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Tr1 cells kill primary pediatric acute myeloid leukemia cells
with immunosuppressive drugs, but these treatments also impair donor-derived cells from clearing residual leukemia cells (graft-versus-leukemia effect [GvL]),7 thereby increasing the risk of relapse. Thus, new treatments that preserve GvL while preventing GvHD are urgently needed.
In order to address this need, we devised a novel cell ther- apy with engineered type 1 regulatory T (Tr1) cells, called LV-10, made by lentiviral transduction of interleukin-10 (IL10) into peripheral CD4+ T cells. Tr1 cells are a FOXP3– subset of peripherally inducible regulatory T cells8 that cor- relate with induction of peripheral tolerance in transplanted patients9–12 and prevent xeno-GvHD in mice.13–16 In addi- tion, Tr1 cells directly lyse and kill malignant myeloid cells via perforin and granzyme B.13,14,17 This killing is not dependent on T-cell receptor (TCR) engagement13,18 and human leukocyte antigen (HLA) class II antigen presenta- tion, but rather on the target cell expression of HLA class I and several other molecules that facilitate target cell and T- cell interaction.13,14 Importantly, LV-10 Tr1 cells were shown to kill primary adult AML blasts and to impair leukemia progression in humanized mouse models of AML.13
The sensitivity of pAML to Tr1-mediated killing has not been tested. pAML have significant genetic, epigenetic, and molecular differences in comparison to adult AML.19–23 Understanding if pAML are also sensitive to Tr1-mediated killing is thus a critical step in LV-10 cell therapy develop- ment. Herein, we used LV-10 Tr1 cells to test 23 primary pAML blasts for their sensitivity to killing.13,14 We found that over 80% of pAML could be killed by LV-10 cells, with three levels of sensitivity to killing ranging from sensitive, intermediate resistant, and resistant. Sensitive pAML were enriched for gene signatures of leukocyte chemotaxis and expressed mature myeloid markers including CD64 and CD11c, suggesting a more mature phenotype. When ana- lyzed together with the large National Cancer Institute (NCI) Therapeutically Applicable Research to Generate Effective Treatments (TARGET) sensitive pAML dataset, pAML formed three clusters with TARGET samples, including one enriched for sensitive pAML samples with French-American-British (FAB) M5 acute monocytic leukemia and pAML with mixed lineage leukemia (MLL) rearrangement, while resistant and intermediate resistant pAML clustered with pAML bearing core binding factor translocations inv(16) or t(8;21)(RUNX1-RUNX1T1) cyto- genetic abnormalities. In addition, we identified that resist- ant pAML may evade LV-10 killing by upregulating CD200, which has been associated with poor prognosis of adult AML.24,25 Overall, we determine that a majority of pAML are sensitive to killing by LV-10 cells, and that resistance to killing is associated with loss of a mature myeloid signature and upregulation of CD200.
Methods
Subjects
De-identified pAML bone marrow aspirates were collected under written informed consent as part of a study approved by the Stanford University Institutional Review Board (IRB, #11062 and #11977) and obtained from the Stanford School of Medicine's Bass Childhood Cancer Center (CA, USA) tissue bank. Patient demo- graphics are listed in Table 1. Human peripheral blood mononu- clear cells (PBMC) were obtained from de-identified healthy donors (Stanford Blood Center, CA, USA) in accordance with IRB guidelines.
Table 1. Patient characteristics.
Age (months) (at sample acquisition) % Female
Average white blood cell count
Risk stratification High risk Standard risk Low risk
Average time of follow-up (months)
Progression to HSCT (%)
Overall survival (%)
HSCT: hematopoietic stem cell transplantation.
Cytotoxicity assays
137 (5-267) 43.50%
83.7 K/mL (0.4-347.6)
12/23 5/23 6/23
33.5 43.50 73.90
Killing assay was performed as previously described.13,14 Briefly, target cells were co-cultured at a 1:1 effector to target (E:T) ratio for 3 days. For primary pAML, blasts were thawed and incubated for 2 hours (h) in complete X-VIVO15 supplemented with IL-3 (20 ng/mL, Peprotech, NJ, USA) and G-CSF (20 ng/mL, Peprotech). After incubation, blasts were co-cultured for 4 days. Surviving cells were enumerated by fluorescence-activated cell sorting (FACS) using CountBright beads (Thermo Fisher, MA, USA). %
Elimination efficiency equals 1 minus (number of targets remaining in LV-10 co-culture/number of targets remaining alone) *100 with two to four LV cell lines per pAML.
Degranulation was measured as previously described.13,14 Briefly, T cells were co-cultured with target cells at a 10:1 E:T ratio with anti-CD107a antibody. After 1 h, brefeldin A (3 mg/mL) and monensin (2 mM) (eBioscience, CA, USA) were added and incubat- ed for 5 h. Cells were stained for surface markers, fixed, permeabi- lized (BD Fixation/Permeabilization kit, BD Biosciences), and stained for intracellular granzyme B as described in the Online Supplementary Table S1. Data was analyzed by flow cytometry. For CD200R1 blocking, 25 ug/mL of CD200R1 or isotype antibody was added to T cells for 30 minutes at 37°C prior to co-culturing with targets.
RNA sequencing
Complete computational methods for RNA sequencing (RNA- Seq) processing, analysis, and raw data are available at Gene Expression Omnibus (GEO) under accession number GSE140960. For differential gene expression, differential gene expression analy- sis (DESeq2) was used to normalize the counts and perform exploratory analysis (e.g., clustering, principal component analy- sis).26 Genes with low expression across all samples, sum (gene) <10 reads, were filtered out before performing differential gene expression. The design matrix was defined as design = ~condition, where the condition variable was composed of the following three levels: sensitive, intermediate resistant, and resistant. Transcripts were hierarchically clustered using Euclidean distance and com- plete linkage function. The heatmaps were created using ComplexHeatmap v2.0.0.27 Gene ontology (GO) terms were col- lapsed using EnrichmentMap v3.2.128 in Cytoscape v3.8.0. Correlation graph was plotted in R version 4.0.0. Enrichment analysis was performed using a binomial test for a one-tailed P-value, and Confidence Interval (CI) was calculated using Wilson/Brown test.
Statistical analysis
For the non-RNA-seq-derived data, analysis was performed using GraphPad Prism 7. As applicable, center bars and whiskers represent the mean with standard deviation, or median with range/interquartile range. The data was analyzed using non-para-
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