Biomedical subjects

K L Rock

Publications and source records attributed to K L Rock.

At least 19 recordsLinked to original sources

PubMed1999

Cytotoxic T-cell immunity to virus-infected non-haematopoietic cells requires presentation of exogenous antigen.

Cytotoxic T lymphocytes (CTLs) are thought to detect viral infections by monitoring the surface of all cells for the presence of viral peptides bound to major histocompatibility complex (MHC) class I molecules. In most cells, peptides presented by MHC class I molecules are derived exclusively from proteins synthesized by the antigen-bearing cells. Macrophages and dendritic cells also have an alternative MHC class I pathway that can present peptides derived from extracellular antigens; however, the physiological role of this process is unclear. Here we show that virally infected non-haematopoietic cells are unable to stimulate primary CTL-mediated immunity directly. Instead, bone-marrow-derived cells are required as antigen-presenting cells (APCs) to initiate anti-viral CTL responses. In these APCs, the alternative (exogenous) MHC class I pathway is the obligatory mechanism for the initiation of CTL responses to viruses that infect only non-haematopoietic cells.

Animals↗

PubMed1999

Class II antigen processing defects in two H2d mouse cell lines are caused by point mutations in the H2-DMa gene.

The molecular nature of the defect in two mouse antigen processing-defective cell lines was examined. Both mutants were derived from the A20 (BALB/c, H2d) B cell line, and both were found to have defects in the H2-DMa gene. Mutant 3A5 exhibits severely reduced amounts of H2-DMa message, and no detectable DMalpha protein. cDNA sequence revealed a C-->T transition at nucleotide 118, introducing a premature stop codon in exon 2 of the H2-DMa gene. In contrast, mutant 2A2 exhibits reduced but detectable levels of H2-DMa message and DMalpha protein only after treatment with IL-4, which induces the expression of both the H2-DMa and the H2-DMb genes in B cells. In this mutant the cDNA sequence revealed a missense mutation in exon 3 resulting in the conversion of a conserved proline residue in the Ig-like domain to serine. Stable transfection with full-length H2-DMa cDNA reconstitutes the antigen processing capacity of both mutants, as demonstrated by the ability to present native antigen to T cell clones, and by restored class II SDS stability.

Animals↗

PubMed1998

The role of B7-1 and B7-2 costimulation for the generation of CTL responses in vivo.

The role of B7-1 and B7-2 costimulatory molecules in the generation of Ag-specific CD8+ CTLs is not well understood. In this paper, we analyze the role of both B7-1 and B7-2 in the generation of CTLs to nonliving, exogenous Ag and to live virus. To analyze the role of B7 costimulation in the induction of CTLs, we blocked B7-1 and/or B7-2 in vivo by injecting C57BL/6 mice with anti-B7-1 and/or anti-B7-2 mAbs; the mice were subsequently immunized with either chicken OVA that had been cross-linked to beads as a model of exogenous Ags or with wild-type and recombinant vaccinia virus expressing different forms of chicken OVA as models of viral Ags. Our results indicate that B7 costimulation is necessary in the generation of CTLs for all of these Ags. Since the B7 molecules could be costimulating CD8+ and/or CD4+ T cells in wild-type animals, we also examined the role of costimulation in the generation of CTLs to exogenous and viral Ag in MHC class II-deficient mice lacking most CD4+ T cells. In these animals, a combination of both mAbs also blocked all CTL responses, indicating that the Th cell-independent activation of CTLs is dependent upon the B7-costimulatory signals supplied to the CD8+ cell. These findings contribute to the understanding of the role of costimulation for the generation of CTLs. We also discuss the implications of these findings on the role of professional APCs in the initiation of CTL responses.

Animals↗

PubMed1998

Interferon-gamma can stimulate post-proteasomal trimming of the N terminus of an antigenic peptide by inducing leucine aminopeptidase.

Most antigenic peptides presented on major histocompatibility complex class I molecules are generated during protein breakdown by proteasomes, whose specificity is altered by interferon-gamma (IFN-gamma). When extended versions of the ovalbumin-derived epitope SIINFEKL are expressed in vivo, the correct C terminus is generated by proteasomal cleavage, but distinct cytosolic protease(s) generate its N terminus. To identify the other protease(s) involved in antigen processing, we incubated soluble extracts of HeLa cells with the 11-mer QLESIINFEKL, which in vivo is processed to the antigenic 8-mer (SIINFEKL) by a proteasome-independent pathway. This 11-mer was converted to the 9-mer by sequential removal of the N-terminal residues, but surprisingly the extract showed little or no endopeptidase or carboxypeptidase activity against this precursor. After treatment of cells with IFN-gamma, this N-terminal trimming was severalfold faster and proceeded to the antigenic 8-mer. The IFN-treated cells also showed greater aminopeptidase activity against many model fluorogenic substrates. Upon extract fractionation, three bestatin-sensitive aminopeptidase peaks were detected. One was induced by IFN-gamma and was identified immunologically as leucine aminopeptidase (LAP). Purified LAP, like the extracts of IFN-gamma-treated cells, processed the 11-mer peptide to SIINFEKL. Thus, IFN-gamma not only promotes proteasomal cleavages that determine the C termini of antigenic peptides, but also can stimulate formation of their N termini by inducing LAP. This enzyme appears to catalyze the trimming of the N terminus of this and presumably other proteasome-derived precursors. Thus, susceptibility to LAP may be an important influence on the generation on immunodominant epitopes.

Antigens↗

PubMed1998

Poliovirus vaccine vectors elicit antigen-specific cytotoxic T cells and protect mice against lethal challenge with malignant melanoma cells expressing a model antigen.

Recombinant polioviruses expressing foreign antigens may provide a convenient vaccine vector system to induce protective immunity against diverse pathogens. Replication-competent chimeric viruses can be constructed by inserting foreign antigenic sequences within the poliovirus polyprotein. When inserted sequences are flanked by poliovirus protease recognition sites the recombinant polyprotein is processed to mature and functional viral proteins plus the exogenous antigen. It previously has been shown that poliovirus recombinants can induce antibody responses against the inserted sequences but it is not known whether poliovirus or vaccine vectors derived from it can elicit effective cytotoxic T lymphocyte (CTL) responses. To examine the ability of the recombinant poliovirus to induce CTL responses, a segment of the chicken ovalbumin gene, which includes the H2-Kb-restricted CTL epitope SIINFEKL, was cloned at the junction of the P1 and P2 regions. This recombinant virus replicated with near wild-type efficiency in culture and stably expressed high levels of the ovalbumin antigen. Murine and primate cells infected with the recombinant virus appropriately processed the SIINFEKL epitope and presented it within major histocompatibility complex class I molecules. Inoculation of mice with recombinant poliovirus that expresses ovalbumin elicits an effective specific CTL response. Furthermore, vaccination with these recombinant poliovirus induced protective immunity against challenge with lethal doses of a malignant melanoma cell line expressing ovalbumin.

Animals↗

PubMed1998

CD4+ T cells mature in the absence of MHC class I and class II expression in Ly-6A.2 transgenic mice.

The TCRs expressed on T lymphocytes recognize foreign peptides bound to MHC molecules. This reactivity is the basis of specific immune response to the foreign Ag. How such specificities are generated in the thymus is still being debated. Signals generated through TCR upon interaction with self MHC-peptide complexes are critical for maturation of the CD4+ helper and CD8+ cytotoxic subsets. We have observed maturation of CD4+ but not CD8+ T cells in Ly-6A.2 transgenic MHC null mice. Since there can be no interactions with MHC molecules in these mice, these CD4+ cells must express the T cell repertoire that exists before positive and negative selection. Interestingly, despite an absence of selection by MHC molecules, the CD4+ cells that mature recognize MHC molecules at a frequency as high as in CD4+ cells in normal mice. These results demonstrate that: 1) the germline sequences encoding TCRs are biased toward reactivity to MHC molecules; and 2) CD4+ cells as opposed to CD8+ cells have distinct lineage commitment signals. These results also suggest that signals originating from Ly-6 can promote or substitute for signals generated from TCR that are required for positive selection. Moreover, this animal model offers a system to study T cell development in the thymus that can provide insights into mechanisms of lineage commitment in developing T cells.

Animals↗

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↗