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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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Accelerated formation of memory B cell compartment in IL-21– and IL-21R–Deficient Mice. (A) Representative flow cytometric plots resolving GC and memory compartments among NP-specific B cells in spleens of C57BL/6 and IL-21R−/− mice 28 d after immunization. Splenocytes, previously gated to include only isotype-switched B cells, were assessed for expression of IgG1 and binding of NP (left). Expression of CD38 on NP+IgG1+ B cells resolved memory (CD38hi) and GC (CD38lo) B cells (right). The percentages are the proportion of displayed events falling within the indicated NP+IgG1+ and CD38hi gates. (B) Proportion of NP-specific B cells with a memory phenotype at times indicated after immunization of C57BL/6, IL-21−/−, and IL-21R−/− mice. (C) Absolute numbers of NP-specific IgG1 memory B cells in spleens of C57BL/6, IL-21−/−, and IL-21R−/− mice at the indicated times after immunization. Symbols in B and C indicate the mean of between 5 and 11 mice at each time point, ± SEM, and derived from at least three independent experiments. Differences were examined using a Student’s t test and only marked if significant.
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fig3: Accelerated formation of memory B cell compartment in IL-21– and IL-21R–Deficient Mice. (A) Representative flow cytometric plots resolving GC and memory compartments among NP-specific B cells in spleens of C57BL/6 and IL-21R−/− mice 28 d after immunization. Splenocytes, previously gated to include only isotype-switched B cells, were assessed for expression of IgG1 and binding of NP (left). Expression of CD38 on NP+IgG1+ B cells resolved memory (CD38hi) and GC (CD38lo) B cells (right). The percentages are the proportion of displayed events falling within the indicated NP+IgG1+ and CD38hi gates. (B) Proportion of NP-specific B cells with a memory phenotype at times indicated after immunization of C57BL/6, IL-21−/−, and IL-21R−/− mice. (C) Absolute numbers of NP-specific IgG1 memory B cells in spleens of C57BL/6, IL-21−/−, and IL-21R−/− mice at the indicated times after immunization. Symbols in B and C indicate the mean of between 5 and 11 mice at each time point, ± SEM, and derived from at least three independent experiments. Differences were examined using a Student’s t test and only marked if significant.

Mentions: The discrepancy between histologically reduced GC at day 28 yet similar numbers of NP-reactive B cells in the spleen was reconciled by resolving NP-specific IgG1+ B cells into GC and memory compartments using expression of CD38 (Oliver et al., 1997; Ridderstad and Tarlinton, 1998) measured by flow cytometry (Fig. 3 A). These analyses revealed the proportion of NP-specific B cells with a memory phenotype to increase in control mice from <10% at day 14 to ∼20% at day 28 (Fig. 3 B), which is consistent with other examples of this immunization protocol (Takahashi et al., 2001). Memory B cell representation in the IL-21 and IL-21R knockout spleens, which was already significantly increased at day 14 compared with controls, reached >70% of antigen-specific B cells by day 28 (Fig. 3 B). The absolute number of NP+IgG1+ memory B cells in the knockout mice was significantly different from controls at days 14 (IL-21 and IL-21R) and 28 (IL-21R), despite not increasing over that period (Fig. 3 C). Thus, the increasing representation of memory B cells seen by flow cytometry in immunized IL-21 and IL-21R knockout mice was a result of the accelerated loss of GC B cells, as indicated by histology. Importantly, the memory B cells formed in the knockout strains persisted and were functional, in that boosting at day 53 with soluble antigen increased the frequency of ASC (Fig. S3). The formation of GC in the absence of IL-21R or IL-21 supports a previous study (Ozaki et al., 2002) and contradicts others (Nurieva et al., 2008; Vogelzang et al., 2008), whereas the subsequent premature dissolution reveals a unique role for IL-21 signaling in the maintenance of GC. In considering whether other potential Tfh cytokines had effects similar to those of IL-21, we immunized IL-4–deficient mice. These mice showed proportions of GC and memory B cells similar to controls at all time points measured, albeit at significantly reduced frequencies (Fig. S4), suggesting distinct roles for IL-21 and IL-4 in the GC (Reinhardt et al., 2009).


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)

Accelerated formation of memory B cell compartment in IL-21– and IL-21R–Deficient Mice. (A) Representative flow cytometric plots resolving GC and memory compartments among NP-specific B cells in spleens of C57BL/6 and IL-21R−/− mice 28 d after immunization. Splenocytes, previously gated to include only isotype-switched B cells, were assessed for expression of IgG1 and binding of NP (left). Expression of CD38 on NP+IgG1+ B cells resolved memory (CD38hi) and GC (CD38lo) B cells (right). The percentages are the proportion of displayed events falling within the indicated NP+IgG1+ and CD38hi gates. (B) Proportion of NP-specific B cells with a memory phenotype at times indicated after immunization of C57BL/6, IL-21−/−, and IL-21R−/− mice. (C) Absolute numbers of NP-specific IgG1 memory B cells in spleens of C57BL/6, IL-21−/−, and IL-21R−/− mice at the indicated times after immunization. Symbols in B and C indicate the mean of between 5 and 11 mice at each time point, ± SEM, and derived from at least three independent experiments. Differences were examined using a Student’s t test and only marked if significant.
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fig3: Accelerated formation of memory B cell compartment in IL-21– and IL-21R–Deficient Mice. (A) Representative flow cytometric plots resolving GC and memory compartments among NP-specific B cells in spleens of C57BL/6 and IL-21R−/− mice 28 d after immunization. Splenocytes, previously gated to include only isotype-switched B cells, were assessed for expression of IgG1 and binding of NP (left). Expression of CD38 on NP+IgG1+ B cells resolved memory (CD38hi) and GC (CD38lo) B cells (right). The percentages are the proportion of displayed events falling within the indicated NP+IgG1+ and CD38hi gates. (B) Proportion of NP-specific B cells with a memory phenotype at times indicated after immunization of C57BL/6, IL-21−/−, and IL-21R−/− mice. (C) Absolute numbers of NP-specific IgG1 memory B cells in spleens of C57BL/6, IL-21−/−, and IL-21R−/− mice at the indicated times after immunization. Symbols in B and C indicate the mean of between 5 and 11 mice at each time point, ± SEM, and derived from at least three independent experiments. Differences were examined using a Student’s t test and only marked if significant.
Mentions: The discrepancy between histologically reduced GC at day 28 yet similar numbers of NP-reactive B cells in the spleen was reconciled by resolving NP-specific IgG1+ B cells into GC and memory compartments using expression of CD38 (Oliver et al., 1997; Ridderstad and Tarlinton, 1998) measured by flow cytometry (Fig. 3 A). These analyses revealed the proportion of NP-specific B cells with a memory phenotype to increase in control mice from <10% at day 14 to ∼20% at day 28 (Fig. 3 B), which is consistent with other examples of this immunization protocol (Takahashi et al., 2001). Memory B cell representation in the IL-21 and IL-21R knockout spleens, which was already significantly increased at day 14 compared with controls, reached >70% of antigen-specific B cells by day 28 (Fig. 3 B). The absolute number of NP+IgG1+ memory B cells in the knockout mice was significantly different from controls at days 14 (IL-21 and IL-21R) and 28 (IL-21R), despite not increasing over that period (Fig. 3 C). Thus, the increasing representation of memory B cells seen by flow cytometry in immunized IL-21 and IL-21R knockout mice was a result of the accelerated loss of GC B cells, as indicated by histology. Importantly, the memory B cells formed in the knockout strains persisted and were functional, in that boosting at day 53 with soluble antigen increased the frequency of ASC (Fig. S3). The formation of GC in the absence of IL-21R or IL-21 supports a previous study (Ozaki et al., 2002) and contradicts others (Nurieva et al., 2008; Vogelzang et al., 2008), whereas the subsequent premature dissolution reveals a unique role for IL-21 signaling in the maintenance of GC. In considering whether other potential Tfh cytokines had effects similar to those of IL-21, we immunized IL-4–deficient mice. These mice showed proportions of GC and memory B cells similar to controls at all time points measured, albeit at significantly reduced frequencies (Fig. S4), suggesting distinct roles for IL-21 and IL-4 in the GC (Reinhardt et al., 2009).

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