty acids, which are necessary in the reproduction of higher plants. The CD1 family of nonpolymorphic major histocompatibility complex-related molecules is highly conserved in mammals, and has been shown to present microbial and self lipids to T cells. Here, we provide evidence that pollen lipids may be recognized as antigens by human T cells through a CD1-dependent pathway. Among phospholipids extracted from cypress grains, phosphatidyl-choline and phosphatidyl-ethanolamine were able to stimulate the proliferation of T cells from cypress-sensitive subjects. Recognition of phospholipids involved multiple cell types, mostly CD4(+) T cell receptor for antigen (TCR)alphabeta(+), some CD4(-)CD8(-) TCRgammadelta(+), but rarely Valpha24i(+) natural killer-T cells, and required CD1a(+) and CD1d(+) antigen presenting cell. The responding T cells secreted both interleukin (IL)-4 and interferon-gamma, in some cases IL-10 and transforming growth factor-beta, and could provide help for immunoglobulin E (IgE) production. Responses to pollen phospholipids were maximally evident in blood samples obtained from allergic subjects during pollinating season, uniformly absent in Mycobacterium tuberculosis-exposed health care workers, but occasionally seen in nonallergic subjects. Finally, allergic, but not normal subjects, displayed circulating specific IgE and cutaneous weal and flare reactions to phospholipids.

Adult↗
PubMed2005

The human CD1-restricted T cell repertoire is limited to cross-reactive antigens: implications for host responses against immunologically related pathogens.

The repertoires of CD1- and MHC-restricted T cells are complementary, permitting the immune recognition of both lipid and peptide Ags, respectively. To compare the breadth of the CD1-restricted and MHC-restricted T cell repertoires, we evaluated T cell responses against lipid and peptide Ags of mycobacteria in leprosy, comparing tuberculoid patients, who are able to restrict the pathogen, and lepromatous patients, who have disseminated infection. The striking finding was that in lepromatous leprosy, T cells did not efficiently recognize lipid Ags from the leprosy pathogen, Mycobacterium leprae, or the related species, Mycobacterium tuberculosis, yet were able to efficiently recognize peptide Ags from M. tuberculosis, but not M. leprae. To identify a mechanism for T cell unresponsiveness against mycobacterial lipid Ags in lepromatous patients, we used T cell clones to probe the species specificity of the Ags recognized. We found that the majority of M. leprae-reactive CD1-restricted T cell clones (92%) were cross-reactive for multiple mycobacterial species, whereas the majority of M. leprae-reactive MHC-restricted T cells were species specific (66%), with a limited number of T cell clones cross-reactive (34%) with M. tuberculosis. In comparison with the MHC class II-restricted T cell repertoire, the CD1-restricted T cell repertoire is limited to recognition of cross-reactive Ags, imparting a distinct role in the host response to immunologically related pathogens.

Adult↗