Advanced

B cell activation in vitro: thymus-dependent immune responses and bacterial cell surface proteins

Axcrona, Karol LU (1998)
Abstract
In this thesis aspects of peripheral B lymphocyte differentiation were studied. Through in vitro experiments, B lymphocyte activation was assessed. B lymphocytes express immunoglobulin (Ig) specific for a certain antigen on the cell surface, and when surface Ig is crosslinked by an antigen, the cell is activated. Antigens giving rise to a T cell dependent immune response are internalized by the B cell, degraded, and presented on MHC II. In order to differentiate, an activated B cell has to interact with a T cell bearing a T cell receptor specific for the same antigen. During B cell/T cell interaction a crosstalk mediated by cell surface molecules is initiated. For example, the CD40/CD40 ligand (CD40L) receptor interaction plays a critical... (More)
In this thesis aspects of peripheral B lymphocyte differentiation were studied. Through in vitro experiments, B lymphocyte activation was assessed. B lymphocytes express immunoglobulin (Ig) specific for a certain antigen on the cell surface, and when surface Ig is crosslinked by an antigen, the cell is activated. Antigens giving rise to a T cell dependent immune response are internalized by the B cell, degraded, and presented on MHC II. In order to differentiate, an activated B cell has to interact with a T cell bearing a T cell receptor specific for the same antigen. During B cell/T cell interaction a crosstalk mediated by cell surface molecules is initiated. For example, the CD40/CD40 ligand (CD40L) receptor interaction plays a critical role in B cell activation; genetically engineered animals deficient for either the CD40L or CD40, lack germinal centres, Ig switch and immunological memory.



When B cells were activated with anti-CD40 monoclonal antibodies (mAbs) together with anti-CD21 mAbs coupled to Sepharose differentiation to Ig secretion was detected. Addition of anti-CD40 mAbs to lipopolysaccharide (LPS) stimulated B cells was, however, shown to inhibit B cell differentiation in the same manner as addition of anti-Ig mAbs to LPS treated cultures. Further, B cells prestimulated with anti-CD40 were shown to undergo apoptosis when restimulated with anti-Ig.



Because of the pivotal role of the CD40L/CD40 interaction in T cell dependent immune responses, the CD40L promoter was analyzed. The CD40L promoter and deletants of that promoter were cloned into an expression vector containing the structural gene for luciferase, as a reporter gene. Upon transient transfection into Jurkat T cells CD40L promoter function was shown to be dependent on signaling via the T cell receptor and CD28. A 270 bp region upstream of the translational start was shown to suffice for full promoter activity. A putative CD28 responsive DNA element distinct from the one in the IL-2 promoter was identified.



Streptococcal Ig-binding surface proteins L, M1, and H were shown to bind various cells of the hematopoetic lineage; proteins L and H coupled to Sepharose induced B cell proliferation, whereas protein H coupled to Sepharose also induced B cell differentiation. Soluble protein H was shown to be taken up by T and B cells and transported to the nucleus. Protein H was found to interact with nucleophosmin/B23 (NPM), a protein previously shown to traffic from the cytoplasm to the nucleus, but which was isolated from a membrane preparation from Jurkat T cells. In the nucleus protein H was found to interact with hnRNP A2/B1 and the SET protein. Affinities of protein H with NPM and a nuclear extract prepared from Jurkat T cells were determined. Lastly, when murine B cells were stimulated with LPS, addition of protein H was shown to exert a cytostatic effect. (Less)
Please use this url to cite or link to this publication:
author
opponent
  • Doc Pettersson, Sven, CGR, Karolinska Institute, Solna, Sweden
organization
publishing date
type
Thesis
publication status
published
subject
keywords
nuclear transport, bacterial Ig-binding proteins, CD40L promoter, CD21, Ig, B lymphocyte activation in vitro, CD40, Immunology, serology, transplantation, Immunologi, serologi
pages
144 pages
publisher
Center for Molecular Biomedicine, Lund University
defense location
Föreläsningssalen, Wallenberglaboratoriet, Sölvegatan 33, Lund, 09.15 am
defense date
1998-05-22 09:15
external identifiers
  • Other:ISRN: LUMEDV/MECM--98/1013--SE
ISBN
91-628-2940-8
language
English
LU publication?
yes
id
d6d51bc8-b526-4e41-b0b5-092558e818ec (old id 38585)
date added to LUP
2007-06-20 10:41:44
date last changed
2016-09-19 08:45:03
@phdthesis{d6d51bc8-b526-4e41-b0b5-092558e818ec,
  abstract     = {In this thesis aspects of peripheral B lymphocyte differentiation were studied. Through in vitro experiments, B lymphocyte activation was assessed. B lymphocytes express immunoglobulin (Ig) specific for a certain antigen on the cell surface, and when surface Ig is crosslinked by an antigen, the cell is activated. Antigens giving rise to a T cell dependent immune response are internalized by the B cell, degraded, and presented on MHC II. In order to differentiate, an activated B cell has to interact with a T cell bearing a T cell receptor specific for the same antigen. During B cell/T cell interaction a crosstalk mediated by cell surface molecules is initiated. For example, the CD40/CD40 ligand (CD40L) receptor interaction plays a critical role in B cell activation; genetically engineered animals deficient for either the CD40L or CD40, lack germinal centres, Ig switch and immunological memory.<br/><br>
<br/><br>
When B cells were activated with anti-CD40 monoclonal antibodies (mAbs) together with anti-CD21 mAbs coupled to Sepharose differentiation to Ig secretion was detected. Addition of anti-CD40 mAbs to lipopolysaccharide (LPS) stimulated B cells was, however, shown to inhibit B cell differentiation in the same manner as addition of anti-Ig mAbs to LPS treated cultures. Further, B cells prestimulated with anti-CD40 were shown to undergo apoptosis when restimulated with anti-Ig.<br/><br>
<br/><br>
Because of the pivotal role of the CD40L/CD40 interaction in T cell dependent immune responses, the CD40L promoter was analyzed. The CD40L promoter and deletants of that promoter were cloned into an expression vector containing the structural gene for luciferase, as a reporter gene. Upon transient transfection into Jurkat T cells CD40L promoter function was shown to be dependent on signaling via the T cell receptor and CD28. A 270 bp region upstream of the translational start was shown to suffice for full promoter activity. A putative CD28 responsive DNA element distinct from the one in the IL-2 promoter was identified.<br/><br>
<br/><br>
Streptococcal Ig-binding surface proteins L, M1, and H were shown to bind various cells of the hematopoetic lineage; proteins L and H coupled to Sepharose induced B cell proliferation, whereas protein H coupled to Sepharose also induced B cell differentiation. Soluble protein H was shown to be taken up by T and B cells and transported to the nucleus. Protein H was found to interact with nucleophosmin/B23 (NPM), a protein previously shown to traffic from the cytoplasm to the nucleus, but which was isolated from a membrane preparation from Jurkat T cells. In the nucleus protein H was found to interact with hnRNP A2/B1 and the SET protein. Affinities of protein H with NPM and a nuclear extract prepared from Jurkat T cells were determined. Lastly, when murine B cells were stimulated with LPS, addition of protein H was shown to exert a cytostatic effect.},
  author       = {Axcrona, Karol},
  isbn         = {91-628-2940-8},
  keyword      = {nuclear transport,bacterial Ig-binding proteins,CD40L promoter,CD21,Ig,B lymphocyte activation in vitro,CD40,Immunology,serology,transplantation,Immunologi,serologi},
  language     = {eng},
  pages        = {144},
  publisher    = {Center for Molecular Biomedicine, Lund University},
  school       = {Lund University},
  title        = {B cell activation in vitro: thymus-dependent immune responses and bacterial cell surface proteins},
  year         = {1998},
}