Introduction
B-cell non-Hodgkin lymphoma (B-NHL) is a highly heterogeneous group of lymphopoietic malignancies, accounting for 85–90% of all Non-Hodgkin lymphomas (NHL) [ 1 – 4 ]. For patients with newly diagnosed DLBCL and other aggressive B-NHL, rituximab combined with anthracycline-based chemotherapy (R-CHOP) is the standard first-line treatment for most patients. Although most patients are cured with standard first-line immunochemotherapy, about one-third of patients do not achieve remission, or relapse or progress after treatment [ 5 ].
CD19 CAR T cell therapy has achieved good results in B-cell hematological tumors, but the efficacy in B-NHL has not been obvious than in acute lymphocytic leukemia (ALL) [ 6 , 7 ]. In addition, with the increasing experience of using CD19 CAR T cells to treat B-NHL, the investigators found that a subset of patients experienced disease recurrence even after the initial response was significant [ 8 ]. Failure of CD19 CAR T cell preparation, inability to efficiently expand and sustain, loss or regulation of target antigens, and toxicity after infusion are the three most common limitations of CD19 CAR T cell therapy [ 9 ]. Therefore, CD19 CAR T cells still face a very big challenge.
Epigenetic drugs can improve the preparation of CD19 CAR T cell products, promote the expansion and persistence of CD19 CAR T cells, and enhance the efficacy of CD19 CAR T cells in B-NHL treatment [ 10 – 12 ]. Chidamide is the only selective histone deacetylase inhibitor (HDACi) that can be taken orally to activate the tumor suppressive immune microenvironment [ 13 , 14 ]. In this study, we investigated whether the anti-B-NHL ability of CD19 CAR T cells could be enhanced by the pretreatment of chidamide. It was found that the pretreatment of B-NHL cells with chidamide could enhance the anti-tumor ability of CD19 CAR T cells by stimulating the secretion of more cytokines by CD19 CAR T cells and increasing the expression of CD19 on the surface of B-NHL cells. This study provides a new direction and theoretical foundation for CD19 CAR T cell therapy in B-NHL.
Methods
Cell lines and cell culture
The diffuse large B-cell lymphoma cell line SU-DHL-4, as well as the Burkitt lymphoma cell lines RAMOS and RAJI, were sourced from ATCC, maintained in our laboratory, and thoroughly validated before use in subsequent experiments. All cell lines were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS, Gibco, USA), and incubated in a humidified atmosphere with 5% CO 2 at 37 °C.
CAR T-cell generation
Peripheral blood mononuclear cells (PBMCs) were isolated from donors using Ficoll density gradient centrifugation. CD3+ T cells were then separated from the PBMCs using magnetic cell sorting (MACS) with CD3 microbeads (Miltenyi Biotec, Inc). These CD3+ T cells were stimulated using anti-CD3/anti-CD28 antibody-coated beads (Thermo Fisher Scientific, Inc) and cultured in X-Vivo 15 medium (Lonza Group, Ltd.) with 250 IU/mL of interleukin-2 (IL-2; Novartis International AG). On the second day, 3 × 10 6 T cells were transduced with a lentiviral vector encoding anti-CD19 CAR (Creative Biolabs, Inc) and continued to be cultured in IL-2 supplemented medium. By the 12th day, transduction efficiency was assessed using flow cytometry to determine the percentage of CAR-T 19 cells within the CD3+ T cell population.
Xenograft tumor model
To assess the functionality of CD19 CAR T cells in vivo, xenograft models were established using 4–6-week-old BALB/c mice (Shanghai Laboratory Animal Center, Shanghai, China) and 6–8-week-old NSG mice (Gempharmatech, Nanjing, China). Raji cells were subcutaneously injected into BALB/c mice divided into four groups at a concentration of 2 × 10 6 cells in 100 μl of PBS. The volume of the implanted tumor was calculated using the formula: tumor volume = length × width 2 × 0.5. The maximal tumor size permitted by our institutional ethics committee for subcutaneous tumors in experimental animals is 20 mm in diameter or 2000 mm 3 in volume. Our experiment did not exceed the limit. NSG mice received an intravenous injection of 1 × 10⁶ GFP + Luciferase + Raji cells on day − 10. Seven days later, the mice were divided into four groups and treated with CD19-CAR-transduced T cells. Lymphoma burden was monitored by injecting luciferin-D (YEASEN, Shanghai, China) into the mice via the caudal vein, followed by imaging 8 min later using the in vivo imaging system (IVIS) (Caliper Life Sciences, USA). Bioluminescence was quantified using Living Image Version 4.1 software. All animal experiments were conducted in accordance with relevant guidelines, approved by the Institutional Committee of Animal Care at Tongji Hospital, and performed at the Tongji Hospital animal experimental center. Reporting followed the ARRIVE guidelines.
Chidamide preparation and administration
Chidamide was obtained from Chipscreen Biosciences, dissolved in DMSO as per the manufacturer’s instructions, and stored at −80 °C. For in vitro experiments, Chidamide was added to cells at the specified concentration, with the medium and drug refreshed every 3 days. The control group received an equal volume of DMSO. For tumor and T cell pretreatment, Chidamide or DMSO was administered at the indicated concentration, followed by washing the cells three times in sterile PBS before co-culture. In in vivo experiments, Chidamide was diluted in DMSO, PEG300, Tween80, and saline, then administered orally at 3 mg/kg. Control groups received an equal volume of diluted DMSO in the same way.
Flow cytometry
Flow cytometry was conducted using a CytoFLEX S flow cytometer (Beckman Coulter, CA, USA) to assess cell surface CAR and protein expression. CD19-CAR was detected by incubating cells with APC-F(ab′) 2 (Jackson ImmunoResearch, USA). CD19 site density was measured using Quantity-PE beads (BD Biosciences, USA) following the manufacturer’s instructions. The detailed procedure followed the provided guidelines.
Proliferation and apoptosis assays
Cell proliferation in both cell lines and primary cells was evaluated using the CCK8 kit (Vazyme Biotech, Nanjing, China). The cell proliferation inhibition rate was calculated as follows: (OD treated – OD blank)/(OD control − OD blank) × 100%. Apoptosis was assessed using the Annexin V/7-AAD apoptosis assay kit (BD Biosciences, USA), with the apoptosis rate determined by flow cytometry following cell staining.
In vitro cytotoxicity
B-NHL cells and CD19 CAR T cells were co-cultured at different effector-to-target ratios. Flow cytometry, along with absolute counting beads, was used to determine the number of viable tumor cells remaining after co-culture.
Detection of intracellular cytokines
Quantification of cytokines was performed using flow cytometry-based bead array technology (Cytometric Bead Array, CBA). The fluorescence intensity detected by the flow cytometer is proportional to the concentration of cytokines in the sample. By using standard curves from cytokine standards, the quantitative values of various cytokines, including IL-2, TNF-α, and IFN-γ, were determined in the samples.
Data analysis
All statistical analyses were conducted with GraphPad Prism 8. Differences in quantitative data between groups were evaluated using Student’s t test, one-way ANOVA, or nonparametric tests as appropriate. Statistical significance was set at P < 0.05, with significance levels indicated as follows: * P < 0.05, ** P < 0.01, and *** P < 0.001.
Results
CD19 CAR T cells can effectively kill B-NHL cells in vitro
We successfully constructed a CD19 CAR T culture and amplification system from peripheral blood mononuclear cell (PBMC) [ 15 ]. We randomly selected peripheral blood of 3 donors to construct CD19 CAR T cells, and detected the expression of CD19 CAR on the cell surface by flow cytometry. We successfully constructed CD19 CAR T cells with transfection efficiency of 57.7%, 54.1% and 55.5%, respectively (Fig. 1 A). At the same time, we detected the expression of CD19 molecule on the surface of Raji, Ramos and SU-DHL-4 cells. The three kinds of cells were CD19 positive cells, and the positive rate of CD19 was nearly 100% (Fig. 1 B). CD19 CAR T cells could effectively kill B-NHL cells with positive CD19 expression, and the ability of CD19 CAR T cells to kill Raji, Ramos and SU-DHL-4 gradually increased with the increase of efficacy target ratio (Fig. 1 C).
Low-dose chidamide did not affect the viability, proliferation and anti-tumor ability of CD19 CAR T cells during preparation
To investigate whether chidamide could improve the product function of CD19 CAR T cells, we transfected T cells with lentivirus with CD19 CAR on the second day, followed immediately by different concentrations chidamide (0.001 μM, 0.01 μM, 0.1 μM and 1 μM) (Fig. 2 A). The experimental results confirmed that with the extension of culture time, the cells in all groups showed different degrees of expansion, but the viability and proliferation of T cells and CD19 CAR T cells were significantly inhibited after the addition of 1 μM of chidamide ( P < 0.05). Other concentrations of chidamide (0.001 μM, 0.01 μM, and 0.1 μM) did not inhibit the viability of T cells and CD19 CAR T cells, but did not promote their proliferation (Fig. 2 B, C). We co-cultured CD19 CAR T cells pretreated with three concentrations (0.001 μM, 0.01 μM, 0.1 μM) of chidamine and B-NHL cells at a 1:4 effect-to-target ratio, and found that their tumor killing ability did not change (Fig. 2 D).
Low-dose chidamide did not affect the proliferation, apoptosis and anti-tumor ability of prepared CD19 CAR T cells
Then, we explored the interaction between chidamide and prepared CD19 CAR T cells. We used CCK-8 assay to detect the effects of different concentrations of chidamide (0 μM, 0.25 μM, 0.5 μM, 1 μM, 2 μM, 4 μM and 8 μM) on the proliferation of CD19 CAR T cells. We observed a negligible inhibitory effect of chidamide on CD19 CAR T cells at 0.5 μM for 3 days (Fig. 3 A, B). In addition, we found that 0.25 μM and 0.5 μM of chidamide treated CD19 CAR T cells did not cause significant apoptosis, while 1 μM of chidamide treated CD19 CAR T cells increased apoptosis (Fig. 3 C, D). Therefore, we selected 0.5 μM of chidamide to pretreat CD19 CAR T cells. The results showed that the ability of CD19 CAR T cells to dissolve B-NHL cells could not be improved by pretreatment with low concentration of chidamide (Fig. 3 E).
The killing ability of CD19 CAR T cells was enhanced by the pretreatment of B-NHL cells with chidamide
Further experiments showed that the proliferation inhibition of chidamide on Raji, Ramos and SU-DHL-4 B-NHL cells was concentrate-dependent and time-dependent. After 3 days of treatment with 0.5 μM of chidamide, the proliferation inhibition of three B-NHL cells was negligible (Fig. 4 A). In addition, 0.5 μM chidamide treated Raji, Ramos and SU-DHL-4 cells for 3 days did not affect apoptosis of these three tumor cells (Fig. 4 B). Therefore, in the following in vitro experiments, we selected 0.5 μM concentration of chidamide to pretreat tumor cells. B-NHL tumor cells pretreated with chidamide were more likely to be killed by CD19 CAR T cells (Fig. 4 C). We found that the mean fluorescence intensity of CD19 on the surface of B-NHL cells was significantly increased after treatment with chidamide (Fig. 4 D). Furthermore, after co-culture with CD19 CAR T cells, CD19 CAR T cells were stimulated to secrete more cytokines (IL-2, TNF-α, and IFN-γ) (Fig. 4 E). These results indicated that chidamide could enhance the anti-tumor ability of CD19 CAR T cells against B-NHL.
Chidamide bridging intervention promotes the antitumor effect of CD19 CAR T cells in B-NHL subcutaneously transplanted tumor mice
We also confirmed the function of chidamide in vivo experiment of subcutaneous tumor transplantation in mice (Fig. 5 A). We found that tumor growth was slower in the Vehicle + CD19 CAR T group and Chidamide + CD19 CAR T group than in the Vehicle and Chidamide groups (Fig. 5 B). There was no significant difference in tumor size between the Vehicle group and the Chidamide group. Compared with the Vehicle + CD19 CAR T cell group, the subcutaneous tumor volume of Chidamide + CD19 CAR T cell group was smaller (Fig. 5 C). Average tumor weight was calculated after 21 days (Fig. 5 D).
Chidamide bridging intervention promotes the antitumor effect of CD19 CAR T cells in B-NHL tail vein grafted tumor mice
We injected NSG mice with Raji-Luciferase cells through the tail vein (Fig. 6 A). Our experimental results showed that the tumor burden of NSG mice in the first group of Vehicle group showed a gradually increasing trend, and one mouse died within Day 7 days, and from Day 7 to Day 14, all mice in this group died. The tumor burden of NSG mice in the Chidamide group also showed a gradually increasing trend. No deaths occurred in NSG mice in this group within 7 days of Day 7, but all NSG mice died from Day 7 to Day 14. NSG mice in the Vehicle + CD19 CAR T group showed a decrease in the rate of tumor load increase after CD19 CAR T cell treatment, but 1 mouse died from Day 7 to Day 14, and 2mouse died from Day 14 to Day 21. The tumor load of NSG mice in the last group of Chidamide + CD19 CAR T group was effectively controlled after CD19 CAR T cell therapy, and no mice died within 21 days after CD19 CAR T cell therapy (Fig. 6 B, C ). Kaplan–Meier survival analysis found that the Chidamide + CD19 CAR T group had the highest survival rate compared with the other three groups (Fig. 6 D).
Discussion
CD19 CAR T cell therapy has effectively improved the prognosis of B-NHL, brought a revolutionary breakthrough, and revolutionized the treatment pattern of B-NHL. However, the therapy still has many limitations, and many challenges and dilemmas remain [ 16 ]. JULIET, ZUMA-1 and TRANSCEND are three clinical trials of three CD19 CAR T cells for the treatment of R/R B-NHL, respectively [ 17 – 19 ]. The results of these three clinical trials suggest that the CR of CD19 CAR T cells for B-NHL is 40–54%, and the 1-year OS rate is 48–59%, which is significantly lower than the efficacy of CD19 CAR T in the treatment of ALL [ 20 ]. Results of a meta-analysis of R/R DLBCL treated with CD19 CAR T cell therapy showed a CR rate of 49.2% in the CD19 CAR T cell therapy group of 263 patients included [ 21 ].
Single CD19 CAR T cell therapy has limited efficacy for most B-NHL patients, while epigenetic drugs interact with CD19 CAR T cell immunotherapy [ 22 ]. As a first-generation epigenetic drug, decitabine can significantly improve the phenotype and function of CD19 CAR T cells, and low-dose decitabine can increase the invasion of CD4+ T cells and its antitumor activity [ 12 ]. As a selective epigenetic regulator of HDAC subtype, Chidamide can activate the tumor inhibitory immune microenvironment, and is studied as monotherapy or combined chemotherapy therapy for NHL, ALL, Acute myelogenous leukemia (AML) and Multiple myeloma (MM)[ 13 ]. A multicenter, real-world study of chidamide confirmed that combined chemotherapy with chidamide had a better ORR compared to monotherapy (73.2vs. 58.6%, P < 0.001), but there was no difference in 2-year OS between the two groups (combination therapy 35.2%, monotherapy 36.5%) [ 23 ]. In combination with prednisone, etoposide and lenalidomide, the ORR reached 73.5% and the median OS was 19.3 months in elderly patients with R/R DLBCL [ 24 ]. It has been reported that chidamide can significantly increase the surface expression of CD20 in DLBCL cell lines. R/R DLBCL patients achieved CR after 3 cycles of a combination of chidamide and rituximab [ 25 ].
Our study showed that the efficacy of CD19 CAR T cell therapy for B-NHL was enhanced after the intervention of chidamide bridging. The results of this study have important theoretical significance and clinical application value to improve the treatment rate of B-NHL patients and improve the quality of life of B-NHL patients. This study did not clarify the changes of CD19 CAR T cell subsets after pretreatment of B-NHL cells with chidamide. The mechanism of enhancing CD19 CAR T cells after pretreatment with B-NHL by chidamide needs further investigation.
Conclusions
In summary, low concentration of chidamide directly acted on CD19 CAR T cells, did not inhibit the proliferation of CD19 CAR T cells, but could not improve the anti-B-NHL ability of CD19 CAR T cells. However, the B-NHL cells were inhibited by low concentration of chidamide pretreatment. Our results show that B-NHL cells pretreated with low concentration of chidamide can enhance the expression of CD19 on the cell surface and promote the secretion of cytokines by D19 CAR T cells. The ability of CD19 CAR T cells to kill B-NHL cells was enhanced through the above pathways.
Author contributions
P.L. and A.B.L. designed the study. Y.Ni. and Q.Z. wrote the manuscript and performed molecular biological experiment. X.C.T., X.C.L., S.G.Y. and Y.L. performed statistical analysis. P.L. and A.B.L. revised the manuscript. All authors read and approved the final manuscript.
Funding
This work was supported by funds from National Key R&D Program of China (2021YFA1100800), National Natural Science Foundation of China (Nos. U23A20418 and 82470225), and Foundation of Shanghai Shen Kang Hospital Development Center (SHDC 2023CRD021).
Data availability
All data are available in the main text.
Declarations
Competing interests
The authors declare no competing interests.
Footnotes
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Ying Ni and Qun Zhang contributed equally to this work.
Contributor Information
Aibin Liang, Email: lab7182@tongji.edu.cn.
Ping Li, Email: lilyforever76@126.com.
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Associated Data
Data Availability Statement
All data are available in the main text.