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Biomedical subjects

A M Benham

Publications and source records attributed to A M Benham.

8 recordsLinked to original sources
PubMed1998

Allelic differences in the relationship between proteasome activity and MHC class I peptide loading.

MHC class I molecules are cell surface glycoproteins that play a pivotal role in the response to intracellular pathogens. The loading of MHC class I molecules with antigenic substrates takes place in the endoplasmic reticulum. This requires a functional TAP transporter, which translocates peptides into the endoplasmic reticulum from the cytosol. The generation of antigenic peptides from polypeptide precursors is thought to be mediated in the cytosol by the proteasome. Previously, we have demonstrated that inhibiting the proteasome with the specific covalent inhibitor lactacystin results in a direct reduction of peptide-loaded MHC class I molecules. This indicates that the proteasome is the limiting step in the MHC class I pathway. In this study we use isoelectric focusing to demonstrate that two related MHC class I alleles, HLA-A3 and HLA-A11, as well as HLA-B35 do not follow this behavior. In contrast to other class I alleles expressed by the same cells, these alleles are loaded with peptides and mature normally when proteasome activity is severely inhibited. Our observations highlight a new level of diversity in the MHC class I system and indicate that there are allele-specific differences in the linkage between proteasome activity and MHC class I peptide loading.

Alleles↗
PubMed1997

Proteasome activity limits the assembly of MHC class I molecules after IFN-gamma stimulation.

For an effective CD8+ cytotoxic T cell response to occur during infection, MHC class I molecules must be loaded with antigenic peptides in the endoplasmic reticulum. The cytosolic factor responsible for peptide generation is believed to be the proteasome, with the TAP heterodimer mediating peptide transport into the endoplasmic reticulum. However, the rate-determining step(s) in this intracellular pathway of Ag presentation is currently unresolved. The availability of a specific and irreversible proteasome inhibitor called lactacystin has enabled us to determine the amount of proteasomes required for the peptide loading of MHC class I molecules in four cell types. In the absence of the IFN-gamma-inducible proteasome subunits LMP2 and LMP7, the trypsin-like (but not the chymotrypsin-like) activity of the proteasome is directly related to MHC class I peptide loading. However, IFN-gamma stimulation or assimilation of catalytic LMP2 and LMP7 subunits into proteasomes causes both chymotrypsin- and trypsin-like activities of the proteasome to become limiting for the loading of class I molecules. Our data suggest that upon full IFN-gamma stimulation, peptide supply by the proteasome is the limiting step in the assembly of MHC class I polypeptides. This mechanism may enable the cell to prevent competition between novel Ags and the pool of endogenous proteins for binding to MHC class I molecules.

Cell Line, Transformed↗
PubMed1997

Dynamics of proteasome distribution in living cells.

Proteasomes are proteolytic complexes involved in non-lysosomal degradation which are localized in both the cytoplasm and the nucleus. The dynamics of proteasomes in living cells is unclear, as is their targeting to proteins destined for degradation. To investigate the intracellular distribution and mobility of proteasomes in vivo, we generated a fusion protein of the proteasome subunit LMP2 and the green fluorescent protein (GFP). The LMP2-GFP chimera was quantitatively incorporated into catalytically active proteasomes. The GFP-tagged proteasomes were located within both the cytoplasm and the nucleus. Within these two compartments, proteasomes diffused rapidly, and bleaching experiments demonstrated that proteasomes were transported slowly and unidirectionally from the cytoplasm into the nucleus. During mitosis, when the nuclear envelope has disintegrated, proteasomes diffused rapidly throughout the dividing cell without encountering a selective barrier. Immediately after cell division, the restored nuclear envelope formed a new barrier for the diffusing proteasomes. Thus, proteasomes can be transported unidirectionally over the nuclear membrane, but can also enter the nucleus upon reassembly during cell division. Since proteasomes diffuse rapidly in the cytoplasm and nucleus, they may perform quality control by continuous collision with intracellular proteins, and degrading those proteins that are properly tagged or misfolded.

Antigen Presentation↗
PubMed1996

A three-cell cluster hypothesis for noncognate T-B collaboration via direct T cell recognition of allogeneic dendritic cells.

In this article, we propose that T cell help for B cells can occur via an unusual three-cell cluster, with recipient CD4+ T helper cells interacting via direct allorecognition with donor dendritic cell class II MHC antigens, recipient B cells interacting with MHC class I (or any other) antigen on the donor dendritic cell surface, and noncognate (i.e., antigen nonspecific) T-B collaboration. In this noncognate pathway, antigen processing by B cells is not required and T cell help is potent because of the high precursor T cell frequency for direct recognition of allogeneic class II MHC molecules. The data supporting this hypothesis are: 1. LEW rat strain recipients of interstitial dendritic cell-free (DAxLEW)F1 kidney allografts were shown to have no detectable antibody to donor class I MHC antigens at day 7 after grafting. By contrast, LEW recipients of normal (DAxLEW)F1 kidneys had strong antibody responses. 2. Consistent wih important role for donor dendritic cells in the early antibody response to donor class I MHC antigens was the finding that it was dependent on donor class II MHC antigens. PVG recipients, previously immunized with pure DA RT1.B class II MHC antigens, had virtually no antibody response to the class I MHC antigens of DA kidney allografts. 3. We confirmed the low and high responder status of PVG and LEW rats, respectively, to DA class I antigens by studying antibody responses to pure DA class I antigens. However, PVG and LEW recipients of DA kidney allografts did not differ in their antibody response to the donor DA class I MHC antigens. This is consistent with this response not requiring the processing and presentation of DA class I antigen by PVG recipients. 4. LEW recipients of interstitial dendritic cell-free (DAxLEW)F1 kidney allografts did eventually develop a strong antibody response to DA class I antigens, but this was delayed by several weeks. That this delayed antibody response was probably mediated by conventional T-B collaboration and that T help was rate limiting in this situation, was demonstrated by immunizing LEW recipients with a DA class I peptide. This markedly accelerated the kinetics of the antibody response to the dendritic cell-free (DAxLEW)F1 kidneys.

Amino Acid Sequence↗
PubMed1995

Indirect T cell allorecognition of donor antigens contributes to the rejection of vascularized kidney allografts.

This report demonstrates for the first time that indirect T cell allorecognition of donor antigens can contribute to the effector mechanism of rejection of vascularized organ allografts. LEW (RT1(1)) rats were primed for indirect T cell allorecognition of DA (RT1av1) classical class I MHC molecules by immunization with synthetic 22-24 amino acid peptides corresponding to the alpha-helices of the RT1-A class I molecule. These rats received (DA x LEW) F1 kidney grafts that had been depleted of donor interstitial dendritic cells to minimize the direct T cell allorecognition response to the graft. The peptide-immunized rats rejected their grafts more rapidly than did control immunized rats, in terms of both graft function and survival. Moreover, the kinetics of antibody production to intact donor class I molecules after kidney transplantation was much more rapid in the peptide-immunized rats, suggesting that T cell help is the rate-limiting factor for antibody production to donor antigens in this model. It was of interest that we could not detect an antibody response to donor peptides after kidney graft rejection.

Amino Acid Sequence↗