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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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Loss of IL-21 signaling reduces B cell proliferation in GCs. (A) Representative flow cytometric plots for the identification and isolation of NP-specific B cells within the spleens of immunized mice. Spleen cells were enriched for Ig lambda (λ1) expression using magnetic separation then fractionated into isotype switched B cells and the percent of Igλ+ NP binding among those cells determined. Expression of CD38 was used to differentiate germinal GC (CD38lo) from memory B cells (CD38hi) and gates for sorting NP-specific GC B cells set accordingly. (B) Proportion of GC B cells (NP+λ+CD38lo) cells in (S + G2) phase in C57BL/6 and IL-21−/− mice at times indicated. Values are derived from two experiments and are the mean of between three and eight mice (±SD) at each time. Significant difference at day 14 was determined by a Student’s t test. Day-21 data for IL-21–deficient mice are from a pool of three animals because low numbers of GC B cells prevented individual analysis.
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fig6: Loss of IL-21 signaling reduces B cell proliferation in GCs. (A) Representative flow cytometric plots for the identification and isolation of NP-specific B cells within the spleens of immunized mice. Spleen cells were enriched for Ig lambda (λ1) expression using magnetic separation then fractionated into isotype switched B cells and the percent of Igλ+ NP binding among those cells determined. Expression of CD38 was used to differentiate germinal GC (CD38lo) from memory B cells (CD38hi) and gates for sorting NP-specific GC B cells set accordingly. (B) Proportion of GC B cells (NP+λ+CD38lo) cells in (S + G2) phase in C57BL/6 and IL-21−/− mice at times indicated. Values are derived from two experiments and are the mean of between three and eight mice (±SD) at each time. Significant difference at day 14 was determined by a Student’s t test. Day-21 data for IL-21–deficient mice are from a pool of three animals because low numbers of GC B cells prevented individual analysis.

Mentions: We next determined if the loss of GC B cells in the absence of IL-21R was associated with altered proliferation by quantifying the fraction of such B cells in cell cycle at varying times after immunization. Control and IL-21R−/− mice, immunized with NP-KLH in alum 7, 14, or 21 d earlier, were sacrificed and the isotype switched, and NP-binding immunoglobulin light chain lambda (λ1+) B cells were purified from spleen by cell sorting after magnetic enrichment for λ1+ B cells (Fig. 6 A), which together with VH186.2 comprises the canonical anti-NP BCR in C57BL/6 mice (Cumano and Rajewsky, 1985). The representation of λ1+ B cells among splenocytes from control and IL-21R−/− mice at these times ranged from 2.0 ± 0.2 to 1.6 ± 0.4% (mean ± SEM) with no significant differences. NP-specific GC (CD38lo) B cells were identified by flow cytometry as indicated (Fig. 6 A) and the proportion of these cells in cell cycle (DNA content > 2n) determined by staining the nuclei with propidium iodide. There was no significant difference in the proportion of antigen-specific GC B cells in cell cycle in IL-21−/− compared with control mice at day 7 (Fig. 6 B). By day 14, however, a significant difference was apparent between the two groups with a mean of <20% of IL-21−/− GC B cells in cell cycle compared with ∼40% in controls (Fig. 6 B). This difference persisted through to day 21 (Fig. 6 B). Diminished proliferation of IL-21R–deficient GC B cells was observed using the alternative approach of measuring BrdU incorporation into antigen-specific B cells at days 7, 14, and 28 after immunization (Fig. S6). Thus, in the absence of IL-21 signaling, GC B cell proliferation was curtailed significantly at later time points.


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)

Loss of IL-21 signaling reduces B cell proliferation in GCs. (A) Representative flow cytometric plots for the identification and isolation of NP-specific B cells within the spleens of immunized mice. Spleen cells were enriched for Ig lambda (λ1) expression using magnetic separation then fractionated into isotype switched B cells and the percent of Igλ+ NP binding among those cells determined. Expression of CD38 was used to differentiate germinal GC (CD38lo) from memory B cells (CD38hi) and gates for sorting NP-specific GC B cells set accordingly. (B) Proportion of GC B cells (NP+λ+CD38lo) cells in (S + G2) phase in C57BL/6 and IL-21−/− mice at times indicated. Values are derived from two experiments and are the mean of between three and eight mice (±SD) at each time. Significant difference at day 14 was determined by a Student’s t test. Day-21 data for IL-21–deficient mice are from a pool of three animals because low numbers of GC B cells prevented individual analysis.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2822601&req=5

fig6: Loss of IL-21 signaling reduces B cell proliferation in GCs. (A) Representative flow cytometric plots for the identification and isolation of NP-specific B cells within the spleens of immunized mice. Spleen cells were enriched for Ig lambda (λ1) expression using magnetic separation then fractionated into isotype switched B cells and the percent of Igλ+ NP binding among those cells determined. Expression of CD38 was used to differentiate germinal GC (CD38lo) from memory B cells (CD38hi) and gates for sorting NP-specific GC B cells set accordingly. (B) Proportion of GC B cells (NP+λ+CD38lo) cells in (S + G2) phase in C57BL/6 and IL-21−/− mice at times indicated. Values are derived from two experiments and are the mean of between three and eight mice (±SD) at each time. Significant difference at day 14 was determined by a Student’s t test. Day-21 data for IL-21–deficient mice are from a pool of three animals because low numbers of GC B cells prevented individual analysis.
Mentions: We next determined if the loss of GC B cells in the absence of IL-21R was associated with altered proliferation by quantifying the fraction of such B cells in cell cycle at varying times after immunization. Control and IL-21R−/− mice, immunized with NP-KLH in alum 7, 14, or 21 d earlier, were sacrificed and the isotype switched, and NP-binding immunoglobulin light chain lambda (λ1+) B cells were purified from spleen by cell sorting after magnetic enrichment for λ1+ B cells (Fig. 6 A), which together with VH186.2 comprises the canonical anti-NP BCR in C57BL/6 mice (Cumano and Rajewsky, 1985). The representation of λ1+ B cells among splenocytes from control and IL-21R−/− mice at these times ranged from 2.0 ± 0.2 to 1.6 ± 0.4% (mean ± SEM) with no significant differences. NP-specific GC (CD38lo) B cells were identified by flow cytometry as indicated (Fig. 6 A) and the proportion of these cells in cell cycle (DNA content > 2n) determined by staining the nuclei with propidium iodide. There was no significant difference in the proportion of antigen-specific GC B cells in cell cycle in IL-21−/− compared with control mice at day 7 (Fig. 6 B). By day 14, however, a significant difference was apparent between the two groups with a mean of <20% of IL-21−/− GC B cells in cell cycle compared with ∼40% in controls (Fig. 6 B). This difference persisted through to day 21 (Fig. 6 B). Diminished proliferation of IL-21R–deficient GC B cells was observed using the alternative approach of measuring BrdU incorporation into antigen-specific B cells at days 7, 14, and 28 after immunization (Fig. S6). Thus, in the absence of IL-21 signaling, GC B cell proliferation was curtailed significantly at later time points.

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