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CD4+ T cells mediate CAR-T cell-associated immune-related adverse events after BCMA CAR-T cell therapy

Nature Medicine (2026)Cite this article

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Abstract

B cell maturation antigen (BCMA)-targeted chimeric antigen receptor (CAR) T cell therapy has revolutionized the treatment of multiple myeloma but can cause unique toxicities, including cranial nerve palsy, parkinsonism and enterocolitis, which we refer to collectively as CAR T cell therapy-associated immune-related adverse events (CirAEs). Among 198 patients treated with ciltacabtagene autoleucel or idecabtagene vicleucel (June 2021–December 2024), 27 (13.6%) developed CirAEs. This included one remarkable case with three distinct CirAEs in association with an extreme CD4+ CAR T cell expansion (peak lymphocytes: 197 × 103 per microliter), which was abrogated in vitro by CCR5 inhibition. CirAEs were associated with significantly higher non-relapse mortality (hazard ratio = 5.2, P = 0.006), and independent risk factors included ciltacabtagene autoleucel (odds ratio = 4.5, P = 0.058), peak absolute lymphocyte count ≥ 2.4 × 103 per microliter in the first 14 days post-infusion (odds ratio = 4.3, P < 0.001) and apheresis CD4:CD8 ratio > 1 (odds ratio = 2.6, P = 0.048). We identified marked CD4+ CAR T cell infiltration in all available CirAE tissues, including cerebrospinal fluid during neurologic CirAEs, implicating CD4+ CAR T cell therapy as a key mediator of these toxicities.

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Fig. 1: CirAEs after BCMA-targeted CAR T cell therapy.
Fig. 2: Factors linked to the development of CirAEs after BCMA-targeted CAR T cell therapy.
Fig. 3: Hyperleukocytosis in cilta-cel patient 4: evidence of polyclonal CD4+ CAR T cell expansion.
Fig. 4: Maraviroc abrogates cilta-cel patient 4 CAR T cell proliferation in vitro.
Fig. 5: Cilta-cel-associated CirAEs are mediated by CD4+ T cells.

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Data availability

All requests for raw and analyzed data and materials are reviewed within 4 weeks by the University of Pennsylvania and the corresponding authors, to determine whether they are subject to intellectual property or confidentiality obligations. Patient-related data not included in the paper may be subject to patient confidentiality. The email addresses for the corresponding authors are as follows: mruella@upenn.edu, adam.cohen@pennmedicine.upenn.edu and jfrai@upenn.edu. Any data and materials that can be shared will be released via a material transfer agreement. Single-cell RNA sequencing data for matched peripheral blood and CSF are available at the NCBI Gene Expression Omnibus under accession code GSE309403. The integration site sequencing data have been deposited in the SRA hosted by the NCBI. The accession number for the SRA dataset is PRJNA1235822. Source data are provided with this paper.

Code availability

All analyses were performed with RStudio version 4.1.1. Packages used in analyses included the following: AUCell (1.25.2), celldex (1.14.0), cowplot (1.1.3), dplyr (1.1.4), enrichR (3.4), flowCore (2.16.0), flowViz (1.68.0), flowVS (1.36.0), flowWorkspace (4.16.0), FlowSOM (2.12.0), ggplot2 (3.5.2), limma (3.60.6), Matrix (1.7.3), msigdbr (24.1.0), Nebulosa (1.14.0), patchwork (1.3.0), pheatmap (1.0.12), RColorBrewer(1.1-3), Seurat (5.0.3), SingleR (2.6.0), tidyverse (2.0.0), VennDiagram (1.7.3), viridis (0.6.5) and writexl (1.5.4). All code needed for reproduction of analyses is available via GitHub at https://github.com/jhnoll/BMCA_CAR_irAE_NatMed.git.

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Acknowledgements

We thank all the patients and their families. We acknowledge the work of the clinical research staff of the Clinical Research Unit and clinical nursing and support staff at the Abramson Cancer Center of the University of Pennsylvania. We acknowledge the laboratories and staff in the Division of Precision and Computational Diagnostics (Center for Personalized Diagnostics and Molecular Pathology Laboratory at the Hospital of the University of Pennsylvania) for performing additional next-generation sequencing assays to support the study. We also acknowledge the Viral Molecular High Density Sequencing core at the University of Pennsylvania (RRID: SCR_022433) for their assistance with integration site analysis. M.H. is supported by a National Human Genome Research Institute T32 training grant (5T32HG009495) and the Guerry Career Development Award. A.R.-F. receives funding from the AIDS Malignancy Consortium and the Alfonso Martín Escudero Foundation. B.C. receives funding from the PhiladElphia PRogram FOR Mentored Research Training in Kidney, Urologic and Hematologic Diseases (PERFORM-KUH) TL1. C.R.H. is supported by National Institutes of Health (NIH) F31 grant CA274961. S.A.A. is supported by a Scientist Development Award from the Rheumatology Research Foundation. C.D. is supported by NIH National Cancer Institute (NCI) grant 1K08CA286762-01, the Canadian Institute for Health Research Fellowship Award and the Alex’s Lemonade Stand Fund ‘A’ Award. A.L.G. is supported by a Scholar in Clinical Research Award from the Leukemia & Lymphoma Society. F.D.B. and J.A.F. are supported by NIH R01 grant CA241762. J.A.F. also acknowledges funding from the National Science Foundation Engineering Research Center for Cell Manufacturing Technologies, the Alliance for Cancer Gene Therapy Investigator Award in Cell and Gene Therapy for Cancer, the Bob Levis Funding Group and the Chambers Centurion gift (U01 AG066100) from the Samuel Waxman Cancer Research Foundation and funding for correlative data science through a Parker Institute for Cancer Immunotherapy Innovation Challenge Award. M.R. is supported by the Colton Center Pilot Award, the Laffey-McHugh Foundation and the Berman and Maguire Funds for Lymphoma Research at Penn. This research was funded by P01 grant CA214278 (to M.R. and J.A.F). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

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Author notes

  1. These authors contributed equally: Matthew Ho, Luca Paruzzo, Julia Han Noll.

Authors and Affiliations

  1. Division of Hematology-Oncology, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Matthew Ho, Luca Paruzzo, Adam Waxman, Shivani Kapur, Vrutti Patel, Audrey C. Bochi-Layec, Shan Liu, Heta Patel, Alberto Carturan, Peter Michener, Janna Minehart, Neel R. Nabar, Zainul S. Hasanali, Bryan T. Ciccarelli, Putzer Hung, Patrizia Porazzi, Dan T. Vogl, David L. Porter, Edward A. Stadtmauer, Sandra P. Susanibar-Adaniya, Alfred L. Garfall, Marco Ruella & Adam D. Cohen

  2. Center for Cellular Immunotherapies, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Matthew Ho, Luca Paruzzo, Julia Han Noll, Federico Stella, Sonia Ndeupen, Yael A. Day, Ivan J. Cohen, Adam Waxman, Fang Chen, Rong Xu, Andrew Huff, Danuta Jarocha, Vrutti Patel, Audrey C. Bochi-Layec, Ranjani Ramasubramanian, Shan Liu, Riemke Bouvier, Vitor B. de Souza, Ziyu Li, Alberto Carturan, Peter Michener, Caitlin R. Hopkins, Owen Koucky, Janna Minehart, Alex Dimitri, Neel R. Nabar, Zainul S. Hasanali, Bryan T. Ciccarelli, Putzer Hung, Erik Williams, Robert Bartoszek, Maya Lavorando, Vanessa E. Gonzalez, Patrizia Porazzi, Vijay G. Bhoj, John Scholler, Marco Ruella & Joseph A. Fraietta

  3. Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Matthew Ho, Luca Paruzzo, Gregory M. Chen, Angel Ramirez-Fernandez, Shivani Kapur, Vrutti Patel, Audrey C. Bochi-Layec, Shan Liu, Alberto Carturan, Peter Michener, Janna Minehart, Neel R. Nabar, Zainul S. Hasanali, Bryan T. Ciccarelli, Putzer Hung, Patrizia Porazzi, Dan T. Vogl, David L. Porter, Edward A. Stadtmauer, Sandra P. Susanibar-Adaniya, Alfred L. Garfall, Marco Ruella, Adam D. Cohen & Joseph A. Fraietta

  4. Lymphoma Program, Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA, USA

    Luca Paruzzo, Adam Waxman, Vrutti Patel, Alberto Carturan, Peter Michener, Patrizia Porazzi & Marco Ruella

  5. Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Pooja Devi, Gregory M. Chen, Angel Ramirez-Fernandez, Vijay G. Bhoj & Joseph A. Fraietta

  6. Department of Radiology, Division of Neuroradiology, University of Pennsylvania, Philadelphia, PA, USA

    Suyash Mohan

  7. Division of Rheumatology, Department of Medicine, University of Pennsylvania, Philadelphia, USA

    Sokratis A. Apostolidis & Marco Ruella

  8. Center for Cell Therapy and Transplant, University of Pennsylvania, Philadelphia, PA, USA

    David L. Porter, Edward A. Stadtmauer, Sandra P. Susanibar-Adaniya, Alfred L. Garfall, Adam D. Cohen & Joseph A. Fraietta

  9. Department of Pediatrics, Children’s Hospital of Philadelphia, University of Pennsylvania, Philadelphia, PA, USA

    Caroline Diorio

  10. Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Aoife M. Roche, John K. Everett & Frederic D. Bushman

  11. Division of Translational Medicine and Human Genetics, University of Pennsylvania, Philadelphia, PA, USA

    Katherine L. Nathanson

  12. Institute for Immunology and Immune Health, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA

    Marco Ruella

  13. Parker Institute for Cancer Immunotherapy, University of Pennsylvania, Philadelphia, PA, USA

    Joseph A. Fraietta

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Contributions

M.R., A.D.C. and J.A.F. contributed equally. M.H., L.P., J.H.N., F.S., G.M.C., S.L., A.L.G., M.R., A.D.C. and J.A.F. participated in the design, execution and/or interpretation of the reported experiments or results. M.H., L.P., J.H.N., F.S., S.N., Y.A.D., I.J.C., P.D., G.M.C., A.R.-F., A.W., S.K., F.C., R.X., A.H., V.P., A.B.-L., R.R., S.L., D.J., R. B., V.B.D.S., Z.L., H.P., A.C., P.M., C.R.H., O.K., J.M., A.D., N.R.N., Z.S.H., B.T.C., E.W., R.B., M.L., S.M., V.E.G., P.P., V.G.B., S.A.A., D.T.V., D.L.P., J.S., C.D., A.M.R., F.D.B., K.L.N., J.K.E., E.A.S. and S.P.S.-A. participated in the acquisition or analysis of data. M.H., L.P., J.H.N., M.R., A.L.G., A.D.C. and J.A.F. wrote the manuscript, with all authors contributing to writing and providing feedback. A.L.G., M.R., A.D.C. and J.A.F. supervised all aspects of the research.

Corresponding authors

Correspondence to Marco Ruella, Adam D. Cohen or Joseph A. Fraietta.

Ethics declarations

Competing interests

D.T.V. has received research funding from Takeda and Active Biotech and consulting fees from Takeda, Karyopharm, GlaxoSmithKline, Genentech and Sanofi. A.D.C. has received research support from Novartis, GlaxoSmithKline, Genentech and Janssen; consulting fees from Janssen, Bristol Myers Squibb, GlaxoSmithKline, Genentech, Legend, Sanofi, Pfizer, AbbVie, Regeneron, Moderna, AstraZeneca, iTeos, Prothena, Kite, Novartis and Ichnos; and has patents related to CAR T cells. A.L.G. declares research support from Johnson & Johnson, Novartis, Tmunity and CRISPR Therapeutics; consultancies/honoraria from Johnson & Johnson, Novartis, Bristol Myers Squibb, Regeneron, AbbVie, AstraZeneca, Smart Immune and Gracell Bio; and data and safety monitoring board membership for Johnson & Johnson. E.A.S. declares an affiliation with Oncopeptides and consultancy for Amgen, Bristol Myers Squibb/Celgene, GlaxoSmithKline, Janssen and AbbVie. J.A.F. has patents and intellectual property in T-cell-based cancer immunotherapy with royalties; funding from Tmunity Therapeutics and Danaher Corporation; consultancy with Retro Biosciences; and scientific advisory board membership with Cartography Bio, Shennon Biotechnologies, CellFe Biotech, OverT Bio and Tceleron Therapeutics. D.L.P declares research funding from Novartis and Bristol Myers Squibb, membership on an entity’s board of directors (National Marrow Donor Program) and advisory committee or honoraria from Novartis, Kite/Gilead, Angiocrine, Mirror Biologics, Sana Biotechnology and Verismo. D.L.P. is a current equity holder in Genentech and has patents and royalties with Novartis and Tmunity/Kite. C.D. declares consultancy with Merck. V.B. declares consulting fees from Alexion and has patents related to CAR T cell platforms for treatment of alloimmune conditions and autoimmune diseases. S.M. has received research funding from Novocure and Galileo CDS and is an advisory board member for Servier Pharmaceuticals and Guerbet. F.D.B. has engaged with Sana Biotechnology. S.P.S.-A. declares consultancies/honoraria from Johnson & Johnson. M.R. has patents related to CD19 CAR T cells; consults for GLG, Guidepoint, AbClon, Acera and Vittoria Bio; received research funding from AbClon, Oxford NanoImaging, Vittoria Biotherapeutics, CURIOX and Beckman Coulter; and is the scientific founder of Vittoria Biotherapeutics. M.H., L.P., J.H.N., A.L.G., M.R., A.D.C. and J.A.F. have filed patent applications related to this work. All other authors declare no competing interests.

Peer review

Peer review information

Nature Medicine thanks Adolfo Aleman, Julio Chavez and Aaron Rapoport for their contribution to the peer review of this work. Primary Handling Editor: Ulrike Harjes, in collaboration with the Nature Medicine team.

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Extended data

Extended Data Fig. 1 Stratified analysis of ciltacabtagene autoleucel (cilta-cel) vs idecabtagene vicleucel (ide-cel).

(a) Cilta-cel and ide-cel infusion dates at our institution. (b) Response rate of the entire BCMA-CART cohort, and by cilta-cel and ide-cel. Categorical variables were compared with Fisher’s exact test. (c) Progression-free survival analysis (PFS) of the entire cohort. (d) Overall survival (OS) analysis of the entire cohort. (e) Progression-free survival analysis stratified by product. Survival distributions were compared with the log-rank test. (f) Overall survival analysis stratified by product. Survival distributions were compared with the log-rank test. (g) Cytokine release syndrome (CRS) grade stratified by product. Categorical variables were compared with Fisher’s exact test. (h) Immune effector cell-associated neurotoxicity syndrome (ICANS) grade stratified by product. Categorical variables were compared with Fisher’s exact test. (i) Absolute lymphocyte count (ALC; median with interquartile range) within the first 24 days following BCMA-CART, and at Day 28, Month 3, and Month 6, stratified by product received. Data are presented as mean values ± 95% confidence interval. Statistical comparisons between groups were performed using the two-tailed Wilcoxin test. Exact P-values are provided in Supplementary Table S9. (j) Peak absolute lymphocyte count (ALCPeak) at apheresis, Day 0, Day 7, Day 14, and Day 21 stratified by product. Data are presented as median values ± interquartile range. Statistical comparisons between groups were performed using the two-tailed Wilcoxin test. Exact P-values are provided in Supplementary Table S9. (k) CD4+ and CD8+ proportion of T-cells at apheresis, Days 7 and 28, Months 3 and 6 post-BCMA-CART, stratified by product. Bar graphs show the median and the error bars denote the 25th and 75th percentiles (top). Area under the curve (AUC) analysis of CD4⁺ T-cell proportions from apheresis to Month 6 in the cilta-cel cohort, stratified by product. Data are shown as median values with error bars representing the 25th and 75th percentiles. Shaded regions indicate the area under the curve (AUC) for each group (bottom). Statistical comparisons between groups were performed using the two-tailed Wilcoxin test. * Indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001. (l) Non-relapse mortality of the entire cohort. (m) Non-relapse mortality analysis stratified by product. Survival distributions were compared with the log-rank test. ALCPeak: peak absolute lymphocyte count; AUC: area under curve; CART: CAR T-cell; cilta-cel: ciltacabtagene-autoleucel; CI: confidence interval; CirAE: CART-associated immune-related adverse events; CRS: cytokine release syndrome; ICANS: immune effector cell-associated neurotoxicity syndrome; NRM: non-relapse mortality; ORR: overall response rate; OS: overall survival; ide-cel: idacabtagene-vicleucel; PFS: progression free survival; ≥ VGPR: very good partial response or better.

Source data

Extended Data Fig. 2 CART-associated immune-related adverse events (CirAE) Clinical correlates in the cilta-cel cohort.

(a) Response rate in the cilta-cel cohort stratified by CART-associated immune-related adverse events, CirAE. Categorical variables were compared with Fisher’s exact test. (b) Progression-free survival (PFS) analysis of the cilta-cel cohort, stratified by CirAE. Survival distributions were compared with the log-rank test. (c) Overall survival (OS) analysis of the cilta-cel cohort, stratified by CirAE. Survival distributions were compared with the log-rank test. (d) Non-relapse mortality (NRM) analysis of the cilta-cel cohort, stratified by CirAE. Survival distributions were compared with the log-rank test. (e) Pre-infusion M-spike levels of the cilta-cel cohort, stratified by CirAE. Data are shown as median values with error bars representing the 25th and 75th percentiles. Statistical comparisons between groups were performed using the two-tailed Wilcoxin test. (f) Pre-infusion differential free light chain (dFLC) levels of the cilta-cel cohort, stratified by CirAE. Data are shown as median values with error bars representing the 25th and 75th percentiles. Statistical comparisons between groups were performed using the two-tailed Wilcoxin test. (g) Cytokine release syndrome (CRS) grade of the cilta-cel cohort, stratified by CirAE. Categorical variables were compared with Fisher’s exact test. (h) Immune effector cell-associated neurotoxicity (ICANS) grade of the cilta-cel cohort, stratified by CirAE. Categorical variables were compared with Fisher’s exact test. (i) Top: absolute lymphocyte count (ALC; median with interquartile range) within the 28 days following BCMA-CART, and at Day 28, Month 3 and Month 6, in the cilta-cel cohort, stratified by CirAE. Data are presented as mean values ± 95% confidence interval. Bottom: peak absolute lymphocyte count (ALCPeak) at apheresis, Day 0, Day 7, Day 14, and Day 21 of the cilta-cel cohort, stratified by CirAE (bottom). Data are presented as median values ± interquartile range. Day 14 ALCPeak of 2.9 falls within the third quartile of the cilta-cel non-CirAE cohort distribution. Statistical comparisons between groups were performed using the two-tailed Wilcoxin test. * Indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001. Exact P-values are provided in Supplementary Table S9. (j) Top: CD4+ and CD8+ proportion of T-cells in the cilta-cel cohort, stratified by CirAE. Bar graphs show the median and the error bars denote the 25th and 75th percentiles. Bottom: area under the curve (AUC) analysis of CD4⁺ T-cell proportions from apheresis to Month 6 in the cilta-cel cohort, stratified by CirAE. Data are shown as median values with error bars representing the 25th and 75th percentiles. Statistical comparisons between groups were performed using the two-tailed Wilcoxin test. Exact P-values are provided in Supplementary Table S9. (k) Univariable logistic regression was performed using a binomial generalized linear model to assess associations between clinical factors and CirAE (left), neurologic CirAE (neuro-CirAE, middle), and non-neuro CirAE (right). Statistical significance of model coefficients was assessed using two-tailed Wald tests. (l) Multivariable logistic regression analysis of risk factors for CirAE development in the cilta-cel cohort. Statistical significance of model coefficients was assessed using two-tailed Wald tests. ALCPeak: peak absolute lymphocyte count; AUC: area under curve; CART: CAR T-cell; cilta-cel: ciltacabtagene-autoleucel; CI: confidence interval; CirAE: CART-associated immune-related adverse events; CRS: cytokine release syndrome; ICANS: immune effector cell-associated neurotoxicity syndrome; NRM: non-relapse mortality; ORR: overall response rate; OS: overall survival; ide-cel: idacabtagene-vicleucel; PFS: progression free survival; ≥ VGPR: very good partial response or better.

Source data

Extended Data Fig. 3 Clinical imaging of CART-associated immune-related adverse events (CirAE) and colon biopsy results in patients who developed immune effector cell-associated enterocolitis.

(a) Clinical imaging of post-BCMA-CART CirAE: Cilta-cel#21: Magnetic resonance imaging (MRI) of the head and orbits illustrates enhancement within the left orbital apex extending to the left superior orbital fissure. There is thickening and edema in the left prechiasmatic optic nerve and left optic chiasm and enhancement within and along the left intraorbital, intracanalicular, and prechiasmatic optic nerve/sheath complex, consistent with optic neuritis. Chimeric antigen receptor (CAR) transgene was detectable at 59596 copies/µg gDNA in the cerebrospinal fluid (CSF). Cilta-cel#14: MRI of the head shows a rim-enhancing lesion in bilateral medial thalami extending to the hypothalamus measuring approximately 2.8 ×2.7 ×2.2 cm, with surrounding FLAIR signal abnormality extending bilaterally into the surrounding thalami, gangliocapsular regions, mammillary bodies, left greater than right midbrain, and optic chiasm. Ide-cel#1: CT chest showing multifocal tree-in-bud ground-glass opacities consistent with pneumonitis. Cilta-cel#4: endoscopy revealed many non-bleeding, cratered duodenal ulcers, and erosions without stigmata of bleeding beyond the second portion of the duodenum. Colonoscopy did not show any areas of ulcerations or erosions within the terminal ileum and colon. (b) H&E shows duodenal mucosa with graft-versus-host and Celiac disease-like patterns of injury, changes compatible with treatment (ciltacabtagene autoleucel) effects, and mild acute inflammation. Circle highlights villous atrophy and crypt hyperplasia. Arrow highlights prominent crypt apoptosis. RNAscope shows CART infiltration. BCMA was not detected. Representative micrographs from a single experiment; imaging was performed once and not independently replicated due to limited available tissue. (c) H&E shows colonic mucosa with mild crypt architectural distortion and markedly reduced plasma cells in the lamina propria. RNAscope shows CART infiltration. BCMA was not detected. Circle highlights a paucity of plasma cells in the lamina propria, resembling histologic features observed in common variable immunodeficiency. Representative micrographs from a single experiment; imaging was performed once and not independently replicated due to limited available tissue. BCMA: B-cell maturation antigen; CART: chimeric antigen receptor T-cell; Cy5: cyanine 5; FITC: fluorescein isothiocyanate; MRI: magnetic resonance imaging.

Extended Data Fig. 4 in vitro functionality analysis of Cilta-cel#4’s CARTs.

(a) Heatmap shows z-scores of the log2 fold changes for each marker across each row. Left: antigen-specific degranulation and cytokine production in CD4+ and CD8+ CARTs from peripheral blood (PB) and cerebrospinal fluid (CSF) from Cilta-cel#4 following coculture with MM.1S, compared to PB CARTs from three normal donors and Cilta-cel#22. Right: fold change of CD4+/CD8+ CART degranulation and cytokine production in the same samples. (b) In vitro cytotoxicity assay comparing Cilta-cel#4 PB CARTs at Day 13 with CARTs manufactured from three normal donors, using MM.1S as target. An effector: tumor ratio of 1:1 was used. Data are shown as mean values with error bars representing the standard error of the mean (SEM) from n = 3 biological replicates. Statistical comparisons between groups were performed using the two-tailed Student’s T-test. * Indicates P < 0.05, ** indicates P < 0.01. Exact P-values are provided in Supplementary Table S9. (c) In vitro cytotoxicity assay comparing Cilta-cel#4 PB and CSF CARTs at Month 2 (M2) with to Cilta-cel#22 PB CARTs, using RPMI-8226 as target. A range of effector: tumor ratios between 2:1 and 1:2 was used. Data are shown as mean values with error bars representing the standard error of the mean (SEM) from n = 3 biological replicates. Statistical comparisons between groups were performed using the two-tailed Student’s T-test. Exact P-values are provided in Supplementary Table S9. (d) Serum cytokine profiling of Cilta-cel#4 from pre-lymphodepletion (Pre-LD), Day 7, peak expansion (Day 13), to Month 2. Left: heatmap shows z-score normalized cytokine concentration. Right, top: temporal kinetics of cytokines associated with T-cell proliferation (IL-1b, IL-2, IL-4, IL-7, IL-15, IL-18). Right, bottom: Temporal kinetics of cytokines implicated in Cytokine release syndrome (CRS; IL-6, TNF-α, GM-CSF, IL-8, IL-10). ^ represents cytokine concentration above upper limit of quantification of the LUMINEX assay. (e) CellTrace Violet-stained CARTs from three normal donors were seeded into 96-well plates, stimulated with IL-2: 10 ng/mL, IL-7: 20 g/mL, or IL-15: 20 ng/mL, and incubated for five days. Left: proliferation was assessed by flow cytometry analysis of CellTrace Violet dilution. Right: phosphorylated STAT5 (pSTAT5+) activation following 15 min of activation with IL-2 (10 ng/mL), IL-7 (20 ng/mL), or IL-15 (20 ng/mL) in 3 normal donors. Data are shown as mean values with error bars representing the standard error of the mean (SEM) from n = 3 biological replicates. (f) Baseline IL2, IL7, and IL15 receptor-alpha (RA) chain expression in Cilta-cel#4 compared to 3 normal donors before cytokine stimulation. Data are shown as mean values with error bars representing the standard error of the mean (SEM) from n = 3 biological replicates. Statistical comparisons between groups were performed using the two-tailed Student’s T-test. * Indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001. Exact P-values are provided in Supplementary Table S9. (g) CCR5 median fluorescence intensity (MFI) in three normal donors and Cilta-cel#4 following cytokine stimulation with IL-2 (10 ng/mL), IL-7 (20 ng/mL), or IL-15 (20 ng/mL). Data are shown as mean values with error bars representing the standard error of the mean (SEM) from n = 3 biological replicates. Statistical comparisons between groups were performed using the two-tailed Student’s T-test. * Indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001. Exact P-values are provided in Supplementary Table S9. (h) Absolute count (left) and viability (right) of CD3+ T-cells from 3 normal donors after 3 days of CD3/28 stimulation in the presence of increasing maraviroc concentrations (2.5 μM, 5 μM, 10 μM, 20 μM, 40 μM). Data are shown as mean values with error bars representing the standard error of the mean (SEM) from 3 biological replicates. Statistical comparisons between groups were performed using the two-tailed Student’s T-test. * Indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001. CSF: cerebrospinal fluid; E:T: effector: tumor ratio.

Source data

Extended Data Fig. 5 Cilta-cel and Ide-cel in vitro functional differences.

(a) Overlay histogram of tNGFR and BCMA-CAR expression in SUP-T1 cell line stably transduced with ide-cel CAR (ide-cel), cilta-cel CAR (cilta-cel), mesothelin-directed CAR (M5) or untransduced control cells (UTD). (b) Comparison of BCMA-binding avidity between ide-cel vs cilta-cel, with M5 and UTD as controls. Left: Percentage of MM.1S cells bound across a range of applied forces. Right: Percentage of MM.1S binding at 1000 pN force; data are presented as mean values ± the standard error of the mean (SEM) from 2–5 biological replicates. Statistical comparisons between groups were performed using the two-tailed Student’s T-test. (c) Overlay histogram of tNGFR and VHH-CAR expression in Jurkat triple parameter reporter (Jurkat_TPR) cell line stably transduced with the ScFv-based ide-cel CAR (ide-cel) or VHH-based cilta-cel CAR (cilta-cel). (d) Baseline (tonic) NFAT and NKFB activation status in Jurkat_TPR cells expressing ide-cel and cilta-cel in the absence of antigen stimulation; data are presented as mean values ± the standard error of the mean (SEM) from n = 3 biological replicates. Statistical comparisons between groups were performed using the two-tailed Student’s T-test. (e) Left: fold-change from unstimulated in NFAT and NKFB % positive cells (top left) and median fluorescence intensity (MFI, bottom left) in Jurkat_TPR expressing ide-cel or cilta-cel following 18 and 24 h of stimulation with MM.1S with a range of effector: tumor ratios between 5:1 to 1:5. Right: fold-change from unstimulated in NFAT and NKFB % positive cells (top right) and median fluorescence intensity (MFI, bottom right) in Jurkat_TPR expressing ide-cel or cilta-cel following 1:1 stimulation with MM.1S across multiple timepoints ranging from 3 to 72 h. Data are shown as mean values with error bars representing the standard error of the mean (SEM) from 3 biological replicates. (f) Jurkat_TPR expressing ide-cel or cilta-cel were co-cultured 1:1 with irradiated MM.1S for 24 to 48 h, then separated from MM.1S using tNGFR-PE magnetic bead (positive) selection. The contour plot confirms high post-sort purity of Jurkat_TPR cells used for subsequent Seahorse metabolic analysis. (g) Mitochondrial respiration, (h) Maximal and spare respiratory capacity, (i) Glycolytic rate, (j) Total ATP production (bioenergetic capacity), and (k) ATP production rate in Jurkat_TPR expressing ide-cel or cilta-cel assessed at baseline (unstimulated), and after 24 and 48 h of stimulation with MM.1S. Data are presented as mean values ± the standard error of the mean (SEM) from 3–6 biological replicates. Statistical comparisons between groups were performed using the two-tailed Student’s T-test. APC: allophycocyanin; ATP: adenosine triphosphate; ECAR: extracellular Acidification Rate; MFI: median fluorescence intensity; NFAT: nuclear factor of activated T-cells; NFkB: nuclear factor kappa-light-chain-enhancer of activated B-cells; NS: not significant; OCR: oxygen consumption Rate; PE: phycoerythrin; ScFV: single-chain variable fragment; SRC: spare respiratory capacity; tNGFR: truncated nerve growth factor receptor; VHH: camelid single-domain antibody/nanobody.

Source data

Extended Data Fig. 6 T-Cell immunophenotype of CirAE patients Analyzed by Flow Cytometry.

(a) Left: uniform Manifold Approximation and Projection (UMAP) visualization of flow cytometry characterization of CD3+ T-cells from 9 patients at peak of expansion, stratified by CirAE. Middle: CAR percentage and median fluorescence intensity (MFI) in CD3+ T-cells at peak expansion, stratified by CirAE. Violin plots show the mirrored probability density of the data; dashed line indicates the median, and dotted lines denote the 25th and 75th percentiles. Right: proportions of CD4, CD8, and double-negative CART subsets at peak expansion, stratified by CirAE. Bar graphs show the median and the error bars denote the 25th and 75th percentiles. Violin plots show the mirrored probability density of the data; dashed line indicates the median, and dotted lines denote the 25th and 75th percentiles. Statistical comparisons between groups were performed using the two-tailed Wilcoxin test. (b) Flow cytometry analysis of peripheral blood mononuclear cells from 10 patients during CirAE and 6 non-CirAE patients at matched timepoints. CAR+ percentage (top) and MFI (bottom) of CART during CirAE in CD3+ (left), CD3+CD4+ (middle), CD3+CD8+ (right) T-cells. The top rightmost panel depicts absolute CART count (%CAR+ x ALC), stratified by CirAE. Violin plots show the mirrored probability density of the data; dashed line indicates the median, and dotted lines denote the 25th and 75th percentiles. Statistical comparisons between groups were performed using the two-tailed Wilcoxin test. (c) Comparison of peripheral blood (PB) CD4⁺ T-cell proportions between CirAE patients at the time of CirAE and time-matched non-CirAE controls. Left: unpaired comparison of CD4⁺ T-cell proportions at pre-lymphodepletion and CirAE/matched timepoints, stratified by CirAE; bar graphs show the median and the error bars denote the 25th and 75th percentiles. Right: comparison of CD4⁺ T-cell proportions at pre-lymphodepletion and CirAE/matched timepoints in paired longitudinal samples, stratified by CirAE. Statistical comparisons between groups were performed using the two-tailed paired-sample T-test. (d) Flow cytometry immunophenotyping of T-cells from matched PB and cerebrospinal fluid (CSF) samples during Cilta-cel#4’s delayed ICANS episode.

Source data

Extended Data Fig. 7 Single-cell analysis of matched peripheral blood and CSF samples in patients with neuro-CirAE (n = 4).

(a) Uniform Manifold Approximation and Projection (UMAP) visualization of CART cells in peripheral blood (PB) and cerebrospinal fluid (CSF), colored by T-cell clusters in each of the samples. (b) AUCell enrichment score for Gene Ontology Biological process (GOBP): leukocyte-mediated cytotoxicity score in each of the clusters. Box plots show the median (center line), interquartile range (bounds of box; 25th–75th percentiles), and minimum and maximum values (whiskers). Statistical comparisons between groups were performed using two-tailed Wilcoxin test. (c) Density plot of representative genes involved in T-cell cytotoxic function (Granzyme K,B,A: GZMK,GZMB,GZMA; Granulysin: GNLY; interferon gamma: IFNG; perforin 1: PRF1; CD107a) in PBMC and CSF samples.

Extended Data Fig. 8 Early pro-inflammatory cytokines are associated with CirAE development.

(a) Serum cytokine profile of CirAE compared to non-CirAE. Heatmap shows the log2 (median CirAE/ median no CirAE) for pre-lymphodepletion (Pre-LD), Days 0, 7, 14, during CirAE. Statistical significance was determined using two-tailed Mann–Whitney tests. Punadj indicates unadjusted P values (without correction for multiple comparisons); Padj indicates adjusted P values (with Holm–Šidák correction for multiple comparisons); * indicates p-values < 0.05. (b) Serum proteins with significantly different concentrations at each time point (Pre-LD, days 0, 7, 14, and during CirAE) in patients who developed CirAE compared with those who did not. Box plots show the median (center line), interquartile range (bounds of box; 25th–75th percentile), and minimum and maximum values (whiskers). Statistical significance was determined using two-tailed Mann–Whitney tests. Punadj indicates unadjusted P values (without correction for multiple comparisons); Padj indicates adjusted P values (with Holm–Šidák correction for multiple comparisons); * indicates p-values < 0.05. (c-d) Line graphs show the dynamic changes of pro-inflammatory and anti-inflammatory cytokines in CirAE patients from Pre-LD to the time of CirAE.

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Extended Data Table 1 Detailed descriptions of patient characteristics and CirAE presentation, management and course
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Supplementary Information

Supplementary Figs. 1–11, supplementary figure legends and Supplementary Tables 1–9.

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Supplementary Table 10

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Ho, M., Paruzzo, L., Noll, J.H. et al. CD4+ T cells mediate CAR-T cell-associated immune-related adverse events after BCMA CAR-T cell therapy. Nat Med (2026). https://doi.org/10.1038/s41591-025-04121-8

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