Selinexor Reduces the Immunosuppression of Macrophages and Synergizes With CD19 CAR‐T Cells Against B‐Cell Lymphoma

Wenjing Luo; Jia Xu; Chenggong Li; Lu Tang; Yingying Li; Xindi Wang; Zhuolin Wu; Yinqiang Zhang; Heng Mei; Yu Hu

doi:10.1111/cas.70123

. 2025 Jun 17;116(9):2388–2399. doi: 10.1111/cas.70123

Selinexor Reduces the Immunosuppression of Macrophages and Synergizes With CD19 CAR‐T Cells Against B‐Cell Lymphoma

Wenjing Luo ^1,^2,³, Jia Xu ^1,^2,³, Chenggong Li ^1,^2,³, Lu Tang ^1,^2,³, Yingying Li ^1,^2,³, Xindi Wang ^1,^2,³, Zhuolin Wu ^1,^2,³, Yinqiang Zhang ^1,^2,³, Heng Mei ^1,^2,^3,^✉, Yu Hu ^1,^2,^3,^✉

PMCID: PMC12400065 PMID: 40528279

ABSTRACT

CD19 chimeric antigen receptor (CAR)‐T cell therapy has achieved high response rates in patients with B‐cell lymphoma. However, treatment failure and relapse can be attributable to CAR‐T cell dysfunction and the immunosuppression of the tumor microenvironment. Combination therapy emerges as a solution strategy, and selinexor might be a potential candidate. In this study, we first established the ex vivo tumor microenvironment model by coculturing tumor cells and macrophages, followed by coculture with CAR‐T cells, and identified that selinexor decreased CAR‐T cell exhaustion and enhanced its cytotoxicity. Moreover, selinexor upregulated NGFR expression and boosted CAR‐T cell proliferation. The ex vivo and in vivo results showed that selinexor prevented macrophages from polarizing to M2 populations. In the xenograft animal model, the sequential use of selinexor and CAR‐T cells significantly reduced the tumor burden compared with selinexor or CAR‐T cell monotherapies. In summary, our findings suggest that selinexor mitigates the immunosuppression of macrophages and improves CAR‐T cell functionality, and the combination of selinexor and CAR‐T cells may be a promising therapeutic strategy for B‐cell lymphoma.

Keywords: chimeric antigen receptor‐T cell, combination therapy, immunomodulation, macrophages, selinexor

In the tumor microenvironment, selinexor suppresses tumor cell growth and prevents macrophages from polarizing to M2 populations. The lower concentration of selinexor decreases CAR‐T cell exhaustion, enhances its cytotoxicity, and upregulates NGFR expression to prompt CAR‐T cell proliferation.

graphic file with name CAS-116-2388-g005.jpg

Abbreviations

AEs: adverse effects
BCL: B‐cell lymphoma
BM: bone marrow
CAR: chimeric antigen receptor
CRS: cytokine release syndrome
DEGs: differentially expressed genes
FCM: flow cytometry
GSEA: Gene Set Enrichment Analysis
HUST: Huazhong University of Science and Technology
RT‐qPCR: real‐time quantitative PCR
SEL: selinexor
TAMs: tumor‐associated macrophages
TME: tumor microenvironment
XPO‐1: exportin‐1

1. Introduction

The remarkable advancements of chimeric antigen receptor (CAR)‐T cell therapy in hematological malignancies have revolutionized anti‐cancer immunotherapy, resulting in the first FDA approval for gene‐editing therapies. Nonetheless, approximately 40% of patients with B‐cell lymphoma (BCL) failed to achieve complete responses after CD19 CAR‐T cell treatment, and over 60% of patients suffered from progression or relapse [1, 2, 3, 4]. These clinical challenges underscore the urgent need for strategies to improve CAR‐T cell therapy.

The expression of inhibitory receptors, such as PD‐1 and LAG‐3, was positively associated with CAR‐T cell exhaustion and would impair their functionality [5, 6]. Preclinical and clinical studies showed that PD‐1‐disrupted CAR‐T cells exerted effective antitumor abilities [7, 8]. Importantly, the tumor microenvironment (TME) represents another therapeutic barrier, where M2 populations dominate immunosuppressive niches that promote tumor progression and subvert immune surveillance [9, 10]. Consequently, concurrent prevention of CAR‐T cell exhaustion and M2 macrophage suppression would potentiate CAR‐T cell efficacy.

Exportin‐1 (XPO‐1), a nuclear export mediator for tumor suppressor proteins and growth regulators, is associated with a poor prognosis in patients with BCL [11, 12], making it a compelling therapeutic target. Selinexor (SEL), an XPO‐1 inhibitor, is clinically effective in patients with multiple myeloma and BCL [13, 14]. Intriguingly, SEL could upregulate the BCMA expression on plasma cells ex vivo, and the combination of SEL and BCMA CAR‐T cells benefited patients with multiple myeloma [15]. Moreover, emerging studies have shown that SEL combined with ibrutinib or mitochondria‐targeting lonidamine could reprogram macrophages [16, 17]. Additionally, the second‐generation XPO‐1 inhibitor eltanexor was reported to preferentially sensitize leukemia cells to CD19 CAR‐T cell killing ex vivo [18]. Nevertheless, no study has comprehensively investigated the impacts of the XPO‐1 inhibitor on CAR‐T cell efficacy against BCL with the presence of macrophages, and the detailed mechanisms underlying the combined effects have not yet been elucidated.

To lay the experimental foundations for the rational application of CAR‐T cells combined with XPO‐1 inhibitors in BCL patients, we selected SEL, the only XPO‐1 inhibitor approved by FDA, for further analysis based on its clinical relevance. In this study, we developed the ex vivo TME and in vivo models and clarified that SEL mitigated the immunosuppression of M2 macrophages and improved CAR‐T cell proliferation and functionality. Furthermore, our results suggested that the combination therapy of SEL and CAR‐T cells was feasible in patients with BCL.

2. Material and Methods

All in vivo experiments were approved by the Experimental Animal Ethics Committee of Huazhong University of Science and Technology (HUST; IACUC No. 3810). Experiments involving healthy donors (REC ref. no. [2023]0754‐01) and patients (REC ref. no. [2019]008) were approved by the Ethics Committee of the Union Hospital affiliated with HUST.

2.1. CAR‐T Cell Preparation

The CD19 CAR construct used throughout this study has been previously described (Figure S1) [19]. Detailed information can be found in Appendix S1.

2.2. Coculture of Tumor Cells and CAR‐T Cells With or Without Macrophages

We established two models: (a) the ex vivo TME model, BCL cells and an equal number of THP‐1 cells were cultured in the administration of PMA and DMSO or SEL for 2 days [20], and (b) the control model, BCL cells were cultured in the administration of PMA and DMSO or SEL for 2 days. On Day 2, we evaluated the polarization of macrophages in the ex vivo TME model by flow cytometry (FCM). Additionally, we performed a half‐medium change and added an equal number of CAR‐T cells. After another 2 days of coculture, we assessed the phenotypes of CAR‐T cells and the residual target cells, quantified as the percentage of CD19⁺CD3⁻ in alive cells [21]. Moreover, we isolated CAR‐T cells by fluorescence‐activated cell sorting to conduct bulk RNA sequencing and real‐time quantitative PCR (RT‐qPCR) analysis.

2.3. Tumor Cells Phagocytized by Macrophages

THP‐1 cells were stained with CytoTell Red 650 (AAT Bioquest, Sunnyvale, CA, USA), and BCL cell lines were labeled with 1 μM CFSE. Then, THP‐1 cells were cocultured with BCL cell lines at a 1:1 ratio (2 × 10⁵/well) in 12‐well plates with the addition of PMA and SEL. We performed a half‐medium change 2 days later and added 2 × 10⁵ CAR‐T cells. After another 2 days, we harvested cells and determined the percentage of phagocytized tumor cells, which was calculated as: phagocytized (%) = $\frac{(CytoTell Red 650^{+} cells) % + ({CFSE}^{+} cells) %}{({CFSE}^{+} cells) %}$ .

2.4. Mice Macrophages Coculture With Tumor Cells

To obtain bone marrow‐derived macrophages, we flushed the femurs of mice and seeded them in 6‐well plates after lysing erythrocytes. The bone marrow (BM) cells (2 × 10⁶/well) were cultured in alpha minimal essential medium (Gibco, Grand Island, New York, USA) supplemented with 10% fetal bovine serum, 1% penicillin–streptomycin, and 30 ng/mL macrophage colony‐stimulating factor (BioLegend, San Diego, CA, USA). After 5 days, A20 cells (5 × 10⁵/well) and SEL were added [17]. After 2 days of coculture, we harvested the cells to perform RT‐qPCR and FCM analysis.

2.5. In Vivo Mice Experiments

We utilized BALB/c mice to evaluate the effects of SEL on the immune landscape. Furthermore, we developed xenograft animal models with SCID‐beige and NXG mice to investigate the safety and efficacy of CAR‐T cells combined with SEL, respectively. Detailed information can be found in Appendix S1.

2.6. Clinical Outcomes Measurement and Immunofluorescence Staining of Patients' Tumor Tissues

Clinical specimens, including peripheral blood and tumor tissues, were obtained from patients who provided informed consent. Clinical responses were evaluated per Lugano criteria [22]. Tumor specimens before CAR‐T cell infusion were made into paraffin sections, followed by incubation with the corresponding antibodies listed in Table S1.

2.7. SYBR Green RT‐qPCR

Total RNA was extracted with Ultrapure RNA Kit (CWBIO, Taizhou, China). Complementary DNA was synthesized using HiScript III RT SuperMix for qPCR (Vazyme, Nanjing, China). The RT‐qPCR was performed using ChamQ SYBR qPCR Master Mix (Vazyme) and run on a CFX96 real‐time system (Bio‐Rad, CA, USA). The primers were shown in Table S2. The β‐actin was used as an internal control, and the mRNA expression was calculated as 2^−∆CT. The relative expression was quantified as 2^−∆∆CT after normalization to the control group.

2.8. Bulk RNA Sequencing and Analysis

RNA was isolated from the sorted CAR‐T cells and mice BM cells. The subsequent complementary DNA library construction and Illumina sequencing with paired‐end reads were performed by the Beijing Novogene Company (Beijing, China). Gene Set Enrichment Analysis (GSEA) and Gene Set Variation Analysis were implemented by the clusterProfiler package v4.8.2.

2.9. Statistical Analysis

All statistical analyses and plots were performed with GraphPad Prism 9 (San Diego, CA, USA) or R 4.0.3. Data were presented as means ± standard deviations. Categorial data was analyzed by Fisher's exact test. Inter‐group comparisons were determined by two‐side Student's t test for unpaired or paired data, the Mann‐Whiney U test, or two‐way ANOVA. Survival differences were calculated by the Kaplan–Meier method and compared using the log‐rank test. p values < 0.05 were considered as statistically significant (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant).

Additional materials and methods regarding cell lines, agents, cell counting kit‐8 assay, cytotoxicity analysis, NGFR overexpression, enzyme‐linked immunosorbent assay, and western blotting analysis are provided in Appendix S1.

3. Results

3.1. Higher Expression of XPO‐1 Correlated With Poor Prognosis in Patients With BCL

Using the median 2^−∆CT of XPO‐1 in pre‐infusion peripheral blood (0.00274) as a cutoff, we stratified 36 BCL patients into higher and lower expression groups. The overall response was 77.78% versus 55.56% in patients with lower and higher XPO‐1 levels, respectively (Figure 1A). Patients with lower expression of XPO‐1 obtained longer overall survival and progression‐free survival, highlighting that inhibiting XPO‐1 might be beneficial for the clinical outcomes of BCL patients receiving CD19 CAR‐T cells (Figure 1B,C).

The association of *XPO‐1* expression with clinical outcomes. (A) Clinical response of patients with lower or higher expression of *XPO‐1*. (B, C) Kaplan–Meier curves of the OS (B) and PFS (C) of patients with lower or higher expression of *XPO‐1*. CR, complete response; OS, overall survival; PD, progression disease; PFS, progression free survival; PR, partial response; SD, stable disease. *p < 0.05; ns, not significant.

3.2. SEL Enhanced the Cytotoxicity of CAR‐T Cells in the Ex Vivo TME

Combing our results and the previous studies [15, 18], we determined the optimal concentration of SEL as 50 and 100 nM in this study (Figure S2A). SEL upregulated XPO‐1 mRNA expression levels (Figure S2B). The possible reason is that the inhibitory effects of SEL on XPO‐1 protein may induce the transcription of XPO‐1 [23, 24]. However, SEL pretreatment did not affect the CD19 expression on BCL cells (Figure S2C) or impact the susceptibility of BCL cells to CAR‐T cells (Figure S2D).

Next, we investigated the direct effects of SEL on the phenotype and functionality of CAR‐T cells. During CAR‐T cell culturing ex vivo, 100 nM SEL increased the effector memory (CD62L⁻CD45RA⁻) populations but decreased the central memory (CD62L⁺CD45RA⁻) subsets, without impacts on other immunophenotypes (Figure S3A,B). SEL treatment impaired the specific cytotoxicity of CAR‐T cells against tumor cells (Figure S3C), suggesting that SEL toxicity might compromise CAR‐T cell functionality. Taken together, we considered that the sequential use of SEL and CAR‐T cells would be a more optimal strategy.

Based on the reports that SEL decreased M2 macrophages [16, 17], we hypothesized that SEL would modulate the tumor‐associated macrophages (TAMs) and consequently enhance CAR‐T cell antitumor capacity in the TME. Accordingly, we developed the ex vivo TME and control models (Figure 2A). CAR‐T cells in the ex vivo TME model displayed pronounced exhaustion markers and impaired functionality compared to those in the control model (Figure S4). Pretreatment with SEL markedly decreased PD‐1 single‐positive expression and PD‐1/LAG‐3 double‐positive populations of CAR‐T cells in the ex vivo TME model (Figure 2B–D), while only significantly suppressing PD‐1 expression in the control model (Figure S5). SEL pretreatment resulted in more tumor reduction, which was more prominent in the ex vivo TME model than in the control model (Figure 2E; Figure S6). Except for macrophages, the factors contributing to tumor cell reduction were consistent in the two models. Therefore, we further evaluated the effects of SEL on the phagocytized proportion of tumor cells by macrophages in the ex vivo TME model, but no statistically significant differences were noted (Figure 2F). Furthermore, we found that SEL prevented M2 polarization in the ex vivo TME model (Figure S7). In conclusion, we deduced that SEL could impede the immunosuppression of macrophages and augment the effectiveness of CAR‐T cells in the ex vivo TME model.

The ex vivo synergistic effects of SEL and CAR‐T cells. (A) A schematic illustration of the ex vivo models. Tumor cells with or without THP‐1 cells were cocultured in supplementation with SEL and PMA, and an equal number of CAR‐T cells were added 2 days later. After another 2 days, the phenotypes of CAR‐T cells, the residual CD19⁺CD3⁻ tumor cells accounting for alive cells, and the percentage of tumor cells phagocytized by macrophages were assessed. (B) The expression of PD‐1 on CAR‐T cells in the ex vivo TME (n = 3). (C) The percentage of PD‐1⁺ LAG‐3⁺ CAR‐T cells in the ex vivo TME (n = 3). (D) Heatmap analysis of CAR‐T cell phenotypes in the ex vivo TME. (E) The relative percentage of residual CD19⁺CD3⁻ tumor cells accounting for alive cells on Day 4 (n = 3). (F) Flow cytometry analysis of the CA46 cells phagocytized by macrophages in the ex vivo TME (left). Quantitative analysis of the phagocytized tumor cells in the ex vivo TME (n = 6; right). Image A was created with BioRender.com. TME, tumor microenvironment. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant.

3.3. SEL Increased NGFR Expression and Promoted CAR‐T Cell Proliferation in the Ex Vivo TME

We conducted bulk RNA sequencing of CAR‐T cells (n = 3) that were isolated from the ex vivo TME model containing Raji cells (Figure S8). GSEA analysis exhibited that 100 nM SEL upregulated cell cycle‐related signaling pathways (Figure 3A; Figure S9A).

The mechanisms of SEL improving CAR‐T cell proliferation in the ex vivo TME model. (A) GSEA analysis of CAR‐T cells in the ex vivo TME. (B) Volcano plot of DEGs. Genes with |Log₂(FC)| > 0 and p value < 0.05 were defined as DEGs. (C) *NGFR*‐positive associated genes from dataset GSE281230. (D) T and CD19 CAR‐Jurkat cells were transfected with Empty or NGFR^OE lentivirus. The expression of NGFR was detected by RT‐qPCR (n = 3 or 4; left) and western blotting (right). (E) FCM was used to measure the MFI of CytoTell Red 650 in Empty‐ or NGFR^OE‐T cells on Day 4 following 2 days of CD3/CD28 stimulation (n = 4). (F) FCM was used to measure the MFI of CytoTell Red 650 in Empty‐ or NGFR^OE‐CAR‐Jurkat cells after coculturing with BCL cells for 2 days (n = 3). The representative FCM figure (left) was from Empty‐ or NGFR^OE‐CAR‐Jurkat cells after coculturing with Ramos cells for 2 days. DEGs, differentially expressed genes; FC, fold change; FCM, flow cytometry; MFI, mean fluorescence intensity; TME, tumor microenvironment. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant.

Furthermore, CFSE‐labeled CAR‐T cells in the ex vivo TME model pretreated with SEL acquired the enhanced proliferation capacity, as evidenced by the decreased fluorescence intensity (Figure S9B). Among the upregulated differentially expressed genes (DEGs; Figure 3B), NGFR was associated with T cell proliferation in TME [25]. Moreover, the bulk RNA sequencing analysis of CAR‐T cells from dataset GSE281230 [26] verified that the expression of NGFR was positively related to TP53, HMGB2, and the genes associated with CAR‐T cell proliferation, including DBI, MAPK3, MKI67, and BCL2 (Figure 3C; Figure S10). To investigate the effects of SEL on the NGF‐NGFR communication, we quantified the NGF levels in the coculture supernatants but detected no differences (Figure S11).

Building on previous findings demonstrating SEL‐mediated NGFR upregulation in glioma cells [27], we wondered whether SEL could directly increase NGFR expression in CAR‐T cells. Subsequently, we cocultured CAR‐T cells with (stim setting) or without (no‐stim setting) 100 Gy‐irradiated Raji cells in the administration of SEL. After 2 days, we observed that SEL exposure markedly elevated NGFR expression under both settings (Figure S12A) but did not improve the proliferative capacity (Figure S12B), possibly due to the abrogation of SEL toxicity.

Next, we employed T cells and CD19 CAR‐Jurkat cells (Figure S13A) to preliminarily explore the role of NGFR in CAR‐T cell proliferation. Since endogenous NGFR expression was relatively low in T cells and CAR‐Jurkat cells, we overexpressed NGFR in both cells (Figure 3D). Notably, while NGFR overexpression alone failed to promote T or CAR‐Jurkat cell basal proliferation (Figure S13B,C), it significantly enhanced T cell expansion on Day 4 following a 2‐day CD3/CD28 stimulation (Figure 3E; Figure S13D) and accelerated CAR‐Jurkat cell proliferation on Days 2 and 4 when cocultured with BCL cells (Figure 3F; Figure S13E). Collectively, these data suggested that upregulating NGFR had the potential for boosting CAR‐T cell expansion upon either TCR or CAR stimulation.

3.4. SEL Reduces M2 Macrophages in Mice

Immunofluorescence analysis of baseline tumor biopsies revealed greater infiltration of CD68⁺CD206⁺ macrophages in the CAR‐T cell therapy non‐responder compared to the complete responder (Figure 4A). Consistent findings were also observed in the previous studies reporting that M2 macrophages were associated with a poor prognosis in BCL patients receiving CAR‐T cells [10, 28].

SEL modulated immunosuppressive TME. (A) Representative immunofluorescence analysis of M2 macrophages in patients with B‐cell lymphoma receiving CD19 CAR‐T cell therapy. Scale bar: 50 μm. (B) Schematic illustration of the experiment in the syngeneic animal model (n = 4). (C) Comparisons of macrophages and the sub‐populations in tumor tissues of mice treated with vehicle or SEL for 28 days. (D) Schematic illustration of BMDMs coculturing with A20 cells in the administration of SEL for 2 days. (E) FCM analysis of the M2 population accounting for macrophages in the coculture of BMDMs and A20 cells treated with SEL (n = 4). (F) The relative mRNA levels of *CD80* and *CD206* in the coculture system (n = 4). Images B and C were created with BioRender.com. BMDMs, bone marrow‐derived macrophages; CR, complete response; FCM, flow cytometry; NR, no response; TME, tumor microenvironment. *p < 0.05; **p < 0.01; ns, not significant.

Furthermore, we explored the impacts of SEL on TAMs as well as the immune landscape in the animal model (Figure 4B). SEL treatment for 28 days decreased the ratio of M2 macrophages to CD45⁺ populations in tumor specimens (Figure 4C) but did not alter the tumor‐infiltrating lymphocytes (Figure S14A), the inhibitory receptors (PD‐1 and TIM‐3) on tumor‐infiltrating T cells, or the tumor‐infiltrating myeloid‐derived suppressor cells (Figure S14B). We monitored the weight of mice and found no significant differences, implying that the oral SEL did not mediate severe adverse effects (AEs; Figure S15). Next, we cocultured bone marrow‐derived macrophages and A20 cells ex vivo for 2 days in supplementation with DMSO or SEL (Figure 4D). FCM results showed that SEL decreased M2 macrophages (Figure 4E), consistent with RT‐qPCR analysis (Figure 4F).

3.5. The Safety Profiles of CAR‐T Cells Combined With SEL In Vivo

The sequential strategy of SEL (5 or 10 mg/kg, 3 times a week) and CAR‐T cells was adopted in SCID‐beige mice engrafted with Raji‐luciferase cells (Figure 5A). SEL markedly increased TNF‐α levels, but not IL‐6 or IFN‐γ levels (Figure 5B). The administration of 10 mg/kg SEL reduced hemoglobin levels on Day 21 after CAR‐T cell infusion, whereas other hematological parameters remained comparable across all groups (Figure 5C). Moreover, 10 mg/kg SEL treatment induced more severe weight loss (Figure 5D), reminding us of the combination therapy‐related AEs.

The effects of SEL on cytokine release and the transcriptional modulation of bone marrow cells. (A) Schematic illustration of the experiment in SCID‐beige mice (n = 3). (B) The cytokine levels in peripheral blood of mice after CAR‐T cell infusion. (C) The hematological parameters of mice after CAR‐T cell infusion. (D) Relative weight alteration of mice after CAR‐T cell infusion. (E) Volcano plot of DEGs in bone marrow cells between mice treated with CAR‐T monotherapy and those receiving CAR‐T cells and SEL (10 mg/kg, 3 times a week; n = 3). Genes with |Log₂(FC)| > 0 and p value < 0.05 were defined as DEGs. (F) Gene Set Variation Analysis of signaling pathways between the monotherapy and combination therapy groups. Image A was created with BioRender.com. DEGs, differentiated expressed genes; FC, fold change. *p < 0.05; **p < 0.01.

Twenty‐eight days after CAR‐T cell infusion, we harvested bone marrow (BM) cells for bulk RNA sequencing and found that the SEL treatment (10 mg/kg, 3 times a week) upregulated 823 genes and downregulated 1278 genes (Figure 5E). Among these, GATA1 and SOD1 played vital roles in hematopoiesis [29, 30], and ATP6V0D2 could mitigate the tumor‐protective factors of tumor‐associated macrophages [31]. We conducted Gene Set Variation Analysis and found that, compared with CAR‐T cell monotherapy, the combination of SEL and CAR‐T cells induced cell cycle arrest and augmented the TGF‐β signaling pathway (Figure 5F), which would suppress hematopoietic functionality [32]. SEL upregulated the T cell receptor signaling pathway and natural killer cell‐mediated cytotoxicity, the important gene signatures positively associated with immunotherapy efficacy [33]. Moreover, SEL decreased oxidative phosphorylation, essential for M2 macrophage differentiation [34]. Furthermore, GSEA analysis showed that combination therapy upregulated glycolysis/gluconeogenesis (Figure S16A), the M1 macrophage‐related signaling pathway [35], accompanied by the downregulation of oxidative phosphorylation (Figure S16B).

3.6. The Efficacy of CAR‐T Cells Combined With SEL In Vivo

To establish the xenograft tumor model (Figure 6A), we subcutaneously injected 3 × 10⁶ Raji‐luciferase cells in NXG mice with severe functional deficiency of T, B, and NK cells [36, 37]. The sequential use of lower‐dose SEL (5 mg/kg, 3 times a week) and CAR‐T cells was adopted to investigate their synergistic efficacy in vivo. SEL increased the count of CD3⁺ T cells 21 days after CAR‐T cell infusion (Figure 6B) and the combination therapy markedly suppressed tumor growth compared to either SEL or CAR‐T cell monotherapies (Figure 6C–E). SEL did not lead to obvious weight loss after CAR‐T cell infusion (Figure S17), suggesting that no severe cytokine release syndrome (CRS) happened in the combination group [21]. The percentage of tumor‐infiltrating T cells was not altered by SEL (Figure S18A,B). Additionally, FCM analysis revealed that SEL significantly decreased the LAG‐3 expression on CAR‐T cells in tumor tissues (Figure 6F), with no effects on other immunophenotypes (Figure S18B).

SEL improved CAR‐T cell efficacy in vivo. (A) Schematic illustration of the experiment in the immunodeficiency NXG mice (n = 3). (B) CD3⁺ T cells counting on Days 7, 14, 21, and 28 after CAR‐T cell infusion. (C) Bioluminescence imaging assessment on Days 0, 7, 14, 21, and 28 after oral SEL treatment. (D) Total radiance analysis of bioluminescence on the indicated days. (E) Tumor weight on Day 28 after CAR‐T cell infusion. (F) Flow cytometry analysis of LAG‐3 and PD‐1 expression on CAR‐T cells in tumor tissue 28 days after CAR‐T cell infusion. Image A was created with BioRender.com. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant.

4. Discussion

CAR‐T cell therapy has resulted in a paradigm shift in hematological malignancies, but treatment failure and recurrence remain obstacles and need to be further overcome. Combination therapies provided solution strategies for patients with BCL [38, 39, 40], aiming to magnify CAR‐T cell expansion, suppress its exhaustion, and modulate TME. Here, we discovered that SEL inhibited M2 macrophage polarization and enhanced CAR‐T cell functionality in BCL (Figure 7).

In the tumor microenvironment, selinexor suppresses tumor cell growth and prevents macrophages from polarizing to M2 populations by inhibiting oxidative phosphorylation. The lower concentration of selinexor decreases CAR‐T cell exhaustion, enhances its cytotoxicity, and upregulates the NGFR signaling pathway to prompt CAR‐T cell proliferation.

Our ex vivo data exhibited that simultaneous coculturing of SEL impaired CAR‐T cell functionality, without impacts on their exhaustion or expansion. To mimic the sequential use of SEL and CAR‐T cells in vivo, we developed the ex vivo TME model. Intriguingly, SEL dramatically increased the cytotoxicity and proliferation of CAR‐T cells and decreased M2 macrophages in the ex vivo TME. The discrepancies between the two experimental conditions might be due to the distinct SEL administration regimens and the inhibitory effects of SEL on M2 macrophages. The reduction in M2 macrophages would enhance CAR‐T cell activity [10], thereby augmenting the functionality of CAR‐T cells. Moreover, our data indicated that SEL upregulated NGFR expression in CAR‐T cells and further accelerated CAR‐T cell expansion upon the stimulation of TCR or CAR. However, simultaneous SEL treatment failed to enhance CAR‐T cell amplification regardless of antigen stimulation, possibly due to its toxicity counteracting NGFR‐mediated proliferative benefits. NGF, the ligand for NGFR, is predominantly secreted by activated immune cells [41], and their interaction could regulate cell proliferation [25]. Consequently, NGFR upregulation would increase NGF consumption. In our ex vivo TME model, while SEL‐mediated upregulation of NGFR expression was observed, NGF concentrations in coculture supernatants remained unaltered, implying that SEL might potentially enhance NGF release. Nevertheless, the underlying mechanisms of how SEL regulated NGF‐NGFR crosstalk to potentiate CAR‐T cell expansion remained to be deciphered.

As the main factor of the immunosuppressive TME, M2 macrophages could hinder the effectiveness of immunotherapy [9, 10]. SEL with concurrent ibrutinib and the combination of SEL and mitochondria‐targeting lonidamine nanoparticles significantly reprogrammed macrophages into M1 subtypes, but the potential mechanisms have not been characterized [16, 17]. Our results showed that SEL reduced M2 macrophages in ex vivo coculture of human BCL cells, as well as murine A20 BCL cells. Moreover, SEL inhibited M2 macrophages in A20‐bearing BALB/c mice. Bulk RNA analysis of BM cells revealed that SEL markedly downregulated oxidative phosphorylation, associated with M2 macrophage differentiation [34]. However, these findings require further experimental validation.

Oral SEL (5 or 10 mg/kg) was well tolerated in the syngeneic mouse model. However, the sequential use of SEL (10 mg/kg) and CAR‐T cells in SCID‐beige mice led to higher levels of TNF‐α, lower concentrations of hemoglobin, and dramatic weight reduction, suggesting the combination therapy‐related AEs. TNF‐α, primarily secreted by activated monocytes/macrophages, NK cells, and T cells [42], played a crucial role in inducing tumor cell death and promoting macrophages to polarize into M1 subpopulations [43]. Meanwhile, TNF‐α was responsible for CRS [44], and CRS was positively associated with cytopenia after CAR‐T cell therapy [45]. Additionally, the bulk RNA analysis of BM cells elucidated that the sequential use of SEL and CAR‐T cells might aggravate the hematological toxicities, manifesting as the cell cycle arrest and upregulation of the TGF‐β signaling pathway [32]. Given the recognized hematologic toxicity risks of both CAR‐T therapy and SEL treatment [13, 46], we speculated that their combination would lead to more severe anemia. Therefore, we considered that a lower dose of SEL should be adapted in combination with CAR‐T cells. Encouragingly, the sequential use of SEL (5 mg/kg) and CAR‐T cells exerted good safety and efficacy profiles in NXG mice engrafted with Raji‐luciferase cells. Moreover, SEL treatment boosted CAR‐T cell proliferation in vivo and attenuated their exhaustion, supporting the ex vivo results.

Based on previous studies [15, 16, 17, 18], we systematically conducted this first investigation about the effects of SEL on BCL cells, macrophages, and CAR‐T cells ex vivo and in vivo. SEL elevated BCMA expression on tumor cells, improving their susceptibility to BCMA CAR‐T cells [15]. BCL cells were less sensitive to XPO‐1 inhibitors than leukemia cell lines, and the cytotoxicity of CD19 CAR‐T cells did not increase when Raji cells were pretreated with eltanexor [18]. In this study, we observed no effects of SEL on CD19 expression on BCL cells or their vulnerability to CAR‐T cell‐mediated killing. Notably, we innovatively clarified that SEL inhibited M2 macrophages in all tested experimental models (ex vivo and in vivo) and newly proposed that SEL upregulated NGFR expression, further boosting CAR‐T cell proliferation. Taken together, we concluded a reasonable assumption that SEL treatment might enhance the CAR‐T cell efficacy across multiple malignancies, including those insensitive to SEL, through the modulation of both tumor‐intrinsic and tumor‐extrinsic factors. Moreover, our animal experiments originally indicated that the combination of a higher dose of SEL and CAR‐T cells mediated higher cytokine release and hematological toxicities, providing theoretical support for the trial design that a lower dose of SEL (20 or 40 mg per week) after platelet recovery was adopted in patients receiving CAR‐T cells [15]. Nevertheless, in the present study, SEL was administered short‐term across CAR‐T cell therapy, and the capacity of cytokine release was limited in immunodeficient mice. Therefore, attention should be paid to potential AEs when combining CAR‐T cells with SEL in clinical practice.

Limitations existed in our present study. First, how SEL regulated NGFR expression and thereby enhanced CAR‐T cell proliferation warrants future studies to investigate. Second, the mechanisms underlying SEL suppression of M2 macrophages require further experiments to clarify. Additionally, this study provided experimental evidence that CAR‐T cells in combination with a higher dose of SEL might increase the risk of AEs, but the precise dosing strategies and treatment regimen need to be determined through dose‐escalation clinical trials.

In summary, our research first demonstrated that SEL treatment mitigated the immunosuppression of M2 macrophages, alleviated the exhaustion levels of CAR‐T cells, and enhanced their cytotoxicity and proliferative capacity. These phenotypic and mechanistic findings establish a strong rationale for combining CAR‐T cells with SEL in BCL treatment and lay the foundation for future clinical translation of this synergistic approach.

Author Contributions

Wenjing Luo: conceptualization, data curation, formal analysis, methodology, software, validation, visualization, writing – original draft. Jia Xu: investigation, methodology, software, validation, visualization. Chenggong Li: supervision. Lu Tang: supervision. Yingying Li: investigation. Xindi Wang: investigation. Zhuolin Wu: investigation. Yinqiang Zhang: investigation. Heng Mei: conceptualization, funding acquisition, project administration, supervision, writing – review and editing. Yu Hu: conceptualization, project administration, resources, supervision, writing – review and editing.

Disclosure

The authors have nothing to report.

Ethics Statement

Approval of the research protocol by an Institutional Reviewer Board: Experiments involving healthy donors (REC ref. no. [2023]0754–01) and patients (REC ref. no. [2019]008) were approved by the Ethics Committee of the Union Hospital affiliated with HUST. Animal studies: All animal experiments were approved by the Experimental Animal Ethics Committee of HUST (IACUC No. 3810).

Consent

Informed consent was obtained from all patients before enrollment.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Appendix S1.

CAS-116-2388-s001.docx^{(27.7KB, docx)}

Figure S1. The CAR structure used in this study.

Figure S2. The effects of SEL on CAR‐T cell viability and tumor cells.

Figure S3. The effects of SEL on the phenotype and functionality of CAR‐T cells.

Figure S4. Comparative analysis of CAR‐T cell reactivity and functionality in two ex vivo models.

Figure S5. Immunophenotypes of CAR‐T cells in the control model.

Figure S6. Flow cytometry analysis of alive tumor cells ex vivo.

Figure S7. The effects of SEL on M2 macrophages ex vivo.

Figure S8. Schematic illustration of the flow cytometry sorting for bulk RNA sequencing and the derived flow cytometry analysis.

Figure S9. The effects of SEL on CAR‐T cell proliferation in the ex vivo TME model.

Figure S10. Correlation between the expression of NGFR and the expression of SELL, MKI67, TCF7, HMGB2, GZMK, and LAMP from dataset GSE281230.

Figure S11. The effects of SEL on NGF release in the ex vivo TME model.

Figure S12. The effects of SEL on NGFR mRNA levels and the proliferation of CAR‐T cells during culturing simultaneously.

Figure S13. The proliferation analysis of NGFROE‐ and Empty‐T and CAR‐Jurkat cells.

Figure S14. Flow cytometry analysis of tumor‐infiltrating lymphocytes.

Figure S15. The changes of weight in mice bearing A20 tumors after SEL treatment.

Figure S16. GSEA analysis of bone marrow cells from SCID‐beige mice.

Figure S17. The changes of weight in NXG mice bearing Raji tumors after CAR‐T cell infusion.

Figure S18. The effects of SEL on CAR‐T cells in tumor issue isolated from mice 28 days after CAR‐T cell infusion.

CAS-116-2388-s002.pdf^{(12.7MB, pdf)}

Table S1. Detailed information of antibodies used in the study.

Table S2. Primers used in the study.

CAS-116-2388-s003.docx^{(19.6KB, docx)}

Acknowledgments

We thank all authors for contributing to this study, Wuhan Sian Medical Technology Co. Ltd. for CD19 CAR‐T cell manufacture, and Hefei PreceDo Pharmaceuticals Co. Ltd. for advice on SEL usage.

Funding: This work was supported by grants from the National Natural Science Foundation of China (Nos. 82330005 and 82350103 to H.M.) and the National Key R&D Program of China (No. 2022YFC2502704 to H.M.).

Wenjing Luo and Jia Xu contributed equally to this work.

Contributor Information

Heng Mei, Email: hmei@hust.edu.cn.

Yu Hu, Email: dr_huyu@126.com.

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