Map3k14 as a Regulator of Innate and Adaptive Immune Response during Acute Viral Infection

The replication of virus in secondary lymphoid organs is crucial for the activation of antigen-presenting cells. Balanced viral replication ensures the sufficient availability of antigens and production of cytokines, and both of which are needed for virus-specific immune activation and viral elimination. Host factors that regulate coordinated viral replication are not fully understood. In the study reported here, we identified Map3k14 as an important regulator of enforced viral replication in the spleen while performing genome-wide association studies of various inbred mouse lines in a model of lymphocytic choriomeningitis virus (LCMV) infection. When alymphoplasia mice (aly/aly, Map3k14aly/aly, or Nikaly/aly), which carry a mutation in Map3k14, were infected with LCMV or vesicular stomatitis virus (VSV), they display early reductions in early viral replication in the spleen, reduced innate and adaptive immune activation, and lack of viral control. Histologically, scant B cells and the lack of CD169+ macrophages correlated with reduced immune activation in Map3k14aly/aly mice. The transfer of wildtype B cells into Map3k14aly/aly mice repopulated CD169+ macrophages, restored enforced viral replication, and resulted in enhanced immune activation and faster viral control.


Introduction
The spleen is a highly organized lymphoid organ that harbors a discrete population of immune cells with versatile functions [1]. Microanatomically, the spleen is composed of two distinct compartments, the red pulp and the white pulp, which are separated by a specialized interface, called the marginal zone (MZ) in rodents and the perifollicular zone in humans [2]. The red pulp zone plays a crucial role in filtering the blood, recycling iron and producing antibodies from plasmablasts and plasma cells. The white pulp zone contains B-and T-cell compartments as sheaths. Between these compartments, the MZ mainly contains B cells and macrophages [3].
Marrginal zone B cells (MZ B) cells are described as innate-like lymphocytes, because they can generate an antibody response against commensal and foreign pathogens much more rapidly than follicular B cells. MZ B cells can mount T cell dependent and independent antigens, and they express non-mutated immunoglobulin-variable (IgV) genes, some of which encode B-cell receptors (BCRs). MZ B cells are professional antigen (Ag)-presenting cells (APCs) that can present Ag to activate naïve CD4 + T cells after reactivity [4][5][6][7].
Being strategically located in the marginal interface, the marginal zone macrophages (MZMs) and marginal metallophilic macrophages (MMMs) are key components during type I interferon (IFN-I)-mediated containment of the virus after phagocytosis, which results in viral clearance. Nevertheless, these cells can prime the adaptive immune response by presenting the antigens directly to B cells [8] or by capturing the antigen via resident dendritic cells (DCs) from the conduits that connect the MZ and the white pulp, culminating in presenting these antigens to T cells [9,10]. These macrophages play a prominent role in protection against fulminant infection with vesicular stomatitis virus (VSV) or lymphocytic choriomeningitis virus (LCMV) [11]. For instance, the ablation of MZMs by clodronate leads to the dissemination of virus to the peripheral organs and to T-cell exhaustion during LCMV infection [12]. Similarly, mice that are devoid of MZMs are highly prone to VSV infection [13]. Moreover, studies using MZM-depleted mice showed that MZMs contribute in controlling Listeria monocytogenes infections [11].
Maintaining intact splenic architecture is important in guaranteeing immune surveillance. The orchestration between B cells and MZMs is crucial for the architecture and quality of the MZ [14]. For example, the absence of B cells results in the ablation of MMMs and MZMs [15]. Another study showed that the integrity and function of organized MZ critically depend on the existence of B cells, as documented in studies using CD70TG mice, in which the B cells were steadily depleted because the high expression of the tumor necrosis factor (TNF) family member CD70, and subsequent loss of splenic marginal zone [16]. On the other hand, the disruption of Src homology 2-containing inositol 5-phosphatase (SHIP) in myeloid cells demonstrates that MZMs are necessary for the retention and trafficking of MZ Bs [14,17].
A mouse strain called alymphoplasia (aly/aly) mice, mitogen-activated protein kinase 14 (Map3k14 aly/aly ) mice, or NF-κB-inducing kinase (NIK aly/aly ) mice is defined by the complete absence of lymph nodes (LN) and Peyer's patches (PP). These mice have no MZ and they exhibit disorganized splenic and thymic architecture and, thus, immune intolerance against viral infections. The atrophic lymphoid structure in these murine models is caused by an autosomal recessive point mutation in the aly locus situated on chromosome 11, which encodes Nik. Nik is a key mediator of Nf-κB activation by the TNF receptor family and it is essential in the development and maintenance of B cells. Nik interacts with the TNF receptor-associated factor (TRAF) family of proteins and its downstream molecules, such as lymphotoxin-β receptor (Ltβr) and CD40 [18][19][20][21][22].
We implemented a genome-wide association study (GWAS) of inbred mouse strains to determine the mechanisms that regulate early viral replication in the spleen. We found that Map3k14 is a key mediator of immune surveillance during viral infection, as it promotes the immune activation, which is dependent on viral replication in the spleen. Map3k14 aly/aly mice showed limited early replication of LCMV and VSV and had a blunted innate and adaptive immune activation. We attributed the underlying mechanism to the deficiency of marginal zone B cells, which are prominent regulators of the integrity of lymphoid organ architecture, with the help of transfer experiments and generation of bone marrow chimeric mice.

Genome-Wide Association Study Shows That Map3k14 Is a Regulator of Viral Replication in the Spleen
We executed genome-wide association study (GWAS) using different inbred mouse lines which have genetic variations due to single nucleotide polymorphisms (SNPs) present in introns and exons of various genes to gain insight about the novel host factors that determine immune activation during virus infection [23]. We infected these inbred mouse lines with lymphocytic choriomeningitis virus (LCMV) and determined the early viral titers in the spleen after three days. We observed remarkable differences in the virus replication between the tested mouse lines ( Figure 1A). Next, we correlated the biological response (viral titers) and genotype (SNPs) for these mouse lines while using efficient mixed-model association (EMMA), as described previously [24,25]. EMMA analysis revealed the SNP mm37-11-103083091 at position 11:103,089.4k in mitogen-activated protein kinase 14 (Map3k14) gene as one of the top rank candidates among all of the SNPs ( Figure 1B,C). We systemically infected wild-type (WT) and Map3k14 aly/aly mice with the acute strain of LCMV and analyzed viral replication early during the course of infection to validate our GWAS screening results concerning the relevance of Map3k14 for LCMV infection. Infectious LCMV particles were strongly reduced in the spleen and serum of Map3k14 aly/aly mice as compared to the WT mice ( Figure 1D, left panel & Figure S3A), whereas the viral burden in the liver and lung tissues was comparable between WT and Map3k14 aly/aly mice ( Figure S3B), which indicated that the spleen represents the early target for LCMV infection. Immunohistologic analysis exhibited virtually no staining for LCMV nucleoprotein in the spleen of Map3k14 aly/aly mice ( Figure 1D right panel).
Next, we questioned whether the Map3k14 mutation affects another RNA virus strain. For this, we infected WT and Map3k14 aly/aly mice with VSV, which is an acute model for cytopathic virus infection, and examined viral multiplication in the spleen qualitatively and quantitatively. Virus titers determined in spleen tissues were significantly reduced in Map3k14 aly/aly mice when compared to the controls ( Figure 1E left panel). Consistently, immunohistologic staining of spleen sections from VSV infected mice showed lower viral replication in Map3k14 aly/aly as compared to WT mice ( Figure 1E right panel). These findings suggest that Map3k14 is a prominent mediator of acute viral replication in the spleen.

Map3k14 Promotes Innate and Adaptive Immune Activation
We assessed the IFNα serum levels in WT and Map3k14 aly/aly mice to address the impact of Map3k14 dysfunction on innate and adaptive immune activation during LCMV and VSV infection. Consistent with decreased replication of LCMV and VSV in spleen tissue, Map3k14 aly/aly mice experienced lower serum IFNα levels after infection with either virus strain than WT mice did (Figure 2A), a finding indicating that the vanished viral titer in the spleen of Map3k14 aly/aly mice results in a lower threshold for sparking IFNα production. Nevertheless, the curtailed IFNα levels in the serum of Map3k14 aly/aly mice was not due to the numeric reduction in DCs subsets, as we showed comparable frequencies of splenic DCs in Map3k14 aly/aly mice when compared to the competent mice ( Figure 2B). Next, we generated bone marrow chimera (BMC) mice by reconstituting lethally irradiated WT mice with bone marrow cells from control mice or from Map3k14 aly/aly mice to know whether Map3k14 expression in immune cells is necessary for virus replication in the spleen and systemic IFN-I production. After reconstitution, the mice were infected with the acute strain of LCMV and we found that WT mice that received BMCs from Map3k14 aly/aly mice demonstrated reduced viral replication and accompanying lower serum IFNα levels as compared to the control group, which exhibited increased splenic virus replication and systemic IFN-I response ( Figure 2C), a finding indicating that systemic defects in Map3k14 aly/aly mice exist on lymphocytes or hematopoietic cells, and not exclusively on stromal cells or deficiency in lymph node. Subsequently, we determined how reduced virus replication and systemic IFN-I response in Map3k14 aly/aly mice affect the adaptive immune response, as our previous studies showed that reduced innate immune activation is accompanied with impaired adaptive immune activation [13,26,27]. Intriguingly, the absence of replicating LCMV and the fact that IFNα levels were lower in Map3k14 aly/aly mice culminate in a blunted adaptive immune response, as demonstrated by a diminished total CD8 + T cells and virus-specific T cell response, which indicates the key role of efficient innate immunity to prime the subsequent adaptive immune response ( Figure 2D). Similarly, the systemic infection of WT mice reconstituted with BMCs from alymphoplasia mice with VSV resulted in a lower viral titer in the spleen and lymph nodes and subsequently in lower serum IFNα levels at an early time point when compared to the littermates ( Figure 2E). Taken together, these findings indicate that Map3k14 plays an intrinsic role in regulating the innate and adaptive immune response in the context of LCMV and VSV infections by mediating enforced viral replication [13]. . ns, not significant; * equals p < 0.05; ** equals p < 0.01; *** equals p < 0.001; **** equals p < 0.0001.

Map3k14 Signaling Maintains Splenic Marginal Zone Architecture
Next, we explored the mechanism for reduced virus replication and limited immune activation in Map3k14 aly/aly mice. Previous reports showed that the initial virus replication in spleen is mainly localized to CD169 + macrophages [13,27]. Further, we investigated whether Map3k14 affects splenic organization. In analyzing splenic histologic sections, we noticed generalized defect in the splenic architecture ( Figure 3A), and more specifically we found that CD169 + cells were absent from Map3k14 aly/aly mice, but were found in WT mice. Moreover, the B cells are substantially reduced in the splenic sections that are harvested from Map3k14 aly/aly mice. Nevertheless, the scarcity of B cells in Map3k14 aly/aly mice exerted no substantial effect on red pulp macrophages ( Figure 3B). Consistently, bone marrow transplantation experiments have shown lower numbers of MMMs in the spleen and lymph nodes of WT mice that were reconstituted with bone marrow derived from Map3k14 aly/aly mice, a finding that indicates that Map3k14 signaling is important in maintaining splenic CD169 + macrophages development ( Figure 3C). More importantly, the functional lack of the Map3k14 resulted in general B cell lymphopenia, with no MZ B cells being detected; however, the follicular B cells were mainly unaffected ( Figure 3D). Further, the F4/80 + macrophages in the liver and lung tissues that were retrieved from WT and Map3k14 aly/aly mice were unaffected by aly mutation, unlike the splenic CD169 + macrophages that are deprived in Map3k14 aly/aly mice ( Figure 3E). These findings indicate that the Map3k14 aly/aly mice were associated with absence of splenic CD169 + cells and MZ B cells, a condition that consequently diminishes early viral replication.

B Cells Are Required for the Development and Immune Activation of Splenic CD169 Macrophages in a Map3k14 Dysfunctional State
Next, we investigated whether the dramatic reduction in the number of CD169 + cells in Map3k14 aly/aly mice was a consequence of B cell lymphopenia [16]. To this end, we supplemented Map3k14 aly/aly mice with WT B cells. After 20 days of transfer, we checked the presence of CD169 + cells in the harvested spleen. Intriguingly, the CD169 + cells were restored in splenic tissue from Map3k14 aly/aly animal that were reintroduced with B cells, and these CD169 + cells subsequently enhanced VSV replication in the marginal zone ( Figure 4A). Moreover, the adoptive transfer of the B cells into Map3k14 aly/aly mice specifically restores the MZ B cells not the follicular, indicating the exclusive role of MZ B cells in maintaining the CD169 + macrophages ( Figure S1). Furthermore, IFN-I production after VSV infection was partially rescued by the transfer of WT B cells into alymphoplasia mice ( Figure 4B). In addition, the repopulation of CD169 + macrophages enhanced the induction of the adaptive immune system qualitatively and quantitatively, as seen in enhanced CD4 + and CD8 + T cells response in terms of number and functionality when compared to the control group ( Figure 4C). Similarly, the neutralizing antibody titers of alymphoplasia mice that were given WT B cells were dramatically higher than those in the non-transferred group ( Figure 4D). In parallel, experimental LCMV infection of WT and Map3k14 aly/aly mice supplemented with WT B cells led to enhanced viral multiplication in splenic tissues that were retrieved from the Map3k14 aly/aly mice and subsequent improved antiviral CD8 + T cells response in terms of frequency and functionality as compared to the untreated group ( Figure 4E,F). These findings indicate that B cells maintain the CD169 + cells and consequently contribute to improved innate and adaptive immunity in the context of VSV and LCMV infection. Consequently, viral replication in CD169 + macrophages is necessary for the promotion of T-cell priming and the production of neutralizing antibodies.  (F) For the same treated mice groups, here we challenged the animals with 2 × 10 4 plaque-forming units (PFU) of LCMV WE strain, and then the mice were put into death after eight days. Shown are the frequencies of polyclonal CD8 + T cells, LCMV-specific CD8 + T cells, and IFNγ producing CD8+ T cells (n = 3 per group). * equals p < 0.05; ** equals p < 0. 01; *** equals p < 0.001; **** equals p < 0.0001.

Discussion
Viral replication in immune cells is a key process for immune activation. Replication of virus is necessary, even during vaccination with attenuated pathogens, such as is the case with measles, rubella, chicken pox, yellow fever, and mumps [28]. Identifying the regulators of viral replication in the spleen and the implications of viral replication during infection is an important challenge to an understanding of the outcome of viral infection. We performed a GWAS of inbred mouse lines so that we could identify genes that regulate viral replication in immune cells. We found that Map3k14 is an important regulator of acute viral replication in the spleen.
In the study reported here, we found that Map3k14 plays a crucial role in the maintenance of MZ B cells and CD169 + macrophages. Map3k14 aly/aly mice exhibited diminished innate immune activation and subsequent curtailed priming for the adaptive immunity, which leads to death ( Figure  S2). Moreover, we found that LCMV persists in the spleen, liver, and lung of Map3k14 aly/aly mice, but is controlled by WT mice ( Figure S4). This could be explained by the fact that the lack of early enforced viral replication in the spleen of Map3k14 aly/aly mice led to impaired innate and adaptive immune activation, which results in fast viral dissemination at the late time point, when the virus control is dependent on T cells response, which are exhausted in Map3k14 aly/aly mice. Mechanistically, the perturbation of MZ B cells impairs the development and maintenance of CD169 + macrophages.
The spleen is endowed with a variety of immune cells, being considered as an integral lymphoid organ, and it is uniquely compartmentalized to form the intact architecture that ensures efficient function upon exposure to virus. MMMs (CD169 + macrophages) are exquisite constituents of the spleen; they mount the innate immune response and exacerbate viral replication, which results in the activation of the adaptive immune response and, thus, control of the virus. Likewise, splenic MZ B cells are key components in the spleen and they exhibit a unique characteristic by bridging with macrophages developmentally and functionally.
A number of host factors dictate the outcome of acute viral infections. Examples of such factors abound: the integrity of the splenic architecture, the number of antigens presented to the adaptive immune system, and IFN-I production. We have recently found that MMMs, but not red pulp macrophages, play a crucial role in augmenting viral particles to induce IFN-I production and trigger adaptive immune priming. This immune phenomenon is called enforced viral replication [13,26]. Furthermore, we found that defects in Map3k14 signaling blunt innate immunity during acute infection, as documented by reduced viral replication and subsequent IFNα production. The role of Map3k14 on T cells was reported in different studies. One study shows the importance of T cell-dependent function of the noncanonical NF-κB pathway in the modulating of autoimmune encephalomyelitis [29]. Further studies showed that, during LCMV infection, antiviral CD8 + T cells are rapidly exhausted after initial activation, leading to systemic viral dissemination into peripheral organs, with no CTL-dependent immunopathology [21,30]. In current study, we showed reduced adaptive immunity in Map3k14 aly/aly mice, as mirrored by reduced antiviral CD8 + T cells immunity upon acute strain of LCMV infection.
Intriguingly, our B cell transfer experiments showed that giving supplemental B cells to defective mice leads to the repopulation of CD169 + macrophages, and this repopulation, in turn, improves the innate response and adaptive priming. The finding that B cells offer a helping hand to CD169 + macrophages might be explained by the fact that a deficiency of B cells in the spleen of alymphoplasia mice might contribute to insufficient lymphotoxin expression, so that normal levels of CD169 + cells can be maintained [31][32][33]. Likewise, the lack of Nik, a unique member of the mitogen-activating protein 3 (MAP3) kinase family that contributes to non-canonical NF-κB signaling, in Map3k14 aly/aly mice, supports the defective B cells and the impaired secondary lymphoid organogenesis [22]. In addition, Nik mediates important steps in the signal transduction cascade of Ltβr and of CD40 [18,34]. Ltβr is indispensable for the ontogeny of secondary lymphoid tissues [33]; accordingly, the lack of B cells or B cell-derived expression of Ltβ restrains the innate immune response and delays the adaptive immune response [32]. Moreover, lymphotoxins induce innate IFN-I production during viral infection [35,36].
In summary, in this study, we used alymphoplasia mice (aly/aly) as a model of disorganized splenic architecture, so that we could determine the effect of splenic architecture disorganization on the early immune response during LCMV and VSV infections. Our findings showed that MZ B cells offer a helping hand to CD169 + macrophages. Further, we observed a restoration of CD169 + macrophages after the adoptive transfer of splenic B cells into aly/aly mutant mice. CD169 + macrophages propagate the virus, central to their function as professional antigen-presenting cells, resulting in the priming of the adaptive immune response.

Mice
Map3k14 aly/aly mice were obtained from (Professor Shibata, Kyoto, Japan) and maintained on C57BL/6 genetic background. Littermate WT or heterozygous mice (Map3k14 aly/+ ) served as immunocompetent controls for all of the experiments. Mice expressing CD45.1 were used to track cells in adoptive transfer experiments. All the mice were matched in sex, age, and weight to competent mice. All animals were housed in single ventilated cages. The animal experiments were authorized by the Landesamt für Natur, Umwelt und Verbraucherschutz (LANUV) Nordrhein-Westfalen and in accordance with the German law for animal protection and/or according to institutional guidelines at the Ontario Cancer Institute of the University Health Network.

Genome-Wide Association Study
The GWAS was performed for 4,000,000 SNPs present in different inbred mouse strains (publicly available from http://mouse.cs.ucla.edu/mousehapmap/full.html), and the total amount of PFU from spleen of LCMV infected inbred mouse lines (5-8 individuals per line) as a biological readout. First, the phenotype was log10 transformed to make it conform to normality, followed by an association test between each SNP and the phenotype while using efficient mixed-model association (EMMA) as previously described [24,25]. In short, we uploaded text file of our data (see attached Supplementary  Table S1) to EMMA webserver, which is available at http://mouse.cs.ucla.edu. The EMMA webserver performed an association test within 24 h and provided a link to download file containing p-value associated to each SNP. The power of genotype-phenotype association was reviewed with p-values. A Manhattan plot for all SNPs was prepared while using the SNP & Variation Suite v8.7.2 (Golden Helix, Inc., Bozeman, MT, www.goldenhelix.com).

Viruses
VSV (Indiana strain, Mudd-Summers isolate) was brought from Prof. D. Kolakofsky (University of Geneva, Geneva, Switzerland). Virus was seeded on BHK-21 hamster kidney fibroblasts at a multiplicity of infection (MOI) of 0.01 and it was plaqued onto Vero cells [37]. The mice were intravenously challenged with 2 × 10 8 PFU VSV for virus quantitation purposes and histological analysis, and 2 × 10 7 PFU VSV for survival assay or with 2 × 10 6 PFU VSV for the neutralizing antibodies assessment. The LCMV-WE strain was originally obtained from F. Lehmann-Grube (Heinrich Pette Institute, Hamburg, Germany) and it was propagated on L929 cells (obtained from American Type Culture Collection [ATCC], National Collection of Type Cultures [NCTC] clone 929).

Bone Marrow Chimeras
C57BL/6 (WT) mice (recipient) were irradiated with a total of 1050 rad to generate BMCs. After 24 hours, irradiated mice were reconstituted by i.v injection of 5 × 10 6 bone marrow cells from the donor mice (Map3k14 aly/aly or Map3k14 aly/+ ) to the recipient. The mice were subjected to the experiments and infected with VSV or LCMV virus was after 30 days of reconstitution.

Plaque Assay
The viral load of VSV were quantified in lymphoid and non-lymphoid organs by means of plaque assays, as previously described [38]. In short, the organs smashed in Dulbecco's modified Eagle medium (DMEM) supplemented with 2% fetal calf serum (FCS) were titrated 1:3 over 12 steps and plaqued onto Vero cells (provided by the Ontario Institute for Cancer Research, Toronto, Ontario, Canada). After incubation for one hour at 37 • C, a methylcellulose overlay was added. After 24 hours, the plaques were counted by crystal violet staining. LCMV viral titers were measured on MC57 fibroblasts (provided by the Ontario Institute for Cancer Research) by a plaque-forming assay, as previously reported [39].

Neutralizing Antibody Assay
Serum was diluted (1:40) to measure the neutralizing IgG antibodies. The complement system was inactivated for 30 min. at 56 • C. For analysis of IgG kinetics, diluted samples were treated with β-mercaptoethanol (0.1 M) to get rid of IgM and IgA. Serum was titrated 1:2 over 12 steps and incubated with 5 × 10 2 PFU VSV. After 90 min. of incubation, the virus-serum mixture was 'plaqued' on Vero cells. I hour later, overlay was added. Plaques were counted 24 h later by crystal violet staining.

Purification and Adoptive Transfer of B Cells
The B cells were harvested while using the mouse B-cell isolation kit (130-090-862; Milteny Biotec, Bergisch-Gladbach, Germany) according to the manufacturer. For adoptive transfer experiments, 10 7 sorted B cells were transferred into Map3k14 aly/aly or Map3k14 aly/+ mice, and the mice were challenged with VSV after 20 days of reconstitution.
For intracellular cytokine staining, splenocytes from mice that were infected with LCMV or VSV were cultured for 1 h at 37 • C in an incubator with DMEM that was supplemented with 5% FCS supplemented with LCMV or VSV peptide (5 µg/mL) or not supplemented. One hour later, brefeldin A (25 µg/mL) (B7651, Sigma, Neustadt, Germany) was added and the cells were further incubated for four hours at 37 • C. Splenocytes were washed with FACS buffer, stained for surface anti-mouse CD8 antibody at 4 • C for 30 min., and then fixed with 2% formalin in phosphate-buffered saline (PBS) at 4 • C for 10 min. For intracellular staining, the cells were incubated in FACS buffer for 30 min. at 4 • C with antibodies to IFNγ and TNFα in 0.1% saponin (S4521, Sigma), washed, and analyzed by flow cytometry with FlowJo V10 software (FlowJo, Ashland, OR, USA).

Cell Preparation and Staining for Dendritic Cell Subsets Study
Splenic tissues were injected with DMEM media containing 0.1 mg/mL DNAse (grade II, Sigma Aldrich) and 1 mg/mL collagenase D (Sigma Aldrich) and incubated at 37 • C for 30 min. After incubation, spleens were smashed with syringe plunger to obtain single cell suspension and then washed with FACS buffer. The cellular pellet was then treated with ACK buffer for two minutes to get rid of red blood cells. After one more washing step, cell were processed for antibody staining. Cells were first blocked for Fc receptors using anti-mouse CD16/32 (2.4G2) for 10 min at 4 • C. Next, cells were stained for biotin conjugated antibodies against lineage markers CD3e (145-2C11), CD19 (6D5), NK1.1 (PK136) and Ly6G (1A8) for 15 minutes then washed. Next, a cocktail of antibodies against CD11c (N418), CD11b (M/70), B220 (RA3-6B2), CD8a, PDCA1 (eBio927), and fluorescent labelled Streptavidin and DAPI were added for 20 minutes. After washing, cells were acquired for FACS.

ELISA
The IFNα serum levels were measured while using enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's protocol (PBL Assay Science, Piscataway, NJ, USA).

Statistical analysis
Data are expressed as arithmetic mean ± SEM. Student's t-test (Unpaired, two-tailed). P values of 0.05 or less were deemed statistically significant. Statistical analyses and graphical presentations were performed using Graph Pad Prism (GraphPad Software, San Diego, CA, USA).

Supplementary Materials:
The following are available online at http://www.mdpi.com/2076-0817/9/2/96/s1, Figure S1: Adoptive B cells transfer specifically repopulate the marginal zone B cells, but not the follicular B cells. Figure S2: Map3k14aly/aly mice succumb to VSV infection. Figure S3: Map3k14aly/aly mice experience low viral load in serum and spleen, but not liver and lung. Figure S4: LCMV persists in spleen, Liver and lung of Map3k14aly/aly mice, but is controlled by WT mice. Table S1: Raw data containing average viral titers transformed to log10 from spleens of their corresponding mouse lines as shown in ( Figure 1A). Funding: This study was funded by Deutsche Forschungsgemeinschaft (DFG) grants LA1419/7-1, LA1419/10-1, LA1558/5-1, and SI1558/3-1. This study was further supported by the Sonderforschungsbereich SFB974, the Transregio TRR60, and the Research Training Groups RTG1949 and RTG2098.

Conflicts of Interest:
The authors declare no competing interests.