Page 67 - 2021_10-Haematologica-web

Tr1 cells kill primary pediatric acute myeloid leukemia cells
Figure 4. CD200 expression is upregulated in resistant pediatric acute myeloid leukemia and can impair LV-10-mediated degranulation and cytotoxicity. (A) Expression of 395 genes positively or negatively correlating with pediatric acute myeloid leukemia (pAML) sensitivity. The Spearman correlation of the expression of each gene to the median elimination efficiency (E.E.) of each pAML was calculated and plotted with genes represented as bars. 2,181 genes had a correlation with P<0.05, 395 of which had an absolute (R) ≥0.7 (red bars). (B) Data-mining strategy to identify genes conferring pAML resistance to LV-10 killing. Genes expressed 4-fold or more in the resistant pAML from the differentially expressed gene (DEG) analysis between sensitive vs resistant and the list of genes negatively correlated with E.E. with P<0.05 and R≤-0.7 were used to identify overlap in a Venn diagram. Genes appearing in both enriched in resistant and negatively correlated with E.E. were overlaid with genes encoding surface proteins identified in the Cell Surface Protein Atlas,35 resulting in ten pAML genes encoding surface proteins. These genes were manually annotated for potential interaction with T-cell surface proteins, identifying CD200. (C) CD200 gene expression in pAML blasts; log2 counts. Error bars: median and interquartile range. (D) CD200 protein expression on pAML, flow cytometry. Left panel: representative plots for one sensitive and one resistant pAML blast; right panel: cumulative data. Values from resistant (R) and intermediate resistant (IR) pAML are grouped, with IR pAML in red. Line represents mean. (E) CD200 receptor (CD200R1) is expressed on both LV-10 and LV-GFP cells. CD200R1 expression was measured in the CD3+CD4+NGFR+ population by flow cytometry, n=8. Line represents mean; error bar standard deviation. (F) CD200 overexpression impairs LV-10 degranulation. LV-10 were co-cultured with target myeloid tumor cell lines at a 10:1 E:T ratio for 6 hours. LV-10 cell line (n=7) degranulation, as measured by %CD107a+GZMB+ co-expression, was determined in co-culture with untrans- duced U937 and ALL-CM cells (No Vector), sorted GFP+ U937 and ALL-CM cells transduced with an empty vector (Empty Vector), or sorted GFP+CD200+ U937 and ALL-CM cells transduced with CD200 (CD200 Vector). *P<0.05, **P<0.01, Friedman ANOVA with Dunn’s post hoc test. (G) Cumulative CD107a+GZMB+% degranu- lation of LV-10 against U937 overexpressing CD200 when pre-treated with isotype control or anti-CD200R1 blocking antibody prior to co-culture, n=6; *P<0.05, Wilcoxon test. (H) CD200 reduces LV-10-mediated killing. LV-10 cells were co-cultured with empty vector- or CD200-overexpressing U937 or ALL-CM cells at a 1:1 E:T ratio for 3 days. Surviving cells were enumerated by flow cytometry. Ratio of remaining pAML was calculated as: (# remaining cells in CD200-overexpressing AML +LV-10/# remaining cells in CD200-overexpressing AML alone)/(# remaining cells in empty vector- AML +LV-10/# remaining cells in empty vector AML alone). (n=8, *P<0.05, Wilcoxon test.) GZMB: granzyme B.
this, we constructed a bicistronic lentiviral vector contain- ing CD200 together with ZsGreen1, a green fluorescent protein (Online Supplementary Figure S6A). Both cell lines transduced with the CD200-containing vector displayed significant upregulation of CD200 protein compared to empty vector-transduced cells (Online Supplementary Figure S6B). First, we tested the impact of CD200R1 signaling on LV-10 degranulation using CD107a degranulation assay coupled with granzyme B intracellular staining.39 In com- parison to the LV-10 cells co-cultured with control cell lines, LV-10 co-cultured with CD200-overexpressing cell lines degranulated significantly less (Figure 4F). In order to test if we could rescue the reduction in degranulation induced by CD200-overexpressing cells, we blocked CD200R1 on the LV-10 with a neutralizing antibody prior to co-culture with U937 or U937 CD200-overexpressing cell lines (Online Supplementary Figure S7A). Blocking CD200R1 partially restored LV-10 degranulation when co-cultured with CD200-overexpressing U937 (Figure 4G), while it had a non-significant effect on LV-10 degranulation when co-cul- tured with wild-type U937 (Online Supplementary Figure S7B). LV-10 degranulation was not fully restored to levels induced by wild-type U937, likely because the CD200R1 neutralizing antibody only blocked approximately 50% of available CD200R1 (Online Supplementary Figure S7C).
We next tested if CD200 overexpression in myeloid leukemia cell lines could confer resistance to LV-10 killing. In comparison to the empty vector-transduced control cells, CD200 overexpression significantly reduced killing of ALL- CM cells, but not of U937 cells (Figure 4H). This may be due to U937 cells’ increased robustness in vitro, as they have an average 1.34-fold higher proliferation rate than ALL-CM cells (not shown) that could compensate for killing in a 3-day culture. LV-GFP degranulation and killing, which are less potent than in LV-10 cells (Online Supplementary Figure S8), was also impaired by CD200, indicating that the CD200R1 signaling-induced inhibition of cytotoxicity is not Tr1-spe- cific. Altogether, these data suggest that resistant pAML can evade LV-10 killing by impairing their degranulation via CD200 expression.
Discussion
AML is a highly diverse hematopoietic cancer with over 20 different WHO sub-classifications,29 with suboptimal responses to conventional therapy and an urgent need for
novel treatments.40 Our previous study revealed that four of eight primary adult AML were sensitive to LV-10 cell killing. Importantly, LV-10 cells could inhibit myeloid leukemia pro- gression in vivo while preventing the induction of GvHD when co-injected with CD4+ T cells,13 suggesting that LV-10 cells can represent an innovative cell therapy for AML. Since pAML differ substantially from adult AML at the molecular, epigenetic, and genetic levels,19-23 herein we determined the pAML sensitivity to LV-10 killing, characterized the sensitive and resistant pAML molecular profiles, and identified CD200 expression as one of the mechanisms of pAML resistance to LV-10 killing.
While previously tested adult AML had only two levels of sensitivity to LV-10 killing,13 resembling the intermediate resistant and resistant pAML we measured, we also observed a subset of pAML that were highly sensitive to elimination by LV-10 cells. This additional sensitivity cate- gory may reflect the intrinsic genetic and epigenetic differ- ences between adult and pediatric AML,19-23 which could affect expression of markers required for LV-10-mediated killing. Interestingly, we observed that the expression of CD13, CD54, or CD112, which positively correlated with sensitivity to LV-10-mediated killing in adult AML,13 did not correlate to pAML sensitivity to killing (data not shown), fur- ther supporting the hypothesis that pediatric and adult AML interact differently with LV-10 cells.
The range of sensitivities we observed in pAML was underscored by significant differences in gene expression and cytogenetics. These analyses revealed that sensitivity to killing was linked to differentiation status. Sensitive pAML resembled more mature differentiated myeloid cells, with an enrichment of monocytic genes and high levels of CD64 and CD11c protein, which are frequently described on more differentiated AML subtypes.32,33,41,42 Conversely, resistant pAML did not have as distinct a gene signature. We found the maturation signature we observed in our sensitive subset present in the 187-sample NCI TARGET-pAML dataset.19 Whether we clustered the combined Stanford and TARGET data sets using only the top variably expressed genes or with our filtered S v R DEG list, the S pAML independently clustered away from the IR/R pAML. This was partially because the top 10% variability expressed genes in the combined Stanford and TARGET pAML datasets incorporated around half of the 335 DEG discriminating S v R pAML, yet this also sug- gests that the S v R DEG may represent underlying distin- guishing features among pAML. In addition to the genes driving the clustering, cytogenetic abnormalities, specifi-
haematologica | 2021; 106(10)
2595