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Tr1 cells kill primary pediatric acute myeloid leukemia cells
Figure 3. Sensitive and resistant pediatric acute myeloid leukemia (pAML) signatures group TARGET pAML into three clusters. Euclidean clustering of the differen- tially expressed genes (DEG) between sensitive and resistant pediatric acute myeloid leukemia (pAML) (false discovery rate [FDR] <0.05, and absolute (log2 fold change [FC]) ≥2) detected in sequencing data from our Stanford dataset and the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) pAML dataset. Measured LV-10 killing sensitivity, risk group, and French-American-British (FAB) diagnosis are matched to each pAML when applicable. The TARGET dataset does not have associated LV-10 killing assay outcomes. Expression color is scaled per gene row. ND: not determined.
on their expression of the identified DEG grouped the sam- ples into three primary clusters: two ‘sensitive’ clusters that grouped with S pAML and 57% of TARGET pAML, and a ‘resistant’ cluster that grouped with the IR and R pAML and 43% of TARGET pAML (Figure 3). As we observed that CBF pAML were highly represented in IR and R pAML, we examined their distribution in the combined Stanford-TAR- GET dataset. Both pAML with t(8;21)(RUNX1-RUNX1T1) and pAML with inv(16)(CBFB-MYH11) translocations were enriched in the ‘resistant’ cluster (P<0.0001, P<0.0001 respectively). In line with our GSEA analysis results, one of the ‘sensitive’ clusters was highly enriched for M5 mono- cytic pAML (P<0.0001), while the ‘resistant’ cluster was enriched for M4 myelomonocytic pAML that also dis- played rearrangement inv(16) (P<0.0001).
Resistant pediatric acute myeloid leukemia express high levels of CD200, which can impair LV-10
cytotoxicity
In order to identify genes linked with pAML sensitivity or resistance to LV-10 cell killing, we correlated gene expression to the median elimination efficiency for each pAML blast. The expression of 2,181 genes significantly correlated to killing with P<0.05 (Figure 4A; Online Supplementary Table S6), 395 of which had an absolute R≥0.7 (Figure 4A, genes
shown as red bars). We hypothesized that the resistant pAML expressed inhibitory markers that protected them from killing. Therefore, we overlaid the genes that signifi- cantly and negatively correlated with killing, R≤-0.7 (189 genes), with the genes that were overexpressed 4-fold or more in the resistant pAML from the DEG analysis of sensi- tive versus resistant pAML (899 genes). We found 60 genes that were both negatively correlated with killing and prefer- entially expressed in resistant pAML (Figure 4B). Since per- forin and granzyme B-mediated killing requires cell-to-cell interaction,17,34 we filtered this gene list for genes encoding surface proteins35 and identified 10 genes (Figure 4B).
Next, we manually examined the functions of the ten genes to uncover proteins that have known interacting receptors expressed on T cells, and identified CD200, a type 1 membrane glycoprotein. CD200 is upregulated on resist- ant pAML (Figure 4C and D), and LV-10 express the CD200 receptor (CD200R1) (Figure 4E), an inhibitory receptor of immunoglobulin superfamily.36 CD200 expression is associ- ated with poor prognosis in adult AML.24,25 Moreover, CD200R1 signaling has been previously shown to impair mast cell37 and CD8+ T-cell degranulation.38
In order to determine if CD200 expression confers resist- ance to LV-10-mediated killing, we overexpressed CD200 in killing-sensitive ALL-CM and U937 myeloid cell lines. For
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