Lenalidomide overcomes the resistance to third-generation CD19-CAR-T cell therapy in preclinical models of diffuse large B-cell lymphoma

Chimeric antigen receptor (CAR)-T cells against CD19 have been proven to be effective in treating B-cell hematological malignancies. However, the efficacy of this promising therapy is limited by many factors. In this study, the germinal center B-cell-like diffuse large B-cell lymphoma (GCB-DLBCL) cell line OCI-Ly1, and patient-derived xenografted (PDX) mice (CY-DLBCL) were used as the CAR-T cell-resistant model. Meanwhile, the activated B-cell-like (ABC) DLBCL cell line OCI-Ly3 and PDX mice (ZML-DLBCL) were defined as the CAR-T sensitive model. The enhancement of CAR-T cell function by lenalidomide (LEN) was examined in vitro and in vivo. Lenalidomide effectively enhanced the function of third-generation CD19-CAR-T cells by polarizing CD8+ CAR-T cells to CD8 early-differentiated stage and Th1 type, reducing CAR-T cell exhaustion and improving cell expansion. It was further demonstrated that CAR-T cells combined with LEN substantially reduce the tumor burden and prolong the survival time in various DLBCL mouse models. LEN was also found to promote the infiltration of CD19-CAR-T cells into the tumor site by modulating the tumor microenvironment. In summary, the results of the present study suggest that LEN can improve the function of CD19-CAR-T cells, providing a basis for clinical trials using this combination therapy against DLBCL.


Introduction
Remarkable progress has been achieved in the treatment of hematological malignancies by using CD19 chimeric antigen receptor (CAR)-T cells. An increasing number of patients with refractory or relapsed acute lymphocytic leukemia, Zhen Jin, Rufang Xiang, Kai Qing and Dan Li contributed equally to this work. chronic lymphocytic leukemia, and B-cell non-Hodgkin's lymphoma have benefited from this new therapeutic strategy. However, landmark clinical trials have shown that the complete response rates achieved in patients with B-cell non-Hodgkin's lymphoma (B-NHL) and chronic lymphocytic leukemia (CLL) are not as high as in patients with acute lymphocytic leukemia (ALL) [1,2]. The efficacy of CAR-T cell treatment has been found to correlate with many factors, such as antigen loss, expansion or persistence of CAR-T cells, as well as CAR-T cell exhaustion [3][4][5][6]. In addition, physical barriers, such as blood vessels and lymphatics, as well as the immunosuppressive microenvironment, also hamper CAR-T cell infiltration and activity, making it more difficult to achieve a high remission rate for CAR-T treatment in patients with lymphoma [7]. Efforts have been made to improve the efficacy of CAR-T cells against lymphoma by designing new CARs, improving the manufacturing process, and overcoming the suppression of the tumor microenvironment [8][9][10]. Third-generation CD19-CAR-T cells have been reported to have a greater expansion in vivo than the second generation of CD19-CAR-T cells [11].
Lenalidomide is one of the immunomodulatory drugs that have a direct antitumor effect in multiple myeloma cases by directly binding to E3 ubiquitin ligase cereblon and inducing Ikaros and Aiolos degradation [12]. This drug exerts its immunomodulatory effects by regulating the immune cells in the tumor environment, including T-cells, B-cells, dendritic cells, and natural killer cells [13]. Previous studies have shown that lenalidomide could skew T-cells towards the Th1 antitumor immune response [14], enhance immune synapse formation between T-cells and natural killer cells, and reduce the number of regulatory T-cells [13].
The present study investigated the effectiveness of combining third-generation CD19-CAR-T cells with lenalidomide administration. The results of this study suggest that this combinatorial strategy is superior to CAR-T monotherapy against DLBCL. Previous studies from our research group have shown that lenalidomide enhances third-generation CD19-CAR-T cells by polarizing CAR-T cells toward CD8 + memory type T-cells and Th1 cells in vitro [15]. In this study, we further utilized in vitro and in vivo models to investigate the effect of lenalidomide on overcoming DLBCL resistance to CAR-T-cells.

CAR-T cell generation
The CAR construct contained a CD19-specific scFv, a CD28 costimulatory domain, a 4-1BB costimulatory domain, and a CD3ζ chain. The genes of mCherry fluorescent protein and firefly luciferase were linked with CAR using the P2A sequence peptide to monitor CAR expression in vitro and in vivo. Both genes were cloned into the pHR lentiviral vector.
Blood samples were obtained from patients with DLBCL and healthy volunteers following a protocol approved by the University Institutional Review Board. The T-cells were isolated from the collected samples using a Human CD3 + T-cell Enrichment Kit (Stem Cell, USA) and cultured in AIM-V (Invitrogen, USA) supplemented with 6% AB human serum (GEMINI, US) and 30 IU/mL human interleukin (IL)-2 (Peprotech, US). The T-cells were transduced with lentiviral vectors at a multiplicity of infection (MOI) ≈ 10 after stimulation with CD3/CD28 Dynabeads (Invitrogen, USA) for 72 h. The CAR expression was detected by flow cytometry either with mCherry or anti-F(ab) staining (Jackson Immu-noResearch, USA). The median transduction efficiency was 66%, ranging from 44.2% to 71.6%.

Flow cytometry
Cells were harvested from in vitro experiments and animals, washed twice with phosphate-buffered saline (PBS), and stained with antibodies (Table S1) for 15-20 min. The cell phenotypes were analyzed using FlowJo 10.0 software (Tree Star, USA).

Cytotoxicity assay
Lenalidomide (Selleck, USA) was dissolved in DMSO at a concentration of 2 μM, since the therapeutic dose of lenalidomide could not exceed 2.2 μM [16]. The target cells, including OCI-Ly3, OCI-Ly1, Su-DHL2, and Su-DHL4, were cocultured with CAR-T cells for 7 h. The effector-to-target cell (E/T) ratio was based on the count of CAR-positive cells obtained by flow cytometry. The antitumor effect of the treatment was assessed through the lactate dehydrogenase (LDH) assay, performed using the CytoTox96 Kit (Promega, WI, USA) following the manufacturer's protocol. Cytotoxicity was calculated in the form of LDH release as follows:

Degranulation assay
The CAR-T cells were co-cultured with OCI-Ly1 or OCI-Ly3 cells at the effector-to-target ratio of 5:1. Thereafter, a CD107a antibody (BioLegend, USA) was added to each well, followed by the addition of monensin solution (Sigma-Aldrich, USA) after a 1 h incubation. Flow cytometry was performed 5 h later to determine the percentage of CAR + CD107a + cells.

Cell counting kit-8 assay
The cell counting kit-8 (CCK-8) assay (Dojindo, Japan) was used to measure the proliferation of DLBCL and EA.hy926 cells. Cells were seeded in a 96-well plate and cultured in different media, including lenalidomide-supplemented medium and medium supplemented with macrophage cell culture supernatant with or without lenalidomide. Growth inhibition was determined from absorbance measurements obtained using the CCK-8 assay, following the manufacturer's protocol.

Carboxyfluorescein succinimidyl ester (CFSE) assay
CAR-T cells were stained with CellTrace CFSE staining solution (ThermoFisher, USA) before co-culturing with DLBCL cells (with or without lenalidomide administration). After 72 h or 96 h of incubation, the cells were harvested and flow cytometry was performed to detect proliferation.

In vitro migration assay
EA.hy926 cells were first seeded in Transwell inserts (Corning, USA) at a density of 1 × 10 4 cells per well and incubated for 4 h. These cell-seeded Transwell inserts were transferred into a 24-well plate seeded with macrophages. The co-cultures were cultured in medium with or without 2 μM lenalidomide. After 72 h, the Transwell inserts were moved to a new 24-well plate and incubated in complete medium supplemented with either CLL19 or CLL21. Thereafter, CAR-T cells were seeded above the EA.hy926 cell layer at a density of 5 × 10 5 cells per insert. After 5 h, the CAR-T cells that had migrated through the EA.hy926 cell layer and reached the bottom wells were harvested, counted, and subjected to flow cytometry for identification. This protocol has been illustrated in Figure S10.

Real-time polymerase chain reaction analysis
RNA content of the CAR-T cells was extracted using the ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech, China), and the corresponding cDNA was synthesized. Genes related to T-cell function were selected and identified using a polymerase chain reaction (PCR) array (Qiagen, Germany). The relative cDNA expression was quantified after normalization.

Animal experiments
NOD-SCID mice, aged 6-8 weeks, were obtained from the Shanghai Laboratory Animal Center (Shanghai, China).
All the experiments were performed following the protocol approved by the Animal Care and Use Committee of Ruijin Hospital, affiliated to the School of Medicine at the Shanghai Jiao Tong University. The mice used in the experiments were sex-matched. Bone marrow (BM) involvement occurs in 11-34% of DLBCL patients at initial diagnosis [17,18], which is associated with poor prognosis [19]. Thus, the "lymphomaleukemia xenograft mouse model" was established in the present study to simulate DLBCL progression by injecting luciferase-positive OCI-Ly3 cells via tail vein. Primary cells isolated from patients or OCI-Ly3 cells were also subcutaneously injected into the right flank of each mouse. Tumorbearing mice were randomized by tumor burden and infused either with saline, lenalidomide 10 mg/(kg ⋅ d), or CAR-T cells with or without LEN 10 mg/(kg ⋅ d). Detailed protocol is presented in Figs. 4 and 5. Bioluminescent imaging was performed using an in vivo imaging system (PerkinElmer, USA) and analyzed with the help of the Living Imaging software (PerkinElmer, USA) to determine the extent of infiltration of CAR-T cells and the tumor burden. To establish the ABC-DLBCL and GCB-DLBCL patient-derived xenograft mouse model, primary ZML-ABC and CY-GCB cells obtained from patients were subcutaneously injected in the right flanks of NOD-SCID mice. The tumors in PDX mouse mimicked the histology and gene expression observed in ABC/GCB-DLBCL patients.

Statistical analysis
Statistical analysis was performed with GraphPad Prism Software (GraphPad, CA, USA). The t test was used for comparing two groups, while one-way analysis of variance (ANOVA) was used for comparing more than two groups. Survival analysis was conducted using the Kaplan-Meier method and comparisons were made using the log-tank test. Comparisons for which P < 0.05 were considered to be statistically significant.

Efficacy of lenalidomide in killing ABC-DLBCL cells was moderate in vitro and in vivo
Since ABC and GCB are two major subtypes of DLBCL, two cell lines (namely, Su-DHL2 and OCI-Ly3) were chosen to represent the ABC subtype, while the cell line OCI-Ly1 and primary CY-GCB cells were used to represent the GCB subtype. All cell types showed a dosedependent growth inhibition with an increase in the concentration of lenalidomide. The ABC subtype was more susceptible to LEN-induced cell death compared to the GCB subtype, and showed a cell viability of 80% upon treatment with 2 μM LEN (Fig. 1a), consistent with previous findings [20]. Of note, the IC 50 of LEN was not reached in the concentration range used, even in sensitive cell lines. Higher doses were not examined because the maximum observed plasma concentration (C max ) for patients treated with lenalidomide cannot be greater than 2.2 μM. For in vivo experiments, two PDX mouse models were established, both of which had characteristics that were consistent with tumors in patients (Fig. S1). Meanwhile, the lymphoma-leukemia model was established by injecting firefly-luciferase-positive OCI-Ly3 cells via the tail vein. All tumor-bearing mice were treated with 10 mg/ kg LEN daily. The ZML-engrafted mice responded to LEN treatment with a significant suppression of tumor growth and c. Luciferase-positive DLBCL cells (1 × 10. 6 cells/mouse) were injected through the tail vein to establish the lymphoma-leukemia model. On day 5, the mice were randomized according to the total bioluminescence (BLI) and lenalidomide was administered at a dose of 10 mg/kg (five mice in each group). A single treatment of lenalidomide did not show any anti-lymphoma effect as there was neither a significant decrease in tumor burden (d; t-test on day 3 and 16, P > 0.05) nor a significant increase in the overall survival (e; logrank test, P > 0.05). Representative data of seven in vivo experiments are presented. (* P < 0.05) (P < 0.05) (Fig. 1b). However, the CY-GCB tumors were resistant to LEN treatment (Fig. 1c). In the OCI-Ly3-lucbearing mice, LEN treatment neither prolonged the overall survival nor decreased the tumor burden (Fig. 1d, e). In conclusion, LEN alone exerted a moderate antitumor effect against ABC-DLBCL in vitro, but did not significantly affect tumor growth in GCB-DLBCL-bearing mice.

DLBCL cells responded to treatment with third-generation CD19-CAR-T-cells
Third-generation CD19-CAR was transduced into T-cells to evaluate the cytotoxic effect of CD19-CAR-T cells.
More than 50% of the CD19-CARs were successfully expressed on the T-cell surface (Fig. 2a). Given the results obtained above, OCI-Ly1 and OCI-Ly3 cells were selected The activation markers were dramatically increased when CAR-T cells were exposed to DLBCL cell lines stimulation. The expression of CD69 and CD137 was assessed by flowcytometry since the CAR-T cells were co-cultured with cell lines for 5 h. The IL2 and IFN-γ secretion was also identified after CAR-T cell co-culturing with cell lines overnight. (g) CAR-T cell proliferation assay. CAR-T cells were labeled with CFSE staining solution and co-cultured with K562 (CD19-negative), OCI-Ly1 and OCI-Ly3 cells at an E/T ratio of 1:1. Three or four days later, CFSE expression was assessed by flow cytometry. Data are presented as mean ± SD. Three replicates per condition. (* P < 0.05, ** P < 0.01, *** P < 0.001) to represent the major subtypes of DLBCL (OCI-Ly1 for GCB-DLBCL, OCI-Ly3 for ABC-DLBCL) in the following experiments. The cytotoxicity of CAR-T cells increased with an increase in the E/T ratio, and this trend was more obvious in OCI-Ly3 cells than in OCI-Ly1 cells (Fig. 2b). This suggests that DLBCL cell lines have different sensitivities to CAR-T cells. Thereafter, the anti-lymphoma effect induced by CD19-CAR-T cells was assessed in the lymphoma-leukemia mouse model and the PDX mouse model. As shown in Figs. 2c and d, CD19-CAR-T cells showed a dose-dependent killing effect. Notably, the ZML-DLBCL mice were more sensitive to CAR-T treatment compared to the CY-DLBCL mice. CD19 expression, degranulation, and CAR-T cell activation markers were also investigated. The results revealed that CAR-T cell could be successfully activated by DLBCL cell lines (Fig. 2f). OCI-Ly3 and OCI-Ly1 cells had comparable CD19 expression, degranulation level and CAR-T cell expansion ( Fig. 2e, g, and Fig. S2b), suggesting that the antigen expression or CAR-T cell activation may not contribute to tumor resistance to CAR-T cell treatment.

LEN overcame DLBCL resistance to third-generation CD19-CAR-T cells by regulating the differentiation of CD8 + CAR-T cells.
The cytolytic effect of CD19-CAR-T cells was further examined in the presence of varying concentrations of LEN within the physiological range. LEN was found to substantially improve the antitumor efficacy of CD19-CAR-T cells against both types of DLBCL cells (P < 0.05) (Fig. 3a,  Fig.S3). A 3-day proliferation assay as well as degranulation and cytokine release assays were conducted to investigate the contribution of LEN in overcoming the resistance of DLBCL to CAR-T treatment. Upon LEN treatment, the CAR-T cells experienced slight growth, but the proliferation was mitigated when the concentration was higher than 2 µM (Fig. 3b). Moreover, it was found that CAR-T cell degranulation can be enhanced by LEN (Fig. 3c). LEN also increased the secretion of IL-2, IFN-γ, and TNF-α, and decreased the secretion of IL-6 and IL-10, without significantly affecting IL-4 secretion (Fig. 3d). These results suggest that LEN can promote the Th1-polarizing-like phenotype of CAR-T cells.
Since LEN has previously been demonstrated to impact T-cell function by modulating a subset of T-cells by directly targeting cereblon protein [21], the effect of LEN on the differentiation of CD19-CAR-T cells involved in DLBCL cell death was further investigated. Although the proportion of the CD8 + T CM subset of T-cells in the process of killing OCI-Ly1 cells was smaller than that in killing OCI-Ly3 cells, the percentage of CD8 + T CM (central memory T cell) subtype displayed a consistent increase in population as the concentration of LEN increased, with a comparable T CM proportion between OCI-Ly1 and OCI-Ly3 cells at a LEN concentration of 2 μM. The CD4 + Th1 subset also experienced a gradual increase in population under LEN treatment during OCI-Ly3 cell death (P < 0.05) (Fig. 3e, Fig  S5a, b.). In contrast, the change in the population of T EM (effector memory T cell) affected by LEN was not significant (Fig. S5c). CAR-T cells derived from patients were subsequently tested and similar results were observed, suggesting that LEN has consistent effects on CAR-T cells obtained from different sources (Figs. S4, S5d, e). The CAR-T cells were then divided into CD8 + and CD4 + subgroups and cocultured with OCI-Ly3 or OCI-Ly1 cells. LEN improved the cytolytic effect of CD8 + CAR-T cells under the physiological concentrations (Fig. 3f, Fig S6a), suggesting that a change in the subsets of CD19-CAR-T cells, especially in the CD8 + subgroup, could be the underlying reason for their enhanced antitumor function in the presence of LEN. Further, the phosphorylation of glycogen synthase kinase-3 (GSK-3) and MEK, both suggested to regulate CD8 differentiation [22,23], was downregulated upon LEN treatment (Fig. 3g, Fig S5b). LEN also induced a series of changes in the gene expression profile of CD19-CAR-T cells. Genes such as EOMES and TCF1, which are related to terminal differentiation, were downregulated, while genes related to the oxidative metabolism, such as CPT1A and FABP5, were upregulated by LEN treatment (Fig. 3h). Collectively, LEN at therapeutic doses may enhance the antitumor function of CAR-T cells mainly by polarizing CD8 + differentiation in vitro.

Resistance of DLBCL to CD19-CAR-T-cell treatment could be overcome by LEN treatment in vivo
The effect of LEN on enhancing the antitumor potency of third-generation CD19-CAR-T-cells was further examined in the lymphoma-leukemia mouse models. A schema of the treatment protocol was described in Fig. 4a. Tumor growth was substantially suppressed by the combination therapy, compared to LEN or CAR-T monotherapies ( Fig. 4b and 4c). CAR-T combined with LEN treatment also significantly prolonged the survival time (Fig. 4d). Since CAR-T cell expansion in patients has previously been hypothesized to be associated with the overall response to the CAR-T treatment [5], the number of CAR-T cells in the peripheral blood was also counted 5 days after CAR-T cell infusion. LEN promoted CAR-T cell expansion in vivo (Fig. 4e), without significantly altering the CD8 + subpopulation (Fig. 4f). LEN also increased the T CM proportion, but reduced the T E proportion of CAR-T cells in vivo (Fig. 4g). Furthermore, the expression of both programmed death 1 (PD-1) and T-cell immunoglobulin and mucin domain 3 (TIM-3) proteins on CAR-T cells was substantially reduced under daily LEN treatment, whereas no difference was observed in the expression of CD4 + and CD8 + CAR-T cells were isolated from the bulk CAR-T cells treated with 2 μM lenalidomide by negative selection using the EasySep Human CD4 + /CD8 + T-cell Enrichment Kit. The LDH assay was conducted 7 h after the CD4 + subset of CAR-T cells or CD8. + cells were co-cultured with OCI-Ly3 with or without lenalidomide. (g) Protein levels of GSK3β/MEK and their phosphorylation as noted through Western blot analysis. GAPDH was used as a loading control. (h) Genes related to memory function and metabolic status were selected and identified with real-time PCR. Data are presented as mean ± SD. Three replicates per condition. (* P < 0.05, ** P < 0.01, *** P < 0.001) LAG-3 and CTLA4 proteins (Fig. 4h). To determine if the memory subpopulation was functionally established, NOD-SCID mice bearing OCI-Ly3 cells were injected with CAR-T cells (group A) or CAR-T cells with daily treatment of LEN (group B), as described above (Fig. 4a). At day 23, the mice in groups A and B were rechallenged with OCI-Ly3 cells via tail vein injection. As shown in Fig S7a, survival benefit was observed in the combination treatment group (P < 0.05). After observing the enhancement of anti-DLBCL function of CD19-CAR-T cells by LEN in the lymphoma-leukemia mouse model, further investigations were performed in the PDX mouse models. As shown in Figs. 5a and b, CY-DLBCL in the PDX mouse model grew more aggressively than ZML-DLBCL, with a larger tumor volume at the same time-point. The infusion of CAR-T cells delayed tumor growth, and additional LEN treatment enhanced this antitumor efficiency. Among the entire population of CD8 + CAR-T cells, only the subpopulation infiltrating the CY-DLBCL tumor site increased due to daily treatment with LEN (Fig. S8d). In the context of the impact of LEN on CD8 + CAR-T cell differentiation, the proportions of earlydifferentiated CAR-T cells, like T N and T CM , were upregulated in the peripheral blood as well the tumor sites (Fig. 5d,  g and Fig. S8b, e). Moreover, PD-1 expression in the infiltrating and peripheral CAR-T cells was substantially reduced in the presence of LEN (Fig. 5e, h and Fig. S8c, f). Additionally, the expression of both PD-1 and TIM3 was downregulated by LEN in the T CM subset of peripheral CAR-T cells in both CY-DLBCL and ZML-DLBCL mouse models. LEN also reduced the expression of PD-1 and TIM3 in the T CM subset of intra-tumoral CAR-T cells in the ZML-DLBCL PDX mouse model, but no significant differences were found in the CY-DLBCL model (Fig. S9). Collectively, the findings suggest that LEN helped overcome the resistance of DLBCL to CAR-T cell treatment by potentially reducing CAR-T cell exhaustion and promoting the earlier differentiated proportion of CAR-T cells.

LEN enhances the potency of CAR-T cells to infiltrate into tumor sites
CAR-T cells expressing firefly luciferase were generated to further investigate the impact of LEN on CAR-T cell infiltration. The CAR-T cells were infused via the tail vein with or without daily LEN treatment 30 days after the ZML-DLBCL cells (ABC type) were engrafted. As shown in Figs. 6a and b, LEN treatment led to an increase in the bioluminescent signal intensity, indicating that the number of infiltrating CAR-T cells was increased by LEN. The same conclusion was reached for the CY-DLBCL (GCB type) mouse model ( Fig. 6c and d). The vascular endothelial growth factor C (VEGF-C), generated from tumor-associated macrophages, plays an essential role in the formation of tumor lymphatic vessels, which provide a physical barrier that prevents T-cell infiltration into the lymphoma site [24]. Therefore, CAR-T cell migration was assessed using the Transwell assay to investigate if LEN can improve CAR-T cell infiltration by regulating VEGFC secretion by macrophages. The endothelial cell line EA.hy926 in the Transwell inserts was co-cultured with human macrophages with or without LEN in the medium. Thereafter, the Transwell inserts were transferred into new bottom wells containing CCL19 or CCL21, the key homing chemokines secreted by lymph nodes. The number of migrated CAR-T cells was significantly higher in the LEN-treated wells than the untreated controls (Fig. 6e). Also, the migrated (infiltrating) cells exhibited a memory-T-biased phenotype (Fig. 6f), suggesting more intercellular gaps in the Transwell inserts generated by EA.hy926. When cell viability was evaluated, it was found that LEN could not directly inhibit EA.hy926 cell proliferation. On the contrary, the viability of EA.hy926 cells increased in the presence of the macrophage cell culture supernatant, and decreased to the basal level with LEN pretreatment (Fig. 6g). As shown in Fig. 6h, the secretion of VEGFC by macrophages was indeed downregulated with LEN treatment. Collectively, physiological levels of LEN downregulated VEGFC secretion by macrophages, which may lead to the suppression of endothelial cell viability and facilitate the infiltration of CAR-T cells.

Discussion
Over the years, the complete remission (CR) rate achieved by CD19-CAR-T-cell treatment in B-NHL has improved, but it is still around 50% according to landmark clinical trials, which is significantly lower than that for B-ALL [5,25,26]. Different strategies, from bench to bedside, are being used to improve the CAR-T cell functionality and antitumor efficacy. In the present study, it was found that LEN can substantially increase the cytotoxicity of third-generation CD19-CAR-T cells against DLBCL by directly modulating CD8 + CAR-Tcell differentiation and potentially impacting the DLBCL microenvironment.
In various clinical trials, nearly one-third of the patients receiving CAR-T cell therapy relapsed owing to the loss of the CD19 antigen [5,27,28]. However, in the present study, no significant difference was observed in CD19 expression between OCI-Ly1 and OCI-Ly3 cells, even in the presence of LEN or CAR-T cells, indicating that other factors may potentially impact the cytolytic effects of CAR-T cells. Furthermore, in the present study, the response of the GCB cell line OCI-Ly1 to CD19-CAR-T cells was not as strong as that of the ABC cell line OCI-Ly3. This effect was also confirmed in the PDX mouse model, wherein CY-DLBCL (GCB-DLBCL subtype) showed greater resistance to CAR-T cell therapy than ZML-DLBCL (ABC-DLBCL subtype). It is well known that the GCB-DLBCL subtype tends to have better clinical outcomes than the non-GCB subtype. However, a recent study reported a novel classification of DLBCL according to genetic aberrations, consisting of the EZB (based on EZH2 mutations and BCL2 translocations), BN2 (based on BCL6 fusion and NOTCH2  (a and b) Tumor growth curve of the ZML-DLBCL and CY-DLBCL PDX mouse models. The ZML-DLBCL (ABC-DLBCL primary cells, 1 × 10 6 cells per mouse) cells were subcutaneously injected into the right flank of the NOD-SCID mouse. On day 7, the animals were randomized into four groups and administered lenalidomide (10 mg/(kg •d) per mouse), 1 × 10 7 CAR-T cells per mouse, or CAR-T cells with lenalidomide (five mice in each group). The mice were culled on day 23 after CAR-T cell infusion. Images of the tumors are shown in the left panel of (a). Tumor volume was estimated using the following formula: length × width 2 /2, and is depicted in the right panel of (a). For the GCB-DLBCL PDX mouse model, NOD-SCID mice were engrafted with CY-DLBCL primary cells (1 × 10 6 in each mouse). After random grouping on day 7, the mice were treated with CAR-T cells or lenalidomide at the same dose as for the ABC-DLBCL PDX mouse model (five mice in each group). Mice were killed on day 15, and the tumor volume is shown in (b). (c) Percentage of peripheral blood CD8 + CAR-T cells in the ZML-DLBCL PDX mouse model. Fourteen days after CAR-T cell infusion, the peripheral blood was collected and stained with antibodies, and flow cytometry was performed to assess CD8 + CAR-T cell expression. The EZB subtype is dominated by GCB cases with the hallmark of BCL2, while nearly 41% of the ABC cases belong to the BN2 subtype [29]. Another recent study identified the molecular features of DLBCL in CAR-T clinical trials. Unexpectedly, the molecular features which are predictive of an inferior response to first-line treatment (A53, MCD) tend to have favorable CAR-T treatment outcomes. Mutations in BTG2, MYD88 and CD79B have been shown to indicate favorable progression free survival (PFS). Meanwhile, driver mutations, including MYC,BCL2, CDKN2A, and KLHL6, have been found to be associated with inferior PFS [30].
MCD is found exclusively in the ABC subtype, featuring the MYD88 L265P and CD79B mutations. Cell lines in the present study were also representative: the OCI-Ly3 cell line carries the MYD88 L265P mutation, while the OCI-Ly1 cell line overexpresses BCL2 [31,32], which could explain why CY and OCI-Ly1 (the GCB subtype) are resistant to CAR-T cell treatment. In summary, the subtypes of DLBCL (or special genetic aberrations) may control the CAR-T cell response, but more pre-clinical work and clinical trials are needed to elucidate this.
It is known that lenalidomide affects T-cell function by promoting immune synapse formation and stimulating the cytotoxic CD8 + and helper CD4 + T-cells [13]. Moreover, DLBCL patients in the maintenance therapy stage are known to respond favorably to LEN treatment [33]. Hence, the present study tested if a therapeutic dose of LEN could enhance Transwell inserts coated with equal numbers of endothelial cells were first cultured in the macrophage supernatant with or without lenalidomide. Thereafter, the inserts were coated with equal numbers of CAR-T cells and moved to pre-seeded wells with indicated stimulants. Five hours later, the cells that had migrated to the bottom wells were harvested and counted. Data are presented as mean ± SD. Three replicates per condition. (f) Migrated CAR-T cells presented with memory type based on flow cytometry. Cells were pre-gated for live CD3 + CD8. + CAR-T cells. (g) Endothelial cell proliferation under different conditions. Equal numbers of EA.hy926 cells were placed in a 96-well plate and cultured for 72 h with either 2 µM lenalidomide, macrophage cell culture supernatant, and a mixture of macrophage cell culture supernatant and lenalidomide. Three days later, the CCK8 assay was performed to measure the viability of the EA.hy926 cells. Data are presented as mean ± SD. Three replicates per condition. (h) Lenalidomide decreased VEGF-C secretion by macrophages. Supernatants in (h) were subjected to an ELISA to evaluate VEGF-C secretion. Data are presented as mean ± SD. Three replicates per condition. (* P < 0.05, ** P < 0.01) the antitumor effect of third-generation of CD19-CAR-T cells. This study demonstrated that while a therapeutic dose of LEN barely suppressed DLBCL growth, it still helped to overcome the resistance of DLBCL cells to CAR-T cells by directly improving degranulation, regulating cytokine production, and affecting the distribution of early differentiated CAR-T cells. An analysis of the redistribution of CAR-T cell subsets revealed that the populations of CD8 + T CM and Th1 CAR-T cells gradually increased in the presence LEN, while the terminally differentiated CAR-T cells were distributed randomly. Noticeably, the differences between the CD8 + T CM subpopulations of OCI-Ly1 and OCI-Ly3 during the cytolytic process decreased during LEN treatment, indicating that the early differentiated CAR-T cells contribute to the enhancement of the anti-lymphoma effect of the combinatorial therapy.
It is possible to arrest T-cell differentiation by inhibiting Wnt signaling or the MEK pathway [8,22,34]. This results in increased oxidative metabolism [35]. In the present study, it was observed that LEN suppresses GSK-3β and MEK phosphorylation in CD8 + CAR-T cells, and also upregulates the CPT1A and FABP5 mRNA levels for memory CD8 + CAR-T cell maintenance.
It has been reported that T CM has superior antitumor activity compared with T EM due to better persistence and proliferation [36,37]. This leads to a superior CR rate among CD19-CAR-T-treated DLBCL patients [38]. In the present study, LEN was consistently found to boost CAR-T cells expansion in vivo with the phenotypes of an early differentiation stage (T N or T CM ) in the peripheral blood and tumors, resulting in the prolonged overall survival and reduction of tumor burden in both the lymphoma-leukemia model and the leukemia rechallenge model. Hence, a lenalidomide-induced increase in the population of CAR-T cells at early differentiation stages helped enhance the overall antitumor efficacy of the combinatorial therapy.
This study did not compare third-generation CAR with second-generation CAR (with a costimulatory domain of 41BB or CD28) to identify the specificity of third-generation CARs. It is well accepted that the costimulatory domain shapes CAR-T cell metabolism and affects its function: CD28 is associated with early response, 4-1BB is related to long-term survival [35]. Compared to CD28, 4-1BB co-stimulation promoted nuclear factor κB (NF-κB) activation [39]. Although the CAR combining CD28 and 4-1BB exhibited deficient NF-κB signaling [40], it could enhance PI3K/AKT/ Bcl-XL activation [41] with better lymphoma-infiltration [42] and superior anti-lymphoma efficacy [40]. In clinical trials, third-generation CAR-T cells were also found to have superior expansion, longer persistence, and modest toxicity when treating B-NHL, compared to the second-generation CAR-T cells (with CD28 only). Moreover, this difference was the most striking in patients with a low tumor burden [43,44]. The present study did not confirm if lenalidomide affected NF-κB signaling. Previous reports have suggested that LEN directly induces CD28 phosphorylation, leading to the activation of PI3K pathways [45]. This could be one of the potential mechanisms for the enhanced antitumor effect of third-generation CAR-T cells in the presence of LEN.
Inhibitory receptors, such as PD-1, negatively regulate T-cell activation and represent one of the major mechanisms of immune escape in CAR-T cell treatment [46]. PD-L1 was found to be upregulated in lymphoma patients treated with CD19-CAR-T cells [47]. This results in an inhibition of the antitumor immune response of CAR-T cells, which can be reversed by blocking PD-1 [48]. In DLBCL patients, a high percentage of LAG3 + T-cells has been correlated with limited response to CD19-CAR-T cell therapy [25]. It has been reported that LEN inhibits the expression of PD-1 in T-cells, thereby augmenting its antitumor capability against myeloma [49]. In the present study, LEN dramatically reduced PD-1 expression in peripheral CAR-T cells as well as tumor-infiltrated CAR-T cells, which could be due to GSK-3β inactivation and MEK inhibition [50,51]. With a deeper understanding of "exhausted" T-cells, it is now clear that PD-1 expression cannot simply represent the dysfunction of T-cells. Recent studies have found that T-cells in the precursor-exhausted stage (T PEX , phenotype of CXCR5 + TCF1 + TIM3 − PD1 int ) retain their antitumor and self-renewal functions. These "memory-like" T-cells could differentiate into final exhausted T-cells (CXCR5 − TCF1 − TIM3 + PD1 high ), which lose their anti-tumor ability [52]. Although the proportion of T PEX induced by lenalidomide was not measured in the present study, CAR-T cells were found to have a lower expression of PD-1 and TIM3 and a higher expression of TCF1 with lenalidomide treatment. This highlights the precursor-exhausted characteristics of CAR-T cells, which likely sustain the immune response in the antitumor activity [52,53].
A suppressive tumor microenvironment also limits the immune response elicited by CAR-T treatment [54]. In this study, lenalidomide dramatically improved the infiltration of CAR-T cells into tumors. Lenalidomide has been reported to inhibit lymphangiogenesis in mantle cell lymphoma in a preclinical model [55]. Accordingly, the function of tumorassociated macrophages may have been suppressed by lenalidomide, leading to the inhibition of lymphangiogenesis, which could have paved the way for the entry of effector and memory CAR-T cells into the tumor site [56].
In summary, lenalidomide could overcome the resistance of DLBCL to third-generation CD19-CAR-T treatment in the present study. The augmentation of antitumor function of the CAR-T cells was attributed to LEN-promoted CD8 + T CM and Th1 CAR-T cell distribution, improved number of tumor-infiltrating CAR-T cells, and reduced exhaustion of CAR-T cells. This study supports the potential benefits of a combinatorial treatment of DLBCL patients with thirdgeneration CD19-CAR-T cells and lenalidomide, even in patients who respond poorly to the currently available commercial CAR-T treatment.