Biomedical subjects
Publications and source records attributed to K L Rock.
Proteins presented to the immune system must first be cleaved to small peptides by intracellular proteinases. Proteasomes are proteolytic complexes that degrade cytosolic and nuclear proteins. These particles have been implicated in ATP-ubiquitin-dependent proteolysis and in the processing of intracellular antigens for cytolytic immune responses.
Ag in the extracellular fluids can be internalized, processed, and presented in association with class I MHC molecules on specialized APC in normal spleen. We examine the fate of these APC after they present Ag to a CTL. When splenocytes present exogenous OVA to CTL, their ability to subsequently present native Ag in association with both class I and class II molecules is inhibited. CTL do not inhibit the ability of splenocytes to present processing independent peptides with class I or class II molecules. Inhibition of Ag presentation is only observed in the presence of the specific Ag recognized by the CTL. This inhibition is MHC-restricted. In the presence of specific Ag, CTL inhibit the ability of APC to present unrelated Ag. However, bystander APC are not affected by activated CTL. Taken together these results indicate that when APC present exogenous Ag to CTL, they are inhibited or killed. The CTL that mediates this activity has a conventional CD4-CD8+ phenotype and utilizes a TCR-alpha beta. The potential significance of these findings and their possible relationship to phenomena associated with Ts cells are discussed.
Class I heterodimers on the surface of cells are generally unreceptive to binding peptides in the absence of exogenous beta 2-microglobulin. Paraformaldehyde covalently cross-links beta 2-microglobulin to class I heavy chains in situ and stabilizes empty class I heterodimers. Functionally, this cross-linking creates receptive class I peptide binding sites by acting on beta 2-microglobulin-associated molecules. The presentation of preexisting peptide-class I complexes is also enhanced. These findings support a model whereby a structural alteration, the dissociation of beta 2-microglobulin, limits the existence of receptive class I molecules on normal cells and may control the half-life of active class I molecules.
LB27.4 cells (a B lymphoblastoid APC) were transfected with a plasmid containing an OVA cDNA. Functional analysis of six independent clones yielded three patterns of MHC-restricted presentation of the endogenously synthesized OVA. A clone displayed either: 1) strong class I and class II-restricted presentation, 2) strong class I but little or no class II-restricted presentation or, 3) only a modest class I-restricted presentation. There was no clonal variation in class II-restricted presentation of exogenous Ag or in the amount of surface class I or II molecules. Heat shock increased the presentation of endogenous but not exogenous Ag with class II. These results indicate that an endogenously synthesized Ag both constitutively and during heat shock can gain access to the class II, MHC-restricted, presentation pathway. The amount of OVA synthesized by a cell correlated with whether OVA-class II complexes were detected. However, the amount of OVA secreted into the extracellular fluid was not sufficient to sensitize APC, which suggests that endogenously synthesized OVA enters the class II pathway of Ag presentation by an intracellular route rather than by an extracellular/reuptake route. Also, the functional and quantitative analysis of the clones suggests that endogenously synthesized OVA was presented more efficiently with class I as compared to class II-MHC molecules. Leupeptin and chloroquine inhibited the class II-restricted presentation of endogenously synthesized OVA. Together these results indicate that endogenously synthesized OVA can gain access to an endosomal/lysosomal compartment via an intracellular route and be processed and presented in association with class II-MHC molecules.
We have used a T-T hybridoma, RF33.70, to detect the MHC class I-restricted presentation of exogenous native OVA by thymic APC in vitro. Presentation of OVA with class I molecules by thymic APC requires intracellular processing. Phenotypic analyses indicated that low bouyant density, MHC class II+, FcR+ cells are capable of using this presentation pathway. In order to determine whether thymic APC have this function in vivo, thymic APC were isolated from mice after i.v. injection of native OVA. We find that OVA is presented in association with MHC class I, but not class II, molecules in the thymus. This is in contrast to splenic APC, which present exogenous OVA with both class I and class II molecules under these conditions. Our findings have implications for the repertoire of self-peptides that might be presented by thymic APC to developing T lymphocytes.
Ly-6A.2 (T cell-activating protein, TAP) and Thy-1 are glycosyl-phosphatidyl-inositol (GPI)-anchored proteins expressed on the surface of murine T lymphocytes. We have found that Ly-6A.2 (TAP) and Thy-1 are internalized by T cells. In the present study we have investigated whether these GPI-anchored proteins enter cells by endocytosis through coated pits. Two lines of evidence argue against the involvement of coated pits in the internalization of Ly-6A.2 (TAP) and Thy-1. First, drugs that effectively blocked the endocytosis of transferrin receptor and H-2 class I molecules, (which are known to be internalized via coated pits) did not inhibit the internalization of the GPI-anchored proteins. Second, in ultrastructural analyses, Ly-6A2 (TAP) and Thy-1, in contrast to the transferrin receptor, were rarely found in coated pits or vesicles. These observations suggest that the GPI-anchored proteins on T lymphocytes are internalized by a distinct pathway that does not involve endocytosis through coated pits.
Lines of mice selected for high or low antibody production to sheep red blood cells (H-I and L-I) were studied for their ability to process and present ovalbumin to a panel of 12 T-T hybridomas in two different H-2 haplotypes. When H-I and L-I spleen cells were used as antigen-presenting cells, no difference could be observed in the peptide generation by these mice compared to H-2-compatible B.10.Q and B.10.S spleen cells, respectively. Neither normal splenic L-I B-cells nor L-I thioglycolate-induced peritoneal macrophages were defective at presenting native ovalbumin, to six and eight different I-As-restricted T-T hybrids, respectively. Altogether, these results differ from previous findings which had indicated a deficiency in the processing and presentation of antigen by the low line, L-I.
Monoclonal antibody crosslinking of phosphatidylinositol-anchored Ly-6A.2 molecules on the surface of murine T lymphocytes leads to cell activation and secretion of IL-2. To examine the potential activity of these molecules in human T cells we transfected the Ly-6A.2 gene into Jurkat cells. Transfection of Jurkat cells with genomic Ly-6A.2 sequences results in low levels of Ly-6A.2 on the cell surface. However, linking the Ly-6A.2 sequences to the enhancer from the human CD2 gene results in greatly increased expression of Ly-6A.2. These molecules are anchored to the membrane via a phosphatidylinositol linkage. Crosslinking of Ly-6A.2 molecules with soluble mAb stimulates the transfected Jurkat cells to produce IL-2. This stimulation is abrogated by treatment with phosphatidylinositol-specific phospholipase C. The transfected human T cells displayed the same unusual crosslinking requirements for stimulation with anti-Ly-6A.2 mAbs as previously observed for murine T cells. Crosslinking of Ly-6A.2 with soluble antibodies is stimulatory, whereas immobilized antibodies are inactive. The crosslinking requirements for antiCD3 mAb stimulation display a reciprocal pattern. These data demonstrate that the Ly-6A.2 pathway for T cell activation is conserved between human and murine T cells.
The ability of splenic APC and a B cell hybridoma (LS.102.9) to process and present OVA to a panel of T-T hybridomas with different fine specificities was investigated. Splenic APC process and present OVA to all the T-T hybrids. The B cell hybridoma could similarly process and present OVA to some T-T hybrids but was very inefficient in stimulating two of the T cell hybridomas. The presentation of native OVA to these two T-T hybrids was significantly increased by leupeptin. Pulsing experiments demonstrated that leupeptin acted on the APC at a step before the processed Ag was displayed on the cell surface in association with MHC molecules. Leupeptin has no effect on the presentation of OVA peptides by LS.102.9 to the T-T hybrids. Leupeptin inhibits the generation of the epitopes of OVA that LS.102.9 produces under basal conditions. We also surveyed the effect of other protease inhibitors and observed similar augmenting and inhibitory effects on the presentation of selected OVA epitopes. The augmentation of processing by a protease inhibitor indicates that in the lysosomal/endosomal compartment proteases have capacity to both generate and destroy immunogenic peptides. Our data suggest that protease inhibitors could potentially be used as immunomodulators and are discussed in terms of physiology of the lysosomal/endosomal compartment.
E710.2 is a cloned T cell line that was isolated from an AKR/J thymic tumor. This clone expresses Thy-1, heat-stable Ag, and the CD3/TCR complex but does not express CD4 or CD8. When the E710.2 cell line is injected into syngeneic mice, it grows as a malignant tumor in lymphoid organs and the thymus. In contrast, this cell line does not grow in vitro under standard culture conditions. This latter property allowed us to analyze the in vitro responsiveness of this CD4-CD8- cell line to stimulation by pharmacologic agents and cellular elements from the spleen and thymus. E710.2 cells proliferate when stimulated with phorbol esters or when cocultured with thymocytes or splenocytes. We could not detect soluble stimulatory factors in cultures of E710.2 and/or lymphoid cells, suggesting that cell contact might be required for this response. The stimulatory activity in thymus and spleen appears to be broadly expressed, because all cell subsets that were examined from these tissues stimulate this cell line. The stimulation of E710.2 cells is not MHC-restricted and is not inhibited by anti-MHC mAb. Furthermore, the responsiveness of these cells is not decreased when the TCR/CD3 complex is modulated from the cell surface. Similarly, TCR/CD3-deficient E710.2 variant clones retain their responsiveness to thymic and splenic cell stimulation. These findings suggest that there is a TCR-independent pathway of activation in E710.2 that is stimulated by a broadly expressed, non-MHC-encoded molecules(s).
A large pool of free class I heavy chains is detected in situ on the plasma membrane of living cells. These chains are present on cells of different MHC genotypes and appear to exist under physiological conditions in vivo. These molecules arise from the dissociation of previously assembled class I heterodimers at the cell surface. The ratio of intact to dissociated heterodimers is strongly affected by the occupancy of the peptide-binding site of the class I molecule. Upon dissociation of the heterodimer, the class I molecule is functionally inactive. These findings may help to explain why class I molecules on the cell surface are unreceptive to binding peptides yet readily associate with peptides in the presence of exogenous beta 2-microglobulin. These results have implications for understanding the distinct functions of class I versus class II molecules and how the immunological identity of cells is preserved.
RMA-S murine cells have a mutation that interferes with the assembly of class I major histocompatibility complex (MHC) heterodimers and are deficient in the expression of class I molecules on the cell surface. The mutant phenotype has been reported to be normalized upon incubation of RMA-S cells at 25 degrees C. We find that much of the increased expression of class I heterodimers is dependent on culturing RMA-S cells in bovine serum or with purified bovine beta 2-microglobulin. Furthermore, epitopes that are associated with class I MHC molecules that have bound xenogeneic beta 2-microglobulin are preferentially formed on RMA-S cells cultured at 25 degrees C. These heterologous class I molecules are thermolabile. Increased expression of class I molecules has also been observed on RMA-S cells incubated at 37 degrees C in the presence of class I-restricted peptides. We find that the increased expression of Db molecules induced by influenza virus nucleoprotein residues 365-380 is similarly dependent on culturing RMA-S cells in bovine serum or with purified bovine beta 2-microglobulin.
Engagement of the surface Ig receptor with anti-IgM antibodies stimulates murine B lymphocytes to markedly increase their expression of the cell adhesion molecules ICAM-1 and LFA-1. Stimulated B cells display increased homotypic adhesiveness and form spontaneous heterotypic conjugates with T lymphocytes. This latter T-B cell interaction is further enhanced if T cells have been previously activated with phorbol esters. In all cases, the formation of cell-cell conjugates is dependent on LFA-1-ICAM-1-mediated interactions as assessed in mAb blocking experiments. B lymphocytes stimulated with anti-IgM display a marked increase in binding to ICAM-1-transfected L cells. This cell-cell interaction is inhibited by anti-LFA-1 mAb binding to the B lymphocyte. Together, these results demonstrate that there is an induction of both ICAM-1 and LFA-1 on stimulated B cells and a corresponding increase in the adhesiveness of these cells. These findings suggest that Ag binding to the surface Ig receptor could prepare a B lymphocyte for subsequent interaction with a T lymphocyte. This provides insight into how efficient T-B collaboration may occur between very infrequent Ag-specific lymphocytes.
The murine Ly-6 locus controls the expression of a number of genes. One of the products of the Ly-6 locus, Ly-6A.2, has been implicated in the process of T cell activation. We have identified the chromosomal sequences encoding the Ly-6A.2 molecule using very stringent hybridization and washing conditions. We confirmed that this gene encoded the Ly-6A.2 molecule by transfection studies using a cell line genetically negative for the Ly-6A.2 gene as a DNA recipient. Sequence analysis showed that the Ly-6A.2 gene is made up of four exons. The start site of transcription was determined by primer extension analysis. The first exon does not contain protein coding sequences. The structure of the Ly-6A.2 gene supports previous speculation that various Ly-6 RNA can be generated by alternate splicing events. The Ly-6A.2 chromosomal gene is closely related to the previously characterized Ly-6C.1 chromosomal gene in the intron, exon, and 5' flanking regions. This analysis indicates that these genes have arisen as a consequence of gene duplication. Although endogenous Ly-6A.2 and Ly-6C genes are IFN responsive, only the latter contains a clearly identifiable IFN responsive element. A transfected Ly-6A.2 chromosomal gene that contains 3.9 kb of 5'-untranslated sequence is IFN responsive. However, a shorter chromosomal clone containing only 940 bp of 5' sequence is constitutively expressed in transfectants but does not respond to IFN.
This report describes the effects of NH4Cl, CH3NH2, and chloroquine on class I and II MHC-restricted Ag presentation. OVA-specific T-T hybridomas were used to detect processed OVA in association with class I, H-2Kb, and class II, I-Ad/b, molecules on a B lymphoblastoid APC. OVA, internalized by APC under hypertonic conditions, was presented in association with class I and II MHC molecules. Treating the APC with NH4Cl or CH3NH2 inhibited class I- and II-restricted Ag presentation. In contrast, chloroquine markedly inhibited class II, but not class I-restricted Ag presentation. Controls indicated that drug-treated APC were fully competent to interact with T cells and present processing-independent antigenic peptides in association with both class I and II MHC molecules. NH4Cl and CH3NH2 did not inhibit the uptake of radiolabeled Ag by the APC. After the proteolytic removal of H-2Kb from the surface of APC, NH4Cl and CH3NH2-treated and control APC regenerated identical amounts of surface H-2Kb and this regeneration required de novo protein synthesis. These latter results indicate that NH4Cl and CH3NH2 can inhibit Ag presentation without affecting the synthesis, transport, or surface expression of H-2Kb. Also, NH4Cl did not affect the transport of H-2Db to the surface of mutant RMA-S cells that were cultured with exogenous peptides. Taken together these results strongly suggest that NH4Cl and CH3NH2 but not chloroquine can inhibit a critical and early intracellular step in class I-restricted Ag presentation while simultaneously inhibiting class II-restricted Ag presentation.
A synthetic peptide corresponding to residues 365-380 of the influenza nucleoprotein (NP365-380) has been previously shown to associate with class I major histocompatibility complex-encoded molecules and to stimulate cytotoxic T lymphocytes [Townsend, A. R. M., Rothbard, J., Gotch, F. M., Bahadur, G., Wraith, D. & McMichael, A. J. (1986) Cell 44, 959-968]. We find that intact Db class I heterodimers on the cell surface are unreceptive to binding this antigen. However, NP365-380 readily associates with Db molecules on the plasma membrane in the presence of exogenous beta 2-microglobulin. In addition, there is a second pathway through which this peptide associates with class I molecules that requires energy and de novo protein synthesis. These findings have implications for maintaining the immunological identity of cells and for the use of peptides as vaccines for priming cytolytic T-cell immunity.
NIH3T3 cells were transfected with a vector containing c-H-ras under the transcriptional control of the mouse metallothionein-I promoter. When c-H-ras expression was induced with heavy metal ions there was a marked reduction in the expression of two glycosylphosphatidylinositol (GPI) anchored proteins, TAP/Ly-6A.2 and Thy-1, on the plasma membrane of the transformed cells. In contrast the cell-surface expression of other non-GPI-anchored proteins was unaltered. The major loss of Thy-1 induced by ras activation occurred from the pool of molecules expressed on the cell surface. The Thy-1 molecules that were preferentially lost from the surface of the ras-transformed cells could not be recovered from the extracellular fluid by immunoprecipitation. In contrast, the rate of internalization of Thy-1 was increased approximately 69% subsequent to ras activation. The possible significance of these findings to the function of c-H-ras is discussed.
Soluble antigens (Ags) in the extracellular fluids are excluded from the class I major histocompatibility complex (MHC)-restricted pathway of Ag presentation in most cells. However, an exogenous Ag can be internalized, processed, and presented in association with class I MHC molecules on specialized Ag-presenting cells (APCs). These APCs express class II molecules and can simultaneously present exogenous Ags to both class I and class II MHC-restricted T cells. These APCs may be important participants in the regulation of host immune responses. This APC activity may explain several phenomena of cytotoxic T lymphocyte (CTL) priming in vivo and might be exploited for eliciting CTL responses to protein vaccines.