Missing-In-Metastasis / Metastasis Suppressor 1 modulates B cell receptor signaling, B cell metabolic potential and T cell-independent immune responses

Efficient generation of antibodies by B cells is one of the prerequisites of protective immunity. B cells, activated by cognate antigens via B cell receptors (BCR) or pathogen-associated molecules through pattern-recognition receptors, such as TLRs, initiate signaling cascades leading to transcriptional and metabolic changes that ultimately transform B cells into efficient producers of antibodies. BCR signaling is also critical for normal development, as well as survival, of B cells, and critical for survival of different types of lymphomas. A number of steps initiated downstream of BCR rely on coordinated action of actin cytoskeleton, which is in turn dependent on concerted action of multiple actin-regulatory proteins, some of them exclusive to B cells. In our study we dissect the role of Missing-In-Metastasis, or Metastasis suppressor 1 (MIM, or MTSS1), a membrane- and actin cytoskeleton-associated I-BAR domain protein, in the regulation of B cell-mediated immunity. Differential expression of MIM has been associated with variety of cancers, whereas loss of MIM was found to lead to development of B cell lymphomas in adult mice, suggesting important functions in B cells. We take advantage of MIM knockout mouse strain and show normal B cell development and composition of B cell compartments in the periphery. However, we found that MIM-deficient B cells are defected in BCR signaling in response to surface-bound antigen and have higher metabolic activity after activation with LPS or CpG. In vivo MIM knockout animals exhibit impaired IgM antibody responses to immunization with T cell-independent antigen. This study provides first comprehensive characterization of MIM in B cells and highlights its modulatory role for B cell-mediated immunity.


Introduction
MIM -/and WT mice. Again, we found no significant differences in the proportions of CD5 + (B1a), CD5 -(B1b) or mature peritoneal B cells (B2) (Fig. 1E). These results demonstrate that normal development of different B cell subsets, both in the bone marrow, as well as in the periphery, do not depend on MIM. On the other hand, these findings also highlight possible variation between the two different published MIM-deficient mouse models (Saarikangas et al., 2011;Yu et al., 2012).

MIM-deficient B cells are defected in BCR signaling upon activation with surface-bound antigens
While previous cell biological studies in other cell types, supported by various biochemical assays, have proposed a role for MIM at the interface of the plasma membrane / actin cytoskeleton interface, which regulates receptor function in B cells, we went to examine the role of MIM in BCR signaling.
First, we analyzed the mobilization of intracellular calcium in mature B cell populations of spleen and peritoneal cavity upon surrogate antigen stimulation. Splenic B cells were loaded with Fluo-4 and Fura-Red, stimulated with anti-IgM antibodies and analyzed by flow cytometry. Ratiometric analysis of Fluo-4/Fura-Red fluorescence intensities revealed similar elevation of intracellular Ca 2+ levels in both WT and MIM -/-B cells ( Fig. 2A). Peritoneal cavity B cells were loaded with Fluo-4, prestained with CD23-Ax594 just before acquisition and stimulated with anti-IgM-Ax633 antibodies, which allowed distinction between B1 (IgM + CD23 -) and B2 (IgM + CD23 + ) cells. Intracellular Ca 2+ mobilization was comparable between WT and MIM -/peritoneal cavity B cells in these two B cell subsets (Suppl. Fig. 2).
Next, we analyzed activation of individual components of BCR signaling pathway by looking at phosphorylation levels of downstream effector molecules. Splenic B cells were stimulated with soluble or surface-bound anti-IgM antibodies for 3, 7 and 15 min and analyzed by immunoblotting. As expected, both stimulatory conditions induced rapid activation of BCR signaling components, as seen from their respective phosphorylation levels. MIM -/-B cells showed clear defects in signaling in response to surface-bound anti-IgM. Activation of most of the analyzed molecules, including Syk, CD19, Btk, p65 NF-κB and MAPK1/2 was significantly reduced (Fig. 2C). In addition, phospho-PI3K showed a trend of reduction, however, not statistical significance. The extent to which early BCR signaling defect was influencing downstream cascades appeared to alternate. The defects in PI3K pathway were largely salvaged at the level of Akt. At the same time, the levels of pp65 NF-kB and pMAPK1/2 were decreased, suggesting that MIM -/-B cells were inefficient in triggering the diacylglycerol (DAG)-PKC module, targets of which both NF-kB and MAPK1/2 are (Mérida et al., 2010). Interestingly, when we studied activation of BCR by soluble ligand, anti-IgM surrogate antibodies in solution, MIM -/-B cells only showed significant defects in the activation of the proximal kinase Syk, but normal activation of other signaling components (Suppl. Fig. 2). These results place MIM as a regulator of, specifically, BCR signaling by surface-bound antigen, function that clearly depends on the fine-tuned activities of the actin cytoskeleton (Bolger-Munro et al., 2019) and could fit well with the previously postulated role of MIM as an organizer of the actin cytoskeleton-membrane interface.

The morphology and formation of the immunological synapse is unaltered in MIM-deficient B cells
To see if MIM-deficient B cells showed any changes in the overall morphology, we visualized them using scanning electron microscopy (SEM), either in steady-state or after 10 min activation by surfacetethered anti-IgM, mimicking formation of the immunological synapse. The morphology of MIM -/-B cells appeared similar to WT cells ( Fig 3A). Next, inspired by the diminished activation of BCR signaling pathways upon surface-bound antigen stimulation in MIM -/cells, we activated B cells on anti-IgM-coated coverslips and analyzed the area of spreading using TIRF microscopy. TIRF microscopy is a method that allows visualization of the membrane-cover slip interface with approximately 150 nm depth providing better signal to noise in this region without disturbance by the signals from deeper parts of the cell. We noted that MIM -/-B cells had a tendency to spread less than their WT counterparts (Fig. 3B). To measure overall phosphorylation at the contact region we stained the cells with anti-phospho-Tyrosine (pTyr) antibodies. Mean fluorescence intensities of pTyr staining were also reduced in MIM -/-B cells, however, the difference to WT appeared smaller as compared to the difference observed in immunoblotting ( Fig. 2B, C). These results indicate that lower amount of engaged antigen due to inefficient spreading response may contribute to the ultimate signaling defect observed in MIM -/ -B cells upon activation with surface-bound anti-IgM antibodies. At the same time, based on the F-actin staining on these cells, we concluded that despite of diminished area of spreading, MIM -/cells did not exhibit major morphological differences in their actin cytoskeleton (Fig. 3B).
surrogate antigen in the center of the IS as WT B cells, displaying normal IS organization at 10 min timepoint (Fig. 3D). These results suggest that while MIM modulates BCR signaling and spreading responses upon antigen contact on stiff substrates, it appears largely dispensable for overall immunological synapse formation upon engagement of membrane-bound mobile antigens.

MIM is required for efficient antibody responses against T-independent antigen
To test if the defected signaling in MIM-deficient B cells leads into problems in mounting of the immune responses, we went on to examine antibody levels of our mice first at the basal state and then upon immunization with T cell-independent or T cell-dependent model antigens. We saw no significant changes in the basal antibody levels in the sera of WT and MIM -/mice, although especially IgG1 appeared slightly diminished (Fig. 4A). To study development of antibody responses towards T-independent antigens, we immunized mice with NP-FICOLL (Fig. 4B). Notably, we found a clear reduction in the levels of both total and NP-specific IgM in MIM-deficient mice (Fig. 4B). Interestingly, we also detected impaired responses in the total levels of IgG subtypes, most profound in IgG2b, while generation of NP-specific IgG responses was normal.
For immunizations with T-dependent protein antigen, we used NP-KLH in Alum and followed up the primary response for four weeks. Four months later, we stimulated a memory response and followed that for 2 weeks (Suppl. Fig. 4A). Somewhat surprisingly, we found that MIM -/mice were as efficient in mounting antibody responses to a protein antigen as their WT counterparts (Suppl. Fig. 4B). Analysis of the memory responses also showed equal levels of NP-specific IgG for all subclasses (Suppl. Fig.   4C). Furthermore, we analyzed affinity maturation by comparing binding to low and high densities of NP-epitopes in ELISA, and found also no significant differences between WT and MIM-/-mice (Suppl. In the light of defects in BCR signaling and T-independent immune responses in MIM-deficient mice, normal antigen-specific IgG immune responses may point towards compensation by other signals in the system, such as T cells help. To dissect the B cell intrinsic features linked to IgG antibody responses in more detail, we set up an in vitro assay for class-switch recombination (CSR). Here, we provoke B cells to change the isotype of the produced Ig molecules, by mimicking cellular events of pathogen encounter or T cell help during maturation of IgG antibody responses. As expected, activation of B cells with LPS or CD40L in combination with cytokines for 3 days induced switching of surfaceexpressed Ig molecules to different IgG isotypes, as detected by flow cytometry. Consistent with the in vivo data on generation of NP-specific IgG antibody responses, MIM -/-B cells switched normally in all tested conditions, producing similar percentages of IgG + cells, indicating that MIM is not required for CSR in response to TLR ligands or CD40L and cytokines (Suppl. Fig 5A, B, C). In fact, switching rates were even slightly higher for MIM KO B cells in LPS cultures (Suppl. Fig. 5A, C). We also loaded the cells with Cell Trace Violet dye, which allowed us to analyze fluorescence profiles of dividing splenic B cells in response to these stimuli. We found robust proliferation of MIM -/-B cells when they were activated with LPS, CD40L or anti-IgM + CD40L (Suppl. Fig. 5D). Analysis of the proliferation indices (PI) showed equal proliferation of MIM -/and WT cells, however, the division indices (DI) of MIM -/-B cells were moderately increased in CD40L + IFNγ cultures, reflecting smaller numbers of undivided cells detected in cultures on day 3. Taken together, our immunization studies suggest that MIM plays a role in IgM antibody responses to T cell-independent antigens, however, is dispensable for the development of antibodies against T celldependent protein antigens.

MIM -/-B cells show higher metabolic profile upon LPS and CpG stimulation
Immunity-related signaling pathways are tied to general cell metabolism, which not only responsible for energy production but also has an impact on signal propagation and transcription through abundance of energy sources and actions of key metabolic enzymes and metabolites (Jellusova, 2018;O'Neill et al., 2016). We used Seahorse platform to analyze metabolic reprogramming of B cells upon activation of BCR or TLRs. To this end, splenic B cells from WT and MIM -/mice were cultured for 24 h with either anti-IgM with IL-4, or TLR agonists LPS or CpG. The rates of cellular oxygen consumption (OCR), an estimate of mitochondrial respiration, and extracellular acidification (ECAR), an estimate of glycolysis, were measured with a Seahorse Mito Stress test. In this assay, serial injections of oligomycin, FCCP and antimycin A / rotenone mixture, sequentially inhibit ATP production, collapse the proton gradient membrane potential and finally shut down mitochondrial respiration. This system allows measurement of the basal OCR, maximal and spare respiratory capacities, as well as determination of non-mitochondrial respiration.
In general, B cell activation is expected to result in increase in both, OCR and ECAR as the cells shift from resting state into activated phenotype (Akkaya et al., 2018;Caro-Maldonado et al., 2014;Price et al., 2018). We activated WT and MIM -/-B cells with LPS, CpG and IgM + IL-4 for 24 h or kept them largely resting with IL-4 alone. Notably, we saw a significant increase in the metabolic profile of MIM-/-cells as compared to WT counterparts upon activation with TLR ligands, LPS or CpG (Fig. 5A, B). We observed approximately 30% increase in the basal metabolic activity as well as maximal respiratory capacity. The spare respiratory capacity showed over two-fold increase in MIM -/cells upon LPS activation, while with CpG activation, we found variability too high to conclude on the possible difference. In contrast to TLR stimulations, BCR activation with soluble anti-IgM showed no difference between WT and MIM -/cells, neither at the basal or maximum respiration levels. Similarly, the levels of glycolytic metabolism, reflected by ECAR values obtained from the same assay, showed elevated levels of glycolytic metabolism in MIM -/-B cells when stimulated with TLR ligands but not IgM + IL-4 (Suppl. Fig. 6). While B cells cultured with IL-4 alone, IgM + IL-4 or LPS showed no change in the predominant route of generated ATP, we observed that basal metabolic activity was shifted to oxidative metabolism in MIM -/-B cells stimulated with CpG (Fig. 5C).
Next we asked if the higher metabolic activity of MIM -/-B cells reflects a bona fide change in the mitochondrial activity or is a result of elevated mitochondrial biogenesis upon TLR-mediated metabolic reprograming. To this end, we stained B cell mitochondria with antibodies against TMRE and Tom20 to assess mitochondrial membrane potential and mitochondrial mass, respectively. Already in freshly isolated splenic MIM -/-B cells, we found normal levels of mitochondrial potential, however, slightly elevated mitochondrial mass without noticeable increase in mitochondrial membrane potential (Fig. 5D).
However, TLR-activated B cells from WT and MIM -/-B cells had comparable levels of both mitochondrial membrane potential and mitochondrial content, suggesting that higher metabolic activity in TLR-stimulated MIM -/-B cells was not a result of increased mitochondrial biogenesis, but reflected increased mitochondrial activity (Fig. 5E).
Thus, we showed that MIM -/-B cells have higher metabolic activity after short-term culture with LPS or CpG TLR ligands and, although these differences do not persist after 3d cultures (data not shown), we believe that such metabolic change may influence, for instance, kinetics of antibody responses to TI-1 antigens.

DISCUSSION
Predisposition of another MIM-deficient mouse strain to lymphomagenesis, reported by Zhan and colleagues, is intriguing (Yu et al., 2012). We have not performed aging experiments to follow lymphomagenesis in our MIM -/strain, however, BCR signaling is one of the main survival pathways in different types of lymphoma and dysregulated wiring of BCR signaling could play a role in lymphomagenesis (Young and Staudt, 2013). Unlike originally reported by Zhan and colleagues (2012), B cell development appeared normal in our MIM -/strain also when gating strategy identical to Zhan and colleagues was applied (Suppl. Fig. 1-2). Confusingly, in the first studies with their MIM-deficient strain, Zhan and colleagues reported abberant levels of total CD19 + or CD19 + IgM + cells in the lymphoid organs (Yu et al., 2012). However, in the later studies, the same group failed to find the phenotype anymore (Zhan et al., 2016). The observed discrepancies may, thus, potentially arise from the differences in strain maintenance or, for instance, from different age of mice at the time of immunophenotyping. The mouse strain generated by Zhan and colleagues used embryonic stem cells (ES; clone CSC156, BayGenomics) with insertion of gene trap sequence between exons 3 and 4 (Yu et al., 2011). Notably, an independent attempt to recapitulate the generation of MIM knock out strain with the same clone of ES cells (CSC156, BayGenomics) resulted in inefficient use of delivered splicing acceptor (SA) site and considerable expression of full-length Mtss1 mRNA as well as protein expression (Fahrenkamp et al., 2017). This data warrants careful interpretation of the studies made with mouse model based on CSC156 gene trap ES cells. However, we cannot rule out the possibility that different genetic background of the mouse strains used for backcrossing in these studies (C57Bl/6 vs FVB/N) may influence activity of the spliceosome. Furthermore, variation in the remaining expression levels between the animals seems notable, indicating lability of the splicing. Lack of protein MIM expression in analyzed cells was, however, shown by Zhan and colleagues (Yu et al., 2011(Yu et al., , 2012. In our strain, based on the insertion of neomycin cassette with several stop codons in the first exon of Mtss1 sequence, low levels of alternative splicing generating a transcript has been previously observed. However this transcript translates into a N-terminally truncated protein that has functionally inhibited critical I-BAR domain, and which also did not lead to detectable protein expression in MIM -/animals (Saarikangas et al., 2011). We confirmed clear and repeatable lack of detectable protein expression in our substrain using two different commercial antibodies (Suppl. Fig. 1-1).
In this study, by observing consistently diminished phosphorylation of several BCR effector molecules, we show that MIM -/-B cells are defected in their ability to signal upon stimulation of BCR with surface-bound antigens (Fig. 2B). Signaling upon soluble antigen stimulation, on the other hand, was mostly normal showing only slight diminution in the levels of pSyk (Suppl. Fig. 2). Although, diminished spreading may contribute to the signaling defect seen in response to surface-bound antigen, the area of spreading was not drastically lower (Fig. 3B). This suggests that MIM participates in the ability of B cells to discriminate different types of antigens by playing a specific role in B cell activation on surfaces. Although the differential responses of B cells to different forms of antigen are nowadays widely accepted (Snapper, 2018), to our knowledge there are only few molecules, such as CD19 (Depoil et al., 2008), that have been reported to specifically regulate stimulation by surface-linked antigens with mechanisms likely separate from structural roles in cell adhesion or spreading.
Our experiments on supported lipid bilayers (SLB) showed, however, that MIM -/-B cells are able to form signaling-competent BCR-Ag microclusters and gather normal amounts of antigen in the center of the IS (Fig. 3D). This suggests that coupling of BCR to actin and microtubule cytoskeleton, required for the cell spreading and antigen gathering, respectively (Liu et al., 2012;Schnyder et al., 2011), is not notably defected in these settings. However, results from SLB, which are laterally fluid, cannot be directly compared with B cell stimulation on immobilized surrogate antigens or a situation where B cell need to overcome frictional coupling of antigen-presenting molecules to the membrane skeleton of the APC. In addition, SLBs in our experiments were functionalized with ICAM-1, which is known to lower the threshold for B cell activation (Carrasco et al., 2004).
We found that stimulation with surface-bound antigen results to a certain degree in reduced phosphorylation of all the tested BCR effectors in MIM -/-B cells, but also noted that defects in early BCR signaling, exemplified by reduced pSyk and pCD19, did not propagate evenly downstream.
Reduced pCD19 seemed to largely spare PI3K pathway as pAkt levels are on par with those of WT (Otero et al., 2001) (Fig. 2B, C). At the same time, levels of both pNF-kB and pMAPK1/2 at later time points (7 and 15 min) are significantly reduced, suggesting that defect lies at the level of DAG-PKC signaling module (Coughlin et al., 2005;Mérida et al., 2010;Su et al., 2002). This is interesting in the light of study pointing on the role of PKC-FAK axis in the regulation of force-dependent B cell activation (Shaheen et al., 2017), which we think is consistent with our results of specific defects in response to surface-bound antigens.
We found that MIM-deficient mice developed impaired IgM antibody responses to NP-FICOLL immunizations. On the other hand, the responses appeared normal in NP-KLH immunization, indicating that the defect in the antibody production is specific to the nature of the antigen. FICOLL polysaccharide haptenated with NP, or other small molecule epitope, is a typical T cell-independent type 2 (TI-2) antigen and, in contrast to TI-1 antigens, is unable to induce humoral immune responses in Btk-deficient CBA/N strain and normal neonatal mice reflecting dependence on mature B cell populations (Scher, 1982). Marginal zone B cells and peritoneal cavity B1b cells are, together, thought to be responsible for hapten-specific antibody production upon immunization with FICOLL conjugates (Girkontaite et al.; Guinamard et al., 2000;Haas, 2011). We show that MIM -/mice have comparable proportions of B1b, B1a and B2 cells in the peritoneal cavity (Fig. 1F) and normal composition of splenic B cell compartments, perhaps with slight tendency to have elevated MZ B cell numbers (Fig. 1D), suggesting that disproportions in mature B cells populations of spleen and peritoneal cavity are unlikely to cause the reduced anti-NP IgM levels (Fig, 4B). As the numbers of CD4 + T cells (Fig. 1C) and antibody responses to T cell-dependent antigen NP-KLH (Suppl. Fig. 4), as well as the levels of class-switched IgG antibodies to NP-FICOLL itself, appeared normal (Fig. 4B), we also consider the defects in possible indirect T cell help very unlikely. Therefore, we suggest that the defected NP-FICOLL responses in MIM -/mice are likely to be caused by defected B cell receptor-mediated signaling. Indeed, we observe that MIM-deficient splenic B cells exhibit diminished BCR signaling on anti-IgM-coated surfaces ( Fig. 2B, C). Thus, our results would be in line with the observations that in vivo B cells recognize and respond to antigens that are immobilized on the surface of other cells in the secondary lymphoid organs (Carrasco and Batista, 2007). FICOLL is a long chain polysaccharide with high molecular weight of ~400 kDa, which is unable to enter conduit system of the spleen for direct access to follicular areas (Nolte et al., 2003). However, both, i.v. and i.p. delivery of FICOLL conjugates results in its accumulation in the marginal zone and later in the follicular regions of the spleen (Cinamon et al., 2008;García de Vinuesa et al., 1999). Such follicular localization of TD and TI antigens has been described as a result of complement-dependent antigen capturing by non-cognate MZ B cells and delivery to follicular areas for deposition onto follicular dendritic cells (FDC) for continuous display to antigenspecific B cells (van den Eertwegh et al., 1992;Ferguson et al., 2004;Heesters et al., 2013;El Shikh et al., 2010). As it was shown by other studies, antigen presenting surface as well as substrate stiffness prominently influences BCR signaling strength (Wan et al., 2013;Zeng et al., 2015) and, in this respect, FDCs have been reported to serve as rather stiff antigen presenting substrate (Spillane and Tolar, 2017).
Thus, we suggest that MIM -/-B cells are defected in being triggered by FDC-displayed NP-FICOLL molecules, similar to BCR stimulation on anti-IgM-coated surface, which results in poor plasmablast differentiation and reduced IgM antibody response.
We also noticed decreasing total IgG levels in the course of the NP-FICOLL response (Fig. 4B).
The cause of it is unclear but may include, for instance, inhibition of constitutive antigen presentation in B cells by high load of NP-FICOLL, which MIM -/-B cells are unable to cope with efficiently (González-Fernández et al., 1997). Our data show relatively equal fold-increase of NP-specific IgG of all isotypes in response to NP-FICOLL immunization (Fig. 4B). This is interesting as some TI-2 antigens, including TNP-FICOLL, are reported to elicit IgG3-restricted class switching (Mongini et al., 1981(Mongini et al., , 1984Perlmutter et al., 1978;Slack et al., 1980) (García de Vinuesa et al., 1999. However, mouse strain or experimental differences may exist and this might not be generally applied to all TI-2 antigens, for which however, only the levels of IgM and IgG3 are typically tested. While naive B cells have low metabolic profile, upon activation, they elevate their metabolic rates typically manifested by an increase in oxygen consumption and glycolysis (Caro-Maldonado et al., 2014;Jellusova, 2018). The mitogenic stimuli IgM, LPS and CpG have been shown to dramatically increase metabolic requirements in B cells and thus, play a key role in this transition, essential for the immune responses and differentiation into antibody secreting cells (Boothby and Rickert, 2017;Jellusova, 2018). Interestingly, we found that after 24h culture with either LPS or CpG, respective stimulators for TLR4 and TLR9, MIM -/-B cells exhibited approximately 30% increased metabolic activity as measured by basal oxygen consumption rate as well as by maximum and spare respiratory capacities (Fig. 5A, B). This finding indicates increased metabolic demands in cells lacking MIM.
Further supporting the higher cellular energetics of MIM -/cells, already at resting state, MIM-deficient B cells had slightly more mitochondrial mass, as detected by Tom20 levels. At the same time, LPS and CpG induced similar levels of increase in mitochondrial mass and membrane potential in both WT and MIM -/cells (Fig. 5D, E), indicating that metabolic differences do not simply arise from enhanced mitochondrial biogenesis. We also did not observe differences in metabolism when B cells were stimulated with anti-IgM + IL-4, suggesting specificity in the role of MIM towards TLR signalinginduced metabolic changes. The higher energetics in the absence of MIM might indicate that its involvement in TLR4/9 signaling might negatively affect metabolism. Such increased metabolic capacity could also compensate the effects of compromised BCR signaling upon in vivo B cell differentiation and antibody responses to TI-1 antigens. The exact mechanism of such involvement and its role for cellular activation remains to be further investigated, but it is tempting to speculate of a possible link between altered cellular metabolism and the loss of MIM in several cancers.

Antibodies and chemicals
Full list of antibodies and reagents used in the study can be found in supplementary file N.

Mice, immunizations and serum collection
MIM knockout mouse colony in Turku was established by breedings of HEZ founder mice and had no apparent health problems until the age of 8 months when mice were sacrificed due to possible damaging effects of reported lymphomas later in life (Yu et al., 2012, Mattila unpublished). However, we observed that among 18 genotyped pups that developed hydrocephaly over the study period, 17 were knockout and 1 was heterozygote. None of the mice that developed hydrocephaly were genotyped as wild-type.

ELISA
Total and NP-specific antibody levels were measured by ELISA on half-area 96-well plates. Capture antibodies (2 µg/mL) or NP-conjugated carrier proteins (NP (1-9) -BSA or NP (>20) -BSA at 50 µg/mL) were coated at +4°C overnight in 25 µL of PBS. Non-specific binding sites were blocked for 2 hours in 150 µL of blocking buffer (PBS, 1%BSA, 0.05 NaN 3 ). Appropriate dilutions of 50 µL serum samples in blocking buffer were added for overnight incubation at +4°C. Biotin-conjugated detection antibodies (2 µg/mL) in 50 µL of blocking buffer are added for 1 hour followed by 50 µL AP (alkaline phosphatase)streptavidin (1:5000) in blocking buffer for 1 hour at room temperature (RT). In between all incubation steps, plates were washed with 150 µL washing buffer (PBS, 0.05% Tween-20) 3 times (steps before sample addition) and 6 times (after). The final wash was completed with 2 times wash with 150 µL of mQ water. Finally, 50 µL of AP-substrate solution was added and and OD was measured at 405 nm.
Appropriate serum dilutions were determined experimentally to fall into linear part of the dose-response curve of the absorbance measurements for any given isotype and typical values are as follows: IgM levels (1:3000-1:4000), IgG levels (1:20000-1:80000). Different dilutions of AP-streptavidin were used where necessary. Typical time for AP-substrate incubation before measurement was about 30 min at RT.
All ELISA samples were run in duplicates, OD values were averaged and blank background was subtracted. Absolute concentrations of total antibody levels were extrapolated from calibration curves prepared by serial dilution of mouse IgM or subclasses of IgG from C57Bl/6 immunoglobulin panel.
Relative NP-specific antibody levels were extrapolated from reference curves prepared by serial dilution of pooled serum, in which the highest dilution step received an arbitrary unit of 0.5.
Membranes were blocked with 5% BSA in TBS (TBS, pH ~7.4) for 1 hour and incubated with primary antibodies (~1:1000) in 5% BSA in TBST (TBS, 0.05% Tween-20) at +4°C, overnight. Secondary antibody incubations (1:20000) were done for 2 hours at RT in 5% milk in TBST for HRP-and with addition of 0.01% SDS for far-red-conjugated antibodies. Washing steps were done in 10 mL of TBST for 5 × 5 min. Membranes were scanned with Odyssey CLx (LI-COR) or visualized with Immobilon Western Chemiluminescent HRP Substrate and ChemiDoc MP Imaging System (Bio-Rad). Phosphoantibodies were stripped in 25mM Glycine-HCl buffer, pH ~2.5 for 10 min, membranes were blocked and probed again for evaluation of total protein levels. Images were background subtracted and raw integrated densities for each band were measured in ImageJ. Ratios of phosphorylated-vs-total protein levels were analyzed with ratio paired t test. For data presentation these ratios were normalized to WT value at 0 min.

Equal amounts of unstained and labeled cells of different genotypes were mixed and loaded onto coated
MatTek dishes at 35 µL/well. Cells were incubated for 10 min (+37°C, 5% CO 2 ), fixed in pre-warmed (+37°C) 4% formaldehyde/PBS for 10 min (RT), permeabilized in 0.1% Triton X-100/PBS for 5 min (RT), washed once with PBS and blocked in blocking buffer (PBS, 1% BSA) at +4°C (overnight). Cells were stained with 1:50 Phalloidin-Alexa-555 and 1:500 anti-pTyr primary antibody (4G10) in blocking buffer for 1 hr (RT), washed 4 times with PBS, and stained with 1:500 Alexa-488 secondary anti-mouse IgG2b in blocking buffer for 1 hr (RT), washed 4 times in PBS and imaged in PBS with DeltaVision OMX Imaging System (GE Healthcare). TIRF images of cortical actin and pTyr were processed with ImageJ macro using B220 and bright field channels to discriminate between attached WT or MIM -/cells. Mean fluorescence intensities of phalloidin and pTyr staining and spreading area (determined on pTyr channel) of each cell were extracted from ~50-340 cells per experiment. Geometric means of WT and MIM -/fluorescence levels and spreading area from each experiment were analyzed with paired ratio t test.

Ca 2+ flux on supported lipid bilayers
Splenic WT or MIM -/-B cells (3.2×10 6 ) were resuspended in 75 µL of L-15 medium and labeled by addition of 75 µL of HBS (HEPES buffered saline, supplemented with 2.5 µM probenecid and 20 µM Fluo4 for 5 minutes at +37°C. Cells were washed in 1ml HBS-probenecid and resuspended in 500 µL HBS-probenecid for immediate injection into FCS2 chambers. Acquired movies were preprocessed with ImageJ and analyzed with CalQuo 2 software (Lee et al., 2017). Cells were categorized as single peak, oscillatory or not triggering, whereas cells showing more than two intensity peaks are classified as oscillatory.

Metabolic assay
B cells, isolated from spleens of WT and MIM -/mice as described above, were seeded at a density of 10 6 cells / mL in complete RPMI. Cells were treated with IL-4 (10 ng/mL) ; anti-mouse IgM (10 µg/mL) + IL-4; LPS (4 µg/mL) or CpG (10 µg/mL) for 24 h (+37°C, 5% CO 2 ). Cells were then spun and resuspended in Seahorse XF RPMI, supplemented with 1 mM pyruvate, 2 mM L-glutamine and 10 mM D-glucose. Cell number was adjusted and 0.15x10 6 cells were seeded per well on a 96-well XF plate, pre-coated with CellTak. Plate coating was done with 22.4 µg/mL CellTak in NaHCO 3 , pH 8.0 at +4°C overnight, followed by two washings with mQ water. Seeded cells were spun at 200 g for 1 min with no break and left for 1 h at 37°C in a humidified incubator without CO 2 to attach to coated wells. Seahorse XF96 was used, following the manufacturer's instructions for XF Cell Mito Stress Test Kit.
Sequentially, 1 µM oligomycin, 2 µM FCCP and 0.5 µM rotenone / antimycin A were added to the media. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) data were recorded by WAVE software (Agilent). OCR and ECAR data were normalized to cell count and first baseline For Tom20 staining, B cells were stained with Zombie Violet as described above, fixed with 1.6% formaldehyde in PBS for 10 min, washed 2 × 150 µL PBS, permeabilized with 0.5% Triton X-100 in PBS for 5 min at RT, blocked for 1 h at RT. Incubation with primary Tom20 antibodies was done at 1:500 dilution for 30 min, followed by 3x 150 µL wash step and staining with 1:1000 dilution of antirabbit-Ax488 secondary antibodies. After 3 × 150µL wash step, cells were resuspended in 130 µL and acquired on BD LSR Fortessa. Antibody incubations, blocking and washings were done in flow cytometry buffer I on ice. Geometric mean fluorescence intensities were extracted with FlowJo software.
All statistical analyses for metabolic data were done with ratio paired t test.  Flow cytometry analysis of the bone marrow, spleen and peritoneal cavity cell populations of WT and MIM-/-mice. Gating strategy is shown in Supplementary Fig. 1-1