DR.BERCOVICH: AVANCES EN AUTOINMUNIDAD

AVANCES EN CONOCIMIENTO DE ENFERMEDADES AUTOINMUNES

Boston Children´s Hospital

¿Por qué nuestro cuerpo puede volverse contra nosotros mismos? El desencadenante era un misterio. Hasta ahora.
Un equipo de científicos del Boston Children’s Hospital y de la Escuela de Medicina de la Universidad de Harvard (EE. UU.) ha identificado -gracias a experimentos con ratones- una reacción en cadena que explica por qué nuestros propios cuerpos pueden volverse contra las células sanas. Este descubrimiento podría transformar nuestro conocimiento sobre las enfermedades autoinmunes y la forma en que las tratamos.

¿Cuál es el desencadenante?

La reacción, descubierta después de cuatro años de investigación en ratones, ha sido descrita como un “tren fuera de control” donde un error conduce al organismo a desarrollar una forma muy eficiente de atacarse a sí mismo.

El estudio se centró en las células B (debido a que los linfocitos B tienen un papel importante en la regulación del sistema inmunitario, tanto en condiciones fisiológicas como patológicas). Normalmente estas células producen anticuerpos y programan las células inmunitarias para atacar antígenos no deseados (o sustancias extrañas), pero los expertos encontraron un “interruptor de anulación” en las células B de los roedores que distorsionó este comportamiento y causó ataques autoinmunes.

Las enfermedades autoinmunes serían como el ‘fuego amigo’ en el combate
Según uno de los autores del trabajo, Michael Carroll, “una vez que se pierde la tolerancia del cuerpo a sus propios tejidos, la reacción en cadena es como un tren fuera de control. La respuesta inmune contra las proteínas del propio cuerpo, o antígenos, se ve exactamente como si el organismo estuviera respondiendo a un patógeno extraño”.

Estas células B podrían a su vez explicar el fenómeno biológico conocido como epítopo (porción de una macromolécula que es reconocida por el sistema inmunitario) donde nuestros cuerpos comienzan a cazar diversos antígenos que no deben estar en la “lista de limpieza/caza del sistema inmune”.

La difusión de epítopos en el organismo sí que se ha observado durante mucho tiempo en laboratorio, pero los científicos desconocían de qué forma sucedía y por qué las enfermedades autoinmunes evolucionan con el tiempo para dirigirse a un catálogo en constante expansión de órganos y tejidos sanos.
Por ello, los investigadores examinaron un modelo de ratón con la enfermedad autoinmune del lupus, considerada una modalidad “clásica” de la enfermedad autoinmune en la que se basan muchas otras.

“El lupus es conocido como el gran imitador, porque la enfermedad puede tener tantas presentaciones clínicas diferentes que se asemeja a otras condiciones comunes. Es una enfermedad de múltiples órganos afectados y con una plétora de potenciales objetivos antigénicos”, comenta Søren Degn, coautor del trabajo.
Los científicos utilizaron proteínas marcadoras fluorescentes para rastrear distintas células B en el cuerpo de los roedores. Cuando las células B detectan un cuerpo extraño -o algo sano que parece ser un cuerpo extraño- se mueven en grupos llamados centros germinales (es por este motivo por el que los ganglios linfáticos se hinchan cuando tenemos un resfriado, por ejemplo).

“Una vez que se pierde la tolerancia del cuerpo a sus propios tejidos, la reacción en cadena es como un tren fuera de control”
Los clones de las células B luchan entre sí dentro de estos centros para que el cuerpo pueda determinar qué anticuerpo es el más adecuado para combatir la amenaza y, en el caso de este estudio, significaba que un color ‘ganaba’ frente a los demás.

El problema surge cuando el cuerpo identifica incorrectamente una proteína normal como una amenaza. Cuando eso sucede, se producen autoanticuerpos que resultan muy eficaces para dañar nuestros propios cuerpos.

Los investigadores han descubierto que “con el tiempo, las células B que inicialmente producen los autoanticuerpos comienzan a reclutar otras células B para producir autoanticuerpos adicionales dañinos”, dice Degn.

La enfermedad celíaca, la hepatitis autoinmune, la tiroiditis de Hashimoto, la artritis reumatoide, el lupus eritematoso sistémico y discoide, la psoriasis o el síndrome de Guillain-Barré, son algunas de ellas
Hasta ahora esto solo se ha observado en ratones, pero los investigadores quieren utilizar esta técnica de ‘confeti’ para ver de qué forma se regula y se acelera esta producción de células B de autoanticuerpos.

El bloqueo de los centros germinales podría suponer una ruptura en el círculo vicioso que crean las enfermedades autoinmunes pues bloquearía eficazmente la memoria a corto plazo del sistema inmunológico.

“Este hallazgo fue una sorpresa. No sólo nos dice que las células B autorreactivas están compitiendo dentro de los centros germinales para diseñar un autoanticuerpo, sino que también vimos que la respuesta inmune se amplía para atacar a otros tejidos del cuerpo, llevando a que el epítopo se extienda a la velocidad de un tren fuera de control”, aclara Carroll.
Referencia: Søren E. Degn, Cees E. van der Poel, Daniel J. Firl, Burcu Ayoglu, Fahd A. Al Qureshah. Clonal Evolution of Autoreactive Germinal Centers. Cell. 2017. DOI: http://dx.doi.org/10.1016/j.cell.2017.07.026

CELL

Volume 170, Issue 5, p913–926.e19, 24 August 2017

Clonal Evolution of Autoreactive Germinal Centers

Summary

Germinal centers (GCs) are the primary sites of clonal B cell expansion and affinity maturation, directing the production of high-affinity antibodies. This response is a central driver of pathogenesis in autoimmune diseases, such as systemic lupus erythematosus (SLE), but the natural history of autoreactive GCs remains unclear. Here, we present a novel mouse model where the presence of a single autoreactive B cell clone drives the TLR7-dependent activation, expansion, and differentiation of other autoreactive B cells in spontaneous GCs. Once tolerance was broken for one self-antigen, autoreactive GCs generated B cells targeting other self-antigens. GCs became independent of the initial clone and evolved toward dominance of individual clonal lineages, indicating affinity maturation. This process produced serum autoantibodies to a breadth of self-antigens, leading to antibody deposition in the kidneys. Our data provide insight into the maturation of the self-reactive B cell response, contextualizing the epitope spreading observed in autoimmune disease.

Introduction

Systemic lupus erythematosus (SLE) is characterized by the production of antibodies to nucleic acid antigens (Ags) (Rahman and Isenberg, 2008), with >75% of patients having serum autoantibodies to double-stranded DNA (compared to ∼0.5% of healthy controls), which typically appear a few years before SLE is diagnosed (Arbuckle et al., 2003). SLE patients at or after disease onset drift in their serological reactivities toward a variety of nuclear, nucleolar, and protein-DNA complexes: a process known as epitope spreading. Although the mechanism is not well understood, epitope spreading is thought to be driven by chronic immune responses causing inclusion of new autoreactive B cell clones (Arbuckle et al., 2003, Cornaby et al., 2015, Vanderlugt and Miller, 2002).

Affinity-matured antibodies (Abs) arise in germinal centers (GCs), wherein B cell clones cycle between division, somatic hypermutation (SHM) and selection based on Ag affinity (Victora et al., 2010). This process of random mutation can generate BCRs that recognize self-Ags (Mietzner et al., 2008, Tiller et al., 2007); however, it has long been thought that GCs are able to limit the affinity maturation of autoreactive cells (Han et al., 1995, Pulendran et al., 1995, Shokat and Goodnow, 1995, Vinuesa et al., 2009). In fact, display of self-Ag by follicular dendritic cells (FDCs) within GCs can delete autoreactive cells (Yau et al., 2013), and autoreactive B cells can mutate away from autoreactivity (Reed et al., 2016, Sabouri et al., 2014). Therefore, it has remained questionable whether autoreactive B cells in GCs follow the same rules of Ag engagement with FDCs, clonal evolution, and affinity maturation as those in foreign-Ag-elicited GCs.

Many autoimmune mouse models have spontaneous GC formation (Luzina et al., 2001), but as these models are based on genetically modified B or T cells or have uncharacterized, complex genetic backgrounds, they are poorly suited to studying natural autoreactive GC behavior. Here, we developed a chimera mouse model that has spontaneous autoreactive GCs composed of self-reactive B cells from the wild-type (WT) repertoire in a genetically normal context. Surprisingly, we find that there is no limit on the evolution of autoreactive GCs—that is, they generate B cells targeting other self-Ags once tolerance is broken.

Results

564Igi Mice Display Spontaneous GCs, VDJ Diversification, SHM, and CSR

The 564Igi mouse is a murine model of SLE on C57BL/6 (B6) background, generated by knock in of the heavy (H) and kappa light (K) chain of an autoreactive B cell clone targeting ribonuclear complexes (Berland et al., 2006). In heterozygous 564Igi mice, which carry a single copy of the knockin H and K chain, ∼50% of circulating B cells express the knockin BCR (identified by anti-idiotype [Id] Ab staining). The remaining half are Id negative (Id⁻) due to receptor editing or allelic inclusion (Berland et al., 2006, Chatterjee et al., 2013, Das et al., 2017, Luning Prak et al., 2011). The mice have high titers of IgG against nuclear-associated Ags, but do not exhibit disease until late in life.

Already at 6 weeks of age, heterozygous 564Igi mice (564het) harbored robust GC B cell populations in spleen and cutaneous lymph nodes (LN) (Figure 1A). Littermates that had the H chain knockin but lacked the K chain (564het K⁻) had baseline GC B cell frequencies comparable to B6 controls (Figure 1A). The presence of spontaneous GC B cells correlated with the presence of circulating Id⁺ (knockin BCR) cells (Figures 1B and S1A, Spearman correlation, p = 0.0002 and p < 0.0001, for spleen and LN, respectively). As reported previously, GC structures were found in the spleens of heterozygous 564Igi mice (Chatterjee et al., 2013) (Figure 1C).

Strikingly, Id⁺ cells were underrepresented in GCs compared to the circulating repertoire (Figure 1D). GC B cells were sorted from the spleens of two heterozygous 564Igi mice, and sequencing their BCR repertoire revealed that ∼95% of the IgM and ∼75% of the IgG sequences were not derived from the knockin allele (Figure 1E; Table S1) but were derived by V(D)J recombination of the other allele or by receptor editing of the knockin allele (essentially recombining it again). These “WT-derived B cells” had undergone SHM, as judged by the mutations in their heavy chain VDJ (Figure S1B), and the mutation frequency was higher in IgG sequences than in IgM sequences (two-tailed Mann-Whitney test, p < 0.0001), suggesting affinity maturation and class-switch recombination (CSR), but it was unclear whether these WT-derived clones were autoreactive.

We next assessed the reactivity of WT-derived serum antibodies to nucleolar autoAg. The 564Igi heavy chain locus is derived from the IgH-1a allotype (that encodes IgG2a), whereas the endogenous B6 heavy chain locus is IgH-1b (that encodes IgG2c). Therefore, we can distinguish antibodies produced by 564Igi B cells versus WT based on their respective IgG2a and IgG2c allotypes. We screened for IgG2c antibodies against nucleolar autoAg in the sera of heterozygous 564Igi mice. Samples from B6 (IgG2c only), heterozygous 564Igi kappa negative (564het K⁻) (IgG2a and IgG2c), and homozygous 564Igi (IgG2a only) mice were included as controls. As expected, heterozygous and homozygous 564Igi mice harbored IgG antibodies targeting nucleoli, whereas B6 and 564het K⁻ littermates did not (Figure 1F). Strikingly, heterozygous 564Igi mice harbored IgG2c Ab targeting nucleoli (Figure 1G), meaning that a subset of their WT-derived B cells had become autoreactive.

To investigate the trajectory of Id frequencies in spontaneous GCs, the 564Igi strain was crossed to an Aid-Cre^ERT2 EYFP reporter (Dogan et al., 2009). In this model, GC B cells will constitutively express YFP upon tamoxifen administration, allowing us to track these cells and their progeny (Figure 1H). Following tamoxifen injection, YFP⁺ cells represented 2/3 to 3/4 of the total GC B cell population (day 8, ∼1,500 YFP⁺ cells per 10⁵ B cells, with most YFP⁺ cells being GC B cells, compared with a total GC B cell frequency of ∼2,000 per 10⁵) (Figures 1I–1K and S1C; Movies S1 and S2). The fidelity and tamoxifen dependence of the YFP expression were verified in control experiments (Figures S1D–S1J).

The YFP⁺ GC population and its progeny were followed for 3 months, showing an overall steady decrease in YFP⁺ cells with an increase in Id⁻ YFP⁺ cells (Figures 1J and 1K), despite circulating Id frequencies remaining constant (Figure 1L). Bulk sorting and sequencing of YFP⁺ cells 90 days after tamoxifen revealed that >80% of IgM sequences and >90% of IgG sequences were not derived from the 564 knockin allele (Figure S1K), confirming that high numbers of WT-derived B cells were included in the GC and downstream plasma and memory cell population over time. These cells had undergone SHM, with more mutations in IgG sequences than in IgM sequences (two-tailed Mann-Whitney test, p < 0.0001; Figure S1L), supporting affinity maturation and CSR. SHM is classically associated with GC responses, but also occurs in extrafollicular foci (Herlands et al., 2008, William et al., 2002). However, the observed mutation rates of the YFP cells were an order of magnitude above those expected from extrafollicular foci. Taken together, these results demonstrate that in heterozygous 564Igi mice, autoreactive WT-derived cells are maturing in spontaneous GCs, thus undergoing affinity maturation, SHM, and CSR.

564Igi Mixed Chimeras Have Spontaneous GCs Composed of WT B Cells

In heterozygous 564Igi mice, spontaneous GCs were predominantly composed of GC B cells with WT-derived BCRs. However, all developing B cells in these mice harbor the knockin allele, thus they are not strictly WT B cells. To follow true WT cells in spontaneous GCs, we used a mixed chimera approach. WT B6 mice were irradiated, ablating their immune system, and reconstituted with a mixture of bone marrows (BM) from WT B6 and homozygous 564Igi donors. In this scenario, the 564Igi cells still kick-start autoimmunity, but they are “locked in,” as they are bi-allelically pre-rearranged, and they are genetically segregated from the WT B cell response (Figure S2A). The cells were followed using two allotypes of leukocyte marker CD45 (CD45.1 for WT cells and CD45.2 for 564Igi cells) (Figure 2A). For controls, we used mixed chimeras in which homozygous 564Igi donors were K-chain negative (CD45.2 564K⁻) (Figure 2A). T cells should be unaffected by the knockin status, as they do not express the BCR, and all chimeras had the predicted ratios of CD45.1 to CD45.2 T cells.

The mixed chimeras had fewer-than-expected 564Igi B cells (CD45.2) when compared to 564Igi T cells, likely because the autoreactive 564Igi cells were selected against (Figures S2B–S2E). A smaller, but significant negative selection was observed in the BM of 564K⁻ mixed chimeras, likely because of higher tonic BCR signal in the double-heavy-chain-expressing 564K⁻ B cells (Figure S2B). However, in spleen, skin-draining LN, and blood, the reduced frequency of 564Igi B cells was more drastic than for 564K⁻ B cells (Figures S2C–S2E). This finding agrees with the previously reported negative selection of self-reactive B cells at the transitional stage observed in 564Igi mice (Berland et al., 2006, Chatterjee et al., 2013). The chimeras displayed normal and consistent total B cell frequency (Figures S2F–S2I).

Despite negative selection against 564Igi B cells, the input BM ratios had a titration effect on circulating Id frequencies (Figure 2B). Weaker effects on GCs were also observed in spleen and skin-draining LN (Figures 2C–2E). The frequency of Id⁺ cells in blood correlated with GC B cell frequencies in spleen (Spearman r = 0.73, p < 0.0001) and skin-draining LN (Spearman r = 0.65, p = 0.0006) (Figure S2J). Furthermore, the ratios impacted the level of circulating anti-nucleolar IgG and IgG2c (produced by WT cells) (Figures 2F and 2G). Strikingly, at 6–8 weeks post reconstitution, GCs were almost exclusively composed of WT-derived cells (Figures 2H and 2I), while the 564K⁻ chimera controls had no GCs. Because the observed phenotypes were most robust in the 1:2 chimeras, this BM ratio was chosen for most subsequent experiments.

B Cells of Spontaneous GCs in 564Igi Mixed Chimeras Are Autoreactive and Converge on Similar BCRs

To analyze the WT-derived GC B cells further, mixed chimeras were generated using a mixture of BM from Aid-Cre^ERT2 EYFP WT and homozygous 564Igi donors transferred into irradiated WT recipients (Figure 3A). Following reconstitution, mice were pulsed with tamoxifen, and 4 weeks later, the BCR repertoire of the YFP⁺ GC B cells was sequenced (Figures 3A and 3B), capturing a total of 275 heavy chains from 4 chimeras, representing a total of 46 distinct V segments (the V segment determines most of the Ag specificity). The most prevalent V segment accounted for over 1/5 of the total sequences, while the second- and third-most accounted for ∼1/6 and ∼1/15, respectively. The most prevalent and the second-most prevalent V segment were observed in 3 and 4 out of 4 mice, respectively (Figure 3B), indicating that the WT-derived GC B cells converged on particular heavy chain V segments, suggesting they were targeting similar Ags. The sequences had many mutations, indicating a significant degree of SHM, as compared to naive mature B cells from B6 mice (Kruskal-Wallis test with Dunn’s multiple comparison, p < 0.0001 for pairwise comparison of mouse A-D with B6) (Figure S3C).

This analysis is a gross under-sampling of a vast pool of B cells and may not adequately reflect clonal selection processes at the level of a single GC. To improve the analysis, chimeras were generated based on mixing WT PA-GFP BM with homozygous 564Igi BM in WT recipients (Figure 3C). Following reconstitution, single GCs from fresh spleen explants were photoactivated as previously described (Victora et al., 2010) and sorted into single cells (Figures 3C and 3D). Again, clonal expansion and selection were observed in the heavy-chain-derived V segments (Figures 3E, 3F, S3A, and S3B). Specifically, the VHQ52.a27.79 (IGHV2-9^∗02) and J558.33 (IGHV1S81^∗02) segments occurred in 3/4, 4/4, and 4/4 GCs analyzed in 3 independent chimeras (Figure S3B). Surprisingly, these sequence elements were shared, not only among clones of distinct GC within the same mice, but also among clones of different mice. Again, mutation analysis indicated a significant degree of SHM, both when comparing all sequences from each PA-GFP mouse to naive mature B cells from B6 mice (Kruskal-Wallis test with Dunn’s multiple comparison, p < 0.0001 for pairwise comparison of E-G with B6) (Figure S3D) and when comparing sequences of individual GC with those from B6 (Kruskal-Wallis test with Dunn’s multiple comparison, p ≤ 0.0001 for pairwise comparison of G1–G4 with B6) (Figure S3E).

Of note, the observed V segments were also prominent in Aid-Cre^ERT2 EYFP chimeras. Furthermore, we identified similar sequences in YFP⁺ cells from tamoxifen-pulsed Aid-Cre^ERT2 EYFP heterozygous 564Igi mice, indicating that this convergence across independent animals was not an artifact of the chimera approach. An unbiased BLASTP search of CDR3 amino acid sequences (the CDR3 region being the most critical for Ag recognition) of clones carrying the VHQ52.a27.79 (IGHV2-9^∗02), J558.22.112 (IGHV1-22^∗01), and J558.33 (IGHV1S81^∗02) elements yielded hits for anti-DNA binding antibodies (including the original 564 VH clone) and anti-phosphocholine antibodies. These findings indicate that WT-derived GC B cells converged on specific autoreactive sequence elements.

To definitively determine whether BCRs from WT-derived clones were autoreactive, paired H and K chains from 16 clones and 1 inferred unmutated common ancestor (UCA) from 4 GCs of a PA-GFP 564Igi mixed chimera were synthesized, cloned, and expressed (Table S2). As controls, we cloned and expressed the original 564 Ab, the inferred 564 UCA, and an influenza hemagglutinin (HA)-specific Ab (6649). An overview of the sequence diversity of the 17 PA-GFP-derived clones and the 564 clone is shown in the phylogram in Figure 3G, which was rooted on the 564 UCA. The 20 total recombinant antibodies and the control 564 Ab derived from a hybridoma were assayed for cross-reactivity with cellular components in human epithelial (HEp-2) cells (Figures 3H–3J). Representative examples of the different staining patterns observed for cloned sequences from single GCs are shown in Figure 3I: perinuclear (G3_M25), predominantly cytoplasmic (G2_M05), cytoplasmic + nucleolar (G4_G22), and nuclear stain with nucleolar exclusion (G4_G55). Staining intensities were quantified in CellProfiler and are shown in Figure 3J.

Out of 4 GCs analyzed, autoreactivity was confirmed in 3. We further validated our findings in nucleolar ELISA-type assays (Figure S3F). The analysis was extended and refined by screening with autoAg arrays (Ayoglu et al., 2016), where each clone was assayed against 241 Ags (227 unique targets) and 30 controls (10 unique antibodies) (Table S3), and overall, results were congruent with the HEp-2 and nucleolar ELISA results (Figures S3G–S3K). The 564 clone, 564 UCA, G4_G22, and UCA GC2 were strongly reactive with single-stranded DNA (ssDNA) (Figure S3H). G2_M05 also reacted to ssDNA, but reacted most strongly with macrophage-derived chemokine (MDC, CCL22). Clones G2_G87, G2_G23, and G3_M25 displayed unique reactivity, albeit weaker, to this target (Figure S3I). These differences suggested a link between subcellular targeting and finer Ag specificity, because in the HEp-2-assay, G4_G22 and G2_M05 stained nucleolar + cytoplasmic and cytoplasmic, respectively, whereas G3_M25 had perinuclear staining. G4_G55, which had nuclear staining with nucleolar exclusion, was strongly reactive with Smith D2 Ag (Figure S3J). Interestingly, UCA_GC2 had additional reactivity to TGFβ-RII, not present in the other ssDNA-reactive clones (Figure S3K). The finding that the inferred UCA of the IgG-switched clones in GC2 was autoreactive suggests that germline precursors are autoreactive and enter GCs as such. Furthermore, there was CSR, as both autoreactive IgMs and IgGs were observed. Taken together, these results confirm that the spontaneous WT-derived GC B cells were autoreactive and were the likely source of the WT-derived, affinity-matured IgG2c autoantibodies observed in the chimeras.

GCs of 564Igi Mixed Chimeras Are T-Dependent with a Prominent Follicular Helper T Cell Population

GCs that form in response to foreign Ag depend on T cell help. To test whether those in 564Igi mice and 564Igi mixed chimeras do as well, we treated mice with anti-CD40L Ab to block T cell interaction. Similar to foreign Ag GCs, CD40L blockade ablated the GCs in spleen, cutaneous LN, and mesenteric LN of both heterozygous 564Igi mice and 564Igi mixed chimeras (Figures S4A–S4D). Splenic Id frequencies were unaffected, whereas those of cutaneous and mesenteric LN were marginally decreased. T follicular helper (Tfh, CXCR5^hiPD-1^hi) cell populations were present in both heterozygous 564Igi mice and 564Igi mixed chimeras, and they were similarly ablated by CD40L blockade, as previously shown (Durie et al., 1993, Yusuf et al., 2014). In contrast, overall CD4 T cell levels were unaffected (Figures S4E–S4H).

WT-Derived GC B Cells in 564Igi Mixed Chimeras Depend on TLR7 Signals

B cell-intrinsic TLR7 signaling is a driver of escape of tolerance for self-reactive B cells, as observed for Id⁺ cells in the 564Igi model (Berland et al., 2006, Green et al., 2012, Lau et al., 2005, Leadbetter et al., 2002). To test whether the WT-derived GC B cells in 564Igi mixed chimeras similarly depended on intrinsic TLR7 signaling, three-part mixed chimeras were generated using irradiated CD45.1/2 recipients. One part was 564Igi BM (identified by CD45.2 and PA-GFP congenic markers), one part was WT (CD45.1) BM, and the last part was either TLR7^−/− or TLR7^+/+ BM (both CD45.2) (Figure 4A). Both TLR7^−/− and TLR7^+/+ BM mixtures reconstituted mice equally, as demonstrated by near-expected (50:50) ratios of CD45.1 to CD45.2 T and B cells after excluding CD45.1/2 double-positive (recipient-derived) and PA-GFP-positive cells (564Igi-derived) (Figures S5A–S5H). Normal and comparable B and T cell frequencies were confirmed (Figures S5I–S5K). Robust and comparable GC frequencies were observed in both spleen and mesenteric LN for TLR7^−/− and TLR7^+/+ chimeras, both in terms of total GC B cells and GC B cells after exclusion of PA-GFP and CD45.1/2 cells (Figures S5L and S5M).

There were significantly fewer TLR7^−/− GC B cells than TLR7^+/+ GC B cells in the spleens of mixed chimeras (Figure 4B), meaning that WT B cells needed intrinsic TLR7 signaling for entry or persistence in autoreactive GCs. In mesenteric LN, there was a less dramatic, albeit significant, reduction in TLR7^−/− cell frequency (Figure S5N). The reduced dependence on the TLR7 pathway in intestinal LN GCs is likely due to multiple other TLR ligands originating from gut flora or food Ags.

Spontaneous GCs of 564Igi Mixed Chimeras Become Self-Propagating

Because of the low frequency of 564-derived cells in the autoreactive GCs of mixed chimeras, we asked whether the GCs were dependent on 564Igi cells for their persistence or whether the epitope spreading was sufficient to maintain them. We crossed 564Igi mice with UBC-Cre^ERT2 mice and a conditional attenuated-diphtheria toxin (DTA) line (564 UBC-Cre^ERT2 DTA). The BM from these mice (CD45.2) were mixed with BM from PA-GFP WT mice (CD45.2) and transferred to irradiated WT recipients (CD45.1) (Figure 4C). This set-up allowed specific ablation of 564-derived cells in a tamoxifen-induced manner. Following reconstitution, a high degree of chimerism was verified, and the expected frequencies of donor and normal B cells were observed (Figures S5O–S5R).

Half the cohort was treated with tamoxifen and mice were analyzed 2 weeks later. As expected, overall CD4 T cell, CD8 T cell, and CD19 B cell frequencies were unaffected (Figures 4D–4F). Because the frequency of 564-derived B cells was already low and therefore harder to quantify, CD8 T cells were used to measure the ablation strategy’s effect on the 564-derived hematopoietic compartment. In the 1:2 chimeras, the 564-derived cell frequency was reduced ∼80% (from ∼30% down to ∼6%); whereas in the 1:9 chimeras, it was reduced ∼90% (from ∼12% down to ∼1.2%) (Figure 4G). Despite this dramatic reduction in 564-derived cells, splenic and mesenteric GC B cell frequencies were only marginally reduced (Figure 4H). Therefore, 564-derived cells were not required once autoreactive GCs had been established.

Autoreactive GCs Evolve toward Pauciclonality

To analyze autoreactive GC dynamics globally, we generated mixed 564Igi and Aid-Cre^ERT2 Confetti BM chimeras. In this set-up, 564-driver cells elicit autoreactive GCs largely composed of WT-derived Aid-Cre^ERT2 Confetti B cells. Upon tamoxifen induction, Aid⁺ (GC) cells and their progeny express 1 of 10 different color combinations, allowing the visualization of clonal selection, as previously done for foreign Ag (Tas et al., 2016).

Robust induction of autoreactive GCs was observed, and the autoreactive GCs were composed of WT clones as evidenced by Confetti positivity (Movies S3 and S4). As an internal control, “Confetti only” chimeras without 564 driver BM were generated and immunized with a foreign Ag (NP-CGG) analogous to that of Tas et al. (2016))(Figure 5A). Over 4 weeks, foreign-Ag-elicited GCs lost color diversity, as evidenced by the frequencies of the most dominant and second-most dominant color and the sum of the two relative to the remainder of the GC (Figures 5B and 5E). Unlike for Tas et al. (2016)), these data do not account for unrecombined cells, and therefore, the results are not directly comparable.

However, we found that the autoreactive GCs evolved in a similar manner as foreign-Ag GCs, losing color diversity at only a slightly decreased rate (Figures 5C and 5E), which was confirmed by comparing the divergence index for the two groups (Figure 5D). Of note, whereas foreign-Ag-induced GCs are synchronized and timed, the 564Igi GCs were spontaneous and chronic, making it difficult to determine GC age. However, the effect of GC age could be controlled for by comparing to mesenteric LN GCs, which are chronic, in both groups. Notably, we observed no difference between the mesenteric LN GCs of foreign-Ag-immunized Confetti-only mice and those of 564Igi chimeric mice (Figures S6A–S6I), indicating that there is no effect from GC age. We conclude that autoreactive GCs, once tolerance has been broken, evolve toward pauciclonality—that is, the dominance of a single or a few clones and their progeny.

Mixed 564Igi Chimeras Undergo Epitope Spreading

Given the differential Vh usage and clonal selection of WT-derived GC B cells, we hypothesized that the WT-derived response may target more self-Ags than the original 564Igi clone. IgG (total), IgG2a (564-derived), and IgG2c (WT-derived) from 9 heterozygous 564Igi, 7 homozygous 564Igi, 6 1:2 564 mixed chimeras, 2 1:9 564 mixed chimeras, 6 1:2 564K⁻ mixed chimeras, and 5 B6 mice were assayed for binding to 241 Ags (227 unique targets) and 30 controls (10 unique Abs) (Figures S7A–S7C; Table S3). Individual mice within each group varied in reactivity, but the IgG antibodies of heterozygous 564Igi mice, homozygous 564Igi mice, and 564Igi mixed chimeras had stronger reactivity across a broad range of self-Ags than did IgG from 564K⁻ mixed chimeras and B6 mice (Figure S7A). IgG2a (564-derived) was strongly reactive in homozygous 564Igi and to a lesser extent heterozygous 564Igi mice (Figure S7B), whereas IgG2c (WT-derived) was strongly reactive in 564Igi mixed chimeras (Figure S7C), agreeing with the preceding experiments. A shift, or broadening, of reactivities was notable when comparing 564Igi mixed chimeras and homozygous 564Igi mice, which have B cells that are doubly pre-arranged and so cannot easily acquire new targets (Figure 6A). TNF-β (tumor necrosis factor beta), GBM (glomerular basement membrane), and numerous other targets were significantly overrepresented in the mixed chimeras (upper right quadrant, Figure 6A). Many of these targets are well-established autoimmune targets (GBM, collagen type VI, U1-snRNP A, etc.) including cytokines (APRIL, VEGF, interleukin [IL]-17, etc.) (Cappellano et al., 2012).

The original 564 clone did not react with interferon (IFN)γ, GBM, SmD2 (Smith D2), or TNF-β, suggesting that these are targets of WT-derived autoreactive B cells (IgG and IgG2c) (Figures 6B, 6C, S7D, and S7E). The 564 clone reacts with ssDNA (Berland et al., 2006, Gavalchin et al., 1987) (compare also with Figure S3), so not surprisingly, ssDNA and Ro60/SSA were the most notable 564-associated targets (upper left quadrant, Figure 6A). Homozygous 564Igi and to a lesser extent heterozygous 564Igi mice had a strong anti-ssDNA profile for IgG and IgG2a (564-derived), but not for IgG2c (WT-derived) (Figure S7D). 564Igi mixed chimeras had a significant but lower level of anti-ssDNA IgG and IgG2a than homozygous and heterozygous 564Igi mice, whereas 564K⁻ mixed chimeras had none (Figure S7E). A similar pattern was noted for Ro60/SSA (Figures 6B and C), in line with the polyreactive nature of the parental 564 clone. Taken together, these results suggest that inclusion of WT B cells in chronic autoreactive GCs drives epitope spreading.

Mixed 564Igi Chimeras Have Ab Deposits in Their Kidneys

The reactivity of 564Igi mixed chimeras toward GBM and collagen type IV suggested autoAb deposition in their kidneys, as those antigens are often targeted in autoimmune renal disease. We found that aged (9–12 months old) heterozygous 564Igi mice and 564Igi mixed chimeras had prominent IgG2c (WT-derived) deposits in the kidney; whereas, homozygous 564Igi and homozygous 564Igi K⁻ mice did not (Figures 7A and 7B). As expected, IgG2a (knockin allele) deposits were prominent in homozygous 564Igi mice, as well as 564Igi mixed chimeras, but were not detected in the homozygous 564Igi K⁻ mice (Figures 7A and 7C). This IgG2c deposition suggests a contribution of the epitope spreading process to pathogenesis.

Discussion

We made the unexpected observation of widespread epitope spreading in the 564Igi model. In heterozygous 564Igi mice, upward of half of circulating naive mature B cells carried an affinity-matured autoreactive BCR derived from the knockin. Yet, these cells failed to win in the GC reaction, giving way to WT-derived cells. This was surprising, because in mice that have a knockin BCR that is specific for foreign Ag, the knockin B cells dominate the GC response. For example, in B1-8 mice, 5% of the pool harbor a BCR specific for the hapten NP, and upon NP-carrier immunization, these cells dominate the response. However, this is arguably an oversimplified system as the hapten response is very narrow. Our findings in the autoreactive setting may reflect those of Kuraoka et al. (2016)), whereby the process of SHM increases the breadth of the response to complex Ag.

Although the maturation of autoreactive BCRs is generally restricted by T cell support, this check may be bypassed by linked recognition. In fact, autoreactive IgG memory antibodies in patients with SLE may arise from nonreactive and polyreactive precursors (Mietzner et al., 2008). However, mechanisms may exist to prevent the emergence of autoreactive B cells in GCs (Reed et al., 2016, Sabouri et al., 2014), perhaps relying in part on FDC presentation of autoAg (Yau et al., 2013). The nature of the self-Ag may be an important factor, as many nuclear self-Ags, such as the 564Igi Ag, trigger FDCs to secrete IFNα via endosomal TLR7 (Das et al., 2017), potentially leading to increased recruitment of self-reactive B cells from the immature repertoire.

To circumvent artifacts that might arise from transgenic B cells, we developed a mixed chimera model with spontaneous, autoreactive GCs composed of WT B cells with 564Igi cells initiating autoimmunity. We found that these autoreactive GCs are largely composed of WT B cells and are dependent on intrinsic TLR7 signaling, similarly to 564Igi B cells (Berland et al., 2006). Moreover, we found that the GCs became self-sustained and gained independence from the initial 564Igi trigger, explaining how autoreactive GCs become chronic and how disease is propagated.

We found that autoreactive GCs are polyclonal, but evolve toward pauciclonality at varying rates, agreeing with the observation that affinity maturation can occur in the absence of homogenizing selection, with many clones maturing in parallel within the same GC (Tas et al., 2016). This “clonal permissiveness” is in line with earlier reports that transient foreign-Ag-driven GCs are dynamic and open structures (Schwickert et al., 2007, Schwickert et al., 2009). We found that clonal evolution was largely similar between foreign Ag and autoAg GCs. Our observations suggest that once tolerance is broken, an increasingly broader array of self-Ags will be targeted.

We observed clonal convergence and selection leading to the predominance of autoreactive V segments in the WT-derived GC B cell population, similar to previous observations in autoimmunity and cancer (Hershberg and Luning Prak, 2015, Shlomchik et al., 1987). Serum antibodies displayed epitope spreading, further supported by our finding of WT-derived Ab deposits (IgG2c) in the kidneys of 564Igi mixed chimeras.

Our findings support that autoreactive GCs are at the heart of the autoimmune response. The chronic, progressive nature of epitope spreading calls for early interventions, even before disease onset. In light of the failure of CD40L blockade in humans, alternative GC checkpoints should be explored. We have focused here on the evolution of the B cell response, but questions remain. Although we demonstrated T cell dependence in our model, the extent and nature of T cell involvement has not been addressed. Future experiments will determine whether the T cell pool is not only necessary, but also sufficient, to sustain and confer autoreactivity.

We conclude that self-reactive 564Igi B cells are sufficient to break tolerance and induce the formation of autoreactive GCs, which are predominantly composed of WT-derived cells. We dub this novel tool, which can be used to study WT epitope spreading and autoreactive GC responses, the “ARTEMIS model” to signify autoreactive B cell driven T-dependent epitope migration toward immunity to self.

Author Contributions

S.E.D. conceived the project. S.E.D., C.E.v.d.P., D.J.F., B.A., F.A.A.Q., and G.B. planned and performed experiments and analyzed data. C.E.v.d.P. and D.J.F. contributed equally. L.M., C.-A.R., J.-C.W., P.J.U., and G.D.V. provided crucial reagents and expertise. P.J.U., G.D.V., and M.C.C. oversaw the project. S.E.D. wrote the manuscript. All authors provided critical feedback.

Acknowledgments

We thank H. Leung of the Optical Microscopy Core and N. Barteneva and K. Ketman of the Flow and Imaging Cytometry Resource at the PCMM for technical assistance. We also thank the SciLifeLab Autoimmunity Profiling Facility for access to FlexMap3D instrument. We are grateful to E. Alicot for technical assistance, D. Weseman for helpful discussion of the topic, and C. Usher for help editing the manuscript. F. Alt provided the attenuated DTA strain and A. Sharpe the UBC-Cre^ERT2 line. S.E.D. was supported by the Benzon Foundation and by a Marie Curie International Outgoing Fellowship within the 7th European Community Framework Programme ( PIOF-GA-2012-330134 ). C.E.v.d.P. was supported by a fellowship from GSK. D.J.F. was supported by the HHMI Medical Research Fellows Program. B.A. was supported by the Knut and Alice Wallenberg Foundation Postdoctoral Scholarship Program ( KAW 2014.0412 ). This research was supported by NIH grants R01AI039246 , R21AI117737 (M.C.C.), R01AI119006 (G.D.V.), U19AI110491 , UM1AI110498 , and R01 AI125197 (P.J.U.), by the Donald E. and Delia B. Baxter Foundation (Career Development Award to P.J.U.), the Henry Gustav Floren Trust (gift to P.J.U.), and a gift from Elizabeth F. Adler (to P.J.U.).