Bone marrow Single cell sequencing data of CAR-T treated IL-2Ra -/- mice

Goala, Payal 1 ; Cortes Gomez, Eduardo 1 ; Wang, Jianmin1; Davila, Marco1

Published Sep 12, 2025 on Dryad. https://doi.org/10.5061/dryad.69p8cz9f3

Abstract

Despite breakthrough research and success in CAR-T therapy in heme malignancies, adverse events caused during CAR-T infusion remain a challenge. Among the various toxicities, the co-occurrence of cytokine release syndrome (CRS) along with neutropenia is the leading cause of severe life-threatening infections, increased hospitalizations, and the leading cause of non-relapse mortality. To this end, we developed an IL-2R alpha knockout model (where exons 2 and 3 that bind to IL-2 are replaced by a neomycin resistance gene) that showed the recapitulation of CRS and neutropenia seen in the clinic. This novel pre-clinical model helped us identify IFNg (one of the CRS-associated cytokines) to drive this co-occurrence. Through adoptive transfer studies, we show that excess IFNg released during CAR-T expansion triggers the inflammatory cascade that fuels the etiology of both CRS and neutropenia by triggering Th1/Th17 imbalance. IFNg is a Th1 cytokine that inhibits Th17 differentiation, which is crucial for neutrophil regulation. To corroborate the impact of IFNg on CRS-neutropenia, we processed bone marrow samples of tumor-bearing IL-2Rα -/- mice treated with CAR-T (Group 1) and IFNγ -/- CAR-T (Group 2) in the presence or absence of Th17 (Group 4) and Th1 cells (Group 5). The samples were processed for sequencing at week 4 post CAR-T infusion to study the mechanistic underpinnings of the impact of IFNg blockade on neutrophils and macrophages (M1 macrophages are known to drive CRS) that are involved in CRS and neutropenia co-occurrence during CAR-T therapy.

Dataset DOI: 10.5061/dryad.69p8cz9f3

Description of the data and file structure

CAR-T therapy has shown tremendous success; however, several clinical trials have witnessed the co-occurrence of adverse events or toxicities such as Cytokine release syndrome (CRS) and neutropenia. Their co-occurrence is associated with more infections, hospitalizations, and cases of non-relapse mortality. To this end, we developed an IL-2R alpha knockout (IL2-Ra -/-) model that recapitulated both CRS and neutropenia by simulating the hyper proliferation of CAR-T cells (where endogenous T cells lack the ability to bind IL-2, thereby providing all IL-2 to the CAR-T cells for expansion) that triggers cytokine-mediated inflammatory cascades implicated in CRS. We found IFNg among the CRS-associated cytokines to be the major driver of this co-occurrence, and its blockade ameliorated both CRS and neutropenia in our in vivo model. IFNg, a Th1 cytokine, is a known inhibitor of Th17 cells essential for neutrophil production. We posited that the imbalance of the Th1/Th17 axis caused by excess IFNg production, post-CAR-T infusion (and during CRS), led to CRS-neutropenia co-occurrence. To corroborate this, we used bone marrow samples of IL-2Rα -/- mice treated with CAR-T (Group 1) and IFNγ -/- CAR-T (Group 2) in the presence or absence of Th17 (Group 4) and Th1 cells (Group 5). The samples were processed for sequencing at week 4.

Files and variables

File: RS-04318056_436-GEX_RS-04310592_S6_L001_I1_001.fastq.gz

Description: IL-2Ra -/- mouse # 436 was injected with Emu- ALL cancer cells (B-ALL cell line) a week prior to CAR-T administration. IL-2Ra -/- have their exons 2 and 3 (that bind to IL-2) replaced by a Neomycin resistance gene. After confirming for tumor establishment, 2 to 3 million IFNg knockout (IFNg -/-) CAR-T cells were intravenously administered into the mouse (Group 2). Bone marrow samples were processed for single-cell sequencing at week 4 post-CAR-T infusion.

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Description: IL-2Ra -/- mouse # 418 was injected with Emu- ALL cancer cells (B-ALL cell line) a week prior to CAR-T administration. IL-2Ra -/- have their exons 2 and 3 (that bind to IL-2) replaced by a Neomycin resistance gene. After confirming for tumor establishment, 2 to 3 million IFNg knockout (IFNg -/-) CAR-T cells were intravenously administered into the mouse (Group 2). Bone marrow samples were processed for single-cell sequencing at week 4 post-CAR-T infusion.

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File: RS-04318058_459-GEX_RS-04310594_S8_L002_R2_001.fastq.gz

Description: IL-2Ra -/- mouse # 459 was injected with Emu- ALL cancer cells (B-ALL cell line) a week prior to CAR-T administration. IL-2Ra -/- have their exons 2 and 3 (that bind to IL-2) replaced by a Neomycin resistance gene. After confirming for tumor establishment, 2 to 3 million CAR-T cells were intravenously administered into the mouse (Group 1). Bone marrow samples were processed for single-cell sequencing at week 4 post-CAR-T infusion.

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File: RS-04318055_426-GEX_RS-04310591_S5_L002_R1_001.fastq.gz

Description: IL-2Ra -/- mouse # 426 was injected with Emu- ALL cancer cells (B-ALL cell line) a week prior to CAR-T administration. IL-2Ra -/- have their exons 2 and 3 (that bind to IL-2) replaced by a Neomycin resistance gene. After confirming for tumor establishment, 2 to 3 million CAR-T cells were intravenously administered into the mouse (Group 1). Bone marrow samples were processed for single-cell sequencing at week 4 post-CAR-T infusion.

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File: RS-04318053_415-GEX_RS-04310589_S3_L002_R2_001.fastq.gz

Description: IL-2Ra -/- mouse # 415 was injected with Emu- ALL cancer cells (B-ALL cell line) a week prior to CAR-T administration. IL-2Ra -/- have their exons 2 and 3 (that bind to IL-2) replaced by a Neomycin resistance gene. After confirming for tumor establishment, 2 to 3 million IFNg -/- CD1928z CAR-T cells were intravenously administered into the mouse along with Th1 cells (differentiated in vitro using Th1-inducing cytokines) (Group 5). Bone marrow samples were processed for single-cell sequencing at week 4 post-CAR-T infusion.

File: RS-04318053_415-GEX_RS-04310589_S3_L001_R1_001.fastq.gz

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File: RS-04318051_416-GEX_RS-04310587_S1_L002_R2_001.fastq.gz

Description: IL-2Ra -/- mouse # 416 was injected with Emu- ALL cancer cells (B-ALL cell line) a week prior to CAR-T administration. IL-2Ra -/- have their exons 2 and 3 (that bind to IL-2) replaced by a Neomycin resistance gene. After confirming for tumor establishment, 2 to 3 million IFNg -/- CD1928z CAR-T cells were intravenously administered into the mouse along with Th1 cells (differentiated in vitro using Th1-inducing cytokines) (Group 5). Bone marrow samples were processed for single-cell sequencing at week 4 post-CAR-T infusion.

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File: RS-04318054_465-GEX_RS-04310590_S4_L002_R2_001.fastq.gz

Description: IL-2Ra -/- mouse # 465 was injected with Emu- ALL cancer cells (B-ALL cell line) a week prior to CAR-T administration. IL-2Ra -/- have their exons 2 and 3 (that bind to IL-2) replaced by a Neomycin resistance gene. After confirming for tumor establishment, 2 to 3 million CD1928z CAR-T cells were intravenously administered into the mouse along with Th17 cells (differentiated in vitro using Th17-inducing cytokines) (Group 4). Bone marrow samples were processed for single-cell sequencing at week 4 post-CAR-T infusion.

File: RS-04318054_465-GEX_RS-04310590_S4_L001_R2_001.fastq.gz

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File: RS-04318052_446-GEX_RS-04310588_S2_L002_R2_001.fastq.gz

Description: IL-2Ra -/- mouse # 446 was injected with Emu- ALL cancer cells (B-ALL cell line) a week prior to CAR-T administration. IL-2Ra -/- have their exons 2 and 3 (that bind to IL-2) replaced by a Neomycin resistance gene. After confirming for tumor establishment, 2 to 3 million CD1928z CAR-T cells were intravenously administered into the mouse along with Th17 cells (differentiated in vitro using Th17-inducing cytokines) (Group 4). Bone marrow samples were processed for single-cell sequencing at week 4 post-CAR-T infusion.

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Code/software

Code available upon request

Access information

Other publicly accessible locations of the data:

Data was derived from the following sources:

Single cells were isolated and then processed using the 10X Genomics Single Cell 3' v3 kit according to the manufacturer's instructions. Libraries were sequenced on the Illumina NovaSeq 6000 instrument (RRID:SCR_016387). Raw sequencing data were processed using the Cell Ranger (CR) (v6.0.0) pipeline (RRID:SCR_017344) to generate fastq files. Fastq files were aligned and quantified, generating feature-barcode count matrices. Gene-barcode matrices containing Unique Molecular Identifier (UMI) counts are filtered using CR's cell detection algorithm. Downstream analyses were performed mainly using Seurat (v5.0.0) single-cell analysis R package (RRID:SCR_016341). Eight single-cell RNA seq samples were individually read into a Seurat object (RRID:SCR_016341) to examine feature number, mitochondrial percentage, and read count distributions within each sample. Cells with fewer than 500 features or greater than 7500 features or >15% mitochondrial content were filtered out. After normalizing and finding variable features from individual samples, all samples are then integrated using FindIntegrationAnchors function. The resulting data would serve for visualization purposes. Separately, samples were merged in a SingleR (v1.6.1), a Bioconductor package (RRID:SCR_006442), which was then used to annotate individual immune cell types using the ImmGen reference database (RRID:SCR_021792) from the celldex (v1.12.0) R package. Principal component analysis was used to detect and visualize highly variable genes. Using the RNA assay, data normalization and scaling were performed using Seurat's (RRID:SCR_016341) SCTransform function regressing against mitochondrial percentage. The data were then dimension-reduced via UMAP (RRID:SCR_018217) and clustered using the Louvain algorithm for downstream visualization. Normalization, scaling, dimension-reduction, clustering, and quantification are performed on specific cell populations (i.e., Neutrophils and Macrophages) to further detect subpopulation clusters based on their expression profiles. Differential gene expression analysis was performed using Seurat's (RRID:SCR_016341) FindMarkers function on groups of interest.

Subsequent gene-set enrichment analyses were performed on comparisons of interest using the GSEApreranked procedure implemented by the clusterProfiler package (v4.6.2) (RRID:SCR_016884) using the msigDB database found in the msigdbr (v7.5.1) package (RRID:SCR_022870). Mouse versions of Hallmark, C2, and C5-GO gene sets were used to perform pathway analyses. The avg_log2FC values (obtained from the FindMarkers results) are utilized as ranked values to perform pathway analyses using prerankedGSEA. Specific cell-level signature scores are obtained using Seurat’s (RRID:SCR_016341) AddModuleScore function. The custom code generated for all single-cell sequencing analysis is available upon request.