Highlights d Patients with severe COVID-19 accumulate HLA-DR Low monocytes and immature neutrophils in blood/lungs d Calprotectin level positively correlates with neutrophil count and disease severity d Loss of non-classical monocytes could identify high risk of severe COVID-19

TET2 converts 5-methylcytosine to 5-hydroxymethylcytosine (5-hmC) in DNA and is frequently mutated in myeloid ma-lignancies, including myeloproliferative neoplasms. Here we show that the level of 5-hmC is decreased in granulocyte DNA from myeloproliferative neoplasm patients with TET2 mutations compared with granulocyte DNA from healthy patients. Inhibition of TET2 by RNA interference decreases 5-hmC levels in both human leukemia cell lines and cord blood CD34 cells. These results confirm the enzymatic function of TET2 in human hematopoietic cells. Knockdown of TET2 in cord blood CD34 cells skews progenitor differentiation toward the granulo-monocytic lineage at the expense of lym-phoid and erythroid lineages. In addition, by monitoring in vitro granulomonocytic development we found a decreased granulocytic differentiation and an increase in monocytic cells. Our results indicate that TET2 disruption affects 5-hmC levels in human myeloid cells and participates in the pathogenesis of my-eloid malignancies through the disturbance of myeloid differentiation. (Blood. 2011;118(9):2551-2555)

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Ten-eleven-translocation 2 (TET2) belongs to the TET protein family that catalyzes the conversion of 5-methylcytosine into 5-hydroxymethylcytosine and plays a central role in normal and malignant adult hematopoiesis. Yet the role of TET2 in human hematopoietic development remains largely unknown. Here, we show that TET2 expression is low in human embryonic stem cell (ESC) lines and increases during hematopoietic differentiation. shRNA-mediated TET2 knockdown had no effect on the pluripotency of various ESCs. However, it skewed their differentiation into neuroectoderm at the expense of endoderm and mesoderm both in vitro and in vivo. These effects were rescued by reintroducing the targeted TET2 protein. Moreover, TET2-driven differentiation was dependent on NANOG transcriptional factor. Indeed, TET2 bound to NANOG promoter and in TET2-deficient cells the methylation of the NANOG promoter correlated with a decreased in NANOG expression. The altered differentiation resulting from TET2 knockdown in ESCs led to a decrease in both the number and the cloning capacities of hematopoietic progenitors. These defects were due to an increased apoptosis and an altered gene expression profile, including abnormal expression of neuronal genes. Intriguingly, when TET2 was knockdown in hematopoietic cells, it increased hematopoietic development. In conclusion, our work suggests that TET2 is involved in different stages of human embryonic development, including induction of the mesoderm and hematopoietic differentiation.

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Very few recurrent chromosomal abnormalities have been identified in T-cell non-Hodgkin lymphomas. These involve the TRA@/TRD@ gene at chromosome band 14q11 in up to 15% of cases. We recently reported a novel and recurrent translocation, t(14;19)(q11;q13), in peripheral T-cell lymphoma (PTCL). Fluorescence in situ hybridization analysis performed in three cases suggested an involvement of the TRA@/TRD@ locus at 14q11 and of a region telomeric to BCL3 on 19q13. We now report the molecular cloning of these translocations. Sequence analysis confirmed the involvement of the TRA@/TRD@ and indicated that the breakpoints were located mainly in the TRAJ region. On chromosome 19, our results revealed a new clustering of breakpoints outside the region involved in t(14;19)(q32;q13)-positive B-cell malignancies. Remarkably, all three breaks were located downstream or within the PVRL2 gene, in a small 10.3 kb interval, suggesting a nonrandom location of the breakpoints. For two patients, a high mRNA expression of both PVRL2 and BCL3 was found. In conclusion, we identified PVRL2 as a new recurrent partner gene of the TRA@ locus in PTCL. These results suggest that both BCL3 and PVRL2 may participate in the pathogenesis of these PTCLs, but further studies should be undertaken to investigate the precise role of these genes.

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1584 TET2, a gene frequently mutated in myeloid malignancies, encodes an oxygenase that may convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in hematopoietic cells. Using high performance liquid chromatography coupled to tandem mass sprectrometry (HPLC-MS-MS), we found lower levels of 5hmC in erythroblasts from patients with myeloproliferative neoplasms and TET2 mutations than in erythroblasts from non mutated patients. To study the function of TET2 during myelopoiesis, we used lentiviruses expressing short hairpin RNA (shRNA) designed to inhibit TET2 expression in CD34+ cells from normal bone marrow or umbilical cord blood. TET2 mRNA was knocked down by 56 to 74% in several human leukemia cell lines expressing shRNA-TET2, compared to cell lines expressing scramble shRNA. TET2 expression was then assessed in these cell lines by western blot, which showed a 49% reduction of TET2 protein in TF1 cell line, and 80 to 90% reduction in Kasumi, Uke1, and Mo7E cell lines. In addition, knock down of TET2 led to a 73% decrease in 5hmC in Mo7E cell line DNA whereas 5mC and cytosine contents remained unchanged as measured by HPLC-MS-MS. These results indicate that TET2 has a role in the hydroxylation of 5mC in human cells of hematopoietic origin. We then studied the consequences of TET2 knock-down in umbilical cord blood CD34+ cells in vitro. We observed a skewing of CD34+CD38- progenitor differentiation toward the myeloid lineage (52 +/− 4% of the TET2 knock down cells versus 35 +/− 3% of the control cells, n=3, p=0.001) at the expense of lympho-myeloid development and B cell and natural killer (NK) lymphoid lineages in a B/myeloid/NK culture condition. Methylcellulose in the presence of EPO, IL3, SCF, G-CSF showed greater numbers of CFU-G/GM (66 +/− 4 versus 58 +/− 3 colonies per 1000 input CD34+ cells) and lower numbers of BFU-E (60 +/−19 versus 78 +/− 19) in TET2 knock-down samples than in control samples (n= 4, p<0.05) indicating that there was also a skewing toward the granulo-monocytic differentiation at the expense of the erythroid lineage. In addition, in presence of G-CSF, IL3, and FLT3-ligand granulocytic differentiation was delayed in TET2 knock-down cells with a relative excess of monocytic cells at day 10 of culture as assessed by the analysis of cell morphology (47 +/− 4% monocytic cells versus 37 +/− 3%, n=5, p=0.006) and immunophenotype (52 +/− 3% of CD14+ cells versus 39+/−8%, n=5, p=0.002). We then analyzed the expression of a set of transcription factors at the mRNA level that confirmed that some transcription factors specific to granulocytic differentiation were under-expressed in TET2 knocked down cells between day 5 and day 10 of culture. Together, our results show that TET2 participates to the conversion of 5mC to 5hmC in hematopoietic cells, and suggest that TET2 inactivation may have a role in the pathogenesis of myeloid malignancies through the disturbance of myeloid differentiation. Disclosures: No relevant conflicts of interest to declare.