Pomalidomide enhances CAR-T cell therapeutic efficacy and Remodels Immune Microenvironment in Lymphoid Malignancies

Pomalidomide enhances CAR-T cell therapeutic efficacy and Remodels Immune Microenvironment in Lymphoid Malignancies | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Pomalidomide enhances CAR-T cell therapeutic efficacy and Remodels Immune Microenvironment in Lymphoid Malignancies Yan Yu, Yi Zhou, Linzhi Xie, Liwen Wang, Yuhan Yan, Qian Cheng, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6571044/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Dec, 2025 Read the published version in Cancer Immunology, Immunotherapy → Version 1 posted 16 You are reading this latest preprint version Abstract Background Despite the remarkable efficacy of Chimeric Antigen Receptor T-cell (CAR-T) therapy in hematological malignancies, challenges including limited persistence and T cell exhaustion hinder long-term responses. Pomalidomide, an immunomodulatory drug, shows potential to synergize with CAR-T therapy, yet its mechanistic basis remains unclear. Methods We performed CCK8, LDH, qPCR, ELISA, flow cytometry and bulk RNA-sequencing to explore the effects and mechanisms of pomalidomide on CAR-T cell in vitro . Myeloma xenograft murine models were established to evaluate the synergetic effects of CAR-T and pomalidomide in vivo . Single-cell RNA sequencing was employed to delineate the effects of pomalidomide on immune microenvironment. Results Pomalidomide (1–5 µg/mL) enhances CAR-T cell proliferation and cytotoxicity in an activation-dependent manner, upregulates the expression of interleukin-2 (IL-2), interferon-γ (IFN-γ), and chemokines CXCL9-CXCL11, and promotes the maintenance of central memory T cells (Tcm). In vivo , combination therapy induced tumor regression (p < 0.001) and extended survival (median OS: 30 days vs. unreached, p < 0.01). Transcriptomic analysis revealed metabolic reprogramming (glycolysis, fatty acid degradation) and reduced exhaustion markers (PD-1, LAG3). Single-cell sequencing demonstrated the immune microenvironment remodeling post pomalidomide treatment, including increased T/NK cells proportion and immune activity, as well as reduced myeloid-derived suppressor cell (MDSC) signatures within monocytes/macrophages. Conclusions Pomalidomide amplifies CAR-T efficacy by enhancing memory formation, cytokine production, and metabolic fitness while reshaping the immune microenvironment. These findings provide a rationale for clinical optimization of pomalidomide-CAR-T combinations in refractory hematologic malignancies. CAR-T therapy Pomalidomide Immune microenvironment Memory T cell Lymphoid Malignancies Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Chimeric Antigen Receptor T-cell (CAR-T) therapy has emerged as a revolutionary approach in the treatment of hematological malignancies, particularly demonstrating remarkable efficacy in lymphoid malignancies [ 1 , 2 ] . However, while CAR-T therapy achieves high initial remission rates, its long-term efficacy remains suboptimal, with many patients experiencing disease relapse or rapid progression shortly after treatment [ 2 , 3 ] . This underscores the critical need to enhance CAR-T cell persistence and anti-tumor activity through innovative combination strategies [ 4 , 5 ] , which has become a key focus in current research. Pomalidomide, a third-generation immunomodulatory drug (IMiDs), has been widely utilized in treating multiple myeloma and lymphomas, particularly demonstrating significant anti-tumor efficacy in lenalidomide-resistant patients [ 6 , 7 ] . In addition to directly inducing tumor cell apoptosis, pomalidomide also modulates the immune microenvironment to enhance T-cell functionality [ 8 , 9 ] . Emerging evidence suggests potential synergistic effects between pomalidomide and CAR-T therapy [ 10 ] . Our preliminary study [ 11 ] revealed that pomalidomide significantly prolongs progression-free survival (PFS) and overall survival (OS) in relapsed/refractory multiple myeloma (R/R MM) patients. Nevertheless, the precise mechanisms underlying this synergy remain incompletely understood. This study aims to systematically investigate the synergistic mechanisms of pomalidomide in CAR-T therapy, focusing on its dual effects on both CAR-T cells and tumor microenvironment modulation. We anticipate that our findings will contribute to the development of clinical protocols, particularly regarding the optimal timing and duration of pomalidomide administration during CAR-T therapy, thereby improving treatment outcomes and offering new strategies for managing refractory hematological malignancies. 2. Materials and Methods 2.1. Cell culture Human myeloma cell lines ARP-1 (kindly provided by Prof. Wen Zhou, Central South University), RPMI 8226, U266 and lymphoma cell line Raji (provided by Prof. Minghong Jiang, Peking Union Medical College), and BCMA/CD19-expressing K562 cells (Yucadi, China) were maintained in RPMI-1640 medium (Gibco, USA) supplemented with 10% FBS (Clark Bioscience, USA) and 1% penicillin/streptomycin (Gibco, USA). BCMA CAR-T and CD19 CAR-T cells (Yucadi, China) were cultured in RPMI-1640 containing 10% FBS, 1% penicillin/streptomycin, 300 IU /mL IL-2 (Abmole, USA) [ 12 , 13 ] , and 50 µM β-mercaptoethanol (Gibco, USA) [ 14 ] . Freshly thawed CAR-T cells received additional IL-7 and IL-15 (10 ng/mL each, Abmole, USA) [ 13 , 15 ] . All cells were maintained at 37°C with 5% CO₂ with medium replenishment every 48 h. 2.2. Reagent Pomalidomide (Selleck Chemicals, USA) was dissolved in DMSO (Sigma, USA) (2 mg/mL) and stored at -80°C. 2.3. PBMCs isolation Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood using Ficoll-Paque PLUS (1.077 g/mL; Solarbio, China) density gradient centrifugation. After centrifugation (1,000 ×g, 20 min, RT), the PBMC layer was collected, washed twice with PBS, and treated with 1× RBC lysis buffer (4–5 min, RT) if erythrocyte contamination persisted. 2.4. Proliferation Assays Working concentrations were prepared in RPMI-1640 before use. Cells were seeded in 96-well U-bottom plates: tumor cells (5×10⁴/well) or CAR-T (2×10⁶/well) with pomalidomide (0.01–100 µg/mL) or DMSO control. After 48 h culture, cells were washed and incubated with 10% CCK-8 reagent (Abbkine, USA) for 3 h (tumor cells) or 6 h (CAR-T). When required, CAR-T cells were activated with 5 µg/mL anti-human CD3 monoclonal antibody (BioLegend, USA) and anti-human CD28 monoclonal antibody (BioLegend, USA) at the beginning of the experiment. Absorbance was measured at 450 nm. Viability was calculated as: [(ODSample – ODBlank)/ (ODControl – ODBlank)] × 100%. 2.5. Cytotoxicity Assay CAR-T cells (pre-treated with 1–5 µg/mL pomalidomide or DMSO control for 24 h or not) were co-cultured with target cells at E:T ratios of 1:1 to 5:1 for 4 h. Cytotoxicity was assessed using LDH Release Assay Kit (Beyotime, China) following manufacturer's protocol. Absorbance was read at 490 nm with 600 nm reference. Cytotoxicity (%) = [(ODTreated – ODSample Control)/ (ODMax – ODSample Control)] × 100%. 2.6. Enzyme-Linked Immunosorbent Assay Supernatants from 24 h CAR-T/tumor co-cultures (E:T ratios 1:1–5:1) were analyzed using a Human IFN-γ ELISA Kit (ZCIBIO, China) following manufacturer protocols. 2.7. RNA Isolation and qPCR Analysis Total RNA was extracted from CAR-T cells or tumor tissues using the RNA rapid extraction kit (RNAfast200, Fastagen, China). cDNA synthesis and qPCR were performed with HiScript III RT SuperMix (Vazyme, China) and Taq Pro Universal SYBR Master Mix (Vazyme, China). Primer sequences for target genes ( IL2, IFNG, CXCL9/10/11 ) are listed in Supplementary Table 1. Relative mRNA expression was normalized to β-actin and calculated via the 2 −ΔΔCt method. 2.8. Flow Cytometry T-cell memory phenotype: CAR-T cells (3×10⁶/well) were treated with 2.5 µg/mL pomalidomide or DMSO control for 7 days. When required, CAR-T cells were activated with 5 µg/mL anti-human CD3 monoclonal antibody and anti-human CD28 monoclonal antibody at the beginning of the experiment. CAR-T cells were collected and stained with anti-CD3-FITC (BioLegend, USA), anti-CD45RA-PE (eBioscience, USA), anti-CD127-PerCP-Cy5.5 (BioLegend, USA), anti-CCR7-APC (eBioscience, USA), anti-CD8-APC-Alexa Fluor 750 (BioLegend, USA), anti-CD4-Pacific Blue (BioLegend, USA), and anti-CD25-PE-Cy7(BioLegend, USA) antibodies for 15 minutes at RT [ 16 , 17 ] . CAR + T cells CD4+/CD8 + ratio: CD19 CAR-T cells activated with anti-CD3/CD28 antibodies (5 µg/mL) were treated with 2.5 µg/mL pomalidomide for 48 h. Cells were collected and stained with anti-FMC63-FITC (for CD19 CAR detection, Acro, USA) antibody in the dark at 4℃ for 60 minutes, and stained with anti-CD3-APC (BioLegend, USA), anti-CD8-APC-Alexa Fluor 750, anti-CD4-Pacific Blue antibodies for 15 minutes at RT. Data were acquired on Gallios-Analyzer (Beckman Coulter, USA) and analyzed by using FlowJo v10. 2.9. Multiple myeloma cell xenograft murine model Female NOD-SCID mice [ 18 ] (5–6 weeks, Hunan SJA Laboratory Animal Co, China) were subcutaneously inoculated [ 18 , 19 ] with U266 cells (1×10⁷ cells/mouse in Matrigel) (Corning, USA). When tumors reached 100–150 mm³, mice were randomized into four groups (n = 5/group): 1)Control: PBS (i.v., 150µL) + 1% DMSO (i.p., 5 times/week) 2) Poma monotherapy: PBS (i.v., 150µL) + 2.5 mg/kg pomalidomide (i.p., 5 times/week) 3) CAR-T monotherapy: 1×10⁷ CAR-T cells (i.v., 150µL) + 1% DMSO (i.p., 5 times/week) 4) Combination therapy: 1×10⁷ CAR-T cells (i.v., 150µL) + 2.5 mg/kg pomalidomide (i.p., 5 times/week) Tumor volume (V = 0.5 × L × W 2 ) [ 18 , 19 ] and body weight were monitored every 2 days. The experimental endpoint was defined as the time when the subcutaneous tumor diameter reached ≥ 19 mm in any single dimension. Mice meeting this criterion were humanely euthanized. Tumors were weighed, snap-frozen in RNAlater (servicebio, China), and stored at − 80°C for molecular analysis. 2.10. Bulk RNA-sequencing and data analysis BCMA CAR-T cells derived from three donors were activated with anti-CD3/CD28 antibodies (5 µg/mL) and treated with pomalidomide (2.5 µg/mL) for 24 hours. Total RNA was extracted from 1 × 10⁶ CAR-T cells using RNAfast200 (Fastagen, China). Subsequent bulk RNA sequencing was performed at BGI Tech Solutions Co. (Shenzhen, Guangdong, China). Transcriptome sequencing was conducted on the DNBSEQ platform with paired-end (PE150) cycles. Raw FASTQ files were trimmed and filtered using Fastp (version 0.19.5), and subsequently aligned to the human reference genome (GRCh38) with Bowtie2 (version 2.4.1). Reads were counted with FeatureCounts (version 2.0.6). Differentially expressed genes (DEGs) were identified using the DESeq2 (version 1.42.0) package in R, with genes defined as differentially expressed if the log2-fold change was > 1 or < -1, and the adjusted P value was < 0.05. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses on DEGs were performed using the clusterProfiler package. Gene Set Enrichment Analysis (GSEA) of the Hallmark, GO: BP and IMMUNESIGDB (C7) gene sets was conducted using the “enrichr”, “GseaVis” and “GSEABase” R packages. 2.11. Single-cell RNA sequencing and data analysis To investigate the impact of pomalidomide on the tumor immune microenvironment, single-cell sequencing analysis was performed on a lymphoma patient who had undergone CD19 CAR-T infusion. Peripheral blood samples were collected at three time points: 1 day prior to pomalidomide treatment, and 10 and 30 days after the start of pomalidomide treatment. PBMCs were isolated from the collected blood samples using Ficoll gradient separation, and subsequently subjected to single-cell sequencing. Single-cell libraries were prepared using the DNBSEQ platform and the DNBelab C4 Single-Cell Library Prep Set, with paired-end sequencing performed on the DIPSEQ T1 platform. Raw FASTQ files underwent quality control, read alignment, and feature quantification using the DNBC4Tools pipeline ( https://github.com/MGI-tech-bioinformatics/DNBelab_C_Series_HT_scRNA-analysis-software ). Cell barcodes, gene names, and feature matrices were standardized across datasets to ensure uniform annotation. Initial quality control metrics were calculated for each dataset, and datasets were merged using the merge function in Seurat after filtering. Principal component analysis (PCA) was performed on the top 2000 variable genes in the merged dataset for dimensionality reduction. Batch correction and dataset integration were performed using Harmony (version 1.2.1). Clustering was performed using the Louvain algorithm based on shared nearest neighbors (SNN), and cell clusters were visualized using uniform manifold approximation and projection (UMAP). Clusters were annotated based on known marker genes and enriched pathways, and cluster-specific marker genes were identified using the FindMarkers function. For immune signature scoring, the ssGSEA were used to evaluate the function of NK cells, and AddModuleScore were used to evaluate the function of T cells [ 22 , 23 ] and the MDSC signature of monocytes/macrophages [ 24 ] . Pseudobulk analysis was performed by aggregating NK cell single-cell RNA-seq data. Following this, the GOBP enrichment score was calculated by GSVA analysis to assess the biological functional states of the NK cells. 2.12. Statistical Analysis All data are presented as mean ± standard error of the mean (SEM) from three independent experiments. Statistical significance was determined using GraphPad Prism 9.5.1 (GraphPad Software, USA). Comparisons between two groups were analyzed by unpaired Student’s t-test. For multiple group comparisons, one-way ANOVA followed by Tukey’s post hoc test was applied. Survival curves in animal studies were generated using the Kaplan-Meier method, and differences were assessed by log-rank (Mantel-Cox) test. A p-value < 0.05 was considered statistically significant. 3. Results 3.1 Pomalidomide promotes proliferation via Activation-Dependent Synergy and enhances Cytotoxicity and Cytokines Production of CAR-T Cells To establish a pharmacologically relevant concentration range, we first systematically evaluated pomalidomide's dual effects on tumor and CAR-T cells. While myeloma cell lines (U266, RPMI 8226, ARP-1), lymphoma cell line (Raji) and K562 controls showed dose-dependent growth suppression above 10 µg/mL (Fig. 1 A, Supplementary Fig. 1A), resting CAR-T cells maintained > 95% viability at ≤ 5 µg/mL (Fig. 1 B). Strikingly, upon CD3/CD28-mediated activation, subtoxic pomalidomide concentrations (2.5-5 µg/mL) induced a proliferation boost compared to activation alone (Fig. 1 B). This activation-dependent synergy suggested pomalidomide amplifies costimulatory signaling cascades rather than exerting generic proliferative effects. Guided by these dose-response relationships, we established 1–5 µg/mL as the operative concentration range for subsequent experiments. In tumor co-culture models, real-time pomalidomide exposure showed no immediate cytotoxicity enhancement (LDH release at three effector-to-target (E: T) ratios). However, 24-hour CAR-T pretreatment with pomalidomide induced profound functional augmentation, elevating specific lysis from 8.27–10.61% at E: T = 1:1 and 18.36–23.12% at E: T = 5:1 (Fig. 1 C, Supplementary Fig. 1B). This pretreatment efficacy gradient implies pomalidomide requires sufficient exposure time to prime CAR-T effector machinery. To further elucidate mechanisms underlying the synergistic antitumor effects of pomalidomide and CAR-T cells, we analyzed the expression of cytokines and chemokines in CAR-T cells activated via anti-CD3/CD28 antibodies or tumor co-culture. 24-hour CAR-T pretreatment with pomalidomide significantly upregulated mRNA levels of IL2 , IFNG , CXCL9 , CXCL10 , and CXCL11 compared to DMSO controls (Fig. 1 E). Correspondingly, IFN-γ protein levels in co-culture supernatants showed marked elevation (Fig. 1 D). These findings suggest that pomalidomide may exert its synergistic antitumor effects by regulating the transcriptome and protein expression of CAR-T cells, promoting the secretion of key cytokines, enhancing CAR-T cell activation, proliferation, and immune functions, and modulating the recruitment and infiltration of immune cells in the tumor microenvironment. 3.2 Pomalidomide Synergizes with CAR-T Cells to Amplify Anti-tumor Efficacy in Myeloma Xenograft Models The translational potential of this combinatorial approach was rigorously validated in U266 myeloma xenografts (Fig. 2 A). Longitudinal tracking revealed that pomalidomide pretreatment combined with CAR-T infusion induced profound tumor regression (p < 0.001, Fig. 2 B-C). This therapeutic synergy translated into a marked survival benefit, with median overall survival increasing from 30 days in the CAR-T-only group to an unachieved endpoint in the combination group (p < 0.01), while pomalidomide monotherapy showed no discernible benefit versus untreated controls (p = 0.39; Fig. 2 D). Molecular dissection of residual tumors unveiled that pomalidomide augmented intra-tumoral IL2 (4.04-fold) and IFNG (4.78-fold) expression (p < 0.05 vs CAR-T alone), concurrently inducing a chemokine storm characterized by 19.73 to 26.11-fold upregulation of CXCL9-11 (Fig. 2 F). Importantly, the absence of significant body weight fluctuations ( 0.05; Fig. 2 E) and preserved vital signs confirmed the regimen's safety profile. These findings suggest that pomalidomide potentiates CAR-T cell functionality by enhancing cytokine production and promoting immune cell infiltration and activation within the tumor microenvironment, thereby amplifying anti-tumor efficacy. 3.3 Pomalidomide Orchestrates CAR-T Cell Memory Programming and Subset Readjusting To investigate the effects of pomalidomide on CAR-T cell memory phenotypes and immune subset dynamics, CAR-T cells were cultured under resting or anti-CD3/CD28 antibody-activated conditions in the presence of pomalidomide for 7 days (Fig. 3 A). Following activation, CAR-T cells exhibited a significant increase in CD25 expression (early activation marker of T cell) at day 2, which subsequently declined over time (Supplementary Fig. 2B). Notably, the pomalidomide treatment maintained elevated CD25 levels throughout the observation period (Supplementary Fig. 2B), suggesting sustained CAR-T activation and functional persistence. Under resting conditions, pomalidomide significantly increased the proportion of central memory T cells (Tcm, CCR7 + CD45RA−) in CD8 + CAR-T cells from 19.60–26.45%, while showing no significant effects on other phenotypes or CD4 + CAR-T cells (Supplementary Fig. 2A). In anti-CD3/CD28-activated cultures, prolonged pomalidomide exposure markedly reduced CD8 + naïve T cells (CCR7 + CD45RA+) and increased both CD4 + and CD8 + Tcm populations compared to DMSO controls. Tcm frequencies rose from baseline levels of 11.4% (CD4+) and 11.6% (CD8+) to 34.8% and 50.65%, respectively, representing 1.42-fold and 1.34-fold increases over the DMSO control group (Fig. 3 B). Given reports linking CAR-T cell CD4/CD8 ratios to clinical prognosis, we evaluated pomalidomide's impact on this parameter. Due to lentiviral transduction bias favoring CD4 + T cells [ 25 ] , CAR + CD4 + T cells predominated in CAR-T products, yielding a resting CD4/CD8 ratio of 11.92. Activation reduced this ratio to 4.378. Notably, pomalidomide significantly increased the CD4/CD8 ratio under both resting and activated conditions, both in total and CAR + T cell populations (Fig. 3 C). 3.4 Pomalidomide Enhances CAR-T Cell Immune Function and Regulates Metabolism at the Transcriptomic Level To investigate the transcriptional mechanisms underlying the effects of pomalidomide on CAR-T cells, we first analyzed bulk RNA-seq data from BCMA CAR-T cells treated with either DMSO control or pomalidomide for 24h after CD3/CD28 antibody stimulation in vitro . The PCA results revealed a marked transcriptional divergence between pomalidomide treated CAR-T cells and the control group (Fig. 4 A). Consistent with the phenotypic plasticity observed in memory subsets, pomalidomide treatment upregulated expression of T cell memory-associated genes including IL7R , CCR7 , RORC , LEF1 , BACH2 and BCL6 . Concurrently, elevated expression levels of genes involved in T cell activation ( IL2RA , IT K, ZAP70 ) and cytokine/chemokine ( IL2 , TNF , CXCL9 , CCL2 , CCL4 , CCL20 ) were observed in pomalidomide treated CAR-T cells. Particularly, the expression of exhaustion and immunosuppressive-related markers ( PDCD1 , LAG3 , TIGIT , TOX , CTLA4 , FOXP3 ) was reduced in pomalidomide-treated CAR-T cells compared to controls (Fig. 4 B), suggesting transcriptional reprogramming towards a memory and effector competent state. Subsequent differential expression analysis identified 226 significantly upregulated and 23 significantly downregulated genes in the treated samples compared to controls (Fig. 4 C). KEGG analysis of upregulated genes revealed significant enrichment in metabolic pathways including fatty acid degradation, glycolysis, cGMP-PKG signaling, and tyrosine metabolism (Fig. 4 D), while GO analysis highlighted enrichment in immune-related pathways such as T cell activation, inflammatory response, cell adhesion, and ERK1/2 signaling (Fig. 4 E). GSEA analysis revealed that pomalidomide treatment induced transcriptional activation of multiple immune-related pathways in CAR-T cells. Specifically, cytokine related pathways including IFN-γ, IL-6, IL-2, and TNF-α signaling, along with inflammatory response pathways, were significantly enriched (Fig. 4 F). Concurrently, GSEA identified upregulation of T cell activation, proliferation, migration, immune response functions, and type II interferon responses in pomalidomide-treated CAR-T cells (Fig. 4 G). Importantly, the prominent activation of memory T cell signatures in pomalidomide treated CAR-T cells (Fig. 4 H), aligning with our prior observations of memory phenotype induction in vitro . 3.5 Pomalidomide Improves Anti-Tumor Immunity Through Remodeling the Tumor Immune Microenvironment To investigate the impact of pomalidomide on the tumor immune microenvironment, PBMCs were collected from a lymphoma patient at three time points: 1 day before pomalidomide treatment, 10 days and 30 days after pomalidomide treatment, and subsequently subjected to scRNA-seq analysis. The PBMCs were primarily classified into CD8 + T cells, monocytes/macrophages, NK cells, CD4 + T cells, pDCs and platelets (Figs. 5 A-B). Notably, as pomalidomide treatment progressed, the proportion of T cell and NK cell subpopulations gradually increased, while the proportion of monocyte/macrophage subpopulations decreased (Fig. 5 C). To further evaluate the impact of pomalidomide on T cells, secondary clustering analysis was performed, which identified six distinct clusters based on specific markers: CD8 + effector T cells (CD8 + effector), CD4 + naïve and central memory T cells (CD4 + Tn/Tcm), CD8 + exhausted T cells (Tex), CD8 + progenitor exhausted T cell (Tpex), CD4 + regulatory Tcells (Treg), and a cluster characterized by high expression of proliferation-related genes (Tprolif) (Fig. 5 C-D). The results revealed that with continuous pomalidomide treatment, the proportion of CD8 + effector, CD4 + Tn/Tcm increased, whereas the proportion of CD8 + Tex and Tpex cells decreased (Fig.E). The average gene expression of different T cell subpopulations was further evaluated. As expected, the expression of exhaustion-related genes ( PDCD1 , LAG3 , and TOX ) was decreased in CD8 + Tex and Tpex cells following pomalidomide treatment. Furthermore, the expression of effector function-related genes ( GZMB , IFNG ) and memory-associated genes ( KLF2 , IL7R ) was transcriptionally upregulated across multiple T cell subsets, which likely contributed to sustained expansion and enhanced cytotoxicity (Fig.F). Subsequently, immune function phenotype scoring was performed on T cell subsets. Of these, the activation, cytotoxicity, inflammation − promoting, memory/stemness and type II IFN response signatures of T cells increased, whereas exhaustion-related signatures exhibited a significant downregulation (Fig. 5 G). To investigate the metabolic regulatory effects of pomalidomide on Tex, we performed KEGG pathway analysis focusing on metabolic pathways. Prolonged treatment with pomalidomide persistently suppressed glycolysis in Tex cells, while OXPHOS was initially inhibited at day 10 but exhibited significant upregulation at day 30 (Fig. 5 H), suggesting that pomalidomide maintenance therapy might reverse exhaustion-associated metabolic signatures through dynamic metabolic reprogramming. Additionly, immune scoring of NK cells revealed a significant increase in cytotoxicity and a decrease in exhaustion signatures following pomalidomide treatment (Fig. 5 K). Featureplot visualization results also showed an upregulation of cytotoxicity-related genes after pomalidomide treatment (Fig. 5 I). Furthermore, GSVA for GOBP gene set scoring indicated that pomalidomide treatment enhanced NK cell proliferation, cytotoxicity, chemotaxis, and activation while inhibited differentiation (Fig. 5 J). Additionally, Myeloid-derived suppressor cells (MDSCs) are known to play a critical role in the suppression of T cell antitumor responses [ 26 ] . We assessed MDSC characteristics in the monocyte/macrophage clusters, and the results showed that the MDSC signature score in these cells significantly decreased after pomalidomide treatment (Fig. 5 L-M), suggesting that pomalidomide may enhance T cell anti-tumor responses by inhibiting MDSCs. 4. Discussion Given the therapeutic limitations of CAR-T cells, which are primarily attributed to exhaustion and limited persistence [ 27 , 28 ] , identifying effective strategies to overcome these challenges remains a critical and ongoing focus in current research. Our previous studies demonstrated that the combination of CAR-T cells and pomalidomide enhanced anti-tumor efficacy, significantly prolonging OS and time to progression in R/R MM patients who received oral pomalidomide following CAR-T infusion [ 11 ] . In the current study, we confirmed that pomalidomide positively regulates the antitumor efficacy of CAR-T cells while reshaping the immune microenvironment, reflecting the multiple benefits of combining pomalidomide with CAR-T therapy in lymphoid malignancies. Previous studies have shown that pomalidomide promotes T cell activation and function by selectively ubiquitinating and degrading Ikaros and Aiolos, leading to increased IL-2 expression [ 29 – 31 ] , which consistent with our data that pomalidomide rapidly enhances CAR-T cell proliferation and upregulates IL-2 and IFN-γ production during early activation. As known, tumor cells could secrete various cytokines/chemokines (e.g., CXCL9-11) to recruit cytotoxic T/NK cells expressing corresponding receptors via ligand-receptor interactions [ 32 , 33 ] . In our in vivo model, the tumor tissues of combination therapy group exhibited elevated CXCL9, 10 and 11 levels, suggesting that that pomalidomide might remodel the tumor microenvironment to enhance CAR-T cell infiltration, which is a pivotal step for effective antitumor immunity. Moreover, upregulation of CXCL9 could further enhance the expression of IFN-gamma in tumor-infiltrated T cells, which amplifies the effector function of T cells [ 34 ] . T cell exhaustion, which is driven by persistent antigen exposure, is the major barrier to durable CAR-T responses [ 27 , 28 , 35 , 36 ] , while Tcm, characterized by self-renewal capacity, long-term survival, and lymphoid homing properties, represent a promising solution to this challenge [ 37 , 38 ] . Our results indicated that pomalidomide not only enhances CAR-T persistence through Tcm enrichment but also reverses T cells exhaustion programs, creating a self-sustaining immunological memory compartment critical for long-term antitumor control. In particular, energy metabolism of T cells could be reprogrammed according to the functional status, that T cells in the early effector phase are characterised by enhanced glycolysis to meet the energy demands of rapid proliferation and cytokine production [ 39 ] . Our results showed that pomalidomide-treated CAR-T cells exhibited early glycolytic activation, consistent with enhanced effector functions. However, CAR-T cells relying predominantly on glycolysis tend to differentiate into short-lived effector memory T cell (Tem) and exhibit accelerated exhaustion [ 40 – 42 ] , while CAR-T cells with OXPHOS-predominant metabolism are more likely to differentiate into Tcm [ 42 , 43 ] . This late-phase metabolic shift complements the early glycolytic burst, revealing a temporal metabolic strategy: initial glycolysis supports rapid effector function, while OXPHOS pathways enable long-term survival and memory formation [ 42 , 43 ] . Intriguingly, the metabolic shift triggered by pomalidomide, might balance rapid effector function with long-term survival and memory formation, thereby mitigating exhaustion. Our prior clinical trial demonstrated that long-term pomalidomide maintenance after CAR-T infusion reduced relapse rates and prolonged PFS/OS in RRMM patients [ 11 ] . However, the universal applicability of this combination remains unclear, as there is no standardized consensus to define the initiation timing, dosing regimens, and maintenance duration of pomalidomide administration following CAR-T infusion, particularly regarding the optimal scheduling and persistence of this therapeutic regimen. Pomalidomide exerts maximal early stimulatory effects on CAR-T activation, with scRNA-seq demonstrating significant T cell subset expansion within 10 days following pomalidomide administration, suggesting that initiating pomalidomide therapy might be particularly beneficial for patients with suboptimal early expansion or declining CAR-T copy numbers. Cytokine release syndrome (CRS), driven by hypersecretion of proinflammatory cytokines (e.g., IL-6, IL-1β, and IFN-γ) from activated T cells and myeloid cells, is the main cause of CAR-T therapeutic failure [ 44 ] . It cannot be discounted that pomalidomide further enhances the expression of these cytokines of CAR-T cells in our study, raising concerns about potential exacerbation of CRS in certain patient populations. Therefore, patients with high-risk factors for CRS should either avoid combination therapy with pomalidomide capsules or implement rigorous monitoring protocols (including serial measurement of IL-6 levels, ferritin). Furthermore, while pomalidomide is currently approved for the treatment of MM, our data reveal that its combination with CAR-T cells demonstrates comparable efficacy in lymphoma, suggesting broad applicability across hematologic malignancies. Further studies are needed to confirm its efficacy in multiple Hematologic Malignancies. Our study systematically investigated pomalidomide's effects on CAR-T cell function, phenotype, and immune microenvironment. While these findings highlight the therapeutic seting potential of pomalidomide-CAR-T combinations, critical limitations must be addressed. First, immunodeficient mouse xenograft models inherently fail to recapitulate tumor microenvironment complexity and tumor-host immune dynamics. Furthermore, given the insufficient CAR-T cell abundance in scRNA-seq samples, deeper analysis of pomalidomide’s effects on CAR-T cells is limited. The inclusion of larger, more diverse samples of patients treated with pomalidomide-CAR-T combination therapy would enhance the generalizability of findings, particularly regarding treatment response variability, safety profiles, and long-term outcomes. 5. Conclusion Our multi-level evidence establishes the scientific rationale for pomalidomide-CAR-T synergy in lymphoid malignancies, providing theoretical guidance for treatment scheduling, and lays foundation for broader hematologic malignancy applications. Abbreviations CAR-T, Chimeric Antigen Receptor T-cell; IL-2, interleukin-2; IFN-γ, interferon-γ; Tcm, central memory T cells; MDSC, myeloid-derived suppressor cell; IMiDs, immunomodulatory drug; PFS, prolongs progression-free survival; OS, overall survival; R/R MM, relapsed/refractory multiple myeloma; poma, pomalidomide; Tpex, progenitor exhausted T cell; Tem, effector memory T cell. Declarations Ethics approval All human peripheral blood samples were obtained with informed consent and ethical approval from the Third Xiangya Hospital Medical Ethics Committee (Ethics Approval Number: 24767). Animal experiments were conducted in compliance with the guidelines estab lished by Central South University Ethics Committee (Ethics Approval Number: CSU-2024-0042). CRediT authorship contribution statement: Yan Yu: Conceptualization, Data curation, Formal analysis, Methodology, Software, Visualization, Writing – original draft. Yi Zhou: Conceptualization, Data curation, Formal analysis, Methodology, Software, Validation, Writing – original draft. Linzhi Xie: Data curation, Formal analysis, Methodology. Liwen Wang: Formal analysis, Software, Writing – review and editing. Yuhan Yan: Formal analysis, Writing – review and editing. Qian Cheng: Writing – review and editing. Jing liu: Supervision. Xin Li: Funding acquisition, Resources, Supervision, Writing – review and editing. Declaration of Competing Interest: The authors declare that they have no competing interest with the contents of this article. Funding This project has been supported by the National Natural Science Foundation of China (grant no. 82170204 to Xin Li). Acknowledgments Not applicable. Data availability Data will be made available on request. References SHI M, LI L, WANG S, et al. Safety and efficacy of a humanized CD19 chimeric antigen receptor T cells for relapsed/refractory acute lymphoblastic leukemia[J]. Am J Hematol, 2022,97(6): 711-718. ROEX G, TIMMERS M, WOUTERS K, et al. Safety and clinical efficacy of BCMA CAR-T-cell therapy in multiple myeloma[J]. J Hematol Oncol, 2020,13(1): 164. MIRVIS E, BENJAMIN R. Are we there yet? CAR-T therapy in multiple myeloma[J]. Br J Haematol, 2024,205(6): 2175-2189. RUELLA M, KORELL F, PORAZZI P, et al. Mechanisms of resistance to chimeric antigen receptor-T cells in haematological malignancies[J]. Nat Rev Drug Discov, 2023,22(12): 976-995. 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COLLINS S M, ALEXANDER K A, LUNDH S, et al. TOX2 coordinates with TET2 to positively regulate central memory differentiation in human CAR T cells[J]. Sci Adv, 2023,9(29): eadh2605. LANITIS E, ROTA G, KOSTI P, et al. Optimized gene engineering of murine CAR-T cells reveals the beneficial effects of IL-15 coexpression[J]. J Exp Med, 2021,218(2). AKBARI B, HOSSEINI Z, SHAHABINEJAD P, et al. Metabolic and epigenetic orchestration of (CAR) T cell fate and function[J]. Cancer Lett, 2022,550: 215948. ROSTAMIAN H, FALLAH-MEHRJARDI K, KHAKPOOR-KOOSHEH M, et al. A metabolic switch to memory CAR T cells: Implications for cancer treatment[J]. Cancer Lett, 2021,500: 107-118. ZHANG M, JIN X, SUN R, et al. Optimization of metabolism to improve efficacy during CAR-T cell manufacturing[J]. J Transl Med, 2021,19(1): 499. RIAL S J, VÖLKL S, AIGNER M, et al. Role of CAR T Cell Metabolism for Therapeutic Efficacy[J]. Cancers (Basel), 2022,14(21). KAWALEKAR O U, O'CONNOR R S, FRAIETTA J A, et al. Distinct Signaling of Coreceptors Regulates Specific Metabolism Pathways and Impacts Memory Development in CAR T Cells[J]. Immunity, 2016,44(2): 380-390. FISCHER J W, BHATTARAI N. CAR-T Cell Therapy: Mechanism, Management, and Mitigation of Inflammatory Toxicities[J]. Front Immunol, 2021,12: 693016. Additional Declarations No competing interests reported. Supplementary Files SupplementaryTable.docx SFig1.eps Supplementary Fig.1 Pomalidomide Exerts CAR-T-Potentiating Effects via Activation-Dependent Synergy (A) Survival curves in myeloma cell line ARP-1 and RPMI 8226. (B) Target-specific cytotoxicity of CAR T-cells against tumor cells (top: BCMA CAR-T + BCMA-K562; bottom: CD19 CAR-T + CD19-K562) during 4-hour co-culture at different effector-to-target (E: T) ratios (1:1, 2:1, and 5:1) under real-time pomalidomide exposure or 24-hour pretreatment conditions. Data represent mean ± SEM from ≥3 independent experiments. ns: not significant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. SFig2.eps Supplementary Fig.2 Effects of Pomalidomide on CAR-T Cell activation and Memory Programming (A) Representative flow cytometry profiles (left) and subset frequencies (right) of CAR-T cell populations on days 2 post-pomalidomide treatment. (B) Representative flow cytometry profiles (left) and subset frequencies (right) of CD25+ CAR-T cell populations on days 2, 4, and 7 post-pomalidomide treatment. Data represent mean ± SEM from ≥3 independent experiments. ns: not significant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Cite Share Download PDF Status: Published Journal Publication published 18 Dec, 2025 Read the published version in Cancer Immunology, Immunotherapy → Version 1 posted Editorial decision: Revision requested 19 May, 2025 Reviews received at journal 16 May, 2025 Reviews received at journal 13 May, 2025 Reviews received at journal 12 May, 2025 Reviews received at journal 09 May, 2025 Reviews received at journal 07 May, 2025 Reviewers agreed at journal 06 May, 2025 Reviewers agreed at journal 06 May, 2025 Reviewers agreed at journal 06 May, 2025 Reviewers agreed at journal 05 May, 2025 Reviewers agreed at journal 05 May, 2025 Reviewers agreed at journal 05 May, 2025 Reviewers invited by journal 05 May, 2025 Editor assigned by journal 03 May, 2025 Submission checks completed at journal 03 May, 2025 First submitted to journal 01 May, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6571044","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":452884451,"identity":"f7c7974d-b28d-4d83-a1ba-d07de098bc25","order_by":0,"name":"Yan Yu","email":"","orcid":"","institution":"Third Xiangya Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"Yu","suffix":""},{"id":452884452,"identity":"c0c8e69f-732a-4234-b65c-79bf24db99ba","order_by":1,"name":"Yi Zhou","email":"","orcid":"","institution":"Third Xiangya Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yi","middleName":"","lastName":"Zhou","suffix":""},{"id":452884453,"identity":"227bb803-6c02-414d-812e-66351c7e3aee","order_by":2,"name":"Linzhi Xie","email":"","orcid":"","institution":"Third Xiangya Hospital","correspondingAuthor":false,"prefix":"","firstName":"Linzhi","middleName":"","lastName":"Xie","suffix":""},{"id":452884454,"identity":"cd5c0068-c47a-4f5a-a257-aa01e394cbef","order_by":3,"name":"Liwen Wang","email":"","orcid":"","institution":"Third Xiangya Hospital","correspondingAuthor":false,"prefix":"","firstName":"Liwen","middleName":"","lastName":"Wang","suffix":""},{"id":452884455,"identity":"904f3841-db39-4e32-b1db-efc0ee75a392","order_by":4,"name":"Yuhan Yan","email":"","orcid":"","institution":"Third Xiangya Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yuhan","middleName":"","lastName":"Yan","suffix":""},{"id":452884456,"identity":"56893fe5-7abe-4138-9ae1-61f1296c5f00","order_by":5,"name":"Qian Cheng","email":"","orcid":"","institution":"Third Xiangya Hospital","correspondingAuthor":false,"prefix":"","firstName":"Qian","middleName":"","lastName":"Cheng","suffix":""},{"id":452884457,"identity":"c56206d6-8710-4e48-9028-78b4fa0f60ea","order_by":6,"name":"Jing Liu","email":"","orcid":"","institution":"Third Xiangya Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Liu","suffix":""},{"id":452884458,"identity":"22c69286-bea1-4233-a84c-29004dcf8171","order_by":7,"name":"Xin Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2UlEQVRIie3PsQrCMBCA4ZNAXE7j2ILoK1SEToKvckVwUii4dOjQQeogOvcxHB1bApni7iT6BCIuTmIFN8XUzSHfckt+LgdgWX+oyQDycqLonk9HimJzwl9J200mzDtqVSF5zYGXT7h7mrMKSR17MowO6OU7FQUJB7FYkuFjnGSmZ+gW6/E+2LbB0buNIWG5bKSETQn+PtAcPGdqSmqJbNwJQYEfBimrkjy3JIQtjT5UTMpbUBG6GR85pBUabxFC968Y01A4rLjcorgjFqvvyRv87bllWZb10QOAmkLNu7+y5gAAAABJRU5ErkJggg==","orcid":"","institution":"Third Xiangya Hospital","correspondingAuthor":true,"prefix":"","firstName":"Xin","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2025-05-01 10:08:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6571044/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6571044/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00262-025-04247-1","type":"published","date":"2025-12-18T15:58:34+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":82259159,"identity":"51e85cbc-cbb5-445c-a070-334f059d8164","added_by":"auto","created_at":"2025-05-08 11:40:53","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":125999,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of pomalidomide on proliferation, cytotoxicity and effector molecules of CAR-T cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A\u003c/strong\u003e-\u003cstrong\u003eB)\u003c/strong\u003e Cell viability following pomalidomide treatment at various concentrations (0.01–100 μg/mL). (\u003cstrong\u003eA)\u003c/strong\u003eSurvival curves in myeloma cell line U266, leukemia cell lines BCMA-K562, lymphoma cell line Raji, and CD19-K562 (from left to right). (\u003cstrong\u003eB)\u003c/strong\u003eSurvival curves of CAR T-cells under resting conditions (left) or activated with anti-CD3/CD28 antibodies (right) over 48 hours of treatment.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eC)\u003c/strong\u003eTarget-specific cytotoxicity of CAR T-cells against tumor cells (left: BCMA CAR-T + U266; right: CD19 CAR-T + Raji) during 4-hour co-culture at different effector-to-target (E: T) ratios (1:1, 2:1, and 5:1) under real-time pomalidomide exposure or 24-hour pretreatment conditions.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eD)\u003c/strong\u003eRelative mRNA expression levels of \u003cem\u003eIL2\u003c/em\u003e, \u003cem\u003eIFNG\u003c/em\u003e, and \u003cem\u003eCXCL9/10/11\u003c/em\u003ein anti-CD3/CD28-activated CAR T-cells following 24-hour treatment with pomalidomide or DMSO control.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eE)\u003c/strong\u003eIFN-γ protein levels measured by ELISA in culture supernatants after 48 hours of co-culture.\u003c/p\u003e\n\u003cp\u003eData represent mean ± SEM from at least three independent experiments. ns = not significant; *p\u0026lt;0.05; **p\u0026lt;0.01; ***p\u0026lt;0.001; ****p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-6571044/v1/9a2b86aae2c41f500a56bb7e.png"},{"id":82259160,"identity":"52d07dcb-b6c1-473d-9cfc-41b44d4fe5be","added_by":"auto","created_at":"2025-05-08 11:40:53","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":107259,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePomalidomide Synergizes with CAR-T Cells to Amplify Anti-Tumor Efficacy in Myeloma Xenograft Models\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Experimental design: Female NOD-SCID mice were subcutaneously inoculated with U266 cells to establish myeloma xenograft models. Mice were randomized into four groups (n=5/group) for treatment when tumor volumes reached 100–150 mm³.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eB)\u003c/strong\u003e Representative images of mice under anesthesia at Day 28 post-tumor inoculation: CAR-T (top) vs. CAR-T + pom (bottom).\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eC)\u003c/strong\u003e Subcutaneous tumor growth curves.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eD) \u003c/strong\u003eSurvival analysis by Kaplan-Meier methodology.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eE)\u003c/strong\u003eBody weight trajectories during therapeutic intervention.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eF)\u003c/strong\u003e Relative mRNA expression levels of \u003cem\u003eIL2\u003c/em\u003e, \u003cem\u003eIFNG\u003c/em\u003e, and \u003cem\u003eCXCL9/10/11\u003c/em\u003e in subcutaneous tumor tissues.\u003c/p\u003e\n\u003cp\u003eData represent mean ± SEM. ns: not significant; *p\u0026lt;0.05; ***p\u0026lt;0.001; ****p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-6571044/v1/b145da47e2b660d8fd140bb2.png"},{"id":82259585,"identity":"a0397e44-5ba2-48c4-8044-e4995fd08843","added_by":"auto","created_at":"2025-05-08 11:48:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":157648,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of pomalidomide on CAR-T Cell Memory Programming and Subset distribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Experimental design: CAR-T cells were treated with pomalidomide or DMSO control under resting or anti-CD3/CD28 antibody-activated conditions. Medium was replenished every 48 hours. Memory phenotypes were assessed by flow cytometry on days 2, 4, and 7.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eB)\u003c/strong\u003e Representative flow cytometry profiles (top) and subset frequencies (bottom) of CAR-T cell populations on days 2, 4, and 7 post-pomalidomide treatment.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eC)\u003c/strong\u003e Flow cytometry characterization of CD4+ and CD8+ CAR-T cell subsets on day 2. Quantified CD4/CD8 ratios and CAR+ CD4/CD8 ratios (right).\u003c/p\u003e\n\u003cp\u003eData represent mean ± SEM from ≥3 independent experiments. ns: not significant; *p\u0026lt;0.05; **p\u0026lt;0.01; ***p\u0026lt;0.001; ****p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-6571044/v1/18cb49aa20f2834901106d65.png"},{"id":82259584,"identity":"e1735d9a-c1a6-45d2-952c-02c239d2e3a4","added_by":"auto","created_at":"2025-05-08 11:48:53","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":181640,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePomalidomide Enhances CAR-T Cell Immune Function and Mediates Metabolism at the Transcriptomic Level\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003ePCA-based dimensionality reduction of bulk RNA-seq data from CD3/CD28-stimulated CAR-T cells treated with pomalidomide (poma) versus CD3/CD28-stimulated CAR-T cells treated with DMSO (control).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B)\u003c/strong\u003e Heatmap depicting Z-score-normalized expression levels of genes associated with CAR-T cell function and phenotype.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C) \u003c/strong\u003eVolcano plot visualizing differentially expressed genes (DEGs) in pomalidomide treated CAR-T cells compared with DMSO. FDR \u0026lt; 0.05 and |logFC| ≥ 1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D) \u003c/strong\u003eKEGG pathway enrichment analysis of upregulated DEGs. P‐value \u0026lt; 0.05.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(E)\u003c/strong\u003e GO enrichment analysis of upregulated DEGs. P‐value \u0026lt; 0.05.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(F-H)\u003c/strong\u003e Gene set enrichment analysis (GSEA) was performed using \u003cstrong\u003e(F)\u003c/strong\u003e Hallmark pathways, \u003cstrong\u003e(G)\u003c/strong\u003e C5: GO categories, and \u003cstrong\u003e(H)\u003c/strong\u003e CD8+T cell memory-related gene signatures (GSE41867_MEMORY_VS_EXHAUSTED_CD8_TCELL_DAY30_LCMV_UP). Above gene sets were conducted through the MSigDB platform.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-6571044/v1/62f74115c76c3576fcb3fdc7.png"},{"id":82259167,"identity":"d459778f-b860-4661-b1ee-107b2b6e3bc3","added_by":"auto","created_at":"2025-05-08 11:40:53","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":205325,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePomalidomide treatment Remodels Peripheral Circulation Immune Microenvironment in a lymphoma patient\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eSingle-cell RNA-seq data from experimental groups (one day before treatment, day10 and day30 post- pomalidomide treatment) were visualized using UMAP.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B)\u003c/strong\u003e The Sankey diagram illustrates differential composition of PBMC immune subsets across three time points.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C) \u003c/strong\u003eA dot plot presents the expression levels of signature genes in distinct T cell clusters identified through single-cell RNA-seq analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D-E) \u003c/strong\u003eUMAP visualization of T cells across all samples\u003cstrong\u003e(D)\u003c/strong\u003e and Sankey diagram revealing temporal changes in T cell subsets across three time points\u003cstrong\u003e(E)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(F)\u003c/strong\u003e Line plots depict dynamic expression patterns of T cell effector (\u003cem\u003eGZMB, IFNG\u003c/em\u003e), exhaustion (\u003cem\u003ePDCD1, LAG3, TOX\u003c/em\u003e), and memory/stemness (\u003cem\u003eIL7R, KLF2\u003c/em\u003e) genes across T cell subsets and time points.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(G)\u003c/strong\u003e Violin plots illustrate the differences in activation, cytotoxicity, memory/stemness, exhaustion, inflammatory response, and type II interferon response scores (calculated using AddModuleScore) among T cell subsets across groups.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(H)\u003c/strong\u003e A dot plot depicts KEGG metabolic pathway enrichment scores computed via ssGSEA in CD8+ T exhausted (Tex) cell subsets.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(I) \u003c/strong\u003eA feature plot visualizes the distribution of NK cell effector function-related genes (\u003cem\u003eGZMB, GNLY, IFNG, CCL5\u003c/em\u003e) across distinct time points.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(J)\u003c/strong\u003e A heatmap displays gene set variation analysis (GSVA) scores for NK cell subgroups using predefined GO gene sets.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(K)\u003c/strong\u003e Violin plots illustrate the differences in cytotoxicity and exhaustion scores (calculated via AddModuleScore) among NK cell subsets across groups.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(L-M)\u003c/strong\u003e Violin plots \u003cstrong\u003e(L)\u003c/strong\u003e and Feature plot \u003cstrong\u003e(M)\u003c/strong\u003e show the differences in MDSC-signature scores (calculated via AddModuleScore) between monocyte/macrophage subgroups across groups.\u003c/p\u003e\n\u003cp\u003ens: not significant; *p\u0026lt;0.05; ***p\u0026lt;0.001; ****p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-6571044/v1/f5f647f1b15056a52d2d94c7.png"},{"id":98814034,"identity":"75431b3a-938c-4654-bcf6-675277fbc5c7","added_by":"auto","created_at":"2025-12-22 16:09:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1988354,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6571044/v1/951f4a94-bcad-4e48-9e76-c7f228e622f4.pdf"},{"id":82259161,"identity":"4785d3e3-381f-4ccc-a35c-5d40be04bfa6","added_by":"auto","created_at":"2025-05-08 11:40:53","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":19908,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable.docx","url":"https://assets-eu.researchsquare.com/files/rs-6571044/v1/328cfc84b1200fb4b7405621.docx"},{"id":82259162,"identity":"8210dec0-1256-4b3e-b059-35776c2681d3","added_by":"auto","created_at":"2025-05-08 11:40:53","extension":"eps","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2242170,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Fig.1\u003c/strong\u003e \u003cstrong\u003ePomalidomide Exerts CAR-T-Potentiating Effects via Activation-Dependent Synergy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Survival curves in myeloma cell line ARP-1 and RPMI 8226.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B)\u003c/strong\u003e Target-specific cytotoxicity of CAR T-cells against tumor cells (top: BCMA CAR-T + BCMA-K562; bottom: CD19 CAR-T + CD19-K562) during 4-hour co-culture at different effector-to-target (E: T) ratios (1:1, 2:1, and 5:1) under real-time pomalidomide exposure or 24-hour pretreatment conditions.\u003c/p\u003e\n\u003cp\u003eData represent mean ± SEM from ≥3 independent experiments. ns: not significant; *p\u0026lt;0.05; **p\u0026lt;0.01; ***p\u0026lt;0.001; ****p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"SFig1.eps","url":"https://assets-eu.researchsquare.com/files/rs-6571044/v1/7d62470a669acc934cedb7aa.eps"},{"id":82259173,"identity":"163da694-8c9c-46ee-86ef-46b24ea4cf9b","added_by":"auto","created_at":"2025-05-08 11:40:54","extension":"eps","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":3838802,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Fig.2 Effects of Pomalidomide on CAR-T Cell activation and Memory Programming\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Representative flow cytometry profiles (left) and subset frequencies (right) of CAR-T cell populations on days 2 post-pomalidomide treatment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B) \u003c/strong\u003eRepresentative flow cytometry profiles (left) and subset frequencies (right) of CD25+ CAR-T cell populations on days 2, 4, and 7 post-pomalidomide treatment.\u003c/p\u003e\n\u003cp\u003eData represent mean ± SEM from ≥3 independent experiments. ns: not significant; *p\u0026lt;0.05; **p\u0026lt;0.01; ***p\u0026lt;0.001; ****p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"SFig2.eps","url":"https://assets-eu.researchsquare.com/files/rs-6571044/v1/9152d9e94e5971ab3c2242aa.eps"}],"financialInterests":"No competing interests reported.","formattedTitle":"Pomalidomide enhances CAR-T cell therapeutic efficacy and Remodels Immune Microenvironment in Lymphoid Malignancies","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eChimeric Antigen Receptor T-cell (CAR-T) therapy has emerged as a revolutionary approach in the treatment of hematological malignancies, particularly demonstrating remarkable efficacy in lymphoid malignancies\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. However, while CAR-T therapy achieves high initial remission rates, its long-term efficacy remains suboptimal, with many patients experiencing disease relapse or rapid progression shortly after treatment\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. This underscores the critical need to enhance CAR-T cell persistence and anti-tumor activity through innovative combination strategies\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e, which has become a key focus in current research.\u003c/p\u003e \u003cp\u003ePomalidomide, a third-generation immunomodulatory drug (IMiDs), has been widely utilized in treating multiple myeloma and lymphomas, particularly demonstrating significant anti-tumor efficacy in lenalidomide-resistant patients\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. In addition to directly inducing tumor cell apoptosis, pomalidomide also modulates the immune microenvironment to enhance T-cell functionality\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. Emerging evidence suggests potential synergistic effects between pomalidomide and CAR-T therapy\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. Our preliminary study\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e revealed that pomalidomide significantly prolongs progression-free survival (PFS) and overall survival (OS) in relapsed/refractory multiple myeloma (R/R MM) patients. Nevertheless, the precise mechanisms underlying this synergy remain incompletely understood.\u003c/p\u003e \u003cp\u003eThis study aims to systematically investigate the synergistic mechanisms of pomalidomide in CAR-T therapy, focusing on its dual effects on both CAR-T cells and tumor microenvironment modulation. We anticipate that our findings will contribute to the development of clinical protocols, particularly regarding the optimal timing and duration of pomalidomide administration during CAR-T therapy, thereby improving treatment outcomes and offering new strategies for managing refractory hematological malignancies.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1. Cell culture\u003c/h2\u003e\n \u003cp\u003eHuman myeloma cell lines ARP-1 (kindly provided by Prof. Wen Zhou, Central South University), RPMI 8226, U266 and lymphoma cell line Raji (provided by Prof. Minghong Jiang, Peking Union Medical College), and BCMA/CD19-expressing K562 cells (Yucadi, China) were maintained in RPMI-1640 medium (Gibco, USA) supplemented with 10% FBS (Clark Bioscience, USA) and 1% penicillin/streptomycin (Gibco, USA). BCMA CAR-T and CD19 CAR-T cells (Yucadi, China) were cultured in RPMI-1640 containing 10% FBS, 1% penicillin/streptomycin, 300 IU /mL IL-2 (Abmole, USA)\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e, and 50 \u0026micro;M \u0026beta;-mercaptoethanol (Gibco, USA)\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. Freshly thawed CAR-T cells received additional IL-7 and IL-15 (10 ng/mL each, Abmole, USA)\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. All cells were maintained at 37\u0026deg;C with 5% CO₂ with medium replenishment every 48 h.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e2.2. Reagent\u003c/h2\u003e\n \u003cp\u003ePomalidomide (Selleck Chemicals, USA) was dissolved in DMSO (Sigma, USA) (2 mg/mL) and stored at -80\u0026deg;C.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003e2.3. PBMCs isolation\u003c/h2\u003e\n \u003cp\u003ePeripheral blood mononuclear cells (PBMCs) were isolated from whole blood using Ficoll-Paque PLUS (1.077 g/mL; Solarbio, China) density gradient centrifugation. After centrifugation (1,000 \u0026times;g, 20 min, RT), the PBMC layer was collected, washed twice with PBS, and treated with 1\u0026times; RBC lysis buffer (4\u0026ndash;5 min, RT) if erythrocyte contamination persisted.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003e2.4. Proliferation Assays\u003c/h2\u003e\n \u003cp\u003eWorking concentrations were prepared in RPMI-1640 before use. Cells were seeded in 96-well U-bottom plates: tumor cells (5\u0026times;10⁴/well) or CAR-T (2\u0026times;10⁶/well) with pomalidomide (0.01\u0026ndash;100 \u0026micro;g/mL) or DMSO control. After 48 h culture, cells were washed and incubated with 10% CCK-8 reagent (Abbkine, USA) for 3 h (tumor cells) or 6 h (CAR-T). When required, CAR-T cells were activated with 5 \u0026micro;g/mL anti-human CD3 monoclonal antibody (BioLegend, USA) and anti-human CD28 monoclonal antibody (BioLegend, USA) at the beginning of the experiment. Absorbance was measured at 450 nm. Viability was calculated as: [(ODSample \u0026ndash; ODBlank)/ (ODControl \u0026ndash; ODBlank)] \u0026times; 100%.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003e2.5. Cytotoxicity Assay\u003c/h2\u003e\n \u003cp\u003eCAR-T cells (pre-treated with 1\u0026ndash;5 \u0026micro;g/mL pomalidomide or DMSO control for 24 h or not) were co-cultured with target cells at E:T ratios of 1:1 to 5:1 for 4 h. Cytotoxicity was assessed using LDH Release Assay Kit (Beyotime, China) following manufacturer\u0026apos;s protocol. Absorbance was read at 490 nm with 600 nm reference. Cytotoxicity (%) = [(ODTreated \u0026ndash; ODSample Control)/ (ODMax \u0026ndash; ODSample Control)] \u0026times; 100%.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003e2.6. Enzyme-Linked Immunosorbent Assay\u003c/h2\u003e\n \u003cp\u003eSupernatants from 24 h CAR-T/tumor co-cultures (E:T ratios 1:1\u0026ndash;5:1) were analyzed using a Human IFN-\u0026gamma; ELISA Kit (ZCIBIO, China) following manufacturer protocols.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003e2.7. RNA Isolation and qPCR Analysis\u003c/h2\u003e\n \u003cp\u003eTotal RNA was extracted from CAR-T cells or tumor tissues using the RNA rapid extraction kit (RNAfast200, Fastagen, China). cDNA synthesis and qPCR were performed with HiScript III RT SuperMix (Vazyme, China) and Taq Pro Universal SYBR Master Mix (Vazyme, China). Primer sequences for target genes (\u003cem\u003eIL2, IFNG, CXCL9/10/11\u003c/em\u003e) are listed in Supplementary Table\u0026nbsp;1. Relative mRNA expression was normalized to \u0026beta;-actin and calculated via the 2\u003csup\u003e\u0026minus;\u0026Delta;\u0026Delta;Ct\u003c/sup\u003e method.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003e2.8. Flow Cytometry\u003c/h2\u003e\n \u003cp\u003eT-cell memory phenotype: CAR-T cells (3\u0026times;10⁶/well) were treated with 2.5 \u0026micro;g/mL pomalidomide or DMSO control for 7 days. When required, CAR-T cells were activated with 5 \u0026micro;g/mL anti-human CD3 monoclonal antibody and anti-human CD28 monoclonal antibody at the beginning of the experiment. CAR-T cells were collected and stained with anti-CD3-FITC (BioLegend, USA), anti-CD45RA-PE (eBioscience, USA), anti-CD127-PerCP-Cy5.5 (BioLegend, USA), anti-CCR7-APC (eBioscience, USA), anti-CD8-APC-Alexa Fluor 750 (BioLegend, USA), anti-CD4-Pacific Blue (BioLegend, USA), and anti-CD25-PE-Cy7(BioLegend, USA) antibodies for 15 minutes at RT\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. CAR\u0026thinsp;+\u0026thinsp;T cells CD4+/CD8\u0026thinsp;+\u0026thinsp;ratio: CD19 CAR-T cells activated with anti-CD3/CD28 antibodies (5 \u0026micro;g/mL) were treated with 2.5 \u0026micro;g/mL pomalidomide for 48 h. Cells were collected and stained with anti-FMC63-FITC (for CD19 CAR detection, Acro, USA) antibody in the dark at 4℃ for 60 minutes, and stained with anti-CD3-APC (BioLegend, USA), anti-CD8-APC-Alexa Fluor 750, anti-CD4-Pacific Blue antibodies for 15 minutes at RT. Data were acquired on Gallios-Analyzer (Beckman Coulter, USA) and analyzed by using FlowJo v10.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e2.9. Multiple myeloma cell xenograft murine model\u003c/h2\u003e\n \u003cp\u003eFemale NOD-SCID mice\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e (5\u0026ndash;6 weeks, Hunan SJA Laboratory Animal Co, China) were subcutaneously inoculated\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e with U266 cells (1\u0026times;10⁷ cells/mouse in Matrigel) (Corning, USA). When tumors reached 100\u0026ndash;150 mm\u0026sup3;, mice were randomized into four groups (n\u0026thinsp;=\u0026thinsp;5/group):\u003c/p\u003e\n\u003c/div\u003e\n\u003cp\u003e1)Control: PBS (i.v., 150\u0026micro;L)\u0026thinsp;+\u0026thinsp;1% DMSO (i.p., 5 times/week)\u003c/p\u003e\n\u003cp\u003e2) Poma monotherapy: PBS (i.v., 150\u0026micro;L)\u0026thinsp;+\u0026thinsp;2.5 mg/kg pomalidomide (i.p., 5 times/week)\u003c/p\u003e\n\u003cp\u003e3) CAR-T monotherapy: 1\u0026times;10⁷ CAR-T cells (i.v., 150\u0026micro;L)\u0026thinsp;+\u0026thinsp;1% DMSO (i.p., 5 times/week)\u003c/p\u003e\n\u003cp\u003e4) Combination therapy: 1\u0026times;10⁷ CAR-T cells (i.v., 150\u0026micro;L)\u0026thinsp;+\u0026thinsp;2.5 mg/kg pomalidomide (i.p., 5 times/week)\u003c/p\u003e\n\u003cp\u003eTumor volume (V\u0026thinsp;=\u0026thinsp;0.5 \u0026times; L \u0026times; W\u003csup\u003e2\u003c/sup\u003e) \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e and body weight were monitored every 2 days. The experimental endpoint was defined as the time when the subcutaneous tumor diameter reached\u0026thinsp;\u0026ge;\u0026thinsp;19 mm in any single dimension. Mice meeting this criterion were humanely euthanized. Tumors were weighed, snap-frozen in RNAlater (servicebio, China), and stored at \u0026minus;\u0026thinsp;80\u0026deg;C for molecular analysis.\u003c/p\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003e2.10. Bulk RNA-sequencing and data analysis\u003c/h2\u003e\n \u003cp\u003eBCMA CAR-T cells derived from three donors were activated with anti-CD3/CD28 antibodies (5 \u0026micro;g/mL) and treated with pomalidomide (2.5 \u0026micro;g/mL) for 24 hours. Total RNA was extracted from 1 \u0026times; 10⁶ CAR-T cells using RNAfast200 (Fastagen, China). Subsequent bulk RNA sequencing was performed at BGI Tech Solutions Co. (Shenzhen, Guangdong, China). Transcriptome sequencing was conducted on the DNBSEQ platform with paired-end (PE150) cycles. Raw FASTQ files were trimmed and filtered using Fastp (version 0.19.5), and subsequently aligned to the human reference genome (GRCh38) with Bowtie2 (version 2.4.1). Reads were counted with FeatureCounts (version 2.0.6). Differentially expressed genes (DEGs) were identified using the DESeq2 (version 1.42.0) package in R, with genes defined as differentially expressed if the log2-fold change was \u0026gt;\u0026thinsp;1 or \u0026lt; -1, and the adjusted P value was \u0026lt;\u0026thinsp;0.05. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses on DEGs were performed using the clusterProfiler package. Gene Set Enrichment Analysis (GSEA) of the Hallmark, GO: BP and IMMUNESIGDB (C7) gene sets was conducted using the \u0026ldquo;enrichr\u0026rdquo;, \u0026ldquo;GseaVis\u0026rdquo; and \u0026ldquo;GSEABase\u0026rdquo; R packages.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003e2.11. Single-cell RNA sequencing and data analysis\u003c/h2\u003e\n \u003cp\u003eTo investigate the impact of pomalidomide on the tumor immune microenvironment, single-cell sequencing analysis was performed on a lymphoma patient who had undergone CD19 CAR-T infusion. Peripheral blood samples were collected at three time points: 1 day prior to pomalidomide treatment, and 10 and 30 days after the start of pomalidomide treatment. PBMCs were isolated from the collected blood samples using Ficoll gradient separation, and subsequently subjected to single-cell sequencing. Single-cell libraries were prepared using the DNBSEQ platform and the DNBelab C4 Single-Cell Library Prep Set, with paired-end sequencing performed on the DIPSEQ T1 platform. Raw FASTQ files underwent quality control, read alignment, and feature quantification using the DNBC4Tools pipeline (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/MGI-tech-bioinformatics/DNBelab_C_Series_HT_scRNA-analysis-software\u003c/span\u003e\u003c/span\u003e). Cell barcodes, gene names, and feature matrices were standardized across datasets to ensure uniform annotation. Initial quality control metrics were calculated for each dataset, and datasets were merged using the merge function in Seurat after filtering. Principal component analysis (PCA) was performed on the top 2000 variable genes in the merged dataset for dimensionality reduction. Batch correction and dataset integration were performed using Harmony (version 1.2.1). Clustering was performed using the Louvain algorithm based on shared nearest neighbors (SNN), and cell clusters were visualized using uniform manifold approximation and projection (UMAP). Clusters were annotated based on known marker genes and enriched pathways, and cluster-specific marker genes were identified using the FindMarkers function. For immune signature scoring, the ssGSEA were used to evaluate the function of NK cells, and AddModuleScore were used to evaluate the function of T cells\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e and the MDSC signature of monocytes/macrophages\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. Pseudobulk analysis was performed by aggregating NK cell single-cell RNA-seq data. Following this, the GOBP enrichment score was calculated by GSVA analysis to assess the biological functional states of the NK cells.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003e2.12. Statistical Analysis\u003c/h2\u003e\n \u003cp\u003eAll data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM) from three independent experiments. Statistical significance was determined using GraphPad Prism 9.5.1 (GraphPad Software, USA). Comparisons between two groups were analyzed by unpaired Student\u0026rsquo;s t-test. For multiple group comparisons, one-way ANOVA followed by Tukey\u0026rsquo;s post hoc test was applied. Survival curves in animal studies were generated using the Kaplan-Meier method, and differences were assessed by log-rank (Mantel-Cox) test. A p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Pomalidomide promotes proliferation via Activation-Dependent Synergy and enhances Cytotoxicity and Cytokines Production of CAR-T Cells\u003c/h2\u003e \u003cp\u003eTo establish a pharmacologically relevant concentration range, we first systematically evaluated pomalidomide's dual effects on tumor and CAR-T cells. While myeloma cell lines (U266, RPMI 8226, ARP-1), lymphoma cell line (Raji) and K562 controls showed dose-dependent growth suppression above 10 \u0026micro;g/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, Supplementary Fig.\u0026nbsp;1A), resting CAR-T cells maintained\u0026thinsp;\u0026gt;\u0026thinsp;95% viability at \u0026le;\u0026thinsp;5 \u0026micro;g/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Strikingly, upon CD3/CD28-mediated activation, subtoxic pomalidomide concentrations (2.5-5 \u0026micro;g/mL) induced a proliferation boost compared to activation alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). This activation-dependent synergy suggested pomalidomide amplifies costimulatory signaling cascades rather than exerting generic proliferative effects. Guided by these dose-response relationships, we established 1\u0026ndash;5 \u0026micro;g/mL as the operative concentration range for subsequent experiments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn tumor co-culture models, real-time pomalidomide exposure showed no immediate cytotoxicity enhancement (LDH release at three effector-to-target (E: T) ratios). However, 24-hour CAR-T pretreatment with pomalidomide induced profound functional augmentation, elevating specific lysis from 8.27\u0026ndash;10.61% at E: T\u0026thinsp;=\u0026thinsp;1:1 and 18.36\u0026ndash;23.12% at E: T\u0026thinsp;=\u0026thinsp;5:1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, Supplementary Fig.\u0026nbsp;1B). This pretreatment efficacy gradient implies pomalidomide requires sufficient exposure time to prime CAR-T effector machinery.\u003c/p\u003e \u003cp\u003eTo further elucidate mechanisms underlying the synergistic antitumor effects of pomalidomide and CAR-T cells, we analyzed the expression of cytokines and chemokines in CAR-T cells activated via anti-CD3/CD28 antibodies or tumor co-culture. 24-hour CAR-T pretreatment with pomalidomide significantly upregulated mRNA levels of \u003cem\u003eIL2\u003c/em\u003e, \u003cem\u003eIFNG\u003c/em\u003e, \u003cem\u003eCXCL9\u003c/em\u003e, \u003cem\u003eCXCL10\u003c/em\u003e, and \u003cem\u003eCXCL11\u003c/em\u003e compared to DMSO controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Correspondingly, IFN-γ protein levels in co-culture supernatants showed marked elevation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). These findings suggest that pomalidomide may exert its synergistic antitumor effects by regulating the transcriptome and protein expression of CAR-T cells, promoting the secretion of key cytokines, enhancing CAR-T cell activation, proliferation, and immune functions, and modulating the recruitment and infiltration of immune cells in the tumor microenvironment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Pomalidomide Synergizes with CAR-T Cells to Amplify Anti-tumor Efficacy in Myeloma Xenograft Models\u003c/h2\u003e \u003cp\u003eThe translational potential of this combinatorial approach was rigorously validated in U266 myeloma xenografts (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Longitudinal tracking revealed that pomalidomide pretreatment combined with CAR-T infusion induced profound tumor regression (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-C). This therapeutic synergy translated into a marked survival benefit, with median overall survival increasing from 30 days in the CAR-T-only group to an unachieved endpoint in the combination group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), while pomalidomide monotherapy showed no discernible benefit versus untreated controls (p\u0026thinsp;=\u0026thinsp;0.39; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMolecular dissection of residual tumors unveiled that pomalidomide augmented intra-tumoral \u003cem\u003eIL2\u003c/em\u003e (4.04-fold) and \u003cem\u003eIFNG\u003c/em\u003e (4.78-fold) expression (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 vs CAR-T alone), concurrently inducing a chemokine storm characterized by 19.73 to 26.11-fold upregulation of \u003cem\u003eCXCL9-11\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). Importantly, the absence of significant body weight fluctuations (\u0026lt;\u0026thinsp;5% variation across groups, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE) and preserved vital signs confirmed the regimen's safety profile. These findings suggest that pomalidomide potentiates CAR-T cell functionality by enhancing cytokine production and promoting immune cell infiltration and activation within the tumor microenvironment, thereby amplifying anti-tumor efficacy.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Pomalidomide Orchestrates CAR-T Cell Memory Programming and Subset Readjusting\u003c/h2\u003e \u003cp\u003eTo investigate the effects of pomalidomide on CAR-T cell memory phenotypes and immune subset dynamics, CAR-T cells were cultured under resting or anti-CD3/CD28 antibody-activated conditions in the presence of pomalidomide for 7 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Following activation, CAR-T cells exhibited a significant increase in CD25 expression (early activation marker of T cell) at day 2, which subsequently declined over time (Supplementary Fig.\u0026nbsp;2B). Notably, the pomalidomide treatment maintained elevated CD25 levels throughout the observation period (Supplementary Fig.\u0026nbsp;2B), suggesting sustained CAR-T activation and functional persistence. Under resting conditions, pomalidomide significantly increased the proportion of central memory T cells (Tcm, CCR7\u0026thinsp;+\u0026thinsp;CD45RA\u0026minus;) in CD8\u0026thinsp;+\u0026thinsp;CAR-T cells from 19.60\u0026ndash;26.45%, while showing no significant effects on other phenotypes or CD4\u0026thinsp;+\u0026thinsp;CAR-T cells (Supplementary Fig.\u0026nbsp;2A). In anti-CD3/CD28-activated cultures, prolonged pomalidomide exposure markedly reduced CD8\u0026thinsp;+\u0026thinsp;na\u0026iuml;ve T cells (CCR7\u0026thinsp;+\u0026thinsp;CD45RA+) and increased both CD4\u0026thinsp;+\u0026thinsp;and CD8\u0026thinsp;+\u0026thinsp;Tcm populations compared to DMSO controls. Tcm frequencies rose from baseline levels of 11.4% (CD4+) and 11.6% (CD8+) to 34.8% and 50.65%, respectively, representing 1.42-fold and 1.34-fold increases over the DMSO control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eGiven reports linking CAR-T cell CD4/CD8 ratios to clinical prognosis, we evaluated pomalidomide's impact on this parameter. Due to lentiviral transduction bias favoring CD4\u0026thinsp;+\u0026thinsp;T cells\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e, CAR\u0026thinsp;+\u0026thinsp;CD4\u0026thinsp;+\u0026thinsp;T cells predominated in CAR-T products, yielding a resting CD4/CD8 ratio of 11.92. Activation reduced this ratio to 4.378. Notably, pomalidomide significantly increased the CD4/CD8 ratio under both resting and activated conditions, both in total and CAR\u0026thinsp;+\u0026thinsp;T cell populations (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Pomalidomide Enhances CAR-T Cell Immune Function and Regulates Metabolism at the Transcriptomic Level\u003c/h2\u003e \u003cp\u003eTo investigate the transcriptional mechanisms underlying the effects of pomalidomide on CAR-T cells, we first analyzed bulk RNA-seq data from BCMA CAR-T cells treated with either DMSO control or pomalidomide for 24h after CD3/CD28 antibody stimulation \u003cem\u003ein vitro\u003c/em\u003e. The PCA results revealed a marked transcriptional divergence between pomalidomide treated CAR-T cells and the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Consistent with the phenotypic plasticity observed in memory subsets, pomalidomide treatment upregulated expression of T cell memory-associated genes including \u003cem\u003eIL7R\u003c/em\u003e, \u003cem\u003eCCR7\u003c/em\u003e, \u003cem\u003eRORC\u003c/em\u003e, \u003cem\u003eLEF1\u003c/em\u003e, \u003cem\u003eBACH2\u003c/em\u003e and \u003cem\u003eBCL6\u003c/em\u003e. Concurrently, elevated expression levels of genes involved in T cell activation (\u003cem\u003eIL2RA\u003c/em\u003e, \u003cem\u003eIT\u003c/em\u003eK, \u003cem\u003eZAP70\u003c/em\u003e) and cytokine/chemokine (\u003cem\u003eIL2\u003c/em\u003e, \u003cem\u003eTNF\u003c/em\u003e, \u003cem\u003eCXCL9\u003c/em\u003e, \u003cem\u003eCCL2\u003c/em\u003e, \u003cem\u003eCCL4\u003c/em\u003e, \u003cem\u003eCCL20\u003c/em\u003e) were observed in pomalidomide treated CAR-T cells. Particularly, the expression of exhaustion and immunosuppressive-related markers (\u003cem\u003ePDCD1\u003c/em\u003e, \u003cem\u003eLAG3\u003c/em\u003e, \u003cem\u003eTIGIT\u003c/em\u003e, \u003cem\u003eTOX\u003c/em\u003e, \u003cem\u003eCTLA4\u003c/em\u003e, \u003cem\u003eFOXP3\u003c/em\u003e) was reduced in pomalidomide-treated CAR-T cells compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), suggesting transcriptional reprogramming towards a memory and effector competent state.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSubsequent differential expression analysis identified 226 significantly upregulated and 23 significantly downregulated genes in the treated samples compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). KEGG analysis of upregulated genes revealed significant enrichment in metabolic pathways including fatty acid degradation, glycolysis, cGMP-PKG signaling, and tyrosine metabolism (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD), while GO analysis highlighted enrichment in immune-related pathways such as T cell activation, inflammatory response, cell adhesion, and ERK1/2 signaling (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003eGSEA analysis revealed that pomalidomide treatment induced transcriptional activation of multiple immune-related pathways in CAR-T cells. Specifically, cytokine related pathways including IFN-γ, IL-6, IL-2, and TNF-α signaling, along with inflammatory response pathways, were significantly enriched (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). Concurrently, GSEA identified upregulation of T cell activation, proliferation, migration, immune response functions, and type II interferon responses in pomalidomide-treated CAR-T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). Importantly, the prominent activation of memory T cell signatures in pomalidomide treated CAR-T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH), aligning with our prior observations of memory phenotype induction \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Pomalidomide Improves Anti-Tumor Immunity Through Remodeling the Tumor Immune Microenvironment\u003c/h2\u003e \u003cp\u003eTo investigate the impact of pomalidomide on the tumor immune microenvironment, PBMCs were collected from a lymphoma patient at three time points: 1 day before pomalidomide treatment, 10 days and 30 days after pomalidomide treatment, and subsequently subjected to scRNA-seq analysis. The PBMCs were primarily classified into CD8\u0026thinsp;+\u0026thinsp;T cells, monocytes/macrophages, NK cells, CD4\u0026thinsp;+\u0026thinsp;T cells, pDCs and platelets (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-B). Notably, as pomalidomide treatment progressed, the proportion of T cell and NK cell subpopulations gradually increased, while the proportion of monocyte/macrophage subpopulations decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further evaluate the impact of pomalidomide on T cells, secondary clustering analysis was performed, which identified six distinct clusters based on specific markers: CD8\u0026thinsp;+\u0026thinsp;effector T cells (CD8\u0026thinsp;+\u0026thinsp;effector), CD4\u0026thinsp;+\u0026thinsp;na\u0026iuml;ve and central memory T cells (CD4\u0026thinsp;+\u0026thinsp;Tn/Tcm), CD8\u0026thinsp;+\u0026thinsp;exhausted T cells (Tex), CD8\u0026thinsp;+\u0026thinsp;progenitor exhausted T cell (Tpex), CD4\u0026thinsp;+\u0026thinsp;regulatory Tcells (Treg), and a cluster characterized by high expression of proliferation-related genes (Tprolif) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC-D). The results revealed that with continuous pomalidomide treatment, the proportion of CD8\u0026thinsp;+\u0026thinsp;effector, CD4\u0026thinsp;+\u0026thinsp;Tn/Tcm increased, whereas the proportion of CD8\u0026thinsp;+\u0026thinsp;Tex and Tpex cells decreased (Fig.E). The average gene expression of different T cell subpopulations was further evaluated. As expected, the expression of exhaustion-related genes (\u003cem\u003ePDCD1\u003c/em\u003e, \u003cem\u003eLAG3\u003c/em\u003e, and \u003cem\u003eTOX\u003c/em\u003e) was decreased in CD8\u0026thinsp;+\u0026thinsp;Tex and Tpex cells following pomalidomide treatment. Furthermore, the expression of effector function-related genes (\u003cem\u003eGZMB\u003c/em\u003e, \u003cem\u003eIFNG\u003c/em\u003e) and memory-associated genes (\u003cem\u003eKLF2\u003c/em\u003e, \u003cem\u003eIL7R\u003c/em\u003e) was transcriptionally upregulated across multiple T cell subsets, which likely contributed to sustained expansion and enhanced cytotoxicity (Fig.F). Subsequently, immune function phenotype scoring was performed on T cell subsets. Of these, the activation, cytotoxicity, inflammation\u0026thinsp;\u0026minus;\u0026thinsp;promoting, memory/stemness and type II IFN response signatures of T cells increased, whereas exhaustion-related signatures exhibited a significant downregulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). To investigate the metabolic regulatory effects of pomalidomide on Tex, we performed KEGG pathway analysis focusing on metabolic pathways. Prolonged treatment with pomalidomide persistently suppressed glycolysis in Tex cells, while OXPHOS was initially inhibited at day 10 but exhibited significant upregulation at day 30 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH), suggesting that pomalidomide maintenance therapy might reverse exhaustion-associated metabolic signatures through dynamic metabolic reprogramming.\u003c/p\u003e \u003cp\u003eAdditionly, immune scoring of NK cells revealed a significant increase in cytotoxicity and a decrease in exhaustion signatures following pomalidomide treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eK). Featureplot visualization results also showed an upregulation of cytotoxicity-related genes after pomalidomide treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI). Furthermore, GSVA for GOBP gene set scoring indicated that pomalidomide treatment enhanced NK cell proliferation, cytotoxicity, chemotaxis, and activation while inhibited differentiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eJ). Additionally, Myeloid-derived suppressor cells (MDSCs) are known to play a critical role in the suppression of T cell antitumor responses\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. We assessed MDSC characteristics in the monocyte/macrophage clusters, and the results showed that the MDSC signature score in these cells significantly decreased after pomalidomide treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eL-M), suggesting that pomalidomide may enhance T cell anti-tumor responses by inhibiting MDSCs.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eGiven the therapeutic limitations of CAR-T cells, which are primarily attributed to exhaustion and limited persistence\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e, identifying effective strategies to overcome these challenges remains a critical and ongoing focus in current research. Our previous studies demonstrated that the combination of CAR-T cells and pomalidomide enhanced anti-tumor efficacy, significantly prolonging OS and time to progression in R/R MM patients who received oral pomalidomide following CAR-T infusion\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. In the current study, we confirmed that pomalidomide positively regulates the antitumor efficacy of CAR-T cells while reshaping the immune microenvironment, reflecting the multiple benefits of combining pomalidomide with CAR-T therapy in lymphoid malignancies.\u003c/p\u003e \u003cp\u003ePrevious studies have shown that pomalidomide promotes T cell activation and function by selectively ubiquitinating and degrading Ikaros and Aiolos, leading to increased IL-2 expression\u003csup\u003e[\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e, which consistent with our data that pomalidomide rapidly enhances CAR-T cell proliferation and upregulates IL-2 and IFN-γ production during early activation. As known, tumor cells could secrete various cytokines/chemokines (e.g., CXCL9-11) to recruit cytotoxic T/NK cells expressing corresponding receptors via ligand-receptor interactions\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. In our \u003cem\u003ein vivo\u003c/em\u003e model, the tumor tissues of combination therapy group exhibited elevated CXCL9, 10 and 11 levels, suggesting that that pomalidomide might remodel the tumor microenvironment to enhance CAR-T cell infiltration, which is a pivotal step for effective antitumor immunity. Moreover, upregulation of CXCL9 could further enhance the expression of IFN-gamma in tumor-infiltrated T cells, which amplifies the effector function of T cells\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eT cell exhaustion, which is driven by persistent antigen exposure, is the major barrier to durable CAR-T responses\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e, while Tcm, characterized by self-renewal capacity, long-term survival, and lymphoid homing properties, represent a promising solution to this challenge\u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e. Our results indicated that pomalidomide not only enhances CAR-T persistence through Tcm enrichment but also reverses T cells exhaustion programs, creating a self-sustaining immunological memory compartment critical for long-term antitumor control. In particular, energy metabolism of T cells could be reprogrammed according to the functional status, that T cells in the early effector phase are characterised by enhanced glycolysis to meet the energy demands of rapid proliferation and cytokine production\u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e. Our results showed that pomalidomide-treated CAR-T cells exhibited early glycolytic activation, consistent with enhanced effector functions. However, CAR-T cells relying predominantly on glycolysis tend to differentiate into short-lived effector memory T cell (Tem) and exhibit accelerated exhaustion\u003csup\u003e[\u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e, while CAR-T cells with OXPHOS-predominant metabolism are more likely to differentiate into Tcm\u003csup\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e. This late-phase metabolic shift complements the early glycolytic burst, revealing a temporal metabolic strategy: initial glycolysis supports rapid effector function, while OXPHOS pathways enable long-term survival and memory formation\u003csup\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e. Intriguingly, the metabolic shift triggered by pomalidomide, might balance rapid effector function with long-term survival and memory formation, thereby mitigating exhaustion.\u003c/p\u003e \u003cp\u003eOur prior clinical trial demonstrated that long-term pomalidomide maintenance after CAR-T infusion reduced relapse rates and prolonged PFS/OS in RRMM patients\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. However, the universal applicability of this combination remains unclear, as there is no standardized consensus to define the initiation timing, dosing regimens, and maintenance duration of pomalidomide administration following CAR-T infusion, particularly regarding the optimal scheduling and persistence of this therapeutic regimen. Pomalidomide exerts maximal early stimulatory effects on CAR-T activation, with scRNA-seq demonstrating significant T cell subset expansion within 10 days following pomalidomide administration, suggesting that initiating pomalidomide therapy might be particularly beneficial for patients with suboptimal early expansion or declining CAR-T copy numbers. Cytokine release syndrome (CRS), driven by hypersecretion of proinflammatory cytokines (e.g., IL-6, IL-1β, and IFN-γ) from activated T cells and myeloid cells, is the main cause of CAR-T therapeutic failure\u003csup\u003e[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/sup\u003e. It cannot be discounted that pomalidomide further enhances the expression of these cytokines of CAR-T cells in our study, raising concerns about potential exacerbation of CRS in certain patient populations. Therefore, patients with high-risk factors for CRS should either avoid combination therapy with pomalidomide capsules or implement rigorous monitoring protocols (including serial measurement of IL-6 levels, ferritin). Furthermore, while pomalidomide is currently approved for the treatment of MM, our data reveal that its combination with CAR-T cells demonstrates comparable efficacy in lymphoma, suggesting broad applicability across hematologic malignancies. Further studies are needed to confirm its efficacy in multiple Hematologic Malignancies.\u003c/p\u003e \u003cp\u003eOur study systematically investigated pomalidomide's effects on CAR-T cell function, phenotype, and immune microenvironment. While these findings highlight the therapeutic seting potential of pomalidomide-CAR-T combinations, critical limitations must be addressed. First, immunodeficient mouse xenograft models inherently fail to recapitulate tumor microenvironment complexity and tumor-host immune dynamics. Furthermore, given the insufficient CAR-T cell abundance in scRNA-seq samples, deeper analysis of pomalidomide\u0026rsquo;s effects on CAR-T cells is limited. The inclusion of larger, more diverse samples of patients treated with pomalidomide-CAR-T combination therapy would enhance the generalizability of findings, particularly regarding treatment response variability, safety profiles, and long-term outcomes.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eOur multi-level evidence establishes the scientific rationale for pomalidomide-CAR-T synergy in lymphoid malignancies, providing theoretical guidance for treatment scheduling, and lays foundation for broader hematologic malignancy applications.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eCAR-T, Chimeric Antigen Receptor T-cell; IL-2, interleukin-2; IFN-\u0026gamma;, interferon-\u0026gamma;; Tcm, central memory T cells; MDSC, myeloid-derived suppressor cell; IMiDs, immunomodulatory drug; PFS, prolongs progression-free survival; OS, overall survival; R/R MM, relapsed/refractory multiple myeloma; poma, pomalidomide; Tpex, progenitor exhausted T cell; Tem, effector memory T cell.\u003c/p\u003e\n"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll human peripheral blood samples were obtained with informed consent and ethical approval from the Third Xiangya Hospital Medical Ethics Committee (Ethics Approval Number: 24767). Animal experiments were conducted in compliance with the guidelines estab lished by Central South University Ethics Committee (Ethics Approval Number: CSU-2024-0042).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eYan Yu:\u003c/strong\u003e Conceptualization, Data curation, Formal analysis, Methodology, Software, Visualization, Writing \u0026ndash; original draft. \u003cstrong\u003eYi Zhou:\u003c/strong\u003e Conceptualization, Data curation, Formal analysis, Methodology, Software, Validation, Writing \u0026ndash; original draft. \u003cstrong\u003eLinzhi Xie:\u003c/strong\u003e Data curation, Formal analysis, Methodology. \u003cstrong\u003eLiwen Wang:\u003c/strong\u003e Formal analysis, Software, Writing \u0026ndash; review and editing. \u003cstrong\u003eYuhan Yan:\u003c/strong\u003e Formal analysis, Writing \u0026ndash; review and editing. \u003cstrong\u003eQian Cheng:\u003c/strong\u003e Writing \u0026ndash; review and editing. \u003cstrong\u003eJing liu:\u003c/strong\u003e Supervision. \u003cstrong\u003eXin Li:\u003c/strong\u003e Funding acquisition, Resources, Supervision, Writing \u0026ndash; review and editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Competing Interest:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interest with the contents of this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis project has been supported by the National Natural Science Foundation of China (grant no. 82170204 to Xin Li).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available on request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSHI M, LI L, WANG S, et al. 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Role of CAR T Cell Metabolism for Therapeutic Efficacy[J]. Cancers (Basel), 2022,14(21).\u003c/li\u003e\n\u003cli\u003eKAWALEKAR O U, O\u0026apos;CONNOR R S, FRAIETTA J A, et al. Distinct Signaling of Coreceptors Regulates Specific Metabolism Pathways and Impacts Memory Development in CAR T Cells[J]. Immunity, 2016,44(2): 380-390.\u003c/li\u003e\n\u003cli\u003eFISCHER J W, BHATTARAI N. CAR-T Cell Therapy: Mechanism, Management, and Mitigation of Inflammatory Toxicities[J]. Front Immunol, 2021,12: 693016.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cancer-immunology-immunotherapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ciim","sideBox":"Learn more about [Cancer Immunology, Immunotherapy](http://link.springer.com/journal/262)","snPcode":"262","submissionUrl":"https://submission.nature.com/new-submission/262/3","title":"Cancer Immunology, Immunotherapy","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"CAR-T therapy, Pomalidomide, Immune microenvironment, Memory T cell, Lymphoid Malignancies","lastPublishedDoi":"10.21203/rs.3.rs-6571044/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6571044/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eDespite the remarkable efficacy of Chimeric Antigen Receptor T-cell (CAR-T) therapy in hematological malignancies, challenges including limited persistence and T cell exhaustion hinder long-term responses. Pomalidomide, an immunomodulatory drug, shows potential to synergize with CAR-T therapy, yet its mechanistic basis remains unclear.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eWe performed CCK8, LDH, qPCR, ELISA, flow cytometry and bulk RNA-sequencing to explore the effects and mechanisms of pomalidomide on CAR-T cell \u003cem\u003ein vitro\u003c/em\u003e. Myeloma xenograft murine models were established to evaluate the synergetic effects of CAR-T and pomalidomide \u003cem\u003ein vivo\u003c/em\u003e. Single-cell RNA sequencing was employed to delineate the effects of pomalidomide on immune microenvironment.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003ePomalidomide (1\u0026ndash;5 \u0026micro;g/mL) enhances CAR-T cell proliferation and cytotoxicity in an activation-dependent manner, upregulates the expression of interleukin-2 (IL-2), interferon-γ (IFN-γ), and chemokines CXCL9-CXCL11, and promotes the maintenance of central memory T cells (Tcm). \u003cem\u003eIn vivo\u003c/em\u003e, combination therapy induced tumor regression (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and extended survival (median OS: 30 days vs. unreached, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Transcriptomic analysis revealed metabolic reprogramming (glycolysis, fatty acid degradation) and reduced exhaustion markers (PD-1, LAG3). Single-cell sequencing demonstrated the immune microenvironment remodeling post pomalidomide treatment, including increased T/NK cells proportion and immune activity, as well as reduced myeloid-derived suppressor cell (MDSC) signatures within monocytes/macrophages.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003ePomalidomide amplifies CAR-T efficacy by enhancing memory formation, cytokine production, and metabolic fitness while reshaping the immune microenvironment. These findings provide a rationale for clinical optimization of pomalidomide-CAR-T combinations in refractory hematologic malignancies.\u003c/p\u003e","manuscriptTitle":"Pomalidomide enhances CAR-T cell therapeutic efficacy and Remodels Immune Microenvironment in Lymphoid Malignancies","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-08 11:40:48","doi":"10.21203/rs.3.rs-6571044/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-05-19T07:02:41+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-16T13:57:22+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-13T13:57:03+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-12T08:47:28+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-09T04:12:17+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-08T01:57:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"248194667946416484510859371939528617275","date":"2025-05-06T20:50:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"282073386087815630221760856478094172265","date":"2025-05-06T20:45:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"162514370801695195598280030755656965775","date":"2025-05-06T20:22:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"92274995218788535728490267642487707571","date":"2025-05-06T02:17:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"232189941175759646483477523032890702800","date":"2025-05-06T01:06:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"125507452292383296874064204889223765316","date":"2025-05-05T23:42:15+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-05T22:18:34+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-03T09:05:13+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-03T09:00:46+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cancer Immunology, Immunotherapy","date":"2025-05-01T09:59:20+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cancer-immunology-immunotherapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ciim","sideBox":"Learn more about [Cancer Immunology, Immunotherapy](http://link.springer.com/journal/262)","snPcode":"262","submissionUrl":"https://submission.nature.com/new-submission/262/3","title":"Cancer Immunology, Immunotherapy","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"b569965a-71c7-438c-a732-c02e6b575695","owner":[],"postedDate":"May 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-22T16:03:20+00:00","versionOfRecord":{"articleIdentity":"rs-6571044","link":"https://doi.org/10.1007/s00262-025-04247-1","journal":{"identity":"cancer-immunology-immunotherapy","isVorOnly":false,"title":"Cancer Immunology, Immunotherapy"},"publishedOn":"2025-12-18 15:58:34","publishedOnDateReadable":"December 18th, 2025"},"versionCreatedAt":"2025-05-08 11:40:48","video":"","vorDoi":"10.1007/s00262-025-04247-1","vorDoiUrl":"https://doi.org/10.1007/s00262-025-04247-1","workflowStages":[]},"version":"v1","identity":"rs-6571044","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6571044","identity":"rs-6571044","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}