A 39-year-old male patient presented to a local hospital in December 2019 with flu-like symptoms lasting 2 weeks. Blood routine examination revealed the following results: white blood cell count (WBC), 84.82 × 10⁹/L; hemoglobin (Hb), 74 g/L; and platelet count (Plt), 14 × 10⁹/L. Bone marrow (BM) examination showed blast cells accounting for 88.5% of the total cell population. Further immunophenotypic analysis by flow cytometry (FCM) revealed that blast cells comprising 96.3% of the population, expressing CD7, CD34, CD38, CD99, cyCD3, cCD3, and sCD3. Karyotype analysis revealed a normal karyotype (46, XY) [20]. The patient was diagnosed with mature T-ALL and classified as the high-risk group based on his age >35 years. Initially, he received two cycles of vinodixin, daunorubicin, cyclophosphamide, pegaspargase, and dexamethasone (VDCLP) and achieved complete remission with minimal residual disease negativity (CR_MRD−), as verified by FCM. Subsequently, the patient received six cycles of consolidation chemotherapy (Figure 1) and four lumbar punctures with intrathecal chemotherapy, with no abnormal white blood cells detected in the cerebrospinal fluid (CSF). Unfortunately, shortly after the final consolidation cycle, the disease relapsed, with BM morphology revealing 8.5% blasts on 30 January 2021.

The patient then received three courses of salvage chemotherapy. However, the disease continued to progress, with 88% lymphoblasts detected in the BM. The leukemic blasts were immunophenotypically characterized as cCD3, CD7, CD99, CD5^dim, and cTdT positive; CD34 partially positive; and CD3, CD4, CD8, and CD2 negative. Thus, the leukemia was refractory to salvage chemotherapy, and the relapsed blasts exhibited immunophenotypes similar to those of the primary blasts. Therefore, CD7 CAR T-cell therapy was introduced.

Peripheral blood (PB) of the patient was collected to prepare the autologous humanized naturally selected anti-CD7 CAR (NS7CAR) T cells. The CAR T cells were engineered by Hebei Senlangio Technology Co., Ltd. (Shijiazhuang, China). Lymphodepleting chemotherapy with the FC regimen (fludarabine 30 mg/m², days −4 to −2; cyclophosphamide 500 mg/kg, days −3 to −2) was initiated on May 27 2021 (day −4). On day 0, the patient received an infusion of CD7 CAR T cells at a dose of 1 × 10⁶ cells/kg. However, MRD in the BM increased to 95.73% on day 18 after CAR T-cell infusion. After one cycle of cytarabine, idarubicin, and granulocyte colony-stimulating factor (CIG) for tumor reduction, the patient received a second CD7 CAR T-cell infusion on June 21 2021 at a dose of 2 × 10⁶ cells/kg. As expected, with the expansion of the reinfused CAR T cells (Figures 2A, B), the leukemic blasts gradually decreased (Figure 2C).

Following the second CD7 CAR T-cell infusion, a series of clinical changes occurred. Scattered rashes appeared on day 2, and the lesions gradually expanded. Fever developed on day 2, peaked at 39.5 °C, and persisted for 1 week (Figure 2D). On day 8, hypotension occurred, requiring norepinephrine or dopamine for blood pressure support. Low percutaneous oxygen saturation was detected, and oxygen inhalation at 10 L/min via mask was initiated. Peak serum levels of interleukin-6 (IL-6; 297-fold above the baseline) and monocyte chemoattractant protein-1 (MCP-1) were detected on day 10 after the infusion (Figure 2E). In addition, the patient developed some signs of organ toxicity, including elevated transaminases, serum creatinine, lactate dehydrogenase, and prolonged activated partial thromboplastin time (Supplementary Figure S1). Immune effector cell-associated neurotoxicity syndrome (ICANS), with an ICE score of 8 (the patient was unable to count backward from 100 by 10 or write a standard sentence), was observed on day 13. According to the ASTCT grading criteria for CRS and CRES (12), grade 3 cytokine release syndrome (CRS) and grade 1 CAR T-cell–related encephalopathy syndrome (CRES) were diagnosed. Accordingly, tocilizumab (anti–IL-6 receptor monoclonal antibody; 4 mg/kg once daily on days +8 and +14 days), dexamethasone (10 mg once daily on day +7; 10 mg every 6 h on days +8 to +9; 10 mg every 8 h on day +10; 10 mg every 12 h on day +11; and 10 mg once daily on days +13 to +14), and supportive care were administered. By day 15, the patient’s vital signs were stable, and the symptoms of ICANS had completely resolved, and the symptoms and indicators of CRS were gradually improving. On day 25, the patient’s WBC count remained close to zero, and unfortunately, localized skin and soft tissue infection developed in the anterior iliac region (Figure 2F).

Given the severe bone marrow suppression, refractoriness to platelet transfusion, dependence on blood transfusions, and skin and soft tissue infection with little chance of improvement without reestablishment of hematopoiesis, allo-HSCT (6/10 HLA-matched related donor, son-to-father transplantation) was recommended as an intervention to achieve disease control. Accordingly, on day 34 after the second CAR T-cell infusion, immediately following improvement of CRS and CRES and restoration of organ function, the patient received myeloablative busulfan plus cyclophosphamide (BU+CY) conditioning (Figure 3). Anti-thymocyte globulin (ATG, rabbit), cyclosporine A and methotrexate were administered for GVHD prophylaxis.

The patient still had a generalized rash when the conditioning regimen was initiated, and the absolute count of CD7 CAR T cells in the peripheral blood on day −7 was 1.61 × 10⁴/L. New neuropsychiatric symptoms, including hallucinations and incoherent speech, as well as cardiac insufficiency, developed from day −5, and symptomatic supportive treatment was provided. The patient’s vital signs remained stable prior to the transplantation. However, he developed fever on the first day of stem cell infusion and diarrhea (up to 2010 g/day) on the second day, which was considered as hyperacute gastrointestinal GVHD. The cyclosporine A dose was adjusted to achieve therapeutic concentrations, and mycophenolate mofetil (MMF), methylprednisolone, and basiliximab were administered. Diarrhea subsequently improved, but the patient developed extensive skin desquamation and peeling, along with elevated bilirubin levels.

Unfortunately, the patient gradually developed worsening neuropsychiatric symptoms, including a suspected seizure episode and coma, as well as persistent fever peaking at 42 °C that was unresponsive to antibiotics. On day +3 after allo-HSCT, the serum sC5b-9 level was 512 ng/mL, indicating a high risk of transplant-associated thrombotic microangiopathy (TA-TMA). Subsequently, red cell fragments were detected on peripheral blood smear. Additional abnormal findings included elevated levels of serum lactate dehydrogenase (LDH) and serum creatinine levels, increased requirements for red blood cell transfusion, continued ineffective platelet transfusion refractoriness, and proteinuria (2+). TA-TMA was diagnosed and treated with rituximab, eculizumab and supportive care. Despite these interventions, the patient’s clinical condition did not improve, and multiple organ functions continued to deteriorate. Hemodynamic instability developed on day +6 after transplantation. The patient was discharged on day +8 and died on the same day.

A 17-year-old male presented with a 10-day history of swollen lymph nodes and poor appetite in July 2020. Blood cell counts revealed WBC of 103.34 × 10⁹/L, Hb of 147 g/L, and Plt of 38 × 10⁹/L. Bone marrow smears showed lymphoblastic cells accounted for 97.5%. Immunophenotyping identified 69.3% of abnormal T cells expressing CD7, CD4, CD5, sCD3, CD99, CD2, CD38, and cCD3, with partial expression of CD1a. Karyotype analysis revealed 46, XY, der (1;7)(p22;p13) [20]. Genetic testing identified NOTCH1 mutation. The patient was diagnosed with T-ALL and classified as the high risk group based on WBC count >100 × 10⁹/L. The patient received induction therapy with vinodixin, daunorubicin, cyclophosphamide, pegaspargase, and dexamethasone (VDCLP) and achieved CR with MRD positivity, followed by one cycle of high-dose methotrexate (HD-MTX), two cycles of high-dose methotrexate plus cytarabine (hyper-CVAD B), and two cycles of cyclophosphamide, vinodixin, doxorubicin, and dexamethasone (hyper-CVAD A) as consolidation therapy (Figure 1). The patient maintained CR but never achieved MRD negativity in the BM by FCM during induction and consolidation therapy. Leukemic cells were detected in the CSF during the first course of consolidation chemotherapy, and the patient was diagnosed with central nervous system leukemia (CNSL). After one lumbar puncture with intrathecal chemotherapy, leukemic cells became undetectable in the CSF. Thereafter, the patient received additional lumbar punctures and intrathecal chemotherapy.

Unfortunately, one month after the final consolidation cycle, the patient experienced relapse, with WBC increasing to 231 × 10⁹/L. Leukemic blasts accounted for 87.6% of BM cells and were immunophenotypically characterized as CD99^bri, CD7, cTdT, CD2, CD5, CD34/CD1a, and CD3^dim positive, and CD56, CD4, and CD8 negative. However, the patient failed to achieve second complete remission after one cycle of salvage chemotherapy with cytarabine, aclarubicin, and granulocyte colony-stimulating factor (CAG). Because relapse occurred within one year, so the leukemia in the patient was considered relapsed and refractory. Given the high expression of CD7 on leukemic blasts, CD7 CAR T-cell therapy was recommended to achieve remission and bridge to allo-HSCT.

After intrathecal injection and FC regimen preconditioning, the patient received an infusion of autologous humanized NS7CAR T cells (Hebei Senlangio Technology Co., Ltd., Shijiazhuang, China). The dose of the effective CD7 CAR T cells in the infusion was 5 × 10⁵ cells/kg. The WBC count in peripheral blood (PB) briefly declined and then rapidly increased to 425 × 10⁹/L soon on day 3 after CAR T-cell infusion, indicating ongoing disease progression. Cyclophosphamide, dexamethasone, and idarubicin were administered for tumor burden reduction. Subsequently, the patient received second CD7 CAR T-cell infusion, with an effective dose of 2.5 × 10⁵ cells/kg. As observed in case 1, approximately two weeks later, the absolute number of CAR T cells reached a peak (Figures 4A, B), and the patient achieved complete remission (CR) with minimal residual disease negativity (MRD−) in the bone marrow (BM), as verified by flow cytometry (FCM) (Figure 4C).

Grade 1 cytokine release syndrome (CRS), manifested by fever and muscle aches, was observed after CD7 CAR T-cell infusion and was successfully managed. CAR T-cell–related encephalopathy syndrome (CRES) events were not detected during therapy. Several inflammatory biomarkers, including interleukin-18 (IL-18) and monocyte chemoattractant protein-1 (MCP-1), peaked approximately 1–2 weeks after the CD7 CAR T-cell infusion (Figure 4D), likely associated with CRS. Neutrophil counts exceeded 0.5 × 10⁹/L on day 41. Red blood cell and platelet transfusion dependence persisted prior to allo-HSCT (Supplementary Figure S2).

On day 78 after the second CAR T-cell therapy, the patient received myeloablative conditioning consisting of total body irradiation (TBI), cytarabine, cladribine, and idarubicin (Figure 3). Anti-thymocyte globulin (ATG), cyclosporine A and methotrexate were administered for GVHD prophylaxis. The patient then underwent infusion of allogeneic hematopoietic stem cells from his father on July 6, 2021 during the second CR. Neutrophil and platelet engraftment occurred on days +12 and +13, respectively. Short tandem repeat (STR) analysis using fluorescence-labeled polymerase chain reaction (PCR) demonstrated complete donor chimerism on day +30.

On day +37 after allo-HSCT, the patient was readmitted because of headache and fever. Epstein–Barr virus (EBV) DNA was detected in the PB and cerebrospinal fluid (CSF) at levels of 5.19 × 10⁵ copies/mL and 2.76 × 10³ copies/mL, respectively. Concurrently, multiple enlarged lymph nodes were observed in the bilateral cervical, supraclavicular, axillary, and inguinal regions. Brain magnetic resonance imaging (MRI) revealed small patchy hyperintense lesions near the posterior horn of the right lateral ventricle on T2-weighted images (Supplementary Figure S3). Based on these findings, the patient was diagnosed with EBV disease, EBV encephalitis, and probable EBV-associated post-transplant lymphoproliferative disease (EBV-PTLD). Glucocorticoids were reduced rapidly tapered, and rituximab was administered via intrathecal injection (40 mg, three doses) and intravenous infusion (375 mg/m² weekly, three doses). Clinical symptoms resolved completely, and EBV-DNA became undetectable after the first rituximab administration. Two weeks later, follow-up brain MRI showed a marked reduction in lesion size of the lesions.

Subsequently, skin rash and diarrhea developed on day +97 after allo-HSCT. The dosage of cyclosporine A was appropriately adjusted based on serum concentration monitoring. On day +104 after allo-HSCT, the patient developed afebrile convulsions accompanied by dizziness and headache. The serum of cyclosporine A at that time was 406 ng/mL. Brain MRI demonstrated resolution of the previous periventricular lesions near the right ventricle disappeared, but revealed new patchy lesions in the bilateral frontal and parietal cortices, consistent with reversible posterior encephalopathy syndrome (Supplementary Figure S3). Cyclosporine A was discontinued, and glucocorticoids and mycophenolate mofetil (MMF) were administered for treating GVHD management. The patient’s clinical symptoms resolved completely following treatment.

On day +184, CSF analysis revealed leukemic cells, indicating central nervous system leukemia (CNSL). Residual leukemic cells in the CSF became undetectable after two courses of intrathecal chemotherapy. Oral venetoclax (100 mg daily) was subsequently initiated to prevent disease recurrence. However, on day +388, abnormal T cells accounted for 54.0% in the CSF and 23.0% in the BM. Leukemic cells initially decreased but later increased, and the patient has developed progressive central nervous system symptoms, primarily manifesting with paraplegia, despite receiving further anti-leukemia treatments, including azacitidine (100 mg subcutaneously on days 1–7) combined with venetoclax (400 mg orally on days 1–14), CD7 CAR T-cell infusion, hematopoietic stem cell infusion from his father, and multiple courses of intrathecal chemotherapy. Ultimately, treatment was discontinued for financial reasons, and the patient eventually died of leukemia on day +561.