Lyn governs the establishment and maintenance of B cell anergy by suppressing PI3K signaling

Domain: chemistry
Published: 2026-03-08
Editorial outcome: major_revision

Abstract
The Src family kinase Lyn is known to be involved in the induction and maintenance of peripheral B cell tolerance; however, mechanistic separation of tolerogenic functions from the role of Lyn in B cell and myeloid cell development and activation is challenging. Here we utilize a system in which Lyn deletion is tamoxifen inducible and B cell restricted, which allows acute elimination of Lyn in B cells only, minimizing confounding factors. This genetic tool is employed in conjunction with immunoglobulin transgenic mice in which peripheral B cells are autoreactive. DNA reactive Ars/A1 B cells require continuous inhibitory signaling, mediated by the inositol phosphatase SHIP-1 and the tyrosine phosphatase SHP-1, to maintain an unresponsive (anergic) state. Here we show that Ars/A1 B cells require Lyn to establish and maintain B cell unresponsiveness via restricting PI3K-dependent signaling pathways. This Lyn-dependent mechanism complements the impact of reduced mIgM B cell receptor (BCR) expression to restrict BCR signaling in Ars/A1 B cells. Our findings thus suggest that a subset of autoreactive B cells requires Lyn to become anergic and that the autoimmunity associated with dysregulated Lyn function may, in part, be due to an inability of these autoreactive B cells to become tolerized.

Introduction
The Src family kinase Lyn plays dual roles in B cell activation 1, 2. It is important early in B cell receptor (BCR) signaling, phosphorylating the Immunoreceptor Tyrosine-based Activation Motifs (ITAM) of CD79A and B, as well as Syk and CD19 3, 4. Lyn is also important in regulatory signaling, phosphorylating Immunoreceptor Tyrosine-based Inhibition Motifs (ITIM) on inhibitory receptors, such as Fc\u03b3RIIB 5, 6, 7, 8, when these are brought into proximity of actively signaling BCRs, as well as by additional less well-defined mechanisms 9. In the absence of Lyn, other Src family kinases, such as Fyn, compensate for Lyn\u2019s role in activating BCR signals, but not inhibitory signaling 5, 7, 10. Consequently, B cell-restricted Lyn deficiency results in B cell hyperresponsiveness and a loss of B cell tolerance 11. The importance of Lyn-mediated inhibitory signaling in B cell tolerance is further demonstrated by the observation that besides Lyn deficiency 8, 11, 12, 13, deficiencies of upstream inhibitory receptors (e.g. CD22 14, 15, Fc\u03b3RIIB 16, 17 and CD72 18) as well as deficiencies of downstream effector phosphatases (SHP-1 19 and SHIP-1 20, 21) result in a loss of B cell tolerance and lupus-like autoimmunity. This pathway is often dysregulated in individuals that develop B cell-dependent autoimmunity 22, 23, 24, 25, 26, 27, 28, 29.

How Lyn contributes to peripheral B cell tolerance is not fully understood 11, 30, 31. B cell anergy is the most prevalent peripheral B cell tolerance mechanism 32. Anergic B cells exist in a state of functional unresponsiveness in the periphery 33. This anergic state is thought to result from continuous BCR stimulation with self-antigen in the absence of T cell help 34, 35. One of the characteristics of anergic B cells is that they are hyporesponsive to BCR stimulation 36. Both reduced IgM BCR surface expression 37, 38 and inhibitory signaling mechanisms 39, 40 contribute to this state of hyporesponsiveness, although the precise contribution of each mechanism is unclear. Since Lyn plays a critical role in establishing inhibitory signaling circuits in B cells, one would expect that Lyn is involved in establishing B cell anergy, but previous studies were inconclusive 30, 31.

To address this question, we used the Ars/A1 model of B cell anergy in conjunction with genetic models that allowed us to delete Lyn in a B cell specific and inducible manner. Ars/A1 B cells express a transgenic BCR that binds the hapten p-azophenylarsonate (Ars) but cross-reacts with lower affinity with DNA-containing self-antigens in vivo, rendering Ars/A1 B cells anergic 41. Previously, we demonstrated that Ars/A1 B cells require continuous activity of the inositol phosphatase SHIP-1 and the tyrosine phosphatase SHP-1 to establish and maintain an anergic state 40. Given its dependence on inhibitory signaling, we asked whether this model of B cell anergy, representing anergic B cells that depend strongly on inhibitory signaling, requires Lyn to become anergic.

We show that autoreactive Ars/A1 B cells require Lyn to establish and maintain an anergic state. Mechanistically, this is due to Lyn-dependent restriction of PI3K-dependent signaling. Impaired activation of PI3K-independent signaling events in Ars/A1 B cells is primarily a consequence of strongly reduced IgM antigen receptor expression. Taken together these findings show that Lyn plays an important role in establishing an anergic state in a subset of autoreactive B cells. It further suggests that the development of autoimmune disease associated with dysregulation of Lyn function may in part be due to compromised anergic B cells.

Results
Ars/A1 B cells require Lyn to become anergic.
To determine if Lyn is required to establish B cell anergy in the Ars/A1 model, we crossed Ars/A1 BCR transgenic mice with mice that have a B cell-restricted Lyn deficiency (mb1Cre x Lynflox/flox). In Ars/A1 BCR transgenic mice >97% of the B cells express the transgene-encoded BCR (Suppl. Fig 1C), therefore in our analysis we defined B220+ cells as Ars/A1 B cells. Compared to Lyn sufficient littermates (Lynflox/flox x Ars/A1), B cell intrinsic loss of Lyn in Ars/A1 mice (mb1cre x Lynflox/flox x Ars/A1) resulted in a drastic reduction of splenic Ars/A1 B cells (Fig 1A). This reduction is not due to aberrant B cell development, since Lyn sufficient and deficient Ars/A1 mice have comparable numbers of immature B cells in their bone marrow (Fig 1B, Suppl Fig 1A-B). Consistent with a peripheral defect the number of mature recirculating B cells was reduced in the bone marrow of Lyn-deficient Ars/A1 mice (Fig 1C). Despite having fewer B cells in the periphery, Lyn deficient Ars/A1 mice have significantly more autoantibody secreting B cells in the spleen compared to Lyn-sufficient littermates, measured both as absolute number of antibody forming cells/spleen (Fig 1D, Suppl Fig 1D) as well as normalized to the number of Ars/A1 B cells/spleen (Fig 1E). This suggests that there is a defect in the ability to tolerize Lyn-deficient Ars/A1 B cells. Phenotypically, Lyn deficient Ars/A1 B cells have slightly elevated levels of surface antigen receptors and the activation markers CD69, CD86 and CD95 (Fig 1F). Upon BCR crosslinking, Lyn-deficient Ars/A1 B cells were able to efficiently elevate intracellular calcium concentrations (Fig 1G), consistent with an inability to acquire an anergic state. Further analysis of BCR signaling revealed that some signaling events were significantly enhanced (phosphorylation of Akt and Erk) in Lyn deficient Ars/A1 B cells while other signaling events were minorly affected (phosphorylation of Syk) (Fig 1H). The B cell-restricted cre recombinase used in these studies (mb1-cre) is inserted in the CD79A locus, resulting in CD79A heterozygosity in the Lyn deficient B cells. Consistent with previous reports 21, CD79A heterozygosity does not impact the anergic state of Ars/A1 B cells (Suppl. Fig 2). Taken together these data demonstrate that Ars/A1 B cells require Lyn to establish an anergic state.

Lyn is required for the maintenance of B cell unresponsiveness by suppressing PI3K-dependent signaling events in anergic Ars/A1 B cells.
Our analysis of mb1cre x Lynflox/flox Ars/A1 mice suggests that loss of Lyn does not affect all signaling events downstream of the BCR equally (Fig 1H). To gain more insight into the mechanisms by which Lyn restricts BCR signaling in anergic B cells, we analyzed the impact of Lyn deficiency on BCR signaling in Ars/A1 B cells in more detail. To minimize confounding effects due to a lack of proper tolerization of Ars/A1 B cells during development (Fig 1), and possible adaptation to compensatory mechanisms by other Src family kinases, we used an inducible cre to delete Lyn in mature anergic Ars/A1 B cells. Ars/A1 mice were crossed with mice that express a B cell-restricted inducible cre (hCD20-CreTAM) and have a YFP reporter gene (Rosa26-stopflox YFP) and floxed Lyn locus (Fig 2A, Lyn KO Ars/A1). Seven days after tamoxifen treatment, splenic YFP+ B cells from these mice were Lyn deficient (Fig 2B). We detected predominately the LynA form (upper Lyn band) in Ars/A1 B cells. Previous reports have described a bias towards expression of the LynB (lower Lyn band) form in B cells 8, 42, an isoform that is associated with regulatory function 42. This bias towards LynA detection was due to the polyclonal anti-Lyn that we used, a commonly used anti-Lyn monoclonal, LYN-01, revealed a similar bias towards LynB expression in Ars/A1 B cells as wild type C57BL/6 B cells (Suppl. Fig 3). For controls we generated Ars/A1 mice that express an inducible Cre and have a YFP reporter and WT Lyn locus. Following tamoxifen treatment, these Ars/A1 B cells express YFP but remain Lyn sufficient (Fig 2A-B, WT Ars/A1). For comparison we also included mice with a WT repertoire of BCRs that upon tamoxifen treatment express YFP but remain Lyn sufficient (Fig 2A, WT).

Inducible deletion of Lyn did not significantly alter surface expression of IgM BCRs on Ars/A1 B cells (Fig 2C). Therefore, any observed changes in signaling in these Ars/A1 B cells are due to a loss of Lyn function and not to changes in the degree of BCR crosslinking. In our experiments we used a polyclonal F(ab’)2 anti-IgM as a stimulus to avoid any confounding effects due to Fc\u03b3RIIB recruitment. As observed in Ars/A1 mice that lost Lyn expression early during B cell development (Fig 1G), acute loss of Lyn restores the ability of Ars/A1 B cells to efficiently mobilize calcium following BCR stimulation (Fig 2D, Suppl Fig 4B). Induced Lyn-deficiency also restored (or enhanced) phosphorylation of Akt (Fig 2E, Suppl Fig 4C), S6 (Fig 2G) and Erk (Fig 2F), as well as degradation of I\u03baB\u03b1 (Fig 2H), in Ars/A1 B cells. In contrast, other signaling events (phosphorylation of Ig\u03b1 ITAM tyrosine 182 (Fig 2I), Syk (Fig 2J), BLNK (Fig 2K) and PLC\u03b32 (Fig 2L)) remained unchanged when compared to Lyn sufficient Ars/A1 B cells. These events were significantly reduced compared to na\u00efve (WT) B cells (Fig 2I-L). Lyn plays an important role in establishing inhibitory signaling pathways. Accordingly, we observed that both basal phosphorylation and induced phosphorylation, following BCR stimulation, of the phosphatases SHIP-1 and SHP-1 was impaired in Lyn deficient Ars/A1 B cells (Fig 2M).

To determine if the selective impact of induced Lyn deficiency on BCR signaling was unique to anergic B cells, we did a similar experiment using mice that have a diverse BCR repertoire (Fig 3A). We observed a similar pattern in Lyn deficient B cells where B cells have equal surface IgM expression (Fig 3B) yet have enhanced calcium responses (Fig 3C), albeit with a small delay, and increased phosphorylation of Akt (Fig 3D, Suppl Fig 5), S6 (Fig 3F) and Erk (Fig 3E), as well as degradation of I\u03baB\u03b1 (Fig 3G) , while the phosphorylation of Ig\u03b1 (Fig 3H), Syk (Fig 3I), BLNK (Fig 3J) and PLC\u03b32 (Fig 3K) is reduced and delayed following IgM crosslinking.

The enhancement of select signaling events, as a function of Lyn deficiency, suggests that Lyn plays an active role in suppressing these events. Lyn has previously been implicated in regulating PI3K-dependent signaling 9, 43. Suppression of PI3K signaling is important for B cell tolerance 21, 39, including B cell anergy. To determine which of the monitored signaling events are dependent on PI3K signaling, we treated B cells from WT C57BL/6 mice with the PI3K inhibitor Ly294002 and performed a similar analysis of BCR signaling. Interestingly, all the signaling events that were restored in Lyn deficient Ars/A1 B cells were either completely (phosphorylation of Akt (Fig 3N, Suppl Fig 6), S6 (Fig 3P) and degradation of I\u03baB\u03b1 (Fig 3Q)) or mostly (phosphorylation of Erk (Fig 3O) and calcium mobilization (Fig 3M)) dependent on PI3K signaling. The signaling events that were not impacted by Lyn deletion in Ars/A1 B cells (phosphorylation of Ig\u03b1 ITAM tyrosine 182 (Fig 3R), Syk (Fig 3S), BLNK (Fig 3T) and PLC\u03b32 (Fig 3U)) were similarly not impacted by chemical inhibition of PI3K. The small significant change in Ig\u03b1 phosphorylation (Fig 3R) upon BCR crosslinking was not a reproducible finding (Suppl Fig 7A), while BLNK (Fig 3T) phosphorylation does appears to have a minor dependence on PI3K activity (Suppl Fig 7B). Taken together these data show that continuous expression of Lyn is required to maintain BCR unresponsiveness in Ars/A1 B cells and suggests that Lyn primarily operates by regulating PI3K-dependent signaling in anergic Ars/A1 B cells. Consistent with a primary role for Lyn in regulating PI3K dependent signaling in Ars/A1 B cells we found that the presence of a PI3K inhibitor (Ly294002) suppressed the restored signaling events in Lyn-deficient Ars/A1 B cells (Suppl Fig 8).

IgM downregulation and suppression of PI3K signaling are responsible for the suppressed BCR signaling phenotype of Ars/A1 B cells.
Some signaling events are reduced in Ars/A1 B cells regardless of the presence or absence of Lyn (Fig 2I-L). Acute deletion of Lyn did not alter the surface expression of IgM BCR on Ars/A1 B cells, which remained significantly lower than most B cells within the WT repertoire (Fig 2C). Downregulation of surface IgM BCR is one of the ways by which autoreactive B cells are thought to reduce antigen receptor signaling strength to stay below an activation threshold. To determine how differences in IgM expression contribute to the difference in signaling between WT repertoire B cells and Ars/A1 B cells, we used a protocol that allows us to compare subpopulations of B cells that have equal levels of surface IgM BCR and therefore equal BCR crosslinking. Prior to stimulation with a polyclonal F(ab’) anti-IgM, we stained B cells with a non-stimulatory, fluorescently tagged Fab fragment of an anti-IgM monoclonal antibody, clone b-7-6 (Fig 4A). Analysis of the phosphorylation of the Y182 ITAM residue of CD79A shows that at a fixed dose of crosslinking antibody there is a direct correlation between the amount of IgM BCR expressed on a B cell and the magnitude of induced phosphorylation (Suppl Fig 9A). BCR internalization following BCR crosslinking did not affect the detection of fluorescently tagged Fab anti-IgM during the first 45 minutes following BCR crosslinking (Suppl Fig 9B). Therefore, we assume in our analysis that the Fab anti-IgM signal measured following stimulation represents the IgM surface expression levels at the time of stimulation.

As reported previously 41, when we compared BCR signaling in the total population of B cells, all signaling events monitored were significantly reduced in Ars/A1 B cells compared to na\u00efve WT C57BL/6 B cells (Fig 4C-K, total B cell plots). However, when we analyze subpopulations with equal IgM BCR expression, differences emerge. Some signaling events remain significantly lower in Ars/A1 B cells such as, calcium mobilization (Fig 4C), Akt phosphorylation (Fig 4D, Suppl Fig 9C). S6 phosphorylation (Fig 4F) and I\u03baB\u03b1 degradation (Fig 4G). In contrast, other events such as BLNK (Fig 4J) or PLC\u03b32 (Fig 4K) phosphorylation were comparable between Ars/A1 B cells and WT C57BL/6 B cells expressing equal surface IgM, and phosphorylation of Ig\u03b1 (Fig 4H) and Syk (Fig 4I) are even slightly increased in Ars/A1 B cells. Gating on a slice of higher equal IgM expressing B cells gave similar results (Suppl Fig 10). These results suggest that downregulation of surface IgM BCR accounts for much of the impaired BCR signaling phenotype we observe in Ars/A1 B cells, while additional active mechanisms suppress PI3K dependent signaling further.

One caveat of this analysis is that the subpopulation of B cells within the WT repertoire that express low IgM are enriched for autoreactive B cells 44. These autoreactive B cells may have their own active suppression mechanisms that could mask the detection of inhibitory mechanisms in Ars/A1 B cells. To circumvent this potential confounding factor, we did a parallel experiment using B cells from MD4 mice. MD4 mice express a transgenic BCR that binds Hen Egg Lysozyme (HEL) with a high affinity and has little cross reactivity with self-antigen. MD4 B cells express very high surface IgM levels (Fig 4L), with virtually no overlap with Ars/A1 B cells, making it unfeasible to compare subpopulation with equal IgM expression. However, IgD BCR expression levels are more comparable between MD4 and Ars/A1 B cells (Fig 4M), with Ars/A1 B cells expressing slightly higher IgD levels. When we analyzed signaling induced following IgD crosslinking, we found that while most signaling events were comparable between MD4 and Ars/A1 B cells (Fig 4S-V, Suppl. Fig 11), PI3K dependent events were significantly suppressed (Fig 4N-R). Taken together these results suggest that downregulation of IgM BCR levels contributes significantly to the suppressed BCR signaling phenotype of anergic Ars/A1 B cells, but that active suppression of PI3K signaling via Lyn also plays an important role.

Acute deletion of Lyn in Ars/A1 B cells results in inefficient autoantibody responses.
Acute deletion of Lyn in anergic Ars/A1 B cells restores their ability to signal following BCR stimulation (Fig 2). Previously, we used an adoptive transfer approach 40 to determine if SHP-1 and SHIP-1 were required to maintain B cell anergy and showed that following acute deletion of either of these phosphatases, Ars/A1 B cells mounted an autoantibody response. Using a similar adoptive transfer approach (Fig 5A), we observed that upon acute deletion of Lyn Ars/A1 B cells also respond to self-antigen (Fig 5B-E), although there were significant differences in that response compared to the previously reported response upon acute SHIP-1 or SHP-1 deletion 40, 45. While the size of the YFP+ Ars/A1 population remains similar or expands upon SHIP-1 deletion 40, 45, we observed a significant reduction in the number of YFP+ (Lyn-deficient) Ars/A1 B cell in the spleen over time following acute Lyn deletion (suppl Fig 12A-B and Fig 5E). At day 14 post tamoxifen treatment the majority of SHIP-1 deficient YFP+ have proliferated and a significant portion of divided cells have differentiated into plasmablasts 40, 45, instead at day 14 post tamoxifen-treatment the remaining YFP+ Lyn-deficient Ars/A1 B cells had a mixed phenotype (Fig 5B,D). In some of the recipient mice few YFP+ Lyn KO Ars/A1 B cells had proliferated, while in other mice the majority had proliferated and a fraction of those had differentiated into plasmablasts. The absolute numbers of cells were so low that in most mice it did not result in a detectable antibody secreting cell response (Fig 5C). We did 4 replicate experiments in which we adoptively transferred variable numbers of Ars/A1 B cells (Suppl Fig 12C-F). In all experiments we observed a considerable variation in the response between mice within each group. There was a trend towards more pronounced autoantibody responses when more Ars/A1 B cells were transferred (Suppl Fig 12), but the overall cell recovery remains low.

To exclude the possibility that there is an inherent problem in mounting an antibody response following acute Lyn deletion, we compared the ability of Lyn sufficient or deficient MD4 B cells to respond to SRBC-HEL immunization (Fig 5F-J). To be able to directly compare antibody secreting cell responses we sorted YFP+ MD4 B cells that were Lyn sufficient or deficient and adoptively transferred equal numbers. Following immunization, we observed that Lyn deficient MD4 B cells have a higher frequency of plasmablasts (Fig 5H, J) but overall mounted an equal antibody forming cell response (Fig 5G). These results suggest that while acute loss of Lyn results in a loss of B cell anergy, the resultant autoantibody responses are inefficient, possibly due to reduced fitness of B cells. Finally, we determined if induced Lyn haploinsufficiency promotes autoantibody responses by itself or when combined with induced haploinsufficiency of SHIP-1 (Fig 5K-O). While induced haploinsufficiency of either Lyn or SHIP-1 in isolation did not induce significant proliferation of Ars/A1 B cells or accumulation of autoantibody secreting cells, induced double haploinsufficiency of Lyn and SHIP-1 in Ars/A1 B cells did (Fig 5O, Suppl Fig 13). Under these conditions no significant loss of YFP+ Ars/A1 B cells was observed (Fig 5N). Collectively these data suggest that Lyn function is required for the maintenance of B cell anergy and that reductions in Lyn function can synergize with reductions of other proteins in the pathway to result in a loss of tolerance of anergic B cells.

Methods
Mice
Six- to twenty-week-old mice were used in all experiments. In each experiment, mice were age-matched and sex-matched. Both sexes were used in our experiments with similar results. As described previously 40, hCD20-TamCre animals 98 (donated by Dr. M. Shlomchik, University of Pittsburgh, PA) were intercrossed with mice carrying the rosa26-flox-STOP-YFP allele 99 (Jax mice strain 006148) generating mice in which YFP is expressed in B cells upon Cre activation. These mice were crossed with Ars/A1 41 (donated by Dr L. Wysocki, National Jewish Health, CO) and MD4 56 (Jax mice strain 002595) B cell antigen receptor transgenic mice to generate hCD20-TamCre x rosa26-flox-STOP-YFP x Ars/A1 mice and hCD20-TamCre x rosa26-flox-STOP-YFP x MD4 mice, respectively. B cells from these mice will be referred to as WT Ars/A1 and WT MD4. To generate mice in which Lyn deletion can be induced in B cells, these mice were crossed with Lynflox/flox mice (donated by Dr. C. Lowell, UCSF, San Francisco, CA) 46. To generate mice in which SHIP-1 and Lyn haploinsufficiency can be induced in Ars/A1 B cells, these mice were crossed with previously described mice 40 in which SHIP-1 deletion can be induced in B cells (hCD20-TamCre x rosa26-flox-STOP-YFP x SHIP-1flox/flox x Ars/A1 mice). SHIP-1flox/flox mice were donated by Dr J. Ravetch (Rockefeller University, NY) In parallel, lines were generated that do not express a transgenic BCR to determine the effect of acute Lyn deletion in B cells from mice that express a WT repertoire of BCRs. Mice in which Lyn deficiency is constitutively restricted to the B cell lineage from early B cell development were generated by crossing Lynflox/flox mice with mb1cre mice 100 (Jax mice strain 020505). This line was crossed with Ars/A1 mice to generate mb1cre x Lynflox/flox x Ars/A1 mice. Lynflox/flox x Ars/A1 mice littermates were used as Lyn sufficient controls. In mb1cre mice the cre recombinase is inserted into the mb1 locus, which encodes CD79A, disrupting the expression of CD79A from that locus. To control for the impact of CD79A heterozygosity on the anergic phenotype we also crossed mb1cre mice with Ars/A1 mice and compared Ars/A1 littermates that did or did not contain the mb1cre construct.

In adoptive transfer experiments, C57BL/6J mice (Jax mice strain 000664) were used as recipients. For some of the signaling experiments, Ars/A1 and MD4 B cell antigen receptor transgenic mice were used without additional strains crossed in. At the end of each experiment the mice were euthanized by CO inhalation. Mice were housed and bred at the University of Colorado Denver Anschutz Medical Campus Vivarium (Aurora, CO) under ESPF conditions. C57BL/6J mice were purchased from Jackson Laboratories for experiments. All experiments with mice were performed in accordance with the regulations and with approval of the University of Colorado SOM Institutional Animal Care and Use Committee.

Tamoxifen treatments, adoptive transfers and immunizations.
Cre activity was induced by tamoxifen treatment. Tamoxifen (Sigma, T-5648) was dissolved in 10% ethanol (Decon Laboratories) and 90% corn oil (Sigma) at 20 mg/mL. In experiments in which we analyzed the impact of induced Lyn deletion on BCR signaling of splenic B cells, mice were injected once i.p. with 100 \u03bcL (2 mg) of tamoxifen/corn oil 7 days prior to analysis. Following a single tamoxifen injection we observed a deletion efficiency (measured as % YFP+ B cells) of 61.1 \u00b121.6%.

In adoptive transfer experiments in which we analyzed the impact of induced Lyn deletion on the ability of Ars/A1 B cells to maintain B cell tolerance, recipient mice were irradiated with 200 rads four hours prior to adoptive transfer to provide space for transferred cells as described previously 40. B cells from donor mice were enriched by depletion of CD43+ cells with anti-CD43-conjugated magnetic beads (CD43 (Ly-48), mouse; Miltenyi Biotec). Resultant populations were >97% B cells based on B220 staining and FACS analysis. Prior to transfer, donor B cells were labeled with CellTrace Violet Proliferation Kit, *for flow cytometry* (Invitrogen by Thermo Fisher) at 5 \u03bcM for 3 min at RT in complete medium (IMDM supplemented with 10% fetal calf serum (FCS), 1 mM Sodium Pyruvate, 2 mM L-Glutamine, 100 U/mL Pen/Strep, 50 \u00b5g/mL gentamicin and 0.1 mM 2-Me). The labeling was stopped by adding an excess of FCS, followed by 3 washes with Phosphate-Buffered Saline (PBS). 1-8x106 B cells in 200 \u03bcL PBS were adoptively transferred by i.v. injection. Twenty-four hours after transfer, Cre activity was induced by 100 \u03bcL (2 mg) tamoxifen injections i.p. on two consecutive days.

In experiments in which we analyzed the impact of induced Lyn deletion on the ability of MD4 B cells to mount an antibody response to Sheep Red Blood Cells-Hen Egg Lysozyme (SRBC-HEL) immunization, donor hCD20-TamCre x rosa26-flox-STOP-YFP x Lynflox/flox x MD4 mice and hCD20-TamCre x rosa26-flox-STOP-YFP x MD4 mice were injected i.p. with 100 \u03bcL (2 mg) of tamoxifen/corn oil. Six days post tamoxifen treatment, splenocytes were stained with B220-Alexa647 (clone RA3-6B2, Biolegend, 1:500) and B220+ YFP+ cells were sorted using a Beckman Coulter Astrios EQ cell sorter. 1.3x105 YFP+ B220+ cells were adoptively transferred by i.v. injection in 200 \u03bcL PBS into C57BL/6 recipient mice. Twenty-four hours post adoptive transfer the recipient mice were immunized by i.p. injection with 200 \u03bcL 1% SRBC-HEL. SRBC were purchased from the Colorado Serum Company and stored in sterile Alsever\u2019s solution at 4 \u00b0C. HEL was chemically coupled to SRBC. One mL packed SRBC was resuspended in 15 mL PBS containing 5 mg/mL HEL (Sigma). To cross-link, 1mL of 50 mg/mL 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (Sigma) in PBS was added, mixed and incubated for 1 h at RT, with occasional mixing. Afterwards, the cells were washed four times in PBS and the conjugation of HEL to the SRBC cell surface was confirmed by flow cytometry after staining with Dylight650 conjugated anti-HEL (clone D1.3, produced and conjugated in house, 1:400). Five days post immunization splenocytes were harvested and analyzed for MD4 anti-HEL responses.

ELISPOT
For detection of IgMa Ars antibody secreting cells, microtiter plates were coated with 10 \u03bcg/mL Bovine Serum Albumin (BSA)-Ars or 10 \u03bcg/mL Ovalbumin (OVA)-Ars in PBS. For detection of IgMa MD4 antibody secreting cells, microtiter plates were coated with 10 \u03bcg/mL HEL in PBS. Before use, the plates were washed twice with PBS-0.05% Tween-20 and blocked with complete 10% FCS IMDM medium. Two-fold serial dilutions were made of splenic single cell suspensions starting at 1/50th of a spleen in the first well. The plates were incubated at 37\u00b0C at 7% CO2 overnight. MD4 and Ars/A1-derived antibody secreting cells were detected with Biotin-conjugated Mouse Anti-Mouse IgM[a] (Clone:DS-1; BD Biosciences, 1:2000), followed by Streptavidin-AP (SouthernBiotech, 1:2000). Between each step, the plates were washed 4 times with PBS-0.05% Tween-20. The plates were developed by incubating with ELISPOT development buffer (25 \u03bcM 5-bromo-chloro-3-indolyl phosphate p-toluidine, 100 mM NaCl, 100 mM Tris, 10 mM MgCl2 [pH 9.5]) for 1 h. The reaction was stopped by washing the plate once with double-distilled H2O. The number of spots at a cell dilution in the linear range was determined, and the number of antibody-secreting cells per spleen was calculated.

Flow cytometry
Single cell suspensions of splenic cells or bone marrow cells, flushed out of the two hindleg femurs, were prepared in 10% FCS complete medium, and red blood cells were lysed using Ammonium Chloride Potassium Lysis buffer (15 mM NH4Cl, 10 mM KHCO3, 0.1 mM Na2EDTA, pH 7.4). Cells were resuspended in PBS containing 1% BSA and 0.05% sodium azide and incubated with an optimal amount of directly fluorochrome labeled antibodies. Antibodies directed against the following molecules were used: B220-BV421 (clone RA3-6B2, Biolegend, 1:500), CD43-APC (clone S11, Biolegend, 1:200), CD69-BV786 (clone H1.2F3, BD Biosciences, 1:200), CD86-BV605 (clone GL1, BD Biosciences, 1:200), CD95-FITC (clone Jo2, BD Biosciences, 1:200), IgD-PE (clone 11-26c.2a, Biolegend, 1:300), and IgM (b-7-6, 1:200). b-7-6 was produced in our own laboratory and was directly conjugated to DyLight (Pierce/Thermo Scientific) fluorochromes, according to the manufacturer\u2019s protocol. The MD4 BCR was stained with its antigen HEL (Sigma) conjugated to Dylight 650 (Pierce/Thermo Scientific, 1:300) fluorophore, according to the manufacturer\u2019s protocol.

To determine the phenotype of Ars/A1 B cells following adoptive transfer, splenocytes were fixed and permeabilized with BD Cytofix/Cytoperm\u2122 and stained with Dylight650-E4 anti-Ars/A1 idiotype (E4 was produced and conjugated to DyLight\u2122 650 NHS Ester (Pierce/Thermo Scientific) in our laboratory, 1:300), Dylight488 anti-GFP (polyclonal Goat anti-GFP Antibody (600-101-215, Rockland) conjugated to DyLight\u2122 488 NHS Ester (Pierce/Thermo Scientific) in our laboratory, 1:500), and CD138-PE-Cy7 (Clone: 281-2; Biolegend, 1:300). Anti-GFP-Dylight488 was used to detect YFP in experiments in which we used fixatives that kill the fluorescence of YFP. The cells were washed twice, and events were collected on a CyAn ADP (Dakocytomation), a BD LSRFortessa\u2122 X-20 Cell Analyzer (BD Biosciences) or a Cytek Northern Lights (Cytek Biosciences) and analyzed using FlowJo software (Becton Dickinson & Company (BD). In all experiments, the same gating strategy was used. We gated on the lymphocyte population based on FSC/SSC properties but including larger cells to capture plasmablasts, followed by doublet exclusion, and gating on YFP+ E4+ events (Suppl Fig 13A). To calculate the absolute number of YFP+ Ars/A1 B cells per spleen in different states (undivided, proliferated and plasmablasts (proliferated and CD138+), we multiplied the total number of splenocytes (counted with a Beckman Coulter Vi-CELL) with the frequency of YFP+ E4+ events and the frequency of violet proliferation dyehi CD138lo (undivided), violet proliferation dyelo (proliferated), violet proliferation dyelo CD138hi (plasmablasts).

Analysis of BCR signaling by flow cytometry
To analyze protein phosphorylation by flow cytometry, splenocytes were resuspended in serum-free IMDM at a concentration of 107 cells/mL and incubated at 37\u02daC for 20-30 minutes prior to stimulation. In some experiments the PI3K inhibitor Ly294002 (Cell Signaling Technology), dissolved in DMSO, was added at 5 or 10 \u03bcM during the last 10 minutes of prewarming. Control tubes received DMSO only at an equal dilution. In some experiments the splenocytes were prestained with a Fab fragment of b-7-6 conjugated to Dylight 488 (1:250) at 4 \u02daC and washed twice with serum free IMDM prior to incubation at 37 \u02daC to allow for gating on subpopulations of B cells with equal IgM expression. B cells were stimulated with 10 \u03bcg/mL polyclonal F(ab\u2019)2 goat anti-mouse IgM (anti-\u03bc) (Jackson ImmunoResearch) or 5 \u03bcg/mL anti-IgDa (clone AMS 9.1, Santa Cruz Biotechnology) for the indicated time. Prior to stimulation with anti-IgDa the splenocytes were incubated with 10 \u03bcg/mL clone 2.4G2 to block binding to Fc\u03b3RIIB.

Signaling was stopped by adding formaldehyde (Tousimis) at a final concentration of 2%. If a methanol permeabilization method was used the cells were incubated at 37\u02daC for 10 min with the 2% formaldehyde. Afterwards the cells were spun down and resuspended in -80 \u02daC 100% MeOH (Fisher Scientific) and incubated on ice for 30 min. After permeabilization the cells were washed three times in PBS containing 1% BSA. Cell staining and washes were done in PBS/1% BSA. If BD Cytofix/Cytoperm permeabilization was used, the cells were immediately spun down following formaldehyde addition and resuspended in BD Cytofix/Cytoperm. The cells were incubated at 4 \u02daC for 20 minutes, followed by three washes with BD Perm/Wash buffer. Cell staining and washes were done in BD Perm/Wash buffer.

Methanol permeabilized cells were stained for: B220-BV421 (clone RA3-6B2 Biolegend, 1:500), Dylight488 anti-GFP (if cells express YFP, 1:500), pCD79A (pY182)-Alex647 (clone D189, Cell Signaling Technology, 1:50), pSyk (pY352)-Alex647 (Clone 17A/P-ZAP70, BD Biosciences, 1:5), pErk (pT202/pY204)-Alex647 (clone 20A, BD Biosciences, 1:5), pPLC\u03b32 (pY759)-Alex647 (clone K86-689.37, BD Biosciences, 1:5), pBLNK (pY84)-Alex647 (clone J117-1278, BD Biosciences, 1:5), Rabbit isotype control-Alex647 (Cell Signaling Technology, 1:200), mouse IgG1 isotype control-Alex647 (clone MOPC-21, BD Biosciences, 1:5). BD permeabilized cells were stained for: B220-BV421 (RA3-6B2 Biolegend, 1:500), Dylight488 anti-GFP (if cells express YFP, 1:500), pAkt (pS473)-Alex647 (clone M89-61, BD Biosciences, 1:5), I\u03baB\u03b1-Alex647 (clone L35A5, Cell Signaling Technology, 1:100), pS6 (pS235/pS236)-PE-Cy7 (clone cupk34k, eBioscience/Invitrogen, 1:100), Rabbit isotype control-Alex647 (Cell Signaling Technology, 1:200), mouse IgG1-Alexa647 isotype control (clone MOPC-21, BD Biosciences, 1:100) and mouse IgG1-PE-Cy7 isotype control (clone P3.6.2.8.1, eBioscience/Invitrogen, 1:200). The cells were stained for 1hr at RT and washed 3 times prior to analysis. Events were collected on a BD LSRFortessa\u2122 X-20 Cell Analyzer (BD Biosciences) or a Cytek Northern Lights (Cytek Biosciences) and analyzed using FlowJo software (Becton Dickinson & Company (BD). In all experiments, the same gating strategy was used. We gated on the lymphocyte population based on FSC/SSC properties, followed by doublet exclusion and gating on YFP+ B220+ events or B220+ events if the cells did not express YFP (Suppl Figs 4A, 5A and 6A).

Analysis of calcium mobilization
For measurements of intracellular free calcium concentration ([Ca2+]i), splenocytes (2*107/mL in IMDM medium containing 2% FCS) were simultaneously stained with anti-B220-Alexa647 (clone RA3-6B2 Biolegend, 1:500) and loaded with 5 \u03bcM Indo-1 acetoxymethyl (Indo1-AM) (Molecular Probes) for 30 min at RT. In some experiments 5 \u03bcM Ly294002 or b-7-6 Fab conjugated to Dylight 488 (1:250) was added. After washing once with IMDM with 2% FCS, the cells were resuspended at 107 cells/mL in warm IMDM with 2% FCS in a 500\u03bcL volume. Indo-1 was excited with a 355 nm laser, Ca2+-bound Indo-1 was detected using a 379/28 bandpass filter and unbound Indo-1 was detected using a 450/50-410LP bandpass filter. Relative free calcium concentration was determined by the ratio bound/unbound Indo-1. After the baseline was established by analysis for 30 seconds, cells were stimulated with 2-10 \u03bcg/mL F(ab\u2019)2 goat anti-mouse IgM (anti-\u03bc) (Jackson ImmunoResearch) or 5 \u03bcg/mL anti-IgDa (clone AMS 9.1, Santa Cruz Biotechnology) for 2 minutes. Prior to stimulation with anti-IgDa the splenocytes were incubated with 10 \u03bcg/mL clone 2.4G2 to block binding to Fc\u03b3RIIB. The bound/unbound Indo-1 ratio was measured using a BD LSRFortessa\u2122 X-20 Cell Analyzer (BD Biosciences) and analyzed using FlowJo software (Becton Dickinson & Company (BD). The area under the curve was calculated in FlowJo.

Immunoblotting
Splenic B cells were enriched by depletion of CD43+ cells with anti-CD43-conjugated magnetic beads (MACS anti-mouse CD43; Miltenyi Biotec). Resultant populations were >97% B cells based on B220 staining and FACS analysis. B cells were resuspended at 107/mL in serum-free IMDM and incubated at 37 \u02daC for 20-30 minutes. B cells were stimulated with 10 \u03bcg/mL F(ab\u2019)2 goat anti-mouse IgM (anti-\u03bc) (Jackson ImmunoResearch). At the indicated times, the B cells were pelleted by centrifugation at 3500g for 30 seconds in a tabletop centrifuge and lysed in reducing SDS sample buffer or cells were lysed in 1% NP-40 lysis buffer (25 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 1 mM EDTA, 1 mM DTT, with protease and phosphatase inhibitors (100 \u00b5g/mL aprotinin, 100 \u00b5g/mL \u03b1-1-antitrypsin, 100 \u00b5g/mL leupeptin, 1 mM PMSF, 10 mM NaF, 2 mM NaVO3, and 10 mM tetrasodium pyrophosphate). For analysis, whole cell lysates (WCLs) were mixed with a quarter final volume of 4X reducing Laemmli SDS-PAGE sample buffer. In some experiments we sorted YFP+ B cells post tamoxifen treatment to validate Lyn deletion and determine the impact of Lyn deficiency on phosphatase phosphorylation. Seven-days post tamoxifen treatment splenocytes were stained with B220-Alexa647 (clone RA3-6B2, Biolegend, 1:500) and B220+ YFP+ cells were sorted using a Beckman Coulter Astrios EQ cell sorter. The sorted cells were counted and either lysed in reducing Laemmli SDS-PAGE sample buffer or resuspended in serum free medium and stimulated and processed as described above.

Samples (0.5-1 x 106 cell equivalents) were boiled for 5 minutes and proteins were separated by SDS-PAGE, and transferred to PVDF membranes, which were then blocked with 5% BSA in TBST (TBS-0.05% tween-20). Blots were probed with antibodies diluted in 3% BSA-TBST. Antibodies specific for pAkt (S473) (Cell Signaling, 1:1000), p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (Cell Signaling Technology, 1:2000), pan Erk (BD, 1:1000), Akt (Cell Signaling, 1:1000), pSyk (Tyr525/Tyr526) (Cell Signaling Technology, 1:1000), Syk (Cell Signaling, 1:1000), pSHIP-1 (Tyr1022, Cell Signaling, 1:1000), SHIP-1 (clone D1163, Cell Signaling, 1:1000), pSHP-1 (Tyr564, Cell Signaling, 1:1000), SHP-1 (clone C14H6, Cell Signaling, 1:1000), Lyn (LYN-01, Abcam, 1:1000), Lyn (affinity purified polyclonal rabbit IgG raised against Lyn 1-131, 1:1000) or actin (clone C-2, Santa Cruz, 1:10,000) were used. Following incubation with primary antibodies, the blots were washed in TBS-0.05% tween-20 and incubated with a secondary antibody HRP-conjugated Goat anti-Rabbit IgG (Cell Signaling Technology, 1:20,000). The anti-actin antibody was directly conjugated to HRP. The blots were washed in TBS-0.05% tween-20 and incubated in SuperSignal West Pico PLUS HRP Chemiluminescent substrate (Thermo Fisher Scientific) and visualized on a G:BOX by Syngene or a ChemiDoc XRS Imagining system (BioRad). Quantification was done in Image Studio Lite v5.2. Blots were stripped using a guanidine hydrochloride-based (GnHCl) stripping solution (6 M GnHCl, 0.2% Nonidet P-40 (NP-40), 0.1 M \u03b2-mercaptoethanol, 20 mM Tris-HCl, pH7.5) 101.

Statistics
Statistical analyses were performed in GraphPad Prism 10 using a paired and unpaired Student\u2019s t test or a unpaired Mann-Whitney U test as indicated in the figure legends. P-values <0.05 were considered statistically significant. P-values >0.05 are marked as not significant (ns).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript titled The Src-family kinase Lyn plays a critical role in establishing and maintaining B cell anergy by suppressing PI3K-dependent signaling by Fiske et al explores the role of Lyn in the Ars/A1 mice, which carry a transgenic BCR specific for the hapten p-azophenylarsonate (Ars) and serve as a model for anergy. Using both developmental (mb1-Cre) and inducible Cre (hCD20-TamCre) models to delete Lyn specifically in B cells, the authors demonstrate that Lyn-deficient Ars/A1 B cells fail to become anergic and produce autoantibodies, concluding that Lyn is required for the development and maintenance of anergic B cells in these mice. The authors link this failure to a dysregulation of PI3K-dependent signaling pathways and show that Lyn deficiency leads to enhanced phosphorylation of signaling components critical for B cell activation. These findings suggest that Lyn plays a crucial role in suppressing PI3K signaling, maintaining B cell anergy, and preventing autoimmunity.\nGenerally, this is a well-conceived study with little to criticize among the presented results; however, Lyn deficiency in B cells has been widely explored in previous publications, and although it was not directly in the context of anergy, there is a certain lack of novelty to this study. While I appreciate the thorough analysis and the complex experiments presented here, I personally find that the content feels somewhat redundant and does not captivate my interest.", "reviewer_2": "Review of the Nature Communications manuscript NCOMMS-24-30899 (Fiske et al.)\nLyn is a Src family, non-receptor tyrosine kinase, found predominantly in myeloid cells and B lymphocytes. In B cells, antigen binding to BCR induces a variety of signaling cascades initiated by Lyn. The role of Lyn in generating the positive signaling cascade can be compensated for by other Src family protein tyrosine kinases; however, its role in the initiation of negative feedback loops that limit the BCR-signalling cascade is unique. Consistent with this, mice lacking Lyn develop symptoms of autoimmunity, produce high titers of antinuclear antibodies, and develop glomerulonephritis. Thus, activation of Lyn-dependent inhibitory signaling in B cells is essential for maintaining B cell tolerance. Fiske et al. used the Ars/A1 model of B cell anergy and specific deletion of Lyn in B cells to demonstrate that Lyn is required to maintain the anergic state of Ars/A1 B cells. The authors performed elegant experiments with tamoxifen-induced Lyn inactivation to conclusively demonstrate that Lyn deletion in Ars/A1 anergic or WT B cells does not affect IgM BCR expression levels, but does disturb PI3K- dependent signalling events upon BCR stimulation. Proposing that limiting PI3K-dependent signalling events is the main function of Lyn in preserving B cell anergy is the novel aspect of this manuscript. The last part of the manuscript tries to show that Lyn-deficient Ars/A1 B cells or combined Lyn and SHIP-1 haploinsufficiency in Ars/A1 B cells leads to the loss of anergy, differentiation into antibody-secreting cells, and production of autoantibodies. The manuscript should be improved on the basis of the comments provided below.\nAbstract\n-Lines 28-29: Tamoxible-inducible Lyn deletion in B cells does not rule out off-target effects. The statement in lines 28-29 is incorrect.\nMaterial and Methods\n-Lines 200, 240, 378, Fig 4B: The anti-IgM Fab fragment B-7-6 appears in the manuscript with three different names (B-7-6, B76, b76)\nResults\nArs/A1 B cells require Lyn to become anergic\n-mb1 encodes the Ig  signaling subunit of the B cell antigen receptor. Mice carrying the mb1-cre allele are heterozygous for Ig  (CD79A). The experiments described in this section used as control mice Lyn+/+ x Ars/A1 and experimental mice mb1cre × Lyn flox/flox × Ars/A1. Could Ig  heterozygosity has an influence on the phenotype observed? Would mb1cre × Lyn+/+ × Ars/A1 be better controls in these experiments?\n-Line 302 contains a typo (B cell --- B cells)\nLyn is required for the maintenance of B cell unresponsiveness by suppressing PI3K-dependent signaling events in anergic Ars/A1 B cells.\n-Lines 342-343 states that signaling events showed in Fig2I-L were significantly reduced for LynKO Ars/A1 compared to naïve WT B cells. However, the figure does not contain statistics for LynKO Ars/A1 versus WT comparisons after BCR stimulation.\n-In Lyn-deficient Ars/A1 B cells, would it be possible to restore anergy (reduced Ca2+ mobilization, Akt, pS6, Erk phosphorylation, and I B  degradation) by inhibiting PI3K-signalling prior to BCR stimulation?\nAlternatively, in Lyn-deficient B cells from mice with a diverse BCR repertoire. Would inhibition of PI3K-signalling prior to BCR stimulation make these cells respond similarly to Lyn-sufficient cells? These experiments might further support the idea that Lyn acts by limiting PI3K-dependent signalling events.\n-Fig 3A and B are not mentioned or described in the manuscript.\nAcute deletion of Lyn in Ars/A1 B cells results in inefficient autoantibody responses.\n-Figures 5B, C, and E have not been mentioned or described in the manuscript.\n-The results in this section of the manuscript try to show that Lyn-deficiency in Ars/A1 B cells affects B cell survival and promotes plasmablast differentiation and autoantibody secretion. The authors performed similar experiments in a previous study on inositol phosphatase SHIP-1 (Reference 40), with impactful results.\n-It is interesting to combine Lyn and SHIP-1 haploinsufficiency in Ars/A1 B cells to avoid the effect of Lyn deficiency on B cell survival. Although Fig 5N shows a tendency towards an increased number of plasmablasts and the ELISPOT assay showed an increased number of IgM-secreting cells, I am not so sure the results are very convincing. Are the results of several experiments combined, or just one experiment? Perhaps an additional replicate experiment would help confirm this finding.\n-The results for MD4 mice shown in Fig5 F-J, are clearer to me, although Fig 5G does not show an increased number of IgM-secreting cells.\nFigures\n-Fig 1E: The y-axis label should be Ars/A1 AFC/ 106 Ars/A1 B cells or something similar, but not 106 splenocytes.\n-Fig 1G: Graph from below, y-axis, value 1 is moved.\n-Fig 2I-L: Statistics of comparison of LynKO Ars/A1 versus WT B cells might be missing, as it is mentioned in the results.\n-Fig 3D,E,H-K: below F(Ab‘)2 anti-IgM is written 2 min, and the time of stimulation was 2 and 10 min.\nSupplementary Figures\n-In the legend of Supplementary Fig 3A, the description of Supplementary Fig 3B should be eliminated.", "reviewer_3": "This well-crafted paper probes the relative contributions of IgM downregulation and Lyn-mediated negative feedback in maintaining anergy in autoreactive B cells. Previous work from this group has shown that reducing or eliminating PI3K and SHIP-1 signaling reverses anergy in B cells. As Lyn kinase has unique functions in regulating these pathways through phosphorylation of ITIMs and SHIP-1, the authors examined the role of Lyn in maintaining B-cell anergy. This study makes elegant use of genetic models, combining the Ars/A1 model of anergy with a tamoxifen-induced, B-cell-specific LynKO, which eliminates contributions of repertoire-skewing and myeloid-cell-intrinsic effects in constitutive LynKO mice. A second inducible knockout (hCD20-CreTAM) restricts deletion even further: to mature anergic B cells. In addition, the authors demonstrate that compensatory mechanisms such as increased surface IgM expression do not affect induced-LynKO Ars/A1 B cells. The major strengths of this paper are in the attention to different pathway components and normalization for IgM expression. The novel contributions are consistent with much of the previous literature but add in an important physiological system and pathway-specific precision. In general, the analysis is rigorous, and the scope and depth are appropriate. A few additional experiments and clarifications would strengthen the link between Lyn and B-cell anergy and further differentiate this work from the previous literature.\nMajor critiques:\n• Figure 1A: It is established that B-cell-specific loss of Lyn leads to developmental blockade after T1 (PMID: 24376269). It is unclear whether the deficit in splenic B cells is due to an unexpected effect of mb1 expression during development or whether this is a decrease in the anergic population. Secondarily, T3 B cells share some markers with anergic B cells (PMID: 17174121), so is it possible that these populations are not resolved in your analysis?\n• Figure 1A-E: Which markers are used to assess total and functional B-cell populations?\n• Figure 1A-E: Descriptions of results and conclusions related to this figure are unclear in the main text.\n• Figure 1F: Which B-cell population is used for analysis of IgM, IgD, and activation markers (BM/Spleen)?\n• Figure 1F, 2C: Clarify how many independent experiment days these data represent. Alternatively, data could be normalized (rather than used as raw MFIs, which can vary by day) and pooled.\n• Figure 1, 5: Is it possible to add representative ELISPOT images to these figures?\n• Figure 1, 5: Is it possible to look at the secretion of IgM anti-chromatin antibodies (as in author’s previous publication PMID: 27114609), as this might be more physiologically relevant?\n• Figure 2: Is the argument that LynKO after tamoxifen exposure reverses anergy or that cells are unable to become anergic in the absence of Lyn? This distinction should be clarified, as it appears to be differently argued in different parts of the manuscript.\n• Figure 2: In a non-anergy-inducing background, LynKO B cells have similarly enhanced PI3K signaling and calcium mobilization following anti-IgM crosslinking (PMIDs: 24376269, 34675079). Although the later IgM normalization experiment is an excellent test of the anergy-specific effects of Lyn deletion, addition of the hCD20-TamCre x Rosa26 stopflox YFP x Lyn fl/fl (not Ars/A1) would be a useful control to show the effects of deleting Lyn only in mature cells. If the small population of anergic cells in the non-Ars/A1 mice became more reactive to stimulus after Lyn deletion, then it would be an important confirmation of the Ars/A1 results.\n• Figure 2B: LynA expression appears to be elevated relative to LynB in Ars/A1 B cells. Is this different from the LynA: LynB ratio in B cells from non-anergic mice? How does this observation fit into previous reports that LynB is more highly expressed than LynA in splenic B cells (PMIDs: 9252121, 35452291)? Discussed in reference to previous literature (w/ citation).\n• Analysis of pSHIP-1 and pSHP-1 would strengthen the link between Lyn and regulation of downstream signaling\n• Since the IgM Fab was used to minimize FcγRIIB recruitment, how is Lyn recruited to dampen signaling in anergic B cells? The author found monophosphorylated ITAMs in anergic cells (PMID: 22078222)—could this be the mechanism? How would this result change with co-crosslinking of an ITIM-containing protein?\n• Figure 4: Is it possible to add a second “equal IgM” gate at higher IgM? The low-IgM WT cells do not express sufficient BCR for pErk induction, yielding an equally negative result for both the WT and Ars/A1 cells. It would be interesting to see whether higher-IgM Ars/A1 cells had restored pErk.\n• Figure 5: Descriptions of results and conclusions related to this figure are unclear in the main text.\nMinor critiques:\n• General: Suggest editing to increase clarity for a readership beyond B-cell biologists (esp. surrounding Figures 1 & 5 and Discussion)\n• Abstract: Confusing sentence “with varied conclusions likely confused by collateral loss of Lyn functions in B cell and myeloid cell development and activation,” considering reference to B-cell-specific KO studies.\n• Figure 1C: Harmonize the identification of the graphed cell population in the y axis, figure legend, and main text.\n• Figure 1H: Quantify immunoblots and add statistical analysis.\n• Figure 2: Perhaps the efficiency of deletion could be reported or included in the supplementary material?\n• Figure 2D: Could kinetic differences in calcium mobilization also be quantified? It is not clear that an absolute concentration of intracellular free calcium is being reported, as suggested by the [Ca2+]i label. If this is an arbitrary fluorescence value, this should be clarified.\n• Figure 3R & T: Re: “The small significant change in Igα (Fig 3R) and BLNK (Fig 3T) phosphorylation upon BCR crosslinking was not a reproducible finding”—could aggregate data be included, normalized to WT t=0 on each day to more accurately reflect the cumulative result?\n• Supplemental Figure 2, 3, 4: The color schemes for the flow plots are difficult to process; the “unstimulated” legend in S2 doesn’t match the flow plots.\n• Figure 4C-K: Why aren’t the Total and Equal IgM populations plotted/analyzed together? This would enable statistical claims about phenotypic rescue.\n• Discussion: “avoiding all the myeloid cell activation” could be edited for precision, as cell-extrinsic effects should activate myeloid cells in B-cell-specific Lyn knockout mice."}, "author_response": "Response to referees for manuscript NCOMMS-24-30899, Fiske et al.\nReviewer 1\nWe thank the reviewer for his/her positive reception of our manuscript and the experimental\nwork presented. The only critique we identified was a concern that the presented work may have\na certain lack of novelty since Lyn has been extensively studied in the context of BCR signaling.\nWe would like to emphasize some key observations that we think are novel and significant,\nwhich we think should be of interest to a larger audience.\nAs recognized by the reviewer, we think that linking Lyn function to B cell anergy is a\nsignificant observation that is of interest to those studying immune tolerance and autoimmune\ndisease. It has puzzled the field that Lyn deficiency in two models of B cell anergy did not result\nin a loss of B cell tolerance. Inhibitory receptors and phosphatases that are dependent on Lyn\nfunction have been shown to be important for B cell anergy. Thus, one would expect a role for\nLyn in establishing and maintaining B cell anergy.\nWhile we cannot say for certain why we observe a dependence of functional B cell anergy on\nLyn expression, while others have not, we suspect that two factors may contribute. The other\nstudies used classical Lyn knock out mice, while we use B cell restricted models. Dysregulation\nof Lyn-deficient myeloid cells has an impact on Lyn-sufficient B cells (PMID: 23940344), which\nmay have confounding effects. Secondly, it is becoming apparent that B cell anergy exists on a\nspectrum that uses several different mechanisms to obtain a sufficient level of unresponsiveness\nto ensure B cell tolerance. We think that the Ars/A1 model used represents a subset of anergic B\ncells in the wild type B cell repertoire that is dependent on Lyn function for functional anergy.\nThe second point that we think is of general interest relates to the mechanisms that an\nautoreactive B cell can use to reduce the likelihood of productive antigen receptor signaling.\nDownregulation of surface IgM is commonly used by autoreactive B cells. It is a characteristic of\nanergic B cells, yet its impact on constraining downstream signaling and contributions to the\nanergic signaling phenotype are not fully understood. Our data suggests that IgM antigen\nreceptor downregulation is responsible for most of the characteristic dampened signaling\npreviously described. However, anergic B cells use additional active suppressive mechanisms to\nlimit PI3K-dependent signaling. Our work demonstrates how multiple mechanisms contribute to\nthe overall suppressed phenotype of anergic B cells and it further emphasizes the importance of\nregulation of PI3K signaling for B cell tolerance.\nReviewer 2\nWe thank the reviewer for his/her insightful comments and positive reception of our manuscript.\nPlease find our replies to your comments below:\n1) Lines 28-29: the term off-target effects is incorrectly used.\nmeant to say. We have edited the text.\n2) Confusing nomenclature of b-7-6\nWe thank the reviewer for pointing this out. We have changed the text to reflect the correct name\nof the hybridoma, b-7-6, as originally described in PMID:6332028\n3) Does CD79A heterozygosity in Mb1cre x Lyn flox/flox x Ars/A1 mice influence the\nphenotype observed?\nThe reviewer raises a valid concern, which was also raised by reviewer 3. We do not think that\nheterozygosity of CD79A in mb1Cre x Lyn flox/flox x Ars/A1 mice has a significant impact on\nthe overall observed phenotype of a loss of B cel tolerance in these mice. We base this on\nhistorical data and a new set of experiments that are presented in the new supplemental Fig 2.\nPreviously, we reported that B cells from mb1cre x Ars/A1 mice had similar suppressed calcium\nresponses as B cells from control Ars/A1 mice (PMID: 22078222), suggesting that the anergic\nstate is unaffected by CD79A heterozygosity. In new experiments we have expanded this line of\nexperimentation and show that CD79A heterozygosity has no significant impact on B cell\nnumbers, surface IgM expression, calcium responses and spontaneous differentiation into IgMa\nanti-Ars/A1 autoantibody secreting cells in the periphery, and development in the bone marrow.\nThese data support the conclusion that B cell-restricted Lyn deficiency in anergic B cells causes a\nloss of B cell tolerance.\n4) Line 302 contains a typo\nThank you for pointing this out. We have corrected this.\n5) The lack of statistical analysis of differences in signaling events between Lyn KO Ars/A1\nvs naïve WT B cells in Fig 2I-L\nThese statistical comparisons have been added to the figure.\n6) Does inhibition of PI3K revert the signaling phenotype of Lyn KO Ars/A1 B cells?\nThat is a great question. We tested this and found that indeed the presence of the PI3K inhibitor\nLy294002 reverts the signaling phenotype of Lyn KO Ars/A1 B cells. This provides further\nsupport to the hypothesis that the signaling phenotype of Lyn-deficient Ars/A1 B cells is due to\ndysregulation of PI3K dependent signaling. These findings are mentioned in lines 389-391 and\nshown in supplemental figure 8.\n7) Fig 3A and 3B are not mentioned in the text\nThank you for pointing this out. This has been corrected\n8) Figs 5B, C and E are not mentioned in the text\n9) Difference in autoantibody response following induced deletion of Lyn in Ars/A1 B cells\nversus the previously described induced deletion of SHIP-1 (PMID: 27114609) in Ars/A1\nB cells\nThe lack of a robust Ars/A1-derived autoantibody response upon adoptive transfer of Ars/A1 B\ncells and subsequent Lyn deletion was unexpected, given the robust Ars/A1 B cell responses we\nobserved following acute deletion of SHIP-1 or SHP-1 using the same experimental approach.\nOur signaling studies demonstrate that the anergic state of Ars/A1 B cells is lost upon acute Lyn\ndeletion. One difference in outcome between the acute SHIP-1 and SHP-1 deletion studies and\nthe current acute Lyn deletion study is that the robust autoantibody responses upon SHIP-1 and\nSHP-1 deletion were associated with an expansion of the B cell population (total YFP+ Ars/A1 B\ncell numbers per spleen) (PMID: 27114609). In contrast, we observe a significant reduction in\nYFP+ Ars/A1 B cells upon acute Lyn deletion, while detecting evidence of proliferation and\ndifferentiation into antibody-secreting cells. We suspect that this is a form of activation induced\ndeath. In a recent paper we identified a critical role for CD40 signaling in Ars/A1 B cell\nresponses upon deletion of negative regulators of BCR signaling (PMID: 38353615). Blocking\nCD40-CD40L interactions cause a similar, but more pronounced, loss of YFP+ SHIP-1 KO\nArs/A1 B cells. This suggests that Ars/A1 B cell require CD40 signaling for survival in order to\nmount an autoantibody response upon loss of tolerance. Ongoing work in the lab identified an\nessential role for Lyn in B cells in the activation of PI3K dependent signaling downstream of\nCD40. We suspect that these divergent roles of Lyn on BCR signaling and CD40 signaling\nimpact the overall outcome, where a loss of B cell tolerance due to Lyn deletion is less likely to\nlead to a successful autoantibody response on a per-cell basis. When there is continuous\nproduction of cells like in the mb1cre-driven model this is less apparent than in a model with a\nfinite number of transferred cells.\n10) Does the data in Fig 5 K-O represent one experiment or are several combined? Showing\nan additional repeat experiment may help convince that these findings are significant\nsince the effects are subtle.\nThe data present in Fig 5K-O are from one experiment. A repeat experiment showing a similar\nresponse (i.e. a small but significant autoantibody response) has been added as supplemental\nfigure 13. We think that the relatively weak autoantibody response is a consequence of two\nrelatively weak genetic “risk factors”, in this case two haploinsufficiencies, that synergize to\nincrease the probability that an individual autoreactive B cells effectively breaks tolerance. The\nprobability of a full loss of B cell tolerance, leading to the successful differentiation into an\nautoantibody-secreting B cell, remains low. In an experimental system where we transfer a finite\namount of B cells it manifests itself as a relatively weak phenotype, however in the life span of\nthe individual these events can accumulate, resulting in detectable autoimmunity (PMID:\n22798664).\n3\n11) MD4 data in Fig 5F-J: no increase in IgM-secreting B cells\nThe purpose of the MD4 experiment was to determine if there are inherent defects in Lyn-\ndeficient B cells to mount an antibody response when we use a similar experimental setup of\ninduced deletion of Lyn but instead examine naive B cells responding to an exogenous antigen.\nPrevious analysis of the ability of Lyn deficient B cells to mount antibody responses was done in\nclassical Lyn KO mice and gave somewhat conflicting results (PMID: 7585947, 7584145,\n22942428). In general, somewhat impaired antibody responses were observed but with an\nincreased plasma cell accumulation. Our results confirm that there is a bias towards plasmablast\ndifferentiation/accumulation (Fig 5H & J). However the overall B cell response trends towards a\nreduced B cell response (Fig 5I). As a result, the overall antibody-secreting B cell response was\nequal when measuring by ELISPOT (Fig 5G). Overall, the results suggest that the phenotype of\nthe autoantibody response observed with transferred Ars/A1 B cells is not a consequence of\ninduced Lyn deficiency per se, but rather something specific about the Ars/A1 response.\n12) Fig 1E: y-axis label is incorrect\nThank you for pointing that out, we have changed the label\n13) Fig 1G. value moved\nThank you for pointing that out, we have fixed this\n14) Fig 2I-L Statistical comparison missing\nThank you for pointing that out, we have added these comparisons to match the text\n15) Fig 3D, E, H-K the time added underneath the stimulus is incorrect\nThank you for pointing that out, we have corrected this\nDescription of Suppl Fig 3B should be removed from legend of Suppl Fig 3A\nWe have removed this description and separated supplemental Figs 3A and 3B into two\nindependent supplemental figures.\nReviewer 3\nWe thank the reviewer for his/her insightful comments and positive reception of our manuscript.\nPlease find our replies to your comments below\n1) Fig 1A: Is the deficit in splenic B cells due to an unexpected effect of mb1 expression\nduring development or is this a decrease in the anergic population?\nWe assume the reviewer has the same concern as reviewer 2, namely that the heterozygosity of\nthe mb1 gene in cre expressing mice may impact the observed phenotypes. As mentioned in our\n4\nresponse to reviewer 2, we did additional experiments to determine if CD79A heterozygosity in\nArs/A1 B cells, due to cre expression, impacts B cell development in the bone marrow, splenic B\ncell numbers and B cell tolerance as measured by calcium mobilization and autoantibody\nsecreting cell numbers. We found no effect of CD79A heterozygosity on anergic Ars/A1 B cells,\nsuggesting that the effects observed in mb1cre+ x Lynflox/flox x Ars/A1 mice on the ability of\nArs/A1 B cells to become anergic are due to Lyn deficiency.\n2) Previous work in mb1cre Lyn flox/flox mice with a WT repertoire identified a block at the\nT1 stage. T3 B cells share markers with anergic B cells, so is it possible that these\npopulations are not resolved in your analysis.\nIn Ars/A1 mice B cells express the same autoreactive antigen receptor that renders the B cell\nanergic with a T3-like phenotype. Using an anti-idiotypic antibody (clone E4) that is specific for\nthe autoreactive BCR encoded by the Ars/A1 transgenic BCR we showed that >97% of B cells\nare E4 positive in the mice we used (Suppl Fig 1C). Therefore, in our analysis we measure\nB220+ cells in the periphery and identify them as Ars/A1 B cells regardless of their phenotype.\n3) Fig 1A-E: which markers were used to assess total and functional B cell-populations?\nIn the bone marrow we gated on B220+ CD43- cells and sub-gated on IgM+ IgD- cells to\nidentify immature B cells and IgM+ IgD+ events to identify mature B cells, as shown in\nsupplemental Fig 1A. As mentioned above, in the periphery we used B220 expression to identify\nArs/A1 B cells. This has been clarified in the text.\n4) Fig 1A-E : description of results and conclusions regarding this figure is unclear in the\ntext.\nWe apologize and have modified the text to hopefully explain our findings in a more accessible\nmanner.\n5) Fig 1F: Which B cell population was used for analysis of IgM, IgD and activation\nmarkers.\nThese were gated on the total splenic B cell population, B220+ events, which captures the total\nArs/A1 B cell population.\n6) Fig 1F, 2C, clarify how many independent experiment days do these data represent.\nAlternatively, data could be normalized (rather than using raw MFI, which can vary day\nby day).\nThese data are from a single experiment (i.e. spleens were harvested on the same day, process\nand run at the same time). Throughout the paper the data presented in each (sub)figure is from a\nsingle collection event. Each data point is a biological replicate. The variation detected in the\nfigures represents the variability within each genotype. Independent repeat experiments show a\nsimilar spread in the data.\n5\n7) Fig 1, 5: can representative ELISPOTS images for these figures be shown\nWe have added scans of ELISPOTs in the supplemental figures (Suppl Figs 1D, 12C and 13D).\n8) Fig 1, 5 is it possible to look at the secretion of IgM anti-chromatin antibodies as was\ndone in a previous publication\nWe do not have the material to rerun these analyses, but we would expect the results to be\nidentical. In the 2016 JEM paper that the reviewer refers to we used the same Ars/A1 BCR\ntransgene in the experiments that they refer to. Since this is a conventional BCR transgene the\nBCR cannot undergo somatic hyper mutation, i.e. the specificity is fixed. The Ars/A1 BCR is of\nthe IgMa allotype allowing us to distinguish antibodies derived from these B cells from\nantibodies derived from C57BL/6 recipient B cells (IgMb) in an adoptive transfer setup. As\nshown in Figs 3G and H of the 2016 JEM publication the number of antibody secreting B cells\nthat make IgMa anti-Ars is the same as those making IgMa anti-chromatin. This is consistent\nwith the initial characterization of the Ars/A1 BCR transgene that identified the BCR to bind\nDNA (PMID: 11163228).\n9) Fig 2: do we believe that upon induced Lyn deletion impacts the maintenance or\nestablishment of anergy? It appears to be differently argued in different parts of the\nmanuscript.\nWe apologize for the unclarity. We interpret our data to show that Lyn is required to both\nestablish and maintain B cell anergy. The data shown in Fig 1 supports a role for Lyn in\nestablishing B cell anergy. The data shown in Fig 2 supports a role for Lyn in maintaining B cell\nanergy. Prior to tamoxifen treatment these Ars/A1 B cells are Lyn sufficient and anergic. Only\nupon induced Lyn deletion do the Ars/A1 B cells regain BCR responsiveness, indicative of a loss\nof B cell anergy. The data presented in Fig 5 further supports the idea that Lyn is required for\nmaintaining B cell anergy. Ars/A1 B cells proliferate and differentiate into autoantibody\nsecreting cells upon induced Lyn deficiency. As discussed above in response to reviewer 2’s\nquestion # 9, the lack of a robust autoantibody response upon acute deletion of Lyn in Ars/A1 B\ncells in Fig 5, compared to what we observed upon acute deletion of SHIP-1 or SHP-1 in Ars/A1\nB cells (PMID: 27114609), made us more cautious in our discussion. However, taken together\nwe think our data shows that Lyn is required to both establish and maintain B cell anergy and we\nhave edited the manuscript to convey this message more clearly.\n10) Fig 2: Loss of tolerance of anergic B cells in the WT repertoire upon loss of Lyn?\nThis is an interesting question. As the reviewer pointed out, the Cambier and Farris labs showed\nthat transitional 3 (T3) B cells are enriched in autoreactive B cells that have an anergic\nphenotype, suggesting that anergic B cells in the wild type repertoire reside in this B cell\ncompartment. We have tried to address this question by studying the effect of acute Lyn deletion\non the calcium response of Transitional 3 B cells in wild type repertoire mice. However, we\nexperienced technical difficulties making a panel work that allowed us to separate the different\n6\ntransitional B cells in indo-1-loaded YFP+ B cells. Going back to the literature we did find two\nreports, one in total Lyn KO and one in B cell restricted Lyn KO that analyzed calcium signaling\nin transitional 3 B cells and their activation state and found these to be enhanced in Lyn deficient\nT3 B cells compared to Lyn sufficient T3 B cells, consistent with a loss of B cell anergy in the\nwild type repertoire. We have added text to the discussion to highlight these data and their\nimplications.\n11) Fig 2B LynA/LynB ratio in anergic B cells is biased towards LynA, while B cells typically\nhave more LynB\nThe reviewer raises an interesting point that we had missed. When we compared Ars/A1 B cells\nwith naïve C57BL/6 we found that both had a similar bias towards LynA expression, contrary to\npublished data. The polyclonal anti-Lyn rabbit IgG that we use was raised against the first 131\namino acid of the LynA form, so we wondered if our reagent preferentially detects LynA. We\nobtained the same anti-Lyn antibody (clone LYN-01) used in PMID:35452291 and compared\nArs/A1 B cells to C57BL/6 B cells. Using this anti-Lyn antibody we detect preferential LynB\nexpression in both Ars/A1 and C57BL/6 B cells as predicted by the literature. The main purpose\nof our Lyn staining is to show the efficiency of deletion in our inducible system. However, we\nhave added a comment regarding the LynA/LynB ratio and added a supplemental figure (Suppl\nFig 3) showing the differential staining pattern depending on the reagent used.\n12) Analysis of pSHIP-1 and pSHP-1 would strengthen the link between Lyn deletion and\nregulation of downstream signaling.\nWe thank the reviewer for this suggestion. We used our inducible deletion model to generate Lyn\nsufficient and deficient YFP+ B cells, sorted the YFP+ B cells and analyzed the phosphorylation\nof SHIP-1 and SHP-1 at a basal state and following BCR crosslinking. As shown in Fig 2M and\ndiscussed in the text (lines 3 64-367) we found that Lyn deficient Ars/A1 B cells have reduced\nphosphorylation of these phosphatases at a basal state. While BCR crosslinking increases their\nphosphorylation in Lyn sufficient Ars/A1 B cells, Lyn deficient Ars/A1 B cels did not\nphosphorylate these phosphatases upon BCR crosslinking. These results are consistent with a\nrole for Lyn in establishing regulatory signaling mechanisms using phosphatases to constrain\nBCR signaling in anergic B cells.\n13) If F(ab’)2 fragments are used to prevent FcgRIIB recruitment, how is Lyn recruited to\ndampen signaling in anergic B cells. Could this have something to do with ITAM\nmonophosphorylation of CD79A ITAMs in anergic B cells? How would this result change\nwith co-crosslinking of an ITIM containing protein.\nThese are excellent questions that we are actively investigating. Our working hypothesis is that\nthe preferential CD79A ITAM monophosphorylation observed in anergic B cells promotes\ndampened downstream signaling in at least 3 ways. Monophosphorylated ITAMs do not recruit\nSyk, limiting downstream activating signaling, but they can recruit Lyn. Lyn can phosphorylate\nBCR thereby biasing signaling towards inhibitory signaling. For example, CD22 interacts with\nsialic acid residues on BCRs. When we compare anergic B cells with naïve B cells, we find that\nSyk phosphoprylation is significantly reduced in anergic B cells compared to naïve B cells. In\ncontrast, CD22 phosphorylation and SHP-1 association is relatively normal, consistent with a\nbias towards inhibitory signaling. Finally, we have evidence that suggests that\nmonophosphorylated CD79A at the Y182 residue can directly recruit SHIP-1 to the BCR to\npromote its phosphorylation. A manuscript describing these findings is in preparation, and we are\ncurrently testing these hypotheses in mice that are restricted in their ability to phosphorylate\nCD79A ITAMs. While these studies are beyond the scope of this manuscript, we have expanded\nour discussion on how Lyn may operate to maintain tolerance.\n14) Fig 4. Could a second gating at higher IgM be added? Does that restore pErk detection?\nWe have added a second gate on Ars/A1 B cells with higher IgM expression and present the data\nin supplemental figure 10. The results are similar to those described in Fig 4. In this\nsubpopulation the pErk response is still very low. In our hands pErk detection is variable and\nusually relatively weak. Most of the detected responses are from the B cells with higher surface\nIgM expression. In Ars/A1 B cell population within the high IgM slice there were some cells that\nhad significant Erk phosphorylation, resulting in a detectible response. We are not sure how to\ninterpret that.\n15) Fig 5 Description and conclusions regarding this figure are unclear in the text.\nThank you for pointing this out, we have edited the text to hopefully do a better job of conveying\nthe main conclusions.\nMinor critiques\n16) General: editing to increase clarity for readership beyond B cell biologists (especially\nFig 1&5)\nWe have made several edits in the text that we hope will increase clarity for a general readership\n17) Abstract: confusing sentence regarding statement on collateral loss\nWe have edited this sentence\n18) Fig 1C Harmonize identification of graphed cell populations in the Y axis, figure legends\nand main text\nThese edited the figures, legends and main text to make it consistent.\n19) Fig 1H Quantify immunoblots and add statistical analysis\nWe have added a figure that summarizes the quantification of several independent experiments\n8\n20) Fig 2: Efficiency of deletion\nWe have looked at the efficiency of deletion (using YFP expression as a proxy) over a range\nof experiments and found it to be in the range of 61.1 ±21.6% of total B cells. This is\nconsistent with what has been reported previously for this inducible cre recombinase (PMID:\n22555432). We added a statement regarding this in the manuscript (lines 118-120)\n21) Fig 2D: Could kinetic differences in calcium mobilization be quantified? Unclear of\nabsolute concentration of free calcium is being reported as suggested by the axis\nWe have added additional analysis of the calcium mobilization in supplemental Fig 4A. We\ndetect a similar delay in the induction of calcium responses as has been described previously in\nother Lyn deficient models. This delay is consistent with the role of Lyn in initiating BCR\nsignaling, a function that is taken over by other Src family kinases such as Fyn if Lyn is deleted.\nRegarding the second point, the reviewer is correct that the labeling of the axis is incorrect and\nmisleading. We did not quantify absolute concentrations of intracellular calcium. We have\nchanged the labeling of the axis to better reflect what was measured.\n22) Fig 3R & T: Could aggregate data be included for pCD79A and pBLNK?\nWe show the BLNK and CD79A data from the replicate experiment, as well as an aggregated\ndata graph using normalized data from both experiments in supplemental figure 7. While CD79A\nY182 phosphorylation does not appear to be dependent of PI3K activity, it seems that there is a\nminor dependence of BLNK phosphorylation. We have adjusted the text accordingly.\n23) Supplemental Figures 2,3,4. Color scheme difficult to process and unstimulated legend in\nFig S2 does not match the flow plot.\nWe have adjusted the color scheme to hopefully make the figures easier to understand. We also\ncorrected the figure legend of suppl Fig 2 (now Suppl Fig 4), thank you for pointing that out.\n24) Fig 4C-K. Why aren’t the total and equal IgM populations plotted/analyzed together?\nWe decided to present the data in this manner to focus the reader’s attention to comparisons that\nwe consider most relevant (stimulated anergic vs non-anergic) within the different gating\nstrategies (total B cells vs gated on similar IgM expression). The data is presented on the same\nscale so comparisons can be made visually between the two different ways of gating B cells,\nhowever we are uncertain if additional statistical comparisons would be informative\n25) Discussion: “avoiding all the myeloid cell activation” should be edited for precision.\nThank you for pointing that out, we have edited the text.\nPoint-by-point response to the reviewers’ comments\nReviewer #1 (Remarks to the Author):\nMany aspects were taken into consideration in the revised form and experiments were\ndone to support the revision. The improved Manuscript is worth to be considered for\npublication.\nWe thank the reviewer for his/her positive evaluation of our revised manuscript!\nReviewer #2 (Remarks to the Author):\nI thoroughly enjoyed reading the revised version of the manuscript. The authors have\nsuccessfully addressed all my comments, and I recommend this manuscript for\npublication.\nWe thank the reviewer for his/her positive evaluation of our revised manuscript!\nReviewer #3 (Remarks to the Author):\nThe authors have made the requested changes and have put to rest our concerns.\nNicely done!\nWe thank the reviewer for his/her positive evaluation of our revised manuscript!"}, {"round": 2, "reviewer_comments": {"reviewer_1": "Many aspects were taken into consideration in the revised form and experiments were done to support the revision. The improved Manuscript is worth to be considered for publication.", "reviewer_2": "I thoroughly enjoyed reading the revised version of the manuscript. The authors have successfully addressed all my comments, and I recommend this manuscript for publication.", "reviewer_3": "The authors have made the requested changes and have put to rest our concerns. Nicely done!"}, "author_response": ""}]

Decision letter