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Jack L Strominger

Publications and source records attributed to Jack L Strominger.

At least 37 records · Page 2Linked to original sources
PubMed2004

Caspases and nitric oxide broadly regulate dendritic cell maturation and surface expression of class II MHC proteins.

The passage of dendritic cells (DC) from immature to terminally differentiated antigen-presenting cells is accompanied by numerous morphological, phenotypic, and functional changes. These changes include, for example, expression of "empty" class II MHC proteins (MHCII) at the surface in immature DC, whereas a much larger amount of peptide-loaded MHCII is expressed at the surface in mature DC. Here we show that, in cultured immature DC derived from murine bone-marrow precursors, a number of molecules involved in intracellular trafficking were present in a cleaved form, degraded by caspase-like proteases. Cleavage was either inhibited or reduced significantly during maturation of DC induced by either LPS and TNF-alpha or by peptides that inhibit caspase activities. Inducible nitric oxide (NO) synthetase up-regulated by LPS was essential for inhibiting the caspase-like activity during the maturation of DC. Moreover, treatment with LPS or caspase inhibitor resulted in expression of MHCII/peptide complexes at the cell surface. Thus, the alteration of the endosomal trafficking pathways during the development of DC that parallels the changes in surface expression of MHCII is regulated at least in part by the activities of caspases, inducible NO synthetase, and its product NO.

Adaptor Protein Complex beta Subunits↗
PubMed2004

Amelioration of proteolipid protein 139-151-induced encephalomyelitis in SJL mice by modified amino acid copolymers and their mechanisms.

Copolymer 1 [Cop1, glatiramer acetate, Copaxone, poly(Y,E,A,K)n] is widely used in the treatment of relapsing/remitting multiple sclerosis in which it reduces the frequency of relapses by approximately 30%. In the present study, copolymers with modified amino acid compositions (based on the binding motif of myelin basic protein 85-99 to HLA-DR2) have been developed with the aim of suppressing multiple sclerosis more effectively. The enhanced efficacy of these copolymers in experimental autoimmune encephalomyelitis (EAE) induced in SJL/J mice with proteolipid protein 139-151 was demonstrated by using three protocols: (i) simultaneous administration of autoantigen and copolymer (termed prevention), (ii) pretreatment with copolymers (vaccination), or (iii) administration of copolymers after disease onset (treatment). Strikingly, in the treatment protocol administration of soluble VWAK and FYAK after onset of disease led to stasis of its progression and suppression of histopathological evidence of EAE. The mechanisms by which these effects are achieved have been examined in several types of assays: binding of copolymers to I-A(s) in competition with proteolipid protein 139-151 (blocking), cytokine production by T cells (T helper 2 polarization), and transfer of protection by CD3(+) splenocytes or, notably, by copolymer-specific T cell lines (induction of regulatory T cells). The generation of these copolymer-specific regulatory T cells that secrete IL-4 and IL-10 and are independent of the immunizing autoantigen is very prominent among the multiple mechanisms that account for the observed suppressive effect of copolymers in EAE.

Adoptive Transfer↗
PubMed2004

Modified amino acid copolymers suppress myelin basic protein 85-99-induced encephalomyelitis in humanized mice through different effects on T cells.

A humanized mouse bearing the HLA-DR2 (DRA/DRB1*1501) protein associated with multiple sclerosis (MS) and the myelin basic protein (MBP) 85-99-specific HLA-DR2-restricted T cell receptor from an MS patient has been used to examine the effectiveness of modified amino acid copolymers poly(F,Y,A,K)n and poly-(V,W,A,K)n in therapy of MBP 85-99-induced experimental autoimmune encephalomyelitis (EAE) in comparison to Copolymer 1 [Copaxone, poly(Y,E,A,K)n]. The copolymers were designed to optimize binding to HLA-DR2. Vaccination, prevention, and treatment of MBP-induced EAE in the humanized mice with copolymers FYAK and VWAK ameliorated EAE more effectively than Copolymer 1, reduced the number of pathological lesions, and prevented the up-regulation of human HLA-DR on CNS microglia. Moreover, VWAK inhibited MBP 85-99-specific T cell proliferation more efficiently than either FYAK or Copolymer 1 and induced anergy of HLA-DR2-restricted transgenic T cells as its principle mechanism. In contrast, FYAK induced proliferation and a pronounced production of the antiinflammatory T helper 2 cytokines IL-4 and IL-10 from nontransgenic T cells as its principle mechanism of immunosuppression. Thus, copolymers generated by using different amino acids inhibited disease using different mechanisms to regulate T cell responses.

Amino Acid Sequence↗
PubMed2004

Crystal structure of HLA-DQ0602 that protects against type 1 diabetes and confers strong susceptibility to narcolepsy.

The MHC class II molecule DQ0602 confers strong susceptibility to narcolepsy but dominant protection against type 1 diabetes. The crystal structure of DQ0602 reveals the molecular features underlying these contrasting genetic properties. Structural comparisons to homologous DQ molecules with differential disease associations highlight a previously unrecognized interplay between the volume of the P6 pocket and the specificity of the P9 pocket, which implies that presentation of an expanded peptide repertoire is critical for dominant protection against type 1 diabetes. In narcolepsy, the volume of the P4 pocket appears central to the susceptibility, suggesting that the presentation of a specific peptide population plays a major role.

Alleles↗
PubMed2004

Monoclonal antibodies specific for the empty conformation of HLA-DR1 reveal aspects of the conformational change associated with peptide binding.

Class II major histocompatibility complex (MHC) proteins bind peptides and present them at the cell surface for interaction with CD4+ T cells as part of the system by which the immune system surveys the body for signs of infection. Peptide binding is known to induce conformational changes in class II MHC proteins on the basis of a variety of hydrodynamic and spectroscopic approaches, but the changes have not been clearly localized within the overall class II MHC structure. To map the peptide-induced conformational change for HLA-DR1, a common human class II MHC variant, we generated a series of monoclonal antibodies recognizing the beta subunit that are specific for the empty conformation. Each antibody reacted with the empty but not the peptide-loaded form, for both soluble recombinant protein and native protein expressed at the cell surface. Antibody binding epitopes were characterized using overlapping peptides and alanine scanning substitutions and were localized to two distinct regions of the protein. The pattern of key residues within the epitopes suggested that the two epitope regions undergo substantial conformational alteration during peptide binding. These results illuminate aspects of the structure of the empty forms and the nature of the peptide-induced conformational change.

Amino Acid Sequence↗
PubMed2004

MLL 5 protein forms intranuclear foci, and overexpression inhibits cell cycle progression.

MLL5 is a mammalian trithorax group (trx-G) gene identified within chromosome band 7q22, a frequently deleted element found in cytogenetic aberrations of acute myeloid malignancies. MLL5 cDNA was linked with the FLAG and V5 tags at the N and C terminus, respectively, and transfected into 293T cells. Immunofluoresence staining of the expressed tagged MLL5 protein showed localization to the nucleus and exclusion from nucleoli, and no surface staining was detected. Both ectopically introduced and endogenous MLL5 protein displayed a speckled nuclear distribution. By using a series of MLL5-truncated mutants fused with enhanced GFP, a domain (residues 945-1,156) required for foci accumulation was identified, and regions containing functional nuclear localization signals were mapped. Ectopic overexpression of GFP-MLL5 induced cell cycle arrest in G(1) phase. This inhibition of cell cycle progression was indicated by delayed progression into nocodazole-induced mitotic arrest and was confirmed by a lack of BrdUrd incorporation. These findings suggest that MLL5 forms intranuclear protein complexes that may play an important role in chromatin remodeling and cellular growth suppression.

Animals↗
PubMed2004

Heterogeneity of TLR-induced responses in dendritic cells: from innate to adaptive immunity.

Toll-like receptors (TLR) mediate recognition of several microbial products. Accumulating evidence indicates that TLR are capable of inducing distinct responses in dendritic cells and other antigen-presenting cells, and can direct T-helper cell differentiation in opposing directions. The generation of such varied responses is achieved through the selective utilization of adaptor molecules that link TLR to distinct signal transduction pathways. The ability of TLR to activate and guide innate and adaptive immunity has the potential to be exploited for practical application that may lead to the development of more successful immunotherapies and vaccination strategies. A review of recent literature, unpublished observations, and future challenges is presented here.

Dendritic Cells↗
PubMed2003

Separate functional domains of human MD-2 mediate Toll-like receptor 4-binding and lipopolysaccharide responsiveness.

Cellular responses to LPS are mediated by a cell surface receptor complex consisting of Toll-like receptor 4 (TLR4), MD-2, and CD14. MD-2 is a secreted protein that interacts with the extracellular portion of TLR4. Site-directed mutagenesis was used to identify the regions of human MD-2 involved in its ability to bind TLR4 and confer LPS responsiveness. A separate region of MD-2 was found to mediate each function. MD-2 binding to TLR4 was dependent on Cys(95) and Cys(105), which might form an intramolecular disulfide bond. Hydrophilic and charged residues surrounding this area, such as R90, K91, D100, and Y102, also contributed to the formation of the TLR4-MD-2 complex. A different region of MD-2 was found to be responsible for conferring LPS responsiveness. This region is not involved in TLR4 binding and is rich in basic and aromatic residues, several of which cooperate for LPS responsiveness and might represent a LPS binding site. Disruption of the endogenous MD-2-TLR4 complex by expression of mutant MD-2 inhibited LPS responses in primary human endothelial cells. Thus, our data indicate that MD-2 interaction with TLR4 is necessary but not sufficient for cellular response to LPS. Either of the two functional domains of MD-2 can be disrupted to impair LPS responses and therefore represent attractive targets for therapeutic interventions.

Antigens, Surface↗
PubMed2003

The mature activating natural killer cell immunologic synapse is formed in distinct stages.

Natural killer (NK) cells form a structure at their interface with a susceptible target cell called the activating NK cell immunologic synapse (NKIS). The mature activating NKIS contains a central and peripheral supramolecular activation cluster (SMAC), and includes polarized surface receptors, filamentous actin (F-actin) and perforin. Evaluation of the NKIS in human NK cells revealed CD2, CD11a, CD11b and F-actin in the peripheral SMAC (pSMAC) with perforin in the central SMAC. The accumulation of F-actin and surface receptors was rapid and depended on Wiskott-Aldrich syndrome protein-driven actin polymerization. The accumulation at and arrangement of these molecules in the pSMAC was not affected by microtubule depolymerization. The polarization of perforin, however was slower and required intact actin, Wiskott-Aldrich syndrome protein, and microtubule function. Thus the process of CD2, CD11a, CD11b, and F-actin accumulation in the pSMAC and perforin accumulation in the central SMAC of the NKIS are sequential processes with distinct cytoskeletal requirements.

Actins↗
PubMed2003

Human decidual natural killer cells are a unique NK cell subset with immunomodulatory potential.

Natural killer cells constitute 50-90% of lymphocytes in human uterine decidua in early pregnancy. Here, CD56(bright) uterine decidual NK (dNK) cells were compared with the CD56(bright) and CD56(dim) peripheral NK cell subsets by microarray analysis, with verification of results by flow cytometry and RT-PCR. Among the approximately 10,000 genes studied, 278 genes showed at least a threefold change with P < or = 0.001 when comparing the dNK and peripheral NK cell subsets, most displaying increased expression in dNK cells. The largest number of these encoded surface proteins, including the unusual lectinlike receptors NKG2E and Ly-49L, several killer cell Ig-like receptors, the integrin subunits alpha(D), alpha(X), beta1, and beta5, and multiple tetraspanins (CD9, CD151, CD53, CD63, and TSPAN-5). Additionally, two secreted proteins, galectin-1 and progestagen-associated protein 14, known to have immunomodulatory functions, were selectively expressed in dNK cells.

CD56 Antigen↗
PubMed2003

CD1d-restricted NKT cells express a chemokine receptor profile indicative of Th1-type inflammatory homing cells.

CD1d-restricted T cells (NKT cells) are innate memory cells activated by lipid Ags and play important roles in the initiation and regulation of the immune response. However, little is known about the trafficking patterns of these cells or the tissue compartment in which they exert their regulatory activity. In this study, we determined the chemokine receptor profile expressed by CD1d-restricted T cells found in the peripheral blood of healthy volunteers as well as CD1d-restricted T cell clones. CD1d-restricted T cells were identified by Abs recognizing the invariant Valpha24 TCR rearrangement or by binding to CD1d-Fc fusion tetramers loaded with alpha-GalCer. CD1d-restricted T cells in the peripheral blood and CD1d-restricted T cell clones expressed high levels of CXCR3, CCR5, and CCR6; intermediate levels of CXCR4 and CXCR6; and low levels of CXCR1, CCR1, CCR2, and CX(3)CR1, a receptor pattern often associated with tissue-infiltrating effector Th1 cells and CD8+ T cells. Very few of these cells expressed the lymphoid-homing receptors CCR7 or CXCR5. CCR4 was expressed predominantly on CD4+, but not on double-negative CD1d-restricted T cells, which may indicate differential trafficking patterns for these two functionally distinct subsets. CD1d-restricted T cell clones responded to chemokine ligands for CXCR1/2, CXCR3, CXCR4, CXCR6, CCR4, and CCR5 in calcium flux and/or chemotaxis assays. These data indicate that CD1d-restricted T cells express a chemokine receptor profile most similar to Th1 inflammatory homing cells and suggest that these cells perform their function in peripheral tissue sites rather than in secondary lymphoid organs.

Animals↗
PubMed2003

CD1d-restricted T cells regulate dendritic cell function and antitumor immunity in a granulocyte-macrophage colony-stimulating factor-dependent fashion.

CD1d-restricted T cells contribute to tumor protection, but their precise roles remain unclear. Here we show that tumor cells engineered to secrete granulocyte-macrophage colony-stimulating factor induce the expansion of CD1d-restricted T cells through a mechanism that involves CD1d and macrophage inflammatory protein 2 expression by CD8 alpha-, CD11c+ dendritic cells (DCs). The antitumor immunity stimulated by vaccination with irradiated, granulocyte-macrophage colony-stimulating factor-secreting tumor cells was abrogated in CD1d- and J alpha 281-deficient mice, revealing a critical role for CD1d-restricted T cells in this response. The loss of antitumor immunity was associated with impaired tumor-induced T helper 2 cytokine production, although IFN-gamma secretion and cytotoxicity were preserved. DCs from immunized CD1d-deficient mice showed compromised maturation and function. Together, these results delineate a role for CD1d-restricted T cell-DC cross talk in the shaping of antitumor immunity.

Animals↗
PubMed2003

Molecular interaction and enzymatic activity of macrophage migration inhibitory factor with immunorelevant peptides.

Disulfide reduction is an important step in antigen processing for HLA class II restricted T cell responses. Migration inhibitory factor (MIF) is a member of the thioredoxin family and has been classically defined as a cytokine. Using enzyme-linked immunosorbent assay and CD analysis, here we describe the binding to MIF of two peptides, hepatitis B surface antigen (HBsAg) and insulin B (InsB) with high affinity for HLA class II allo-types, HLA-DP2 and HLA-DQ8, respectively. At neutral pH, cysteinylated InsB was a substrate for MIF thiol reductase activity, as assessed by mass spectroscopy/electrospray analysis. Finally, a biologically active form of MIF co-immunopurified with mature forms of HLA DP2/15, and a peptide derived from the HLA-DP beta1 helix could be used for affinity purification of MIF. The possibility that MIF participates in class II antigen presentation and/or as a chaperone is discussed.

Antigen Presentation↗
PubMed2003

Interleukin-12 and interleukin-2-induced invariant natural killer T-cell cytokine secretion and perforin expression independent of T-cell receptor activation.

Human invariant natural killer (iNK) T cells expressing an invariant Valpha24-Jalpha15 T-cell receptor (TCR) are thought to be important regulators of autoimmunity and tumour surveillance. Two major subsets of iNK T cells, CD4+ or CD4- CD8- are known to exist, but the in vivo importance of CD4 expression is unclear. Since interleukin-12 (IL-12) is a key iNK T-cell-activating cytokine, the effect of IL-12 plus or minus the T-cell growth factor IL-2 on a large panel of CD4+ versus CD4- CD8- iNK T-cell clones was examined. Strikingly, IL-12 and IL-2 significantly activated iNK T cells to secrete IL-4, interferon-gamma and granulocyte-macrophage colony-stimulating factor, and up-regulated perforin expression in the absence of TCR stimulation. Furthermore, IL-2 and IL-12 treatment resulted in a preferential increase in apoptosis of CD4- CD8- clones. Thus, independent of TCR activation, IL-2 and IL-12 can directly activate iNK T cells and provide a selective advantage to the CD4+ iNK T-cell population.

Apoptosis↗
PubMed2002

A pH-sensitive histidine residue as control element for ligand release from HLA-DR molecules.

Class II MHC molecules undergo conformational changes on shifts of the pH. As a consequence, low-affinity peptides tightly bound at pH 7.0 can be released at pH 5.0. The imidazole group of histidine is the only amino acid side chain affected within this range. At pH 5.0 the group is positively charged, polar, and hydrophilic, whereas at pH 7.4 it is neutral, apolar, and hydrophobic. In this study, we used soluble forms of HLA-DR and substituted conserved histidine residues with tyrosine, an isosteric analogue to the uncharged form of histidine. The goal of this substitution was to identify crucial His residues by an increase in pH stability of the ligand complex. HLA-DM-mediated release experiments revealed that substitution of His-33 in the alpha(1) domain of the HLA-DR molecule almost doubled the half-life of HLA-DR1class II-associated invariant-chain peptide complexes. The divergence in the off-rate of WT and H33Y mutated complex was strictly pH-dependent and correlated with the theoretical titration curve of the imidazole group. For both HLA-DR1 and HLA-DR4 molecules the mutation resulted in a shift of class II-associated invariant-chain peptide release curves by up to 0.5 pH units. His-33alpha1 is present in all HLA-DR and H-2E molecules. It connects the alpha(1) and alpha(2) domains in its noncharged form by hydrophobic interactions with residue Val-136alpha2. It is located in close proximity to the putative interface with HLA-DM and may function as a pH-sensitive "button," which is closed at pH 7.0 but opens below pH 6.0 to allow conformational transitions necessary for ligand exchange.

Amino Acid Sequence↗
PubMed2002

Disulfide bond-mediated dimerization of HLA-G on the cell surface.

HLA-G is a nonclassical class I MHC molecule with an unknown function and with unusual characteristics that distinguish it from other class I MHC molecules. Here, we demonstrate that HLA-G forms disulfide-linked dimers that are present on the cell surface. Immunoprecipitation of HLA-G from surface biotinylated transfectants using the anti-beta2-microglobulin mAb BBM.1 revealed the presence of an approximately equal 78-kDa form of HLA-G heavy chain that was reduced by using DTT to a 39-kDa form. Mutation of Cys-42 to a serine completely abrogated dimerization of HLA-G, suggesting that the disulfide linkage formed exclusively through this residue. A possible interaction between the HLA-G monomer or dimer and the KIR2DL4 receptor was also investigated, but no interaction between these molecules could be detected through several approaches. The cell-surface expression of dimerized HLA-G molecules may have implications for HLA-Greceptor interactions and for the search for specific receptors that bind HLA-G.

Amino Acid Substitution↗
PubMed2002

Uniquely conformed peptide-containing beta 2-microglobulin-free heavy chains of HLA-B2705 on the cell surface.

The human class I MHC molecules are known to generally exist on the cell surface either as peptide-containing complexes of H chain (alpha-chain) and beta(2)-microglobulin (beta(2)m) or as beta(2)m-free H chains incapable of binding peptides. In this study, a uniquely conformed peptide-containing beta(2)m-free HLA-B2705 H chain has been isolated using the recently described highly efficient perfusion-affinity chromatography system for purification of class I MHC protein molecules. This form recognized by the mAb MARB4 is very closely associated with the remainder of the peptide containing HLA-B2705/beta(2)m complex reactive with mAb ME1 and is present to approximately 1-10% of mAb ME1 reactive forms on the cell surface. Also, HLA-B2705 purified using the mAb ME1 affinity column includes this unique mAb MARB4-reactive, unusually stable peptide-containing beta(2)m-free form. A peptide nonamer GRWRGWYTY was isolated and identified from this beta(2)m-free HLA-B2705 H chain and was used to assemble the mAb MARB4 reactive form efficiently on the surface of cells expressing HLA-B2705. The discovery of this form opens new avenues for further investigation of the role of HLA-B27 in spondyloarthropathies.

Antibodies, Monoclonal↗