Expression analysis of YBX1 across various hematopoietic cell types revealed variable expression levels. The lowest YBX1 expression was detected in myeloid cells (Lin^-CD11c^-CD11b⁺) in the bone marrow and in neutrophils (Lin^-CD11c^-CD11b⁺Ly6G⁺) in the spleen ( Supplementary Figure S1 ). Among bone marrow-derived myeloid-derived suppressor cells (MDSCs), the Ly6C^high population exhibited the highest YBX1 expression, whereas the Ly6C^- population displayed the lowest levels.

Due to the embryonic lethality of the Ybx1 knockout, we employed fetal liver cells (FLCs) from E14.5 Ybx1^-/- and Ybx1^+/+ embryos ( Supplementary Figure S2 ) to assess the role of YBX1 in adult hematopoiesis. Ybx1^-/- fetal livers were markedly smaller and showed up to a fivefold reduction in total cellularity compared to controls ( Supplementary Figure S3 ). Flow cytometric analysis revealed that Ybx1^+/+ fetal livers contained significantly higher numbers of common lymphoid progenitors (CLPs; Lin^-c-Kit⁺Sca-1⁺) and myeloid progenitors (MPs; Lin^-c-Kit⁺Sca-1^-) than Ybx1^-/- fetal livers (p < 0.05, Figure 1A ).

To evaluate the differentiation potential of these progenitors, we conducted ex vivo colony-forming unit (CFU) assays. Despite the differences in overall cellularity, the frequencies of multipotent progenitors (CFU-GEMM), pre-B progenitors, and erythroid progenitors (BFU-E) were comparable between genotypes. However, granulocyte/macrophage progenitors (CFU-GM) were significantly increased in Ybx1^-/- FLCs relative to Ybx1^+/+ (p < 0.05, Figure 1B ).

As in vitro differentiation of lineage-committed progenitors appeared largely intact in the absence of YBX1, we next assessed its role in hematopoietic engraftment and reconstitution in vivo. Lethally irradiated C57BL/6J (CD45.1) recipient mice were transplanted with FLCs (CD45.2) from either Ybx1^-/- or Ybx1^+/+ embryos ( Figure 1C ). Four weeks post-transplantation, except for eosinophils, the peripheral blood of both groups showed comparable distributions of lymphocytes, neutrophils, and monocytes to those in age-matched controls (p < 0.05, Figure 1D ). At eight weeks, only donor-derived (CD45.2⁺) B220⁺ B cells were detectable in the periphery ( Figure 1E , Supplementary Figure S4A ), indicating a complete hematopoietic reconstitution.

Histological examination ( Supplementary Figure S4B ) confirmed normal trilineage hematopoiesis in bone marrow sections from all groups, including erythroid and myeloid progenitors as well as megakaryocytes. Splenic architecture was preserved in both genotypes, with intact white and red pulp compartments.

Collectively, these data demonstrate that despite its detectable expression in all hematopoietic subsets analyzed, YBX1 is dispensable for adult hematopoietic engraftment, differentiation, and tissue homeostasis.

Histological and preliminary analyses suggested overall similarities in the major compartments of the hematopoietic system. A detailed quantitative analysis of the myeloid compartment confirmed that there were no significant differences across most cell types studied. Using established flow cytometry panels (14, 15), we analyzed myeloid populations in the spleen and lungs of FLC-reconstituted mice. In the spleen ( Supplementary Figure S5A ) eosinophil (Lin^-CD11b⁺Ly6G^-SSC^high) and monocyte (Lin^-CD11b⁺Ly6G⁺Ly6C^low/high) numbers were unchanged ( Supplementary Table S1 ). However, a significantly higher frequency of neutrophils (Lin^-CD11b⁺Ly6G⁺, p < 0.05) was observed in Ybx1^-/- FLC-reconstituted mice compared to Ybx1^+/+ controls ( Figure 2A ).

Lung single-cell suspensions ( Supplementary Figure S5B ) were analyzed after excluding dead cells and doublets. Using a multicolor panel to distinguish monocytes (inflammatory and resident), macrophages (alveolar and interstitial), neutrophils, eosinophils, and dendritic cell subsets (CD11b⁺ and CD11b^-), we found that the only significant difference among pulmonary myeloid populations was an increased frequency of inflammatory monocytes in Ybx1^-/- reconstituted animals (p < 0.05, Figure 2B ).

We also performed a detailed characterization of the lymphoid compartment, beginning with stage-specific B cell subsets in bone marrow and spleen (gating strategy shown in Supplementary Figure S6 ). Briefly, pro‐B cells (B220 ^+/lowCD43⁺) and pre‐B cells (B220^lowCD43- ) were identified based on B220 and CD43 expression. Recirculating B cells (B220⁺IgM⁺IgD⁺) and immature B cells (B220⁺IgM⁺IgD^-) were quantified in the bone marrow using IgM and IgD staining. In the spleen, we examined naive mature B cells (B220⁺IgM ^+/lowIgD⁺), marginal zone B cells (B220⁺IgM⁺IgD^-), and plasma cells (PC) (B220 ^+/- CD138⁺). No significant differences were observed in the frequencies or absolute numbers of B cells in the bone marrow between Ybx1^-/- and Ybx1 ^+/+ reconstituted mice ( Figure 2C , Supplementary Figure S7A ). Likewise, all splenic B cell subtypes were present at comparable levels across genotypes at eight weeks post-transplantation ( Figure 2D , Supplementary Figure S7B , Supplementary Table S1 ). Furthermore, the B cell compartment remained stable for at least six months following reconstitution ( Supplementary Table S2 ). Analysis of the T cell compartment, defined by CD4⁺ and CD8⁺ expression, also revealed no differences between Ybx1^-/- and Ybx1^+/+ reconstituted mice ( Figure 2E , Supplementary Table S1 ).

Collectively, these results demonstrate that reconstitution of both the myeloid and lymphoid compartments proceeds largely independent of YBX1 expression, with the only significant differences observed in pulmonary inflammatory monocytes and splenic neutrophils, both of which were elevated in Ybx1^-/- FLC-reconstituted animals.

To evaluate the functional properties of the myeloid compartment, we generated MDSCs in vitro as previously described (16, 17). Bone marrow cells were cultured in low-attachment plates with 20 ng/mL M-CSF to induce MDSC differentiation and expansion. After five days, cells were harvested from the supernatant and analyzed for surface marker expression. Following the exclusion of dead cells, doublets, and Lin⁺ cells, CD11b⁺ cells were further classified based on Ly6C and Ly6G expression. No differences in the frequencies of monocytic (M-MDSCs, Lin^-CD11b⁺Ly6G^-Ly6C⁺) and granulocytic (G-MDSCs, Lin^-CD11b⁺Ly6G⁺Ly6C^low/- ) MDSCs were observed between Ybx1^-/- and Ybx1^+/+ cultures ( Figure 3A ).

To assess effector function, MDSCs were stimulated with LPS and IFN-γ for 24 hours, followed by analysis of nitric oxide (NO) and reactive oxygen species (ROS) production. Ybx1^-/- MDSCs secreted significantly higher levels of nitrite compared to Ybx1^+/+ controls (p < 0.005; 20 ± 3.1 µM vs. 12 ± 1.4 µM) ( Figure 3B ), while ROS production was independent of YBX1 ( Supplementary Figure S8 ). Consistent with increased NO production, Ybx1^-/- MDSCs exhibited elevated Nos2 expression ( Figure 3C ).

To test the immunosuppressive capacity of MDSCs, we co-cultured them with splenocytes under CD3/CD28 stimulation to induce T cell proliferation (18). Both Ybx1^-/- and Ybx1 ^+/+ MDSCs efficiently suppressed the proliferation of wild-type CD4⁺ and CD8⁺ T cells in vitro, across different MDSC to T cell ratios (2,500/10,000/200,000 MDSCs; Figures 3D, E ). Therefore, although YBX1 deficiency alters the inflammatory potential of MDSCs as reflected by increased nitric oxide production, it does not improve T cell suppression under the tested conditions.

Next, we evaluated the role of YBX1 in B cells by isolating splenic B cells from Ybx1^-/- and Ybx1^+/+ FLC-reconstituted animals and stimulating them ex vivo for four days with LPS and IL-4. Cell proliferation was comparable between genotypes, and the frequencies of mature (B220⁺CD138^-) and plasmablastic (B220 ^+/- CD138⁺) B cells were similar ( Supplementary Figure S9A ). Following stimulation, 81–84% of cells expressed surface IgM (B220⁺sIgM⁺), and surface IgM levels were comparable between the groups ( Supplementary Figures S9B, C ), indicating that B cell maturation was unaffected by the absence of YBX1.

B cell development and function largely depend on intact BCR signaling, and therefore we looked closely at the resulting intracellular Ca²⁺ changes and the signaling proteins involved. To do so, isolated B cells were stimulated with F(ab’)₂ anti-IgM, and downstream signaling responses were measured. Interestingly, intracellular Ca²⁺ mobilization was significantly reduced in Ybx1^-/- B cells as reflected by a reduced MFI ratio of Ca²⁺-bound Fluo-4 and Ca²⁺-unbound FuraRed ( Figure 4A ). The peak Ca²⁺ amplitude was also significantly lower in Ybx1^-/- cells (p < 0.05; Figure 4B ). Notably, both CD22 mRNA and surface expression (MFI) were reduced in Ybx1^-/- B cells ( Figures 4C, D ), arguing against increased CD22-mediated inhibition as the cause of diminished calcium signaling.

Additionally, mRNA levels of several critical BCR signaling genes (e.g., Blnk, Nfatc1, and Ikzf3, Figure 4D ) were significantly elevated in Ybx1^-/- B cells, yet their corresponding protein levels were markedly reduced ( Figures 4E, F ). To determine whether downstream signaling pathways were affected, we analyzed the expression of NFATC1 targets (Tnf, Cflar, Nfatc3) and IKZF3 targets (Ebf1, Ikzf1, Tcf3) but found no significant differences between genotypes ( Figure 4G ). These findings show that although IgM-mediated BCR signaling is reduced in Ybx1^-/- B cells, compensatory mechanisms, possibly involving reduced CD22 expression, help maintain downstream transcriptional responses despite a reduction in cytoplasmic calcium mobilization upon BCR stimulation with anti-IgM.

Having established stable B cell maturation despite reduced BCR signaling in Ybx1^-/- B cells, we next examined the function of mature B cells, namely PC antibody secretion. Splenic B cells were stimulated with LPS for four days, and IgM secretion was measured. While total cell numbers were similar between Ybx1^-/- and Ybx1^+/+ cultures (data not shown), IgM titers in the supernatant were significantly lower in Ybx1^-/- B cells (p < 0.001; Figure 5A ).

To assess class-switch recombination, B cells were cultured with LPS and IL-4 to induce switching to IgG1. Class-switching frequency, measured by IgG1⁺B220⁺ cells, was comparable between genotypes ( Supplementary Figure S10A ). However, Ybx1^-/- B cells secreted significantly less IgG1 (p < 0.005; Figure 5B ), indicating that while class-switching is intact, antibody secretion was significantly lower in Ybx1^-/- B cells.

To explore the mechanism underlying reduced secretion, we analyzed immunoglobulin RNA processing and stability by quantifying unprocessed and mature Ighm and Ighg1 transcripts and further distinguished between membrane-bound and secreted isoforms of Ighm. Precursor transcript levels were similar between genotypes, indicating that transcriptional initiation is unaffected by YBX1 deficiency. However, we observed a significant increase in secreted Ighm isoform and mature Ighg1 transcripts in Ybx1^-/- B cells (p < 0.05; Figure 5C ). These data demonstrate that Ig RNA maturation is YBX1 independent, as similar precursor RNA levels exclude a role for YBX1 as a transcriptional regulator of Ig genes. Furthermore, the increased mature mRNA levels in Ybx1^-/- B cells argue against the involvement of YBX1 in RNA stabilization.

To investigate whether defective secretion might explain reduced antibody titers, we assessed if YBX1 impacts secretion by using stable Gaussia luciferase (GLuc)-expressing mouse embryonic fibroblasts (MEFs) derived from Ybx1^+/+ and Ybx1^-/- embryos. Comparable GLuc activity in the supernatants of Ybx1^+/+ and Ybx1-/- MEFs after 72 hours of incubation ( Supplementary Figure S10B ) excluded impaired secretion as a contributing factor to the reduced antibody titers. Moreover, intracellular flow cytometry analysis for IgG1 in stimulated splenic B cells revealed no intracellular accumulation ( Figure 5D ), supporting the conclusion that the defect in Ybx1-/- B cells lies at the level of translation of secretory Ig transcripts rather than transcription, splicing, or secretion.

XBP1 is essential for PC antibody production (19), and thus changes in protein levels could be responsible for the decreases in antibody production. However, in our cells Xbp1 mRNA levels were significantly increased in stimulated Ybx1^-/- B cells (p < 0.05; Figure 5E ), but XBP1s protein levels were unchanged ( Figure 5F ), suggesting that Ybx1 may influence Xbp1 transcription but not its splicing or translation. To further assess this, we analyzed XBP1s protein expression following ER stress induction with 2 µg/ml tunicamycin in a CRISPR/Cas9-generated Ybx1-deficient MOPC315.BM cell line. Consistent with our findings in primary B cells, XBP1s levels were comparable between Ybx1^-/- and Ybx1^+/+ cells ( Supplementary Figure S11 ). Thus, the reduced antibody production observed in Ybx1^-/- B cells results neither from altered XBP1 expression nor the availability of subsequent spliced isoforms.

To determine whether YBX1 directly binds immunoglobulin RNA, we performed RNA immunoprecipitation (RIP) in two murine B cell lines: MOPC315.BM, which expresses YBX1 and secretes DNP-specific IgA, and in WEHI-231, a mature B cell line that secretes IgM. RIP followed by RT-PCR revealed that in MOPC315.BM ~30% of input Actb mRNA (a known YBX1 target) and ~11% of Igha mRNA were precipitated using an anti-YBX1 antibody ( Figure 5G ). In WEHI-231 cells, ~4% of Actb and ~2% of Ighm mRNA were recovered in the YBX1 pull-down fraction ( Supplementary Figure S12 ). These results indicate that YBX1 binds to both Igha and Ighm mRNAs, albeit less efficiently than Actb, and suggest a direct role for YBX1 in regulating Ig RNA fate at the translational level.

To confirm this role, we overexpressed YBX1 in the WEHI-231 B cell line and stimulated the cells with LPS. YBX1 overexpression had no effect on cell proliferation ( Figure 5H , Supplementary Figure S13 ) but significantly increased IgM secretion (p < 0.005; Figure 5I , Supplementary Figure S14 ), reinforcing its enhancing role in antibody production.

Collectively, these findings demonstrate that YBX1 is dispensable for B cell maturation and class switching but is important for efficient antigen-specific antibody production. The underlying mechanism likely involves immunoglobulin mRNA translation efficiency, mediated by direct interaction of YBX1 with Ig transcripts.

To evaluate the role of YBX1 in humoral immunity in vivo, we immunized Ybx1^-/- and Ybx1^+/+ FLC-reconstituted mice (four weeks post-transplantation) with either nitrophenyl (NP)-KLH, a T cell-dependent (TD) antigen, or NP-FICOLL, a T cell-independent (TI) antigen. Sera were collected one week after the initial immunization (day 7) and one week after the second antigen exposure (day 14, Figure 6A ).

Ybx1^-/- mice exhibited significantly reduced NP-specific TD Ig responses across multiple isotypes following primary immunization, including IgM (p < 0.0001), IgG1 (p < 0.05), and IgG2b (p < 0.05), while IgG3 levels remained unaffected ( Figures 6C-F ). After the second immunization, NP-specific Ig titers were further diminished in Ybx1^-/- mice, including IgM (p < 0.0001), IgG3 (p < 0.005), IgG1 (p < 0.0001), and IgG2b (p < 0.0001), compared to Ybx1^+/+ controls. Importantly, total numbers of splenic plasma cells (B220⁺/CD138⁺) and the distribution of B cell subsets were comparable between groups ( Figure 6B , Supplementary Table S3 ), indicating that impaired antibody responses were not due to differences in B cell numbers.

To assess T cell-independent responses, we immunized animals with NP-FICOLL and measured NP-specific antibody titers. Seven days after immunization, NP-specific IgM titers were comparable between Ybx1^-/- and Ybx1^+/+ mice, whereas IgG3 (p < 0.0001), IgG1 (p < 0.05), and IgG2b (p < 0.05) titers were significantly lower in Ybx1^-/- animals ( Figures 6G-J ). Following second antigen exposure, NP-specific IgM titers remained unchanged across genotypes ( Figure 6G , Supplementary Figure S15 ), but reductions in IgG1 (p < 0.001), IgG3 (p < 0.005), and IgG2b (p < 0.05) were again observed in Ybx1^-/- mice ( Figures 6H-J ).

Together, these findings demonstrate that YBX1 is essential for robust antigen-specific antibody production during both T cell-dependent and T cell-independent immune responses in vivo.

From C57BL/6J-Ybx1 mice, a kind gift from T. Ley (Washington University, St. Louis, USA), we retrieved embryos of Ybx1^+/+ and Ybx1^-/- genotypes. Additionally, 8- to 10-week-old female C57BL/6J (CD45.1) mice (Charles River, Germany) were used as recipients for transplantation experiments. All mice were bred and housed in the Center for Experimental Molecular Medicine, and experiments were done in compliance with German animal welfare regulations.

Pregnant mice were euthanized, and E14.5 fetal livers were harvested under sterile conditions. Livers were mechanically dissociated using a 40-µm cell strainer (Greiner Bio-One, Germany) and suspended in IMDM medium (Thermo Fisher, Germany) supplemented with 20% (v/v) BIT 9500 Serum Substitute (Stemcell Technologies, Canada), 100 U/mL penicillin-streptomycin, and 0.1 µM β-mercaptoethanol (Merck, Germany).

Recipient C57BL/6J (CD45.1) mice underwent lethal total-body irradiation (TBI) using a Faxitron CP-160 X-ray irradiator (Faxitron X-Ray, USA). Irradiation was administered in two fractions of 7 Gy at a 4 hour interval. Two hours following the second irradiation dose, 1.6x10⁶ fetal liver cells were injected intravenously via the tail vein in a total volume of 150 µL DPBS (Thermo Fisher, Germany). To prevent infections, transplanted mice received neomycin sulfate (2 g/L, Betapharm, Germany) in their drinking water.

Four weeks post-transplantation, mice were immunized intraperitoneally with 50 µg NP-KLH (LGC, Biosearch/Biocat Germany) in 500 µL Imject Alum Adjuvant (Thermo Fisher Germany, 1:1 diluted in DPBS) to induce a T cell-dependent (TD) immune response or 50 µg NP-FICOLL (LGC, Biosearch/Biocat Germany) in 100 µL DPBS to assess T cell-independent (TI) immune responses.

Fetal liver cells at E14.5 were used for colony-forming unit (CFU) assay and Western blot analysis.

Reconstituted and control mice were used for May–Grünwald–Giemsa staining, single-cell suspensions, flow cytometry, and histochemical analyses. For immune function assessments, ELISA was performed using cell supernatants or sera from immunized animals. Functional B cell assays included primary B cell stimulation, Ca²⁺ measurements, RT-qPCR analysis, and RNA immunoprecipitation (RIP). Myeloid cell functional assays included generation of MDSCs (myeloid-derived suppressor cells), T-cell suppression assay, ROS production measurements, and Griess assay for nitric oxide (NO) production. Detailed protocols for all experiments are provided in the Supplementary Methods .

Data were analyzed using Prism GraphPad 8.3.0 (GraphPad Software, USA). Statistical comparisons were performed using unpaired two-tailed t-tests. Results were considered statistically significant at p < 0.05.