Immunology and Cell Biology (2014) 92, 645–646 & 2014 Australasian Society for Immunology Inc. All rights reserved 0818-9641/14 www.nature.com/icb
NEWS AND COMMENTARY Sequential IgG class switching
B cells take their time: sequential IgG class switching over the course of an immune response? Menno C van Zelm Immunology and Cell Biology (2014) 92, 645–646; doi:10.1038/icb.2014.48; published online 17 June 2014
B
cells are unique in their capability to modify their immunoglobulin (Ig) molecules during an immune response. Affinity maturation is established by somatic hypermutations (SHMs) and selection for antigen binding. Furthermore, through class switch recombination (CSR), the Ig constant regions can be changed from IgM into IgD, IgE, one of the two IgA, or one of the four IgG isotypes. In this issue of Immunology and Cell Biology, Jackson et al.1 show a relationship between the extent of affinity maturation and Ig subclass usage through large-scale sequence analysis of IgA and IgG transcripts. SHM levels in variable regions were increased in transcripts switched to more downstream-located IgG constant regions, that is, IgG3oIgG1oIgG2oIgG4 (Figure 1a). A similar pattern was seen for the selection for replacement mutations, except for IgG4. These results confirmed earlier findings and the authors’ temporal model, which states that there is a programmed order of IgG subclass usage during an immune response. Specifically, the laterformed IgG2 and IgG4 might function to restrict inflammation due to their inability to fix complement (Figure 1b).2 The authors’ model for sequential IgG switching is further supported by the presence of IgG3 and IgG1 switch region remnants in IgG1 þ and IgG2 þ B cells, respectively.3 Interestingly, the authors did not find differences in mutation loads between IgA1 and IgA2.1 A possible explanation lies in the fact that IgA can be produced in both T-cell-dependent and T-cell-independent manners. Especially local MC van Zelm is at the Department of Immunology, Erasmus MC, University Medical Center, Rotterdam, The Netherlands E-mail: [email protected]
intestinal responses can generate IgA2 without an intermediate switch via IgG1 or IgA1, excluding the possibility of a temporal mechanism.3,4 Potentially, the same is true for IgE þ B cells, which can be derived in T-cell-dependent responses via IgG1 or directly from IgM in T-cell-independent responses.5 In contrast, IgG responses are completely T-cell dependent, because individuals with inherited mutations in CD40L that lack germinal centers show a complete absence of IgG.6 This makes IgG a better model to study the relation between SHM levels and sequential CSR. SHM levels are directly related to cell divisions in a germinal center,7 and can therefore be used as a measure for the extent of an antibody response. Unexpectedly, urban dwellers did not carry fewer SHMs in their IgA and IgG transcripts than villagers from Papua New Guinea, who live in areas with a high burden of infectious disease.1 Apparently, the repeated antigen exposure did not lead to higher mutation loads per IgA or IgG subclass. Recent studies with Sanger sequencing of transcripts generated with IgA and IgG consensus primers showed similar SHM levels between children (5–14 years) and adults (23–35 years) per Ig subclass.6,8,9 However, adults showed more dominant IgG2 and IgA2 subclass usage (Figure 1d). Thus, repeated antigen exposure might not lead to higher SHM levels per Ig subclass, but rather to more sequential CSR. This would suggest that sequential switching takes place not only in the course of one antigen response, but also in consecutive responses through the recruitment of IgG3 þ and IgG1 þ memory B cells (Figure 1c).3 It would therefore be interesting to study whether the Papua New Guinean individuals carry relatively more IgG2/4 than urban dwellers.
Are SHM levels then independent of the extent of antigen exposure? The observations in urban dwellers vs villagers and in children vs adults suggest that this is the case for healthy individuals.1 B-cell responses can also be driven by chronic inflammation. Indeed, patients with Sarcoidosis, a multi-system disorder involving abnormal collections of inflammatory cells (granulomas), show B-cell involvement with increased IgG2 usages and higher SHM levels per Ig subclass.8 It is therefore entirely possible that SHM levels are tightly regulated, also independent of sequential IgG, and that this mechanism is only disrupted in disease states. Advances in multi-color flow cytometry have enabled the phenotyping of separate memory B cells with distinct extents of antibody maturation. CD27-IgG þ B cells have lower degrees of proliferation, SHM and IgG2 usage than CD27 þ IgG þ B cells.3 These CD27 IgG þ B cells could therefore represent earlier products from an immune response than CD27 þ IgG þ B cells, fitting with the temporal model. However, the higher SHM levels in CD27 þ IgG þ B cells were not related to the increased IgG2 usage, as CD27 B cells carry fewer SHMs in each of the IgG subclasses than CD27 þ B cells.10 Thus, also in distinct antigen-experienced B-cell subsets SHM levels and sequential class switching seem uncoupled. In conclusion, the study by Jackson et al.1 fits with the temporal model of human IgG function. The temporal model is the first to describe how IgG subclasses might act together to provide a strong and ratelimiting immune response. Although SHM levels and sequential IgG class switching are not directly coupled in all types of antibody responses, the authors provide clear indications for a temporal effect of SHM and sequential Ig class switching. Still, in vivo
News and Commentary 646
a
V DJ
Cμ Cδ
Cγ3
Sμ
Sγ3
Cγ1
Ψε
Sγ1
Ψγ
Cα1
Cγ2
Sα1
b
Sγ2
Cγ4
Sγ4
Cε Sε
Cα2 Sα2
c germinal center germinal center T cell
IgG3/1+
naive B cell
T cell naive B cell IgG2/4+ IgG3+
IgG1+
IgG2+
IgG4+ IgG3 IgG4 10% 1%
IgG3 IgG4 5% 5%
d IgA2 22% 102
IgA1 78%
IgA2 46% 183
IgA1 54%
IgG2 22%
107
IgG1 68%
p < 0.01
p < 0.0001 child
IgG2 IgG1 33% 214 56%
adult
child
adult
Figure 1 Ig class switching and SHM levels in children and adults. (a) Schematic representation of the human IGH constant gene regions. (b) The temporal model of IgG class switching, where over the course of a germinal center response sequential switching towards IgG34IgG14IgG24IgG4 occurs with increasing levels of SHMs. (c) Model for sequential IgG switching in secondary germinal center responses, where primary IgG memory B cells re-enter a germinal center in a secondary response, undergo additional SHMs and switch to a more downstream IgG subclass.3 (d) IgA and IgG subclass distributions in transcripts derived with consensus primers in previous studies.5,6,8 Ig subclass distributions were compared between children and adults using the w2 test.
these effects will be blurred by secondary antigen encounters (Figure 1c), the diversity of antigens, anatomical locations of immune responses and the formation of diverse antigen-experienced B-cell subsets in humans. Furthermore, the diversity of antigens, anatomical locations of immune responses and the diversity of antigen-experienced human B-cell subsets can blur the effects of temporal CSR. To overcome these limitations, it would be important to design an analysis strategy for antigen-specific B cells. These could be isolated at several time points following primary and secondary vaccinations of healthy volunteers with a T-cell-dependent antigen (for example, rabies vaccine). Such analyses would provide new insights into the temporal and sequential nature of antibody responses and could advance our understanding of how humoral immunity is regulated by memory B cells, affinity maturation and Ig effector functions.
Immunology and Cell Biology
ACKNOWLEDGEMENTS I would like to thank M van der Burg, ED de Geus, C Grosserichter-Wagener, JJ Heeringa and BG de Jong for critical reading of the manuscript. This work was supported by the Sophia Children’s Hospital Fund (grant S698), and fellowships from the Erasmus University Rotterdam (EUR) and the Erasmus MC.
1 Jackson KJ, Wang Y, Collins AM. Human immunoglobulin classes and subclasses show variability in VDJ gene mutation levels. Immunol Cell Biol 2014; 92: 729–733. 2 Collins AM, Jackson KJ. A temporal model of human IgE and IgG antibody function. Front Immunol 2013; 4: 235. 3 Berkowska MA, Driessen GJ, Bikos V, GrosserichterWagener C, Stamatopoulos K, Cerutti A et al. Human memory B cells originate from three distinct germinal center-dependent and -independent maturation pathways. Blood 2011; 118: 2150–2158. 4 He B, Xu W, Santini PA, Polydorides AD, Chiu A, Estrella J et al. Intestinal bacteria trigger T cellindependent immunoglobulin A(2) class switching by inducing epithelial-cell secretion of the cytokine APRIL. Immunity 2007; 26: 812–826.
5 Berkowska MA, Heeringa JJ, Hajdarbegovic E, van der Burg M, Thio HB, van Hagen PM et al. Human IgE þ B cells are derived from T cell–dependent and T cell–independent pathways. J Allergy Clin Immunol (e-pub ahead of print 13 May 2014; doi:10.1016/ j.jaci.2014.03.036). 6 van Zelm MC, Bartol SJ, Driessen GJ, Mascart F, Reisli I, Franco JL et al. Human CD19 and CD40L deficiencies impair antibody selection and differentially affect somatic hypermutation. J Allergy Clin Immunol (e-pub ahead of print 10 January 2014; doi:10.1016/ j.jaci.2013.11.015 ). 7 Gitlin AD, Shulman Z, Nussenzweig MC. Clonal selection in the germinal centre by regulated proliferation and hypermutation. Nature 2014; 509: 637–640. 8 Kamphuis LS, van Zelm MC, Lam KH, Rimmelzwaan GF, Baarsma GS, Dik WA et al. Perigranuloma localization and abnormal maturation of B cells: emerging key players in sarcoidosis? Am J Respir Crit Care Med 2013; 187: 406–416. 9 Verstegen RH, Driessen GJ, Bartol SJW, van Noesel CJM, Boon L, van der Burg M et al. Defective B-cell memory in patients with Down syndrome. J Allergy Clin Immunol (in press). 10 Wu YC, Kipling D, Leong HS, Martin V, Ademokun AA, Dunn-Walters DK. High-throughput immunoglobulin repertoire analysis distinguishes between human IgM memory and switched memory B-cell populations. Blood 2010; 116: 1070–1078.
NEWS AND COMMENTARY Sequential IgG class switching
B cells take their time: sequential IgG class switching over the course of an immune response? Menno C van Zelm Immunology and Cell Biology (2014) 92, 645–646; doi:10.1038/icb.2014.48; published online 17 June 2014
B
cells are unique in their capability to modify their immunoglobulin (Ig) molecules during an immune response. Affinity maturation is established by somatic hypermutations (SHMs) and selection for antigen binding. Furthermore, through class switch recombination (CSR), the Ig constant regions can be changed from IgM into IgD, IgE, one of the two IgA, or one of the four IgG isotypes. In this issue of Immunology and Cell Biology, Jackson et al.1 show a relationship between the extent of affinity maturation and Ig subclass usage through large-scale sequence analysis of IgA and IgG transcripts. SHM levels in variable regions were increased in transcripts switched to more downstream-located IgG constant regions, that is, IgG3oIgG1oIgG2oIgG4 (Figure 1a). A similar pattern was seen for the selection for replacement mutations, except for IgG4. These results confirmed earlier findings and the authors’ temporal model, which states that there is a programmed order of IgG subclass usage during an immune response. Specifically, the laterformed IgG2 and IgG4 might function to restrict inflammation due to their inability to fix complement (Figure 1b).2 The authors’ model for sequential IgG switching is further supported by the presence of IgG3 and IgG1 switch region remnants in IgG1 þ and IgG2 þ B cells, respectively.3 Interestingly, the authors did not find differences in mutation loads between IgA1 and IgA2.1 A possible explanation lies in the fact that IgA can be produced in both T-cell-dependent and T-cell-independent manners. Especially local MC van Zelm is at the Department of Immunology, Erasmus MC, University Medical Center, Rotterdam, The Netherlands E-mail: [email protected]
intestinal responses can generate IgA2 without an intermediate switch via IgG1 or IgA1, excluding the possibility of a temporal mechanism.3,4 Potentially, the same is true for IgE þ B cells, which can be derived in T-cell-dependent responses via IgG1 or directly from IgM in T-cell-independent responses.5 In contrast, IgG responses are completely T-cell dependent, because individuals with inherited mutations in CD40L that lack germinal centers show a complete absence of IgG.6 This makes IgG a better model to study the relation between SHM levels and sequential CSR. SHM levels are directly related to cell divisions in a germinal center,7 and can therefore be used as a measure for the extent of an antibody response. Unexpectedly, urban dwellers did not carry fewer SHMs in their IgA and IgG transcripts than villagers from Papua New Guinea, who live in areas with a high burden of infectious disease.1 Apparently, the repeated antigen exposure did not lead to higher mutation loads per IgA or IgG subclass. Recent studies with Sanger sequencing of transcripts generated with IgA and IgG consensus primers showed similar SHM levels between children (5–14 years) and adults (23–35 years) per Ig subclass.6,8,9 However, adults showed more dominant IgG2 and IgA2 subclass usage (Figure 1d). Thus, repeated antigen exposure might not lead to higher SHM levels per Ig subclass, but rather to more sequential CSR. This would suggest that sequential switching takes place not only in the course of one antigen response, but also in consecutive responses through the recruitment of IgG3 þ and IgG1 þ memory B cells (Figure 1c).3 It would therefore be interesting to study whether the Papua New Guinean individuals carry relatively more IgG2/4 than urban dwellers.
Are SHM levels then independent of the extent of antigen exposure? The observations in urban dwellers vs villagers and in children vs adults suggest that this is the case for healthy individuals.1 B-cell responses can also be driven by chronic inflammation. Indeed, patients with Sarcoidosis, a multi-system disorder involving abnormal collections of inflammatory cells (granulomas), show B-cell involvement with increased IgG2 usages and higher SHM levels per Ig subclass.8 It is therefore entirely possible that SHM levels are tightly regulated, also independent of sequential IgG, and that this mechanism is only disrupted in disease states. Advances in multi-color flow cytometry have enabled the phenotyping of separate memory B cells with distinct extents of antibody maturation. CD27-IgG þ B cells have lower degrees of proliferation, SHM and IgG2 usage than CD27 þ IgG þ B cells.3 These CD27 IgG þ B cells could therefore represent earlier products from an immune response than CD27 þ IgG þ B cells, fitting with the temporal model. However, the higher SHM levels in CD27 þ IgG þ B cells were not related to the increased IgG2 usage, as CD27 B cells carry fewer SHMs in each of the IgG subclasses than CD27 þ B cells.10 Thus, also in distinct antigen-experienced B-cell subsets SHM levels and sequential class switching seem uncoupled. In conclusion, the study by Jackson et al.1 fits with the temporal model of human IgG function. The temporal model is the first to describe how IgG subclasses might act together to provide a strong and ratelimiting immune response. Although SHM levels and sequential IgG class switching are not directly coupled in all types of antibody responses, the authors provide clear indications for a temporal effect of SHM and sequential Ig class switching. Still, in vivo
News and Commentary 646
a
V DJ
Cμ Cδ
Cγ3
Sμ
Sγ3
Cγ1
Ψε
Sγ1
Ψγ
Cα1
Cγ2
Sα1
b
Sγ2
Cγ4
Sγ4
Cε Sε
Cα2 Sα2
c germinal center germinal center T cell
IgG3/1+
naive B cell
T cell naive B cell IgG2/4+ IgG3+
IgG1+
IgG2+
IgG4+ IgG3 IgG4 10% 1%
IgG3 IgG4 5% 5%
d IgA2 22% 102
IgA1 78%
IgA2 46% 183
IgA1 54%
IgG2 22%
107
IgG1 68%
p < 0.01
p < 0.0001 child
IgG2 IgG1 33% 214 56%
adult
child
adult
Figure 1 Ig class switching and SHM levels in children and adults. (a) Schematic representation of the human IGH constant gene regions. (b) The temporal model of IgG class switching, where over the course of a germinal center response sequential switching towards IgG34IgG14IgG24IgG4 occurs with increasing levels of SHMs. (c) Model for sequential IgG switching in secondary germinal center responses, where primary IgG memory B cells re-enter a germinal center in a secondary response, undergo additional SHMs and switch to a more downstream IgG subclass.3 (d) IgA and IgG subclass distributions in transcripts derived with consensus primers in previous studies.5,6,8 Ig subclass distributions were compared between children and adults using the w2 test.
these effects will be blurred by secondary antigen encounters (Figure 1c), the diversity of antigens, anatomical locations of immune responses and the formation of diverse antigen-experienced B-cell subsets in humans. Furthermore, the diversity of antigens, anatomical locations of immune responses and the diversity of antigen-experienced human B-cell subsets can blur the effects of temporal CSR. To overcome these limitations, it would be important to design an analysis strategy for antigen-specific B cells. These could be isolated at several time points following primary and secondary vaccinations of healthy volunteers with a T-cell-dependent antigen (for example, rabies vaccine). Such analyses would provide new insights into the temporal and sequential nature of antibody responses and could advance our understanding of how humoral immunity is regulated by memory B cells, affinity maturation and Ig effector functions.
Immunology and Cell Biology
ACKNOWLEDGEMENTS I would like to thank M van der Burg, ED de Geus, C Grosserichter-Wagener, JJ Heeringa and BG de Jong for critical reading of the manuscript. This work was supported by the Sophia Children’s Hospital Fund (grant S698), and fellowships from the Erasmus University Rotterdam (EUR) and the Erasmus MC.
1 Jackson KJ, Wang Y, Collins AM. Human immunoglobulin classes and subclasses show variability in VDJ gene mutation levels. Immunol Cell Biol 2014; 92: 729–733. 2 Collins AM, Jackson KJ. A temporal model of human IgE and IgG antibody function. Front Immunol 2013; 4: 235. 3 Berkowska MA, Driessen GJ, Bikos V, GrosserichterWagener C, Stamatopoulos K, Cerutti A et al. Human memory B cells originate from three distinct germinal center-dependent and -independent maturation pathways. Blood 2011; 118: 2150–2158. 4 He B, Xu W, Santini PA, Polydorides AD, Chiu A, Estrella J et al. Intestinal bacteria trigger T cellindependent immunoglobulin A(2) class switching by inducing epithelial-cell secretion of the cytokine APRIL. Immunity 2007; 26: 812–826.
5 Berkowska MA, Heeringa JJ, Hajdarbegovic E, van der Burg M, Thio HB, van Hagen PM et al. Human IgE þ B cells are derived from T cell–dependent and T cell–independent pathways. J Allergy Clin Immunol (e-pub ahead of print 13 May 2014; doi:10.1016/ j.jaci.2014.03.036). 6 van Zelm MC, Bartol SJ, Driessen GJ, Mascart F, Reisli I, Franco JL et al. Human CD19 and CD40L deficiencies impair antibody selection and differentially affect somatic hypermutation. J Allergy Clin Immunol (e-pub ahead of print 10 January 2014; doi:10.1016/ j.jaci.2013.11.015 ). 7 Gitlin AD, Shulman Z, Nussenzweig MC. Clonal selection in the germinal centre by regulated proliferation and hypermutation. Nature 2014; 509: 637–640. 8 Kamphuis LS, van Zelm MC, Lam KH, Rimmelzwaan GF, Baarsma GS, Dik WA et al. Perigranuloma localization and abnormal maturation of B cells: emerging key players in sarcoidosis? Am J Respir Crit Care Med 2013; 187: 406–416. 9 Verstegen RH, Driessen GJ, Bartol SJW, van Noesel CJM, Boon L, van der Burg M et al. Defective B-cell memory in patients with Down syndrome. J Allergy Clin Immunol (in press). 10 Wu YC, Kipling D, Leong HS, Martin V, Ademokun AA, Dunn-Walters DK. High-throughput immunoglobulin repertoire analysis distinguishes between human IgM memory and switched memory B-cell populations. Blood 2010; 116: 1070–1078.
