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Outer periarteriolar lymphoid sheath arrest and subsequent differentiation of both naive and tolerant immunoglobulin transgenic B cells is determined by B cell receptor occupancy.

Cook MC, Basten A, Fazekas de St Groth B - J. Exp. Med. (1997)

Bottom Line: In addition, tolerant B cells were found to relocate from the follicles to the outer PALS of HEL/anti-HEL double Tg mice in which the concentration of soluble self-antigen had been increased by zinc feeding.Thus, subtle variations in antigen concentration resulted in dramatic changes in positioning of B cells within the spleen.A series of mixed bone marrow chimeras in which the effective antigen concentration was inversely related to the number of self-reactive B cells due to absorption of antigen by transgene-encoded membrane and secreted Ig, was used to confirm that alteration in B cell position previously attributed to changes in follicular composition could be explained on the basis of available antigen concentration, rather than the diversity of the repertoire.

View Article: PubMed Central - PubMed

Affiliation: Centenary Institute for Cancer Medicine and Cell Biology, Newtown, Sydney, New South Wales, Australia 2042.

ABSTRACT
T-dependent B cell responses in the spleen are initiated in the outer periarteriolar lymphoid sheath (PALS) and culminate in the generation of proliferative foci and germinal center reactions. By pulsing anti-hen egg lysozyme (HEL) immunoglobulin transgenic (IgTg) B cells with various concentrations of HEL in vitro before adoptive transfer into normal recipients, it was shown that a critical number of B cell receptors (BCRs) must be ligated for B cells to undergo arrest in the outer PALS. T cell help was manipulated independently of the BCR stimulus by incubating B cells expressing the appropriate major histocompatibility complex class II antigen with a peptide recognized by CD4(+) TCR Tg T cells. B cells which either failed to arrest in the outer PALS due to a subthreshold BCR stimulus, or arrested only transiently due to the brevity of the BCR stimulus, underwent an abortive response within the follicles when provided with T cell help. In contrast, naive B cells stimulated by a sustained, suprathreshold concentration of either foreign or self-antigen and given T cell help, proliferated in the outer PALS and then differentiated. Outer PALS arrest was not influenced by the nature of the B cells occupying the follicle, but appeared to be determined solely by the magnitude of BCR stimulation. Thus antigen-pulsed B cells arrested in the outer PALS in an identical manner irrespective of whether the follicles comprised a population of normal B cells with multiple specificities, a monoclonal naive population, or a monoclonal population of tolerant B cells. In addition, tolerant B cells were found to relocate from the follicles to the outer PALS of HEL/anti-HEL double Tg mice in which the concentration of soluble self-antigen had been increased by zinc feeding. Similarly, when anti-HEL Tg mice were crossed with a second HEL Tg strain expressing a higher concentration of soluble HEL, the tolerant anti-HEL Tg B cells were located constitutively in the outer PALS. Thus, subtle variations in antigen concentration resulted in dramatic changes in positioning of B cells within the spleen. A series of mixed bone marrow chimeras in which the effective antigen concentration was inversely related to the number of self-reactive B cells due to absorption of antigen by transgene-encoded membrane and secreted Ig, was used to confirm that alteration in B cell position previously attributed to changes in follicular composition could be explained on the basis of available antigen concentration, rather than the diversity of the repertoire.

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Location of T-dependent B cell proliferation depends on duration of BCR ligation with antigen. Fluorescent micrographs of frozen sections  of spleen. HEL-binding B cells are stained green (fluorescein) and B220+ cells are stained red (Texas red). In (F) the section was also stained blue with  PNA fluoroblue to reveal germinal center cells. (A–C) H-2bk IgTg B cells were pulsed in vitro with MCC87-103 and HEL (100 ng/ml) and transferred  with activated cytochrome-specific TCR Tg T cells into H-2bk non-Tg recipients. The day 1 section (A) shows the HEL-binding B cells located in the  outer PALS. The day 3 section (B) shows that there has been a marked increase in the number of intrafollicular HEL-binding B cells. The day 7 section  (C) shows reduction in the number of intrafollicular B cells and no evidence of differentiation into proliferative foci or germinal centers. (D–F) H-2bk  IgTg B cells were pulsed with MCC87-103, but not HEL, and transferred with activated cytochrome-specific TCR Tg T cells into H-2bk HEL Tg  (ML5) recipients. The day 1 section (D) shows arrest of HEL-binding B cells in the outer PALS. The day 3 section (E) shows a dramatic increase in the  number of HEL-binding B cells in the outer PALS. The day 5 section (F) shows that HEL-binding B cells have differentiated into proliferative foci adjacent to the follicles (P) and germinal centers (G), which costain with PNA (blue) and B220 (red) within the follicles. (G–I) H-2bk IgTg B cells were pulsed  in vitro with MCC87-103 but not HEL, and transferred with activated cytochrome-specific TCR Tg T cell help into H-2bk non-Tg recipients. The day  1 section (G) shows HEL-binding B cells located in the follicles. The day 3 section (H) shows an increase in the number of HEL-binding B cells in the  follicles. The day 7 section (C) shows a reduction in the number of intrafollicular B cells and no evidence of differentiation.
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Figure 2: Location of T-dependent B cell proliferation depends on duration of BCR ligation with antigen. Fluorescent micrographs of frozen sections of spleen. HEL-binding B cells are stained green (fluorescein) and B220+ cells are stained red (Texas red). In (F) the section was also stained blue with PNA fluoroblue to reveal germinal center cells. (A–C) H-2bk IgTg B cells were pulsed in vitro with MCC87-103 and HEL (100 ng/ml) and transferred with activated cytochrome-specific TCR Tg T cells into H-2bk non-Tg recipients. The day 1 section (A) shows the HEL-binding B cells located in the outer PALS. The day 3 section (B) shows that there has been a marked increase in the number of intrafollicular HEL-binding B cells. The day 7 section (C) shows reduction in the number of intrafollicular B cells and no evidence of differentiation into proliferative foci or germinal centers. (D–F) H-2bk IgTg B cells were pulsed with MCC87-103, but not HEL, and transferred with activated cytochrome-specific TCR Tg T cells into H-2bk HEL Tg (ML5) recipients. The day 1 section (D) shows arrest of HEL-binding B cells in the outer PALS. The day 3 section (E) shows a dramatic increase in the number of HEL-binding B cells in the outer PALS. The day 5 section (F) shows that HEL-binding B cells have differentiated into proliferative foci adjacent to the follicles (P) and germinal centers (G), which costain with PNA (blue) and B220 (red) within the follicles. (G–I) H-2bk IgTg B cells were pulsed in vitro with MCC87-103 but not HEL, and transferred with activated cytochrome-specific TCR Tg T cell help into H-2bk non-Tg recipients. The day 1 section (G) shows HEL-binding B cells located in the follicles. The day 3 section (H) shows an increase in the number of HEL-binding B cells in the follicles. The day 7 section (C) shows a reduction in the number of intrafollicular B cells and no evidence of differentiation.

Mentions: To test whether persistence of antigen after the initial stimulus could influence the nature and site of the subsequent T-dependent B cell response, IgTg B cells were pulsed in vitro with both soluble HEL and MCC87-103, and transferred with activated TCR Tg T cells as a source of T cell help (Table 1). In non-Tg hosts, the B cell stimulus was limited by the half-life of the BCR–ligand association, making it unlikely that such a stimulus would be available for a prolonged period of time. As expected, the donor B cells were arrested in the outer PALS on day 1 after transfer (Fig. 2 A). By day 3, the number of donor B cells within the follicles had increased significantly (Fig. 2 B), but the response failed to generate proliferative foci (Fig. 2 C) or germinal centers (not shown) at later times. On the other hand, transfer of MCC87-103– pulsed IgTg B cells together with TCR Tg T cells into soluble HEL Tg recipients resulted in outer PALS arrest on day 1 (Fig. 2 D) and extensive proliferation of the B cells (as indicated by FACS® analysis of CFSE-labeled donor cells, not shown) throughout the T cell zone on day 3 (Fig. 2 E). Furthermore, proliferative foci and germinal centers, the latter indicated by the presence of PNA+ HEL-binding B cells in the follicles, were present on day 5 (Fig. 2 F; see also reference 14). In other words, when exposure to HEL was prolonged rather than transient, the collaborative response between B and T cells could proceed to completion.


Outer periarteriolar lymphoid sheath arrest and subsequent differentiation of both naive and tolerant immunoglobulin transgenic B cells is determined by B cell receptor occupancy.

Cook MC, Basten A, Fazekas de St Groth B - J. Exp. Med. (1997)

Location of T-dependent B cell proliferation depends on duration of BCR ligation with antigen. Fluorescent micrographs of frozen sections  of spleen. HEL-binding B cells are stained green (fluorescein) and B220+ cells are stained red (Texas red). In (F) the section was also stained blue with  PNA fluoroblue to reveal germinal center cells. (A–C) H-2bk IgTg B cells were pulsed in vitro with MCC87-103 and HEL (100 ng/ml) and transferred  with activated cytochrome-specific TCR Tg T cells into H-2bk non-Tg recipients. The day 1 section (A) shows the HEL-binding B cells located in the  outer PALS. The day 3 section (B) shows that there has been a marked increase in the number of intrafollicular HEL-binding B cells. The day 7 section  (C) shows reduction in the number of intrafollicular B cells and no evidence of differentiation into proliferative foci or germinal centers. (D–F) H-2bk  IgTg B cells were pulsed with MCC87-103, but not HEL, and transferred with activated cytochrome-specific TCR Tg T cells into H-2bk HEL Tg  (ML5) recipients. The day 1 section (D) shows arrest of HEL-binding B cells in the outer PALS. The day 3 section (E) shows a dramatic increase in the  number of HEL-binding B cells in the outer PALS. The day 5 section (F) shows that HEL-binding B cells have differentiated into proliferative foci adjacent to the follicles (P) and germinal centers (G), which costain with PNA (blue) and B220 (red) within the follicles. (G–I) H-2bk IgTg B cells were pulsed  in vitro with MCC87-103 but not HEL, and transferred with activated cytochrome-specific TCR Tg T cell help into H-2bk non-Tg recipients. The day  1 section (G) shows HEL-binding B cells located in the follicles. The day 3 section (H) shows an increase in the number of HEL-binding B cells in the  follicles. The day 7 section (C) shows a reduction in the number of intrafollicular B cells and no evidence of differentiation.
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Related In: Results  -  Collection

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Figure 2: Location of T-dependent B cell proliferation depends on duration of BCR ligation with antigen. Fluorescent micrographs of frozen sections of spleen. HEL-binding B cells are stained green (fluorescein) and B220+ cells are stained red (Texas red). In (F) the section was also stained blue with PNA fluoroblue to reveal germinal center cells. (A–C) H-2bk IgTg B cells were pulsed in vitro with MCC87-103 and HEL (100 ng/ml) and transferred with activated cytochrome-specific TCR Tg T cells into H-2bk non-Tg recipients. The day 1 section (A) shows the HEL-binding B cells located in the outer PALS. The day 3 section (B) shows that there has been a marked increase in the number of intrafollicular HEL-binding B cells. The day 7 section (C) shows reduction in the number of intrafollicular B cells and no evidence of differentiation into proliferative foci or germinal centers. (D–F) H-2bk IgTg B cells were pulsed with MCC87-103, but not HEL, and transferred with activated cytochrome-specific TCR Tg T cells into H-2bk HEL Tg (ML5) recipients. The day 1 section (D) shows arrest of HEL-binding B cells in the outer PALS. The day 3 section (E) shows a dramatic increase in the number of HEL-binding B cells in the outer PALS. The day 5 section (F) shows that HEL-binding B cells have differentiated into proliferative foci adjacent to the follicles (P) and germinal centers (G), which costain with PNA (blue) and B220 (red) within the follicles. (G–I) H-2bk IgTg B cells were pulsed in vitro with MCC87-103 but not HEL, and transferred with activated cytochrome-specific TCR Tg T cell help into H-2bk non-Tg recipients. The day 1 section (G) shows HEL-binding B cells located in the follicles. The day 3 section (H) shows an increase in the number of HEL-binding B cells in the follicles. The day 7 section (C) shows a reduction in the number of intrafollicular B cells and no evidence of differentiation.
Mentions: To test whether persistence of antigen after the initial stimulus could influence the nature and site of the subsequent T-dependent B cell response, IgTg B cells were pulsed in vitro with both soluble HEL and MCC87-103, and transferred with activated TCR Tg T cells as a source of T cell help (Table 1). In non-Tg hosts, the B cell stimulus was limited by the half-life of the BCR–ligand association, making it unlikely that such a stimulus would be available for a prolonged period of time. As expected, the donor B cells were arrested in the outer PALS on day 1 after transfer (Fig. 2 A). By day 3, the number of donor B cells within the follicles had increased significantly (Fig. 2 B), but the response failed to generate proliferative foci (Fig. 2 C) or germinal centers (not shown) at later times. On the other hand, transfer of MCC87-103– pulsed IgTg B cells together with TCR Tg T cells into soluble HEL Tg recipients resulted in outer PALS arrest on day 1 (Fig. 2 D) and extensive proliferation of the B cells (as indicated by FACS® analysis of CFSE-labeled donor cells, not shown) throughout the T cell zone on day 3 (Fig. 2 E). Furthermore, proliferative foci and germinal centers, the latter indicated by the presence of PNA+ HEL-binding B cells in the follicles, were present on day 5 (Fig. 2 F; see also reference 14). In other words, when exposure to HEL was prolonged rather than transient, the collaborative response between B and T cells could proceed to completion.

Bottom Line: In addition, tolerant B cells were found to relocate from the follicles to the outer PALS of HEL/anti-HEL double Tg mice in which the concentration of soluble self-antigen had been increased by zinc feeding.Thus, subtle variations in antigen concentration resulted in dramatic changes in positioning of B cells within the spleen.A series of mixed bone marrow chimeras in which the effective antigen concentration was inversely related to the number of self-reactive B cells due to absorption of antigen by transgene-encoded membrane and secreted Ig, was used to confirm that alteration in B cell position previously attributed to changes in follicular composition could be explained on the basis of available antigen concentration, rather than the diversity of the repertoire.

View Article: PubMed Central - PubMed

Affiliation: Centenary Institute for Cancer Medicine and Cell Biology, Newtown, Sydney, New South Wales, Australia 2042.

ABSTRACT
T-dependent B cell responses in the spleen are initiated in the outer periarteriolar lymphoid sheath (PALS) and culminate in the generation of proliferative foci and germinal center reactions. By pulsing anti-hen egg lysozyme (HEL) immunoglobulin transgenic (IgTg) B cells with various concentrations of HEL in vitro before adoptive transfer into normal recipients, it was shown that a critical number of B cell receptors (BCRs) must be ligated for B cells to undergo arrest in the outer PALS. T cell help was manipulated independently of the BCR stimulus by incubating B cells expressing the appropriate major histocompatibility complex class II antigen with a peptide recognized by CD4(+) TCR Tg T cells. B cells which either failed to arrest in the outer PALS due to a subthreshold BCR stimulus, or arrested only transiently due to the brevity of the BCR stimulus, underwent an abortive response within the follicles when provided with T cell help. In contrast, naive B cells stimulated by a sustained, suprathreshold concentration of either foreign or self-antigen and given T cell help, proliferated in the outer PALS and then differentiated. Outer PALS arrest was not influenced by the nature of the B cells occupying the follicle, but appeared to be determined solely by the magnitude of BCR stimulation. Thus antigen-pulsed B cells arrested in the outer PALS in an identical manner irrespective of whether the follicles comprised a population of normal B cells with multiple specificities, a monoclonal naive population, or a monoclonal population of tolerant B cells. In addition, tolerant B cells were found to relocate from the follicles to the outer PALS of HEL/anti-HEL double Tg mice in which the concentration of soluble self-antigen had been increased by zinc feeding. Similarly, when anti-HEL Tg mice were crossed with a second HEL Tg strain expressing a higher concentration of soluble HEL, the tolerant anti-HEL Tg B cells were located constitutively in the outer PALS. Thus, subtle variations in antigen concentration resulted in dramatic changes in positioning of B cells within the spleen. A series of mixed bone marrow chimeras in which the effective antigen concentration was inversely related to the number of self-reactive B cells due to absorption of antigen by transgene-encoded membrane and secreted Ig, was used to confirm that alteration in B cell position previously attributed to changes in follicular composition could be explained on the basis of available antigen concentration, rather than the diversity of the repertoire.

Show MeSH
Related in: MedlinePlus