Biomedical subjects

K L Rock

Publications and source records attributed to K L Rock.

At least 37 records · Page 2Linked to original sources

PubMed1998

Cytotoxic T lymphocytes in resistance to tuberculosis.

Recent experimental evidence has suggested T cells recognizing antigens in the context of both classical MHC class I and nonclassical class I-like molecules contribute to protective responses against Mycobacterium tuberculosis (MTB) infection. Our aims were to characterize both types of T cells, and to explore the basis of communication between the tubercle bacilli and the MHC class I pathway of the host macrophage. A model system was developed using exogenously added ovalbumin as a surrogate antigen to study presentation by MTB-infected macrophages. Viable, virulent MTB and closely related mycobacterial species facilitated the presentation of ovalbumin on MHC class I molecules to CD8+ cytolytic T cells that was dependent upon the cytosolic transport of peptides, implying communication between the MTB phagosome and the host cell cytoplasm. MHC class I presentation of soluble antigens was mimicked by Listeria monocytogenes, which grows within the host cell cytoplasm, as well as its purified hemolysin. We have also characterized T cells that recognize nonpeptide MTB antigens presented by CD1 molecules. CD1-restricted T cells demonstrated to lyse macrophages infected with virulent MTB were divided into distinct subsets based on surface phenotype (CD4-CD8- versus CD8-) and cytotoxicity mechanism (Fas receptor-mediated versus granule exocytosis). A functional consequence of these two mechanisms was observed that while both subsets lysed infected macrophages, only those T cells utilizing the granule exocytosis pathway were able to reduce viability of intracellular MTB.

Animals↗

PubMed1998

Fully mobilizing host defense: building better vaccines.

Developments in methods for identifying antigens from infectious agents and cancers has provided exciting new opportunities in prevention and treatment through vaccination. In many of these situations, however, traditional immunization techniques do not stimulate protective immunity because they fail to fully mobilize the appropriate immune responses. This limitation, together with new insights into the underlying mechanism of immune responses, has spurred development of several new approaches for vaccine delivery. We discuss some of the current efforts being developed to provide effective vaccine delivery systems.

Adjuvants, Immunologic↗

PubMed1997

Two distinct proteolytic processes in the generation of a major histocompatibility complex class I-presented peptide.

Although cellular proteins degraded by proteasomes are the source of most antigenic peptides presented on major histocompatibility complex class I molecules, it is unknown whether the eight- to nine-residue peptides that fit in the binding groove of class I molecules are directly produced by proteasomes alone in vivo. If the eight-residue peptide SIINFEKL from chicken ovalbumin is extended by one or several residues at its C terminus and microinjected into cells or expressed from a minigene, it is processed and presented on major histocompatibility complex class I. However, processing and presentation are inhibited by proteasome inhibitors, such as lactacystin. In contrast, when SIINFEKL is extended by 2 to 25 residues at its N terminus, its presentation is not blocked by proteasome inhibitors. N-terminal processing also can occur when the extended peptide is cotranslationally inserted into the endoplasmic reticulum. Thus, two different proteolytic steps in the generation of an chicken ovalbumin-presented peptide can be distinguished. Cleavage by the proteasome defines the proper C terminus, whereas distinct peptidase(s) in the cytosol or endoplasmic reticulum may generate the appropriate N terminus from extended peptides.

Animals↗

PubMed1997

Lactacystin and clasto-lactacystin beta-lactone modify multiple proteasome beta-subunits and inhibit intracellular protein degradation and major histocompatibility complex class I antigen presentation.

The antibiotic lactacystin was reported to covalently modify beta-subunit X of the mammalian 20 S proteasome and inhibit several of its peptidase activities. However, we demonstrate that [3H]lactacystin treatment modifies all the proteasome's catalytic beta-subunits. Lactacystin and its more potent derivative beta-lactone irreversibly inhibit protein breakdown and the chymotryptic, tryptic, and peptidylglutamyl activities of purified 20 S and 26 S particles, although at different rates. Exposure to these agents for 1 to 2 h reduced the degradation of short- and long-lived proteins in four different mammalian cell lines. Unlike peptide aldehyde inhibitors, lactacystin and the beta-lactone do not inhibit lysosomal degradation of an endocytosed protein. These agents block class I antigen presentation of a model protein, ovalbumin (synthesized endogenously or loaded exogenously), but do not affect presentation of the peptide epitope SIINFEKL, which does not require proteolysis for presentation. Generation of most peptides required for formation of stable class I heterodimers is also inhibited. Because these agents inhibited protein breakdown and antigen presentation similarly in interferon-gamma-treated cells (where proteasomes contain LMP2 and LMP7 subunits in place of X and Y), all beta-subunits must be affected similarly. These findings confirm our prior conclusions that proteasomes catalyze the bulk of protein breakdown in mammalian cells and generate the majority of class I-bound epitopes for immune recognition.

Acetylcysteine↗

PubMed1997

Cloned dendritic cells can present exogenous antigens on both MHC class I and class II molecules.

Pathways for presenting proteins from the extracellular fluids on MHC class I molecules have been described in macrophages. However, it is uncertain whether similar mechanisms exist in dendritic cells, because conventional preparations of these cells can be contaminated with macrophages. We addressed this issue by transducing granulocyte-macrophage CSF into bone marrow cultures followed by supertransfection with myc and raf oncogenes. These immortalized clones displayed dendritic morphology, and many expressed the dendritic cell-specific markers DEC-205 and 33D1 as well as high levels of MHC molecules and costimulatory molecules. Using these cloned dendritic cells, we found that exogenous OVA could be presented on both their MHC class I and class II molecules. This presentation was markedly enhanced when the Ag was particulate and internalized by phagocytosis. Presentation of particulate OVA on MHC class I molecules was insensitive to the weak base chloroquine, but was blocked by peptide aldehyde inhibitors of the proteasome, indicating that the class I-presented peptides were generated in the cytosol. Brefeldin A, which inhibits the exocytosis of newly synthesized proteins from the endoplasmic reticulum, also inhibited Ag presentation. These results establish that dendritic cells can present exogenous Ags on MHC class I molecules and appear to use a similar phagosome to cytosol pathway as macrophages. Therefore, dendritic cells are likely to play an important role in generating immune responses to tissue transplants and tumors in vivo. Furthermore, these findings provide an approach for targeting vaccine Ags into these cells to prime immune responses in vivo.

Animals↗

PubMed1996

Major histocompatibility class I presentation of soluble antigen facilitated by Mycobacterium tuberculosis infection.

Cell-mediated immune responses are essential for protection against many intracellular pathogens. For Mycobacterium tuberculosis (MTB), protection requires the activity of T cells that recognize antigens presented in the context of both major histocompatibility complex (MHC) class II and I molecules. Since MHC class I presentation generally requires antigen to be localized to the cytoplasmic compartment of antigen-presenting cells, it remains unclear how pathogens that reside primarily within endocytic vesicles of infected macrophages, such as MTB, can elicit specific MHC class I-restricted T cells. A mechanism is described for virulent MTB that allows soluble antigens ordinarily unable to enter the cytoplasm, such as ovalbumin, to be presented through the MHC class I pathway to T cells. The mechanism is selective for MHC class I presentation, since MTB infection inhibited MHC class II presentation of ovalbumin. The MHC class I presentation requires the tubercle bacilli to be viable, and it is dependent upon the transporter associated with antigen processing (TAP), which translocates antigenic peptides from the cytoplasm into the endoplasmic reticulum. The process is mimicked by Listeria monocytogenes and soluble listeriolysin, a pore-forming hemolysin derived from it, suggesting that virulent MTB may have evolved a comparable mechanism that allows molecules in a vacuolar compartment to enter the cytoplasmic presentation pathway for the generation of protective MHC class I-restricted T cells.

ATP-Binding Cassette Transporters↗

PubMed1996

Proteasome subunits X and Y alter peptidase activities in opposite ways to the interferon-gamma-induced subunits LMP2 and LMP7.

Most antigenic peptides presented on major histocompatibility complex class I molecules are generated by proteasomes. Interferon-gamma, which stimulates antigen presentation, induces new proteasome beta-subunits LMP2 and LMP7, which replace the homologous beta-subunits Y (delta) and X (epsilon). As a result, the capacity of the proteasome to cleave model peptides increases after hydrophobic and basic residues and falls after acidic residues. To clarify the function of these subunits, we examined the effects of overexpressing subunits X (delta) and Y (epsilon). Transfection of the Y gene into HeLa cells stimulated the proteasomal cleavage after acidic residues without altering other peptidase activities. This effect was proportional to the amount of the Y subunits and opposite to the effect of its homolog, LMP2. Y appears to promote cleavages after acidic residues. Furthermore, in mutants lacking the LMP genes (in contrast to wild-type cells), interferon-gamma treatment increased the proteasome content of Y subunits and enhanced postacidic cleavages. Transfection with cDNA for the X subunit reduced hydrolysis after hydrophobic and basic residues, an effect opposite to transfection of LMP2 and LMP7. Surprisingly, transfection of X increased the amounts not only of X, but also of Y, while decreasing LMP2 content. Thus, the loss of the Y subunit upon interferon-gamma treatment or LMP2 transfection accounts for the suppression of postacidic cleavages, and the loss of X contributes to the increased hydrolysis after hydrophobic and basic residues. These adaptations should favor the production of the kinds of peptides that are presented on major histocompatibility complex class I molecules.

Amino Acid Sequence↗

PubMed1996

Chemical denaturation and modification of ovalbumin alters its dependence on ubiquitin conjugation for class I antigen presentation.

Class I presentation of microinjected native OVA by a temperature-sensitive ubiquitin conjugation mutant, ts85, but not wild-type murine cells, was markedly inhibited following incubation at a nonpermissive temperature. In contrast, the nonpermissive temperature did not affect class I presentation of a minimal OVA peptide expressed in the cytosol. Therefore, these results provide a second example in which a temperature sensitive mutation in the ubiquitin conjugation pathway inhibits MHC class I presentation of native OVA. Surprisingly, incubation at the nonpermissive temperature did not inhibit class I presentation of chemically denatured and alkylated OVA microinjected into the cytosol of mutant cells. Similarly, the presentation of endogenously synthesized OVA (which is expressed from a recombinant vaccinia virus and, presumably, is misfolded in the cytosol) was also not inhibited in both mutant cell lines. Methylation of the lysine groups in denatured OVA, which blocks ubiquitin conjugation, reduced but did not eliminate the presentation of denatured OVA, providing evidence for both ubiquitin-dependent and ubiquitin-independent pathways for class I presentation. In contrast, a proteasome inhibitor blocked class I presentation of all forms of OVA, while a control peptide aldehyde was not inhibitory. These results indicate that modification of the structure of a protein can influence its requirements for ubiquitin conjugation for efficient class I presentation, with the key alteration possibly being the loss of proper conformation. However, regardless of the form of the Ag, the proteasome appears to be required for generating peptides from both endogenously synthesized and microinjected OVA for class I presentation.

Amino Acid Sequence↗

PubMed1996

Analysis of the role of MHC class II presentation in the stimulation of cytotoxic T lymphocytes by antigens targeted into the exogenous antigen-MHC class I presentation pathway.

By conjugation of proteins to beads, Ags can be selectively targeted into the MHC class I pathway of phagocytes in vivo and can stimulate CTL responses. Because phagocytes also present particulate Ag on MHC class II molecules, we examined whether these Ags stimulated concomitant CD4 T cell immunity. Although the priming of CD4 T cells with soluble OVA required adjuvants, particulate Ag was stimulatory when injected in saline. We next examined whether CD4 T cell responses played a role in the generation of CTL to particulate Ag. At low concentrations of Ag, OVA primed CTLs in wild-type mice but not in MHC class II-deficient animals, indicating that MHC class II presentation of Ag was essential for CTL generation. These data both support a model where CD4 T cells collaborate with CTLs as part of a three-cell interaction and identify a phagocyte as the third cell in this reaction. Interestingly, injection of higher concentrations of the same Ag primed equivalent CTL responses in both wild-type and MHC class II-deficient mice. These results indicate that a key variable in determining whether CTL generation is helper cell dependent or independent is the dose of immunogen. This may explain in part why CTL responses to abundant Ags, such as viruses, tend to be helper independent, while responses to less abundant Ags, such as minor histocompatibility Ags, require T helper cells. In addition, these results also point to the potential of using particulate Ags to prime or boost responses in settings with CD4 immunodeficiency.

Animals↗

PubMed1996

Analysis of MHC class II presentation of particulate antigens of B lymphocytes.

To generate Ab responses to most protein Ags, B cells must first degrade proteins in endocytic compartments and then display antigenic peptides bound to MHC class II molecules. T helper lymphocytes recognize these complexes and stimulate the B cell to synthesize Ab. Although Ab play a key role in host defense against bacteria, it is believed that B cells are incapable of internalizing particulate Ags. However, we find that B lymphoblastoid cell lines and LPS-activated B lymphocytes can present particulate Ag up to 10(5)-fold more efficiently compared with soluble Ag. Moreover, particulate Ags are presented efficiently by unstimulated B cells when they bind to surface Ig. In comparison to B cells, macrophages in general presented particulate Ags 10- to 1000-fold more efficiently and could also present Ag from particles of a much wider range of sizes. We document by ultrastructural and immunofluorescence analysis that B lymphoblastoid cell lines bind and internalize these particles. The internalization and presentation of the particulate Ag is inhibited by cytochalasin B. In contrast, a similar morphologic analysis of normal lymphocytes demonstrated that while Ag beads are bound to the cell surface, they are internalized only rarely. These results suggest there may be both surface and intracellular pathways for the presentation of particulate Ags by B cells. Interestingly, for both macrophages and B cells, the epitopes generated from particulate and soluble Ags were not identical quantitatively or qualitatively, indicating that there are differences in how these forms of Ag are processed and presented.

Amino Acid Sequence↗

PubMed1996

A new foreign policy: MHC class I molecules monitor the outside world.

Although most cells exclusively use their major histocompatibility complex (MHC) class I molecules to present peptides from endogenous proteins, phagocytes also use them to present exogenous antigens. Here, Kenneth Rock describes how this novel antigen-presenting pathway may play an important role in immune surveillance for intracellular bacteria or parasites, as well as for viral infections and tumors affecting somatic tissues.

Animals↗

PubMed1996

Antigen processing and presentation by the class I major histocompatibility complex.

Major histocompatibility complex (MHC) class I molecules bind peptides derived from cellular proteins and display them for surveillance by the immune system. These peptide-binding molecules are composed of a heavy chain, containing an antigen-binding groove, which is tightly associated with a light chain (beta 2-microglobulin). The majority of presented peptides are generated by degradation of proteins in the cytoplasm, in many cases by a large multicatalytic proteolytic particle, the proteasome. Two beta-subunits of the proteasome, LMP2 and LMP7, are inducible by interferon-gamma and alter the catalytic activities of this particle, enhancing the presentation of at least some antigens. After production of the peptide in the cytosol, it is transported across the endoplasmic reticulum (ER) membrane in an ATP-dependent manner by TAP (transporter associated with antigen presentation), a member of the ATP-binding cassette family of transport proteins. There are minor pathways for generating presented peptides directly in the ER, and some evidence suggests that peptides may be further trimmed in this location. The class I heavy chain and beta 2-microglobulin are cotranslationally translocated into the endoplasmic reticulum where their assembly may be facilitated by the sequential association of the heavy chain with chaperone proteins BiP and calnexin. The class I molecule then associates with the lumenal face of TAP where it is retained, presumably awaiting a peptide. After the class I molecule binds a peptide, it is released for exocytosis to the cell surface where cytotoxic T lymphocytes examine it for peptides derived from foreign proteins.

Animals↗

PubMed1995

Rate of antigen degradation by the ubiquitin-proteasome pathway influences MHC class I presentation.

The effect on MHC class I Ag presentation of enhancing a protein's rate of degradation by the ubiquitin-proteasome pathway was investigated. In extracts of mouse B-lymphoblasts and reticulocytes, as in rabbit reticulocytes, proteins with acidic or basic N-termini are conjugated to ubiquitin and degraded by the 26S proteasome very rapidly. We found that the rate of MHC class I presentation of microinjected beta-galactosidase was enhanced when this antigenic protein was modified with such a destabilizing amino-terminal residue. This enhanced presentation was inhibited by blocking potential ubiquitination sites on the protein through methylation of amino groups and by peptide aldehyde inhibitors of the proteasome. Furthermore, in B lymphoblast cell extracts, the rapid degradation of these beta-galactosidase constructs required ATP and ubiquitin and was blocked by inhibitors of proteasomes. Their rates of degradation in extracts correlated with their rates of class I Ag presentation in vivo. These results indicate that ubiquitin conjugation is a key rate-limiting step in Ag presentation and provide further evidence for a critical role of ubiquitin and the 26S proteasome in generating MHC class I-presented peptides.

Amino Acid Sequence↗

PubMed1995

Overexpressed Ly-6A.2 mediates cell-cell adhesion by binding a ligand expressed on lymphoid cells.

The Ly-6 locus encodes several cell surface proteins whose functions are unknown. Although it is hypothesized that these proteins may be receptors, there is no direct evidence that they bind a ligand. Herein we present evidence that Ly-6A.2, a Ly-6 protein expressed on T lymphocytes, binds a ligand expressed on normal thymocytes and splenic B and T cells. We find that transgenic thymocytes that overexpress Ly-6A.2 spontaneously aggregate in culture. This homotypic adhesion requires the overexpression of Ly-6A.2 because it is not observed in cultures of nontransgenic thymocytes. The aggregation of Ly-6A.2 transgenic thymocytes is inhibited by phosphatidylinositol-specific phospholipase C (which removes Ly-6A.2 and other glycosylphosphatidylinositol-anchored proteins from the membrane). Some anti-Ly-6A.2 monoclonal antibodies, including nonactivating ones and Fab' fragments, inhibit this aggregation. In contrast, other anti-Ly-6A.2 monoclonal antibodies increase the aggregation of transgenic but not nontransgenic thymocytes. To further examine whether Ly-6A.2 mediates adhesion (versus inducing another adhesion pathway) reaggregation assays were performed with paraformaldehyde-fixed Tg+ thymocytes. Paraformaldehyde-fixed Tg+ thymocytes reaggregate in culture and this aggregation is also blocked by phosphatidyl-inositol-specific phospholipase C and anti-Ly-6A.2 monoclonal antibodies. These results indicate that the homotypic adhesion of cultured Ly-6A.2 transgenic thymocytes is directly mediated by Ly-6A.2 and, more importantly, strongly suggests that Ly-6A.2 binds a ligand that is expressed on thymocytes. Tg+ thymocytes also bind to nontransgenic thymocytes, B cells, and T cells, indicating that normal cells naturally express the Ly-6A.2 ligand.

Animals↗

PubMed1995

Regulated expression of Ly-6A.2 is important for T cell development.

Ly-6A.2 is a surface protein on T cells that may play a role in lymphocyte activation. The regulation of Ly-6A.2 expression during T cell lymphopoiesis has been intriguing. It is one of the earliest markers expressed on pluripotent hemopoietic stem cells and is present on both primitive and mature T cells, but its expression is extinguished in the thymus during key developmental stages. To determine whether Ly-6A.2 is active on developing T cells, as well as the significance of its developmental regulation, Ly-6A.2 was expressed throughout T cell development under control of the T cell-specific human CD2 enhancer in transgenic mice. The constitutive overexpression of Ly-6A.2 in vivo led to a marked impairment in the generation of thymocytes. Development was arrested at the time in thymic development when Ly-6A.2 expression is normally turned off. These results indicate that the regulated expression of Ly-6A.2 in thymocytes may be important for normal development. Moreover, these findings demonstrate that Ly-6A.2 is active in the thymic microenvironment.

Animals↗

PubMed1995

Gamma-interferon causes a selective induction of the lysosomal proteases, cathepsins B and L, in macrophages.

Previous studies have indicated that acid-optimal cysteine proteinase(s) in the endosomal-lysosomal compartments, cathepsins, play a critical role in the proteolytic processing of endocytosed proteins to generate the antigenic peptides presented to the immune system on major histocompatibility complex (MHC) class II molecules. The presentation of these peptides and the expression of MHC class II molecules by macrophages and lymphocytes are stimulated by gamma-interferon (gamma-IFN). We found that treatment of human U-937 monocytes with gamma-IFN increased the activities and the content of the two major lysosomal cysteine proteinases, cathepsins B and L. Assays of protease activity, enzyme-linked immunosorbant assays (ELISA) and immunoblotting showed that this cytokine increased the amount of cathepsin B 5-fold and cathepsin L 3-fold in the lysosomal fraction. By contrast, the aspartic proteinase, cathepsin D, in this fraction was not significantly altered by gamma-IFN treatment. An induction of cathepsins B and L was also observed in mouse macrophages, but not in HeLa cells. These results suggest coordinate regulation in monocytes of the expression of cathepsins B and L and MHC class II molecules. Presumably, this induction of cysteine proteases contributes to the enhancement of antigen presentation by gamma-IFN.

Animals↗

PubMed1995

A phagosome-to-cytosol pathway for exogenous antigens presented on MHC class I molecules.

Peptides from endogenous proteins are presented by major histocompatibility complex class I molecules, but antigens (Ags) in the extracellular fluids are generally not. However, pathogens or particulate Ags that are internalized into phagosomes of macrophages (M phi s) stimulate CD8 T cells. The presentation of these Ags is resistant to chloroquine but is blocked by inhibitors of the proteasome, a mutation in the TAP1-TAP2 transporter, and brefeldin A. Moreover, phagocytosis of a ribosomal-inactivating protein inhibited M phi protein synthesis. These results demonstrate that M phi s transfer Ags from phagosomes into the cytosol and that endogenous and exogenous Ags use a final common pathway for class I presentation.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1995

Targeting antigen into the phagocytic pathway in vivo induces protective tumour immunity.

Cytotoxic T lymphocytes (CTLs) kill neoplastic or virally infected cells after recognizing on their surface antigenic peptides bound to major histocompatibility complex class I molecules. These peptides are derived from antigens that are degraded in the cytosol of the affected cell. Because exogenous proteins cannot enter the cytosol, immunizations with killed pathogens or their proteins do not generally elicit CTLs. However, antigens that are internalized into phagocytic cells can enter the cytosol and be processed for class I presentation. Here we show that immunization with a purified antigen on an avidly phagocytized particle primes CTLs, which in turn protect animals from subsequent challenge with tumours transfected with the antigen gene. Interestingly, these animals also become immune to other antigens expressed by the tumour. This approach could be exploited to develop tumour and viral vaccines.

Amino Acid Sequence↗