DOI: 10.5061/dryad.69p8cz9f3
title: Bone marrow Single cell sequencing data of CAR-T treated IL-2Ra -/- mice

description: 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.
description: 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 &gt;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.
description: # Bone marrow single-cell sequencing data of CAR-T treated IL-2Ra -/- mice
 Dataset DOI: [10.5061/dryad.69p8cz9f3](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. #### File:
 RS-04318056_436-GEX_RS-04310592_S6_L001_I2_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. #### File:
 RS-04318056_436-GEX_RS-04310592_S6_L001_R1_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. #### File:
 RS-04318056_436-GEX_RS-04310592_S6_L001_R2_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. #### File:
 RS-04318056_436-GEX_RS-04310592_S6_L002_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. #### File:
 RS-04318056_436-GEX_RS-04310592_S6_L002_I2_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. #### File:
 RS-04318056_436-GEX_RS-04310592_S6_L002_R1_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. #### File:
 RS-04318056_436-GEX_RS-04310592_S6_L002_R2_001.fastq.gz
 **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. #### File:
 RS-04318057_418-GEX_RS-04310593_S7_L001_I1_001.fastq.gz
 **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. #### File:
 RS-04318057_418-GEX_RS-04310593_S7_L001_I2_001.fastq.gz
 **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. #### File:
 RS-04318057_418-GEX_RS-04310593_S7_L001_R1_001.fastq.gz
 **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. #### File:
 RS-04318057_418-GEX_RS-04310593_S7_L001_R2_001.fastq.gz
 **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. #### File:
 RS-04318057_418-GEX_RS-04310593_S7_L002_I1_001.fastq.gz
 **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. #### File:
 RS-04318057_418-GEX_RS-04310593_S7_L002_I2_001.fastq.gz
 **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. #### File:
 RS-04318057_418-GEX_RS-04310593_S7_L002_R1_001.fastq.gz
 **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. #### File:
 RS-04318057_418-GEX_RS-04310593_S7_L002_R2_001.fastq.gz
 **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. #### 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. #### File:
 RS-04318058_459-GEX_RS-04310594_S8_L001_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. #### File:
 RS-04318058_459-GEX_RS-04310594_S8_L001_R1_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. #### File:
 RS-04318058_459-GEX_RS-04310594_S8_L001_I2_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. #### File:
 RS-04318058_459-GEX_RS-04310594_S8_L002_R1_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. #### File:
 RS-04318058_459-GEX_RS-04310594_S8_L002_I2_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. #### File:
 RS-04318058_459-GEX_RS-04310594_S8_L002_I1_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. #### File:
 RS-04318058_459-GEX_RS-04310594_S8_L001_I1_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. #### 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. #### File:
 RS-04318055_426-GEX_RS-04310591_S5_L001_R2_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. #### File:
 RS-04318055_426-GEX_RS-04310591_S5_L002_R2_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. #### File:
 RS-04318055_426-GEX_RS-04310591_S5_L002_I1_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. #### File:
 RS-04318055_426-GEX_RS-04310591_S5_L001_I2_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. #### File:
 RS-04318055_426-GEX_RS-04310591_S5_L002_I2_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. #### File:
 RS-04318055_426-GEX_RS-04310591_S5_L001_I1_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. #### File:
 RS-04318055_426-GEX_RS-04310591_S5_L001_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. #### 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
 **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_L002_R1_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_L002_I2_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_L002_I1_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_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-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. #### File:
 RS-04318053_415-GEX_RS-04310589_S3_L001_I2_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_I1_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-04318051_416-GEX_RS-04310587_S1_L001_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. #### File:
 RS-04318051_416-GEX_RS-04310587_S1_L001_R1_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. #### File:
 RS-04318051_416-GEX_RS-04310587_S1_L002_R1_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. #### File:
 RS-04318051_416-GEX_RS-04310587_S1_L002_I2_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. #### File:
 RS-04318051_416-GEX_RS-04310587_S1_L002_I1_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. #### File:
 RS-04318051_416-GEX_RS-04310587_S1_L001_I2_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. #### File:
 RS-04318051_416-GEX_RS-04310587_S1_L001_I1_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. #### 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
 **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_L002_R1_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_R1_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_L002_I2_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_L002_I1_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_I2_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_I1_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-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. #### File:
 RS-04318052_446-GEX_RS-04310588_S2_L001_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. #### File:
 RS-04318052_446-GEX_RS-04310588_S2_L002_I1_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. #### File:
 RS-04318052_446-GEX_RS-04310588_S2_L002_I2_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. #### File:
 RS-04318052_446-GEX_RS-04310588_S2_L002_R1_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. #### File:
 RS-04318052_446-GEX_RS-04310588_S2_L001_I2_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. #### File:
 RS-04318052_446-GEX_RS-04310588_S2_L001_I1_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. #### File:
 RS-04318052_446-GEX_RS-04310588_S2_L001_R1_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. ## Code/software Code available upon
 request ## Access information Other publicly accessible locations of the
 data: * N/A Data was derived from the following sources: * N/A