Biomedical subjects
Publications and source records attributed to R Glas.
Psychological and immunological responses of cancer patients to a psychosocial intervention program will be assessed over time. Previously it has been proposed that there are two large divisions in cancer histology (type I and II) and that the psychobiology of patients will vary accordingly.
Cytosolic proteolysis is carried out predominantly by the proteasome. We show that a large oligopeptidase, tripeptidylpeptidase II (TPPII), can compensate for compromised proteasome activity. Overexpression of TPPII is sufficient to prevent accumulation of polyubiquitinated proteins and allows survival of EL-4 cells at otherwise lethal concentrations of the covalent proteasome inhibitor NLVS (NIP-leu-leu-leu-vinylsulfone). Elevated TPPII activity also partially restores peptide loading of MHC molecules. Purified proteasomes from adapted cells lack the chymotryptic-like activity, but still degrade longer peptide substrates via residual activity of their Z subunits. However, growth of adapted cells depends on induction of other proteolytic activities. Therefore, cytosolic oligopeptidases such as TPPII normalize rates of intracellular protein breakdown required for normal cellular function and viability.
Natural killer (NK) cells can spontaneously lyse certain virally infected and transformed cells. However, early in immune responses NK cells are further activated and recruited to tissue sites where they perform effector functions. This process is dependent on cytokines, but it is unclear if it is regulated by NK cell recognition of susceptible target cells. We show here that infiltration of activated NK cells into the peritoneal cavity in response to tumor cells is controlled by the tumor major histocompatibility complex (MHC) class I phenotype. Tumor cells lacking appropriate MHC class I expression induced NK cell infiltration, cytotoxic activation, and induction of transcription of interferon gamma in NK cells. The induction of these responses was inhibited by restoration of tumor cell MHC class I expression. The NK cells responding to MHC class I-deficient tumor cells were approximately 10 times as active as endogenous NK cells on a per cell basis. Although these effector cells showed a typical NK specificity in that they preferentially killed MHC class I-deficient cells, this specificity was even more distinct during induction of the intraperitoneal response. Observations are discussed in relation to a possible adaptive component of the NK response, i.e., recruitment/activation in response to challenges that only NK cells are able to neutralize.
The ubiquitin/proteasome-dependent proteolytic pathway is an attractive target for therapeutics because of its critical involvement in cell cycle progression and antigen presentation. However, dissection of the pathway and development of modulators are hampered by the complexity of the system and the lack of easily detectable authentic substrates. We have developed a convenient reporter system by producing N-end rule and ubiquitin fusion degradation (UFD)-targeted green fluorescent proteins that allow quantification of ubiquitin/proteasome-dependent proteolysis in living cells. Accumulation of these reporters serves as an early predictor of G2/M arrest and apoptosis in cells treated with proteasome inhibitors. Comparison of reporter accumulation and cleavage of fluorogenic substrates demonstrates that the rate-limiting chymotrypsin-like activity of the proteasome can be substantially curtailed without significant effect on ubiquitin-dependent proteolysis. These reporters provide a new powerful tool for elucidation of the ubiquitin/proteasome pathway and for high throughput screening of compounds that selectively modify proteolysis in vivo.
Triggering of a T cell requires interaction between its specific receptor (TCR) and a peptide antigen presented by a self-major histocompatibility complex (MHC) molecule. TCR recognition of self-MHC by itself falls below the threshold of detection in most systems due to low affinity. To study this interaction, we have used a read-out system in which antigen-specific effector T cells are confronted with targets expressing high levels of MHC compared with the selecting and priming environment. More specifically, the system is based on CD8(+) T cells selected in an environment with subnormal levels of MHC class I in the absence of beta2-microglobulin. We observe that the MHC restriction element can trigger viral peptide-specific T cells independently of the peptide ligand, provided there is an increase in self-MHC density. Peptide-independent triggering required at least four times the natural in vivo level of MHC expression. Furthermore, recognition of the restriction element at expression levels below this threshold was still enough to compensate for lack of affinity to peptides carrying alanine substitutions in major TCR contact residues. Thus, the specificity in TCR recognition and T cell activation is fine tuned by the avidity for self-MHC, and TCR avidities for peptide and MHC may substitute for each other. These results demonstrate a functional role for TCR avidity for self-MHC in tuning of T cell specificity, and support a role for cross-reactivity on "self" during T cell selection and activation.
MHC class I molecules can prevent NK cell-mediated cytotoxicity by interacting with inhibitory receptors on the effector cells. Different conclusions have been reached regarding possible peptide selectivity of these receptors. To address whether peptide selectivity is an exclusive feature of human or immunoglobulin-superfamily receptors, we have studied a system based on the murine NK receptor Ly-49C in the lectin-superfamily. Loading of TAP-deficient RMA-S cells with the H-2Kb-restricted, ovalbumin-derived peptide OVA(257 - 264) (pOVA) induced their ability to bind Ly-49C-transfected reporter cells, and also protected them from killing by Ly-49C+ NK cells. Other peptides that bound and stabilized H-2Kb equally well differed in their NK protective capacity. Comparison of the MHC class I peptide complexes (crystal structures and molecular models) revealed a conformational motif encompassing the C-terminal parts of the alpha1 helix (73 - 77) and the bound peptide that was common for the protective complexes. Substitution analysis of pOVA suggested that position 7 in the peptide may be critical for optimal protection as well as for the conformational motif at position 73 - 77. In conclusion, protection mediated by the murine C-type lectin receptor Ly-49C is peptide dependent and selective.
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In order to deduce the ancestral genome arrangement in the karyotypically diverse marsupial family Macropodidae, and to assess chromosome change in this family, chromosome-specific paints from the tammar wallaby (2n = 16) were hybridized to metaphase spreads from the two species proposed to represent the 2n = 22 ancestral karyotype, as well as species with derived 2n = 20 and 2n = 14 karyotypes. Identical patterns were observed in the two 2n = 22 species, from which the rearrangements to form the three derived karyotypes may be easily deduced to be 1, 3 and 4 different fusions, respectively. The identical Thylogale and Dorcopsis genomes may both be used to represent the pleisiomorphic macropodid chromosome complement. Variation in the X chromosome was also investigated by hybridizing an X-Y shared tammar wallaby 12-kb repeat element to chromosomes from the other four macropodid species, finding that it hybridized only to the most closely related species, and therefore is of recent origin.
We obtained mice deficient for major histocompatibility complex (MHC) molecules encoded by the H-2K and H-2D genes. H-2 KbDb -/- mice express no detectable classical MHC class I-region associated (Ia) heavy chains, although beta2-microglobulin and the nonclassical class Ib proteins examined are expressed normally. KbDb -/- mice have greatly reduced numbers of mature CD8+ T cells, indicating that selection of the vast majority (>90%) of CD8+ T cells cannot be compensated for by beta2-microglobulin-associated molecules other than classical H-2K and D locus products. In accord with the greatly reduced number of CD8+ T cells, spleen cells from KbDb -/- mice do not generate cytotoxic responses in primary mixed-lymphocyte cultures against MHC-disparate (allogeneic) cells. However, in vivo priming of KbDb -/- mice with allogeneic cells resulted in strong CD8+ MHC class Ia-specific allogeneic responses. Thus, a minor population of functionally competent peripheral CD8+ T cells capable of strong cytotoxic activity arises in the complete absence of classical MHC class Ia molecules. KbDb -/- animals also have natural killer cells that retain their cytotoxic potential.
Proteolysis is essential for the execution of many cellular functions. These include removal of incorrectly folded or damaged proteins, the activation of transcription factors, the ordered degradation of proteins involved in cell cycle control, and the generation of peptides destined for presentation by class I molecules of the major histocompatibility complex. A multisubunit protease complex, the proteasome, accomplishes these tasks. Here we show that in mammalian cells inactivation of the proteasome by covalent inhibitors allows the outgrowth of inhibitor-resistant cells. The growth of such adapted cells is apparently maintained by the induction of other proteolytic systems that compensate for the loss of proteasomal activity.
Mice lacking beta2-microglobulin (beta2m- mice) express greatly reduced levels of MHC class I molecules, and cells from beta2m- mice are therefore highly sensitive to NK cells. However, NK cells from beta2m- mice fail to kill beta2m- normal cells, showing that they are self tolerant. In a first attempt to understand better the basis of this tolerance, we have analyzed more extensively the target cell specificity of beta2m- NK cells. In a comparison between several MHC class I-deficient and positive target cell pairs for sensitivity to beta2m- NK cells, we made the following observations: First, beta2m- NK cells displayed a close to normal ability to kill a panel of MHC class I-deficient tumor cells, despite their nonresponsiveness to beta2m- concanavalin A (Con A)-activated T cell blasts. Secondly, beta2m- NK cells were highly sensitive to MHC class I-mediated inhibition, in fact more so than beta2m+ NK cells. Thirdly beta2m- NK cells were not only tolerant to beta2m- Con A blasts but also to Con A blasts from H-2Kb-/Db- double deficient mice in vitro. We conclude that NK cell tolerance against MHC class I-deficient targets is restricted to nontransformed cells and independent of target cell expression of MHC class I free heavy chains. The enhanced ability of beta2m- NK cells to distinguish between MHC class I-negative and -positive target cells may be explained by increased expression of Ly49 receptors, as described previously. However, the mechanisms for enhanced inhibition by MHC class I molecules appear to be unrelated to self tolerance in beta2m- mice, which may instead operate through mechanisms involving triggering pathways.
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This study addresses the role of MHC class I molecules in the rejection of tumor grafts by SCID mice. Tumor cell lines, their corresponding MHC class I transfectants, and MHC class I-deficient mutants were inoculated to SCID mice. This allowed a study of tumor rejection responses in an environment with normal numbers of natural killer cells but largely devoid of functional T and B cells. C.B-17 (H-2d) SCID mice were found to reject low (10(2)) but not high (10(4)) doses of allogeneic (H-2b) tumor cells. The introduction of H-2Dd into such allogeneic tumor cells abrogated the rejection response with progressive tumor growth as a consequence. Introduction of H-2Kd or Ld had no or only marginal effects. The protective ability of H-2Dd was mapped to the alpha 1/alpha 2 domains of the molecule. H-2Dd protected allogeneic tumors from rejection also in C3H SCID mice of the H-2k haplotype, demonstrating that this ability was not dependent on H-2Dd expression in the host. Expression of endogenous H-2Kb and/or Db molecules partially protected wild-type allogeneic tumor cells from rejection since mutant allogeneic cells, devoid of class I expression, were rejected even after high-dose inoculation. Introduction of either allogeneic or xenogeneic class I molecules did not lead to rejection of otherwise MHC class I syngeneic (H-2d) tumor cells. The observed tumor cell rejection in SCID mice was dependent on natural killer cells. After depletion of asialo-GM1+ cells, all inoculated tumor cell lines grew progressively, independently of MHC class I expression. These results are compatible with a model where expression of certain, but not all, class I molecules protect from natural killer cell-mediated rejection. There was no evidence for rejection occurring as a consequence of the expression of allogeneic or xenogeneic class I molecules on the grafted cells. MHC class I expression may thus influence tumor cell recognition in mice lacking T-cell receptor expression.
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Beta 2-Microglobulin-deficient (beta 2m -/-) mice are reported to lack cell surface expression of major histocompatibility complex (MHC) class I molecules, CD8+ T cells, and the ability to mount MHC class I-specific T cell responses. We have observed that beta 2m -/- mice possess CD8+ T cells that can be induced to perform strong allospecific cytotoxic responses against nonself-MHC class I by in vivo priming. We report that these beta 2m -/- cytotoxic T lymphocyte (CTL) differ from those induced in beta 2m-positive littermates in that they cross-react and kill cells expressing self-MHC class I at normal ligand density with beta 2m. beta 2m -/- CTL could even be induced in primary mixed lymphocyte culture by self-MHC class I expressing stimulator cells, whereas allogeneic stimulator cells failed to elicit a response under similar conditions. Cells with a reduced cell surface MHC class I expression were less sensitive, while syngeneic beta 2m -/- cells were resistant to the beta 2m -/- CTL. This antiself-MHC reactivity could not be induced when beta 2m -/- T cells matured in an environment with normal MHC class I expression in bone marrow chimeric mice. Antiself-MHC reactivity was also observed against human peptide loading-deficient cells expressing the appropriate murine class I molecules, suggesting that affinity to self-MHC class I may occur irrespective of peptide content. The results fit with a model where positive and negative selection of CD8+ T cells in beta 2m -/- mice is mediated by low levels of MHC class I free heavy chains. In this model, low ligand density on selecting cells leads to positive selection of rare T cells that bind to low levels of MHC class I free heavy chains, resulting in a very small peripheral CD8+ compartment. Due to low density of the selecting ligand, negative selection does not remove T cells recognizing beta 2m-positive cells expressing self-MHC class I at normal ligand density, which generates a T cell repertoire that would be autoreactive in a beta 2m-positive littermate. The first "MHC deficient" animals thus paradoxically provide a tool for direct demonstration and analysis of self MHC bias in the T cell repertoire.
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