Neutralizing antibody responses develop within germinal centers (GCs); histological structures that arise within lymphoid tissues due to antigen-driven clonal B cell expansion. GCs are also thought to represent key sites for breach of tolerance in development of autoimmune disease (1) . GC B cell responses rely on T follicular helper (Tfh) cells, which support B cell expansion, facilitate antibody affinity maturation and eventually select GC B cells into the compartments of long-lived plasma cells or memory B cells (2) .
Type I interferons (IFNs), including a single IFN-b and several IFN-a proteins, are rapidly produced in response to viral and bacterial infections and signal through the common and ubiquitously expressed heterodimeric IFN-a receptor (IFNAR) (3) . Initially discovered for their ability to induce the "antiviral state" in host cells, type I IFNs are also associated with a plethora of immune-regulatory functions essential for antiviral immunity (4) . The importance of type I IFNs in controlling SARS-CoV-2 and their role in preventing severe disease was also recently demonstrated (5, 6) . Furthermore, elevated type I IFN production is a hallmark of systemic autoimmune diseases, with a strong correlation between the IFN gene expression signature and clinical manifestations in SLE (7) (8) (9) (10) . When co-injected with a protein antigen, IFN-a has sufficient adjuvant activity to induce GC formation (11) and GC B cell responses have been shown to depend on type I IFNs both during viral infections (12) and in autoimmune models (13, 14) . While we and others have shown that type I IFN signaling in cDCs augments generation of Tfh cells (15, 16) , direct signaling in B-and T-cells has also been implicated in type I IFN-dependent enhancement of humoral immunity and autoimmunity (11, 12, 14, 17, 18) . The relative contribution of these pathways, and how they interact with the type I IFN -cDC -Tfh cell axis to enhance and modulate the GC response, is however unclear. Different IgG subclasses are associated with distinct effector mechanisms. In C57BL/6 mice, complement-fixing IgG2c (IgG2a in the BALB/c strain) is more efficient than other subclasses in neutralizing viruses (19) and is also associated with more severe pathology in lupus models (20) . The transcription factor T-bet induces class switch recombination (CSR) to IgG2a/c (21) .
T-bet expression in B cells appears to have effects beyond CSR (22) and was recently shown to be required for generation of protective anti-influenza stalk region-specific antibodies (23) .
Mouse IgG2a/c may hence represent a useful surrogate marker for antibody responses driven by T-bet expressing B cells, with relevance for the human setting. Purified IFN-b can enhance production of all IgG subclasses in mice and a similarly broad and type I IFN-dependent isotype distribution is induced through the adjuvant effects of the synthetic dsRNA analogue polyinosinic-polycytidylic acid (poly I:C) (24) . Other studies suggest that type I IFNs preferentially promote IgG2a/c (25, 26) , and B cell-intrinsic type I IFN signaling mediates IgG2a/c CSR in the T cell-independent response to NP-Ficoll (27) . The importance of this pathway in GC B cell responses is however less clear and the IgG2a/c subclass has more frequently been associated with strong Th1 immunity and IFN-g production (28, 29) .
Type I IFNs can both inhibit and promote generation of IFN-g producing Th1 cells, with a general trend that sustained type I IFN responses during chronic infections suppress Th1 immunity (4) . Through binding to endosomal TLR3 and the cytoplasmic RNA helicase MDA5, poly I:C instead triggers a transient systemic type I IFN response that peaks 3-6 hours after injection (30, 31) , and an almost identical pattern in type I IFN production is observed for novel mRNA vaccination regimens (32) . In contrast to the chronic infection models, this short-lived type I IFN response leads to Th1-biased immunity with abundant IFN-g production from CD4
and CD8 T cells and in particular from NK cells (30) . How this IFN-g response develops, and what impact it has on GC B cell differentiation remains to be determined.
In the current study we have identified the pathways by which a succinct type I IFN response induced by poly I:C drives formation of GCs characterized by a broad IgG subclass distribution.
Our experiments also reveal the trajectories downstream of type I IFNs that promote T-bet expression and IgG2c CSR in developing GC B cells.
Reduced GC B cell response in Ifnar1 -/mice involves loss of both IgG1 + and IgG2c + GC B cells.
To determine how type I IFNs affect GC B cell differentiation at the level of individual IgG subclasses, Ifnar1 -/mice were immunized i.p. with OVA and poly I:C. To visualize concurrent CD4 T cell responses (see below), all mice received 5000 CD45 congenic OT-II cells. Thus, all cells are unable to respond to type I IFNs in these recipients except for a small and physiologically relevant number of antigen-specific CD4 T cells. In contrast to the strong splenic GC B cell response detected in wt controls after eight days, immunization of Ifnar1 -/mice did not lead to an increase in percentage or number of GC B cells ( Fig. 1A and B). The percentage and number of GC B cells were however similar between unimmunized wt and Ifnar1 -/mice ( Fig. 1A and B) , which is in agreement with reports of an important role for B cell IFN-g receptor signaling, and not type I IFNs, in driving spontaneous GC formation in wt C57BL/6 mice (33, 34) . The response induced by immunization of wt mice involved equal percentages ( Fig. 1C and D) and numbers (Fig. 1E ) of IgG1 + and IgG2c + GC B cells. Due to the robust magnitude of this response, pre-existing GCs made a very small and negligible contribution to the isotypes expressed by GC B cells in immunized wt mice. In Ifnar1 -/mice, we observed a significantly increased percentage of IgG1 + GC B cells after immunization, indicating that an IgG1 + GC B cell response to some extent had developed also in the absence of type I IFN signaling (Fig. 1 C and D) . However, in this set of experiments we could not detect a corresponding increase in the number of IgG1 + GC B cells (Fig. 1E ). There was no significant increase in either percentage ( Fig. 1C and D) or number (Fig. 1E ) of IgG2c + GC B cells in immunized as compared to non-immunized Ifnar1 -/mice.
The majority of antigen-specific plasma cells (PCs) present in spleen around eight days after immunization are thought to be derived from GCs (35) . The total number of splenic OVAbinding CD138 + PCs was approximately 10-fold lower in Ifnar1 -/compared to wt mice (Fig. 1F and G) , and both IgG1-and in particular IgG2c-producing cells were affected (Fig. 1H ). In addition, the few IgG1 + PCs present in Ifnar1 -/mice appeared to produce antibodies of lower affinity than their counterparts in wt animals, as indicated by fewer OVA binding IgG1 cells binding high levels of OVA ( Fig. 1I and J). These results were further corroborated by serological studies where Ifnar1 -/mice essentially failed to mount an NP-specific IgG2c response after administration of NP-conjugated OVA plus poly I:C whereas their NP-specific IgG1 titers were reduced only 6.9-and 4.2-fold at two and four weeks after immunization, respectively ( Fig. 1K and L). Antigen-specific IgG1 titers however then declined more rapidly in the Ifnar1 -/mice and after one year they had an almost 80-fold lower NP-specific IgG1 titer than wt controls, indicating that the longevity of the specific IgG1 response was affected in the absence of type I IFN signaling (Fig. 1K ). Collectively these results demonstrate that type I IFNs are essential for GC formation induced by poly I:C, affecting both the quantity and quality of the response and, moreover, demonstrate that type I IFN signaling promotes the generation of both IgG1 + and, in particular, IgG2c + GC B cells.
both reduced in Ifnar1 -/mice.
We have previously shown that early Tfh cell fate commitment is reduced in Ifnar1 -/mice (as assessed three days after immunization) (15) . To assess the relationship between Tfh and Th1 cell development, and to determine how type I IFN impacts on the accumulation of these subsets at the peak of the GC reaction, we analyzed transferred OT-II cells in the same cohorts of mice as described above. Ifnar1 -/mice had fewer total OT-II cells in their spleens as compared to wt controls, confirming that type I IFNs enhance expansion and survival of activated CD4 + T cells (36, 37) (Fig. 2A) . Furthermore, Tfh cells were additionally affected in the Ifnar1 -/mice, as evident from a significantly reduced percentage of CXCR5 high Bcl6 + OT-II cells (Fig. 2B and C) . T-bet drives Th1 cell differentiation and is required for IFN-g expression by CD4 + T cells (38) . OT-II cells with detectable T-bet expression were confined to the Bcl6subset in the wt animals and in agreement with previously published results (30) IFN-g expression was essentially lost in the Ifnar1 -/recipients, as was expression of T-bet ( Fig. 2D-G) . Collectively, these results show that Tfh cells and IFN-g producing Th1 cells exist as mutually exclusive subsets at the peak of the GC reaction and that both subsets depend on type I IFNs for their development.
Early IFN-g derived from cognate CD4 + T cells acts directly on B cells to drive IgG2c CSR without enhancing the overall magnitude of the GC B cell response.
IFN-g represents a well-established in vitro switch factor for the IgG2a/c subclass (39) . B cell intrinsic IFN-g signaling can also underlie GC formation in murine autoimmune models (33, 34) . To determine the role of B cell intrinsic IFN-g signaling in the GC B cell response driven by OVA/poly I:C, we made mixed bone marrow (BM) chimeric mice. Irradiated wt mice were grafted with wt BM (CD45.1 + , CD45.2 + ) mixed with Ifngr1 -/or wt control BM (both CD45.2 single positive) at a 1:1 ratio. Analysis of splenocytes eight days after immunization showed that GC B cells developed equally well from Ifngr1 -/and wt B cells ( Fig. 3A and B), demonstrating that IFN-g signaling in B cells is not involved in type I IFN dependent GC B cell expansion. In contrast, CSR to IgG2c was strongly reduced in the Ifngr1 -/as compared to wt GC B cells present in the same mouse ( Fig. 3A and B ). In agreement with the ability of IFN-g to inhibit CSR to IgG1 and IgE (28, 39) , the impaired IgG2c CSR in Ifngr1 -/-B cells was compensated by an increased percentage of IgG1 + GC B cells ( Fig. 3A and B) . Given that IL-27 represents an additional IgG2a/c switch factor (40) and can be produced by myeloid cells in response to type I IFNs (41, 42) , we performed analogous experiments with mixed wt/Il27ra -/-BM chimeras. These experiments did however not reveal any significant differences between wt and Il27ra -/-B cells ( Fig. S1A and B ).
To determine if IFN-g from cognate CD4 + T cells is sufficient for driving IgG2c CSR in B cells, we transferred IFN-g sufficient or deficient OT-II cells into Ifng -/mice and again evaluated the splenic response eight days after immunization. Wt and Ifng -/-OT-II cells had expanded and differentiated into Tfh cells equally efficient in the Ifng -/recipient mice ( Fig. 3C and D).
Additionally, IFN-g-deficient OT-II cells supported a GC B cell response of a comparable magnitude as their wt counterparts (Fig. 3E ). However, compared to mice receiving wt OT-II cells, recipients of Ifng -/-OT-II cells developed GCs with a strong and selective reduction in IgG2c, again compensated by an increase in IgG1 ( Fig. 3F and G). These experiments thus demonstrate an important role for IFN-g derived from cognate CD4 + T cells in stimulating IgG2c CSR, but not for enhancing the overall magnitude of the GCs.
IFN-g producing Tfh cells have been suggested to underpin IgG2a/c CSR within GCs (43) .
Given that Tfh cells largely lacked expression of T-bet at the peak of the poly I:C driven GC response (see Fig. 2 D and E), we considered the possibility that IFN-g stimulates IgG2c CSR at an earlier stage and before evident GC formation. To assess this, we injected an IFN-g neutralizing antibody either before or 72 hours after immunization. Regardless of the timing, this treatment had no impact on the percentage of total GC B cells detected eight days after immunization and staining with fluorescently labeled OVA confirmed an equal percentage of OVA-specific GC B cells in both experimental groups and in mice receiving an isotype control mAb ( Fig. 3H -J). However, treatment with anti-IFN-g before immunization resulted in significantly fewer OVA-specific GC B cells expressing IgG2c as compared to isotype control treated mice ( Fig. 3K and L). This reduction was again reciprocated by an increased percentage of cells expressing IgG1 (Fig. 3K and L) . No such effects on the IgG subclass distribution were observed when treatment with anti-IFN-g instead was started 72 hours after immunization ( Fig.   3K and L). Accordingly, IFN-g acts on B cells within the first 72 hours after immunization to initiate the IgG2c CSR process and thereafter appears to be redundant for the IgG2c associated GC B cell response.
The finding that B cell intrinsic IFN-g signaling involved in IgG2c CSR precedes GC formation predicts that 1) type I IFN dependent priming of IFN-g producing Th1 cells also occurs early in the response and 2) cognate IFN-g producing CD4 T cells appear earlier than detectable GCs.
To test the first prediction, we neutralized type I IFN signaling by injecting an anti-IFNAR mAb either 16 hours before or 24 hours after immunization. Neutralization of type I IFN signaling before, but not after, immunization inhibited OT-II cell expansion and differentiation into Tfh cells as assessed day eight ( Fig. 4A and B). As predicted, IFN-g production by transferred OT-II cells was completely inhibited at this time point (Fig. 4C ). Consistent with the reduced number of Tfh cells, the overall magnitude of the GC B cell response was reduced when anti-IFNAR treatment preceded immunization (Fig. 4D) . Additionally, the numbers of IgG2c + GC B cells were reduced after early but not late IFNAR blockade (Fig. 4E ). In the same type of neutralization experiment we also confirmed that NP-specific IgG2c was reduced > 10fold in sera 14 days after immunization when the mice received anti-IFNAR mAb before, but not 24 hours after, immunization (Fig. 4F ). The type I IFN dependent signaling event that underlies Th1 development and IFN-g dependent IgG2c CSR is therefore initiated during the first 24 hours of the poly I:C driven response.
To confirm that IFN-g producing Th cells appear within the first 72 hours post-immunization, and to verify their dependence on upstream type I IFN signaling, we analyzed early Th cell differentiation in wt and Ifnar1 -/mice, respectively. IFN-g producing OT-II cells were indeed detectable in wt mice 3 days after administration of OVA and poly I:C and the IFN-g response was reduced in the Ifnar1 -/mice also at this earlier time point (Fig. 4G ). This correlated with significantly higher Tbx21 mRNA (Fig. 4H) and T-bet protein expression ( Fig. 4 
OT-II cells recovered from wt compared to Ifnar1 -/recipients. OT-II cells recovered from wt animals did however not express T-bet and Bcl6 in a mutually exclusive manner indicating the absence of clear Th1 versus Tfh cell dichotomy at this early stage of the response (Fig. 4I ).
Based on these results we conclude that type I IFNs drive development of the IFN-g producing CD4 T cells that underpin IgG2c CSR within the first few days after immunization, prior to evident GC formation and appearance of fully committed Th1 and Tfh cells.
To determine how and to what extent direct type I IFN signaling in B cells influences the GC B cell response and IgG subclass selection after immunization with OVA/poly I:C, we again generated mixed BM chimeras, now by reconstituting irradiated wt mice with a 1:1 mix of wt with Ifnar1 -/or wt (control) BM. Analysis of immunized chimeric mice revealed that Ifnar -/-B cells contributed to the GC B cell response to a lesser extent than IFNAR sufficient B cells present in the same animal. In this competitive setting, there was an approximately 2-fold lower number of Ifnar1 -/than wt GC B cells in the spleen ( Fig. 4K and L) . Strikingly, this reduction was mostly caused by a loss in IgG2c + and IgG2b + GC B cells; the frequency of GC B cells expressing either of these IgG subclasses was approximately 2-fold lower in the Ifnar1 -/than wt compartment of the same chimeric animal ( Fig. 4K and L) . Altogether, these results show that type I IFN augments the IgG2c + and IgG2b + GC B cell response through direct signaling in B cells and that, in contrast to IFN-g, this effect goes beyond CSR and enhances the overall magnitude of the GC B cell response. To determine if IFN signaling had altered the core GC B cell program, we first compared these sequencing datasets to the transcriptional changes in GC B cell compared to naïve follicular B cells described in Shi et al. (44) . We found that the GC B cell transcriptional signature was largely intact in the absence of either type I or type II IFN signaling ( Fig. S2A and B) . Key GC B cell transcriptional changes, including mRNA encoding core transcription factors (Bcl6 and Bach2), proteins involved in the somatic hypermutation program (Aicda and Polh), as well as proteins directing migration and localization of the B cells to the GC (Sipr2, Gpr183), were unaffected by the loss of B cell intrinsic IFN signaling. This suggests that type I IFN or IFNg signaling in B cells is not critical for the GC B cell program. We also compared our IFNAR dataset to the early type I IFN induced transcriptional changes in follicular B cells described by Mostavi et al. (45) . Out of 71 genes that showed statistically significant differences in expression with at least a 2-fold change after IFN injection, only six were dysregulated in GC B cells at day eight after immunization with OVA and poly I:C Fig. S2C ). This suggests that while a part of the IFN signature may be robustly conserved after IFN signaling has stopped a majority of the IFN depended transcriptional changes are transient.
For both the IFNAR and IFNGR dataset, we filtered genes that displayed at least a 2-fold and statistically significant difference in expression between the WT and IFN receptor deficient cells ( Fig. 5A and B) . By this approach, both cytokine families were found to influence a relatively limited number of genes, with only 87 and 72 genes being affected by type I IFN and IFN-g signaling, respectively (Fig. 5C ). While the type I and type II IFN gene expression signatures have been difficult to separate when assaying peripheral blood during infections or in autoimmunity (46, 47) , only seven genes were found to be dysregulated in both the IFNAR and IFNGR datasets. Of these, upregulation of three genes, (Ctse, Tbx21, and Igh2c) were dependent on both IFNAR and IFNGR signaling. The downregulation of these genes in IFN receptor deficient B cells was confirmed by qrt-PCR analysis from the same sorted GC B cell populations ( Fig. 5D and E) . This suggests that B cell intrinsic type I IFN and IFN-g signaling predominately affect different aspects of the GC B cell response but that the two pathways synergize to control class switching to IgG2c, likely through the induction of Tbx21 (21) . In addition to IgG2c, the differential gene expression identified in the two data sets indicated that both type I IFN and IFN-g signaling in B cells affect the expression of other Ig isotypes. While both may have a small impact on IgG2b expression, we found type I IFN signaling to be associated with increased Ighg3 mRNA expression. IFN-g signaling was instead required for optimal Igha expression. Thus, while type I IFN and IFN-g signaling collaboratively support switching to IgG2c, and possibly IgG2b, they may have unique roles in controlling switching to other isotypes.
Given that type I IFNs are required for optimal generation of both Tfh and Th1 cells in poly I:C/OVA immunized mice, we next set out to determine the cellular targets for type I IFN signaling in the respective differentiation pathways. To this end we co-transferred CTV-labeled wt and Ifnar1 -/-OT-II cells into BM chimeras, lacking IFNAR in either the hematopoietic, the non-hematopoietic (radio resistant) or both cell compartments. Cell cycle dependent dilution of CTV was examined three days after immunization. The ability of type I IFNs to enhance CD4 T cell proliferation tracked with IFNAR expression by the BM donor cells (i.e. hematopoietic non T cell-intrinsic) ( Fig. 6A and B) , likely reflecting the ability of type I IFNs to improve the antigen-presenting function and co-stimulatory capacity of APCs (15, 30, 48, 49) . No change in CTV dilution was observed when comparing wt and Ifnar1 -/-OT-II cells or wt and Ifnar1 -/irradiated recipient mice, demonstrating that T cell-intrinsic type I IFN signaling or signaling in radio resistant cells has no impact on early T cell proliferation under these conditions ( Fig.   6A and B). The ability of type I IFNs to enhance Th1-associated T-bet and IFN-g ( Fig. 6C and D) as well as Tfh cell associated Bcl6 and CXCR5 ( Fig. 6E and F) expression was also mostly a result of IFNAR expression on the BM donor cells. However, both T-bet and Bcl6 expression was slightly higher in Ifnar1 -/than wt OT-II cells, indicating that type I IFNs to some extent can counteract both Th1 and Tfh cell development through direct effects on T cells (Fig. 6C and E). These effects were however subdominant to the enhanced T-bet and Bcl6 expression driven by T cell extrinsic type I IFN signaling in hematopoietic cells. In conclusion, these results indicate that IFNAR on cognate CD4 T cells or in the non-hematopoietic cell compartment is redundant for both Th1 and Tfh cell fate commitment and hence unlikely to have a major impact on the GC B cell response through these pathways.
We have previously demonstrated reduced Tfh cell differentiation in CD11c-cre.Ifnar1 fl/fl mice with specific deletion of Ifnar1 in cDCs (15) . Here, we wished to determine how conditional deletion of Ifnar1 in cDCs impacts on the IgG subclass composition within GCs. In particular, while results presented so far reveal how B cell intrinsic type I IFN signaling acts in synergy with the switch factor IFN-g, produced from cognate CD4 T cells, to enhance IgG2c associated GC B cell responses, it was still unclear why IgG1 + GC B cells also are strongly reduced in the complete IFNAR knockout (see Fig. 1 ). Selection of the IgG1 subclass is promoted by and within secondary lymphoid organs IL-4 secreting T cells are largely confined to the Tfh cell subset (43, 50) . To visualize active secretion of IL-4 from cognate CD4 T cells, we intercrossed the OT-II strain with KN2 mice, reporting IL-4 secretion through expression of membrane anchored human CD2 (51) . Similar to OT-II cells activated in wild type C57Bl/6 recipients (see Fig. 2D ), donor KN2-OT-II cells developed into mutually exclusive Th1 (Tbet + ) and Tfh (Bcl6 + ) cell subsets eight days after immunization of Ifnar1 fl/fl control mice (hereafter referred to as Crecontrol mice) while in CD11c-Cre.Ifnar1 fl/fl mice both subsets were significantly reduced in frequency and number (Fig. 7 A-C) . In the Crecontrol group, active IL-4 secretion was largely confined to the Bcl6 + Tfh cells and although their capacity to produce IL-4 appeared to be at least partially maintained in CD11c-Cre.Ifnar1 fl/fl recipients ( Fig. 7D and E) , the number of IL-4 secreting Tfh cell was dramatically reduced due to the overall weakened Tfh cell response in these mice (Fig. 7F) . Consistent with the impaired generation of T-bet + Th1 cells, IFN-g producing OT-II cells were also strongly diminished in CD11c-Cre.Ifnar1 fl/fl mice (Fig. 7G) . Collectively, these results demonstrate a bifunctional effect of type I IFN signaling in cDCs to promote the appearance of separable IFN-g producing Th1 and IL-4 producing Tfh cell subsets.
Similar to the complete Ifnar1 knockout, the expansion of GC B cells was strongly reduced after immunization of CD11c-Cre.Ifnar1 fl/fl mice compared with their Crecontrols (Fig. 7H) .
The requirement for IFNAR-expressing cDC was equally evident when analyzing the number of OVA-binding GC B cells in the two groups of mice, confirming that the reduction in GC B cells was related to the immunization rather than differences in pre-existing GC B cell numbers ( Fig. 7I and J). In marked contrast to when B cells specifically lack IFNAR, the reduced GC B cell response in the absence of IFNAR expressing cDCs was not caused by a selective loss of IgG2 expressing GC B cells; IgG1 + GC B cells were now at least equally affected, again confirmed by analysis of OVA-binding GC B cells ( Fig. 7K and L) .
To determine if IFNAR signaling in cDC1 was required for any of the effects that type I IFNs have on Th1, Tfh and GC B cell differentiation, we immunized XCR1-cre.Ifnar1 fl/fl mice (52) as above and examined the OT-II cell response ( Fig. S3A and B) . No difference in OT-II cell expansion or differentiation to Th1 and Tfh cells was observed at day 8 post-immunization.
Similarly, no effects on GC B cell expansion or generation of OVA-specific IgG1 + or IgG2c + GC B cells were observed ( Fig. S3C and D) .
Finally, we examined PC generation in immunized CD11c-Cre.Ifnar1 fl/fl mice. Similar to the GC B cell response in these mice we found reduced numbers of OVA-specific PC at day eight post-immunization (Fig. 7M) . While the total number of OVA-binding PC thus was more than 10-fold lower in CD11c-Cre.Ifnar1 fl/fl mice as compared with Crecontrols, the reduction was most pronounced for the IgG1 producing subset, as evident from a significant reduction in the percentage of IgG1 + but not IgG2c + events when comparing the OVA-specific PC that had developed in the two mouse strains (Fig. 7N and O) . Of note, the reduced affinity of PCs developing in the complete IFNAR knockout was not apparent in the absence of IFNAR on cDCs, indicating that type I IFN signaling in B cells may underlie this effect (Fig. 7P ).
Altogether these results show that expansion of IL-4 secreting Tfh cells, through type I IFN signaling in cDCs, represents a third pathway whereby type I IFNs regulate the GC response, providing an explanation to how type I IFN in addition to its pronounced effect on IgG2 also amplifies the IgG1 response.
Type I IFNs possess a wide range of immune stimulatory properties with relevance for antiviral immunity, vaccination, and systemic autoimmune disease. Here, we have dissected how a succinct type I IFN response induced by dsRNA drives GC formation and IgG subclass specification. By selectively blocking receptor signaling in defined target populations or permitting signaling to occur only during defined time windows, we demonstrate that type I IFNs primarily act on B cells and cDCs to rapidly program the B and T cell responses underlying formation of GCs and class switching to a broad IgG subclass distribution. In addition to enhancing generation of Tfh cells and long-lived IgG1 responses, type I IFNs induce
Our results show that type I IFNs act on cDCs within the first 24 hours after immunization to initiate concurrent Tfh and Th1 cell differentiation. Signaling in the cDC1 subset was redundant for both processes. It therefore seems likely that type I IFN signaling in cDC2 plays an important role for both Th cell fates, although we have not addressed this directly through cDC2 specific deletion of IFNAR. The demonstration that type I IFNs enhance both GC B cell responses and Th1 immunity stands in marked contrast to their immune suppressive role in chronic viral infections (4) . The LCMV clone 13 strain establishes a persistent infection where long-lasting expression of IFN-b and IFN-a in cDCs leads to exhaustion of the protective Th1 cell response by a mechanism involving up-regulation of PD-L1 and IL-10 expression by the cDCs (53, 54) . Likewise, the Th1-promoting property of cDC2 is suppressed by an excessive type I IFN response during severe blood-stage Plasmodium infection (55) . The stimulatory effects of type I IFNs described in the current study are hence likely related to the short duration of the type I IFN response induced by poly I:C, a notion further supported by the beneficial effects these cytokines have in acute viral infections, including vesicular stomatitis virus (VSV) (18, 56) , influenza (17), RSV (57) and adenovirus (12) . Still, the ability of type I IFNs to concomitantly drive Th1 and Tfh cell development is not necessarily recapitulated in the acute viral infection models. While VSV similar to poly I:C gives rise to a rapid and transient peak of type I IFNs that stimulates generation of Tfh cells, this occurs at the expense of Th1 cell polarization (16) . One explanation for this apparent discrepancy could be the mixed proinflammatory response induced by viral sensing through multiple innate receptors (58) . The adjuvant effect of poly I:C is on the other hand completely lost in IFNAR -/mice, as shown herein, and injection of purified IFN-b replicates the immune stimulatory effects of poly I:C when co-injected with a protein antigen (24) . The induction of T-bet associated GCs and longlived IgG responses through the isolated effect of type I IFNs should be of considerable interest for the development of adjuvants to enhance antiviral vaccine efficacy. IgG2a/c represents the dominant anti-viral IgG subclass in mice (59) and B cell-specific T-bet deletion leads to impaired antiviral IgG2a/c production and viral clearance (60), as well as in an inability to contain chronic viral infection (61) . In addition, protective antibody responses against influenza were recently demonstrated to rely on T-bet associated GCs (23) . The current study may also contribute towards understanding the immunogenicity of emerging mRNA vaccine approaches (62) . Similar to poly I:C, mRNA vaccines induce a short-lived type I IFN response (32) , and currently two of twelve SARS-CoV-2 vaccine candidates in phase 3 trials are mRNA-based (63) and induce specific IgG serum concentrations higher or equivalent to the levels detected in convalescent sera (64, 65) .
While antiviral immunity frequently has been associated with both type I IFN and IgG2a/c antibody production, it has not been clear how type I IFNs promote IgG2a/c dominated GCs. B cell-intrinsic type I IFN signaling was recently shown to be crucial for spontaneous development of IgG2c + GCs in lupus prone B6.Sle1b mice (14) . GC formation was however not affected by lack of type I IFN signaling when the same lupus-prone strain or wt mice were immunized with NP-conjugated chicken g-globulin (14) , probably reflecting an insufficient type I IFN response to this particular immunization regimen. In the current study, we show that IFN-g production from cognate CD4 T cells, which requires type I IFN signaling in cDCs, is a critical component of the type I IFN-dependent IgG2c + GC B cell response. This pathway was however not sufficient but acted in concert with direct sensing of type I IFNs by the B cells and both pathways contributed to the induction of T-bet expression in GC B cells. Yet, the effects of type I IFNs and IFN-g differed. While IFN-g acted as a non-redundant IgG2c switch factor, only signaling through IFNAR amplified GC B cell expansion with specific effects on IgG2c + and IgG2b + GC B cells. How this occurs and why the effect was confined to GC B cells expressing the IgG2 subclasses remains to be determined. However only early, and not late, IFNAR neutralization resulted in reduced IgG2c + GC B cell numbers. Likewise, only few of the roughly 70 genes previously shown to be induced in B cells following type I IFN treatment (45) were affected in IFNAR deficient GC B cells eight days after immunization, further supporting that type I IFNs acted on the B cells early in the response, possibly during initial B cell activation. Indeed, type I IFNs have been shown to confer increased sensitivity to BCR stimulation and to promote B cell expansion by both enhancing proliferation and reducing sensitivity to apoptosis (14, 66) .
Whereas targeted deletion of IFNgR in B cells, IFN-g in cognate CD4 T cells or IFNAR in cDCs resulted in similar reduction in IgG2c + GC B cells, IFNAR deletion in cDCs additionally impaired development of IgG1 + GC B cells and resulted in an overall reduced magnitude of the GC response. This coincided with reduced Tfh cell development, with a particularly pronounced effect on IL-4 producing Tfh cells. Tfh cells thus became IL-4 producers also under the strong Th1 polarizing conditions otherwise conferred by poly I:C and downstream type I IFN production. In addition, T-bet was absent from the Tfh cells around the peak of the GC reaction, indicating limited Th1 cell characteristics of the Tfh cell subset. Consistent with this, we demonstrate that both IFN-g and type I IFN acted on B cells very early after immunization and induced IgG2c CSR prior to evident GC formation. These results are in agreement with previous studies, demonstrating that CSR mostly precedes GC formation (67) (68) (69) . Nonetheless, similar to viral infection models (70) (71) (72) , Bcl6 and T-bet were co-expressed by the T cells at this early stage. We could hence not identify divergent Th1 versus Tfh cell commitment at the time when B cells were receiving the IgG2c switch signals. Tfh cells with a history of T-bet expression have been shown to produce IFN-g within established GCs (73) . While it remains possible that equivalent IFN-g producing T-bet -Tfh cells were present within the GCs studied herein, late IFN-g neutralization had no detectable effect on the magnitude or IgG subclass composition of the GC response. This indicates that the IgG2c + GC B cells did not rely on continuous IFN-g signaling within the GCs.
In summary, the current study describes how type I IFNs through at least three separate pathways can enhance and modulate the GC B cell response. Our results provide a detailed roadmap of how this family of cytokines confers long-lived humoral immunity. Exploiting the type I IFN dependent pathways identified herein could provide a means to enhance efficacy of e.g. mRNA vaccine regimens and to prolong the duration of vaccine-induced protection. On the other hand, the relative contribution of these pathways to onset of systemic autoimmune disease warrants further investigations.
This study sought to determine pleiotropic effects and identify the cellular targets of acute type 
To generate mixed BM chimeras, BM cells from age matched (8-12 weeks) wt, Ifnar1 -/-, Il27r -/or Ifngr1 -/donor mice were isolated and re-suspended in sterile PBS. A 1:1 mixture of wt and Ifnar1 -/-, Il27r -/or Ifngr1 -/-BM cells (2-3x10 6 total cells) were transferred into lethally irradiated (900 cGy) recipients (CD45.1 + CD45.2 + B6.SJL x C57Bl/6 or CD45.1 + B6.SJL). Recipient mice were thereafter kept on Ciprofloxacin for 2 weeks. At 8 weeks after transfer, mice were bled to assess reconstitution by flow cytometry. Whole BM chimeric mice were generated by reconstituting irradiated wt (C57Bl/6) and Ifnar1 -/mice with wt or Ifnar1 -/-BM, otherwise as described above.
Flow-cytometry analyses were performed with Abs conjugated to FITC, PE, PerCP-Cy5. 
Single cell suspensions were prepared by mechanical disruption and filtered through 70µm cell strainers. RBCs were lysed with ACK buffer. For IgG analysis, cells were blocked with anti-FcR mAb (2.4G2) in 10% rat serum and thereafter incubated with isotype specific anti-IgG antibodies (see Abs and reagents). Remaining anti-mouse IgG reactivity were subsequently blocked with 10% mouse serum before incubation with fluorophore-conjugated mAbs. CXCR5 was detected as previously described (15) and followed by intracellular staining of Bcl6 and Tbet. Intracellular IFN-g was detected after re-stimulation in complete medium with PMA (50 ng/ml) ionomycin (500 ng/ml; both Sigma-Aldrich, St. Louis, MN, USA), and Brefeldin A (eBioscience) for 3 hours. All intracellular staining was done using the FoxP3 Fixation/Permeabilization kit (eBioscience). Data was acquired on a LSRII or FACS AriaII and analyzed with FlowJo software (BD Bioscience).
NP-specific serum antibodies were measured by ELISA. 96-well EIA/RIA plates (Sigma Aldrich) were coated overnight with 0.5ug/ml NP23-BSA in PBS at room temperature and thereafter washed once in wash buffer (PBS+ 0.1% Tween20) and blocked with sample buffer (1% BSA in PBS) for 1 h and thereafter washed twice. Samples were diluted in sample buffer at 1:100 and 1:5000 and subsequently added in duplicates and incubated for 2 hours. Plates were washed four times before biotinylated anti-mouse IgG, anti-mouse IgG2c (polyclonal;
both Southern Biotech), or anti-mouse IgG1 (RMG1-1) (BioLegend) was added in sample buffer, incubated for 1 h, and subsequently washed four times. Thereafter, Horseradish peroxidase (HRP) conjugated streptavidin was added and incubated for 45 minutes. After four washes, plates were developed with 3,3′,5,5′-Tetramethylbenzidine (TMB) and absorbance values were read at 450 nm on a SPECTROstar Nano (BMG Labtech, Ortenberg. Germany).
NP-specific standard was prepared by pooling day 14 sera from NP-OVA immunized WT mice and used for all ELISAs to calculate serum titers (determined as 4x background OD value).
Total OT-II cells (Live, singlet, CD4 + CD45.1 + B220 -) or GC B cells (Live, singlet, B220 + Fas + CD38 -CD45.1 + or CD45.1 -) were sorted from immunized recipients directly into RLT buffer supplemented with 1% b-ME (Qiagen, Hilden, Germany). mRNA was extracted with an RNeasy Micro Kit (Qiagen) and either used for sequencing or converted into cDNA using a SuperScript III First-Strand cDNA kit according to manufacturer's protocol (Thermo model was used for the differential expression analysis. Differentially expressed genes with a false discovery rate of <5% were considered statistically significant.
Data were analyzed with Prism version 6.0 (GraphPad Software). Analysis of statistical significance was done using one-way ANOVA with Kruskal-Wallis multiple comparison test for tree or more groups, or Mann-Whitney U test for two unpaired groups. Differences were considered significant when p ≤ 0.05 (*p£0.05, **p<0.01and ***p<0.001).