CD64 enables TCR-dependent, MHC-independent CTL cytotoxicity of AML cells
Data files
Jun 29, 2026 version files 5.39 GB
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14A_S5_L001_R1_001.fastq.gz
167.57 MB
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14A_S5_L002_R1_001.fastq.gz
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14A_S5_L003_R1_001.fastq.gz
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14A_S5_L004_R1_001.fastq.gz
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14B_S6_L001_R1_001.fastq.gz
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14B_S6_L002_R1_001.fastq.gz
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14B_S6_L003_R1_001.fastq.gz
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14B_S6_L004_R1_001.fastq.gz
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15A_S7_L001_R1_001.fastq.gz
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15A_S7_L002_R1_001.fastq.gz
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15A_S7_L003_R1_001.fastq.gz
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15A_S7_L004_R1_001.fastq.gz
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15B_S8_L001_R1_001.fastq.gz
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15B_S8_L002_R1_001.fastq.gz
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15B_S8_L003_R1_001.fastq.gz
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15B_S8_L004_R1_001.fastq.gz
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7A_S3_L001_R1_001.fastq.gz
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7A_S3_L002_R1_001.fastq.gz
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7A_S3_L003_R1_001.fastq.gz
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7A_S3_L004_R1_001.fastq.gz
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7B_S4_L001_R1_001.fastq.gz
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7B_S4_L002_R1_001.fastq.gz
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7B_S4_L003_R1_001.fastq.gz
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7B_S4_L004_R1_001.fastq.gz
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README.md
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Ref_S9_L001_R1_001.fastq.gz
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Ref_S9_L002_R1_001.fastq.gz
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Ref_S9_L003_R1_001.fastq.gz
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Ref_S9_L004_R1_001.fastq.gz
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UA_S1_L001_R1_001.fastq.gz
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UA_S1_L002_R1_001.fastq.gz
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UA_S1_L003_R1_001.fastq.gz
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UA_S1_L004_R1_001.fastq.gz
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UB_S2_L001_R1_001.fastq.gz
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UB_S2_L002_R1_001.fastq.gz
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UB_S2_L003_R1_001.fastq.gz
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UB_S2_L004_R1_001.fastq.gz
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Abstract
CD8+ T cell-mediated cytotoxicity classically occurs through engagement of an alpha-beta T cell receptor (TCRab) with a peptide-class I major histocompatibility complex (pMHC). However, it is also known that cytotoxic CD8+ T lymphocytes (CTLs) can kill tumor cells in a pMHC-independent manner. The relative physiologic contribution and biological significance of pMHC-independent CTL killing remains unclear, and a receptor shared between CTLs and NK cells is generally invoked as the mechanism by which this occurs. In this study, we used acute myeloid leukemia (AML) as a model to examine mechanisms of pMHC-independent cytotoxicity and discovered a paradoxical TCR-dependent, MHC-independent mechanism that requires CD64. Utilizing knockouts of potential AML ligands and CTL receptors, we demonstrate that pMHC-independent cytotoxicity is a potent mechanism of CTL-mediating killing of AML cells and is largely restricted to CD64-expressing cells through an IFNg-regulated process. Notably, we found that pMHC-independent CTL killing is not due to activation of commonly implicated NK activating receptors but rather requires an activated TCRab/CD3 complex. Thus, we identify a novel form of CD64-dependent, pMHC-independent, TCR-dependent killing by CTLs that appears highly enriched for in AML.
Dataset DOI: 10.5061/dryad.rr4xgxdps
Description of the data and file structure
All cell lines were on the OCI-AML2 background.
- UA = OCI-AML2 HLA-A KO cells not treated with CTLs
- UB = OCI-AML2 B2M KO cells not treated with CTLs
- 7A = OCI-AML2 HLA-A KO cells treated with WT1 CTLs from donor Hem07
- 7B = OCI-AML2 B2M KO cells treated with WT1 CTLs from donor Hem07
- 14A = OCI-AML2 HLA-A KO cells treated with WT1 CTLs from donor Hem14
- 14B = OCI-AML2 B2M KO cells treated with WT1 CTLs from donor Hem14
- 15A = OCI-AML2 HLA-A KO cells treated with WT1 CTLs from donor Hem15
- 15B = OCI-AML2 B2M KO cells treated with WT1 CTLs from donor Hem15
Please contact Dr. Cassian Yee for more questions EMAIL:cyee@mdanderson.org
Files and variables
File: 7A_S3_L002_R1_001.fastq.gz
Description: OCI-AML2 HLA-A KO cells treated with WT1 CTLs from donor Hem07
File: 7A_S3_L001_R1_001.fastq.gz
Description: OCI-AML2 HLA-A KO cells treated with WT1 CTLs from donor Hem07
File: 7A_S3_L003_R1_001.fastq.gz
Description: OCI-AML2 HLA-A KO cells treated with WT1 CTLs from donor Hem07
File: 7A_S3_L004_R1_001.fastq.gz
Description: OCI-AML2 HLA-A KO cells treated with WT1 CTLs from donor Hem07
File: 7B_S4_L001_R1_001.fastq.gz
Description: OCI-AML2 B2M KO cells treated with WT1 CTLs from donor Hem07
File: 7B_S4_L002_R1_001.fastq.gz
Description: OCI-AML2 B2M KO cells treated with WT1 CTLs from donor Hem07
File: 7B_S4_L003_R1_001.fastq.gz
Description: OCI-AML2 B2M KO cells treated with WT1 CTLs from donor Hem07
File: 7B_S4_L004_R1_001.fastq.gz
Description: OCI-AML2 B2M KO cells treated with WT1 CTLs from donor Hem07
File: 14A_S5_L002_R1_001.fastq.gz
Description: OCI-AML2 HLA-A KO cells treated with WT1 CTLs from donor Hem14
File: 14A_S5_L001_R1_001.fastq.gz
Description: OCI-AML2 HLA-A KO cells treated with WT1 CTLs from donor Hem14
File: 14A_S5_L003_R1_001.fastq.gz
Description: OCI-AML2 HLA-A KO cells treated with WT1 CTLs from donor Hem14
File: 14A_S5_L004_R1_001.fastq.gz
Description: OCI-AML2 HLA-A KO cells treated with WT1 CTLs from donor Hem14
File: 14B_S6_L002_R1_001.fastq.gz
Description: OCI-AML2 B2M KO cells treated with WT1 CTLs from donor Hem14
File: 14B_S6_L001_R1_001.fastq.gz
Description: OCI-AML2 B2M KO cells treated with WT1 CTLs from donor Hem14
File: 14B_S6_L003_R1_001.fastq.gz
Description: OCI-AML2 B2M KO cells treated with WT1 CTLs from donor Hem14
File: 14B_S6_L004_R1_001.fastq.gz
Description: OCI-AML2 B2M KO cells treated with WT1 CTLs from donor Hem14
File: 15A_S7_L002_R1_001.fastq.gz
Description: OCI-AML2 HLA-A KO cells treated with WT1 CTLs from donor Hem15
File: 15A_S7_L001_R1_001.fastq.gz
Description: OCI-AML2 HLA-A KO cells treated with WT1 CTLs from donor Hem15
File: 15A_S7_L003_R1_001.fastq.gz
Description: OCI-AML2 HLA-A KO cells treated with WT1 CTLs from donor Hem15
File: 15A_S7_L004_R1_001.fastq.gz
Description: OCI-AML2 HLA-A KO cells treated with WT1 CTLs from donor Hem15
File: 15B_S8_L001_R1_001.fastq.gz
Description: OCI-AML2 B2M KO cells treated with WT1 CTLs from donor Hem15
File: 15B_S8_L002_R1_001.fastq.gz
Description: OCI-AML2 B2M KO cells treated with WT1 CTLs from donor Hem15
File: 15B_S8_L004_R1_001.fastq.gz
Description: OCI-AML2 B2M KO cells treated with WT1 CTLs from donor Hem15
File: 15B_S8_L003_R1_001.fastq.gz
Description: OCI-AML2 B2M KO cells treated with WT1 CTLs from donor Hem15
File: Ref_S9_L002_R1_001.fastq.gz
Description: Brunello library
File: Ref_S9_L001_R1_001.fastq.gz
Description: Brunello library
File: Ref_S9_L003_R1_001.fastq.gz
Description: Brunello library
File: Ref_S9_L004_R1_001.fastq.gz
Description: Brunello library
File: UA_S1_L002_R1_001.fastq.gz
Description: OCI-AML2 HLA-A KO cells not treated with CTLs
File: UA_S1_L001_R1_001.fastq.gz
Description: OCI-AML2 HLA-A KO cells not treated with CTLs
File: UB_S2_L002_R1_001.fastq.gz
Description: OCI-AML2 B2M KO cells not treated with CTLs
File: UB_S2_L001_R1_001.fastq.gz
Description: OCI-AML2 B2M KO cells not treated with CTLs
File: UA_S1_L003_R1_001.fastq.gz
Description: OCI-AML2 HLA-A KO cells not treated with CTLs
File: UA_S1_L004_R1_001.fastq.gz
Description: OCI-AML2 HLA-A KO cells not treated with CTLs
File: UB_S2_L003_R1_001.fastq.gz
Description: OCI-AML2 B2M KO cells not treated with CTLs
File: UB_S2_L004_R1_001.fastq.gz
Description: OCI-AML2 B2M KO cells not treated with CTLs
Code/software
Read count matrices for each sgRNA barcode and sample were quantified using hisat2 version 2.1.0. Quality control was performed using 1000 non-targeting control sgRNA sequences acquired from the CRISPR-KO pooled library from Addgene (catalog # 73179-LV, one-vector system). HLA-A KO (UA) and B2M KO (UB) groups were used as control groups. Positive selection of gene knockouts was tested using MAGeCK version 0.5.9.4 with the test sub module. MAGeCK computed the FDR from the empirical permutation p-values using the Benjamini–Hochberg method.
Detailed descriptions of cell lines and primary samples used in the study, CTL generation and co-culture assays, generation of genetically engineered cell lines and CTLs, CRISPR screening process, generation of the CD64-Fc protein, antibodies used for Western blot and flow cytometry, TCR sequencing, quantification of TNFa by ELISA, and statistical analyses are provided in the Supplementary Materials and Methods and in the Supplementary Appendix of the associated article.
Generation of CTLs
PBMCs were obtained from HLA-A02:01-positive healthy donors from HemaCare (Northridge, CA, USA). CTLs specific for the HLA-A02:01-restricted WT1, TPX2, and MART1 peptide targets described in Supplementary Table 2 were generated utilizing the previously described endogenous T cell (ETC) protocol (24, 25). The TPX2 peptide was identified via a peptide-elution, tandem mass spectrometry (MS/MS) approach from HLA-A*02:01-expressing K562 cells using a previously described protocol (30). Additional detailed methods are described in the Supplementary Appendix.
Co-culture cytotoxicity assays
Co-culture assays between CTLs and target cells (tumor cell lines, T2 cells, primary AML samples, or PBMCs) were performed as previously described (24). In brief, CTLs were thawed from cryopreservation, rested overnight in IL-15 (5ng/ml), and utilized the following day (after multiple washes to remove residual IL-15) in co-culture assays with target cells. Cytotoxicity was assessed through a flow cytometry-based annexin V / PI assay in which CTLs or target cells were labeled with CellTrace Violet (CTV) (Thermo Fisher Scientific, Waltham, MA, USA). Specifically in the case of co-culture with AML cells and T2 cells (cold-target inhibition assay), AML cells were labeled with CTV and T2 cells were labeled with CellTrace Far Red. Additional detailed methods are described in the Supplementary Appendix.
Flow cytometry
Flow cytometry on cell lines, primary AML samples, PBMCs, and CTLs was performed on a NovoCyte flow cytometer (Agilent Technologies, Hayward, CA, USA) or a BD LSRFortessa flow cytometer (BD Biosciences). Output flow cytometry standard (.fcs) files were analyzed using FlowJo 10 software. Fluorescent compensation was performed with single-color controls. Antibodies utilized for experiments are listed in Supplementary Table 5.