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A fail-safe mechanism for negative selection of isotype-switched B cell precursors is regulated by the Fas/FasL pathway.

Seagal J, Edry E, Keren Z, Leider N, Benny O, Machluf M, Melamed D - J. Exp. Med. (2003)

Bottom Line: Ongoing isotype switching was found in both normal and microMT B cell development as reflected by detection of IgG1 germline and postswitch transcripts as well as activation-induced cytidine deaminase expression, resulting in the generation of IgG-expressing cells.We suggest a novel developmental pathway used by isotype-switched B cell precursors that effectively circumvents peripheral tolerance requirements.This developmental pathway, however, is strictly controlled by Fas/FasL interaction to prevent B cell autoimmunity.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa 31096, Israel. melamedd@techunix.technion.ac.il

ABSTRACT
In B lymphocytes, immunoglobulin (Ig)M receptors drive development and construction of naive repertoire, whereas IgG receptors promote formation of the memory B cell compartment. This isotype switching process requires appropriate B cell activation and T cell help. In the absence of T cell help, activated B cells undergo Fas-mediated apoptosis, a peripheral mechanism contributing to the establishment of self-tolerance. Using Igmicro-deficient microMT mouse model, where B cell development is blocked at pro-B stage, here we show an alternative developmental pathway used by isotype-switched B cell precursors. We find that isotype switching occurs normally in B cell precursors and is T independent. Ongoing isotype switching was found in both normal and microMT B cell development as reflected by detection of IgG1 germline and postswitch transcripts as well as activation-induced cytidine deaminase expression, resulting in the generation of IgG-expressing cells. These isotype-switched B cells are negatively selected by Fas pathway, as blocking the Fas/FasL interaction rescues the development of isotype-switched B cells in vivo and in vitro. Similar to memory B cells, isotype-switched B cells have a marginal zone phenotype. We suggest a novel developmental pathway used by isotype-switched B cell precursors that effectively circumvents peripheral tolerance requirements. This developmental pathway, however, is strictly controlled by Fas/FasL interaction to prevent B cell autoimmunity.

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Blocking the Fas/FasL interaction in vivo rescues isotype-switched B cells in μMT mice. A neutralizing recombinant FasIg protein was purified and engineered into slow releasing PLGA microcapsules. The microcapsules were administered to μMT mice by intramuscular injection of 10 mg/mouse every 10 d for 8 wk. Mice were first injected at 2 wk of age. Control μMT mice were injected with empty microcapsules. 10 d after the last injection, mice were killed. (A) Serum samples from the mice were assayed for the presence of IgG by ELISA. Results are mean ± SEM of three mice in each group. (B) Detection of γH chain in serum samples by Western blotting. Normal serum sample dilution is 1:10. Samples of μMT mice (control and FasIg-treated) were not diluted. Purified IgG was used as control. Line indicates where irrelevant lane was removed digitally. (C) Quantitation of IgG-producing cells in spleens of μMT mice treated with FasIg relative to μMT and normal mice by ELISPOT. Representative ELISPOT membranes and the calculated frequency of IgG-producing cells per 107 spleen cells are shown. Results are mean ± SEM of three mice in each group. (D) FACS® analysis for κ and IgG expression. Spleen cells from the treated mice were stained for CD19, B220, κ, and IgG. Analysis for κ and IgG expression was performed on 10,000 gated CD19+/B220+ cells. The results shown are representative of eight injected mice in three different experiments.
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fig6: Blocking the Fas/FasL interaction in vivo rescues isotype-switched B cells in μMT mice. A neutralizing recombinant FasIg protein was purified and engineered into slow releasing PLGA microcapsules. The microcapsules were administered to μMT mice by intramuscular injection of 10 mg/mouse every 10 d for 8 wk. Mice were first injected at 2 wk of age. Control μMT mice were injected with empty microcapsules. 10 d after the last injection, mice were killed. (A) Serum samples from the mice were assayed for the presence of IgG by ELISA. Results are mean ± SEM of three mice in each group. (B) Detection of γH chain in serum samples by Western blotting. Normal serum sample dilution is 1:10. Samples of μMT mice (control and FasIg-treated) were not diluted. Purified IgG was used as control. Line indicates where irrelevant lane was removed digitally. (C) Quantitation of IgG-producing cells in spleens of μMT mice treated with FasIg relative to μMT and normal mice by ELISPOT. Representative ELISPOT membranes and the calculated frequency of IgG-producing cells per 107 spleen cells are shown. Results are mean ± SEM of three mice in each group. (D) FACS® analysis for κ and IgG expression. Spleen cells from the treated mice were stained for CD19, B220, κ, and IgG. Analysis for κ and IgG expression was performed on 10,000 gated CD19+/B220+ cells. The results shown are representative of eight injected mice in three different experiments.

Mentions: To test whether blocking the Fas/FasL interaction in vivo would rescue isotype-switched B cell precursors, PLGA microcapsules containing recombinant FasIg were injected into μMT mice. As described in Materials and Methods, these capsules allowed continuous slow release of FasIg at a constant amount of 10 ng/ml/day. 10 d after the last FasIg microcapsule injection, mice were tested for the presence of mature B cells and antibody production. The results in Fig. 6 clearly show that inhibition of Fas/FasL interaction by continuous presence of FasIg rescued isotype-switched B cell precursors in μMT mice. Serum analysis revealed a low, but significant amount of IgG production in μMT mice treated with FasIg (Fig. 6 A). This was in contrast to the control μMT mice, which fail to produce any serum IgG. The presence of IgG in sera was confirmed by Western blotting and independent detection of γH chain (Fig. 6 B). The specificity of the ELISA and the Western blot assays was confirmed and no reactivity was found between the anti–mouse IgG reagent and human IgG or purified FasIg (not depicted). Using an ELISPOT assay we were able to detect low frequency of IgG-producing cells in the FasIg-treated μMT mice (total of 10 ± 4/spleen) relative to control (Fig. 6 C). Finally, staining of spleen cells for κ and IgG expression revealed a very low population of mature, isotype-switched B cells in μMT mice treated with FasIg (total of 1,750 ± 585/spleen), but not in the control μMT group (Fig. 6 D). Thus, administration of FasIg to μMT mice rescues isotype-switched B cells from Fas-mediated apoptosis allowing maturation and serum IgG production in these mice, as confirmed by several experimental techniques.


A fail-safe mechanism for negative selection of isotype-switched B cell precursors is regulated by the Fas/FasL pathway.

Seagal J, Edry E, Keren Z, Leider N, Benny O, Machluf M, Melamed D - J. Exp. Med. (2003)

Blocking the Fas/FasL interaction in vivo rescues isotype-switched B cells in μMT mice. A neutralizing recombinant FasIg protein was purified and engineered into slow releasing PLGA microcapsules. The microcapsules were administered to μMT mice by intramuscular injection of 10 mg/mouse every 10 d for 8 wk. Mice were first injected at 2 wk of age. Control μMT mice were injected with empty microcapsules. 10 d after the last injection, mice were killed. (A) Serum samples from the mice were assayed for the presence of IgG by ELISA. Results are mean ± SEM of three mice in each group. (B) Detection of γH chain in serum samples by Western blotting. Normal serum sample dilution is 1:10. Samples of μMT mice (control and FasIg-treated) were not diluted. Purified IgG was used as control. Line indicates where irrelevant lane was removed digitally. (C) Quantitation of IgG-producing cells in spleens of μMT mice treated with FasIg relative to μMT and normal mice by ELISPOT. Representative ELISPOT membranes and the calculated frequency of IgG-producing cells per 107 spleen cells are shown. Results are mean ± SEM of three mice in each group. (D) FACS® analysis for κ and IgG expression. Spleen cells from the treated mice were stained for CD19, B220, κ, and IgG. Analysis for κ and IgG expression was performed on 10,000 gated CD19+/B220+ cells. The results shown are representative of eight injected mice in three different experiments.
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Related In: Results  -  Collection

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fig6: Blocking the Fas/FasL interaction in vivo rescues isotype-switched B cells in μMT mice. A neutralizing recombinant FasIg protein was purified and engineered into slow releasing PLGA microcapsules. The microcapsules were administered to μMT mice by intramuscular injection of 10 mg/mouse every 10 d for 8 wk. Mice were first injected at 2 wk of age. Control μMT mice were injected with empty microcapsules. 10 d after the last injection, mice were killed. (A) Serum samples from the mice were assayed for the presence of IgG by ELISA. Results are mean ± SEM of three mice in each group. (B) Detection of γH chain in serum samples by Western blotting. Normal serum sample dilution is 1:10. Samples of μMT mice (control and FasIg-treated) were not diluted. Purified IgG was used as control. Line indicates where irrelevant lane was removed digitally. (C) Quantitation of IgG-producing cells in spleens of μMT mice treated with FasIg relative to μMT and normal mice by ELISPOT. Representative ELISPOT membranes and the calculated frequency of IgG-producing cells per 107 spleen cells are shown. Results are mean ± SEM of three mice in each group. (D) FACS® analysis for κ and IgG expression. Spleen cells from the treated mice were stained for CD19, B220, κ, and IgG. Analysis for κ and IgG expression was performed on 10,000 gated CD19+/B220+ cells. The results shown are representative of eight injected mice in three different experiments.
Mentions: To test whether blocking the Fas/FasL interaction in vivo would rescue isotype-switched B cell precursors, PLGA microcapsules containing recombinant FasIg were injected into μMT mice. As described in Materials and Methods, these capsules allowed continuous slow release of FasIg at a constant amount of 10 ng/ml/day. 10 d after the last FasIg microcapsule injection, mice were tested for the presence of mature B cells and antibody production. The results in Fig. 6 clearly show that inhibition of Fas/FasL interaction by continuous presence of FasIg rescued isotype-switched B cell precursors in μMT mice. Serum analysis revealed a low, but significant amount of IgG production in μMT mice treated with FasIg (Fig. 6 A). This was in contrast to the control μMT mice, which fail to produce any serum IgG. The presence of IgG in sera was confirmed by Western blotting and independent detection of γH chain (Fig. 6 B). The specificity of the ELISA and the Western blot assays was confirmed and no reactivity was found between the anti–mouse IgG reagent and human IgG or purified FasIg (not depicted). Using an ELISPOT assay we were able to detect low frequency of IgG-producing cells in the FasIg-treated μMT mice (total of 10 ± 4/spleen) relative to control (Fig. 6 C). Finally, staining of spleen cells for κ and IgG expression revealed a very low population of mature, isotype-switched B cells in μMT mice treated with FasIg (total of 1,750 ± 585/spleen), but not in the control μMT group (Fig. 6 D). Thus, administration of FasIg to μMT mice rescues isotype-switched B cells from Fas-mediated apoptosis allowing maturation and serum IgG production in these mice, as confirmed by several experimental techniques.

Bottom Line: Ongoing isotype switching was found in both normal and microMT B cell development as reflected by detection of IgG1 germline and postswitch transcripts as well as activation-induced cytidine deaminase expression, resulting in the generation of IgG-expressing cells.We suggest a novel developmental pathway used by isotype-switched B cell precursors that effectively circumvents peripheral tolerance requirements.This developmental pathway, however, is strictly controlled by Fas/FasL interaction to prevent B cell autoimmunity.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa 31096, Israel. melamedd@techunix.technion.ac.il

ABSTRACT
In B lymphocytes, immunoglobulin (Ig)M receptors drive development and construction of naive repertoire, whereas IgG receptors promote formation of the memory B cell compartment. This isotype switching process requires appropriate B cell activation and T cell help. In the absence of T cell help, activated B cells undergo Fas-mediated apoptosis, a peripheral mechanism contributing to the establishment of self-tolerance. Using Igmicro-deficient microMT mouse model, where B cell development is blocked at pro-B stage, here we show an alternative developmental pathway used by isotype-switched B cell precursors. We find that isotype switching occurs normally in B cell precursors and is T independent. Ongoing isotype switching was found in both normal and microMT B cell development as reflected by detection of IgG1 germline and postswitch transcripts as well as activation-induced cytidine deaminase expression, resulting in the generation of IgG-expressing cells. These isotype-switched B cells are negatively selected by Fas pathway, as blocking the Fas/FasL interaction rescues the development of isotype-switched B cells in vivo and in vitro. Similar to memory B cells, isotype-switched B cells have a marginal zone phenotype. We suggest a novel developmental pathway used by isotype-switched B cell precursors that effectively circumvents peripheral tolerance requirements. This developmental pathway, however, is strictly controlled by Fas/FasL interaction to prevent B cell autoimmunity.

Show MeSH
Related in: MedlinePlus