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2876 Garnett Kelsoe Eur. J. Immunol. 2014. 44: 2876–2879DOI: 10.1002/eji.201445102
Curiouser and curiouser: The role(s) of AID expressionin self-tolerance
Garnett Kelsoe
Department of Immunology and Human Vaccine Institute, Duke University, Durham, NC, USA
Aicda is crucial for antibody diversification by mediating Ig class-switch recombination,V(D)J hypermutation (SHM) and, in some species, gene conversion. Recently, evidencehas accumulated to show that Aicda is expressed during B-cell development and thatthis expression in some unknown way, mediates tolerance in immature and transitionalB cells. In this issue of the European Journal of Immunology, Umiker et al. [Eur. J. Immunol.2014. 44: 3093–3108] show that enforced expression of Aicda during early B-cell devel-opment is associated with self-tolerance. Curiously, constitutive Aicda expression thatbegins early in B cells suppresses the generation of autoreactive IgM but promotes theexpression of self-reactive IgG. In contrast, when Aicda is activated later in B-cell develop-ment, self-reactive IgM is abundant but IgG is not. These observations suggest pathwaysfor self-tolerance that have been little explored.
Keywords: Autoimmunity � Antibodies � B cells � B cell development � Immunopathology
See accompanying Article by Umiker et al.
B lymphocytes exhibit a unique degree of genomic plasticity; notonly are B-cell antigen receptors (BCRs) generated by genomicrearrangements of V, D, and J gene segments, but assembledBCR genes are also modified by class-switch recombination (CSR)and somatic hypermutation (SHM). Whereas V(D)J rearrange-ment occurs during B lymphopoiesis, SHM and CSR are largelyconfined to secondary lymphoid tissues where activated germinalcenter (GC) B cells express high levels of AID, the product of Aicda[1, 2]; however, in amphibians [3], birds [4], and some mam-mals [5, 6], however, AID expression and activity is unequivocallypresent in distinct subsets of developing B cells as well.
AID expression, SHM and CSR in mice and humans can also befound in B cells outside the GC, e.g. in autoimmune plasmablasts[7] and human gut B cells [8]. Type I hyper IgM syndrome patientscannot form GC, but nonetheless support populations of B cellscarrying mutated immunoglobulin (Ig) genes [9]. In addition, lowlevels of AID message, and low levels of CSR, and SHM have
Correspondence: Dr. Garnett Kelsoee-mail: [email protected]
been recovered from detected in mouse and human immatureand transitional 1 B cells [10–14]; this, and evidence for AIDexpression and activity in human fetal liver cells [14–16] suggestthat AID expression during B-cell development may be a commonvertebrate trait [4–6]. Strong, but nonphysiologic, evidence forAID expression early in B-cell development is provided by therescue of B-cell development [17, 18] in some strains of μMT mice[19] by early CSR events that restore pre-BCR/BCR signaling.
Nonetheless, AID expression in developing B cells remains acontroversial topic, in part because the first AID-reporter mouselines only identified GC B cells but not immature and transitional Bcells, as expressing AID [20–22]; however, it should be noted thata later, independent reporter-strain indicated prior AID expres-sion in immature and transitional B cells [23]. There was also ageneral conviction that the low levels of AID expression that hasbeen observed in developing B cells [14, 24] were physiologicallyinsignificant.
Direct tests of this conviction in mice [24] and humans[25] showed it to be unfounded. Remarkably, AID expressioncontrols self-tolerance in B cells by a mechanism that is B-cell
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Eur. J. Immunol. 2014. 44: 2876–2879 HIGHLIGHTS 2877
intrinsic and acts directly on immature and transitional B cells.In mice, Aicda reduces the fitness of hematopoietic progenitors togenerate B cells — but not T lymphocytes or myeloid cells —and this reduced fitness becomes manifest in immature andtransitional 1 B cells, the compartments with the highest levels ofintrinsic AID message [12, 14, 24]. The B lymphopoietic advantageof Aicda−/− progenitor cells is directly linked to impaired B-cell tol-erance, as the arrested development of autoreactive B cells [26] isrestored in Aicda−/− animals [24]. In line with this, the laboratoryof E. Meffre found that recombinant human antibodies made fromthe CD10++CD21loIgMhiCD27− transitional (“new emigrant”)B cells of AICDnull hyper-IgM syndrome patients exhibited aprimary B-cell repertoire skewed to autoreactivity and rich in VDJrearrangements known to encode self-reactive antibodies [25].
In this issue of the European Journal of Immunology, Umikeret al. [27] continue to expand our understanding of the role(s)of AID expression and B-cell autoreactivity in a complex exper-imental model that, unfortunately, may be too clever by half.Essentially, Umiker et al. sought to control the expression of aconditional Aicda transgene (Aicdatg) in 564Igi mice [13] duringB-cell development to discriminate between the effects of AIDexpression early in development versus later expression in matureB cells. On an Aicda−/− genetic background, the Aicdatg wasactivated by stage-dependent expression of Cre-recombinase:controlled by the mb1 promoter, Cre was active at all stages ofB-cell development; under the Cd19 promoter, Cre activityincreased with lineage maturation; under the Cd21 promoter Creexpression was confined to mature B cells. Simply put, in thismodel, AID expression ranges from always on, to, with varyingdegrees, on in maturing/mature B cells. A confounding variableof this approach is that each construct pair supports different levelsof AID message in B220+ spleen and bone marrow cells and thatin all cases, AID expression far exceeds physiological levels.
The 564Igi knockin mice express a polyreactive BCR/antibodythat binds single-strand (ss) DNA, ssRNA, and to nucleosomesand is pathogenic; this BCR/antibody is imprecisely recognizedby a monoclonal anti-idiotope antibody, B6–256 [13]. Curiously,546Igi mice are Janus-like in that whereas B cells bearing the 564BCR are subject to tolerance by deletion, receptor editing, andanergy, 564Igi mice generate substantial titers of serum IgG2a(but not IgM or IgG1) that retains an autoreactive component[13]. The production of autoreactive, 564id+ IgG2a is T-cell inde-pendent but requires TLR-7 [13]. Imanishi-Kari and her colleagueshave proposed [13] that this T-independent, 564 IgG2a is gener-ated when anergic 564 B cells are activated in the periphery bysynergistic BCR and TLR signals [28, 29].
To summarize, the experimental model is a series of geneticallymodified mice that express a polyreactive BCR subject to toler-ization by deletion, editing, and anergy and capable of inducingT-independent but TLR-7-dependent IgG2a serum autoantibodyon a genetic background that directs constitutive but variable lev-els of AID expression early (mb1-Cre) or later (Cd19-Cre and Cd21-Cre) during B-cell development (Fig. 1). As is usual in these typesof experiments, one assumes, but cannot know, that T-cells aretolerant to the self-antigens recognized by the 564 BCR.
An interesting aspect of this study [27] comes from the con-trols. The authors note that in those mice in which Aicdatg isnot activated, i.e., mice fully deficient in AID activity, significantamounts of 564id+, autoreactive IgM appear in the serum. In addi-tion, the authors also note that receptor editing is diminished inthese control mice, implying that in the absence of AID activity,B-cell tolerance is impaired. This mirrors the findings reported ear-lier by Kuraoka et al. [24] and by Meyers et al. [25] in hyper-IgMsyndrome patients.
As might be expected, the results of this study [27] are com-plex and subject to many caveats, however, interestingly there areclear distinctions between the various experimental groups thatare strongly correlated with the timing of AID expression (Fig. 1).Strikingly, 564id+ IgG2a and IgG2b that bound RNA was presentin all mb1-Cre and Cd19-Cre mice, but absent in the great majorityof Cd21-Cre animals. Cd21-Cre mice instead expressed high levelsof 564id−RNA− IgG antibody. Although the presentation of datamakes quantitative analyses difficult, the genetic data presentedby Umiker et al. [27] provide repeated examples to support theirpoint that SHM and CSR are active in immature B cells from mb1-Cre mice and that in many cases, V(D)J mutations reduce or abol-ish RNA binding. In this way, early AID activity acts as a tolerizingmechanism. The nonautoreactive IgG abundant in Cd21-Cre miceis largely (�80%) encoded by VDJ rearrangements that representsecondary recombination events, or mutated 564Igi alleles. It iscurious that the authors observe substantially more H-chain edit-ing than the usually common L-chain replacements. The authorsdo not make much of this unusual reversal but it suggests that thebiology of these 564Igi strains substantially differs from that ofnormal mice.
Expression of 564Id+ RNA-binding IgM was reciprocally cor-related with the IgG autoantibody production; that is, lowerlevels were observed in mb1-Cre and Cd19-Cre mice whereas self-reactive IgM was as high in Cd21-Cre animals as in AID deficientmice. Umiker et al. [27] concludes that this must reflect in someway the absence of AID expression early in B-cell development.Given the authors’ own observations of 564Id+ RNA-binding IgMin Aicda−/− controls and the prior work of other groups [24, 25],this seems a likely hypothesis but leaves dangling the problem ofhow these autoreactive B cells are dealt with later. In this exper-imental model, activation by RNA DAMPs must be frequent inthe bone marrow and appears to drive these cells into terminaldifferentiation as short-lived plasmacytes. But what if theencounter with the DAMP/BCR-ligand was less frequent? In thiscase, autoreactive B cells would either become anergic or escapeto maturity in the periphery.
In a curious reversal of dogma, Goodnow and colleagues [30]recently showed that anergic B cells can readily be activated inGCs and acquire there, mutations that reduce self-reactivity. Thisprocess of “redemption” for autoantibodies has also been observedin knockin mice that carry a BCR that neutralizes many strains ofHIV-1 [31, 32] but is subject to tolerization because it cross-reactswith host determinants [33]. In these mice, a T-independent HIV-1vaccine containing TLR4/TLR9 ligands readily elicited HIV-1neutralizing IgG2b antibody. In contrast, T-dependent protein
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2878 Garnett Kelsoe Eur. J. Immunol. 2014. 44: 2876–2879
Figure 1. Synopsis of the methods and critical results of Umiker et al. [27]. (A) To determine the role of early versus later expression of AID inB-cell development and differentiation, the authors controlled expression of a conditional Aicda transgene (Aicdatg) in 564Igi mice that express anautoreactive BCR. On an Aicda−/− genetic background, Aicdatg was activated by conditional expression of Cre-recombinase. In mb1-Cre mice, Aicdatg
was active at all stages of B-cell development; under the Cd19 promoter, Cre activity increased with lineage maturation, and; Cd21-Cre animalsexpressed Aicdatg only in mature B cells. Control mice that do not express Cre are negative for AID activity. (B) The autoreactive BCR of 564Igimice binds DNA, RNA and to nucleosomes; it is pathogenic and is subject to tolerization. The 564 BCR is recognized by a monoclonal anti-idiotopeantibody that defines 564id+ B cells. Expression of 564Id+ autoreactive IgM was associated with the absence of AID activity during early B-celldevelopment. Normally, 546Igi mice display the unusual phenotype of substantial titers of autoreactive 564id+ serum IgG2a (and IgG2b) that is T-cellindependent but requires TLR-7. 564id+ RNA-binding, 564id+ IgG2a was present in all mb1-Cre and Cd19-Cre mice, but rare in Cd21-Cre animals;instead, Cd21-Cre mice expressed both 564id+ IgM and high levels of 564id−RNA− IgG antibody. Umiker et al. [27] conclude that early AID activity(i) suppresses the functional tolerization of IgM+ 564Ig B cells (no Cre and mb-1-Cre) but (ii) is required for the generation of autoreactive, 564id+
IgG2a (cd19− and cd21−Cre). Finally, AID expression only in mature B cells (iii) constrains the T-independent production of 564id+ RNA-bindingIgG2a but promotes nonautoreactive 564id− IgG, in part, by the introduction of “redemptive” mutations [30] in germinal centers. AID activity actsthen, both early and late in B-cell development to control autoimmunity.
antigens elicit non-neutralizing, nonautoreactive IgG responses —likely by the “redemption” pathway. In both of these cases, theknockin BCR is mutated and exhibits high affinity for its autoli-gand. Under such conditions, V(D)J mutations that decrease affin-ity are likely, more so than in lower affinity, germline rearrange-ments. These experiments may be usefully applied to interpretUmiker et al.
Imanishi-Kari and her colleagues have made fundamental con-tributions to the growing appreciation for the physiological role ofAID activity during B-cell development [10, 11]. The latest contri-bution of this group to understanding this role, namely the articleby Umiker et al. [27], may be distilled to a single and importantconclusion: early AID activity can tolerize activated, autoreactiveB cells by altering specificity through V(D)J hypermutation, andby driving B cells to terminal differentiation as short-lived plasma-cytes. This model is both interesting and testable. A first test mustbe whether CD154 independent, IgG2b antibody can be observedin normal mice. Obviously, the level of antibody and plasmacytesmust be low, but well within the reach of contemporary single-cellcloning methods. Following the 564Igi model, the BCR recovered
from these cells will be specific for DAMPs. These more physiolog-ical tests of Umiker et al. [27] are anxiously awaited.
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Abbreviations: CSR: class-switch recombination · SHM: somatic
hypermutation
Full correspondence: Dr. Garnett Kelsoe, DUMC 3010, Department ofImmunology, Duke University, Durham, NC 27710, USAFax: +1-919-613-7815e-mail: [email protected]
See accompanying Article:http://dx.doi.org/10.1002/eji.201344282
Received: 10/9/2014Revised: 10/9/2014Accepted: 22/9/2014
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