Citations

Abstract Objectives The aim of this work was to develop an American Headache Society position statement addressing diagnostic screening for migraine among girls and women. Background Despite its high prevalence and substantial negative impacts, migraine is underdiagnosed and undertreated. Diagnostic screening for migraine enables more patients to receive timely, appropriate, and effective management. Methods Development of this position statement followed the rules established by the American Headache Society Guidelines Committee. The published literature was reviewed to determine if migraine meets criteria for when disease screening is justified, to guide recommendations for screening tools, and to determine subpopulation(s) for which migraine screening is indicated. After author consensus was reached, the position statement was reviewed and approved by the American Headache Society Board of Directors. Results Migraine fulfills established criteria for conditions in which screening is appropriate since it is highly prevalent, results in significant morbidity, and exerts substantial economic and social costs. Migraine incidence and prevalence are exceptionally high among girls and women during adolescence and through menopause. Furthermore, there are valid and reliable diagnostic screening methods (e.g., ID Migraine) and effective treatments that reduce migraine symptoms and disease impact. Conclusion Yearly diagnostic screening for migraine should be included as part of women's preventive healthcare services, particularly from adolescence to menopause.

Malignant transformation of intracranial nongerminomatous germ cell tumors (NGGCTs) is a rare but clinically relevant phenomenon. We present the case of a 13-year-old boy with a localized, pineal NGGCT. After an initial favorable response, tumor growth was noted while still on chemotherapy. Histopathologic characterization revealed that 40% of the tumor was embryonal rhabdomyosarcoma (RMS), consistent with potential malignant transformation. Due to the rare nature of NGGCT malignant transformation, the best clinical approach to these cases remains unclear. We explore existing cases, treatments, and outcomes of malignant transformation in intracranial NGGCTs to help inform future clinical decision-making and the establishment of treatment guidelines.

Abstract Immune checkpoint inhibitor–associated immune thrombocytopenia (ICI-ITP) has been described in case reports and small case series, but comprehensive data on its incidence, risk factors, clinical features, treatment, and outcomes are lacking. We reviewed medical records of all adults initiating ICI therapy between 2016 and 2023 at 29 US hospitals across 7 major cancer centers to identify patients with ICI-ITP. Multivariable logistic regression was used to identify risk factors, and Cox modeling was performed to assess the association between ICI-ITP, its severity, and mortality. Among 86 467 patients, ICI-ITP occurred in 214 (0.25%). Independent risk factors included lower baseline platelet count, combination ICI therapy, stage IV cancer, and additional immune-related adverse events. ICI-ITP occurred at a median of 8 weeks (interquartile range [IQR], 4-18) after ICI initiation, with a median nadir platelet count of 41 × 109/L (IQR, 17 × 109/L to 64 × 109/L). Patients were treated with glucocorticoids (n = 106 [49.5%]), immune globulin (n = 39 [18.2%]), and thrombopoietin receptor agonists (n = 29 [13.6%]). Recovery occurred in 161 patients (75.2%) at a median of 2.3 weeks (IQR, 1.0-5.3). Of 76 patients rechallenged with ICIs, 23 (30.3%) developed recurrent ICI-ITP. ICI-ITP and its severity were associated with higher all-cause mortality, with a nearly threefold increase in risk among patients with severe ICI-ITP than those without ICI-ITP (adjusted hazard ratio, 2.96 [95% confidence interval, 2.14-4.08]). These findings establish ICI-ITP as a rare but clinically significant complication of ICI therapy, provide, to our knowledge, the first large-scale description of its risk factors and clinical course, and underscore the importance of timely recognition and management.

OBJECTIVE The aim of this study was to compare 3 electrode designs—a novel 22-contact circular grid, standard 6-contact strip, and high-density (HD) grid between 32 and 64 contacts—for the detection of epileptiform activity during awake craniotomy. METHODS This study included patients who underwent functional brain mapping with and without direct electrical stimulation during awake craniotomy. Demographic, clinical, and electrocorticography (ECoG) data were collected. RESULTS A total of 194 patients were included, with 264 instances of electrode use: 113 circular grid, 96 strip electrode, and 55 HD grid. The HD grid (15%) was used less to record ECoG during direct electrical stimulation than the circular grid (80%, p < 0.001) and strip electrodes (69%, p < 0.001). Sporadic interictal epileptiform activity was better detected with circular (45%, p < 0.001) and HD (40%, p = 0.006) grids compared with strip electrodes (16%). Spontaneous seizures were recorded more frequently with the circular grid (16%, p = 0.018) compared with the strip electrode (9.4%) and HD grid (1.8%). Afterdischarges were more frequently detected by the circular grid (69%, p = 0.031) and with circular and HD grids (combined) compared with strip electrodes (67% vs 48%, p = 0.024), at lower median intensity (3 mA vs 5 mA, p < 0.001). Multivariable analyses demonstrated circular grids (p < 0.001) to be more sensitive in detecting spontaneous epileptiform activity than strip and HD grid electrodes, and grids (circular and HD) to be better in recording stimulus-induced epileptiform activity (p = 0.01). CONCLUSIONS This study demonstrates a higher rate of detection of epileptiform activity with circular and standard grids when compared with strip electrodes. The standard HD grid was used less during electrical stimulation, demonstrating the importance of both the number of contacts and design array when considering optimal conditions for ECoG and functional brain mapping.

Abstract BACKGROUND Children exposed to poverty during frontline therapy for B-lymphoblastic leukemia (B-ALL) experience inferior overall survival (OS), higher relapse rates, and earlier relapse than their peers. Real-world data describing treatment patterns and survival after relapse of B-ALL are lacking and, as a result, the impact of poverty following relapse remains poorly understood. This study evaluated the association of poverty exposure, proxied by insurance status, with post-relapse survival and explored potential explanatory mechanisms, including disease biology and therapy utilization. METHODS Data from Retrospective Study of Contemporary Approaches to First Relapse in B-ALL (ReCALL-1), a multicenter retrospective cohort study of patients &lt; 30 years old with first relapse of B-ALL from 2018-2022, were used. The primary exposure was poverty, proxied by insurance status at relapse, categorized as any private insurance (poverty unexposed), public-only insurance (poverty exposed), no insurance, or unknown; the latter two were not included in comparative analyses due to low numbers. The primary outcome was OS. Secondary outcomes included measurable residual disease (MRD)-negative second complete remission (CR2), relapse after CR2, non-relapse mortality (NRM), with relapse treated as a competing risk, and receipt of cellular and immunotherapies. RESULTS Among 462 patients from 31 U.S. institutions, 41% had private insurance, 47% public-only insurance, 2% no insurance, and 10% unknown. Compared to privately insured patients, those with public insurance were more likely to be Hispanic (56% vs 19%, p&lt;0.001) and have non-English preferred language (30% vs 6%, p&lt;0.001). Non-Hispanic Black patients comprised 9% of those with public insurance and 5% of those with private insurance. Disease characteristics that differed by insurance status at relapse included: Ph-like cytogenetics, more common in publicly insured (23% vs 11%, p=0.002), and infant ALL, more common in privately insured patients (10% vs 5%, p=0.06). Publicly insured patients were more likely to have experienced major organ toxicity during frontline therapy (32% vs 18%, p=0.002) and very early relapse (&lt;18m from diagnosis: 43% vs 32%, p=0.07). Other demographic and disease characteristics, including relapse site and cytomolecular features, were similar by insurance status. With a median follow-up of 51 months from relapse, 4-year OS differed by insurance (log-rank p=0.02): 72% for private vs 56% for public insurance. After adjusting for race/ethnicity and language, public insurance increased the hazard of death by 65% (aHR 1.65 95% CI 1.13-2.42, p=0.004). The association remained, but was attenuated, when unbalanced disease characteristics (time to relapse, infant ALL, Ph-like cytogenetics, prior organ dysfunction) were included (aHR 1.47, 95% CI 1.00-2.16, p=0.05). Rates of MRD-negative CR2 were similar (85% public vs 87% private). However, point estimates suggested publicly insured patients had more relapse after CR2 (44% vs 37%, p=0.42) and higher 4-year NRM (16% vs 10%, Gray's test p=0.12), though neither were significant. Treatment choices for first relapse did not differ by insurance status. Utilization of intensive reinduction (91% vs 95% for public vs private, p=0.18) and clinical trial enrollment (22% for both) were similar. Receipt of cellular therapies did not significantly differ by public vs private insurance: Chimeric Antigen Receptor-T cells (CAR-T: 38% vs 34%, p=0.41), hematopoietic cell transplant (HCT: 42% vs 47%, p=0.32). Use of blinatumomab trended higher in those with private insurance (36% vs 45%, p=0.07), and inotuzumab was similar (11% for both, p=0.87). Median time from relapse to CAR-T or HCT was comparable (to CAR-T 102 vs 100 days, to HCT 117 vs 114 days). CONCLUSION This is the first study to demonstrate differential survival by insurance type, a proxy for poverty exposure, after first relapse of B-ALL. In this large multicenter, real-world cohort, patients with public insurance had worse OS after relapse than those with private insurance. The survival disparity existed independent of other SDOH factors. Adjustment for known high-risk disease features only partially accounted for the observed differences in OS. In contrast, rates of MRD-negative CR2 and receipt of CAR-T and HCT were comparable across insurance types. These findings highlight the urgent need to identify effective interventions that will ensure equitable outcomes after relapse.

Abstract Chimeric antigen receptor T (CART) cell therapy has shown unprecedented success in the treatment of hematological malignancies. However, the immunosuppressive tumor microenvironment of both solid and liquid tumors is a major barrier for CART efficacy. Immunosuppressive myeloid cells inhibit CART efficacy. Colony-stimulating factor 1 receptor (CSF1R) promotes macrophage proliferation, survival, and anti-inflammatory response and is upregulated on myeloid cells in many cancers, making CSF1R an appealing therapeutic target. We aimed to test the interactions between CSF1R, myeloid cells, and CART response, and to study CSF1R targeting in the context of CART cell therapy. We utilized CD19-targeted CART cell therapy (CART19) in lymphoma preclinical models and thyroid-stimulating hormone receptor (TSHR)-targeting CART cell therapy (TSHR-CART) in thyroid cancer models. We first measured CSF1R expression on myeloid cells in vitro. Suppressive myeloid cells, polarized from healthy donor monocytes using an anaplastic thyroid cancer (ATC) cell line, 8505c, or mantle cell lymphoma cell line, JeKo-1, significantly upregulate CSF1R (p=0.0002) compared to media control. After polarization, myeloid cells significantly inhibit TSHR-CART and CART19 antigen-specific proliferation compared to freshly isolated monocytes (p=0.012, p&lt;0.01, respectively) indicating that suppressive myeloid cells contribute to CART failure in multiple tumor types. We then performed single cell RNA sequencing on peripheral blood taken at baseline from patients with mantle cell lymphoma treated with CART19. We found a significant increase of CSF1R on monocyte subsets in CART19 non-responders vs responders (padj=0.0037). Next, we determined the impact of CSF1R blockade on CART cell therapy in vivo. Using lymphoma models, B6 mice were engrafted with the luciferase+ CD19+ murine lymphoma cell line TBL-12 (1x106 cells i.v. 1 day after 250 mg/kg cyclophosphamide). Tumor burden was significantly reduced after 7 days of treatment with low dose CART19 (0.5x106 i.v.) + anti-mouse CSF1R antibody (16mg/kg i.p. twice weekly) vs CART19 + IgG control (p=0.0146). We then tested the impact of CSF1R blockade in mice experiencing tumor relapse. After TBL-12 engraftment, mice were treated with anti-mouse CSF1R or IgG control antibody followed two days later with high dose CART19 (1x106 i.v.) to induce remission. Mice were re-challenged with TBL-12 14 days later. Mice treated with anti-mouse CSF1R antibody showed increased tumor control and significantly higher survival (p=0.0416). Using thyroid cancer models, first we identified high macrophage infiltration in biopsy specimens of patients with ATC compared to normal thyroid tissue and benign thyroid tumors through immunohistochemistry (IHC) of patient samples (p&lt;0.01, p&lt;0.0001, respectively). To test if macrophages are associated with CART failure in ATC, NSG mice were subcutaneously engrafted with 1x106 TSHR+ THJ529 tumors, an ATC cell line derived from patient biopsy samples from the Mayo Clinic thyroid cancer biobank, followed by i.v. treatment with 1x107 TSHR-CART or untransduced T cell control (UTD). IHC staining of tumor tissue collected at experimental endpoints revealed a dose-dependent, significant infiltration of human CD3+ T cells into the tumors which inversely correlated with murine F4/80+ myeloid cell and murine YM-1+ M2 macrophage infiltration. This led us to hypothesize that myeloid cell targeting with CSF1R blockade can improve TSHR-CART efficacy. We utilized the same ATC model described above to test CSF1R targeting. When TSHR+ THJ529 tumors reached approximately 100 mm3, mice were randomized by tumor volume and treated with CSF1R inhibitor PLX5622 (600mg/kg) or vehicle control, followed by either UTD or TSHR-CART cells 5x106 i.v. Mice treated with TSHR-CART + PLX5622 showed significantly improved tumor control vs TSHR-CART + vehicle control (p&lt;0.0001) or UTD + PLX5622 (p&lt;0.0001). Together, our results highlight immunosuppressive myeloid cells which express CSF1R as a major cell type limiting CART efficacy in both solid and liquid tumors. We also identified a strategy to overcome myeloid-mediated suppression of CART cells by targeting CSF1R with antibody or small molecule inhibitors.

Abstract Background Chimeric antigen receptor T cell (CART) therapy has revolutionized treatment for hematologic malignancies. However, durable remission following anti-CD19 CART therapy (CART19) remains limited to 40–50% of patients, often due to tumor relapse and CART dysfunction (PMID:31501612). We previously demonstrated that tumor-derived extracellular vesicles (EVs) from patients with chronic lymphocytic leukemia (CLL) can directly impair CART function (PMID:33388419). In this study, we aimed to identify tumor-derived EV microRNA (miRNA) cargo associated with clinical CART failure in B cell malignancies and define how they mechanistically drive CART cell dysfunction. Methods To achieve these aims, we profiled tumor-derived EV miRNA cargo using small RNA sequencing (RNAseq) in two independent patient cohorts. Cohort 1 compared plasma-derived EVs from untreated CLL patients to those from healthy donor EVs to identify miRNAs enriched in CLL (n=23). Cohort 2 included patients with diffuse large B cell lymphoma (DLBCL) treated with axicabtagene ciloleucel (CART19, n=12). Here, plasma EV miRNAs were analyzed at baseline (pre-CART treatment) and 1-month post-CART treatment and compared between CART responders (complete remission at 6 months) and nonresponders (no response at 1 month). Enriched miRNAs from both cohorts were identified following miRNA count normalization and differential expression analysis using adjusted p values of ≤0.05. Functional studies were conducted by transfecting CART19-28ζ cells with synthetic miRNA mimics or scrambled controls. CART cells were also generated with stable overexpression of miRNA via lentiviral shRNA delivery. CART function was assessed via in vitro killing, proliferation, apoptosis assays, RNAseq, and in vivo tumor xenograft models. Results In Cohort 1, we identified several miRNAs including miR-125a to be significantly enriched in CLL-derived EVs relative to healthy controls. Similarly, in Cohort 2, miRNA-125a was also identified among the miRNAs enriched in nonresponders vs. responders to CART treatment; Notably, miR-125a was significantly enriched in nonresponders at baseline (pre-CART treatment) and 1-month post-CART treatment (p=0.0177, p=0.0196). To define the functional effects of miR-125a, we delivered synthetic mimics or stable shRNA constructs encoding miR-125a into CART cells. Delivery of miR-125a was confirmed by RT-qPCR (p&lt;0.0001 for mimic vs. scramble, p=0.0069 for shRNA vs. scramble). Here, miR-125a delivery and overexpression impaired CART cytotoxicity/killing of target tumor cells (~25% reduction, p= 0.0039), decreased antigen-specific proliferation (~47% reduction, p=0.0002), and increased apoptosis following antigen stimulation (1.8-fold increase, p=0.0002) as compared to scramble controls in vitro. We next studied how miR-125a impacts CART functions in vivo. Here, tumor-bearing mice treated with miR-125a–overexpressing CART cells (CART sh125a) exhibited accelerated tumor progression (4.1-fold increase, p=0.0019) and significantly reduced survival as compared to mice treated with scramble control CART treatment (CART shScr) (20% vs 80% survival at day 35, p=0.0064), underscoring the dramatic detrimental impact of miR-125a on therapeutic efficacy. To uncover changes in CART gene expression induced by miR-125a delivery, RNAseq was also performed across stimulated CART sh125a vs. CART shScr cells. CART sh125a cells demonstrated broad transcriptional changes, with gene set enrichment of E2F targets, G2/M checkpoint, and apoptosis pathways (FDR p&lt;0.0001, p&lt;0.0001, and p=0.006) further validating the role of miR-125a in disrupting CART cell cycle control, proliferation, and survival. Conclusion In summary, our study identifies miR-125a as a tumor-derived EV cargo that drives CART dysfunction in two separate B cell malignancies (CLL and DLBCL). miR-125a was enriched in both CLL EVs and nonresponders to CART19 therapy at baseline prior and post CART treatment. Functional studies demonstrate that miR-125a delivery and overexpression directly impair CART cytotoxicity and proliferation, increase CART apoptosis, reduce therapeutic efficacy in tumor xenograft models, and disrupt key pathways related to CART cell cycle regulation and survival. These findings position miR-125a as both a key mechanistic driver of CART failure and a potential predictive biomarker for treatment response, while highlighting it as a possible therapeutic target to improve CART outcomes in the clinic.

Abstract Chimeric antigen receptor T (CART) cell therapy is potentially curative in certain hematological malignancies, resulting in several FDA approvals since 2017. However, widespread adoption is limited by 1) cost and complexity of an autologous platform, 2) severe toxicities, and 3) limited durable remissions. To better predict CART efficacy and toxicity, live CART imaging would allow the realtime assessment of in vivo CART expansion and trafficking. Incorporation of the sodium iodide symporter (NIS)in CART cells (NIS+ CART cells) allows the CART cells to be tracked via PET imaging at high sensitivity. PET signal correlates with CART expansion and toxicity in mouse models (PMID: 34244299). Building on this platform, we aimed to track xenogeneic CART cells to monitor for their rejection, utilizing dogs with spontaneous lymphoma. Canine diffuse large B-cell lymphoma (DLBCL) affects over 250,000 dogs a year and has shared pathologic, biologic, phenotypic, genetic, immunotherapeutic targets, and treatment responses with human DLBCL, providing strong rationale for studying canine lymphoma as a pre-clinical model. We therefore initiated a USDA-CVB regulated pilot study and treated companion dogs with naïve canine CD20 (cCD20)+ DLBCL with human-derived TRAC- and B2M-KO NIS+ CART cells which targeted cCD20. First, we tested and optimized a chemotherapy regimen to achieve optimal lymphodepletion in dogs. Healthy beagles were treated with pentostatin (pen) on days 2-4 (0.125, 0.25, or 0.5 mg/kg i.v.), cyclophosphamide (cy) on day 4 (400 mg/m2 i.v.), or combination cy/pen. The most robust decrease in lymphocytes was seen with combination cy/pen (all cy/pen doses vs all other treatments, p≤0.0001) and in neutrophils with cy or cy/pen (all cy/pen doses vs all pen doses, p≤0.0001; cy vs 0.25 mg/kg pen, p≤0.001; cy vs 0.5 mg/kg pen, p≤0.0001). All lymphodepletion was transient and recovered within two weeks. All regimens were well-tolerated, and we therefore used 0.5 mg/kg pen plus 300 mg/m2 cy for the companion dog pilot study. Next, we assessed the feasibility of generating cCD20-targeted NIS+ human CART cells (CART20-NIS) with double KO (DKO) of TRAC and B2M. Primary T cells from healthy human donors were transduced with bicistronic lentivirus encoding 41BB-costimulated cCD20-targeted CAR and NIS. Two days after transduction, cells were transfected with CRISPR Cas9-gRNA targeted to TRAC and B2M. The resulting CART20-NIS DKO cells were expanded until day 11 and then were further depleted for CD3 and cryopreserved. CART20-NIS DKO cells demonstrated high CAR expression (untransduced T cells (UTD) vs CART20-NIS DKO, p&lt;0.0001) and KO efficiency (TCRαβ and HLA-ABC expression of UTD vs CART20-NIS DKO, p&lt;0.0001). NIS functionality was confirmed via a qualitative in vitro I-125radioactive uptake assay. We then assessed antigen specificity and activity in vitro. CART20-NIS DKO or UTD were co-cultured with either wildtype or cCD20-overexpressing K562 cells for three days. Target cell killing was then quantified via flow cytometry. CART20-NIS DKO cells demonstrated significant cytotoxicity against cCD20+ K562 cells (UTD vs CART20-NIS DKO, p&lt;0.0001). Lastly, we treated two companion dogs in a USDA-CVB regulated pilot study of CART20-NIS DKO cells in naïve cCD20+ DLBCL. Following lymphodepletion on days -5 to -3, on day 0, dogs were i.v. administered 1 x 108 CART20-NIS DKO cells and intratumorally administered 2.5 x 107 CART20-NIS DKO cells in two lymph nodes, with contralateral saline injection as controls. Dogs underwent 18F-TFB-PET imaging on days 1, 3, and 7, which showed evidence of CART20-NIS DKO cells both in the CART-injected lymph nodes and distal lymph nodes, as determined by SUVmax. Both dogs sustained clinical remission for the duration of the 21-day study, as determined by sum of longest diameters and accompanying x-ray radiology and abdominal ultrasound. Histology showed marked reduction of CD20 cells in the lymph nodes. Organ and hematological toxicities were minimal, and dogs remained in remission for 95 and 101 days post-CART. In summary, this study demonstrated that xenogeneic CART cells were well-tolerated, induced remission, and were visualized via PET imaging in companion dogs with lymphoma. These results are promising both for the treatment of companion dogs with novel cell therapies and for the translation of novel CART cell therapies into human clinical trials.

Abstract Background: CD19-directed CART cell (CART19) therapy has transformed the treatment of B-cell malignancies but is limited by life-threatening toxicities, including cytokine release syndrome (CRS) and immune effector cell–associated neurotoxicity syndrome (ICANS), as well as low durable remission rates. Our understanding of the pathogenesis of CART-associated toxicities is limited, and although current treatments are effective, refractory cases remain challenging. Methods and Results: To investigate CRS/ICANS pathogenesis and identify new therapeutic targets, we analyzed plasma cytokine kinetics within the first month of CART19 cell therapy in 25 relapsed/refractory (r/r) large B-cell lymphoma (LBCL) and 14 r/r follicular lymphoma (FL) patients (NCT02030834). In this cohort, 22 (88%) patients developed any-grade CRS (73% LBCL, 27% FL) and 4 (16%) developed any-grade ICANS (50% LBCL, 50% FL). Across 30 analyzed cytokines, IL-5 emerged as the second most enriched cytokine based on the median peak-to-baseline (Day 0) ratio in patients who experienced CRS vs. those who did not. The IL-5 ratio was 6.6-fold higher (p&lt;0.05) and 11.9-fold higher (p=0.093) in patients who experienced CRS or ICANS, respectively. Moreover, IL-5 was elevated in patients experiencing CRS on Days 1 and 2 (p&lt;0.05). Prompted by our results, we evaluated the effect of IL-5 neutralization with a monoclonal antibody (mAb) on CART-associated toxicity in a patient-derived B-acute lymphoblastic leukemia (B-ALL) xenograft model. After engraftment (average of 110 human CD19+ cells/µL peripheral blood), NSG mice were randomized based on tumor burden and treated with 3.5×10⁶ CART19 plus either IL-5 mAb or IgG control (10 mg/kg, weekly), or no treatment. Mice receiving IL-5 mAb showed reduced weight loss (p&lt;0.005, Days 13–16) and neuroscore (p&lt;0.05, Day 15), as assessed by a daily, blinded 18-point assessment, vs. the IgG group. Additionally, while CRS-associated cytokines (GM-CSF, IFN-γ, MIP-1β) were significantly elevated in the IgG group (p&lt;0.05) vs. the untreated group, they were not in the IL-5 mAb group. In this model, all mice treated with CART19 cells were able to clear the tumor burden. However, in a follow-up challenge model generated by engrafting NSG mice with 1x106 luciferase+ JeKo-1 cells, mice treated with a combination of 1x106CART19 cells + IL-5 mAb showed reduced weight loss (p&lt;0.0001) and enhanced overall survival (p&lt;0.01) vs. mice treated with CART19 + IgG. Next, we evaluated IL-5 neutralization in an immunocompetent toxicity model. BALB/c mice were treated with 10×10⁶ murine untransduced T cells, CART19 + IL-5 mAb, or CART19 + IgG (10 mg/kg, weekly). Mice receiving IL-5 mAb exhibited significantly reduced weight loss (p&lt;0.05, Day 1) and lower neuroscore (p&lt;0.05, Days 1, 6, and 8) vs. those receiving IgG control, with a consistent trend observed on other days, supporting a protective role for IL-5 blockade in mitigating CART-associated toxicities. To explore the direct effects of IL-5 on CART19 cells, we disrupted either IL-5 (IL-5KO-CART19) or its receptor (IL-5RαKO-CART19) using CRISPR-Cas9. In NSG mice engrafted with 1×10⁶ luciferase+NALM6 cells, mice receiving 1×10⁶ IL-5KO-CART19 vs. IL-5WT-CART19 showed a trend toward reduced weight loss (p=0.08), significantly increased antitumor activity by Day 20 (p&lt;0.05), and improved overall survival (p&lt;0.05), indicating that IL-5 production can significantly impair CART19 cell efficacy. We next evaluated changes in IL-5Rα gRNA representation in a published genome-wide CRISPR screen (PMID: 39266501). CART19 cells with IL-5Rα-targeting gRNAs were enriched following chronic stimulation, indicating a protective effect for IL-5 pathway interruption. In subsequent studies, IL-5RαKO-CART19 cells showed enhanced killing of JeKo-1 cells following 48 hours of co-culture (p&lt;0.05) and reduced co-expression of multiple inhibitory markers (p&lt;0.01) following chronic stimulation with JeKo-1 cells for one week vs. IL-5RαWT-CART19 cells, suggesting an improvement in CART19 cell activity and phenotype following disruption of IL-5Rα. Conclusion: Our data identify IL-5 as a previously unrecognized mediator of CART–associated toxicities and show significant improvement of CART19 activity with disruption of IL-5 signaling either indirectly with an IL-5 neutralizing antibody or directly with CRISPR gene editing. Thus, IL-5 could serve as a dual-purpose target to improve both safety and efficacy of CART cell therapy.