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IL-21 regulates germinal center B cell differentiation and proliferation through a B cell-intrinsic mechanism.

Zotos D, Coquet JM, Zhang Y, Light A, D'Costa K, Kallies A, Corcoran LM, Godfrey DI, Toellner KM, Smyth MJ, Nutt SL, Tarlinton DM - J. Exp. Med. (2010)

Bottom Line: Germinal centers (GCs) are sites of B cell proliferation, somatic hypermutation, and selection of variants with improved affinity for antigen.The absence of IL-21 or IL-21 receptor does not abrogate the appearance of T cells in GCs or the appearance of CD4 T cells with a follicular helper phenotype.IL-21 thus controls fate choices of GC B cells directly.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Walter and Eliza Hall Institute of Medical Research, Parkville 3052, Australia.

ABSTRACT
Germinal centers (GCs) are sites of B cell proliferation, somatic hypermutation, and selection of variants with improved affinity for antigen. Long-lived memory B cells and plasma cells are also generated in GCs, although how B cell differentiation in GCs is regulated is unclear. IL-21, secreted by T follicular helper cells, is important for adaptive immune responses, although there are conflicting reports on its target cells and mode of action in vivo. We show that the absence of IL-21 signaling profoundly affects the B cell response to protein antigen, reducing splenic and bone marrow plasma cell formation and GC persistence and function, influencing their proliferation, transition into memory B cells, and affinity maturation. Using bone marrow chimeras, we show that these activities are primarily a result of CD3-expressing cells producing IL-21 that acts directly on B cells. Molecularly, IL-21 maintains expression of Bcl-6 in GC B cells. The absence of IL-21 or IL-21 receptor does not abrogate the appearance of T cells in GCs or the appearance of CD4 T cells with a follicular helper phenotype. IL-21 thus controls fate choices of GC B cells directly.

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Intrafollicular response in IL-21– and IL-21R–deficient mice. (A) Representative spleen histology staining revealing the localization of NP-reactive cells (blue) 7 d after immunization in control and IL-21R−/− mice. B cells follicles were stained with IgD (brown). GCs are indicated, as are foci of ASC (open arrows). NP-reactive B cells in follicles are shown by closed arrows in expanded region. Bars, 100 µm. (B) Quantification of the histology staining shown in A from C57BL/6, IL-21−/−, and IL-21R−/− mice of NP-specific plasmablasts (PBs) and plasma cells (PCs) in the red pulp and NP-specific B cell blasts in the follicles. (C) Flow cytometric scheme for the analysis of antigen-specific B cells in the spleens of NP immunized mice. Spleen cells were partitioned into isotype-switched B cells, and the percentage of those cells that were IgG1+ and NP binding was determined to calculate the fraction of total spleen involved in the B cell response. Values shown are the percentage of events displayed and not overall splenic frequencies. (D) Frequency distribution of antigen-reactive B cells in the spleen after immunization of C57BL/6, IL-21−/− and IL-21R−/− mice. (E) Total spleen cell number of antigen-reactive B cells in C57BL/6, IL-21−/−, and IL-21R−/− mice after immunization at the times indicated. Data in D and E are representative of at least three independent experiments of between 5 and 11 mice at each time point. Error bars are ± SEM. Differences were examined using a Student’s t test and only marked if significant. (F) Representative appearance of GC in the spleen by immunohistochemistry, 14 d after immunization, from C57BL/6, IL-21−/−, and IL-21R−/− mice. GC was revealed with PNA (blue). Follicles (F) revealed with B220 (red). Bars, 100 µm.
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fig2: Intrafollicular response in IL-21– and IL-21R–deficient mice. (A) Representative spleen histology staining revealing the localization of NP-reactive cells (blue) 7 d after immunization in control and IL-21R−/− mice. B cells follicles were stained with IgD (brown). GCs are indicated, as are foci of ASC (open arrows). NP-reactive B cells in follicles are shown by closed arrows in expanded region. Bars, 100 µm. (B) Quantification of the histology staining shown in A from C57BL/6, IL-21−/−, and IL-21R−/− mice of NP-specific plasmablasts (PBs) and plasma cells (PCs) in the red pulp and NP-specific B cell blasts in the follicles. (C) Flow cytometric scheme for the analysis of antigen-specific B cells in the spleens of NP immunized mice. Spleen cells were partitioned into isotype-switched B cells, and the percentage of those cells that were IgG1+ and NP binding was determined to calculate the fraction of total spleen involved in the B cell response. Values shown are the percentage of events displayed and not overall splenic frequencies. (D) Frequency distribution of antigen-reactive B cells in the spleen after immunization of C57BL/6, IL-21−/− and IL-21R−/− mice. (E) Total spleen cell number of antigen-reactive B cells in C57BL/6, IL-21−/−, and IL-21R−/− mice after immunization at the times indicated. Data in D and E are representative of at least three independent experiments of between 5 and 11 mice at each time point. Error bars are ± SEM. Differences were examined using a Student’s t test and only marked if significant. (F) Representative appearance of GC in the spleen by immunohistochemistry, 14 d after immunization, from C57BL/6, IL-21−/−, and IL-21R−/− mice. GC was revealed with PNA (blue). Follicles (F) revealed with B220 (red). Bars, 100 µm.

Mentions: The reduced frequency and affinity of NP-specific bone marrow plasma cells indicated a possible GC abnormality in the immune response of IL-21−/− and IL-21R−/− mice. We therefore investigated GC formation in spleen by immunizing mice as before and determining both the distribution of antigen-specific cells by immunohistochemistry and their frequency by flow cytometry (Fig. 2). Resolving the location and nature of NP-reactive B cells revealed substantial differences between knockout and control mice. Although NP-specific cells were identified as ASC in bridging channels and in GC within follicles in spleens of all mice 7 d after immunization, there was an unusual accumulation of NP-binding B cell blasts in the follicular mantles of the knockout strains (Fig. 2, A and B; and Fig. S2 B). Parenthetically, this staining revealed NP-specific cells irrespective of isotype, showing that the deficiency in ASC in the knockout spleens shown in Fig. 1 is a defect in development and not in isotype switching. NP staining also revealed an unexpected role for IL-21 in migration of antigen-activated B cells early in the immune response. Despite their aberrant localization, the frequency and total number of NP-specific B cells in spleens of immunized IL-21– and IL-21R–deficient mice at day 7 were approximately equal to those in control mice (Fig. 2, C–E). In fact the representation of NP+IgG1+ B cells in the spleens of the different groups was similar at all times examined, up to day 53 (Fig. 2, D and E; and not depicted). Despite the early differences in NP-specific B cell location, GC histology in day-14 immunized knockout and control mice was very similar (Fig. 2 F), but by day 28 after immunization, GC in the IL-21/IL-21R−/− spleens were markedly smaller than those in controls (Fig. S2, C and D).


IL-21 regulates germinal center B cell differentiation and proliferation through a B cell-intrinsic mechanism.

Zotos D, Coquet JM, Zhang Y, Light A, D'Costa K, Kallies A, Corcoran LM, Godfrey DI, Toellner KM, Smyth MJ, Nutt SL, Tarlinton DM - J. Exp. Med. (2010)

Intrafollicular response in IL-21– and IL-21R–deficient mice. (A) Representative spleen histology staining revealing the localization of NP-reactive cells (blue) 7 d after immunization in control and IL-21R−/− mice. B cells follicles were stained with IgD (brown). GCs are indicated, as are foci of ASC (open arrows). NP-reactive B cells in follicles are shown by closed arrows in expanded region. Bars, 100 µm. (B) Quantification of the histology staining shown in A from C57BL/6, IL-21−/−, and IL-21R−/− mice of NP-specific plasmablasts (PBs) and plasma cells (PCs) in the red pulp and NP-specific B cell blasts in the follicles. (C) Flow cytometric scheme for the analysis of antigen-specific B cells in the spleens of NP immunized mice. Spleen cells were partitioned into isotype-switched B cells, and the percentage of those cells that were IgG1+ and NP binding was determined to calculate the fraction of total spleen involved in the B cell response. Values shown are the percentage of events displayed and not overall splenic frequencies. (D) Frequency distribution of antigen-reactive B cells in the spleen after immunization of C57BL/6, IL-21−/− and IL-21R−/− mice. (E) Total spleen cell number of antigen-reactive B cells in C57BL/6, IL-21−/−, and IL-21R−/− mice after immunization at the times indicated. Data in D and E are representative of at least three independent experiments of between 5 and 11 mice at each time point. Error bars are ± SEM. Differences were examined using a Student’s t test and only marked if significant. (F) Representative appearance of GC in the spleen by immunohistochemistry, 14 d after immunization, from C57BL/6, IL-21−/−, and IL-21R−/− mice. GC was revealed with PNA (blue). Follicles (F) revealed with B220 (red). Bars, 100 µm.
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Related In: Results  -  Collection

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Show All Figures
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fig2: Intrafollicular response in IL-21– and IL-21R–deficient mice. (A) Representative spleen histology staining revealing the localization of NP-reactive cells (blue) 7 d after immunization in control and IL-21R−/− mice. B cells follicles were stained with IgD (brown). GCs are indicated, as are foci of ASC (open arrows). NP-reactive B cells in follicles are shown by closed arrows in expanded region. Bars, 100 µm. (B) Quantification of the histology staining shown in A from C57BL/6, IL-21−/−, and IL-21R−/− mice of NP-specific plasmablasts (PBs) and plasma cells (PCs) in the red pulp and NP-specific B cell blasts in the follicles. (C) Flow cytometric scheme for the analysis of antigen-specific B cells in the spleens of NP immunized mice. Spleen cells were partitioned into isotype-switched B cells, and the percentage of those cells that were IgG1+ and NP binding was determined to calculate the fraction of total spleen involved in the B cell response. Values shown are the percentage of events displayed and not overall splenic frequencies. (D) Frequency distribution of antigen-reactive B cells in the spleen after immunization of C57BL/6, IL-21−/− and IL-21R−/− mice. (E) Total spleen cell number of antigen-reactive B cells in C57BL/6, IL-21−/−, and IL-21R−/− mice after immunization at the times indicated. Data in D and E are representative of at least three independent experiments of between 5 and 11 mice at each time point. Error bars are ± SEM. Differences were examined using a Student’s t test and only marked if significant. (F) Representative appearance of GC in the spleen by immunohistochemistry, 14 d after immunization, from C57BL/6, IL-21−/−, and IL-21R−/− mice. GC was revealed with PNA (blue). Follicles (F) revealed with B220 (red). Bars, 100 µm.
Mentions: The reduced frequency and affinity of NP-specific bone marrow plasma cells indicated a possible GC abnormality in the immune response of IL-21−/− and IL-21R−/− mice. We therefore investigated GC formation in spleen by immunizing mice as before and determining both the distribution of antigen-specific cells by immunohistochemistry and their frequency by flow cytometry (Fig. 2). Resolving the location and nature of NP-reactive B cells revealed substantial differences between knockout and control mice. Although NP-specific cells were identified as ASC in bridging channels and in GC within follicles in spleens of all mice 7 d after immunization, there was an unusual accumulation of NP-binding B cell blasts in the follicular mantles of the knockout strains (Fig. 2, A and B; and Fig. S2 B). Parenthetically, this staining revealed NP-specific cells irrespective of isotype, showing that the deficiency in ASC in the knockout spleens shown in Fig. 1 is a defect in development and not in isotype switching. NP staining also revealed an unexpected role for IL-21 in migration of antigen-activated B cells early in the immune response. Despite their aberrant localization, the frequency and total number of NP-specific B cells in spleens of immunized IL-21– and IL-21R–deficient mice at day 7 were approximately equal to those in control mice (Fig. 2, C–E). In fact the representation of NP+IgG1+ B cells in the spleens of the different groups was similar at all times examined, up to day 53 (Fig. 2, D and E; and not depicted). Despite the early differences in NP-specific B cell location, GC histology in day-14 immunized knockout and control mice was very similar (Fig. 2 F), but by day 28 after immunization, GC in the IL-21/IL-21R−/− spleens were markedly smaller than those in controls (Fig. S2, C and D).

Bottom Line: Germinal centers (GCs) are sites of B cell proliferation, somatic hypermutation, and selection of variants with improved affinity for antigen.The absence of IL-21 or IL-21 receptor does not abrogate the appearance of T cells in GCs or the appearance of CD4 T cells with a follicular helper phenotype.IL-21 thus controls fate choices of GC B cells directly.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Walter and Eliza Hall Institute of Medical Research, Parkville 3052, Australia.

ABSTRACT
Germinal centers (GCs) are sites of B cell proliferation, somatic hypermutation, and selection of variants with improved affinity for antigen. Long-lived memory B cells and plasma cells are also generated in GCs, although how B cell differentiation in GCs is regulated is unclear. IL-21, secreted by T follicular helper cells, is important for adaptive immune responses, although there are conflicting reports on its target cells and mode of action in vivo. We show that the absence of IL-21 signaling profoundly affects the B cell response to protein antigen, reducing splenic and bone marrow plasma cell formation and GC persistence and function, influencing their proliferation, transition into memory B cells, and affinity maturation. Using bone marrow chimeras, we show that these activities are primarily a result of CD3-expressing cells producing IL-21 that acts directly on B cells. Molecularly, IL-21 maintains expression of Bcl-6 in GC B cells. The absence of IL-21 or IL-21 receptor does not abrogate the appearance of T cells in GCs or the appearance of CD4 T cells with a follicular helper phenotype. IL-21 thus controls fate choices of GC B cells directly.

Show MeSH
Related in: MedlinePlus