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Antigen-unspecific B cells and lymphoid dendritic cells both show extensive surface expression of processed antigen-major histocompatibility complex class II complexes after soluble protein exposure in vivo or in vitro.

Zhong G, Reis e Sousa C, Germain RN - J. Exp. Med. (1997)

Bottom Line: In striking contrast to conclusions drawn from indirect T cell activation studies, this direct method of TCR ligand analysis shows that i.v. administration of HEL protein results in nearly all B cells in lymphoid tissues having substantial levels of HEL 46-61-Ak complexes on their surface.Although the absolute number of displayed ligands is greater on such DC, the relative specific ligand expression compared to total MHC class II levels is similar or greater on B cells.The numerical advantage of the TCR ligand-bearing B cells may permit them to interact first or more often with naive antigen-specific T cells, contributing to the induction of high-dose T cell tolerance or immune deviation.

View Article: PubMed Central - PubMed

Affiliation: Lymphocyte Biology Section, Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892-1892, USA.

ABSTRACT
Intravenous (i.v.) injection of high amounts of soluble proteins often results in the induction of antigen-specific tolerance or deviation to helper rather than inflammatory T cell immunity. It has been proposed that this outcome may be due to antigen presentation to T cells by a large cohort of poorly costimulatory or IL-12-deficient resting B cells lacking specific immunoglobulin receptors for the protein. However, previous studies using T cell activation in vitro to assess antigen display have failed to support this idea, showing evidence of specific peptide-major histocompatibility complex (MHC) class II ligand only on purified dendritic cells (DC) or antigen-specific B cells isolated from protein injected mice. Here we reexamine this question using a recently derived monoclonal antibody specific for the T cell receptor (TCR) ligand formed by the association of the 46-61 determinant of hen egg lysozyme (HEL) and the mouse MHC class II molecule I-Ak. In striking contrast to conclusions drawn from indirect T cell activation studies, this direct method of TCR ligand analysis shows that i.v. administration of HEL protein results in nearly all B cells in lymphoid tissues having substantial levels of HEL 46-61-Ak complexes on their surface. DC readily isolated from spleen also display this TCR ligand on their surface. Although the absolute number of displayed ligands is greater on such DC, the relative specific ligand expression compared to total MHC class II levels is similar or greater on B cells. These results demonstrate that in the absence of activating stimuli, both lymphoid DC and antigen-unspecific B cells present to a similar extent class II-associated peptides derived from soluble proteins in extracellular fluid. The numerical advantage of the TCR ligand-bearing B cells may permit them to interact first or more often with naive antigen-specific T cells, contributing to the induction of high-dose T cell tolerance or immune deviation.

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Both B cells and DC present processed HEL after i.v. injection. Groups of five CBA/J mice were injected i.v. with 8 mg of BSA or  HEL in 100 μl PBS. Spleens were removed 4 h later and were dissociated  into a cell suspension using collagenase. Part of this suspension was fractionated over dense BSA columns to obtain LOD. Whole splenocytes (top  panel) and LOD (middle and bottom panels) were double stained with C4H3  and B220 or C4H3 and N418. Profiles represent the C4H3 fluorescence  of gated B220+ cells (top and middle panels) or gated N418+ cells (bottom  panel); profiles from BSA-injected mice are represented by thin lines and  those from HEL-injected mice by thick lines.
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Figure 4: Both B cells and DC present processed HEL after i.v. injection. Groups of five CBA/J mice were injected i.v. with 8 mg of BSA or HEL in 100 μl PBS. Spleens were removed 4 h later and were dissociated into a cell suspension using collagenase. Part of this suspension was fractionated over dense BSA columns to obtain LOD. Whole splenocytes (top panel) and LOD (middle and bottom panels) were double stained with C4H3 and B220 or C4H3 and N418. Profiles represent the C4H3 fluorescence of gated B220+ cells (top and middle panels) or gated N418+ cells (bottom panel); profiles from BSA-injected mice are represented by thin lines and those from HEL-injected mice by thick lines.

Mentions: The ability of splenic DC to process antigens after i.v. immunization remains controversial (7, 10). To compare directly the presentation of HEL by DC and B cells, we used flow cytometric analysis of cells stained with C4H3. Spleens were removed from groups of mice that had been injected i.v. 4 h before with either BSA or a subsaturating amount of HEL and whole or low density spleen cells were stained with C4H3 followed by double staining with either anti-DC or anti–B cell antibodies. Overlapping the profiles from HEL- and BSA-injected mice demonstrates an HEL-dependent increase in C4H3 staining for splenic B cells (Fig. 4, top panel), as seen before (Fig. 3 and Table 1). As with antigen exposure in vitro (Fig. 1), the increase is similar for whole spleen and low density B cells and for DC when considered as a fraction of total class II expression (Fig. 4). In three out of three experiments, whole spleen B cells were consistently slightly better than either low density B cells or DC by this criterion, whereas the differences between the latter two APCs were small and inconsistent. We conclude that both DC and B cells in the spleen are capable of processing and presenting peptides derived from soluble proteins administered i.v. and that under nonactivating conditions of antigen exposure, do so with similar relative efficiency. As with the in vitro experiments, however, DC again have a higher absolute level of specific ligand expression.


Antigen-unspecific B cells and lymphoid dendritic cells both show extensive surface expression of processed antigen-major histocompatibility complex class II complexes after soluble protein exposure in vivo or in vitro.

Zhong G, Reis e Sousa C, Germain RN - J. Exp. Med. (1997)

Both B cells and DC present processed HEL after i.v. injection. Groups of five CBA/J mice were injected i.v. with 8 mg of BSA or  HEL in 100 μl PBS. Spleens were removed 4 h later and were dissociated  into a cell suspension using collagenase. Part of this suspension was fractionated over dense BSA columns to obtain LOD. Whole splenocytes (top  panel) and LOD (middle and bottom panels) were double stained with C4H3  and B220 or C4H3 and N418. Profiles represent the C4H3 fluorescence  of gated B220+ cells (top and middle panels) or gated N418+ cells (bottom  panel); profiles from BSA-injected mice are represented by thin lines and  those from HEL-injected mice by thick lines.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199022&req=5

Figure 4: Both B cells and DC present processed HEL after i.v. injection. Groups of five CBA/J mice were injected i.v. with 8 mg of BSA or HEL in 100 μl PBS. Spleens were removed 4 h later and were dissociated into a cell suspension using collagenase. Part of this suspension was fractionated over dense BSA columns to obtain LOD. Whole splenocytes (top panel) and LOD (middle and bottom panels) were double stained with C4H3 and B220 or C4H3 and N418. Profiles represent the C4H3 fluorescence of gated B220+ cells (top and middle panels) or gated N418+ cells (bottom panel); profiles from BSA-injected mice are represented by thin lines and those from HEL-injected mice by thick lines.
Mentions: The ability of splenic DC to process antigens after i.v. immunization remains controversial (7, 10). To compare directly the presentation of HEL by DC and B cells, we used flow cytometric analysis of cells stained with C4H3. Spleens were removed from groups of mice that had been injected i.v. 4 h before with either BSA or a subsaturating amount of HEL and whole or low density spleen cells were stained with C4H3 followed by double staining with either anti-DC or anti–B cell antibodies. Overlapping the profiles from HEL- and BSA-injected mice demonstrates an HEL-dependent increase in C4H3 staining for splenic B cells (Fig. 4, top panel), as seen before (Fig. 3 and Table 1). As with antigen exposure in vitro (Fig. 1), the increase is similar for whole spleen and low density B cells and for DC when considered as a fraction of total class II expression (Fig. 4). In three out of three experiments, whole spleen B cells were consistently slightly better than either low density B cells or DC by this criterion, whereas the differences between the latter two APCs were small and inconsistent. We conclude that both DC and B cells in the spleen are capable of processing and presenting peptides derived from soluble proteins administered i.v. and that under nonactivating conditions of antigen exposure, do so with similar relative efficiency. As with the in vitro experiments, however, DC again have a higher absolute level of specific ligand expression.

Bottom Line: In striking contrast to conclusions drawn from indirect T cell activation studies, this direct method of TCR ligand analysis shows that i.v. administration of HEL protein results in nearly all B cells in lymphoid tissues having substantial levels of HEL 46-61-Ak complexes on their surface.Although the absolute number of displayed ligands is greater on such DC, the relative specific ligand expression compared to total MHC class II levels is similar or greater on B cells.The numerical advantage of the TCR ligand-bearing B cells may permit them to interact first or more often with naive antigen-specific T cells, contributing to the induction of high-dose T cell tolerance or immune deviation.

View Article: PubMed Central - PubMed

Affiliation: Lymphocyte Biology Section, Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892-1892, USA.

ABSTRACT
Intravenous (i.v.) injection of high amounts of soluble proteins often results in the induction of antigen-specific tolerance or deviation to helper rather than inflammatory T cell immunity. It has been proposed that this outcome may be due to antigen presentation to T cells by a large cohort of poorly costimulatory or IL-12-deficient resting B cells lacking specific immunoglobulin receptors for the protein. However, previous studies using T cell activation in vitro to assess antigen display have failed to support this idea, showing evidence of specific peptide-major histocompatibility complex (MHC) class II ligand only on purified dendritic cells (DC) or antigen-specific B cells isolated from protein injected mice. Here we reexamine this question using a recently derived monoclonal antibody specific for the T cell receptor (TCR) ligand formed by the association of the 46-61 determinant of hen egg lysozyme (HEL) and the mouse MHC class II molecule I-Ak. In striking contrast to conclusions drawn from indirect T cell activation studies, this direct method of TCR ligand analysis shows that i.v. administration of HEL protein results in nearly all B cells in lymphoid tissues having substantial levels of HEL 46-61-Ak complexes on their surface. DC readily isolated from spleen also display this TCR ligand on their surface. Although the absolute number of displayed ligands is greater on such DC, the relative specific ligand expression compared to total MHC class II levels is similar or greater on B cells. These results demonstrate that in the absence of activating stimuli, both lymphoid DC and antigen-unspecific B cells present to a similar extent class II-associated peptides derived from soluble proteins in extracellular fluid. The numerical advantage of the TCR ligand-bearing B cells may permit them to interact first or more often with naive antigen-specific T cells, contributing to the induction of high-dose T cell tolerance or immune deviation.

Show MeSH
Related in: MedlinePlus