A novel fourth generation of CAR-T cells: CD19 CAR-T cells engineered to express membrane-bound interleukin-15 and CXCR5 for the treatment of lymphoma

Abstract

The efficacy of CD19 CAR-T cells in B-cell lymphoma patients is not as good as that in B-cell acute lymphoblastic leukemia (B-ALL) patients. This might be attributed to the intricate tumor microenvironment of B-cell lymphoma, which leads to CAR-T cell exhaustion, inability to sustain function, and difficulty infiltrating into the tumor interior. We developed CD19 CAR structures that simultaneously produce membrane-bound IL-15 (mbIL-15) and CXCR5. This design aims to enhance the migration of CAR-T cells into CXCL13+ B-cell lymphomas and their long-term antitumor ability. Compared with CD19 CAR-T cells, CD19 mbIL15-CXCR5 CAR-T cells exhibited greater cytotoxicity against CD19+ tumor cell lines in vitro. In particular, when exposed to recurrent tumor antigen stimulation, CD19 mbIL15-CXCR5 CAR-T cells still exerted long-lasting antitumor effects. CD19 mbIL15-CXCR5 CAR-T cells had a greater proportion of central memory T (TCM) and effector memory T (TEM) cells, which allowed them to exhibit more long-lasting antitumor effects. Moreover, in Transwell assays and mouse models, compared with CD19 CAR-T cells, CD19 mbIL15-CXCR5-CAR-T cells exhibited significant chemotaxis toward CXCL13+ tumor cells and superior tumor infiltration ability. The Xenogram animal model demonstrated better and more persistent tumor suppression ability than did the CD19 CAR-T cells. We preliminarily demonstrated the safety of CD19 mbIL15-CXCR5-CAR-T cells in vivo by evaluating liver and kidney function and major organ morphology in mice. In summary, the use of CD19 mbIL15-CXCR5-CAR-T cells is a relatively safe and effective option for the treatment of B-cell malignancies.

Supplementary Information

The online version contains supplementary material available at 10.1007/s10238-025-01860-3.

Introduction

While CD19 CAR-T cells have demonstrated optimal clinical efficacy in the treatment of CD19-positive acute lymphoblastic leukemia, their effectiveness in treating B-cell lymphoma is relatively limited [1]. Findings from international multicenter clinical trials have indicated that the remission rate for patients with B-cell lymphoma following CAR-T-cell therapy ranges from 50 to 80% [2–5]. More than half of patients experience relapse within one year [6, 7]. Various obstacles may impede the function of CAR-T cell therapy in B-cell lymphoma, including antigen escape, CAR-T cell depletion, restricted T cell infiltration, and a complex tumor microenvironment [8]. New therapeutic methods to improve the efficacy of CAR-T cells in treating B-cell lymphoma are needed. We propose that efforts should focus on enhancing the antitumor efficacy of CD19 CAR-T cells, mitigating CAR-T cell depletion, and facilitating their evasion from the constraints of the tumor microenvironment to better target lymphoma cells and counteract factors that inhibit the therapeutic effects of CAR-T cells. Fourth-generation CAR-T cells bring new hope to researchers and clinical practitioners. This enhances the effectiveness of CAR-T cells from the perspective of cytokines, allowing them to exert effects beyond the traditional effects of CAR-T cells. Researchers have promoted the sustained antitumor effect of CAR-T cells by inducing them to secrete cytokines such as IL-12, IL-7, and IL-21 [9–11]. In addition, researchers have improved CAR-T cells to express chemokines or chemokine receptors, such as CCL19, to guide CAR-T cells toward the direction of the tumor [12]. In recent studies, researchers have combined cytokines and chemokines to modify CAR-T cells, such as anti-CD20 CAR-T cells that simultaneously express IL-7 and CCL19, endowing CAR-T cells with multiple functions and expanding their application in clinical practice [13].

Research has shown that T memory stem cells (TSCMs), the least differentiated subset of memory T cells, have the best long-term persistence potential [14, 15]. This memory subset is beneficial for CAR-T-cell therapy [16]. Cytokines encompass a diverse group of small protein molecules with multifaceted biological activities [17, 18]. T cells only receive prosurvival signals through the common γ-chain cytokine family, such as those mediated by IL-2 and IL-15 [19]. IL-2 is commonly used to stimulate CAR-T-cell and T cell proliferation in most associated studies [2, 3, 20]. However, it may promote the production of regulatory T (Treg) cells, leading to the depletion of CAR-T cells [21]. Unlike IL-2, IL-15 does not cause T cell terminal differentiation [19, 21]. Conversely, IL-15 induces the formation of memory cells and enhances the adaptability of T cells by delaying their senescence [22, 23]. These qualities are beneficial for CAR-T cells [24]. IL-15 exists in three different forms: monomeric IL-15, soluble IL-15 bound to the IL-15 receptor alpha chain (IL-15Rα), and membrane-bound IL-15 (mbIL-15) [25]. High doses of IL-15 may cause toxic side effects in patients, such as hypotension, fever, and thrombocytopenia, and may promote the ultrastructural activation of T cells, leading to abnormal proliferation or toxicity of T cells. MbIL-15 avoids such adverse reactions due to the absence of large doses of interleukin-15 infusion. At the same time, due to continuous cell-to-cell contact, mbIL-15 can tightly occupy the receptor for a long time, thus often achieving a more sustained biological function on T cells compared to soluble interleukin-15. [26].

Moreover, increasing amounts of clinical evidence suggest that the efficacy of classic CAR-T cell designs in B-cell lymphoma is not as satisfactory as that in B-ALL because of the lack of CAR-T cell infiltration [27]. Some studies have shown that adding additional chemokines to match the corresponding receptors could improve their migration into tumor sites [28]. CXC chemokine Ligand 13 (CXCL13) is a homeostatic chemokine normally expressed in lymphoid tissues [29]. CXCL13+ Tfh cells are widely distributed in lymphocyte-rich classic Hodgkin lymphoma [30]. In the field of non-Hodgkin's lymphoma research, studies have shown high expression of CXCL13 in follicular lymphoma [31]. A study on chemokine detection in patients with diffuse large B-cell lymphoma, including 21 cases of primary testicular lymphoma (PTL) and 28 cases of systemic lymphoma (sDLBCL) [32], reported that 89.3% of sDLBCL patients and 57.1% of PTL patients presented high expression of CXCL13 in the cytoplasm, whereas 35.7% and 28.6% presented high expression in the nucleus, respectively. Additionally, 7.1% of the sDLBCL group showed strong membrane expression [32]. CXCR5, a unique receptor for CXCL13, can rely on the concentration of CXCL13 to mediate the migration of corresponding cells to secondary lymphoid organs [33]. Therefore, CAR-T cell infiltration into lymphoma may be achieved by increasing the expression of CXCR5 on CD19 CAR-T cells.

In this study, we constructed fourth-generation CAR-T cells expressing both mbIL-15 and CXCR5 (CD19-mIL15-CXCR5 CAR-T cells). We subsequently conducted in vivo and in vitro experiments to verify whether this modification could enhance the function and therapeutic efficacy of CD19 CAR-T cells.

Materials and methods

Patients and study design

This study retrospectively collected data from patients with bulky mass lymphoma treated with CD19 CAR-T cells at Tianjin First Central Hospital from January 1, 2017, to December 31, 2023, and evaluated the efficacy of this special patient population.

(ChiCTR1800019622, ChiCTR1900021353). The inclusion criteria for patients were as follows: (1) were diagnosed with refractory/relapsed (r/r) B-lymphoma according to the 2016 WHO diagnostic criteria [34]; (2) had a maximum tumor diameter of ≥ 7.5 cm [35]; and (3) had complete and accessible disease information and medical records of CAR-T cell therapy.

Cell lines

The cell lines used in this study were all purchased from ATCC. The tumor cell lines were modified with LGP (luciferase [Luc] and green fluorescent protein [GFP]) for subsequent experiments. In addition, U2932 cells were transfected with lentivirus supernatant containing the corresponding gene sequences to produce cells expressing LGP and CXCL13. U2932, Bjab, and Nalm6 cells were grown in RPMI-1640 (Gibco, USA) media supplemented with 10% fetal bovine serum (FBS; Biological Industries). HEK-293 T cells were cultured in DMEM (Gibco, USA) supplemented with 10% FBS. All the cell Lines were cultured at 37 °C, 5% CO₂, and 95% humidity. We regularly tested for mycoplasma.

Transgenic constructs

The single-chain variable fragment (scFv) targeting CD19 was derived from the FMC63 clone. Then, the scFv, CD8 hinge, transmembrane segment, 4-1BB costimulation signaling, and CD3ζ intracellular signaling domains were integrated into the pCDH-MND-MCS-PURO lentiviral plasmid vector. Similarly, the sequences of human mbIL-15 and CXCR5 were inserted into the CD19-CAR lentivirus driven by the MND promoter.

Lentivirus production

According to the manufacturer’s instructions for polyethylenimine (PEI) (#239666–1, Polysciences, USA), 50 µL of this reagent was combined with 500 µL of Opti-MEM. Four micrograms of the target plasmid, 3 µg of the packaging plasmid SPAX2, and 2 µg of the packaging plasmid MD2G were added to another 500 µl of Opti-MEM. Then, these were mixed. After 15 min, the mixture was gently added to the HEK-293 T cells. After 24 h, we used fresh DMEM supplemented with 10% FBS to replace the original medium. After 48 h, the lentivirus supernatant was collected and concentrated via ultracentrifugation (47492xg, 4 °C) for 2 h. The lentivirus was stored at −80 °C for subsequent experiments.

Production of CAR-T cells

Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors (n = 3) via the density gradient centrifuge method, and CD3+ T cells were isolated by CD3+ immunomagnetic beads and then incubated in RPMI-1640 containing 10% FBS, IL-2 (250 IU/mL) and CD3/CD28 magnetic beads. The activated T cells were supplemented with concentrated lentiviral supernatant at 37 °C (multiplicity of infection was 10, MOI = 10) and transferred to T25 cell culture flasks (Corning, USA) after 48–72 h.

Flow cytometry

To validate the expression of CXCR5 and mbIL-15, CXCR5-APC and human IL-15-FITC (R&D) antibodies were used for staining. To assess the expression of CAR molecules, CAR-T cells were labeled with anti-FMC63 antibodies. To evaluate differentiation, CAR-T cells were detected with anti-human monoclonal antibodies against CD62L-APC and CD45RA-KO525.

Proliferation

First, we adjusted the CAR+ /CD3+ ratio by adding untransduced CD3+ cells to ensure equal numbers and proportions of CAR-T cells between each group. Two CAR-T cells were subsequently seeded into a 96-well plate (Corning, USA) with 5 × 10⁵ cells per well. The medium was RPMI-1640 supplemented with 10% FBS and IL-2 (250 IU/ml). We added 100 μl of culture medium to each well. After 24 h, 48 h, and 72 h, 10 μL of CCK8 (Biosharp, China) was added to each well, and the absorbance was measured at a wavelength of 450 nm.

Cytotoxicity

We adjusted the CAR+ /CD3+ ratio by adding untransduced CD3+ cells to ensure equal numbers and proportions of CAR-T cells between each group. CD19 mbIL15-CXCR5 CAR-T cells were cocultured with Nalm6, Bjab, or U2932 cells at an effector-to-target ratio of 1:1. The cytotoxicity was monitored by flow cytometry at different time points and calculated as (number of cells in pure tumor cell wells minus the number of tumor cells remaining in the CAR-T group)/number of cells in pure tumor cell wells. CD19 mbIL15-CXCR5 CAR-T cells, CD19 CAR-T cells, and uninfected T cells were separately coincubated with CD19+ tumor cell lines at different effector: target ratio (E:T). At a specific time point, the number of tumor cells was detected by flow cytometry, and the number of tumor cells in the blank control well was used as a control group to compare the killing effects among all the groups. Additionally, we repeatedly added tumor cells when the cytotoxicity was approximately 80% to simulate the situation where tumor antigens persist in vivo, and the sustained antitumor effect was observed by calculating the remaining number of tumor cells.

Cytokine measurements

When cytotoxicity was evident, supernatants were collected from the cocultures of the two CAR-T cells with the tumor cells separately. Luminex was used to assess the concentrations of the serum inflammatory markers IL-2, IL-6, IL-8, IL-10, TNF-α, and IFN-γ, as recommended by the manufacturer (CBA).

In vitro transwell assay

Before the Transwell assay, CD19 mbIL15-CXCR5 CAR-T cells, CD19 CAR-T cells, and uninfected T cells were starved in serum-free 1640 medium for 24 h. Transwell chambers were placed in 24-well plates, two kinds of CAR-T cells or uninfected T cells (5 × 10⁵ cells/well, with 200 µl of serum-free 1640 medium) were added to the upper chamber, and U2932 cells expressing CXCL13-LGP (1 × 10⁵ cells/well, with 500 μL of 1640 medium supplemented with 10% FBS) or different concentrations of the recombinant human chemokine CXCL13 were added to the lower chamber. The cells were cultured at 37 °C, 5% CO₂, and 95% Humidity for 6 h; the number of cells that migrated from the upper chamber to the lower chamber was assessed by flow cytometry.

Distribution of CAR-T cells in vivo

Nine male NSG mice, aged 6 to 8 weeks, were purchased from Spaf (Beijing) Biotechnology Co., Ltd., and randomly split into three groups. U2932-LGP and U2932-CXCL13-LGP were precooled with PBS (1 × 10⁶ cells/50 μL PBS), and an equal volume of Matrigel (Corning, USA) (1 × 10⁶ cells/50 μL: 50 μL Matrigel) was added. A 100 µl mixture of U2932-LGP was injected into the left ventral side of NSG mice via subcutaneous injection. U2932-CXCL13-LGP, a 100 µl mixture, was injected into the right ventral side of the same mouse via subcutaneous injection. The size of the tumors was monitored, and when the tumor volume was approximately 100–200 mm³, the mice were randomly grouped and subjected to different treatments (CD19mbIL15-CXCR5 CAR-T, CD19 CAR-T, and UT groups). The mice were euthanized 7 days after receiving treatment, and the tumors on both sides were separated, embedded and then stained with a human CD3+ antibody against T cells. The results were assessed on the basis of the staining of the cells observed under a microscope (400×), and inter- and intragroup comparisons were carried out.

Xenograft animal model

Nine male NSG mice were also purchased from Spaf Biotechnology Co., Ltd. (Beijing) and were randomly split into three groups. The xenograft mouse model was constructed with U2932-CXCL13-LGP. All the mice were subcutaneously injected with a mixture of 1 × 10⁶ tumor cells and 50 µl of matrix on day −14. When the tumor volume was measured with a caliper at approximately 50–100 mm³ (the tumor volume was calculated with the formula: length × width × height), 4 × 10⁶ CD19 mbIL15-CXCR5 CAR-T cells, CD19 CAR-T cells, and uninfected T cells were infused through the tail vein. (Day 0). The body weights and tumor sizes of the mice were monitored and recorded every two days; when the tumor size reached 2000 mm³, the mice were killed, and then, their spleens, livers, and kidneys were stained with HE.

Statistical analysis

All experiments were repeated three times independently. The results were statistically analyzed using GraphPad Prism. The data are expressed as the means ± standard deviations (SDs). A paired t test was used to compare the results of the two groups of CD19 CAR-T cells and CD19 mbIL15-CXCR5 CAR-T cells. One way analysis of variance (ANOVA) was used to compare multiple groups of UT cells, CD19 CAR-T cells, and CD19 mbIL15-CXCR5 CAR-T cells. P < 0.05 was considered to indicate statistical significance.

Results

The poor therapeutic effect of CD19 CAR-T cell therapy on bulky mass lymphoma

The treatment of refractory/recurrent B-cell lymphoma has always been a challenging issue. Although researchers have proposed new treatment approaches such as Polatuzumab vedotin plus rituximab and lenalidomide, the objective response rate is still only 31% [36]. Compared with targeted therapy and chemotherapy, CD19 CAR-T cells have better therapeutic effects on refractory/recurrent B-cell lymphoma, but still cannot achieve satisfactory results. This conclusion is particularly evident in bulky mass lymphoma. We included a total of 42 patients with bulky mass lymphoma (n = 42) for evaluation, and their characteristics are shown in Table 1. We found that after the infusion of CD19 CAR-T cells, the objective response rate (ORR) of patients was 57.14%, with a complete response rate (CR) of 28.57%. Among the 42 patients, 32 patients developed CRS, 27 of whom had grade 1–2 CRS, and 5 patients experienced severe grade 3–4 CRS reactions. Three patients experienced ICAN reactions, and no patients experienced ICAN reactions exceeding grade 3 or above. The median survival time of patients with giant lymphoma receiving CD19 CAR-T cell therapy was 297 days. The long-term survival time of these patients is not satisfactory (Supplementary Fig. 1). The underlying reasons may be related to the complex tumor microenvironment, depletion of CD19 CAR-T cells, and difficulty in infiltrating tumors with CD19 CAR-T cells. Therefore, we hope to improve the efficacy of treating lymphoma, especially bulky mass lymphoma, by engineering CD19 CAR-T cells.

Table 1.

Characteristics of patients with bulky mass lymphoma

Characteristics	Patients, n (%)
Diagnosis
DLBCL	42,100%
Age, median(range)	54(44–65.5)
Gender
Male	24(57.1%)
Female	18(42.9%)
Ann Arbor
3	15(35.7%)
4	27(64.3%)
IPI
2	4(9.5%)
3	12(28.6%)
4	21(50%)
5	4(9.5%)
aaIPI
1	4(9.5%)
2	13(31.0%)
3	18(42.9%)
4	5(11.9%)
5	2(4.8%)
NCCN-IPI
2	2(4.8%)
3	8(19.0%)
4	9(21.4%)
5	14(33.3%)
6	8(19.0%)
7	1(2.4%)
Extramedullary accumulation
Yes	37(88.1%)
No	5(11.9%)
bone marrow involvement	8(19.0%)
CNS involvement	2(4.8%)
Chemotherapy, median(range)	3(2.5–6)

Characteristics of patients with bulky mass lymphoma. DLBCL: Diffuse large B-cell lymphoma; CNS involvement: Central Nervous System involvement

Successful generation of CD19 mbIL15-CXCR5 CAR-T cells

First, we successfully prepared a CAR vector targeting CD19. Moreover, we modified the CD19 CAR vector with mbIL-15 and CXCR5 to obtain the viral vector of the CD19 mbIL15-CXCR5 CAR (Fig. 1A). The sequence was verified to be correct by sequencing. Then, CD19 CAR-T cells and fourth-generation CAR-T cells were generated from T cells via a lentiviral vector. The expression of CARs was measured by flow cytometry 72 h after transduction. In addition, anti-CXCR5 and anti-IL-15 antibodies were used to detect whether the modification was successful. Uninfected T (UT) cells were used as a blank control. The results revealed that the median expression of CARs in CD19 mbIL15-CXCR5 CAR-T cells was 25% (range 18–39%) and that in CD19 CAR-T cells was 53% (range 45–60%) (Fig. 1B). Moreover, the expression of mbIL-15 (Fig. 1C) and CXCR5 (Fig. 1D) in fourth-generation CAR-T cells was greater than that in CD19 CAR-T cells and UT cells. After obtaining the two types of CAR-T cells, we evaluated their proliferation using the CCK8 assay. We found that, although we modified CD19 CAR-T cells, this modification did not affect CAR-T cell proliferation (t₂₄ = 1.014, p = 0.3679; t₄₈ = 0.9621, p = 0.3905; t₇₂ = 1.584, p = 0.1885) (Fig. 1E).

Fig. 1.

Construction and transfection of CD19 CAR-T cells and CD19 mbIL15-CXCR5 CAR-T cells. A Structural diagram of the anti-CD19 CAR and anti-CD19 mbIL15-CXCR5 CAR. B Schematic representation of the proportion of cells positive for CAR expression. C Positive rate of CXCR5 expression. D Positive rate of mbIL-15 expression. E The proliferation of the two kinds of CAR-T cells was evaluated using the CCK8 assay. ns indicates no statistical significance, *p < 0.05, **p < 0.01, ***p < 0. 001

CD19mbIL15-CXCR5 CAR-T cells effectively induce cytotoxicity in CD19+ tumor cell lines in vitro

To preliminarily validate the in vitro antitumor activity of the CD19 mbIL15-CXCR5 CAR-T cells, Nalm6, Bjab, and U2932 cells were selected as the target cells. An examination of the expression of CD19 antigens in Nalm6, Bjab, and U2932 cells revealed that all three cell lines presented high levels of CD19 expression. These cells were successfully modified to express LGP (luciferase [Luc] and green fluorescent protein [GFP]). The results demonstrated the capacity of CD19 mbIL15-CXCR5 CAR-T cells to efficiently eliminate CD19+ target cells. And it has been validated in multiple cell lines (Fig. 2A).To compare the differences in the cytotoxicity of the two kinds of CD19 CAR-T cells, we selected U2932-LGP cells and cocultured them with two types of CAR-T cells and UT cells at ratios of 1:3, 1:1, and 3:1. We repeatedly introduced the same tumor cells to replicate the prolonged presence of tumor cells in vivo. The results indicated that, at a lower effector-to-target ratio (1:3), the cytotoxicity of CD19 mbIL15-CXCR5 CAR-T cells was significantly greater than that of CD19 CAR-T cells 72 h after initial exposure to the tumor antigen (t = 12.47, p = 0.0064). And in the subsequent second round of tumor antigen stimulation, CD19 mbIL15-CXCR5 CAR-T cells consistently maintained stronger antitumor ability (Fig. 2B). In addition, at ratios of 1:1 (Fig. 2C) and 3:1 (Fig. 2D), although no difference was shown in the first round, after multiple rounds of stimulation, the killing rate of CD19 mbIL15-CXCR5 CAR-T cells was significantly greater than that of CD19 CAR-T cells. Overall, the CD19 mbIL15-CXCR5 CAR-T cells not only demonstrated effective cytotoxicity against CD19+ tumor cells but also exhibited enhanced antitumor efficacy in the presence of prolonged tumor antigen exposure.

Fig. 2.

A Evaluation of the cytotoxicity of CD19 mbIL15-CXCR5 CAR-T cells against CD19+ tumor cell lines. B Two rounds of tumor cells were added by co-culturing CD19 mbIL15-CXCR5 CAR-T cells or CD19 CAR-T cells with U2932 at a 1:3 ratio. C Three rounds of tumor cells were added at a 1:1 ratio. D Four rounds of tumor cells were added at a 3:1 ratio. E Secretion of cytokine during the killing of U2932 by the two CAR-T cells. F Differentiation of the T cell subsets before and after coculture of the two CAR-T cells with tumor cells. All experiments were independently repeated three times.*p < 0.05, **p < 0.01, ***p < 0. 001

We evaluated various indicators of CD19 mbIL15-CXCR5 CAR-T cells and CD19 CAR-T cells. We examined the levels of cytokines in the supernatant of the coculture system. We found that the levels of IL-4, IL-6, and IFN-γ were lower in the CD19 mbIL15-CXCR5 CAR-T cell group than in the CD19 CAR-T cell group (t_IL-4 = 13.04, P = 0.0487; t_IL-6 = 20.89, P = 0.0304; t_IFN-γ = 27.55, P = 0.0231) (Fig. 2E). In the related research on the secretion of cytokines by CAR-T cells, the secretion of IL-4 is considered to be associated with the exhaustion of CAR-T cells [37]. While IL-6 and IFN-γ are related to adverse toxic and side effects after CAR-T cell therapy, such as CRS [38]. We also compared the subpopulations and differentiation of the two types of CAR-T cells. We found that the proportions of central memory T cells (TCMs) and effector memory T cells (TEMs) were greater, whereas the proportion of effector T cells (TEFFs) was lower (Fig. 2F) in the CD19 mbIL15-CXCR5 CAR-T cell group after experiencing tumor antigen stimulation. In addition, we assessed the exhaustion phenotype of both CAR-T cell types and found no significant disparity between them. These findings indicate that CD19 mbIL15-CXCR5 CAR-T cells can continuously kill tumor cells in a stable and low activated form.

The migratory ability of CAR-T cells toward CXCL13+ tumors is enhanced in the CD19-mbIL15-CXCR5 CAR-T cell group both in vitro and in vivo

Next, we verified whether CXCR5 expression improved CAR-T cell migration. Since CXCL13 is the only ligand for CXCR5, we transfected the U2932 cell line with a lentiviral vector to express LGP and CXCL13 to simulate high expression of CXCL13 in malignant B-cell lymphoma in vivo. We used puromycin for screening and successfully identified its presence through flow cytometry and polymerase chain reaction (Fig. 3A). We subsequently performed in vitro Transwell experiments, where the upper chamber was seeded with CD19 mbIL15-CXCL13 CAR-T cells, CD19 CAR-T cells, and UT cells. In the lower chamber, we placed U2932-CXCL13-LGP cells or recombinant human cytokine CXCL13. Six hours later, the number of migrated T cells in the lower chamber was estimated by flow cytometry. As shown in Fig. 3B, at concentrations of 1 and 5 µg/ml CXCL13, the number of T cells in the CD19mbIL15-CXCR5 CAR-T cell group was significantly greater than that in the CD19 CAR-T cell or UT cell groups (P < 0.05). In addition, chemotaxis increased with increasing CXCL13 concentrations. We placed CXCL13-expressing tumor cells in the lower chamber and observed the same results. To further confirm whether CXCR5 could promote CAR-T cell migration to CXCL13-positive tumors in vivo, U2932-CXCL13-LGP cells and U2932-LGP cells were injected subcutaneously into the right and left lateral abdomens of NSG mice, respectively (Fig. 3C). When the tumors were approximately 100–200 mm³ in size, the mice received different treatments. Seven days later, the mice were euthanized, and the tumor tissues on both sides were dissected, fixed, embedded in paraffin blocks, and stained with a CD3 antibody. As shown in Fig. 3D, the presence of CD3+ T cells was not detected in the right abdominal tumors (U2932-CXCL13-LGP) of the mice in either the CD19 CAR-T or the UT group, whereas it was detected in the CD19 mbIL15-CXCR5 CAR-T group at a rate of approximately 10%. In addition, the presence of CD3+ T cells was not detected in the left abdominal tumors (U2932-LGP) of the mice in any of three treatment groups. Therefore, we believe that CD19 mbIL15-CXCR5 CAR-T cells have a stronger ability to infiltrate tumors and are more likely to migrate toward CXCL13 + tumor cells.

Fig. 3.

Increased migration of CD19 mbIL15-CXCR5 CAR-T cells toward CXCL13+ tumors. A U2932 cells were transfected with lentiviruses expressing both LGP and CXCL13, and their expression was detected by flow cytometry. B The effect of CXCR5-CXCL13 on CAR-T cell migration was assessed in vitro. C To evaluate the effect of CXCL13 expression on CAR-T cell migration in a lymphoma mouse model, 1 × 10⁶ U2932-LGP was injected into the left side of the abdomen of each mouse, and 1 × 10⁶ U2932-CXCL13-LGP was injected into the right side of the abdomen of each mouse. (D) Immunohistochemistry was used to evaluate the tumor infiltration ability of two CAR-T cells. ns indicates no statistical significance, *p < 0.05, **p < 0.01. ***p < 0. 001

CD19 mbIL15-CXCR5 CAR-T cells demonstrate enhanced efficacy in inhibiting tumor growth in xenograft models

To further evaluate the antitumor activity of CD19 mbIL15-CXCR5 CAR-T cells in vivo, we constructed a lymphoma mouse xenograft model using U2393-CXCL13-LGP cells (Fig. 4A). Then, we recorded the weights of the mice (Fig. 4B) and the volumes of the tumors (Fig. 4C) every 48 h. We evaluated the tumor growth rate of the mice by regularly measuring their body weight and tumor volume. We found that the tumors in the UT-cell group grew the fastest among the three groups of mice, and the tumor growth rate of the mice in the CD19 CAR-T cell group was inhibited to some extent; however, the inhibition was not as effective as that in the CD19 mbIL15-CXCR5 CAR-T cell group. Then, the mice were euthanized, and a direct comparison of tumor sizes across all groups was conducted (Fig. 4D). The tumors of the CD19 mbIL15-CXCR5 CAR-T cells were significantly smaller than those of the CD19 CAR-T cells and UT cells, indicating better tumor suppression.

Fig. 4.

Good antitumor effects and safety of CD19 mbIL15-CXCR5 CAR-T cells in mouse models. A Flowchart of the animal experiments. B The weights of the mice were monitored at fixed time points. C Mouse tumor size was monitored at fixed time points. D Hepatic and renal functions of the mice: AST, ALT, TBIL, DBIL, ALB, and CREA levels. E Tumor sizes of the three groups of mice 25 days after receiving treatment. F Schematic representation of HE staining of the liver (200×), spleen (200×) and kidney (200×) of the mice 25 days after receiving treatment. G Spleen size and morphology of the mice

The safety of CD19 mbIL15-CXCR5 CAR-T cell therapy for lymphoma was evaluated. Before the mice were euthanized, several biochemical indices associated with the liver and kidney were detected via the serum. We compared the specific values of alanine aminotransferase, alanine aminotransferase, total bilirubin, indirect bilirubin, direct bilirubin, and creatinine in the serum of the mice in each group and found that the liver and kidney function indicators of the CD19mbIL15-CXCR5 CAR-T cell-treated group were within the normal range and did not show any liver or kidney toxicity (Fig. 4D). Organ tissue samples, such as liver, spleen, and kidney samples, from the mice in the different treatment groups were subjected to HE staining to observe whether there were any abnormal changes in various organs of the mice after they received different treatments. The results revealed that the internal organs of the mice in the UT, CD19 CAR-T, and CD19 mbIL15-CXCR5 CAR-T cell groups were not obviously damaged (Fig. 4F). There was no significant difference in spleen size among the groups (Fig. 4G). These results indicate that CD19 mbIL15-CXCR5 CAR-T cells have a good safety profile for the treatment of lymphoma.

Discussion

In this study, on the basis of the limitations of CAR-T cell therapy for B-cell lymphoma, we constructed fourth-generation CAR-T cells targeting CD19, which express membrane-bound (mb) IL-15 and CXCR5 (CD19mbIL15-CXCR5 CAR-T cells). IL-15 can induce the formulation of memory cells, enhance the adaptability of T cells by slowing their senescence, and preserve the naive/stem cell memory phenotype [22, 23, 39, 40]. These effects are beneficial for the efficacy of CAR-T cell therapy [24, 41]. However, high doses of IL-15 may cause toxic side effects in patients, leading to ultrastructural activation of T cells and causing abnormal proliferation or toxicity of T cells. However, mbIL-15 can avoid these problems [41–43]. CXCR5 is an endogenous B-cell-homing receptor and the only receptor for CXCL13, which helps improve the inability of T cells to penetrate the tumor site. We demonstrated via in vitro and in vivo experiments that CD19mbIL15-CXCR5 CAR-T cells have stronger antitumor ability, better persistence, and better tumor infiltration ability. Unlike traditional CD19 CAR-T cells, CD19 mbIL15 CXCR5 CAR-T cells exert more functions. Moreover, we did not observe abnormal changes in the spleen, liver, or kidney after treatment with CD19 mbIL15-CXCR5 CAR-T cells. There was also no significant difference in the size of the spleen among all the mice.

There are still some limitations in this study. Firstly, through flow cytometry detection, we found that the fluorescence ratio of CXCR5 in CD19 mbIL15-CXCR5 CAR-T cells was lower than that of mbIL-15 and CAR. We speculated that this might be due to partial folding of the CXCR5 protein, which hindered its expression of the same fluorescence as other transgenic products in flow cytometry. Although the functionality of CXCL5 has been preliminarily verified, in subsequent explorations, we will conduct experiments to further confirm its surface localization and signal transduction ability. Second, we did not use primary tumor cells with high expression of CXCL13 for validation. In subsequent studies, we collected primary lymphoma cells from patients to detect the expression of CXCL13 and further validate the function of CD19 mbIL15 CXCR5 CAR-T cells using CD19-positive and CXCL13-positive lymphoma primary cells using in vitro experiments and patient-derived tumor xenograft (PDX) animal models. We will increase the number of experimental mice, test more indicators, and optimize the detection methods, such as using flow cytometry to detect the infiltration ability of CAR-T cells, in order to better evaluate the safety and functionality of CD19 mbIL15-CXCR5 CAR-T cells before clinical translation. Additionally, we found that, compared with that of traditional CD19 CAR-T cells, the transfection rate of CD19 mbIL15 CXCR5 CAR-T cell therapy was significantly lower. This might be due to the lower infection efficiency of the large lentiviral construct, or it could be that the large messenger/protein before T2A cleavage leads to a reduction in protein synthesis efficiency. Nanobody-based CAR-T cells may be able to solve this problem. Compared with scFvs, nanoantibodies have a smaller structure, lower immunogenicity, and better therapeutic effects [44]. In the future, we may choose nanoantibodies to replace the current scFv segment for the preparation of CD19 mbIL15-CXCR5 CAR-T cells. Finally, in subsequent clinical applications, we still need to evaluate the safety of CD19 mbIL15-CXCR5 CAR-T cell therapy. Although there is no research indicating that mbIL15 has adverse effects on CAR-T cells, safety threats are a concern in clinical applications because of the potential adverse effects of high-dose IL-15 on T cells. Moreover, whether the CXCR5/CXCL13 immune axis leads to off-target effects of CD19 mbIL15-CXCR5 CAR-T cell therapy also needs to be validated in subsequent studies. Although this study has initially demonstrated some positive effects of CD19 mbIL15-CXCR5 CAR-T cells, continuous evaluation is still needed for their clinical translation to ensure that patients can benefit from CD19 mbIL15-CXCR5 CAR-T cells.

The tumor microenvironment plays a significant role in the development of drug resistance in B-cell lymphoma. The microenvironment of B-cell malignancies is one of the main factors that restrict the efficacy of immunotherapy. By enriching immunosuppressive cells such as regulatory T cells and upregulating immune checkpoints, it affects the effectiveness and durability of immunotherapy [45]. How to reduce its negative impact on treatment is a key issue facing the treatment of B-cell lymphoma. Currently, researchers have proposed many strategies. For instance, combining small molecule drugs, such as the combined treatment of ibrutinib and focal adhesion kinase inhibitors, to inhibit the generation of focal adhesion kinase and thereby reduce the resistance of mantle cell lymphoma to ibrutinib [46]. Combining PD-1 inhibitors or radiotherapy to reduce the negative impact of the tumor microenvironment and thereby enhance the therapeutic effect of immunotherapy such as CD19 CAR-T cells [47, 48]. This study provides ideas for improving the therapeutic effect of B-cell malignancies. Only through continuous exploration can we find more suitable treatment methods for B-cell lymphoma patients and bring hope to more patients.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contribution

XPC, JLL and MHZ wrote the manuscript draft. MZ and SJG designed the study. XMZ, RS, RTG, YFZ and YZ collected and analyzed the data. MFZ revised the manuscript. All the authors contributed to the article and approved the final version of the manuscript.

Funding

This work is sponsored by Tianjin Health Research Project (Grant No. TJWJ2024QN040); the major special project on public health science and technology in Tianjin (24ZXGZSY00120); Tianjin Key Medical Discipline (Specialty) Construction Project (TJYXZDXK-056B), and the Science and Technology Project of Tianjin Municipal Health Committee (TJWJ2022XK018, TJWJ2022QN030). This work is also funded by Tianjin key Clinical Specialty Construction Project and Tianjin Key Medical Discipline Construction Project (Grant No.TJYXZDXK-3-001A-004).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Competing interest

The authors declare that the research was conducted without any commercial or financial relationships that could be construed as potential conflicts of interest. The authors declare no competing interests.

Ethics approval

This study was approved by the Ethics Committee of Tianjin First Central Hospital. Our study complies with the Declaration of Helsinki. All patients signed informed consent forms. All animal experiments and procedures were approved by the Ethics Committee of Tianjin First Central Hospital. We confirmed that the tumor burden did not exceed the recommended dimensions and that the animals were anesthetized and killed using acceptable methods. The mice were euthanized using cervical dislocation. The death of the experimental animals was determined by paying attention to their respiration, pupils, and neural responses.