- Email: [email protected]
Selection in the germinal center
Available online at www.sciencedirect.com
ScienceDirect Selection in the germinal center Angelica WY Lau1 and Robert Brink1,2 Germinal centers (GCs) are well known for their important role in shaping the secondary B cell repertoire to generate antibodies capable of binding with high-affinity and specificity to foreign antigens. Somatic hypermutation of the Ig variable region genes in GC B cells represents a highly efficient mechanism for generating new antibody variants with increased antigen affinity. To be effective, however, this process needs to be intimately linked with equally efficient processes that positively select highaffinity clones for perpetuation in the GC and, ultimately, for differentiation into plasma cell and memory B cell effector populations. Just as important is the need for mechanisms of negative selection that remove GC B cell clones with unwanted specificities, particularly those that have gained reactivity with self-components. Here, we discuss recent advances in our understanding of the various selective processes that occur within the GC and identify the major questions in this field that remain to be answered. Addresses 1 Immunology Division, Garvan Institute of Medical Research, 384 Victoria St, Darlinghurst, NSW 2010, Australia 2 St Vincent’s Clinical School, UNSW Australia, 390 Victoria St, Darlinghurst, NSW 2010, Australia Corresponding author: Brink, Robert ([email protected])
CD4+ T helper cells. B cell activation and differentiation, including Ig class-switching, is for the most part carried out autonomously by the collaborating B and T cells and can be effectively replicated in a tissue culture dish. However, B cell responses that proceed based solely on the specificities present in the primary repertoire are typically low-affinity, polyreactive and largely ineffective [1,2]. For this reason, higher vertebrates have evolved a powerful elaboration upon T-dependent B cell responses, in which large numbers of responding B cells coalesce into a complex and specialized physiological structure known as the germinal center (GC). GC B cells undergo a unique process of somatic hypermutation (SHM) of their Ig variable regions genes linked with ‘positive’ selection for rare clones that acquire (mutant) B cell antigen receptors (BCRs) with increased affinity for the antigen. Whilst effectively taken ‘offline’ to undergo this process of affinity maturation, some GC B cells are released after their differentiation into either PCs or MBCs to rejoin the pool of effector B-lineage cells (Figure 1). Although GC-independent PCs and MBCs are likely to play their own part in the evolution of immune responses and broadbased protection, the increased affinity and specificity of the GC-derived PC and MBC populations are vital to establishing effective long-term humoral immunity.
Current Opinion in Immunology 2020, 63:29–34 This review comes from a themed issue on Lymphocyte development and activation Edited by Claude-Agnes Reynaud and Stephen Hedrick
https://doi.org/10.1016/j.coi.2019.11.001 0952-7915/ã 2019 Elsevier Ltd. All rights reserved.
In this review, we will examine recent advances to our understanding of how selection in the GC is regulated. This includes not only the positive selection of highaffinity GC B cells but also the selection of GC clones for differentiation into PCs versus MBCs and the ‘negative’ selection of GC clones that acquire undesirable mutations in their BCRs. Whilst mention will be made here of the various cellular components of the GC, their functions, regulation and migratory properties, the reader is referred to several excellent recent articles for more detailed coverage of these topics [3–5].
Positive selection Introduction The fundamental modus operandi of the adaptive immune system is well established — rare, antigen-specific lymphocyte clones are selectively activated, undergo proliferative expansion and ultimately differentiate into effector populations geared to eliminate foreign antigens. In the case of B cells, antibody-secreting plasma cells (PCs) provide the key effector population whilst long-lived memory B cells (MBCs) act as an expanded and readily mobilized population that initiates rapid secondary responses. The generation of these effector populations typically depends on ongoing cognate collaboration between antigen-specific B cells and www.sciencedirect.com
The preferential retention of B cells that have acquired increased affinity for a foreign antigen is the central process around which the GC has evolved. Crucial to this is a persistent source of foreign antigen within the GC so that the constantly evolving BCRs expressed by GC B cells can be tested and re-tested for antigen-binding through iterative rounds of SHM. Most if not all foreign antigen in the GC is maintained in the form of immune complexes on the surface of follicular dendritic cells (FDCs) that reside within the GC light zone (LZ) (Figure 1). It is generally accepted that positive selection operates via the ability of high-affinity GC B cells to compete most successfully for access to this antigen store. Current Opinion in Immunology 2020, 63:29–34
30 Lymphocyte development and activation
Figure 1
B Cell Follicle Ab PC
MBC
Light Zone
Dark Zone IRF4 PC
BCR Low-aff
High-aff
TFH
SHM
Ag
T Zone
FDC High-aff BCR Damage
Self-rx
Low-aff
?
High-aff
TFH
Low-aff
?
Apoptosis Current Opinion in Immunology
Selection, differentiation and death of germinal center B cells. Germinal centers (GCs) form within the B cell follicles of secondary lymphoid tissues. Antigen (Ag) is held on the surface of light zone (LZ)-resident follicular dendritic cells (FDCs) in the form of immune complexes. GC B cells with high-affinity (High-aff.) for antigen preferentially access antigen and receive stimulatory signals from B cell antigen receptor (BCR) engagement with antigen contact and from cognate T follicular helper (TFH) cells following antigen presentation with class II MHC. Delivery of a particularly strong stimulus upregulates IRF4 and initiates differentiation into plasma cells (PCs) which migrate through the dark zone (DZ) and out of the GC where they secrete antibody (Ab). Otherwise, stimulated B cells undergo positive selection whereby they migrate into the DZ, proliferate and undergo somatic hypermutation (SHM) of their Ig variable region genes. Clones that retain or increase their affinity as a result of SHM return to the LZ and can re-engage with FDC-displayed antigen to undergo further rounds of positive selection. Low affinity (Low-aff.) clones and any high affinity clones that do not encounter antigen undergo apoptosis following their return to the LZ. Clones acquiring mutations that result in the damage or loss of their BCR fail to return to the LZ and undergo apoptosis in the DZ. Clones that acquire self-reactive (Self-rx.) BCRs as a result of SHM are purged from the GC. Whether they undergo apoptosis and where this occurs remains unknown. LZ B cells with lower antigen affinity are favoured to undergo differentiation into memory B cells (MBCs), potentially with survival signals provided by TFH cells to protect them from apoptosis in the LZ.
In this way, high-affinity GC B cells preferentially receive pro-survival and mitogenic stimuli through a combination of inputs delivered via contact with the antigen itself and through cognate interactions with the specialized CD4+ T helper cells that reside within the LZ, T follicular helper (TFH) cells [6] (Figure 1). This results in the migration of the activated high-affinity GC B cell to the dark zone (DZ) of the GC, where the clone subsequently undergoes both cell division and SHM. Depending on the impact on the BCR of any mutations introduced into the Ig variable region genes, the daughter cells of the clone originally Current Opinion in Immunology 2020, 63:29–34
dispatched to the DZ can return to the LZ where their new BCR can again be tested for binding to FDC-displayed antigen. Clones that have maintained or even increased their affinity can again compete successfully for antigen access and undergo further rounds of SHM and selection (Figure 1). The exact nature of the signals that drive positive selection has been difficult to define. This is in large part because the two major inputs that likely trigger a selection-advantage for high-affinity GC B cells — that is, www.sciencedirect.com
Life, death and selection in the Germinal Center Lau and Brink 31
signals delivered directly upon antigen engagement and signals delivered by TFH cells after antigen presentation — both depend on the binding of their BCR to FDCdisplayed antigen (Figure 1). Since TFH cell help is unequivocally required for GC B cells to survive, increased antigen presentation and, therefore, preferential access to TFH cell help could theoretically be all that is required to drive the positive selection of high-affinity GC clones. Indeed, it is clear that provision of large amounts of TFH cell help is sufficient to drive low-affinity LZ GC B cells to the DZ [7]. Moreover, increasing the potency of TFH cell help by removing a negative regulatory circuit (mediated by the interaction of the inhibitory TNF receptor superfamily molecule HVEM on GC B cells with its TFH cell-derived ligand BTLA) results in increased competitiveness of low-affinity against highaffinity GC B cells [8]. Arguing against a mechanism based solely on provision of increased TFH cell help, a recent study demonstrated that GC B cells expressing half the normal levels of MHC class II (MHC2) undergo positive selection equally as well as wild-type B cells in the same GC microenvironment [9]. Ultimately, answering the question of whether differential presentation of antigen does drive positive selection may require direct demonstration of a significant difference in the density of peptide-MHC2 complexes on high versus lowaffinity B cells in an unmanipulated GC response. But since as little as a single peptide-MHC2 molecule is capable of mediating a productive CD4+ T cell interaction [10], this remains a significant challenge. Several recent studies have highlighted the fact that BCRs expressed by GC B cells signal poorly compared to those on naı¨ve B cells and possess a higher activation threshold in relation to antigen affinity [11,12]. Whilst this may at least in part be due to negative feedback loops elicited upon triggering of the BCR on GC B cells [13], most proximal BCR-signaling events are also significantly attenuated in GC B cells, with the apparent exception of PI3K-dependent and AKT-dependent phosphorylation of the transcription factor Foxo1 [11]. Although clearly not sufficient to drive positive selection in isolation, the ability of the BCRs on GC B cells to signal indicates that they could nevertheless play a key role in this process. Indeed, a recent study indicated that antigen-independent cross-linking of the BCR augments GC B cell selection under conditions of limiting TFH cell help [14]. This is consistent with the findings of Luo et al. [11] who showed that signals through the BCR and CD40 (the key receptor for the reception of TFH cell help) both complement and synergize with each other to activate the key pathways required for migration and proliferation in the DZ (PI3K, NF-kB, c-Myc). Whilst these data obtained from ex vivo stimulated GC B cells point to a ‘positive selection’ mechanism that is underpinned by collaborative BCR-linked and TFH cellderived signals, definitive demonstration of this awaits www.sciencedirect.com
separation of these distinct signals in an ongoing GC response in vivo. Whatever the relative contribution of these inputs, recent studies point to the strong activation of the PI3K-driven mTORC1 complex and the c-Myc transcription factor as being key to GC B cells undergoing the multiple rounds of replication in the DZ required to drive ongoing positive selection [15–17].
Selection for PC differentiation In addition to positive selection for retention in the GC, B cells can also be selected to exit this structure and enter the peripheral lymphocyte pool following differentiation into either PCs or MBCs (Figure 1). We recently reviewed this area in detail for this journal [18] and so briefly discuss some new insights into the regulation of PC differentiation below. It is notable that the PC differentiation of GC B cells shares three major properties with positive selection of GC B cells. First, it is tightly linked to antigen affinity, with only those GC B cells with high-affinity for antigen selected to undergo PC differentiation [19] (Figure 1). Second, the signals that initiate PC differentiation are delivered in the LZ of the GC [20,21]. Third, GC B cells undergoing PC differentiation migrate into the DZ from the LZ [20,22]. In contrast to positive selection, however, differentiating PCs do not return to the LZ but instead transit out of the GC via the DZ:T zone boundary (Figure 1) [20,22]. Recent work has focused on the signals that trigger PC differentiation among high-affinity LZ B cells. Interestingly, signals directly associated with antigen-binding can initiate PC-differentiation independent of TFH cell input [20]. However, it is also possible for strong TFH cell help to make a key contribution to this process [21]. Similar to proposed mechanisms for initiating positive selection [11], signals derived from antigen engagement and TFH cells may synergize to trigger PC-differentiation of high-affinity GC B cells. Once PC-differentiation is initiated, TFH cell help becomes essential for the completion of this process, including migration into and through the DZ [20]. Interestingly, signals delivered by TFH cell-derived CD40 ligand appear to be redundant in these later stages of PC differentiation [20]. Instead, TFH cell-derived cytokines such as IL-21 may play the dominant role at this point, potentially in conjunction with the PC survival factor APRIL synthesized by fibroblastic reticular cells located at the DZ:T zone interface where PCs exit the GC [22]. It is clear that high-affinity LZ B cells stimulated via antigen-engagement and TFH cell help can undergo either positive selection or PC differentiation (Figure 1). A major question is, therefore, how is the choice between these two response pathways determined? Since the frequency of high-affinity LZ B cells Current Opinion in Immunology 2020, 63:29–34
32 Lymphocyte development and activation
In contrast to PC differentiation, the differentiation of MBCs derives mostly from GC precursors that do not have high-affinity for the antigen [25,26]. Moreover, differentiating MBCs do not adopt a DZ phenotype but seem to pass directly out of the LZ and into the surrounding B cell follicle (Figure 1). Whilst detailed analysis of MBC production from the GC is beyond the scope of this article, two excellent recent reviews in this area are recommended to the reader [27,28].
crippled the expression of their BCR [29]. In a separate study, Stewart et al. also found that GC B cells that had lost BCR expression following SHM were concentrated in the DZ and destined to undergo apoptosis [30]. They also showed that these cells were incapable of returning to the LZ, even when apoptosis was curtailed by expression of a Bcl2 transgene [30]. Indeed our unpublished data have seen the same accumulation of BCR GC B cells in the DZ when they lack expression of the pro-apoptotic BH3-only protein Bim (Figure 2). Stewart et al. concluded that GC B cells replace their BCR with one encoded by their freshly mutated Ig variable region genes before they move back to the LZ. In addition, failure to express a functional BCR at this point prevents this migration as well as triggering apoptosis [30] (Figure 1). Consistent with this idea, the GC B cells identified by Mayer et al. that were undergoing apoptosis in the LZ did not possess damaged BCRs [29]. Interestingly, these BCR-expressing apoptotic LZ B cells consisted of clones with both high-affinity and low-affinity for antigen [29]. It appears, therefore, that the B cells that die in the LZ are those that have failed to encounter FDC-displayed antigen and undergo positive selection either because they fail to compete (low-affinity) or simply for stochastic reasons (high-affinity) (Figure 2).
Figure 2
IgM+
Negative selection
Two recent studies have focused on the identification and characterization of the B cells that undergo apoptosis in the GC. Mayer et al. developed an in vivo reporter of apoptosis which allowed them to calculate that up to half of the B cells in the GC undergo apoptosis every 6 hours [29]. Whilst they found that LZ and DZ GC B cells undergo apoptosis at largely similar rates, the nature of the cells dying in each compartment was found to be quite different. In particular, most of the cells undergoing apoptosis in the DZ had acquired somatic mutations that Current Opinion in Immunology 2020, 63:29–34
IgG+
2%
IgG+
11%
IgG
IgG+
BCRCXCR4
Negative selection in the context of the GC is often viewed as the flip-side of positive selection — that is, GC B cells that return to the LZ expressing a BCR that has not increased or even lowered its affinity for antigen fail to be positively selected and so die ‘by neglect’. Whilst this form of negative selection exists, it represents only part of the story. Thus, clones that acquire somatic mutations that either damage or remove their BCR (e.g. via the introduction of a stop codon) or result in the expression of a BCR with reactivity against a self-antigen must also be removed during the GC response.
Bim-deficient
WT IgM+
IgM
undergoing PC differentiation at any one time is typically low (<1%) [20], it is possible that only those cells that receive the strongest or most sustained stimulus can move past a threshold required to trigger PC differentiation [21]. In a fascinating recent study, Li et al. showed that removal of the homologous E3 ubiquitin ligases Cbl and Cbl-b from GC B cells resulted in the failure of stringent positive selection, with most high-affinity GC B cells instead committing to PC differentiation [23]. It appears, therefore, that the absence of these molecules dramatically lowers the threshold above which PC differentiation is triggered in the GC. The authors also showed that Cbl/Cbl-b normally target the transcription factor IRF4 for degradation and that strong BCR/CD40 signaling in GC B cells decreases Cbl/Cbl-b expression, in turn increasing IRF4 levels [23]. Since high level IRF4 expression promotes PC differentiation in the GC [24], these data support a model where only the delivery of a strong stimulus to high-affinity LZ B cells is sufficient to achieve the downregulation of Cbl/Cbl-b activity required to lift IRF4 levels above the threshold level required to initiate PC differentiation (Figure 1).
93
54
DZ
DZ
3
LZ
41
LZ
CD86 Current Opinion in Immunology
Dying GC B cells that have lost BCR expression accumulate in the DZ. Flow cytometric analysis of GC B cells derived from wild-type versus Bcl2l11 / (Bim-deficient) SWHEL (anti-hen egg lysozyme = HEL) B cells on day 12 of their responses to antigen (HEL3X-SRBC). Cells lacking a BCR (IgM , IgG ) are rare in wild-type GCs but accumulate when responding B cells lack the pro-apoptotic BH3-only protein Bim (top panels). Unlike the predominant BCRexpressing population (IgG+), cells lacking a BCR reside almost exclusively in the CXCR4hiCD86lo dark zone (DZ) compartment (unpublished data), highlighting their inability to return to the light zone (LZ) following SHM-mediated loss of BCR expression in the DZ. Unpublished data from the authors. www.sciencedirect.com
Life, death and selection in the Germinal Center Lau and Brink 33
One of the remaining puzzles of GC biology is how those B cells that acquire self-reactive BCRs as a result of SHM are controlled. Whilst there are many theories of how these cells are disposed of [31], it is generally assumed that at least some undergo rapid apoptosis upon encounter with self-antigen. Since such cells likely arise only infrequently under normal circumstances, self-reactive clones were not detectable in analyses of apoptotic GC B cells [29] and so could not, therefore, be seen to be dying preferentially in the LZ versus the DZ (Figure 1). Nevertheless, it is apparent that GC B cells that have a significant level of self-reactivity can survive long enough in the GC to acquire somatic mutations that remove their self-reactivity as long as they can maintain or increase their affinity for foreign antigen [32]. How self-reactive GC B cells are dealt with is likely to vary according to the strength of the interaction with the autoantigen as well as its location [33]. Identifying how these mechanisms operate and how they fail in the case of autoantibody production and autoimmune disease remains one of the significant challenges in the field.
Conflicts of interest statement Nothing declared.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of outstanding interest
Showed that reduced antigen presentation on MHCII haploinsufficient B cells impairs entry into but not the competitiveness within GCs. 10. Huang J, Brameshuber M, Zeng X, Xie J, Li QJ, Chien YH, Valitutti S, Davis MM: A single peptide-major histocompatibility complex ligand triggers digital cytokine secretion in CD4(+) T cells. Immunity 2013, 39:846-857. 11. Luo W, Weisel F, Shlomchik MJ: B cell receptor and CD40 signaling are rewired for synergistic induction of the c-Myc transcription factor in germinal center B cells. Immunity 2018, 48:313-326 e315 Detailed analysis of signaling via the two key receptors that control GC B cell fate. Experiments are carried out ex vivo but provide many leads to be followed up in vivo. 12. Kwak K, Quizon N, Sohn H, Saniee A, Manzella-Lapeira J, Holla P, Brzostowski J, Lu J, Xie H, Xu C et al.: Intrinsic properties of human germinal center B cells set antigen affinity thresholds. Sci Immunol 2018, 3 eaau6598. 13. Luo W, Hawse W, Conter L, Trivedi N, Weisel F, Wikenheiser D, Cattley RT, Shlomchik MJ: The AKT kinase signaling network is rewired by PTEN to control proximal BCR signaling in germinal center B cells. Nat Immunol 2019, 20:736-746. 14. Turner JS, Ke F, Grigorova IL: B cell receptor crosslinking augments germinal center B cell selection when t cell help is limiting. Cell Rep 2018, 25:1395-1403 e1394. 15. Ersching J, Efeyan A, Mesin L, Jacobsen JT, Pasqual G, Grabiner BC, Dominguez-Sola D, Sabatini DM, Victora GD: Germinal center selection and affinity maturation require dynamic regulation of mTORC1 kinase. Immunity 2017, 46:1045-1058.e1046. 16. Finkin S, Hartweger H, Oliveira TY, Kara EE, Nussenzweig MC: Protein amounts of the MYC transcription factor determine germinal center B cell division capacity. Immunity 2019, 51:324336 e325. 17. Heinzel S, Binh Giang T, Kan A, Marchingo JM, Lye BK, Corcoran LM, Hodgkin PD: A Myc-dependent division timer complements a cell-death timer to regulate T cell and B cell responses. Nat Immunol 2016, 18:96.
1.
Dent AL, Shaffer AL, Yu X, Allman D, Staudt LM: Control of inflammation, cytokine expression, and germinal center formation by BCL-6. Science 1997, 276:589.
18. Suan D, Sundling C, Brink R: Plasma cell and memory B cell differentiation from the germinal center. Curr Opin Immunol 2017, 45:97-102.
2.
Toyama H, Okada S, Hatano M, Takahashi Y, Takeda N, Ichii H, Takemori T, Kuroda Y, Tokuhisa T: Memory B cells without somatic hypermutation are generated from Bcl6-deficient B cells. Immunity 2002, 17:329-339.
19. Phan TG, Paus D, Chan TD, Turner ML, Nutt SL, Basten A, Brink R: High affinity germinal center B cells are actively selected into the plasma cell compartment. J Exp Med 2006, 203:2419-2424.
3.
Victora GD, Nussenzweig MC: Germinal centers. Annu Rev Immunol 2012, 30:429-457.
4.
Luo W, Hawse W, Conter L, Trivedi N, Weisel F, Wikenheiser D, Cattley RT, Shlomchik MJ: The AKT kinase signaling network is rewired by PTEN to control proximal BCR signaling in germinal center B cells. Nat Immunol 2019, 20:736-746.
5.
Shlomchik MJ, Luo W, Weisel F: Linking signaling and selection in the germinal center. Immunol Rev 2019, 288:49-63.
6.
Crotty S: T follicular helper cell biology: a decade of discovery and diseases. Immunity 2019, 50:1132-1148.
7.
Victora GD, Schwickert TA, Fooksman DR, Kamphorst AO, MeyerHermann M, Dustin ML, Nussenzweig MC: Germinal center dynamics revealed by multiphoton microscopy with a photoactivatable fluorescent reporter. Cell 2010, 143:592-605.
8.
Mintz MA, Felce JH, Chou MY, Mayya V, Xu Y, Shui JW, An J, Li Z, Marson A, Okada T et al.: The HVEM-BTLA axis restrains T cell help to germinal center B cells and functions as a cell-extrinsic suppressor in lymphomagenesis. Immunity 2019, 51:310-323 e317 Showed that the engagement of BTLA ligand with its receptor HVEM, normally expressed at low levels on GC B cells and a frequent target of mutation in GC-derived lymphomas, increases competitiveness of low affinity GC B cells during positive selection.
9.
Yeh C-H, Nojima T, Kuraoka M, Kelsoe G: Germinal center entry not selection of B cells is controlled by peptide-MHCII complex density. Nat Commun 2018, 9:928
www.sciencedirect.com
20. Kra¨utler NJ, Suan D, Butt D, Bourne K, Hermes JR, Chan TD, Sundling C, Kaplan W, Schofield P, Jackson J et al.: Differentiation of germinal center B cells into plasma cells is initiated by high-affinity antigen and completed by Tfh cells. J Exp Med 2017, 214:1259 Demonstrates that initiation of differentiation of high affinity GC B cells into plasma cells can be specifically dependent on antigen encounter. 21. Ise W, Fujii K, Shiroguchi K, Ito A, Kometani K, Takeda K, Kawakami E, Yamashita K, Suzuki K, Okada T et al.: T follicular helper cell-germinal center B cell interaction strength regulates entry into plasma cell or recycling germinal center cell fate. Immunity 2018, 48:702-715 e704. 22. Zhang Y, Tech L, George LA, Acs A, Durrett RE, Hess H, Walker LSK, Tarlinton DM, Fletcher AL, Hauser AE et al.: Plasma cell output from germinal centers is regulated by signals from Tfh and stromal cells. J Exp Med 2018, 215:1227 Identifies the importance of stromal cell factors in supporting the differentiation of GC-derived plasma cells. 23. Li X, Gadzinsky A, Gong L, Tong H, Calderon V, Li Y, Kitamura D, Klein U, Langdon WY, Hou F et al.: Cbl ubiquitin ligases control B cell exit from the germinal-center reaction. Immunity 2018, 48:530-541 e536 Fascinating analysis showing how the Cbl ubquitin ligases play a key role in GC B cells differentiation and affinity maturation by regulating expression of the transcription factor IRF4. 24. Ochiai K, Maienschein-Cline M, Simonetti G, Chen J, Rosenthal R, Brink R, Chong Anita S, Klein U, Dinner Aaron R, Singh H et al.: Transcriptional regulation of germinal center B and plasma Current Opinion in Immunology 2020, 63:29–34
34 Lymphocyte development and activation
cell fates by dynamical control of IRF4. Immunity 2013, 38:918929.
showing a fundamental difference between those that dies in the LZ versus the DZ.
25. Shinnakasu R, Inoue T, Kometani K, Moriyama S, Adachi Y, Nakayama M, Takahashi Y, Fukuyama H, Okada T, Kurosaki T: Regulated selection of germinal-center cells into the memory B cell compartment. Nat Immunol 2016, 17:861.
30. Stewart I, Radtke D, Phillips B, McGowan SJ, Bannard O: Germinal center B cells replace their antigen receptors in dark zones and fail light zone entry when immunoglobulin gene mutations are damaging. Immunity 2018, 49:477-489 e477 Showed that GC B cells acquiring damaging BCR mutation genes during SHM cannot cycle back to the LZ and undergo apoptosis in the DZ.
26. Suan D, Kra¨utler NJ, Maag JLV, Butt D, Bourne K, Hermes JR, Avery DT, Young C, Statham A, Elliott M et al.: CCR6 defines memory B cell precursors in mouse and human germinal centers, revealing light-zone location and predominant low antigen affinity. Immunity 2017, 47:1142-1153 e1144. 27. Inoue T, Moran I, Shinnakasu R, Phan TG, Kurosaki T: Generation of memory B cells and their reactivation. Immunol Rev 2018, 283:138-149. 28. Cyster JG, Allen CDC: B cell responses: cell interaction dynamics and decisions. Cell 2019, 177:524-540. 29. Mayer CT, Gazumyan A, Kara EE, Gitlin AD, Golijanin J, Viant C, Pai J, Oliveira TY, Wang Q, Escolano A et al.: The microanatomic segregation of selection by apoptosis in the germinal center. Science 2017, 358 eaao2602 Developed a unique in vivo reporter of apoptosis and carried out a detailed analysis of the GC B cells undergoing apoptosis in the GC,
Current Opinion in Immunology 2020, 63:29–34
31. Brink R, Phan TG: Self-reactive B cells in the germinal center reaction. Annu Rev Immunol 2018, 36:339-357. 32. Burnett DL, Langley DB, Schofield P, Hermes JR, Chan TD, Jackson J, Bourne K, Reed JH, Patterson K, Porebski BT et al.: Germinal center antibody mutation trajectories are determined by rapid self/foreign discrimination. Science 2018, 360:223 Detailed analysis of affinity mutation pathways that can be taken by GC B cells to simultaneously reduce self-reactivity and increase affinity for foreign antigen. 33. Chan TD, Wood K, Hermes JR, Butt D, Jolly Christopher J, Basten A, Brink R: Elimination of germinal-center-derived self-reactive B cells is governed by the location and concentration of selfantigen. Immunity 2012, 37:893-904.
www.sciencedirect.com
ScienceDirect Selection in the germinal center Angelica WY Lau1 and Robert Brink1,2 Germinal centers (GCs) are well known for their important role in shaping the secondary B cell repertoire to generate antibodies capable of binding with high-affinity and specificity to foreign antigens. Somatic hypermutation of the Ig variable region genes in GC B cells represents a highly efficient mechanism for generating new antibody variants with increased antigen affinity. To be effective, however, this process needs to be intimately linked with equally efficient processes that positively select highaffinity clones for perpetuation in the GC and, ultimately, for differentiation into plasma cell and memory B cell effector populations. Just as important is the need for mechanisms of negative selection that remove GC B cell clones with unwanted specificities, particularly those that have gained reactivity with self-components. Here, we discuss recent advances in our understanding of the various selective processes that occur within the GC and identify the major questions in this field that remain to be answered. Addresses 1 Immunology Division, Garvan Institute of Medical Research, 384 Victoria St, Darlinghurst, NSW 2010, Australia 2 St Vincent’s Clinical School, UNSW Australia, 390 Victoria St, Darlinghurst, NSW 2010, Australia Corresponding author: Brink, Robert ([email protected])
CD4+ T helper cells. B cell activation and differentiation, including Ig class-switching, is for the most part carried out autonomously by the collaborating B and T cells and can be effectively replicated in a tissue culture dish. However, B cell responses that proceed based solely on the specificities present in the primary repertoire are typically low-affinity, polyreactive and largely ineffective [1,2]. For this reason, higher vertebrates have evolved a powerful elaboration upon T-dependent B cell responses, in which large numbers of responding B cells coalesce into a complex and specialized physiological structure known as the germinal center (GC). GC B cells undergo a unique process of somatic hypermutation (SHM) of their Ig variable regions genes linked with ‘positive’ selection for rare clones that acquire (mutant) B cell antigen receptors (BCRs) with increased affinity for the antigen. Whilst effectively taken ‘offline’ to undergo this process of affinity maturation, some GC B cells are released after their differentiation into either PCs or MBCs to rejoin the pool of effector B-lineage cells (Figure 1). Although GC-independent PCs and MBCs are likely to play their own part in the evolution of immune responses and broadbased protection, the increased affinity and specificity of the GC-derived PC and MBC populations are vital to establishing effective long-term humoral immunity.
Current Opinion in Immunology 2020, 63:29–34 This review comes from a themed issue on Lymphocyte development and activation Edited by Claude-Agnes Reynaud and Stephen Hedrick
https://doi.org/10.1016/j.coi.2019.11.001 0952-7915/ã 2019 Elsevier Ltd. All rights reserved.
In this review, we will examine recent advances to our understanding of how selection in the GC is regulated. This includes not only the positive selection of highaffinity GC B cells but also the selection of GC clones for differentiation into PCs versus MBCs and the ‘negative’ selection of GC clones that acquire undesirable mutations in their BCRs. Whilst mention will be made here of the various cellular components of the GC, their functions, regulation and migratory properties, the reader is referred to several excellent recent articles for more detailed coverage of these topics [3–5].
Positive selection Introduction The fundamental modus operandi of the adaptive immune system is well established — rare, antigen-specific lymphocyte clones are selectively activated, undergo proliferative expansion and ultimately differentiate into effector populations geared to eliminate foreign antigens. In the case of B cells, antibody-secreting plasma cells (PCs) provide the key effector population whilst long-lived memory B cells (MBCs) act as an expanded and readily mobilized population that initiates rapid secondary responses. The generation of these effector populations typically depends on ongoing cognate collaboration between antigen-specific B cells and www.sciencedirect.com
The preferential retention of B cells that have acquired increased affinity for a foreign antigen is the central process around which the GC has evolved. Crucial to this is a persistent source of foreign antigen within the GC so that the constantly evolving BCRs expressed by GC B cells can be tested and re-tested for antigen-binding through iterative rounds of SHM. Most if not all foreign antigen in the GC is maintained in the form of immune complexes on the surface of follicular dendritic cells (FDCs) that reside within the GC light zone (LZ) (Figure 1). It is generally accepted that positive selection operates via the ability of high-affinity GC B cells to compete most successfully for access to this antigen store. Current Opinion in Immunology 2020, 63:29–34
30 Lymphocyte development and activation
Figure 1
B Cell Follicle Ab PC
MBC
Light Zone
Dark Zone IRF4 PC
BCR Low-aff
High-aff
TFH
SHM
Ag
T Zone
FDC High-aff BCR Damage
Self-rx
Low-aff
?
High-aff
TFH
Low-aff
?
Apoptosis Current Opinion in Immunology
Selection, differentiation and death of germinal center B cells. Germinal centers (GCs) form within the B cell follicles of secondary lymphoid tissues. Antigen (Ag) is held on the surface of light zone (LZ)-resident follicular dendritic cells (FDCs) in the form of immune complexes. GC B cells with high-affinity (High-aff.) for antigen preferentially access antigen and receive stimulatory signals from B cell antigen receptor (BCR) engagement with antigen contact and from cognate T follicular helper (TFH) cells following antigen presentation with class II MHC. Delivery of a particularly strong stimulus upregulates IRF4 and initiates differentiation into plasma cells (PCs) which migrate through the dark zone (DZ) and out of the GC where they secrete antibody (Ab). Otherwise, stimulated B cells undergo positive selection whereby they migrate into the DZ, proliferate and undergo somatic hypermutation (SHM) of their Ig variable region genes. Clones that retain or increase their affinity as a result of SHM return to the LZ and can re-engage with FDC-displayed antigen to undergo further rounds of positive selection. Low affinity (Low-aff.) clones and any high affinity clones that do not encounter antigen undergo apoptosis following their return to the LZ. Clones acquiring mutations that result in the damage or loss of their BCR fail to return to the LZ and undergo apoptosis in the DZ. Clones that acquire self-reactive (Self-rx.) BCRs as a result of SHM are purged from the GC. Whether they undergo apoptosis and where this occurs remains unknown. LZ B cells with lower antigen affinity are favoured to undergo differentiation into memory B cells (MBCs), potentially with survival signals provided by TFH cells to protect them from apoptosis in the LZ.
In this way, high-affinity GC B cells preferentially receive pro-survival and mitogenic stimuli through a combination of inputs delivered via contact with the antigen itself and through cognate interactions with the specialized CD4+ T helper cells that reside within the LZ, T follicular helper (TFH) cells [6] (Figure 1). This results in the migration of the activated high-affinity GC B cell to the dark zone (DZ) of the GC, where the clone subsequently undergoes both cell division and SHM. Depending on the impact on the BCR of any mutations introduced into the Ig variable region genes, the daughter cells of the clone originally Current Opinion in Immunology 2020, 63:29–34
dispatched to the DZ can return to the LZ where their new BCR can again be tested for binding to FDC-displayed antigen. Clones that have maintained or even increased their affinity can again compete successfully for antigen access and undergo further rounds of SHM and selection (Figure 1). The exact nature of the signals that drive positive selection has been difficult to define. This is in large part because the two major inputs that likely trigger a selection-advantage for high-affinity GC B cells — that is, www.sciencedirect.com
Life, death and selection in the Germinal Center Lau and Brink 31
signals delivered directly upon antigen engagement and signals delivered by TFH cells after antigen presentation — both depend on the binding of their BCR to FDCdisplayed antigen (Figure 1). Since TFH cell help is unequivocally required for GC B cells to survive, increased antigen presentation and, therefore, preferential access to TFH cell help could theoretically be all that is required to drive the positive selection of high-affinity GC clones. Indeed, it is clear that provision of large amounts of TFH cell help is sufficient to drive low-affinity LZ GC B cells to the DZ [7]. Moreover, increasing the potency of TFH cell help by removing a negative regulatory circuit (mediated by the interaction of the inhibitory TNF receptor superfamily molecule HVEM on GC B cells with its TFH cell-derived ligand BTLA) results in increased competitiveness of low-affinity against highaffinity GC B cells [8]. Arguing against a mechanism based solely on provision of increased TFH cell help, a recent study demonstrated that GC B cells expressing half the normal levels of MHC class II (MHC2) undergo positive selection equally as well as wild-type B cells in the same GC microenvironment [9]. Ultimately, answering the question of whether differential presentation of antigen does drive positive selection may require direct demonstration of a significant difference in the density of peptide-MHC2 complexes on high versus lowaffinity B cells in an unmanipulated GC response. But since as little as a single peptide-MHC2 molecule is capable of mediating a productive CD4+ T cell interaction [10], this remains a significant challenge. Several recent studies have highlighted the fact that BCRs expressed by GC B cells signal poorly compared to those on naı¨ve B cells and possess a higher activation threshold in relation to antigen affinity [11,12]. Whilst this may at least in part be due to negative feedback loops elicited upon triggering of the BCR on GC B cells [13], most proximal BCR-signaling events are also significantly attenuated in GC B cells, with the apparent exception of PI3K-dependent and AKT-dependent phosphorylation of the transcription factor Foxo1 [11]. Although clearly not sufficient to drive positive selection in isolation, the ability of the BCRs on GC B cells to signal indicates that they could nevertheless play a key role in this process. Indeed, a recent study indicated that antigen-independent cross-linking of the BCR augments GC B cell selection under conditions of limiting TFH cell help [14]. This is consistent with the findings of Luo et al. [11] who showed that signals through the BCR and CD40 (the key receptor for the reception of TFH cell help) both complement and synergize with each other to activate the key pathways required for migration and proliferation in the DZ (PI3K, NF-kB, c-Myc). Whilst these data obtained from ex vivo stimulated GC B cells point to a ‘positive selection’ mechanism that is underpinned by collaborative BCR-linked and TFH cellderived signals, definitive demonstration of this awaits www.sciencedirect.com
separation of these distinct signals in an ongoing GC response in vivo. Whatever the relative contribution of these inputs, recent studies point to the strong activation of the PI3K-driven mTORC1 complex and the c-Myc transcription factor as being key to GC B cells undergoing the multiple rounds of replication in the DZ required to drive ongoing positive selection [15–17].
Selection for PC differentiation In addition to positive selection for retention in the GC, B cells can also be selected to exit this structure and enter the peripheral lymphocyte pool following differentiation into either PCs or MBCs (Figure 1). We recently reviewed this area in detail for this journal [18] and so briefly discuss some new insights into the regulation of PC differentiation below. It is notable that the PC differentiation of GC B cells shares three major properties with positive selection of GC B cells. First, it is tightly linked to antigen affinity, with only those GC B cells with high-affinity for antigen selected to undergo PC differentiation [19] (Figure 1). Second, the signals that initiate PC differentiation are delivered in the LZ of the GC [20,21]. Third, GC B cells undergoing PC differentiation migrate into the DZ from the LZ [20,22]. In contrast to positive selection, however, differentiating PCs do not return to the LZ but instead transit out of the GC via the DZ:T zone boundary (Figure 1) [20,22]. Recent work has focused on the signals that trigger PC differentiation among high-affinity LZ B cells. Interestingly, signals directly associated with antigen-binding can initiate PC-differentiation independent of TFH cell input [20]. However, it is also possible for strong TFH cell help to make a key contribution to this process [21]. Similar to proposed mechanisms for initiating positive selection [11], signals derived from antigen engagement and TFH cells may synergize to trigger PC-differentiation of high-affinity GC B cells. Once PC-differentiation is initiated, TFH cell help becomes essential for the completion of this process, including migration into and through the DZ [20]. Interestingly, signals delivered by TFH cell-derived CD40 ligand appear to be redundant in these later stages of PC differentiation [20]. Instead, TFH cell-derived cytokines such as IL-21 may play the dominant role at this point, potentially in conjunction with the PC survival factor APRIL synthesized by fibroblastic reticular cells located at the DZ:T zone interface where PCs exit the GC [22]. It is clear that high-affinity LZ B cells stimulated via antigen-engagement and TFH cell help can undergo either positive selection or PC differentiation (Figure 1). A major question is, therefore, how is the choice between these two response pathways determined? Since the frequency of high-affinity LZ B cells Current Opinion in Immunology 2020, 63:29–34
32 Lymphocyte development and activation
In contrast to PC differentiation, the differentiation of MBCs derives mostly from GC precursors that do not have high-affinity for the antigen [25,26]. Moreover, differentiating MBCs do not adopt a DZ phenotype but seem to pass directly out of the LZ and into the surrounding B cell follicle (Figure 1). Whilst detailed analysis of MBC production from the GC is beyond the scope of this article, two excellent recent reviews in this area are recommended to the reader [27,28].
crippled the expression of their BCR [29]. In a separate study, Stewart et al. also found that GC B cells that had lost BCR expression following SHM were concentrated in the DZ and destined to undergo apoptosis [30]. They also showed that these cells were incapable of returning to the LZ, even when apoptosis was curtailed by expression of a Bcl2 transgene [30]. Indeed our unpublished data have seen the same accumulation of BCR GC B cells in the DZ when they lack expression of the pro-apoptotic BH3-only protein Bim (Figure 2). Stewart et al. concluded that GC B cells replace their BCR with one encoded by their freshly mutated Ig variable region genes before they move back to the LZ. In addition, failure to express a functional BCR at this point prevents this migration as well as triggering apoptosis [30] (Figure 1). Consistent with this idea, the GC B cells identified by Mayer et al. that were undergoing apoptosis in the LZ did not possess damaged BCRs [29]. Interestingly, these BCR-expressing apoptotic LZ B cells consisted of clones with both high-affinity and low-affinity for antigen [29]. It appears, therefore, that the B cells that die in the LZ are those that have failed to encounter FDC-displayed antigen and undergo positive selection either because they fail to compete (low-affinity) or simply for stochastic reasons (high-affinity) (Figure 2).
Figure 2
IgM+
Negative selection
Two recent studies have focused on the identification and characterization of the B cells that undergo apoptosis in the GC. Mayer et al. developed an in vivo reporter of apoptosis which allowed them to calculate that up to half of the B cells in the GC undergo apoptosis every 6 hours [29]. Whilst they found that LZ and DZ GC B cells undergo apoptosis at largely similar rates, the nature of the cells dying in each compartment was found to be quite different. In particular, most of the cells undergoing apoptosis in the DZ had acquired somatic mutations that Current Opinion in Immunology 2020, 63:29–34
IgG+
2%
IgG+
11%
IgG
IgG+
BCRCXCR4
Negative selection in the context of the GC is often viewed as the flip-side of positive selection — that is, GC B cells that return to the LZ expressing a BCR that has not increased or even lowered its affinity for antigen fail to be positively selected and so die ‘by neglect’. Whilst this form of negative selection exists, it represents only part of the story. Thus, clones that acquire somatic mutations that either damage or remove their BCR (e.g. via the introduction of a stop codon) or result in the expression of a BCR with reactivity against a self-antigen must also be removed during the GC response.
Bim-deficient
WT IgM+
IgM
undergoing PC differentiation at any one time is typically low (<1%) [20], it is possible that only those cells that receive the strongest or most sustained stimulus can move past a threshold required to trigger PC differentiation [21]. In a fascinating recent study, Li et al. showed that removal of the homologous E3 ubiquitin ligases Cbl and Cbl-b from GC B cells resulted in the failure of stringent positive selection, with most high-affinity GC B cells instead committing to PC differentiation [23]. It appears, therefore, that the absence of these molecules dramatically lowers the threshold above which PC differentiation is triggered in the GC. The authors also showed that Cbl/Cbl-b normally target the transcription factor IRF4 for degradation and that strong BCR/CD40 signaling in GC B cells decreases Cbl/Cbl-b expression, in turn increasing IRF4 levels [23]. Since high level IRF4 expression promotes PC differentiation in the GC [24], these data support a model where only the delivery of a strong stimulus to high-affinity LZ B cells is sufficient to achieve the downregulation of Cbl/Cbl-b activity required to lift IRF4 levels above the threshold level required to initiate PC differentiation (Figure 1).
93
54
DZ
DZ
3
LZ
41
LZ
CD86 Current Opinion in Immunology
Dying GC B cells that have lost BCR expression accumulate in the DZ. Flow cytometric analysis of GC B cells derived from wild-type versus Bcl2l11 / (Bim-deficient) SWHEL (anti-hen egg lysozyme = HEL) B cells on day 12 of their responses to antigen (HEL3X-SRBC). Cells lacking a BCR (IgM , IgG ) are rare in wild-type GCs but accumulate when responding B cells lack the pro-apoptotic BH3-only protein Bim (top panels). Unlike the predominant BCRexpressing population (IgG+), cells lacking a BCR reside almost exclusively in the CXCR4hiCD86lo dark zone (DZ) compartment (unpublished data), highlighting their inability to return to the light zone (LZ) following SHM-mediated loss of BCR expression in the DZ. Unpublished data from the authors. www.sciencedirect.com
Life, death and selection in the Germinal Center Lau and Brink 33
One of the remaining puzzles of GC biology is how those B cells that acquire self-reactive BCRs as a result of SHM are controlled. Whilst there are many theories of how these cells are disposed of [31], it is generally assumed that at least some undergo rapid apoptosis upon encounter with self-antigen. Since such cells likely arise only infrequently under normal circumstances, self-reactive clones were not detectable in analyses of apoptotic GC B cells [29] and so could not, therefore, be seen to be dying preferentially in the LZ versus the DZ (Figure 1). Nevertheless, it is apparent that GC B cells that have a significant level of self-reactivity can survive long enough in the GC to acquire somatic mutations that remove their self-reactivity as long as they can maintain or increase their affinity for foreign antigen [32]. How self-reactive GC B cells are dealt with is likely to vary according to the strength of the interaction with the autoantigen as well as its location [33]. Identifying how these mechanisms operate and how they fail in the case of autoantibody production and autoimmune disease remains one of the significant challenges in the field.
Conflicts of interest statement Nothing declared.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of outstanding interest
Showed that reduced antigen presentation on MHCII haploinsufficient B cells impairs entry into but not the competitiveness within GCs. 10. Huang J, Brameshuber M, Zeng X, Xie J, Li QJ, Chien YH, Valitutti S, Davis MM: A single peptide-major histocompatibility complex ligand triggers digital cytokine secretion in CD4(+) T cells. Immunity 2013, 39:846-857. 11. Luo W, Weisel F, Shlomchik MJ: B cell receptor and CD40 signaling are rewired for synergistic induction of the c-Myc transcription factor in germinal center B cells. Immunity 2018, 48:313-326 e315 Detailed analysis of signaling via the two key receptors that control GC B cell fate. Experiments are carried out ex vivo but provide many leads to be followed up in vivo. 12. Kwak K, Quizon N, Sohn H, Saniee A, Manzella-Lapeira J, Holla P, Brzostowski J, Lu J, Xie H, Xu C et al.: Intrinsic properties of human germinal center B cells set antigen affinity thresholds. Sci Immunol 2018, 3 eaau6598. 13. Luo W, Hawse W, Conter L, Trivedi N, Weisel F, Wikenheiser D, Cattley RT, Shlomchik MJ: The AKT kinase signaling network is rewired by PTEN to control proximal BCR signaling in germinal center B cells. Nat Immunol 2019, 20:736-746. 14. Turner JS, Ke F, Grigorova IL: B cell receptor crosslinking augments germinal center B cell selection when t cell help is limiting. Cell Rep 2018, 25:1395-1403 e1394. 15. Ersching J, Efeyan A, Mesin L, Jacobsen JT, Pasqual G, Grabiner BC, Dominguez-Sola D, Sabatini DM, Victora GD: Germinal center selection and affinity maturation require dynamic regulation of mTORC1 kinase. Immunity 2017, 46:1045-1058.e1046. 16. Finkin S, Hartweger H, Oliveira TY, Kara EE, Nussenzweig MC: Protein amounts of the MYC transcription factor determine germinal center B cell division capacity. Immunity 2019, 51:324336 e325. 17. Heinzel S, Binh Giang T, Kan A, Marchingo JM, Lye BK, Corcoran LM, Hodgkin PD: A Myc-dependent division timer complements a cell-death timer to regulate T cell and B cell responses. Nat Immunol 2016, 18:96.
1.
Dent AL, Shaffer AL, Yu X, Allman D, Staudt LM: Control of inflammation, cytokine expression, and germinal center formation by BCL-6. Science 1997, 276:589.
18. Suan D, Sundling C, Brink R: Plasma cell and memory B cell differentiation from the germinal center. Curr Opin Immunol 2017, 45:97-102.
2.
Toyama H, Okada S, Hatano M, Takahashi Y, Takeda N, Ichii H, Takemori T, Kuroda Y, Tokuhisa T: Memory B cells without somatic hypermutation are generated from Bcl6-deficient B cells. Immunity 2002, 17:329-339.
19. Phan TG, Paus D, Chan TD, Turner ML, Nutt SL, Basten A, Brink R: High affinity germinal center B cells are actively selected into the plasma cell compartment. J Exp Med 2006, 203:2419-2424.
3.
Victora GD, Nussenzweig MC: Germinal centers. Annu Rev Immunol 2012, 30:429-457.
4.
Luo W, Hawse W, Conter L, Trivedi N, Weisel F, Wikenheiser D, Cattley RT, Shlomchik MJ: The AKT kinase signaling network is rewired by PTEN to control proximal BCR signaling in germinal center B cells. Nat Immunol 2019, 20:736-746.
5.
Shlomchik MJ, Luo W, Weisel F: Linking signaling and selection in the germinal center. Immunol Rev 2019, 288:49-63.
6.
Crotty S: T follicular helper cell biology: a decade of discovery and diseases. Immunity 2019, 50:1132-1148.
7.
Victora GD, Schwickert TA, Fooksman DR, Kamphorst AO, MeyerHermann M, Dustin ML, Nussenzweig MC: Germinal center dynamics revealed by multiphoton microscopy with a photoactivatable fluorescent reporter. Cell 2010, 143:592-605.
8.
Mintz MA, Felce JH, Chou MY, Mayya V, Xu Y, Shui JW, An J, Li Z, Marson A, Okada T et al.: The HVEM-BTLA axis restrains T cell help to germinal center B cells and functions as a cell-extrinsic suppressor in lymphomagenesis. Immunity 2019, 51:310-323 e317 Showed that the engagement of BTLA ligand with its receptor HVEM, normally expressed at low levels on GC B cells and a frequent target of mutation in GC-derived lymphomas, increases competitiveness of low affinity GC B cells during positive selection.
9.
Yeh C-H, Nojima T, Kuraoka M, Kelsoe G: Germinal center entry not selection of B cells is controlled by peptide-MHCII complex density. Nat Commun 2018, 9:928
www.sciencedirect.com
20. Kra¨utler NJ, Suan D, Butt D, Bourne K, Hermes JR, Chan TD, Sundling C, Kaplan W, Schofield P, Jackson J et al.: Differentiation of germinal center B cells into plasma cells is initiated by high-affinity antigen and completed by Tfh cells. J Exp Med 2017, 214:1259 Demonstrates that initiation of differentiation of high affinity GC B cells into plasma cells can be specifically dependent on antigen encounter. 21. Ise W, Fujii K, Shiroguchi K, Ito A, Kometani K, Takeda K, Kawakami E, Yamashita K, Suzuki K, Okada T et al.: T follicular helper cell-germinal center B cell interaction strength regulates entry into plasma cell or recycling germinal center cell fate. Immunity 2018, 48:702-715 e704. 22. Zhang Y, Tech L, George LA, Acs A, Durrett RE, Hess H, Walker LSK, Tarlinton DM, Fletcher AL, Hauser AE et al.: Plasma cell output from germinal centers is regulated by signals from Tfh and stromal cells. J Exp Med 2018, 215:1227 Identifies the importance of stromal cell factors in supporting the differentiation of GC-derived plasma cells. 23. Li X, Gadzinsky A, Gong L, Tong H, Calderon V, Li Y, Kitamura D, Klein U, Langdon WY, Hou F et al.: Cbl ubiquitin ligases control B cell exit from the germinal-center reaction. Immunity 2018, 48:530-541 e536 Fascinating analysis showing how the Cbl ubquitin ligases play a key role in GC B cells differentiation and affinity maturation by regulating expression of the transcription factor IRF4. 24. Ochiai K, Maienschein-Cline M, Simonetti G, Chen J, Rosenthal R, Brink R, Chong Anita S, Klein U, Dinner Aaron R, Singh H et al.: Transcriptional regulation of germinal center B and plasma Current Opinion in Immunology 2020, 63:29–34
34 Lymphocyte development and activation
cell fates by dynamical control of IRF4. Immunity 2013, 38:918929.
showing a fundamental difference between those that dies in the LZ versus the DZ.
25. Shinnakasu R, Inoue T, Kometani K, Moriyama S, Adachi Y, Nakayama M, Takahashi Y, Fukuyama H, Okada T, Kurosaki T: Regulated selection of germinal-center cells into the memory B cell compartment. Nat Immunol 2016, 17:861.
30. Stewart I, Radtke D, Phillips B, McGowan SJ, Bannard O: Germinal center B cells replace their antigen receptors in dark zones and fail light zone entry when immunoglobulin gene mutations are damaging. Immunity 2018, 49:477-489 e477 Showed that GC B cells acquiring damaging BCR mutation genes during SHM cannot cycle back to the LZ and undergo apoptosis in the DZ.
26. Suan D, Kra¨utler NJ, Maag JLV, Butt D, Bourne K, Hermes JR, Avery DT, Young C, Statham A, Elliott M et al.: CCR6 defines memory B cell precursors in mouse and human germinal centers, revealing light-zone location and predominant low antigen affinity. Immunity 2017, 47:1142-1153 e1144. 27. Inoue T, Moran I, Shinnakasu R, Phan TG, Kurosaki T: Generation of memory B cells and their reactivation. Immunol Rev 2018, 283:138-149. 28. Cyster JG, Allen CDC: B cell responses: cell interaction dynamics and decisions. Cell 2019, 177:524-540. 29. Mayer CT, Gazumyan A, Kara EE, Gitlin AD, Golijanin J, Viant C, Pai J, Oliveira TY, Wang Q, Escolano A et al.: The microanatomic segregation of selection by apoptosis in the germinal center. Science 2017, 358 eaao2602 Developed a unique in vivo reporter of apoptosis and carried out a detailed analysis of the GC B cells undergoing apoptosis in the GC,
Current Opinion in Immunology 2020, 63:29–34
31. Brink R, Phan TG: Self-reactive B cells in the germinal center reaction. Annu Rev Immunol 2018, 36:339-357. 32. Burnett DL, Langley DB, Schofield P, Hermes JR, Chan TD, Jackson J, Bourne K, Reed JH, Patterson K, Porebski BT et al.: Germinal center antibody mutation trajectories are determined by rapid self/foreign discrimination. Science 2018, 360:223 Detailed analysis of affinity mutation pathways that can be taken by GC B cells to simultaneously reduce self-reactivity and increase affinity for foreign antigen. 33. Chan TD, Wood K, Hermes JR, Butt D, Jolly Christopher J, Basten A, Brink R: Elimination of germinal-center-derived self-reactive B cells is governed by the location and concentration of selfantigen. Immunity 2012, 37:893-904.
www.sciencedirect.com