Pomalidomide enhances CAR-T cell therapeutic efficacy and remodels immune microenvironment in lymphoid malignancies

Abstract

Background

Chimeric antigen receptor T cell (CAR-T) therapy achieves high remission rates in lymphoid malignancies, but its long-term efficacy is limited by poor persistence and T cell exhaustion. Pomalidomide, an immunomodulatory drug (IMiD), demonstrates clinical synergy with CAR-T therapy, yet the underlying mechanisms driving this potentiation remain poorly defined. This study aimed to elucidate how pomalidomide enhances CAR-T cell function and remodels the immune microenvironment to overcome therapeutic limitations.

Methods

In vitro assays (CCK-8, LDH, qPCR, ELISA, flow cytometry) and bulk RNA-seq assessed pomalidomide’s effects on human CAR-T cells. In vivo efficacy was evaluated in myeloma xenograft models. Single-cell RNA sequencing (scRNA-seq) of PBMCs from a lymphoma patient post-CAR-T/pomalidomide assessed immune microenvironment remodeling.

Results

Pomalidomide significantly enhanced CAR-T cell proliferation and cytotoxicity in an activation-dependent manner. It upregulated effector molecules (IL-2, IFN-γ) and chemokines (CXCL9-CXCL11), promoted central memory T cells (Tcm), and induced metabolic reprogramming while reducing exhaustion markers. In xenografts, combination therapy induced tumor regression and extended survival vs. CAR-T alone. scRNA-seq revealed pomalidomide-driven remodeling, characterized by increased T/NK cell proportions/activity and reduced myeloid-derived suppressor cell (MDSC) signatures.

Conclusions

Pomalidomide synergizes with CAR-T by directly enhancing CAR-T function (memory, cytokine/chemokine production, metabolic fitness, and reduced exhaustion) and remodeling the suppressive immune microenvironment (increased cytotoxic effectors, diminished MDSC activity). These findings provide a crucial mechanistic rationale for optimizing pomalidomide-CAR-T combinations in refractory lymphoid malignancies.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00262-025-04247-1.

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 (IMiD), 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.

Materials and methods

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.

Reagent

Pomalidomide (Selleck Chemicals, USA) was dissolved in DMSO (Sigma, USA) (2 mg/mL) and stored at – 80 °C.

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 (1000 × 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.

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 antihuman CD3 monoclonal antibody (BioLegend, USA) and antihuman 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%.

Cytotoxicity assay

CAR-T cells (pretreated 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%.

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. Serum of NOD-SCID mice was analyzed using Human IFN-γ, IL-2, IL-10, IL-6, and TNF-α ELISA Kits (ABclonal, China) following manufacturer protocols.

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 (IL-2, 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.

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 antihuman CD3 monoclonal antibody and antihuman 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 min 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 °C for 60 min, and stained with anti-CD3-APC (BioLegend, USA), anti-CD8-APC-Alexa Fluor 750, and anti-CD4-Pacific Blue antibodies for 15 min at RT. The intratumoral infiltration of T cells in tumors: tumor tissues were digested with Type IV collagenase (Yeasen, China) for flow cytometric analysis of CD3 + cell proportion. Data were acquired on Gallios-Analyzer (Beckman Coulter, USA) and analyzed by using FlowJo v10.

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). Five days after U266 cell infusion, mice were randomized into four groups (n = 4/group):

1. Untransduced T cells (UTD) monotherapy: 1 × 10⁷ control UTD cells (i.v., 150μL) + 1% DMSO (i.p., 5 times/week)

2. UTD + Poma therapy: 1 × 10⁷ control UTD cells (i.v., 150μL) + 2.5 mg/kg pomalidomide (i.p., 5 times/week) [20, 21]

3. CAR-T monotherapy: 1 × 10⁷ CAR-T cells (i.v., 150μL) [18] + 1% DMSO (i.p., 5 times/week)

4. CAR-T + Poma therapy: 1 × 10⁷ CAR-T cells (i.v., 150μL) + 2.5 mg/kg pomalidomide (i.p., 5 times/week)

The day of UTD or CAR-T infusion was set as day 0. Tumor volume (V = 0.5 × L × W²) [18, 19] and body weight were monitored every 7 days. Serum was collected on days 7, 14, and 21. On day 21, all the mice were killed and the subcutaneous xenograft tumors were isolated and weighed. Tumor tissues were digested for flow cytometric analysis.

Bulk RNA sequencing and data analysis

BCMA CAR-T cells derived from three donors (patient information is listed in Supplementary Table 2) were activated with anti-CD3/CD28 antibodies (5 μg/mL) and treated with pomalidomide (2.5 μg/mL) for 24 h. 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 were conducted using the “enrichr,” “GseaVis,” and “GSEABase” R packages.

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: one 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 was used to evaluate the function of NK cells, and AddModuleScore was 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.

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.

Results

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. The characteristics of the CAR-T cells we used are shown in Supplementary Fig. 1. 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. 1A, Supplementary Fig. 2A), resting CAR-T cells maintained > 95% viability at ≤ 5 μg/mL (Fig. 1B). Strikingly, upon CD3/CD28-mediated activation, subtoxic pomalidomide concentrations (2.5–5 μg/mL) induced a proliferation boost compared to activation alone (Fig. 1B). 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.

Fig.1.

Effects of pomalidomide on proliferation, cytotoxicity, and effector molecules of CAR-T cells. A–B Cell viability following pomalidomide treatment at various concentrations (0.01–100 μg/mL). A Survival curves in myeloma cell line U266, leukemia cell lines BCMA-K562, lymphoma cell line Raji, and CD19-K562 (from left to right). B Survival curves of CAR-T cells under resting conditions (left) or activated with anti-CD3/CD28 antibodies (right) over 48 h of treatment. C Target-specific cytotoxicity of CAR-T cells against tumor cells (left: BCMA CAR-T + U266; right: CD19 CAR-T + Raji) during 4-h co-culture at different effector-to-target (E/T) ratios (1:1, 2:1, and 5:1) under real-time pomalidomide exposure or 24-h pretreatment conditions. D Relative mRNA expression levels of IL-2, IFNG, and CXCL9/10/11 in anti-CD3/CD28-activated CAR-T cells following 24-h treatment with pomalidomide or DMSO control. E IFN-γ protein levels measured by ELISA in culture supernatants after 48 h of co-culture. Data represent mean ± SEM from at least three independent experiments. ns = not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

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-h CAR-T pretreatment with pomalidomide induced profound functional augmentation, elevating specific lysis from 8.27 to 10.61% at E/T = 1:1 and 18.36–23.12% at E/T = 5:1 (Fig. 1C, Supplementary Fig. 2B). This pretreatment efficacy gradient implies pomalidomide requires sufficient exposure time to prime CAR-T effector machinery.

To explore the synergy mechanism, we measured cytokine and chemokine expression in CAR-T cells activated by anti-CD3/CD28 antibodies or tumor co-culture. 24-h CAR-T pretreatment with pomalidomide significantly upregulated mRNA levels of IL-2, IFNG, CXCL9, CXCL10, and CXCL11 compared to DMSO controls (Fig. 1E). Correspondingly, IFN-γ protein levels in co-culture supernatants showed marked elevation (Fig. 1D). These findings suggest pomalidomide may enhance CAR-T synergy by boosting cytokine secretion, activation, proliferation, immune function, and immune cell recruitment into tumors.

Pomalidomide synergizes with CAR-T cells to amplify anti-tumor efficacy in myeloma xenograft models

Based on the finding that pomalidomide enhanced the proliferative capacity and effector functions of CAR-T cells, we subsequently tested whether pomalidomide combination therapy could further enhance the in vivo anti-tumor efficacy of CAR-T cells. We employed a murine model wherein U266 multiple myeloma cells were inoculated into NOD-SCID mouse subcutaneously (Supplementary Fig. 3A, Fig. 2A). Long-term longitudinal tracking revealed that pomalidomide pretreatment combined with CAR-T infusion induced profound tumor regression (p < 0.001, Fig.S3B-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 end point in the combination group (p < 0.01), while pomalidomide monotherapy showed no discernible benefit versus untreated controls (p = 0.39; Supplementary Fig. 3D). Importantly, the absence of significant body weight fluctuations (< 5% variation across groups, p > 0.05; Supplementary Fig. 3E) and preserved vital signs confirmed the regimen’s safety profile. Furthermore, our results show that mRNA levels of IL-2, IFNG, and chemokine CXCL9-11 were significantly higher in tumor tissues of the CAR-T + Poma group compared to that of the CAR-T group (Supplementary Fig. 3F).

Fig.2.

Pomalidomide synergizes with CAR-T cells to amplify anti-tumor efficacy in myeloma xenograft models. A Experimental schema: NOD-SCID mice were injected with the U266 cell line (1 × 10⁷ cells per mouse i.h.). Five days later, mice were received 1 × 10⁷ BCMA CAR-T or equivalent number of control UTD cells the following day with either pomalidomide or control vehicle (2.5 mg/kg, given i.p. 5 days a week for 21 days, starting on the day of CAR-T injection). B–D The effect of CAR-T, pomalidomide, or combination therapy on tumor size, weight, and volume. E ELISA of serum levels of cytokines. On days 7, 14, and 21 post-UTD or CAR-T injection, blood was collected from mice, and IL-6, IL-2, IL-10, IFN-γ, and TNF-α were measured. F-G The intratumoral infiltration of CD3+ T cells in the tumors was examined by flow cytometry. Data represent mean ± SEM. ns: not significant; *p < 0.05; **p < 0.01; ***p < 0.001

Further, we employed infusion of UTD as the control group to clarify the superior therapeutic efficacy of pomalidomide combined with CAR-T (Fig. 2A) and found that pomalidomide did not enhance the anti-tumor effect of UTD but significantly potentiated that of CAR-T (Fig. 2B-D). Additionally, although serum effector cytokine levels (including IL-2, IFN-γ, and TNF-α) from mice treated by CAR-T therapy were significantly higher than those in the UTD group, and there was no significant difference in these cytokines between the UTD + Poma and UTD groups (Fig. 2E). Notably, pomalidomide further enhanced IL-2 and IFN-γ secretion specifically by CAR-T cells, while it did not increase TNF-α, IL-10, and IL-6 secretion (Fig. 2E). More importantly, pomalidomide treatment significantly increased CAR-T cell infiltration within the tumor (Fig. 2F-G). 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.

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. 3A). 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. 4B). Notably, the pomalidomide treatment maintained elevated CD25 levels throughout the observation period (Supplementary Fig. 4B), 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% to 26.45%, while showing no significant effects on other phenotypes or CD4+ CAR-T cells (Supplementary Fig. 4A). 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. 3B, Supplementary Fig. 1E).

Fig.3.

Effects of pomalidomide on CAR-T cell memory programming and subset distribution. A 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 h. Memory phenotypes were assessed by flow cytometry on days 2, 4, and 7. B Representative flow cytometry profiles (top) and subset frequencies (bottom) of CAR-T cell populations on days 2, 4, and 7 post-pomalidomide treatment. C Flow cytometry characterization of CD4+ and CD8+ CAR-T cell subsets on day 2. Quantified CD4/CD8 ratios and CAR + CD4/CD8 ratios (right). Data represent mean ± SEM from ≥ 3 independent experiments. ns: not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

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. 3C, Supplementary Fig. 1B-D, and Supplementary Fig. 4C).

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. 4A). 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, ITK, ZAP70) and cytokine/chemokine (IL-2, 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. 4B), suggesting transcriptional reprogramming toward a memory and effector competent state.

Fig.4.

Pomalidomide enhances CAR-T cell immune function and mediates metabolism at the transcriptomic level. A PCA-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). B Heat map depicting Z-score-normalized expression levels of genes associated with CAR-T cell function and phenotype. C Volcano plot visualizing differentially expressed genes (DEGs) in pomalidomide-treated CAR-T cells compared with DMSO. FDR < 0.05 and |logFC|≥ 1. D KEGG pathway enrichment analysis of upregulated DEGs. P value < 0.05. E GO enrichment analysis of upregulated DEGs. P value < 0.05. F–H Gene set enrichment analysis (GSEA) was performed using F Hallmark pathways, G C5: GO categories, and H 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

Differential expression analysis identified 226 upregulated and 23 downregulated genes in treated vs. control CAR-T cells (Fig. 4C). KEGG analysis of upregulated genes revealed significant enrichment in metabolic pathways including fatty acid degradation, glycolysis, cGMP-PKG signaling, and tyrosine metabolism (Fig. 4D), while GO analysis highlighted enrichment in immune-related pathways such as T cell activation, inflammatory response, cell adhesion, and ERK1/2 signaling (Fig. 4E).

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. 4F). 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. 4G). Importantly, the prominent activation of memory T cell signatures in pomalidomide-treated CAR-T cells (Fig. 4H), aligning with our prior observations of memory phenotype induction in vitro.

Pomalidomide improves anti-tumor immunity through remodeling the tumor immune microenvironment

To assess pomalidomide’s impact on the immune microenvironment, we performed scRNA-seq on PBMCs from a lymphoma patient collected before treatment (day 1), and 10 and 30 days after treatment initiation. The PBMCs were primarily classified into CD8+ T cells, monocytes/macrophages, NK cells, CD4+ T cells, pDCs, and platelets (Fig. 5A-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. 5C).

Fig.5.

Pomalidomide treatment remodels peripheral circulation immune microenvironment in a lymphoma patient. A Single-cell RNA-seq data from experimental groups (one day before treatment, day10 and day30 post- pomalidomide treatment) were visualized using UMAP. B The Sankey diagram illustrates differential composition of PBMC immune subsets across three time points. C A dot plot presents the expression levels of signature genes in distinct T cell clusters identified through single-cell RNA-seq analysis. D–E UMAP visualization of T cells across all samples D and Sankey diagram revealing temporal changes in T cell subsets across three time points E. F Line plots depict dynamic expression patterns of T cell effector (GZMB, IFNG), exhaustion (PDCD1, LAG3, TOX), and memory/stemness (IL7R, KLF2) genes across T cell subsets and time points. G 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. H A dot plot depicts KEGG metabolic pathway enrichment scores computed via ssGSEA in CD8+ T exhausted (Tex) cell subsets. I A FeaturePlot visualizes the distribution of NK cell effector function-related genes (GZMB, GNLY, IFNG, CCL5) across distinct time points. J A heat map displays gene set variation analysis (GSVA) scores for NK cell subgroups using predefined GO gene sets. K Violin plots illustrate the differences in cytotoxicity and exhaustion scores (calculated via AddModuleScore) among NK cell subsets across groups. L–M Violin plots L and FeaturePlot M show the differences in MDSC signature scores (calculated via AddModuleScore) between monocyte/macrophage subgroups across groups. ns: not significant; *p < 0.05; ***p < 0.001; ****p < 0.0001

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 T cells (Treg), and a cluster characterized by high expression of proliferation-related genes (Tprolif) (Fig. 5C-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. 5E). 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. 5F). 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. 5G). 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. 5H), suggesting that pomalidomide maintenance therapy might reverse exhaustion-associated metabolic signatures through dynamic metabolic reprogramming.

Additionally, immune scoring of NK cells revealed a significant increase in cytotoxicity and a decrease in exhaustion signatures following pomalidomide treatment (Fig. 5K). FeaturePlot visualization results also showed an upregulation of cytotoxicity-related genes after pomalidomide treatment (Fig. 5I). Furthermore, GSVA for GOBP gene set scoring indicated that pomalidomide treatment enhanced NK cell proliferation, cytotoxicity, chemotaxis, and activation while inhibited differentiation (Fig. 5J). Additionally, myeloid-derived suppressor cells (MDSCs) are known to play a critical role in the suppression of T cell anti-tumor 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. 5L-M), suggesting that pomalidomide may enhance T cell anti-tumor responses by inhibiting MDSCs.

Discussion

CAR-T cell therapy is limited by exhaustion and poor persistence [27, 28]. Overcoming these challenges remains a critical research focus. 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 anti-tumor 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 in the combination therapy group exhibited elevated levels of CXCL9-11 and enhanced CAR-T cell infiltration, suggesting that pomalidomide might remodel the tumor microenvironment to enhance CAR-T cell infiltration, which is a pivotal step for effective anti-tumor immunity. Moreover, upregulation of CXCL9 could further enhance the expression of IFN-γ 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], while Tcm, characterized by self-renewal capacity, long-term survival, and lymphoid homing properties, represent a promising solution to this challenge [36–38]. Our results show pomalidomide enhances CAR-T persistence by enriching Tcm cells and reversing exhaustion, establishing a durable memory pool essential for long-term tumor control. T cell energy metabolism adapts to functional status: Early effector T cells rely on enhanced glycolysis to fuel 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 metabolic shift complements the early glycolytic burst: Glycolysis drives initial effector function, while OXPHOS supports 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.

Significantly, CD4⁺ T cells enhance both expansion and persistence of CD8⁺ CAR-T cells. While the optimal CD4/CD8 ratio remains undefined, CD4⁺ CAR-T cells, particularly the TCM and naïve subsets, synergize with CD8⁺ CAR-T cells to augment anti-tumor activity in vitro and in vivo, which enhances cytotoxic efficacy [44]. Clinically, patients receiving CAR-T products with CAR⁺CD4⁺/CD8⁺ ratios exceeding 3 demonstrated significantly prolonged CAR-T persistence [25]. In our findings, pomalidomide elevates the CD4/CD8 ratio while most importantly increasing frequencies of both CD4⁺ and CD8⁺ TCM subsets, suggesting enhanced therapeutic efficacy and potential for sustained CAR-T maintenance. We emphasize, however, that elevated ratios do not invariably confer benefit, as excessively high proportions may increase toxicity risks including cytokine release syndrome (CRS).

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. 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 [45]. 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 approved for MM, our data show pomalidomide combined with CAR-T also works effectively in lymphoma, suggesting potential across hematologic malignancies.

Our study systematically investigated pomalidomide’s effects on CAR-T cell function, phenotype, and immune microenvironment. While these findings highlight the therapeutic setting potential of pomalidomide-CAR-T combinations, critical limitations must be addressed. First, immunodeficient mouse xenograft models cannot fully replicate the complex tumor microenvironment or tumor–host immune interactions. Furthermore, the sample size and low CAR-T cell numbers in the scRNA-seq sample limited deeper analysis of pomalidomide’s direct effects on them. Larger, more diverse patient cohorts receiving pomalidomide-CAR-T therapy are needed to confirm generalizability, especially for treatment response variability, safety, and long-term outcomes.

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.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

CAR-T

Chimeric antigen receptor T cell

IFN-γ

Interferon-γ

IL-2

Interleukin-2

IMiDs

Immunomodulatory drug

MDSC

Myeloid-derived suppressor cell

OS

Overall survival

PFS

Prolongs progression-free survival

Poma

Pomalidomide

R/R MM

Relapsed/refractory multiple myeloma

Tcm

Central memory T cells

Tem

Effector memory T cell.

Tpex

Progenitor exhausted T cell

Author contribution

Yi Zhou: Conceptualization, Data curation, Formal analysis, Methodology, Software, Validation, Writing—original draft, Funding acquisition. Yan Yu: Conceptualization, Data curation, Formal analysis, Methodology, Software, Visualization, 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. Chang Zhang: Data curation, Formal analysis, Methodology, Writing—review and editing. Xin Li: Funding acquisition, Resources, Supervision, Writing—review and editing.

Funding

This project has been supported by the National Natural Science Foundation of China (grant no. 82170204 to Xin Li), Beijing Medical and Health Foundation (JSZ021), and the Fundamental Research Funds for the Central Universities of Central South University (2025ZZTS0194).

Data availability

Data will be made available on request.

Declarations

Conflict of interest

The authors declare no conflict of interest.

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 established by Central South University Ethics Committee (Ethics Approval Number: CSU-2024-0042).