Biomedical subjects

D P Dalke

Publications and source records attributed to D P Dalke.

3 recordsLinked to original sources

PubMed1995

CD40-CD40 ligand interactions stimulate B cell antigen processing.

The interactions between B cell CD40 and T cell CD40 ligand (CD40L) have been shown recently to play an important role in T cell-dependent activation of B cells. Here, we show that the ligation of CD40 stimulates the processing of antigen by B cells. The activation of an antigen-specific T cell hybrid by B cells co-cultured with insect cells expressing recombinant CD40L or with a CD40-specific monoclonal antibody requires less antigen and fewer B cells compared to control cells. The augmentation was observed both for processing initiated by antigen binding to and cross-linking the surface immunoglobulin, and processing of antigen taken up by fluid-phase pinocytosis. CD40 appears to affect a step in the intracellular processing of antigen, as CD40 has no effect on the presentation of an antigenic peptide which does not require processing. In addition, the CD40-induced augmentation of processing is not attributable to the effect of CD40 ligation on the cell surface expression of B7, LFA-1 or CD23. CD40 ligation does not affect the biosynthesis of the class II EK molecules, and although ligation of CD40 induces B cell proliferation, the augmentation of processing does not require proliferation. The ability of CD40 to stimulate B cell antigen processing has the potential to influence significantly the outcome of antigen-dependent T cell-B cell interactions.

Adjuvants, Immunologic↗

PubMed1994

Separation of subcellular compartments containing distinct functional forms of MHC class II.

Antigen processing in B lymphocytes entails initial binding of antigen to the surface Ig and internalization of the antigen into acidic compartments where the antigen is degraded, releasing peptides for binding to major histocompatibility complex class II molecules. Using subcellular fractionation techniques we show that functional, processed antigen-class II complexes capable of activating antigen-specific T cells in vitro are first formed in dense vesicles cosedimenting with lysosomes which are distinct from early endosomes and the bulk of late endosomes. With time, processed antigen-class II complexes appear in vesicles sedimenting with early endosomes and finally cofractionate with plasma membrane. A separate compartment is identified which contains major histocompatibility complex class II receptive to peptide binding but which does not have access to processed antigen in the B cell. These class II molecules are in the so-called "floppy" form in contrast to the class II molecules in the very dense vesicles which are in the "compact" form. These results demonstrate a correlation between the floppy and compact forms of class II molecules and their association with processed antigen and show that floppy and compact forms of class II reside in distinct and physically separable subcellular compartments.

Amino Acid Sequence↗

PubMed1992

Biochemical evidence for the rapid assembly and disassembly of processed antigen-major histocompatibility complex class II complexes in acidic vesicles of B cells.

Helper T cell recognition of antigen requires that it be processed within antigen-presenting cells (APC) to peptide fragments that subsequently bind to major histocompatibility complex (MHC) class II molecules and are displayed on the APC surface. Heretofore, processed antigen-MHC class II complexes have been detected by functional assays, measuring the activation of specific T cells. We now report direct, biochemical evidence for the assembly of processed antigen-MHC class II complexes within splenic B cells as APC. The I-Ek MHC class II molecules were immunoprecipitated from B cells that had processed the model protein antigen cytochrome c radiolabeled across its entire length by reductive methylation of lysine residues and covalently coupled to Ig-specific antibodies, allowing internalization after binding to surface Ig. Our previous studies showed that I-Ek immunoaffinity purified from B cells that had processed cytochrome c contains functional processed antigen--MHC class II complexes and that approximately 0.2% of the I-Ek molecules are specifically associated with one of two predominant processed antigenic fragments. Here we show that these complexes are rapidly assembled, within 30-60 min after antigen binding to surface Ig on splenic B cells. Maximal numbers of complexes are assembled by 2 h in a process that is sensitive to acidic vesicle inhibitors but not to inhibitors of protein synthesis. The processed antigen-I-Ek complexes have a relatively short half-life of 2-4 h and are disassembled or degraded within 8 h after antigen is first internalized. The disassembly or degradation of the processed antigen-I-Ek complexes requires acidic vesicle function, and in the presence of an acidic vesicle inhibitor the complexes are long lived. Thus, using a biochemical assay to monitor processed antigen-I-Ek complexes, we find that, in B cells, processed antigen is relatively rapidly associated in acidic vesicles with preexisting MHC class II molecules, and the complexes are disassembled 4-6 h later in processes that also require acid vesicle function.

Acids↗