Chimeric antigen receptor-T (CAR-T) cell therapy has made considerable advancements in the treatment of malignant tumors; however, its clinical application continues to face challenges such as low response rates and relapse, which are critical issues requiring urgent resolution. The insufficient functionality of CAR-T cells remains a core factor affecting their clinical efficacy. This article provides a systematic review of various strategies to enhance CAR-T cell functionality, including structural modification and gene editing of chimeric antigen receptor CAR molecules, optimization of manufacturing processes, enhancement of CAR-T cells to counteract the inhibitory tumor immune microenvironment, and combination therapies with other drugs. In terms of optimizing CAR molecules, particularly the development of dual-target CARs, this approach not only effectively prevents antigen escape but also significantly enhances the activation, proliferation, and anti-tumor efficacy of CAR-T cells. Gene editing technology offers new opportunities to improve the persistence, proliferative capacity, and anti-tumor activity of CAR-T cells, thereby enhancing their function and reducing disease relapse. Furthermore, epigenetic regulation augments the adaptability of CAR-T cells, strengthening their anti-tumor effects. Simultaneously, combining CAR-T cell therapy with other immunotherapies provides fresh perspectives for improving overall treatment efficacy. However, challenges remain in areas such as the precision of gene editing, reversibility of epigenetic regulation, and optimization of CAR structures. Future research should focus on refining these strategies and exploring their synergistic applications to maximize the therapeutic potential of CAR-T cell therapy. With ongoing technological advancements, CAR-T cell therapy is poised to achieve groundbreaking applications in a broader range of malignant tumor treatments, offering new hope to patients.

Antibodies targeting foreign HLA molecules can be detrimental in transplantation. Nonetheless, existing desensitisation methods for anti-HLA antibodies frequently prove insufficient. We conducted a single-arm pilot study to evaluate CD19/B cell maturation antigen (BCMA) chimeric antigen receptor T (CAR-T) cell therapy for desensitising anti-HLA antibodies prior to transplantation. Seven patients diagnosed with acute leukaemia participated in the study, comprising four individuals with acute myeloid leukaemia (AML) and three with acute B lymphoblastic leukaemia (B-ALL). Among them, one patient was treated with BCMA CAR-T cells, while six received CD19 CAR-T cells. No instances of neurotoxicity or severe cytokine release syndrome (grade ≥ 3) were noted. CAR-T cell expansion was observed in six patients (86%), with the exception of patient 3. Among the seven patients with pre-infusion anti-HLA-I antibodies, five (71%) showed a decrease in mean fluorescence intensity (MFI), including three (43%) who experienced a reduction greater than 75%. Among the six patients confirmed to exhibit CAR-T cell expansion in vivo, three presented with donor-specific anti-HLA antibodies (DSAs) prior to CAR-T cell infusion. The median MFI level of DSA declined from 15797.8 (7750.3-21106.0) to 3831.1 (523.3-13197.0) (p < 0.001) in the three patients studied. Two patients with decreased DSA levels after CD19 CAR-T cell infusion underwent combined desensitisation before haematopoietic stem cell transplantation. This pilot study illustrates the potential safety and efficacy of CD19 and BCMA CAR-T cells in desensitising anti-HLA antibodies in patients with AML or B-ALL. However, only one patient received BCMA CAR-T cells in our cohort, so further research with an expanded sample size is essential for comprehensively evaluating the safety and efficacy of CD19/BCMA CAR-T cell therapy for anti-HLA antibody sensitisation.

Patients with relapsed/refractory B-cell non-Hodgkin lymphoma (R/R B-NHL) typically experience dismal outcomes. Although CD19 chimeric antigen receptor T-cell (CAR-T) therapy has shown considerable efficacy, the factors influencing its long-term efficacy remain unclear. We retrospectively analyzed 79 R/R B-NHL patients treated with CD19 CAR-T cell therapy at our center to evaluate efficacy, long-term survival, and prognostic factors. Among all evaluable patients, the overall response rate was 89.7%, with a 3-year duration of response (DOR) of 44.9% (95% CI, 34.2%-59.0%), a 3-year progression-free survival (PFS) of 40.2% (95% CI, 30.3%-53.4%), and a 3-year overall survival (OS) of 48.3% (95% CI, 38.0%-61.3%). Long-term adverse events (AEs) included hypogammaglobulinemia, which occurred in 73 of 74 evaluable patients (92.4%), viral reactivation (5.1%), and secondary malignancy (5.1%). Notably, high tumor Ki-67 (>60%) was associated with lower peak CAR-T cell expansion (P < .01), reduced area under the curve of expansion from day 0 to 28 (P < .0001), and inferior complete response rate, DOR, and PFS (all P < .05). However, CAR-T cell persistence was not significantly associated with long-term remission or survival (all P > .05). In conclusion, CD19 CAR‑T cell therapy yielded sustained remissions in patients with R/R B-NHL, with manageable long-term AEs. High tumor Ki‑67 expression serves as a poor prognostic factor and is linked to limited CAR‑T cell expansion, whereas CAR‑T cell persistence does not influence long-term prognosis.

Diffuse large B-cell lymphoma (DLBCL) exhibits 30–40

Long-term persistence of chimeric antigen receptor (CAR)-T cells is essential for durable therapeutic efficacy but the mechanisms underlying CAR-T cell dysfunction remain unclear. On the basis of integrated analyses of clinical samples from participants with multiple myeloma and acute lymphoblastic leukemia treated with CAR-T cells, we show that rapid expansion of CAR-T cells after infusion is followed by a 'diminution' phase characterized by ferroptosis-associated features and elevated serum iron levels. In preclinical cancer models in female mice and ex vivo culture systems, excess intracellular iron impaired CAR-T cell function. Mechanistically, iron promoted ferroptosis by increasing mitochondrial reactive oxygen species and lipid peroxidation, in part through acyl-CoA synthetase long-chain family member 4 (ACSL4)-associated lipid remodeling. Targeting ferroptosis, particularly through genetic ablation of ACSL4 in CAR-T cells, substantially enhanced antitumor efficacy. Together, these findings identify iron-driven ferroptosis as a determinant of CAR-T cell dysfunction and a targetable barrier to durable CAR-T efficacy.

Importance:Interleukin (IL)-10 expressing CD19 chimeric antigen receptor (CAR) T cells (META 10-19) have demonstrated encouraging clinical activity in B-cell acute lymphoblastic leukemia, but their safety and efficacy in relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL) remain unknown. Objective:To evaluate the safety and efficacy of META 10-19 in patients with R/R DLBCL. Design, Setting, and Participants:This nonrandomized, phase 1 clinical trial was conducted at the First Affiliated Hospital of Zhejiang University School of Medicine. Patients were enrolled from November 16, 2023, to April 7, 2025. The data cutoff date was January 20, 2026. The data analysis was conducted on January 22, 2026. Twenty patients were screened, and 13 received a META 10-19 infusion. The median duration of follow-up was 15.6 (range, 0.7-25.5) months. Intervention:Following lymphodepletion with fludarabine and cyclophosphamide, patients received META 10-19 at dose levels of 2 × 103, 5 × 103 or 2 × 104 CAR T cells/kg. Main Outcomes and Measures:The primary end points were adverse events, dose-limiting toxic effects, and objective response rate. The secondary end points included complete remission (CR) and a cellular kinetic of META 10-19. Results:Among 13 treated patients (median age, 61 years [range, 35-74 years]; 8 men [61.5%] and 5 women [38.5%]), the objective response rate was 92.3%, including CR in 11 patients (84.6%) and partial remission in 1 patient (7.7%). One patient died before response assessment because of disease-related gastrointestinal complications. Cytokine release syndrome occurred for 12 patients (grade 1: n = 8; grade 2: n = 3; grade 3: n = 1), and immune effector cell-associated neurotoxicity syndrome occurred for 2 patients (grade 1: n = 1; grade 2: n = 1). Robust in vivo CAR T-cell expansion was observed across dose levels, with a median (range) peak expansion of 660.7 (30.7-10 562.3) cells/µL. At data cutoff, 5 patients experienced a maintained CR and 7 experienced disease relapses or progression (2 with CD19 negative relapses). Conclusions and Relevance:The results of this nonrandomized clinical trial suggest that ultralow-dose META 10-19 demonstrated promising antitumor activity and a manageable safety profile in patients with R/R DLBCL. Further investigation in larger cohorts is warranted. Trial Registration:ClinicalTrials.gov Identifier: NCT06120166.

Background Standard immunochemotherapy yields suboptimal outcomes in patients with newly diagnosed high-risk large B-cell lymphoma (LBCL). The ZUMA-12 trial demonstrated the feasibility of CD19 chimeric antigen receptor (CAR) T-cell therapy as a first-line treatment, yet more data are needed to support its broader application in this setting. Furthermore, how to design better integrated strategies to further enhance the efficacy and safety of frontline CAR T-cell therapy remains an open question. Methods In this single-center phase II trial, we enrolled patients with newly diagnosed high-risk LBCL. Patients received two cycles of rituximab, lenalidomide, and Zanubrutinib (ZR 2 ), followed by CD19 CAR T-cell infusion. The primary endpoint was complete response (CR) rate after CAR T-cell therapy. Results A total of 43 patients were enrolled (median age, 69 years; 44% ≥70). After ZR 2 debulking, the overall response rate was 92.9%, with a CR rate of 33.3%. Among the 40 patients who received CAR T‑cell therapy, the primary endpoint was met, with a CR rate of 95.1% (95% CI: 83.5%-99.4%). At a median follow-up of 25.6 months, estimated 2-year progression-free survival, overall survival and duration of response were 84%, 97.6% and 86.2%, respectively. Cytokine release syndrome occurred in 25% of patients (grade 3 in 2.5%); no neurotoxicity or treatment-related deaths occurred. Conclusions In this phase II trial, ZR 2 debulking followed by sequential CD19 CAR T-cell therapy resulted in a high CR rate, durable responses, and a favorable safety profile in patients with newly diagnosed high-risk LBCL. Larger randomized trials are warranted. (ClinicalTrials.gov number, NCT04661020.)

Although CAR-T cell therapy has revolutionized treatment for hematologic malignancies, its application in acute myeloid leukemia remains challenging. CD7 is expressed in approximately 30% of AML cases and represents a promising target. This phase I clinical trial (NCT04599556) evaluated CD7-targeted CAR-T cell therapy in patients with relapsed/refractory CD7-positive AML. Patients received a single infusion of autologous or donor-derived CD7 CAR-T cells using a standard 3 + 3 dose escalation design across two dose levels. The primary endpoint was the incidence of dose-limiting toxicities. Fourteen patients were enrolled. Treatment-related adverse events included cytokine release syndrome (92.9%), grade 3-4 cytopenia (100%), grade 1 neurotoxicity (7.1%), and viral reactivation (78.6%). The objective response rate was 92.3%, with an MRD-negative rate of 84.6%. Despite initial responses, seven patients relapsed with CD7-negative disease. At a median follow-up of 172.5 days, five patients remained in remission. The 1-year overall survival and leukemia-free survival rates were 34.3% and 34.1%, respectively. These initial results indicate that CD7 CAR-T cell therapy exhibits a manageable safety profile and preliminary efficacy in R/R AML patients, supporting further investigation in larger trials.

Relapse after allogeneic hematopoietic stem cell transplantation (allo-HSCT) in patients with B-cell acute lymphoblastic leukemia (B-ALL) is associated with a poor prognosis. Although CD19 chimeric antigen receptor T (CAR-T) cell therapy has shown promise in this population, its long-term efficacy and safety remain unclear. To address this, we conducted a retrospective multicenter study to evaluate the long-term survival outcomes of autologous and allogeneic CD19 CAR-T cell therapies in 55 B-ALL patients who relapsed after allo-HSCT. Following CAR-T cell infusion, 37 patients (67.3%) achieved complete remission (CR). The 3-year leukemia-free survival (LFS) rate among CR patients was 37.8%. The 3-year overall survival (OS) rate for both CR and non-remission (NR) patients was 36.2%. Cytokine release syndrome (CRS) was the most common adverse event, occurring in 74.5% of patients. No significant differences in efficacy or safety were found between autologous and allogeneic CAR-T cell groups (all P > 0.05). This study demonstrates the promising 3-year survival and safety profile of both autologous and allogeneic CD19 CAR-T cell therapies, and further supports CD19 CAR-T cell therapy as a valuable treatment for patients with relapsed B-ALL after allo-HSCT.

Patients progressing after CD19-targeted immunotherapy in r/r B-ALL experience poor outcomes. CD22-targeted therapies, including CD22 CAR-T cells and Inotuzumab Ozogamicin, show promise as alternatives, although data in those patients is limited. This study retrospectively analyzed 43 r/r B-ALL patients who had previously received CD19-targeted therapy at two centers in China. Among these patients, 27.9% received blinatumomab, 58.1% received CD19 CAR-T cells, and 14% received both. After CD19-targeted therapy, 34.9% of patients experienced CD19-negative relapse, while the remaining patients maintained CD19 expression. Subsequent treatments included CD22 CAR-T cells (55.8%) and InO (44.2%). The median age was 39 (24-56) years, with an overall CR/CRi rate of 51.1% and 30.2% achieving MRD negativity. Among the 22 patients achieving CR/CRi, 13 (59.1%) experienced relapse. The median relapse-free survival (RFS) was 236 days (95% CI: 132-unreached), and the median OS has not been reached. Multivariate analysis showed similar remission rates and survival for CD22 CAR-T and Inotuzumab Ozogamicin therapies. Patients with extramedullary disease had worse remission rates, and those previously resistant to CD19-targeted therapy had shorter RFS. CD22-targeted therapies offer a potential option for patients progressing after CD19-targeted immunotherapy, but high relapse rates highlight the need for better strategies for lasting remission.

Relapse after allogeneic hematopoietic stem cell transplantation (allo-HSCT) in patients with B-cell acute lymphoblastic leukemia (B-ALL) is associated with a poor prognosis. Although CD19 chimeric antigen receptor T (CAR-T) cell therapy has shown promise in this population, its long-term efficacy and safety remain unclear. To address this, we conducted a retrospective multicenter study to evaluate the long-term survival outcomes of autologous and allogeneic CD19 CAR-T cell therapies in 55 B-ALL patients who relapsed after allo-HSCT. Following CAR-T cell infusion, 37 patients (67.3%) achieved complete remission (CR). The 3-year leukemia-free survival rate among CR patients was 37.8%. The 3-year overall survival rate for both CR and non-remission patients was 36.2%. Cytokine release syndrome was the most common adverse event, occurring in 74.5% of patients. No significant differences in efficacy or safety were found between autologous and allogeneic CAR-T cell groups (all P > 0.05). This study demonstrates the promising 3-year survival and safety profile of both autologous and allogeneic CD19 CAR-T cell therapies, and further supports CD19 CAR-T cell therapy as a valuable treatment for patients with relapsed B-ALL after allo-HSCT.

e14518 Background: Despite advances in treatment, outcomes for adults with relapsed or refractory B-cell acute lymphoblastic leukemia (r/r B-ALL) remain poor, with conventional chemotherapy achieving complete remission in only 18–45% of patients. Allogeneic CAR-T cells offer an "off-the-shelf" alternative but require genome editing to mitigate host rejection and graft-versus-host disease (GvHD). This study evaluates the safety and efficacy of BRL-301, a healthy donor-derived, multiplex genome-edited allogeneic CD19-targeted CAR-T product, in patients with r/r B-ALL. Methods: In this investigator-initiated trial (NCT05381181), patients with r/r B-ALL received lymphodepletion with etoposide, cyclophosphamide, and fludarabine followed by BRL-301 infusion. Safety and response assessments were conducted per protocol. Results: Five patients (median age 16 years, range 10–45) received BRL-301. All patients achieved an objective response, with a 100% complete response rate. Robust CAR-T expansion was observed in all subjects, peaking between days 7–18 post-infusion. Grade 3/4 adverse events included cytopenias attributable to lymphodepletion. All patients experienced mild (grade 1/2) cytokine release syndrome; 4 patients received tocilizumab and steroids. No immune effector cell-associated neurotoxicity syndrome (ICANS) or GvHD was observed. Conclusions: BRL-301 demonstrated promising efficacy and a manageable safety profile in patients with r/r B-ALL, with no evidence of GvHD or severe neurotoxicity. These results support further development of allogeneic CAR-T therapy as a viable treatment option for r/r B-ALL. Clinical trial information: NCT05381181 .

Sequential allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a critical approach to enhance the efficacy of chimeric antigen receptor T cell (CAR-T) therapy for relapsed/refractory acute lymphoblastic leukemia (R/R ALL). The long-term survival rates following allo-HSCT after CAR-T cell therapy remain controversial. We previously reported the results of 122 patients with R/R B-ALL who achieved minimal residual disease negative (MRD-) complete remission (CR) after anti-CD19 CAR-T, including 67 patients without subsequent transplantation (non-transplant group) and 55 patients with subsequent haplo-HSCT (transplant group). Here, we present the extended follow-up of this cohort, with a median of 72.9 months. Compared with the non-transplant group, transplantation recipients had a higher 5-year LFS (50.5% vs. 25.7%; p < 0.001) and OS (53.5% vs. 25.6%; p < 0.001). Multivariable analysis identified MRD positivity at transplantation as an independent factor associated with worse LFS, OS, and a higher cumulative incidence rate of relapse (CIR). MRD-negative patients before transplant (MRD- group) had a lower 5-year CIR compared to MRD-positive patients (MRD+ group) (20.8% vs. 50.0%; p = 0.014). The 5-year LFS rates in the MRD+ and MRD- groups were 26.7% and 59.4%, respectively (p = 0.003). The 5-year OS of the MRD- group was higher than that of the MRD+ group (62.0% vs. 30.5%; p = 0.006). For patients who achieve CR after CAR-T therapy, haplo-HSCT with pre-transplant MRD negativity was associated with better long-term survival. However, infections emerged as a significant complication that may reduce the long-term survival benefits. Trial Registration: ClinicalTrials.gov identifier: ChiCTR1900023957.

Although chemotherapy-free regimens have improved initial remission rates in patients with newly diagnosed Philadelphia chromosome-positive B-cell acute lymphoblastic leukemia (Ph + B-ALL), outcomes for relapsed or refractory (R/R) Ph + B-ALL remain poor. This study reports the efficacy and safety of CD19 chimeric antigen receptor (CAR) T-cell therapy in R/R Ph + B-ALL. This study retrospectively analyzed 93 patients with R/R Ph + B-ALL who received CD19 CAR T-cell therapy across China between August 2015 and March 2024. We evaluated the overall response rates, long-term efficacy, safety, and prognostic factors associated with CD19 CAR T-cell therapy. Complete remission (CR) or CR with incomplete hematologic recovery rate was 87.1

Patients with relapsed/refractory multiple myeloma (R/R MM) receiving chimeric antigen receptor (CAR) T-cell therapy are at high risk of toxicities, including cytokine release syndrome (CRS). The roles of positron emission-tomography computed tomography (PET/CT) in predicting these toxicities remain unclear. We retrospectively studied 62 patients with R/R MM who received B-cell maturation antigen (BCMA) CAR T-cell therapy at our center. Baseline metabolic tumor volume (MTV) of more than 107.2 cm3 and total lesion glycolysis (TLG) exceeding 406.5 were classified as high MTV and high TLG, respectively. Both high MTV (P = 0.008) and high TLG (P = 0.011) were identified as independent risk factors for the development of severe CRS classified as grade 3 to 4. Moreover, the administration of tocilizumab for the treatment of CRS was associated with high MTV (P = 0.005) or high TLG (P = 0.010). Notably, our multivariate Cox models that incorporated either MTV (P = 0.029) or TLG (P = 0.009) along with plasmacytoma types, high-risk cytogenetics, and high bone marrow plasma cell frequencies demonstrated strong predictive capabilities for the 5.3-year long-term OS. Therefore, baseline high MTV or TLG measured by PET/CT are recognized as adverse prognostic indicators for the incidence of severe CRS and poor outcomes in patients with R/R MM undergoing BCMA CAR T-cell therapy.

Introduction CAR-T cell therapy has revolutionized the therapeutic landscape for relapsed/refractory (R/R) B-cell acute lymphoblastic leukemia (B-ALL), and bridging with allogeneic hematopoietic stem cell transplantation (allo-HSCT) has the potential to lower relapse rates. Nevertheless, the majority of existing studies have exclusively focused on short-term outcomes, resulting in a lack of comprehensive understanding of the long-term sustainability of overall prognosis. Objectives Our study aimed to provide real-world, long-term follow-up data for patients who underwent sequential therapy. Methods Patients with R/R B-ALL who achieved MRD-CR following CAR-T therapy and subsequently underwent allo-HSCT between January 2016 and May 2024 were enrolled. The primary outcomes included overall survival (OS), leukemia-free survival (LFS), non-relapse mortality (NRM) and cumulative incidence of relapse (CIR). Acute and chronic graft-versus-host disease (GVHD) and graft-versus-host disease-free survival (GRFS) were also investigated. Results The median age at transplant of 32.1 years. Of these patients, 88.2 % underwent haploidentical-HSCT, and 11.8 % received either unrelated matched or related matched HSCT. The cumulative incidences of grades I-IV and grade II-IV aGVHD at day 100 were 31.4 % and 15.7 %, respectively. The cumulative incidence of cGVHD at 4 years was 48.3 %. With a median follow-up time of 43.2 months, OS, LFS, and GRFS at 4 years were 68.9 %, 61.4 %, and 39.5 %, respectively. Fifteen cases (29.4 %) experienced relapse, predominantly antigen-positive relapse (n = 11). NRM and CIR at 4 years were 10.6 % and 28.0 %, respectively. In the multivariate analyses, patients over 45 years of age and with poor-risk had significantly dismal OS (P = 0.018; P = 0.038) and LFS (P = 0.01; P = 0.03). Conclusion Our study exhibits favorable long-term outcomes consistent with those reported in clinical trials, with sustained, durable responses observed at the 4-year follow-up. However, these benefits are less pronounced in older patients and those with poor-risk disease characteristics.

Background Epstein-Barr virus (EBV) reactivation has increasingly been recognized as a poor prognostic indicator, as it may progress to severe EBV-associated diseases, including EBV+ lymphomas, EBV-driven HLH, and EBV+ PTLD. Since EBV predominantly infects B lymphocytes, depletion of B cells using anti-CD20 monoclonal antibodies has been adopted to reduce EBV load; however, it fails to eliminate EBV in T or NK cells, which remains a major therapeutic challenge. In this study, we found that anti-CD19/BCMA CAR-T therapies not only cleared EBV-infected B cells but also T/NK cells in B-cell or plasma cell hematologic malignancies, possibly due to immune remodeling during cytokine release syndrome (CRS). Method A post-hoc analysis was conducted of patients with B-cell or plasma cell malignancies who had received CD19- or BCMA-CAR-T cell therapy at our institution (2018–2024; ChiCTR1800015575, ChiCTR1800017404, ChiCTR2100053871, ChiCTR2100052009, NCT04532268). Baseline EBV reactivation status, defined as detectable EBV DNA in fresh whole-blood samples, was evaluated using clinical-use real-time qPCR (Daan Gene) with a detection limit of 2.0×10² copies/ml. Longitudinal EBV DNA levels and clinical outcomes were systematically recorded to evaluate viral clearance dynamics. To further delineate the cellular reactivation of EBV and CAR-T therapy response, cryopreserved baseline PBMCs before lymphodepletion were thawed, with T, B, and NK cell subsets sorted. EBV DNA in each was quantified using research-use-only qPCR (Sansure Biotech) with a detection limit of 4.0×10² copies/ml. Paired PBMC samples (baseline and 2 months post-infusion) were processed for scRNA-seq and TCR-seq (10x Genomics) to explore potential immune remodeling mechanisms. Results Baseline EBV reactivation was identified in 20 patients (B-ALL, n=10; MM, n=6; lymphoma, n=4) receiving CD19 (n=12), BCMA (n=6), or CD19/CD22 (n=2) sequential CAR-T therapy. Median age of this cohort was 56 (range 27–77), with 50.0% having prior transplantation. Baseline plasma EBV DNA levels ranged from 3.65 × 10² to 3.84 × 10⁴ copies/ml. Nineteen patients (95%) achieved undetectable EBV DNA post-CAR-T. All patients experienced CRS, and grade 3–4 CRS occurred in 2 patients (10.5%). The remaining patient (5%) with persistent EBV DNA showed no CAR-T cell expansion, suggesting therapeutic inefficacy. In light of EBV negativity observed, baseline T/B/NK subsets were subsequently analyzed in 17 patients to explore mechanisms. Among 8 with EBV in all subsets, 75% showed the highest levels in B cells, and 25% in T cells, suggesting potential clearance of non-CAR-target EBV-reactivated cells in sustained systemic clearance. During further elucidation of how CAR-T therapy achieved beyond B-cell elimination, scRNA/TCR-seq in 5 patients (B-ALL, n=4; MM, n=1) with paired PBMCs showed decreased overall proportion of exhausted T cells and increased proportion of CD8⁺ T cells with an effector-related phenotypic shift (T_CM to T_EM) post-CAR-T, especially in B-ALL patients. EBV-specific clonotypes expanded after therapy (CALRGGGADGLTF_CASSSGGNEQFF, NA_CASSSGGNEQFF, CAEKTSYDKVIF_CASSESYTQYF), indicating CAR-T-related restored endogenous EBV-specific T cell antiviral responses.Interactome analysis demonstrated that EBV-specific T cells consistently exhibited the strongest interactions with CD8⁺ effector T cells and NK cells, with post-treatment signaling changes favoring enhanced effector function. Mechanistically, these T cells received weaker apoptotic signals (FASLG/TRAIL↓) and engaged in more robust CXCL12-CXCR4 signaling with CD8⁺ effectors, facilitating recruitment to EBV-reactivated sites. Concurrently, NK-derived LTA-TNFRSF14 signaling increased, supporting T cell survival and memory. Myeloid cells shifted from a pro-apoptotic state (FASLG/TRAIL↑) to a costimulatory and supportive state (CD40↑, LIGHT↑, GAS6↑, IL6↑). These immune remodeling events during CRS fostered a permissive microenvironment that enhanced EBV-specific T cell function and contributed to durable EBV clearance. Conclusion CAR-T therapy targeting B or plasma cells effectively cleared EBV reactivation in hematologic malignancies. Beyond B-cell depletion, CAR-T induced systemic EBV clearance, likely mediated by immune remodeling during CRS. Enhanced EBV-specific T cell responses and supportive cellular interactions suggest CAR-T as a promising strategy to overcome EBV-related complications in this population.

Despite advancements in CAR-T therapy, over half of the lymphoma patients still face drug resistance or relapse. Seventy-nine Chinese patients with B-cell lymphoma provided 192 serum samples for circulating tumor DNA (ctDNA) detection to identify the genomic features linked to prognosis during CAR-T cell therapy. Patients in complete remission and noncomplete remission groups were analyzed, and those with >10 ctDNA gene mutations before CAR-T cell therapy had significantly worse overall survival and progression-free survival rates than those with fewer mutations. MYD88, FAT1, and BTG2 mutations were correlated with poorer OS, whereas MUC16 mutations were correlated with better OS. Patients with TP53 mutation pretreatment had significantly lower CR rates than those without TP53 mutations (33.3% vs. 68.1%, P=0.02). However, TP53 mutation pretreatment did not affect long-term patient survival. All patients with TP53 mutations 4 weeks after CAR-T cell therapy failed to achieve CR, with poorer OS (1-year OS rate: 37.5% vs. 66.4%; 2-year OS rate: 12.5% vs. 56.3%, P=0.0023). Among patients with CR, those with BCR mutations at 4 weeks post-treatment exhibited poorer OS (2-year OS rate: 40.9% vs. 76.1%, P=0.035). One week after CAR-T cell therapy, patients without CDKN2A, CBLB, APC, SPEN, KMT2D, CARD11, FOXO1, or PDGFRB mutations were more likely to achieve CR (76.6% vs. 28.6%, P<0.001) and had better OS (1-year OS rate: 81.5% vs. 38.9%, 2-year OS rate: 62.2% vs. 5%, P<0.001) and PFS (1-year PFS rate: 67.2% vs. 0%, P<0.001). This study evaluated the genomic features and screened a gene set to predict CAR-T cell therapy efficacy in B-cell lymphoma, aiding clinicians in accurately evaluating efficacy and treatment decision-making.

Abstract Background Recently, CD7-targeted CAR-T cells have shown both safety and efficacy in early-stage clinical studies. More than 95% of T-cell malignancies and 30% of acute myeloid leukemia cells express CD7, rendering CD7 CAR-T cells to become a promising candidate for the development of next-generation cellular therapy. However, since >90% of normal T cells also express CD7, the potential of compromised T cell function following CD7 CAR-T cell therapy has become a major concern. We and others have reported the expansion of CD7-negative (CD7-) T cells in patients after CD7 CAR-T cell therapy. However, reactivation of EB virus (EBV) or cytomegalovirus (CMV) infection after CD7 CAR-T cell therapy has also been reported. It still remains largely unknown about the origin and function of these post-therapy CD7- T cells, which is critical for understanding the immune reconstitution of these patients. Methods Peripheral blood mononuclear cell (PBMC) samples from 10 patients underwent flow cytometry, single-cell RNA sequencing (scRNA-seq), and TCR sequencing (scTCR-seq). Comparative TCR profiling was performed on flow-sorted pre-treatment CD7+ T cells, pre-treatment CD7- T cells, and post-treatment CD7- T cells from three patients. Mechanistic studies employed immunofluorescence, Western blot, pharmacological inhibitors, and CD7 promoter methylation analysis (-300 to +200 region) via bisulfite sequencing PCR. Functional assessments included PMA/ionomycin and viral peptide (EBV/CMV) stimulation of PBMCs (pre-treatment, 4-week, and 4-month post-treatment timepoints), with evaluation of proliferation capacity, immunophenotypic shifts, and cytokine secretion profiles. Results Longitudinal analysis of 10 CD7 CAR-T cell responders revealed transcriptional and surface CD7 expression in endogenous T cells progressively declined post-infusion, with a transient surface CD7-negative and cytoplasmic CD7-positive (sCD7-cyCD7+) population peaking at 2 weeks before sCD7-cyCD7- T cells dominated by 4 months. TCR sequencing revealed post-treatment CD7- T cells originated from both pre-existing CD7+ and CD7- clones, demonstrating CD7 downregulation from CD7+ precursors as a key immune reconstitution mechanism after CD7 CAR-T cell therapy. Mechanistically, scRNA-seq identified clathrin-dependent endocytosis enrichment in endogenous T cells at 2 weeks post-infusion. In vitro modeling confirmed that upon contact with CD7 CAR-T cells, surface CD7 internalization via clathrin-mediated endocytosis and degradation through lysosome. Persistent CD7- T cells exhibited CD7 gene -300 to +200 region hypermethylation without alternative splicing, indicating epigenetic silencing enabled T cell escape from CAR-T cell-mediated killing. Functionally, single-cell analysis revealed CD7- T cells post-CAR-T cell therapy displayed memory/cytotoxic features with distinct granzyme/cytokine profiles. Despite reduced TCR diversity, patients maintained EBV/CMV-specific TCRs, though EBV-related complications occurred in one case. CD7+-derived clones exhibited enhanced TNF signaling and reduced exhaustion versus CD7--derived counterparts. Despite impaired costimulatory molecule expression (CD25/CD27/CD28), CD7- T cells maintained activation markers (CD69/CD137) and cytokine production (IFNγ/TNFα/IL1β) upon PMA/ionomycin and viral peptide stimulation, confirming preserved immune responsiveness. Conclusions This study was the first to elucidate the dynamic CD7 expression, mechanisms and function of endogenous T cells following CD7 CAR-T cell therapy. Our results have together revealed that normal T cells can escape the killing of CD7 CAR-T cells through clathrin-dependent endocytosis pathway and lysosomal degradation of CD7, followed by hyper-methylation at the CD7 gene locus. We have also shown that the immune function is partially retained in these CD7- T cells. We believe that this study has greatly contributed to the understanding of post-CAR-T cell immune reconstitution, which will be attractive to broad readers in the fields of immunology and oncology.