Abstract Never in mitosis gene A (NIMA)-related kinase 2 (NEK2), a member of the serine-threonine kinase family, is critically involved in the regulation of the cell cycle. Upregulation of NEK2 is associated with aberrant B cell proliferation, a phenomenon potentially driven by NEK2-mediated disruption of the PKM1/PKM2 equilibrium. The overexpression of NEK2 in the B cell lineage may facilitate the maturation processes of B cells. Nonetheless, the precise role of NEK2 in modulating B cell-mediated immunity in autoimmune disorders remains to be fully elucidated. In this study, we demonstrate that NEK2 was significantly upregulated in multiple sclerosis (MS) patients. Pharmacological inhibition of NEK2 resulted in a marked reduction in the expression of co-stimulatory molecules CD80 and CD86 on B cells, concomitant with a suppression of their proliferation and differentiation into antibody-secreting cells (ASCs) and class-switched memory B cells (SWM). Administration of the NEK2 inhibitor INH1 in a murine model of experimental autoimmune encephalomyelitis (EAE) led to notable improvements in neurological function, amelioration of demyelination, and a decrease in the infiltration of inflammatory cells in the central nervous system (CNS) compared to vehicle-treated EAE mice. Mass cytometry analysis revealed that NEK2 inhibition downregulated the expression of co-stimulatory molecules and diminished the proportion of Th1 cells in the CD4 + T cell population. In vitro studies further substantiated that NEK2 blockade attenuated CD4 + T cell proliferation and differentiation into Th1 cells by disrupting B-T cell interactions. Collectively, these findings underscore an immunomodulatory function for NEK2 and highlight its potential as a therapeutic target in the treatment of multiple sclerosis. Supplementary Information The online version contains supplementary material available at 10.1186/s12974-025-03472-w. Keywords: NEK2, Multiple sclerosis, Experimental autoimmune encephalomyelitis, B cells Introduction Multiple sclerosis (MS) is a chronic inflammatory demyelinating disorder of the central nervous system (CNS) characterized by autoimmune dysregulation, leading to demyelination and axonal damage [[46]1, [47]2]. MS is primarily recognized as a T cell-mediated disease, involving an imbalance between regulatory T cells and CNS-reactive effector T cells [[48]3]. Historically, B cells were regarded as a relatively homogeneous and passive cell population, dependent on T cell assistance to differentiate into antibody-secreting cells (ASCs), such as plasmablasts and plasma cells. The contribution of B cells to MS pathogenesis has largely been attributed to their capacity to produce autoreactive antibodies within the CNS [[49]4, [50]5]. However, the molecule CD20, a target of anti-CD20 therapies, is minimally or not expressed on antibody-secreting cells. The demonstrated efficacy of anti-CD20 therapies in reducing relapse rates in MS patients and mitigating disability in secondary progressive MS has prompted a paradigm shift in understanding the immune-pathophysiology of MS [[51]6, [52]7]. This has redirected scientific attention toward the potential antibody-independent functions of B cells in MS pathogenesis. Emerging evidence has elucidated the antibody-independent roles of B cells in MS. For instance, Martin et al. demonstrated that B cells from MS patients carrying the disease-susceptible allele HLA-DR15 present brain-associated antigens, such as RASGRP2, to T cells, thereby promoting T cell activation and the secretion of pro-inflammatory cytokines [[53]8]. Additionally, B cells from MS patients and healthy controls with disease-susceptible genetic variants exhibit elevated expression of the co-stimulatory molecule CD86, which is critical for T cell activation [[54]9]. These findings are consistent with our observations in this study that NEK2 regulates the expression of co-stimulatory molecules on B cells, thereby modulating T cell responses. Furthermore, B cells have been shown to drive CD4 + T cell differentiation into effector subsets by regulating surface receptors/co-stimulators or via cytokine-mediated signaling, a critical mechanism of MS pathogenesis [[55]10, [56]11]. Collectively, these studies underscore the role of B cells in MS, involving both antibody-dependent and antibody-independent mechanisms, and highlight the importance of targeting B cell function in therapeutic strategies for MS. While anti-CD20 therapies currently represent one of the most effective strategies for reducing relapses and disease activity in MS, their broad B cell depletion raises significant clinical concerns. Long-term use of anti-CD20 therapies is associated with an increased risk of severe infections [[57]12], and preclinical evidence from experimental autoimmune encephalomyelitis (EAE) mice demonstrates that pre-onset B cell depletion exacerbates neuroinflammation, likely through depletion of IL-10-producing regulatory B cells (Bregs) [[58]13]. These limitations underscore the clinical need to develop novel B cell-targeting strategies. Never in mitosis gene A (NIMA)-related kinase 2 (NEK2), a member of the serine-threonine kinase family, plays a critical role in cell cycle regulation, gene expression, and the maintenance of centrosome structure and function [[59]14–[60]16]. Elevated NEK2 expression has been associated with excessive B cell proliferation. Mechanistically, this aberrant B-lymphocyte proliferation may be linked to NEK2-mediated PKM1/PKM2 imbalance. Such perturbations in the metabolic regulatory network could potentially drive proliferation through reprogramming cellular energy metabolism pathways, ultimately resulting in the disruption of homeostatic control mechanisms governing B cell proliferation [[61]17–[62]19]. Additionally, NEK2 appears to influence B cell development and maturation. Transgenic mice with conditional overexpression of NEK2 in the B cell lineage exhibit increased populations of immature B cells in the bone marrow and reduced B-1 B cells in the peritoneal cavity [[63]20]. However, the role of NEK2 in regulating B cell immunity in autoimmune diseases, including MS, remains poorly understood. In this study, we observed significant upregulation of NEK2 in B cells from MS patients. We further demonstrated that NEK2 regulates B cell-T cell interactions by modulating the expression of co-stimulatory molecules on B cells, thereby contributing to the severity of EAE. These findings reveal a novel, non-depleting mechanism by which NEK2 mediates B cell responses in MS and suggest that targeting B cell phenotypes may represent a viable therapeutic strategy for mitigating pro-inflammatory B cell activity. Methods and materials Participants and samples Patients diagnosed with MS were identified in accordance with the 2017 McDonald criteria and recruited from Tianjin Medical University General Hospital. Inclusion criteria required that patients be within 12 weeks of a new MS diagnosis and have no prior history of immune therapy. Healthy control participants, who exhibited normal results on basic laboratory tests and neurological examinations, were enrolled from the hospital staff. Written informed consent was obtained from all participants, and the study protocol, along with supporting documentation, was approved by the Institutional Review Board of Tianjin Medical University General Hospital (Tianjin, China). Single-cell RNA sequencing analysis Publicly available single-cell transcriptome data from cerebrospinal fluid (CSF) and peripheral blood mononuclear cells (PBMCs) of MS patients was reanalyzed, specifically focusing on B cells. These samples included seven MS patients and eight controls across three independent studies ([64]GSE138266, [65]GSE133028, Syn21904732). The clinical characteristics of them are listed in Table [66]S1. We firstly performed quality control on all included samples. In the case of [67]GSE138266 and Syn21904732, cells with fewer than 200 detected genes or belonging to the top 1% of cells with an extreme number of detected genes were excluded. For samples from [68]GSE133028, quality control metrics were obtained from the original publications, and only cells that passed the quality assessment were included in downstream analysis. Additionally, for all cells across all studies, those with more than 3% hemoglobin unique molecular identifier (UMI) counts or more than 10% mitochondrial UMI counts were filtered out; meanwhile, genes detected in less than three cells were excluded from further analysis. The analyses were performed using Seurat (V4.4.0). Bulk RNA sequencing The isolation of blood B cells from patients with MS and control individuals was accomplished through the application of flow cytometry sorting (FACS Aria III, BD Biosciences, San Jose, CA, USA). The libraries derived from these samples were sequenced on the Illumina NovaSeq platform. Differential gene analysis (DEGs) was conducted by utilizing DESeq2 (V1.32.0). Regarding the functional enrichment analysis of gene sets, we employed the KEGG rest API ([69]https://www.kegg.jp/kegg/rest/keggapi.html) to acquire the most recent gene annotations of KEGG pathways and took this as the background for mapping the genes to the background set. Enrichment analysis was carried out by using the R software package clusterProfiler (V3.14.3) to obtain the outcomes of gene set enrichment. A minimum gene set of 5 and a maximum gene set of 5000 were established, and a P value < 0.05 and FDR < 0.25 were regarded as statistically significant. Quantitative RT-PCR Total RNA was extracted from isolated human B cells using the EZ-press Cell to cDNA Kit (EZBioscience). Quantitative RT-PCR was performed using SYBR Green PCR Master Mix (Roche Diagnostics) on a Bio-Rad Optical 2 Real-Time PCR Detection System. Gene expression was normalized to β-actin, and relative quantification was performed using the ΔΔCt method. Flow cytometry Single-cell suspensions were prepared from human peripheral blood, mouse spleen tissue, or mouse brain and spinal cord tissues and stained with fluorochrome-conjugated antibodies as previously described [[70]21]. The following antibodies were used: 7AAD viability staining solution, anti-human CD3 (SK7), CD19 (HIB19), CD86 (BU63), CD80 (2D10), CD69 (FN50), HLA-DR (L243), CD38 (HIT2), CD27 (M-T271), CD24 (ML5), IgD (IA6-2); anti-mouse CD11b (M1/70), CD45 (30-F11), CD19 (1D3/CD19), IL-17 A (TC11-18H10.1), IFN-γ (XMG1.2), FOXP3 (QA20A67), IgM (RMM-1), CD43 (S11), IgD (11-26c-2a), CD5 (53 − 7.3). Antibodies were conjugated to fluorescent tags, including FITC, PE, PerCP-Cy5.5, allophycocyanin (APC), PE/Cyanine7, APC/Cyanine7, and Brilliant Violet 421 (BV421). All antibodies were purchased from BioLegend or BD Biosciences. Human B cells culture PBMCs were isolated from human blood by density-gradient centrifugation. B cells were purified using magnetic-activated cell sorting (MACS) with a human B cell isolation kit (EasySep™, STEMCELL) according to the manufacturer’s protocol, achieving > 97% purity. Isolated B cells were cultured in serum-free X-VIVO 15 medium (Lonza) for 24 h to assess activation or for 6 days to evaluate differentiation. Activation stimuli included soluble CD40L (100 ng/mL, R&D Systems), anti-human B-cell receptor F(ab′)2 fragment antibody (2.5 µg/mL, Jackson ImmunoResearch), and R-848 (2.5 µg/mL, R&D Systems). Differentiation stimuli included CD40L (100 ng/mL), R-848 (2.5 µg/mL), IL-2 (100 ng/mL, R&D Systems), IL-21 (100 ng/mL, R&D Systems), and IL-6 (10 ng/mL, R&D Systems). B cell proliferation was quantified using CFSE staining according to the manufacturer’s protocol. Mice Eight- to ten-week-old female C57BL/6 mice were used in this study. Mice were housed in pathogen-free conditions with free access to food and water and a standardized light-dark cycle. Animal surgeries were performed under anesthesia. All animal experiments were approved by the Committee on the Ethics of Animal Experiments of Tianjin Neurological Institute (Tianjin, China). All experiments were conducted in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and were designed and performed according to the Animal Research: Reporting In Vivo Experiments guidelines ([71]www.nc3rs.org.uk/arrive-guidelines). Administration of NEK2 inhibitor Mice were administered the NEK2 inhibitor INH1 [[72]22] (25 mg/kg body weight, Selleck) or vehicle (5% DMSO, 40% PEG300, 5% Tween 80, and 50% distilled water) via intraperitoneal injection daily following the onset of experimental autoimmune encephalomyelitis (EAE) until euthanasia. For in vitro experiments, human B cells were treated with INH1 at gradient concentrations (10 µM, 20 µM, 30 µM, 50 µM, and 100 µM). A concentration of 50µM INH1 was identified as optimal for inhibiting B cell function and was used in subsequent in vitro studies. EAE induction EAE was induced following previously published procedures [[73]23]. Briefly, mice were injected subcutaneously on each side of the spine at the femoral level with 100 µg of MOG[35 − 55] peptide in complete Freund’s adjuvant (CFA) with 5 mg/mL Mycobacterium tuberculosis (Sigma). Mice also received 250 ng of pertussis toxin (LIST Biological Laboratories) intraperitoneally immediately after immunization and again 48 h later. Mice were monitored daily for weight changes and clinical symptoms. Clinical scores were assessed by two blinded investigators as follows: 0, no signs; 1, decreased tail tone; 2, mild monoparesis or paraparesis; 3, severe paraparesis; 4, paraplegia; 5, quadriparesis; and 6, moribund or death. Single cell cytometry by time of flight (CyTOF) Mass Cytometry was performed as previous described [[74]24]. Briefly, single-cell suspensions were prepared from blood and CNS tissues (brain and spinal cord) of EAE mice treated with INH1 or vehicle. Cells were stained with 194Pt-viability dye (1 mM) and an antibody panel, followed by fixation and permeabilization. Intracellular staining was performed using iridium dye (250 nM) and specific antibodies. Samples were acquired on a Helios CyTOF Mass Cytometer. Mass cytometry (CyTOF) data was normalized with bead standards, and then preprocessed with Flowjo to exclude debris, dead cells, and doublets, and then transformed by Acrsinh method (cofactor = 5), clustered using unsupervised clustering algorithm Mena shift Clustering (manual annotation), visualized by dimensionality reduction algorithms T-distributed Stochastic Neighbor Embedding (t-SNE). Mouse T cells and mouse B cells culture Mouse spleen B cells and CD4 + T cells were isolated from naïve mice and EAE mice at day 11 post-immunization using fluorescence-activated cell sorting (FACS). EAE-derived B cells (EAE-B) and CD4 + T cells (EAE-T) were obtained from EAE mice, while Non-EAE B cells were isolated from naïve mice. B cells were defined as CD3 − CD19 + populations, and CD4 + T cells as CD3 + CD4 + populations. For co-culture experiments, isolated CD4 + T cells were stained with CFSE and co-cultured with B cells at a 1:1 ratio (total 1 × 10^5 cells per well: 5 × 10^4 T cells and 5 × 10^4 B cells) in complete RPMI 1640 medium. Cultures were supplemented with either INH1 or vehicle control, along with MOG peptide (10 µg/mL). T cell activation was achieved using anti-CD3 (5 µg/mL; clone 145-2C11, BioLegend) and anti-CD28 (5 µg/mL; clone 37.51, BioLegend). Th1/Th17 differentiation was induced with IL-12 (10 ng/ml; R&D Systems) or IL-6/IL-23 (10 ng/ml, R&D Systems) respectively. Cells were cultured in complete RPMI 1640 medium for 3 days. T cell proliferation was detected by CFSE, and the expressions of IFN-γ (Th1) and IL-17 A (Th17) were analyzed through intracellular staining. Immunostaining Mice were perfused with ice-cold PBS, and lumbar spinal cord tissues were collected, fixed in formalin, and embedded in paraffin. Section (5 μm) were stained with hematoxylin and eosin (H&E) to assess inflammatory cell infiltration and luxol fast blue (LFB) to evaluate demyelination. Inflammation scores were assigned as follows: 0, no inflammation; 1, perivascular and meningeal infiltration; 2, mild infiltration (< 1/3 of white matter); 3, moderate infiltration (> 1/3 of white matter); and 4, extensive infiltration throughout the white matter. Demyelination was quantified using ImageJ software to calculate the proportion of vacuoles in the white matter. Statistics All values are expressed as Means ± SEM or as individual data points, and p values are assessed as appropriate by student’s t test, one-way or two-way ANOVA, with Tukey post hoc test using Graphpad Prism version 9. Flow cytometry data were analyzed by FlowJo V10. Results B cells are significantly elevated in the CSF of MS patients and exhibited elevated NEK2 expression We integrated single-cell transcriptome data from the CSF and PBMCs of 7 MS patients and 8 healthy controls across three public datasets ([75]GSE133028, [76]GSE138266, Syn21904732) (Fig. [77]1A). After quality control to remove unqualified cells, dimensionality reduction by UMAP were performed on total 96,501 cells. We identified 8 immune cell types, including B cells, CD4 + T cells, CD8 + T cells, conventional dendritic cells (cDCs), monocytes, natural killer (NK) cells, plasma cells, and plasmacytoid dendritic cells (pDCs) (Fig. [78]1B) based on expression of marker genes (Fig. [79]1C). Notably, analysis of cell proportions showed a significant increase in B cells and plasma cells in the CSF of MS patients (Fig. [80]1D). However, no significant difference in the proportions of B cells and plasma cells was observed in the peripheral blood of MS patients compared to healthy controls (Fig. [81]1E). The differential distribution of B cells in the CSF and peripheral blood may indicate that B cells play a key role in CNS-compartmentalized inflammation in MS. Besides, the proportion of monocytes was markedly reduced in the CSF (Fig. [82]1D), consistent with previous studies [[83]25, [84]26]. It was demonstrated to associate with different distribution of monocyte subsets in CSF induced by MS. Collectively, these findings underscore the potentially critical role of B cells in MS onset and CNS-compartmentalized inflammation. Considering the impact of NEK2 on B cell proliferation, we conducted additional analysis and discovered that its expression was increased in B cells in peripheral blood and CSF. (Fig. [85]1F). To further explore the expression characteristics of NEK2 in B cells, we performed bulk RNA sequencing with sorted peripheral blood B cells from 4 MS patients and 4 healthy controls (Fig. [86]1A). Through systematic evaluation of the transcriptional profiles and functional characteristics of B cells, our analysis revealed significant upregulation of NEK2 expression in MS-derived B cells (Fig. [87]1G). Besides, KEGG pathway enrichment analysis demonstrated that differentially expressed genes were enriched in the MAPK signaling pathway, chemokine signaling pathway, Th1 and Th2 cell differentation (Fig. [88]1H). To verify this finding, we isolated B cells from the peripheral blood of MS patients and healthy controls and quantified NEK2 mRNA levels. Results confirmed that NEK2 expression was significantly higher in MS patients compared to healthy controls (Fig. [89]1I). These findings suggest that NEK2 may serve as a key regulator of B cell proliferation and function in MS, contributing to disease onset and progression. Fig. 1. [90]Fig. 1 [91]Open in a new tab The proportion of B cells in the CSF of MS patients increased significantly, and the expression of NEK2 was enhanced. A. Schematic diagram illustrating the composition and integration strategy of transcriptomic datasets. B. CSF cells and PBMCs clustered by dimensionality reduction with UMAP shows 8 immune cell types (total cells: 96501 cells). C. Bubble plots shows the expression of marker genes of immune cells. D. Bar graphs showing the immune cell proportions in CSF of controls and MS patients. E. Bar graphs shows the immune cell proportions in peripheral blood of controls and MS patients. F. Bubble plots showing the expression of NEK2 in B cells of CSF and peripheral blood from controls and MS patients. G. Volcano plot illustrating differential gene expression in B cells between MS patients and healthy controls. H. The enrichment analysis results of KEGG pathways are presented by bubble graphs I. RT-qPCR analysis of mRNA levels showing the NEK2 expression in B cells of MS patients (n = 8) or healthy control (n = 12).Unpaired t-tests were used to test the differences between groups, and data are expressed as mean ± standard deviation (SEM) Inhibition of NEK2 limits B cell activation, proliferation and differentiation toward ASCs and SWMs To investigate the functional role of NEK2 in B cells, we treated B cells from MS patients and healthy controls with a NEK2 inhibitor in vitro (Fig. [92]2A). The results demonstrated that NEK2 inhibition reduced the expression of co-stimulatory molecules (CD80 and CD86) (Fig. [93]2B and C) and the proportion of CD80 + B cells and CD86 + B cells (Fig. [94]2D) in live B cells from MS patients in a dose-dependent manner. Similar effects of NEK2 inhibition were observed in B cells from healthy controls (Fig. [95]2E). A concentration of 50 µM INH1 was identified as optimal for suppressing B cell responses without significantly affecting cell viability (Supplemental Fig. [96]1A). However, NEK2 inhibition did not significantly alter the proportions of CD69 + or HLA-DR + B cells and their expression levels in live B cells from MS patients (Supplemental Fig. [97]1B) and healthy controls (Supplemental Fig. [98]1C), suggesting that NEK2 primarily regulates B cell function through modulation of co-stimulatory molecule expression. Fig. 2. [99]Fig. 2 [100]Open in a new tab Blockage of NEK2 limits B cell activation, proliferation, and differentiation toward ASCs and SWMs. A. Schematic shows the experimental design of this part of study utilizing human blood sample. Periphery CD19 + B cell were isolated from venous blood of MS patients or healthy control (purity confirmed by flow cytometry > 97%). Isolated B cells were treated with a concentration gradient of NEK2 inhibitor (INH1) or vehicle and stimulated under various conditions including soluble CD40L (100ng/ml), anti-human B-cell receptor F (ab′) 2 fragment antibody (2.5 µg/m), and R-848 (2.5 µg/ml) for activation of B cells; and CD40L (100ng/ml), R-848 (2.5 µg/ml), IL-2 (100ng/ml); IL-21 (100ng/ml), IL-6 (10ng/ml) for differentiation of B cells. B. Histogram plot showing the expression of CD80 and CD86 in MS B cells receiving indicated treatment. C, D. Flow cytometry analysis showing relative MFI (C) and proportion (D) of CD80 and CD86 in live MS B cells receiving indicated treatment (n = 4–7). E. Flow cytometry analysis showing relative MFI and proportion of CD80 and CD86 in live healthy controls' B cells receiving indicated treatment (n = 3-5). F, G. Flow cytometry plot and quantification result show the proportion of proliferating B cells in live B cell from blank group (without stimulation), sti (stimulated) group and sti + INH1 (stimulated cell treated with 50µM INH1) group (n = 6). Periphery CD19 + B cell were isolated from venous blood of MS patients (F) or healthy control (G) (purity confirmed by flow cytometry > 97%). H. Representative flow cytometry of CD19 + CD27high CD38high (Autoantibodies secreted cells, ASC), CD19 + CD27- IgD+ (naive), CD19 + CD27– IgD- (double negative, DN), CD19 + CD27 + IgD+ (unswitched memory, USW), and CD19 + CD27 + IgD (switched memory, SWM) from MS patients' B cells receiving indicated treatment. I. Quantitative analysis of B cell subsets including ASC, naïve B cell, SWM, DN, Breg cell, and USM (n = 4-6).One-way ANOVA, each line represents paired results from individual donors. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 Notably, NEK2 inhibitor suppressed the proliferation of B cells from MS patients (Fig. [101]2F) and healthy controls (Fig. [102]2G) under stimulation. Furthermore, NEK2 inhibition significantly reduced the differentiation of B cells from MS patients into ASCs and switched memory (SWM) B cells (Fig. [103]2H and I) that are elevated in the peripheral blood of MS patients and implicated in disease pathogenesis [[104]27–[105]29]. Intriguingly, NEK2 inhibition had no significant effect on differentiation of B cells from healthy controls (Supplemental Fig. [106]2A).These results suggest that elevated NEK2 expression in MS patients may contribute to the altered distribution of B cell subsets, thereby promoting disease progression. Inhibition of NEK2 attenuates EAE severity and disease progression To further explore the role of NEK2 in MS, we administrated NEK2 inhibitor in the EAE model, a well-established murine model of MS. After onset, EAE mice were treated with NEK2 inhibitor INH1 or vehicle to assess its effects on neurological function, spinal cord demyelination, and neuroinflammation (Fig. [107]3A). Remarkably, inhibition of NEK2 significantly improved their neurological deficits (Fig. [108]3B). Pathological analysis revealed reduced inflammatory cell infiltration (Fig. [109]3C and E) and demyelination (Fig. [110]3D and F) in the spinal cord white matter of INH1-treated mice compared to controls. Flow cytometry analysis further demonstrated that NEK2 inhibition reduced the infiltration of CD4 + T cells, B cells, and CD11b + myeloid cells into the CNS (Fig. [111]3G). These results indicate that blockage of NEK2 effectively improves neurological function, reduces pathological damage, and attenuates immune cell infiltration in the CNS, thereby attenuating EAE severity and progression. Fig. 3. [112]Fig. 3 [113]Open in a new tab Blockage of NEK2 attenuates the EAE development. A. Schematic showing the regimen of INH1 administration and experimental design. Mice were immunized to induce EAE model and then intraperitoneally injected PT solution (12.5 µg/kg) immediately and 48 h after immunization. The body weight and clinical scores of EAE mice were assessed daily and INH1 or vehicle (25 mg/kg, i.p.) was administered after onset. EAE mice reached the peak of the disease on day 17–20 after immunization. We collected the brain, spinal cord, and spleen tissues or peripheral blood of the EAE mice on day 25 to evaluate demyelination and neuro-inflammatory response. B. Line chart displays the clinical scores of EAE mice given INH1 or vehicle (n = 12–15). C, E. Representative H&E staining images (C) and bar graph (E) show the infiltration of inflammatory cells in white matter of spinal cord from EAE mice receiving INH1 or vehicle (n = 4). Left scale bar: 100 μm; right scale bar: 50 μm. D, F. Representative LFB staining images (D) and bar graph (F) show the demyelination in white matter of spinal cord from EAE mice receiving indicated treatment (n = 4). G. Flow cytometry analysis shows the cell counts of CD4 + T cells, CD8 + T cells, CD11b + cells, and B cells in CNS (brain and spinal cord) tissues of EAE mice receiving indicated treatment (n = 4–6). Data are presented as mean ± SEM. *p < 0.05 and ****p < 0.0001 NEK2 Inhibition regulates B-cell function in EAE Given the observed effects of NEK2 inhibition on human B cells in vitro, we next investigated its impact on B cell function in EAE mice using single-cell CyTOF analysis. Based on cell markers (Fig. [114]4A and B, Supplement Fig. [115]3 and Supplement Fig. [116]4), we identified major immune cell populations in the CNS, including B cells, CD4 + T cells, CD8 + T cells, double-negative T (DNT) cells, neutrophils, MHCII − dendritic cells (DCs), MHCII + DCs, Ly6C + monocytes, F4/80 + macrophages, and microglia (Fig. [117]4C). Similarly, peripheral blood immune cell populations included B cells, CD4 + T cells, CD8 + T cells, neutrophils, DCs, and monocytes-macrophages (Fig. [118]4D). We found that NEK2 inhibition significantly reduced the expression of co-stimulatory molecules CD80 and CD86 on B cells in both the CNS and peripheral blood, along with decreased levels of inflammatory cytokines such as IL-6 and TNF-α (Fig. [119]4E and F). Flow cytometry confirmed reduced proportions of CD80 + and CD86 + B cells in the CNS (Fig. [120]4G) and CD80 + B cells in the spleen of INH1-treated EAE mice (Fig. [121]4H). However, CD86 + B cells in the spleen showed no significant reduction compared to controls. Although the proportion of IL-10 + B cells displayed an increased trend after NEK2 inhibition, this difference was not statistically significant (Fig. [122]4G and H). To fully understand the contribution of B cells to the EAE phenotype and precisely dissect the function of NEK2 in their biology, B cell subpopulations of EAE mice receiving INH1 or vehicle were characterized and no significant changes were observed (Supplement Fig. [123]5). These results suggest that NEK2 primarily regulates B cell function in EAE by modulating the expression of co-stimulatory molecules. Fig. 4. [124]Fig. 4 [125]Open in a new tab Blockage of NEK2 regulates B cell function in EAE. A, B. Heatmap shows the expression of marker genes of immune cells from CNS (brain and spinal cord) (A) and periphery blood (B) of EAE mice given INH1 or vehicle. C, D. Cells from CNS (brain and spinal cord) (C) and periphery blood (D) of EAE mice given INH1 or vehicle clustered by multidimensional reduction with t-SNE shows immune cell types. E, F Heatmaps show the differential protein expression of B cells in CNS (brain and spinal cord) (E) and periphery blood (F) from EAE mice given INH1 or vehicle, detected by single-cell CyTOF. G, H. Flow cytometry analysis shows the proportion of CD80 +, CD86 +, and IL-10 + B cells in CNS (brain and spinal cord) tissues (G) and spleen tissues (H) from EAE mice given INH1 or vehicle. Data are presented as mean ± SEM. *p < 0.05 NEK2 Inhibition suppresses Th1 polarization of CD4 + T cells The imbalance between regulatory T cells (Tregs) and CNS-reactive effector T cells (Th1 and Th17) is a hallmark of MS pathogenesis [[126]30]. Given the role of B cells as antigen-presenting cells (APCs) and their regulation by NEK2, we investigated the effect of NEK2 inhibition on CD4 + T cells polarization using CyTOF and flow cytometry (Fig. [127]5A). CyTOF analysis revealed downregulation of Th1-specific transcription factor T-bet, Th17-specific transcription factor RORγt, and Th1-associated cytokine IFN-γ in CD4 + T cells from INH1-treated EAE mice in the CNS (Fig. [128]5B) and peripheral blood (Fig. [129]5C). Flow cytometry confirmed a significant reduction in the proportion of Th1 cells among CD4 + T cells in the CNS (Fig. [130]5D) and peripheral blood (Fig. [131]5E), while no significant changes were observed in Th17 or Treg proportions (Fig. [132]5F and I). These findings indicated that NEK2 inhibition suppresses Th1 polarization in EAE. Notably, NEK2 inhibition reduced RORγt expression in CD4 + T cells without significantly affecting IL-17 A levels. Although RORγt is the master transcription factor for Th17 cells, its genetic deficiency does not fully suppress IL-17 production [[133]31]. Th17 differentiation relies on the cooperative interaction between RORγt and STAT3. IL-6/IL-23 signaling also can activate STAT3, thereby driving IL-17 A transcription [[134]32]. In this study, despite NEK2 inhibition downregulating RORγt, the core effector function of Th17 differentiation remained unaltered, indicating NEK2’s limited global regulatory role in this process. Fig. 5. [135]Fig. 5 [136]Open in a new tab Blockage of NEK2 inhibits the Th1 polarization of CD4 + T cells. A. Schematic diagram illustrating the experimental workflow for sample detection in EAE mice. B, C. Heatmap displays the differential protein expression of in CD4 + T cells in CNS (brain and spinal cord) and periphery blood of EAE mice given INH1 or vehicle, detected by single-cell CyTOF. D, F, H. Flow cytometry plots and summarized results show the proportion of Th1 cells(D), Th17(F), and Treg (H) cells in CD4 + T cells of CNS tissues from EAE mice treated with INH1 or vehicle. E, G, I. Flow cytometry plots and summarized results show the proportion of Th1 cells (E), Th17 cells (G), and Treg cells (I) in CD4 + T cells of spleen tissues from EAE mice treated with INH1 or vehicle. Data are presented as mean ± SEM. *p < 0.05 NEK2 regulates CD4 + T cell proliferation and polarization by modulating B-T cell interactions B cells contribute to MS pathogenesis through interactions with T cells, mediated by co-stimulatory molecules and cytokine secretion [[137]33]. Based on the regulatory effects of NEK2 inhibition on B-cell function and T-cell polarization, we evaluated the impact of NEK2 on B-T cell interactions using an in vitro coculture system (Fig. [138]6A). The experimental results demonstrated that under three distinct culture conditions including CD4 + T cells cultured alone, co-cultured with naïve mouse B cells, or co-cultured with EAE mouse B cells, NEK2 inhibition significantly reduced CD4 + T cell proliferation (Fig. 6B) and the proportion of Th1 cells (Fig. [139]6C), but showed no significant effect on the Th17 cells (Fig. 6D). Notably, compared to the alone-cultured group and the naïve mouse B cell co-culture group, NEK2 inhibition exhibited a markedly stronger suppressive effect on CD4 + T cell proliferation and Th1 differentiation blockade in the EAE-derived B cell co-culture system. These findings suggest that the mechanism by which NEK2 regulates CD4 + T cell proliferation and Th1 differentiation in EAE is primarily achieved through modulating the interactions between EAE mouse B cells and CD4 + T cells. Fig. 6. [140]Fig. 6 [141]Open in a new tab NEK2 regulates CD4 + T cell proliferation and polarization by modulating B-T cell interactions. A. Schematic displaying the experimental design of the B-T cell co-culture. B-D. Flow cytometry analysis the proportion of proliferating T cells (B), IFNγ + CD4 + T cells (C), and TH17a + CD4 + T cells (D) in CD4 + T cells cultured alone, co-cultured with B cells from naïve mice and cultured with B cells from EAE mice. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 Discussion In this study, we propose that NEK2 represents a critical therapeutic target for modulating immune responses and mitigating disease progression in MS. Specifically, NEK2 regulates the excessive proliferation and pro-inflammatory phenotypic alterations of B cells. Inhibition of NEK2 disrupts B cell-T cell interactions, ultimately suppressing Th1 cell expansion and ameliorating autoimmune-mediated damage. NEK2, a key kinase involved in cell cycle regulation, has been extensively studied in the context of cancer, where it is targeted to inhibit tumor cell activity and overcome drug resistance [[142]18, [143]34–[144]36]. Our findings provide the first evidence of NEK2’s role in modulating aberrant immune responses in B cells. Previous studies have hinted at the potential involvement of NEK2 in immune regulation. For instance, in pancreatic cancer, NEK2 binds to and phosphorylates PD-L1, stabilizing its expression and thereby suppressing anti-tumor immune responses [[145]37]. In hematological malignancies such as multiple myeloma and diffuse large B-cell lymphoma, NEK2 promotes aerobic glycolysis in B cells by regulating the splicing of PKM and inducing an imbalance in the PKM2/PKM1 ratio [[146]38, [147]39]. Clinically, elevated NEK2 expression in multiple myeloma cells correlates with increased CD8 + T effector memory cell counts and heightened T cell activation [[148]40]. Additionally, NEK2 loss reduces tumor-associated macrophages and alleviates T cell exhaustion by upregulating PD-L1 expression in both multiple myeloma cells and myeloid cells [[149]41]. Our study extends these findings by demonstrating that NEK2 inhibition modulates the expression of co-stimulatory molecules on B cells, as well as their proliferation and differentiation, within a pro-inflammatory environment. The traditional view of B cells in MS pathogenesis emphasizes their role in producing CNS-autoreactive antibodies [[150]42, [151]43]. However, the clinical efficacy of anti-CD20 therapies, which deplete B cells but spare antibody-secreting plasmablasts and plasma cells, suggests that B cells contribute to MS through antibody-independent mechanisms [[152]44, [153]45]. Our findings support this paradigm by demonstrating that NEK2 regulates B cell function, particularly their ability to promote antigen-specific pro-inflammatory T cell responses through the expression of co-stimulatory molecules. Previous studies have shown that circulating B cells in MS patients exhibit elevated levels of co-stimulatory molecules CD80 and CD86 [[154]46–[155]48]. Inhibition of Bruton’s tyrosine kinase (BTK), a key component of the B cell receptor (BCR) signaling pathway, has been shown to reduce the expression of these molecules, thereby limiting B cell-T cell interactions in MS patients [[156]49, [157]50]. In vivo studies have further demonstrated that conditional knockout of CD80 and CD86 on B cells attenuates primary and secondary T cell responses [[158]51]. Our study, based on an animal disease model, provides additional evidence that modulating the expression of CD80 and CD86 on B cells can suppress Th1 cell polarization, with NEK2 emerging as a critical regulatory target. Besides, we observed NEK2 inhibition reduced RORγt expression in CD4 + T cells, while IL-17 A levels remained unchanged. Although RORγt serves as the lineage-specific master transcription factor for Th17 cells, its genetic deficiency does not fully abolish IL-17 production. Th17 differentiation requires cooperative RORγt-STAT3 interactions. Notably, even with diminished RORγt, STAT3 directly sustains IL-17 A transcription. In this study, despite NEK2 inhibition downregulating RORγt, the core effector function of Th17 differentiation (IL-17 A secretion) was unaffected, collectively indicating NEK2’s limited global regulatory role in this process. Collectively, these findings underscore the pathogenic role of B cells in initiating CNS autoimmunity. While EAE model has provided valuable insights into MS pathophysiology, it is an imperfect representation of the human disease. Some of the negative results observed in our study may reflect intrinsic properties of NEK2 or limitations of the EAE model in replicating the immunological responses and B cell characteristics seen in MS. Furthermore, the effects of NEK2 on the expression and function of T cells require further exploration and elucidation in our study. Additionally, the effects of NEK2 on T cell expression and function warrant further investigation. Although our study primarily focuses on B cells, the potential role of NEK2 in CNS-resident cells cannot be excluded. Lastly, although the potential role of NEK2-mediated mechanisms in CNS resident cells cannot be excluded. Despite these limitations, our findings highlight a previously unrecognized role for NEK2 in B cell biology and inflammation, suggesting that NEK2 may serve as a target in autoimmune demyelination. Future studies should explore the broader implications of NEK2 inhibition in MS and other autoimmune disorders, as well as its potential interactions with other immune cell populations. Electronic supplementary material Below is the link to the electronic supplementary material. [159]Supplementary Material 1^ (4.6MB, docx) Acknowledgements