Extrafollicular responses are sufficient to drive initiation of autoimmunity and early disease hallmarks of lupus

Many autoimmune diseases are characterized by germinal center (GC) derived affinity-matured, class-switched autoantibodies. Strategies to block GC formation and progression are currently being explored clinically, however, extrafollicular responses may also contribute to early events in autoimmunity. To investigate the relative contribution of these two pathways in autoimmune disease development, we leveraged a transgenic strategy to genetically block the GC pathway. Surprisingly, this accelerated extrafollicular responses and failed to curb autoimmune progression in two lupus models. In vitro, loss of the GC transcription factor Bcl-6 prevented cellular expansion and accelerated plasma cell differentiation, suggesting the in vivo phenotype was caused by rewiring of B cell intrinsic transcriptional programming. In a competitive scenario in vivo, B cells harboring the genetic GC block contributed disproportionately to the plasma cell output. Taken together, this emphasizes the extrafollicular pathway as a key contributor to autoimmune progression, and suggests that strategies aimed at blocking GCs should simultaneously target this pathway to avoid rerouting the pathogenic response. Highlights - A genetic GC block fails to prevent autoimmune progression in two lupus models - An intrinsic GC block drives B cell differentiation into terminally differentiated plasma cells in vitro - B cells harboring a GC block competitively contribute to the plasma cell compartment in an autoreactive setting in vivo - Lupus mice with a GC block display immune complex deposition in kidney glomeruli that is indistinguishable from their wild-type counterparts Summary Affinity-matured autoantibodies generated in germinal centers are a hallmark of autoimmune diseases. Voss et al. block germinal centers in two autoimmune models, but surprisingly find that disease progresses unimpeded. They identify the extrafollicular pathway as a ‘backdoor to autoimmunity’.


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Summary (39/40) 19 Affinity-matured autoantibodies generated in germinal centers are a hallmark of autoimmune 20 diseases. Voss et al. block germinal centers in two autoimmune models, but surprisingly find 21 that disease progresses unimpeded. They identify the extrafollicular pathway as a 'backdoor 22 to autoimmunity'. 23 24 25 Abstract (160/160) 26 Many autoimmune diseases are characterized by germinal center (GC) derived affinity-27 matured, class-switched autoantibodies. Strategies to block GC formation and progression 28 are currently being explored clinically, however, extrafollicular responses may also contribute 29 to early events in autoimmunity. To investigate the relative contribution of these two 30 pathways in autoimmune disease development, we leveraged a transgenic strategy to 31 genetically block the GC pathway. Surprisingly, this accelerated extrafollicular responses and 32 failed to curb autoimmune progression in two lupus models. In vitro, loss of the GC 33 transcription factor Bcl-6 prevented cellular expansion and accelerated plasma cell 34 differentiation, suggesting the in vivo phenotype was caused by rewiring of B cell intrinsic 35 transcriptional programming. In a competitive scenario in vivo, B cells harboring the genetic 36 GC block contributed disproportionately to the plasma cell output. Taken together, this 37 emphasizes the extrafollicular pathway as a key contributor to autoimmune progression, and 38 suggests that strategies aimed at blocking GCs should simultaneously target this pathway to 39 avoid rerouting the pathogenic response. 40 41 42 Highlights: 43 44 -A genetic GC block fails to prevent autoimmune progression in two lupus models 45 -An intrinsic GC block drives B cell differentiation into terminally differentiated plasma cells 46 in vitro 47

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Mice 116 The Bcl-6 flx/flx strain (Hollister et al., 2013) and congenic B6.CD45.1 (B6.SJL-Ptprc a Pepc b /BoyJ) 117 were purchased from Jackson Laboratories (stock no. 023737 and 002014, respectively). 118 Aicda-Cre transgenic mice (Kwon et al., 2008) were kindly provided by Meinrad Busslinger, 119 The Research Institute of Molecular Pathology (IMP), Vienna. Aicda-Cre and Bcl-6 flx/flx strains 120 were intercrossed to generate Aicda-Cre+ and Aicda-Cre-Bcl-6 flx/flx littermates. 564Igi mice 121 (Berland et al., 2006) (B6.Cg-Igh tm1(Igh564)Tik Igk tm1(Igk564)Tik /J) were kindly made available by 122 Theresa Imanishi-Kari, Tufts University, and provided by Michael C. Carroll, Boston Children's 123 Hospital. The 564Igi line was crossed to Aicda-Cre Bcl6 flx/flx to generate 564Igi H +/+ K +/+ Aicda-124 Mice were treated topically on the right ear 3 times per week for 4 weeks with 1 mg R848/mL 137 acetone using a cotton applicator or did not receive any treatment (Untreated/Unt). 138 139 Mixed bone marrow chimeras 140 Recipient mice were irradiated with 9 Gy in a MultiRad 350 (Faxitron), with 350 kV, 11.4 mA, 141 a Thoraeus filter [0.75 mm Tin (Sn), 0.25 mm Copper (Cu), and 1.5 mm Aluminium (Al)], and 142 with a beam-distance of 37 cm. Irradiated recipients were kept on antibiotic water (either 1 143 mg sulfadiazine together with 0.2 mg trimethoprim per mL drinking water, or 0.25 mg 144 amoxicillin per mL drinking water) to avoid any opportunistic infections. On the following day, 145 donor mice were anesthetized with 4% isoflurane and euthanized. Femora, fibulae/tibiae, 146 ossa coxae and humeri were harvested, mechanically cleaned and rinsed in FACS buffer. The 147 Bone marrow (BM) cells were released from the harvested bones by crushing and the cell 148 extract was then passed through a 70 µm cell strainer. The donor BM cells were then counted 149 in a Cellometer K2 cell counter (Nexcelom). Cells were pelleted by centrifugation (200 g, 10 150 min, 4°C) and resuspended to 1*10 8 cells/mL. Donor cells from three different mice were then 151 mixed according to the proportions mentioned in the figure legend. The donor cell mixtures 152 were used to reconstitute the recipient mice by retroorbital injection of 200 µL (containing a 153 total of 20*10 6 cells) into each recipient mouse. The reconstituted recipient mice were placed 154 on antibiotic water the following 14 days. 155 156 Tissue preparation 157 Mice were anesthetized with isoflurane (055226, ScanVet), blood samples from the 158 retroorbital plexus were collected, and mice were euthanized using 100-150 mg/kg sodium 159 pentobarbital (450009, Dechra Veterinary Products). Mesenteric lymph nodes (MesLN) and 160 6/37 inguinal lymph nodes (IngLN) were removed, the splenic artery was clamped with a hemostat,  161  and the spleen was removed. The mice were perfused intracardially with PBS (BE17-515Q,  162 Lonza) to remove the blood, and subsequently perfused with 4% w/v paraformaldehyde (PFA) 163 (1.04005.100, Merck) in PBS to fix the tissues. Finally, kidneys and auricular lymph nodes 164 (AurLNs) were removed. 165 Collected blood samples were centrifuged at 3,000 g for 10 minutes, the supernatant 166 was collected, and centrifuged again at 20,000 g for 3 minutes. were seeded into 6-well plates at a density of 520 cells/cm 2 for IL-4 stimulated wells and 5200 347 cells/cm 2 for the four other conditions. The following day (day 0), B cells were purified 348 10/37 according to the described protocol, and resuspended in B cell medium (BCM) (RPMI-1640  349 supplemented with 10% FCS, 55 µM 2-ME, 1% Pen/Strep, 1% MEM NEAA, 10 mM HEPES, 1 350 mM Sodium Pyruvate). B cells were pre-diluted in BCM with the given cytokine cocktail as 351 indicated in figure legends. From day 2-8, 2/3 of the total volume of BCM was collected and 352 fresh BCM was added to reach the same final volume. One mL of medium from each well of 353 the 6 well plates was collected on the final day (day 10) for TRIFMA analyses. Cells were 354 analyzed using flow cytometry, as described above. and S6H, Mann-Whitney test was used to analyze the data that were not normally distributed. 364 All other datasets were normally distributed, except for the data in Figure 1L, 5F, and S6K 365 which were neither normally, nor log-normally distributed. However, a non-parametric t-test 366 for the isolated data between each group in panel 1L, 5F and S6K showed similar results (for 367 Fig 1L: a significant increase in GC formation in Cre-between Unt and R848. For Fig 5F: a 368 significant increase in CD8 frequencies in the IngLNs but not in other tissues. For S6K: slight, 369 but significant increases across spleen, IngLN and blood, with a trend towards an increase in 370 MesLN when tissues are analyzed individually), indicating that the observed differences, 371 beyond being biologically robust and in agreement with the flow data, were also statistically 372 robust. Parametric tests were used in all analyses, with specific tests indicated in the figure  373 legends. All data is presented as bar graphs with mean ± SD. A p-value <0.05 was considered 374 to be statistically significant. ns = p≥0.05, * = p<0.05, **= p<0.01, *** = p<0. After 4 weeks of treatment, the Bcl-6 flx/flx (Cre-) controls and the Aicda-Cre Bcl-6 flx/flx (Cre+) 397 mice showed similar, significant increases in spleen weight upon R848 treatment (Fig. 1B). 398 Anti-dsDNA autoantibodies of the IgG2c subtype measured in serum were dramatically 399 elevated upon R848 treatment compared with untreated animals. Surprisingly, there was a 400 trend towards higher levels in treated Cre+ mice compared with treated Cre-mice (Fig. 1C).

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A similar trend towards an increase was seen in total IgG2c levels ( Fig. 1D). No statistically 402 significant differences in total IgG1 and total IgG3 levels were seen upon treatment (Fig. 1E  403 and F). 404 405 To validate the effect of R848 treatment and the integrity of the GC block in Cre+ mice, we 406 carried out immunofluorescence staining of spleens to identify GC formation ( Fig. 1G-J). Using 407 the proliferation marker Ki67 and the naïve B cell marker IgD, it was evident that larger and 408 more frequent GCs were observed upon R848 treatment in Cre-animals (Fig. 1G, H and K). 409 Quantification revealed that this difference was statistically significant (Fig. 1L). Cre+ animals 410 did not display any baseline or R848-induced GCs ( Fig. 1I and J). However, in Cre+ treated 411 mice we did observe many proliferating cells at the T-B border and in the red pulp (Fig. 1J), 412 likely abortive primary foci and extrafollicular foci, respectively. 413 414 Flow cytometric analyses of inguinal LNs (IngLNs), mesenteric LNs (MesLN), and the spleen 415 were carried out ( Fig. 1M-Q, Fig S1A). In treated animals, we saw slight increases in monocyte 416 and neutrophil frequencies in some of the tissues (Fig. 1M and N), and a slight increase in B 417 cell frequencies in the skin-draining IngLNs (Fig. 1O), which might be caused by the direct 418 stimulatory effect from the R848 treatment of the ear skin. We observed robust GC B cell 419 frequencies in Cre-R848 treated mice, compared to untreated littermates (Fig. 1P). No GC B 420 cells were found in Cre+ animals, further validating the fidelity of the GC block (Fig. 1P). 421 Surprisingly, despite this, we found a significant increase in PB/PC frequencies upon 422 12/37 treatment in both groups, and the level was significantly higher in the spleens of mice 423 harboring a GC block compared to Cre-R848-treated littermate controls (Fig. 1Q). Splitting 424 up for PB and PC, it was clear that this difference between the two groups for the spleen was 425 attributable to PB (Fig. S1B) rather than PC (Fig. S1C) expansion in Cre+ animals, as compared 426 to Cre-littermates. This observation corresponded well with the increase in plasma IgG2c 427 autoantibody levels as well as total IgG2c levels (Fig 1C and D). Taken together, this 428 surprisingly indicated an exacerbated autoimmune phenotype in GC block mice compared to 429 GC sufficient mice upon R848 treatment.  To further understand the local effects of R848 treatment, we performed 463 immunofluorescence microscopy analyses of draining auricular lymph nodes (AurLNs) from 464 treated mice and untreated controls. We observed gross enlargement of the lymph nodes of 465 treated animals, with a robust induction of GCs in Cre-R848-treated mice (Fig. S2A). In 466 comparison, the Cre+ R848-treated mice had many proliferating cells outside the follicles (Fig.  467  S2A). These dividing cells in the AurLNs overlapped to some extent with the PC marker CD138, 468 pointing towards dividing extrafollicular PCs (Fig. S2B). To further understand the importance of the increased anti-dsDNA IgG2c autoantibodies in 487 Cre+ R848-treated mice, in the face of a complete absence of GCs, we implemented this 488 nanoparticle tracking approach ( Fig. 2A-C). We analyzed superoligomeric complexes in the 489 band from 90-130 nm ( Fig. 2D and E). In agreement with spMBL as a lupus marker, treated 490 mice tended towards higher levels, as compared to untreated mice, but interestingly, we also 491 observed a global trend towards higher levels in Cre+ compared to Cre-animals (Fig. 2F). 492 Although they did not reach statistical significance, these observations were well in line with 493 the previously noted increases in anti-dsDNA and total IgG2c antibodies upon R848 494 treatment, and in Cre+ compared to Cre-animals (Fig. 1C).

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In our prior study on autoimmune mice carrying an autoreactive B cell receptor knock-in 497 (564Igi) on a wild-type background (Juul-Madsen et al., 2021), a significant inverse correlation 498 was found between the frequency of splenic GC B cells and the ratio between the spMBL and 499 anti-dsDNA antibody concentrations measured in serum. This suggested that an excess of 500 spMBL increased GC B cell formation while an excess of anti-dsDNA antibodies decreased GC 501 B cell proliferation, or vice versa, indicating a potential negative feedback loop. In 502 consideration of this, we next asked whether the same correlation could be established in the 503 R848 model, and how this phenomenon was impacted in GC block mice. 504 505 In Cre-treated mice, GC B cell frequencies were positively correlated with the concentration 506 of spMBL particles in serum (Fig. 2G). Total IgG2c levels were similar between treated Cre+ 507 and Cre-mice (Fig. 2H) with a significant positive correlation between anti-dsDNA IgG2c and 508 the level of spMBL particles (Fig. 2I). A significant positive correlation was also observed 509 between the frequency of splenic GC B cells and the ratio between the spMBL and total IgG2c 510 levels in serum (Fig. 2J). Conversely, a significant inverse correlation was found between the 511 frequency of splenic GC B cells and the ratio between the spMBL and anti-dsDNA IgG2c 512 antibody concentrations measured in serum (Fig. 2K). Taken together, these findings now 513 took our original observations from the autoreactive B cell receptor knock-in model (564Igi) 514 into the epicutaneous R848 model on wild-type (Cre-) background. Importantly, in Cre+ mice, 515 there was an uncoupling of the concentration of spMBL particles and GC B cell levels, 516 indicating that the GC block failed to curb the production of spMBL complexes. 517 17/37 518 519

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Enhanced immune complex deposition in kidneys of R848-treated mice 534 To understand the pathological importance of the GC block in R848-treated mice and the 535 elevated serum IgG2c, serum anti-dsDNA IgG2c and spMBL levels, we investigated whether 536 there were any pathological changes associated with lupus nephritis in the kidneys. First, we 537 carried out Periodic acid-Schiff (PAS) staining, which displayed no obvious kidney injury upon 538 R848 treatment (Fig. 3A). Apart from the presence of mesangial and capillary immune 539 deposits, histopathological changes associated with lupus nephritis may include increased 540 matrix or mesangial cellularity, endocapillary proliferation, thickening of capillary walls, 541 glomerular tuft necrosis, extracapillary proliferation (crescents), karyorrhexis, hyaline 542 thrombi (micronodular intracapillary aggregation of immune complexes), and glomerular 543 sclerosis (segmental or global), as well as, rarely, pathognomonic hematoxylin bodies 544 (Gasparotto et al., 2020;Weening et al., 2004). However, no significant histopathologic 545 findings were identified by light microscopy in any of the mice included in any of the groups. 546 In line with the PAS stainings, we did not observe any differences in glomerular nephrin levels 547 among the groups, suggesting normal glomerular podocytes (Fig. 3B). As we could not identify 548 any gross pathological changes nor changes in nephrin levels in the kidneys upon treatment, 549 this verified our short-term treatment strategy in terms of the goal to investigate early 550 immune-driven events in the absence of any secondary pathology. 551 552 To evaluate if immune complex deposition occurred in the kidney glomeruli, and if there were 553 any differences between GC-sufficient and deficient groups, we performed 554 immunofluorescence staining of kidney sections targeting total Ig, C3, and IgG2c (Fig 3B, 3C, 555 3E-G). The total Ig levels in glomeruli were clearly increased upon R848-treatment. 556 Interestingly, we found a trend towards an increase in the total Ig levels of Cre+ mice 557 compared with littermate R848-treated controls ( Fig. 3B and E). We also found that there was 558 a significant increase of antibodies of the pathogenic subtype IgG2c upon R848 treatment 559 ( Fig. 3C and G), and a trend towards an increase in C3 deposition upon R848-treatment ( Fig.  560 3C and F). However, differences between Cre+ and Cre-R848-treated groups were seen 561 neither in C3 nor IgG2c levels ( Fig. 3F and G). 562 563 Taken together, R848-treated mice displayed immune complex deposition in glomeruli, based 564 on an increased level of C3, total Ig and IgG2c (Fig. 3E-G). We corroborated these findings by 565 peroxidase-staining, as a corollary to the immunofluorescence microscopy, and this 566 confirmed the glomerular changes in total Ig, C3 and IgG2c upon R848-treatment (Fig S3).   (Fig. 4A). Naïve B cells purified from 595 Cre-and Cre+ Bcl-6 flx/flx mice by negative magnetic-activated cell sorting were seeded onto 596 fibroblast feeder cells expressing CD40L, IL-21 and BAFF, and stimulated with IL-4 (Fig. 4A). compared to those derived from Cre+ mice (Fig. 4B+E and Fig. S4A+B). This was mirrored by 604 a similar relative increase in PBs (B220+, CD138+) in Cre-cultures ( Fig. 4B and D), but a relative 605 decrease in PCs (B220neg, CD138+) ( Fig. 4B and C). Of interest, the total number of cells in 606 the live gate for Cre-cultures was approximately 4 times higher than that of Cre+ cultures 607 (Cre-: 140,000 vs. Cre+: 33,000, Fig. S4C). To understand this difference in cell numbers, we 608 investigated whether the Cre+, and hence Bcl-6 deficient, B cells had an increased propensity 609 to undergo apoptosis, because Bcl-6 has previously been reported to suppress P53 and inhibit 610 apoptosis in GC B cells (Phan and Dalla-Favera, 2004). Somewhat surprisingly, we found that 611 upon IL-4 stimulation, there was no difference in the frequency of dead cells (Fig. 4F and G), 612 a slight and significant drop in apoptotic cell frequency (Fig. 4F and H), and a corresponding 613 increased relative frequency of live cells in Cre+ cultures compared to Cre-cultures on day 6 614 (Fig. 4F and I). However, at day 10 there were no significant differences in live, apoptotic, nor 615 necrotic cell frequencies between Cre-and Cre+ cultures (Fig. 4J-M). Thus, apoptosis could 616 not account for the dramatic difference in resulting cell numbers between Cre+ and Cre- (Fig.  617  S4C). Taken together, this suggested that the higher overall cell numbers in Cre-cultures was 618 not simply a reflection of increased apoptosis among Bcl-6 deficient cells in Cre+ cultures, but 619 rather represented an improved intrinsic proliferative potential of the Bcl-6 sufficient cells. 620 621 In summary, our iGB experiments revealed a vigorous expansion of B cells and PCs in Cre-622 cultures, but less pronounced PC differentiation, whereas Cre+ cultures conversely displayed 623 a lesser degree of proliferation but more pronounced PC differentiation. This suggested that 624 B cells with an intrinsic GC block may differentiate quicker to PCs, and thereby lose their 625 proliferative capacity, a notion that is in line with the established function of Bcl-6 in 626 repressing     Our observation that Bcl-6 deficient B cells rapidly lost their replicative potential in vitro (Fig.  650  4) was somewhat at odds with our in vivo observations from the R848 model, which displayed 651 a global increase in PCs and autoantibodies (Fig. 1). This is because even if Bcl-6 deficient cells 652 more readily became PCs, their poorer capacity to expand compared to Bcl-6 sufficient cells 653 would be predicted to limit PC output. However either Aicda-Cre+ Bcl-6 flx/flx or Aicda-Cre-Bcl-6 flx/flx BM ( Fig. 5A and S5).

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There were no differences in the basic parameters when comparing Cre-control BM chimera 680 mice with Cre+ BM chimera mice, in which approximately 50% of the B cells harbored a GC 681 block (Fig. 5B-H). That is, aside from a very small but significant relative increase of CD8 T cells 682 in IngLN of Cre+ chimeras, we saw no statistically significant differences in anti-dsDNA IgG2c 683 (Fig. 5B), total anti-dsDNA Ig (Fig. 5C), B cell frequencies (Fig. 5D), CD4 and CD8 T cell 684 frequencies ( Fig. 5E and F, respectively), overall GC B cell frequencies (Fig. 5G) and PB/PC 685 frequencies (Fig. 5H) between the groups. This confirmed that the two groups of chimeras 686 were comparable and had robust GCB and PC compartments. In the total B cell compartment, 687 CD45.2 positive cells were present at levels comparable to that of CD45.1 cells in both Cre-688 (Fig. 5I) and Cre+ (Fig. 5J) chimeras. However, within the GC B cell gate, CD45.2 cells were 689 robustly represented in Cre-chimeras, but virtually absent in Cre+ chimeras ( Fig. 5I and J). 690 When quantifying this effect across chimeras and expressing as the ratio of CD45.2 of GCB 691 relative to CD45.2 of total B cells, it was clear that Bcl-6 deficient cells, as expected from their 692 genetic deficiency, were incapable of contributing to the GC compartment (Fig. 5K). However, 693 when similarly comparing PB/PC ratio over B cells, the cells harboring a GC block remained 694 able to contribute to the final PB/PC pool, albeit underrepresented relative to the competitor 695 cells (Fig. 5L). Taken together, these findings demonstrated that in a GC-sufficient 696 environment, B cells experiencing a block in their ability to partake in the GC reaction readily 697 contributed to the PB and PC compartments.  The extrafollicular pathway is sufficient to drive early hallmarks of autoimmunity in a 723 second, independent lupus model 724 To further evaluate the robustness of our central observation that extrafollicular responses 725 were sufficient to compensate or could even exacerbate autoimmune progression, we set out 726 to test the effect of a complete GC block in a second, independent lupus model, 564Igi. The 727 564Igi model, which we also used as the driver compartment in the mixed chimeras, is a B cell 728 receptor knock-in model, derived from a B cell clone isolated from an autoreactive F1 hybrid 729 cross of Swiss Inbred (SWR) and New Zealand Black (NZB) mice (Berland et al., 2006;Gavalchin 730 et al., 1985). The clone was originally screened for reactivity with ssDNA, but has been found 731 to react more generally with ribonucleic acids and several ribonuclear proteins (Degn et al.,  732 2017b), a polyreactivity likely owing to a high cationic pI in its complementarity determining 733 region (Gavalchin et al., 1987). 564Igi mice present with spontaneous GCs and robust levels 734 of circulating anti-DNA antibodies, but do not develop more severe hallmarks of disease until 735 later in life (at least 9-12 months of age). We crossed the 564Igi line with Aicda-Cre Bcl6 flx/flx 736 mice, and backcrossed the heavy and light chain, as well as Bcl6 flx to homozygosity, retaining 737 hemiparental Aicda-Cre hemizygosity. Hence, Aicda-Cre positive vs. negative offspring, which 738 were invariably 564Igi and Bcl6 flx homozygous, could be compared ( Fig. 6A and S6A). We 739 analyzed this offspring either at adolescence (13-16 weeks) (Figure 6) or in early adulthood 740 (17-23 weeks of age) (Figure S6). At the earlier time point, the two groups had comparable 741 levels of anti-dsDNA of IgG2a isotype (Fig. 6B), whereas GC block 564Igi mice presented with 742 slightly elevated levels of anti-dsDNA IgG2b (Fig. 6C), although this difference did not reach 743 statistical significance. Total anti-dsDNA levels were comparable between groups (Fig. 6D). 744 Total IgG1 was elevated in GC-sufficient littermates (Fig. 6E), whereas total IgG2a (Fig. 6F) and 745 IgG3 (Fig. 6G) were on par. By flow cytometry (Fig. S7), we verified that main lymphocyte 746 subsets were comparable between groups across all tissues (Fig. 6H-J). Furthermore, the 747 frequency of idiotype positive cells, i.e., cells carrying the knock-in B cell receptor (identified 748 by staining with the anti-idiotype antibody 9D11) was comparable between groups (Fig. 6K). 749 We verified that GCs were indeed efficiently blocked in GC block mice (Fig. 6L), but despite 750 this, overall PB/PC frequencies (Fig. 6M) and PB frequencies (Fig. 6N) across tissues were not 751 different between the two groups. PC frequencies were similar in IngLN and MesLN, and 752 slightly elevated in the spleen of GC block mice (Fig. 6O). Thus, overall, the GC block did not 753 prevent any of the autoimmune read-outs. This was also the case at the later time point, 754 where no gross differences were observed (Fig. S6).

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Taken together, this demonstrated that the GC block failed to curb the main hallmarks of 757 autoimmunity presented by the 564Igi model, further corroborating our findings from the 758 Resiquimod model.     GCs are believed to be the nexus of autoreactive responses in a range of autoimmune 804 diseases. Due to their role in potent antibody responses, memory generation, and long-lived 805 PC formation, there has been extensive interest in targeting GCs in autoimmune disease. The 806 strategy has proven useful in autoimmune models, but due to off-target effects, did not 807 initially progress through clinical trials (Degn et  is a key mechanism of anti-DNA autoreactivity (Soni et al., 2020), and it has been argued that 812 more attention should be paid to the non-GC responses, as these may play a critical role in 813 humoral immunity in both mice and men (Jenks et al., 2019). 814 815 Here, we took an unbiased approach and asked to what extent a genetic block of GCs would 816 ameliorate autoreactive manifestations in a lupus-like disease model. To our surprise, we 817 found blocking GCs did not lessen autoreactive manifestations, but in some cases worsened 818 these. Upon autoimmune induction, we observed a trend towards an increase in anti-dsDNA 819 antibodies of the IgG2c isotype (Fig. 1C) and total IgG2c antibody ( Fig. 1D) in GC block mice, 820 compared to WT. These changes were also mirrored by a significantly higher frequency of 821 PB/PCs in the spleens of GC block mice, despite a total absence of GCs. In agreement with 822 this, we observed robust deposition of immune complexes in the kidney glomeruli of GC block 823 mice, at least on par with that of GC sufficient controls (Fig. 3). This indicated that the 824 extrafollicular pathway could compensate, and in some cases even augment, the autoreactive 825 response. We corroborated this central finding in a second, independent model (Fig. 6), 826 confirming that the observed sufficiency of the extrafollicular response was not model-827 specific. 828 829 To understand the B cell intrinsic effect of a GC block, we leveraged an induced GC B cell 830 culture system. It has previously been noted that Bcl-6 expression can inhibit apoptosis in 831 numerous cell types including (GC) B cells (Kumagai et al., 1999;Kurosu et al., 2003; Phan and 832 Dalla-Favera, 2004). Yet, contrary to expectations, GC block B cells did not display a 833 significantly increased propensity to undergo apoptosis ( Fig. 4H and L), rather, they much 834 more readily underwent terminal differentiation to PCs, and had a dramatically reduced 835 capacity to expand compared to their wild type counterparts (Fig. 4). However, this agreed 836 well with the established cross-regulation between Bcl-6 and Blimp-1, the master regulator 837 of the PC fate, also known as Prdm1 (Vasanwala et al., 2002). Although the increased 838 propensity for terminal PC differentiation was, in principle, well in line with our in vivo 839 observations, the lack of proliferative capacity was at the same time at odds with the dramatic 840 PC output in the mice harboring a GC block in B cells. This suggested that the PC differentiation 841 process in mice displaying a global GC block in B cells might be dysregulated, potentially as a 842 consequence of absence of GC-derived antibody feedback, as previously suggested for GC B 843 cells (Zhang et al., 2013). To address this possibility, we asked whether B cells with a GC block 844 would be precluded from contributing to the PC pool in a GC sufficient environment. Our 845 findings demonstrated that this was not the case, although the relative contribution of GC 846 block B cells to the PC pool was smaller than that of GC sufficient B cells (Fig. 5). However, 847 given their inability to expand in GCs, the magnitude of the contribution of GC block B cells to 848 the PC compartment in direct competition with GC-sufficient B cells was remarkable. In the 849