Erythropoietin-stimulating agent-resistant anemia (ESA-RA) is observed in a significant number of hemodialysis (HD) patients. However, the underlying mechanisms remain unclear. We report two HD patients in whom vitamin B6 (VB6) deficiency was thought to be the cause of ESA-RA, and oral administration of pyridoxal phosphate hydrate led to prompt elevation of hemoglobin levels. VB6 is crucial for heme biosynthesis. HD removes VB6 from the serum. Therefore, patients undergoing HD are theoretically prone to VB6 deficiency anemia. However, reports of VB6 deficiency anemia in HD patients are scarce, and we speculate that this is an under-recognized cause of ESA-RA.

The KMT2A gene is frequently altered in acute myeloid leukemia (AML). KMT2A abnormalities include partial tandem duplication (PTD), single-nucleotide variants (SNVs), and chromosomal rearrangements such as t(9;11)(p21.3;q23.3) and t(v;11q23.3). However, with the widespread adoption of next-generation sequencing (NGS), PTD has been assessed less frequently. We evaluated the clinical features and prognostic impact of PTD and other KMT2A abnormalities. We analyzed the KMT2A abnormalities and coexisting genetic alterations in 585 patients with de novo AML diagnosed across 25 institutions (1991–2020). Using bone marrow and/or peripheral blood samples, we identified PTD by targeted polymerase chain reaction-based assay, chromosomal rearrangements by karyotyping, and SNVs and other genetic alterations by targeted NGS. KMT2A abnormalities were detected in 18.3

T follicular helper (TFH)-derived peripheral T-cell lymphomas (PTCLs) harbor frequent mutations in epigenetic regulators and are sensitive to epigenetic therapies. The histone deacetylase inhibitor tucidinostat demonstrated efficacy in relapsed/refractory PTCL, especially angioimmunoblastic T-cell lymphoma (AITL), in a phase IIb trial; however, the lack of TFH phenotyping has prevented assessment of the predictive value of this phenotype across a broader PTCL spectrum. Therefore, we retrospectively analyzed patients originally diagnosed with AITL or PTCL not otherwise specified (NOS) based on 2008 World Health Organization criteria who participated in the aforementioned trial. TFH phenotype was defined as the expression of ≥ 2 TFH markers. Among the 23 evaluable patients, the TFH group (n = 17) included six with AITL and 11 with PTCL-NOS exhibiting TFH features, and the non-TFH group included six patients with PTCL-NOS. The objective response rate was numerically higher in the TFH group (12/17, 70.6

IntroductionCD19-directed chimeric antigen receptor (CD19-CAR) T-cell therapy has markedly improved outcomes in relapsed and refractory B-cell malignancies, but its efficacy remains limited by insufficient in vivo persistence and functional exhaustion. We have generated functionally rejuvenated T-cells (rejTs) by reprogramming antigen-specific cytotoxic T lymphocytes (CTLs) into induced pluripotent stem cells (iPSCs) and redifferentiating them into CTLs with restored proliferative capacity. In this study, we explored a vaccine synergy strategy to enhance the persistence of CAR-rejuvenated CTLs (CARrejTs) through T-cell receptor (TCR) restimulation.MethodsSARS-CoV-2 spike protein-specific rejTs (COVID19-rejTs) were established from iPSCs derived from spike protein-specific CTLs. A CD19-CAR was introduced into these iPSCs to generate dual-antigen recognition CARrejTs targeting CD19 and COVID-19 spike protein (1919-CARrejTs). Subsequently, 1919-CARrejTs were assessed for cytotoxicity, proliferative capacity, and exhaustion phenotype using 51Cr release assays, sequential rechallenge assays, and CFSE-based proliferation analysis with CAR- or TCR-dependent stimulation.Results1919-CARrejTs uniformly expressed both CD19-CAR and spike protein-specific TCRs, retained antigen-specific cytotoxicity, and exhibited a rejuvenated phenotype with higher expression of granzyme B and perforin and lower expression of exhaustion markers compared with conventional CD19-CAR-T cells. Dual-antigen recognition enhanced cytotoxicity under matched antigen presentation, and 1919-CARrejTs maintained durable tumor control in sequential rechallenge assays. CFSE dilution analysis revealed that TCR-mediated stimulation by spike protein-specific peptide provided strong proliferative capacity of 1919-CARrejTs in an HLA-dependent manner.ConclusionThe combination of iPSC-mediated rejuvenation and dual-antigen recognition via CAR and native TCR confers superior cytotoxicity, persistence, and proliferative potential compared to conventional CD19-CAR-T cells. These findings provide a proof-of-concept for a vaccine-synergy strategy in which in vivo TCR restimulation supports selective expansion and sustained antitumor effect of dual-antigen recognition T-cells that can be a promising treatment approach for B-cell malignancies.

The BCL-2 inhibitor venetoclax combined with the hypomethylating agent azacitidine or with low-dose cytarabine significantly improves response rates and overall survival (OS) for newly diagnosed unfit and relapsed/refractory (R/R) acute myeloid leukemia (AML) patients. We retrospectively analyzed our experience with venetoclax combination therapy in 41 unfit AML patients (23 untreated, 18 R/R). Overall response rates were 78.3% for untreated patients and 61.1% for R/R patients. TP53 alterations were observed in 13 patients (31.7%) and were identified as an independent predictor of poor outcome (p = 0.0002). We further conducted single-cell RNA sequencing in bone marrow, sampled before and after venetoclax and azacitidine treatment, of three TP53-mutated AML patients who achieved complete remission (CR) or CR with incomplete hematologic recovery. After treatment, numbers of cells expressing anti-apoptotic genes such as BCL2 and MCL1 decreased. CD4 T cells, cytotoxic CD8 T cells, and NK cells significantly increased both in number and in levels of gene expression associated with cytotoxicity post-treatment, confirming immune recovery in the tumor microenvironment. Residual AML cells expressed CD47 and CLEC12A (CLL1). These results indicate that venetoclax combination therapy of AML is promising in real-world clinical practice and suggest a role for ancillary treatment targeting antigens expressed on residual AML cells as a therapeutic strategy in TP53-mutated AML.

CD4+ T cells are anticipated to enhance the overall immune response, including the anti-tumor activity of chimeric antigen receptor (CAR)-T cell therapy. In the past, we established a culture system to generate CD8+ T cells from iPS cells (iPSCs); however, it was challenging to generate CD4+ T cells. Drawing inspiration from the observation that adult T cell leukemia (ATL) cells are consistently CD4+ and possess Treg characteristics, we successfully generated CD4+ Treg cells by reprogramming ATL cells into iPSCs and then differentiating them into T cells. Gene expression analysis of this generation system suggested that RUNX3 serves as a key regulator in T cell differentiation within the ex vivo generation system. By knocking out RUNX3 , we demonstrated the generation of antigen-specific CD4+ Th1 cells via the iPSC route, thereby enhancing the activity of CD8+ CAR-T cells against GD2-expressing lymphoma. These technologies hold significant promise for contributing to “off-the-shelf” immunotherapies against malignant tumors, including solid tumors. ### Competing Interest Statement H.N. is a co-founder of Century Therapeutics. Japan Society for the Promotion of Science, https://ror.org/00hhkn466, 24K02330 Juntendo University School of Medicine Center for Genomic and Regenerative Medicine, AM425G2309

Background:Bispecific antibodies are effective for relapsed/refractory B-cell lymphoma patients even after CAR-T therapy. Methods:Peripheral blood CAR-T kinetics and functional analysis in vivo were carried out pre- and post-epcoritamab infusion in B-cell lymphoma patients relapsing after CAR-T therapy. Results:CAR copy numbers spiked at relapse and rose again following epcoritamab administration. Expression levels of CAR-T exhaustion markers did not increase, whereas perforin and granzyme B expression increased after epcoritamab induction. Conclusion:Early epcoritamab administration without cytotoxic agents at relapse after CAR-T therapy revives CAR-T cell numbers and cytotoxicity, which may potentially lead to favorable clinical outcomes.

Venetoclax (VEN) with azacitidine (AZA) is the standard treatment for patients with acute myeloid leukemia (AML) who are ineligible for intensive chemotherapy. However, real-world evidence on dosing, scheduling, and outcomes is lacking, particularly for patients with prior myelodysplastic syndrome (MDS) or AZA treatment, who have been excluded from clinical trials. This was a multicenter retrospective study of VEN + AZA in 120 patients newly diagnosed with AML between June 2021 and September 2022. The cohort had a median age of 77 years, 52% had secondary AML, 74% had previously been diagnosed with MDS, and 39% had previously received AZA. During cycle 1, half of the patients received 400 mg of VEN for a median of 27 days, with a median holding period of 12 days. With a median follow-up of 13.6 months, the rate of complete remission (CR) or CR with incomplete blood count recovery was 56.7% in VEN + AZA-treated patients in the overall cohort and 56.5% in patients with prior MDS. Median overall survival was 14.8 months for the overall cohort and 15.4 months for those with prior MDS. The real-world outcomes were comparable to those of clinical trials.

BACKGROUND AND OBJECTIVES:We developed an automated morphological image recognition deep learning system (image recognition DLS) of peripheral blood cells, then constructed the diagnostic assist DLS combining image recognition DLS data with complete blood count (CBC) data. This study aimed to evaluate the clinical performance of the image recognition DLS and the diagnostic assist DLS in routine examinations. METHODS:The image recognition DLS was trained using datasets containing 1 476 727 images of white blood cells (WBCs), nucleated red blood cells (NRBCs), and large platelets to differentiate 14 blood cell types and to recognize 24 morphological characteristics. CBC data were obtained through the automated hematology analyzer (Sysmex XN-9000) and combined with the image recognition DLS data to construct the diagnostic assist DLS. The clinical performance of the image recognition DLS was evaluated using 128 716 blood cell images from 589 smears obtained from healthy subjects, ALL, AML, ML, MPN, and MDS cases in routine examinations. RESULTS:The image recognition DLS classified 14 blood cell types with an accuracy of 97.3%-99.9%. The accuracy of 11 morphological characteristics exceeded 90%. Blast cells were detected accurately on all slides, where they were identified by manual microscopy. Malignant lymphocytes were classified as blasts and/or lymphocytes with the morphological characteristics of each subtype of lymphoma. The diagnostic assist DLS successfully differentiated MDS, achieving an AUC (area under the curve) of 0.99. CONCLUSION:This study demonstrated the potential of the diagnostic assist DLS, utilizing morphological image recognition DLS data combined with CBC parameters, as a promising diagnostic tool.

BACKGROUND:The final manufactured tisagenlecleucel product should meet the commercial product release specifications to ensure the quality in terms of safety, purity, identity, and potency. However, it may occasionally fail to meet these specifications due to the nature of patient-derived cells with variable properties as starting material and the complex manufacturing process. The final product that does not meet at least one of the commercial release specifications is referred to as "out-of-specification" (OOS). However, the benefit-risk profile of OOS tisagenlecleucel has not yet been fully elucidated. AIMS:To evaluate the safety and efficacy of OOS tisagenlecleucel in Japanese patients with relapsed or refractory (r/r) diffuse large B-cell lymphoma (DLBCL) and B-cell acute lymphoblastic leukemia (B-ALL). METHODS:This is a single-arm, open-label, multicenter phase 3b study (NCT04094311). Patients consistent with label indication were enrolled and followed-up for 3 months. RESULTS:Of the 29 patients enrolled between December 2019 and May 2022 across 13 qualified sites in Japan, 28 received tisagenlecleucel, and of these, 23 had r/r DLBCL and 5 had r/r B-ALL. The primary reasons for OOS were low cell viability (15 of 24 batches) and low dose (8 of 23 batches) tisagenlecleucel in the r/r DLBCL group, and high dose (4 of 5 batches) in the r/r B-ALL group. In patients with r/r DLBCL, the grade 3 or 4 cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome occurred in 3 and 1 patients, respectively. Response assessments were performed for 15 of 23 patients with r/r DLBCL: 6 achieved a complete response, and 1 achieved a partial response as the best response within 3 months. CONCLUSIONS:Despite the limited patient sample size, our findings affirm that the infusion of OOS tisagenlecleucel is a viable option, with no observed increase in toxicity and outcomes comparable to those of in-specification products in clinical and real-world studies.

In most cases of anaplastic lymphoma kinase–positive anaplastic large cell lymphoma (ALK + ALCL), long-term survival is achieved using CHOP therapy. However, some cases have a poor prognosis. Here, we investigated the clinical impact of TP53 deletion on adult ALK + ALCL patients via a multicenter, retrospective analysis. TP53 deletion was evaluated by fluorescence in situ hybridization (FISH) using paraffin sections of lymphoma samples. To re-evaluate the FISH results, whole genome copy number changes were analyzed in DNA extracted from paraffin sections using OncoScan analysis. Fourteen patients treated with first-line chemotherapy enrolled at six centers were analyzed. All patients received CHOP-based therapy as initial therapy. The 5-year progression-free survival (PFS) and overall survival (OS) of the 14 patients were 28.6% (median 7 months) and 57.1% (median 99 months), respectively. FISH analysis revealed 6 (43%) patients were positive for TP53 deletion (deletion group) and 8 (57%) were negative (non-deletion group). All six patients in the deletion group were diagnosed at an advanced stage; five were refractory to initial treatment, one relapsed after treatment, and all patients died of ALK + ALCL. The median PFS was 3.5 months in the deletion group and 76 months in the non-deletion group. The median OS was 7 months in the deletion group and has yet to be confirmed in the non-deletion group. OncoScan analysis showed TP53 copy number reduction in the deletion group and no TP53 copy number abnormalities in the non-deletion group. This study suggests that TP53 deletion is a poor prognostic factor in ALK + ALCL treated with CHOP-based therapy.

T cells play a critical role in immune defense by eliminating virus-infected and malignant cells. Their antigen specificity is determined by T cell receptors (TCRs), heterodimers composed of α and β chains, whose diversity is generated through V(D)J recombination and junctional nucleotide insertions. It is well known that, unlike the β chain which follows the principle of allelic exclusion and is expressed from only one allele, the α chain can undergo rearrangement at both alleles and may be expressed from both loci. Identifying the TCRαβ sequences of cytotoxic T lymphocyte (CTL) clones with robust cytotoxicity is essential for advancing TCR-engineered T cell therapies. Although the TCR repertoire is highly diverse, dominant clones occasionally emerge that possess strong reactivity against specific antigens. Analyzing these dominant clones offers opportunities to identify antigen-specific TCRs with therapeutic potential and to understand the principles of immunological memory. We have established a platform to generate rejuvenated antigen-specific CTLs (rejTs) by reprogramming T cells into induced pluripotent stem cells (T-iPSCs) and redifferentiating them into CTLs. These rejTs proliferate vigorously and exhibit strong antigen-specific cytotoxicity, allowing efficient functional evaluation and potential application in adoptive T cell therapies. In our attempt to generate Epstein-Barr virus (EBV) LMP2 (419–427, TYGPVFMSL)-specific rejTs, we obtained CTL clones with identical TCRαβ sequences in two independent experiments. RejTs derived from these clones demonstrated potent cytotoxicity, suggesting that they represent dominant clones. Interestingly, these clones expressed two functionally rearranged TCRα chains (α₁ and α₂), both paired with the same TCRβ chain. To determine which TCRα chain mediated LMP2 recognition, we knocked out endogenous TCRs in HLA-matched donor T cells using CRISPR/Cas9 and transduced them with either TCRα₁β or TCRα₂β. Only TCRα₁β-transduced T cells bound the LMP2/HLA-A24 tetramer and exhibited specific cytotoxicity against LMP2-expressing, HLA-matched lymphoblastoid cell lines (80% vs 0.5% lysis at an E:T ratio of 40:1), indicating that TCRα₁β mediates the observed anti-EBV activity. To explore whether TCRα₂β also recognizes a different epitope of , we applied three deep learning–based TCR specificity prediction tools (MixTCRpred, TCRex, and ERGO-II), which identified candidate EBV epitopes. MHC binding predictions (NetMHC v4.0) confirmed strong binding to the donor's HLA alleles. We are currently validating the predicted epitopes to assess the contribution of TCRα₂β to the cytotoxicity observed in this dominant clone.

COVID-19 is a threat to patients with hematological malignancies (HM) even in the Omicron era, because mortality rates are still high in HM patients, and a significant number of patients develop a protracted disease course called "persistent COVID-19 (pCOVID-19)" which can continue for weeks to months. pCOVID-19 can be life-threatening by itself, but also drastically affects the disease course of the underlying HM by delaying or terminating chemotherapy. Also, patients with pCOVID-19 can be potentially contagious, and timing of ending isolation is a dilemma the hematology ward faces. Furthermore, pCOVID-19 has been reported to lead to acquisition of SARS-CoV-2 multidrug-resistant mutations, which is an alarming issue for both the patient and public health. The optimal management method of pCOVID-19 is currently unknown, and because HM patients are excluded from randomized clinical trials, evidence is limited to case reports and small case series. We carried out a comprehensive literature review of Omicron pCOVID-19 occurring in HM patients, compiled the scattered evidence, and provide practical recommendations which can be of guide to clinicians. Main topics discussed within this review include efficacy of vaccinations in HM patients, risk factors for developing pCOVID-19 (B-cell depleting agents, bendamustine + rituximab therapy, bispecific T-cell engagers, etc.), treatment of pCOVID-19 including extended/sequential/combination therapy incorporating antivirals (nirmatrelvir/ritonavir, remdesivir, molnupiravir, and ensitrelvir) and convalescent plasma/intravenous immunoglobulin therapy, monitoring pCOVID-19 with reverse transcription (RT)-PCR, and optimal target cycle threshold values as goals of therapy.

Venetoclax has demonstrated clinical benefit for newly-diagnosed acute myeloid leukemia (AML), but significant neutropenia is a concern. Data on the time course of neutrophil counts for across treatment cycles in real-world settings remain limited. We report an interim analysis of the VENUS study, which examined neutropenia management in patients with AML receiving venetoclax with azacitidine (VEN/AZA) in Japan. This multicenter (10 sites), retrospective, observational study included adults with newly-diagnosed AML ineligible for intensive chemotherapy and initiating venetoclax treatment. Treatment patterns, granulocyte colony-stimulating factor (G-CSF) use, antifungal prophylaxis, and time course of neutrophil counts were analyzed for patients who received > 1 cycle of venetoclax. Venetoclax was administered for a median 27.0 days in Cycle 1 and then a median 21.0 (range 14.0–22.0) days for subsequent cycles, with median dose holds at the end of each cycle of 8.5–15.0 days. Patients (n = 81) receiving G-CSF were treated with VEN/AZA for a median of 6.0 cycles versus 3.0 in those who did not receive G-CSF (n = 39). In Cycle 1, median neutrophil counts decreased to < 500/µl during Days 8–28 but recovered to > 500/µl by Days 29–35. Median nadir neutrophil count was reached during Days 22–28 in almost all subsequent cycles until Cycle 10. Neutrophil counts decreased to < 500/µl in some cycles but improved to > 500/µl by the next week, suggesting neutrophil levels without higher risk of infection in most patients after Cycle 2 with venetoclax dosing schedule modifications and G-CSF administration. Eighty-eight (73.3

Extranodal NK / T-cell lymphoma, nasal type (ENKL) is a highly aggressive lymphoma invariably infected by Epstein-Barr virus (EBV) with type II latency expressing latent membrane protein (LMP1/LMP2). Therefore, antigen-specific cytotoxic T lymphocyte (CTL) therapy targeting these antigens is a promising strategy to selectively kill these lymphoma cells. However, continuous exposure to their target antigens often exhausts CTLs. To overcome this problem, we have successfully rejuvenated exhausted virus-specific CTLs (rejTs) by iPSC technology, demonstrating that rejTs can more robustly proliferate, show younger memory T cell phenotype, and have stronger antitumor effect than original CTLs (Cell Stem Cell 2013, Stem Cell Reports 2015, Haematologica 2020). Unfortunately, generating rejTs from each patient is time-consuming, which has been an obstacle in clinical application. Therefore, in this study, we generated HLA-class I-edited allogeneic LMP2-specific rejTs from a healthy donor using CRISPR/Cas9 gene editing to evade rejection from patients' immune cells. iPSCs were first established from a healthy donor's HLA-A2402 restricted LMP2-CTL clone. Next, we knocked out B2M gene to eliminate HLA class I antigen expression and introduced B2M-HLA-A24-fusion and trimer peptide-B2M-HLA-E fusion genes to avoid NK-cell attack via bw4 epitope of HLA-A24/KIR3DL1 and HLA-E/NKG2A bindings (Cell Reports Medicine 2023). These HLA-edited iPSCs efficiently differentiated into rejTs and suppressed alloreactivity when cocultured CD8+ T cells in a mixed lymphocyte reaction assay. CD107a expression levels were significantly lower on the allogeneic CD8+ T cells upon culture with HLA-edited rejTs than unedited-rejTs (p=0.0421). HLA-A24 and HLA-E dual knock-in (KI) rejTs significantly suppressed NK cell cytotoxicity compared to either single KI-A24-rejTs (p<0.0001) or KI-E-rejTs (p<0.0001) in a cytotoxicity assay at an effector to target (E:T) ratio of 9:1 (KI-A24&E-rejTs 19.2%, KI-A24-rejTs 31.4%, KI-E-rejTs 45.4%). To analyze the antitumor effect of HLA-edited LMP2-rejTs against ENKL, we performed in vitro cytotoxic assay. The cytotoxicity of HLA-edited-LMP2-rejTs was higher against ENKL cells (NK-YS and ENKL-J1), than that of original LMP2-CTLs (NK-YS; 74.3% vs 59.8%, ENKL-J1; 78.8% vs 34.7%) at an E:T ratio of 2.5 : 1. Next, to observe the tumor suppressive effect of LMP2-rejTs in vivo, firefly luciferase-labeled NK-YS cells were injected into NOG mice. Mice were divided into an untreated group and two treatment groups (original LMP2-CTLs or HLA-edited LMP2-rejTs). Although there was no significant difference in the bioluminescent signal treated with original CTLs compared to untreated mice (p=0.3919), the signal was significantly lower in mice treated with HLA-edited LMP2-rejTs compared to untreated mice on Day 28 (p=0.0219). To compare gene expression in original CTLs and EBV-rejTs, we performed single-cell RNA sequencing. EBV-rejTs expressed higher level of genes associated with cytotoxicity (IFNG, PRF1) and lower level of genes associated with exhaustion (TIGIT, LAG-3) compared to original CTLs. In conclusion, we have successfully generated minimally immunogenic HLA-edited LMP2-rejTs from a healthy donor that can evade allogeneic immune responses while enhancing T cell function. To bring this platform to the clinic, we have established a master cell bank of HLA class I-edited iPSCs derived from a healthy donor-derived LMP2-CTL clone at our cell processing center. We are currently preparing to start investigator-initiated Phase I clinical trials using these cells for HLA-A2402+ patients. Once established, such antigen-specific CTL derived, HLA-edited iPSCs could provide an unlimited source of “off-the-shelf” T cell therapy targeting EBV-associated lymphomas.

Background: Acute graft-versus-host disease (GVHD) is a major complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT) with significant morbidity and mortality, and efficacy of currently available therapeutics are limited. Acute and chronic GVHD are similar in that both are initiated by antigen presenting cells and activation of alloreactive B-cells and T-cells, subsequently leading to inflammation, tissue damage, and organ failure. One difference is that acute GVHD is mostly attributed to T-cell activation and cytokine release, whereas B-cells are the key players in chronic GVHD. Ibrutinib is an irreversible inhibitor of the Bruton's tyrosine kinase (BTK), which is part of B-cell receptor signaling. Ibrutinib is currently used for treating chronic GVHD, but its efficacy towards acute GVHD is unknown. Besides BTK, ibrutinib also inhibits interleukin-2 inducible T-cell kinase (ITK), which is predominantly expressed in T-cells and a crucial enzyme for activating the downstream pathway of TCR signaling. ITK activates PLCγ2 and facilitates signaling through NF-κB, NFAT, and MAPK, leading to activation and proliferation of T-cells and enhanced cytokine production. Therefore, the TCR signaling pathway is indispensable for development of acute GVHD, and ITK inhibition by ibrutinib would be a rational therapeutic approach. Case presentation: A 56-year-old male acute myeloid leukemia patient with Myeloid neoplasms with germline DEAD-box RNA helicase 41 (DDX41) mutation underwent cord blood transplantation and developed severe gastrointestinal (GI) acute GVHD which was refractory to steroids and mesenchymal stem cell therapy. While acute GVHD accommodated by multiple life-threatening GI bleeding events persisted, chronic cutaneous GVHD developed, and ibrutinib 420 mg/day was initiated from day 147 of transplant. Although ibrutinib was commenced targeting the chronic GVHD, unexpected and abrupt remission of acute GVHD along with remission of chronic GVHD was observed. Conclusion: Ibrutinib is a promising therapeutic for treating acute GVHD, and further studies are warranted.

Supplementary Figure S2 showcases the functional and phenotypic analysis of GD2-CARTs and GD2-CARrejTs, including cytokine production (IL-4, IL-6, IL-10, and IL-17A) through cytometric bead assays, flow cytometry gating strategies, memory phenotype subsets, CAR transgene expression, and CD4/8 ratios.