T-ALL and thymocytes : a message of noncoding RNAs

In the last decade, the role for noncoding RNAs in disease was clearly established, starting with microRNAs and later expanded towards long noncoding RNAs. This was also the case for T cell acute lymphoblastic leukemia, which is a malignant blood disorder arising from oncogenic events during normal T cell development in the thymus. By studying the transcriptomic profile of protein-coding genes, several oncogenic events leading to T cell acute lymphoblastic leukemia (T-ALL) could be identified. In recent years, it became apparent that several of these oncogenes function via microRNAs and long noncoding RNAs. In this review, we give a detailed overview of the studies that describe the noncoding RNAome in T-ALL oncogenesis and normal T cell development

Comprehensive miRNA expression profiling in human T-cell acute lymphoblastic leukemia by small RNA-sequencing

T-cell acute lymphoblastic leukemia (T-ALL) is a genetically heterogeneous disease that can be classified into different molecular genetic subtypes according to their mRNA gene expression profile. In this study, we applied RNA sequencing to investigate the full spectrum of miRNA expression in primary T-ALL patient samples, T-ALL leukemia cell lines and healthy donor thymocytes. Notably, this analysis revealed that genetic subtypes of human T-ALL also display unique miRNA expression signatures, which are largely conserved in human T-ALL cell lines with corresponding genetic background. Furthermore, small RNA-sequencing also unraveled the variety of isoforms that are expressed for each miRNA in T-ALL and showed that a significant number of miRNAs are actually represented by an alternative isomiR. Finally, comparison of CD34(+) and CD4(+) CD8(+) healthy donor thymocytes and T-ALL miRNA profiles allowed identifying several novel miRNAs with putative oncogenic or tumor suppressor functions in T-ALL. Altogether, this study provides a comprehensive overview of miRNA expression in normal and malignant T-cells and sets the stage for functional evaluation of novel miRNAs in T-ALL disease biology

Conventional and computational flow cytometry analyses reveal sustained human intrathymic T cell development from birth until puberty

The thymus is the organ where subsets of mature T cells are generated which subsequently egress to function as central mediators in the immune system. While continuously generating T cells even into adulthood, the thymus does undergo involution during life. This is characterized by an initial rapid decrease in thymic cellularity during early life and by a second age-dependent decline in adulthood. The thymic cellularity of neonates remains low during the first month after birth and the tissue reaches a maximum in cellularity at 6 months of age. In order to study the effect that this first phase of thymic involution has on thymic immune subset frequencies, we performed multi-color flow cytometry on thymic samples collected from birth to 14 years of age. In consideration of the inherent limitations posed by conventional flow cytometry analysis, we established a novel computational analysis pipeline that is adapted from single-cell transcriptome sequencing data analysis. This allowed us to overcome technical effects by batch correction, analyze multiple samples simultaneously, limit computational cost by subsampling, and to rely on KNN-graphs for graph-based clustering. As a result, we successfully identified rare, distinct and gradually developing immune subsets within the human thymus tissues. Although the thymus undergoes early involution from infanthood onwards, our data suggests that this does not affect human T-cell development as we did not observe significant alterations in the proportions of T-lineage developmental intermediates from birth to puberty. Thus, in addition to providing an interesting novel strategy to analyze conventional flow cytometry data for the thymus, our work shows that the early phase of human thymic involution mainly limits the overall T cell output since no obvious changes in thymocyte subsets could be observed

Notch/Delta signaling constrains reengineering of pro-T cells by PU.1

PU.1 is essential for early stages of mouse T cell development but antagonizes it if expressed constitutively. Two separable mechanisms are involved: attenuation and diversion. Dysregulated PU.1 expression inhibits pro-T cell survival, proliferation, and passage through β-selection by blocking essential T cell transcription factors, signaling molecules, and Rag gene expression, which expression of a rearranged T cell antigen receptor transgene cannot rescue. However, Bcl2 transgenic cells are protected from this attenuation and may even undergo β-selection, as shown by PU.1 transduction of defined subsets of Bcl2 transgenic fetal thymocytes with differentiation in OP9-DL1 and OP9 control cultures. The outcome of PU.1 expression in these cells depends on Notch/Delta signaling. PU.1 can efficiently divert thymocytes toward a myeloid-like state with multigene regulatory changes, but Notch/Delta signaling vetoes diversion. Gene expression analysis distinguishes sets of critical T lineage regulatory genes with different combinatorial responses to PU.1 and Notch/Delta signals, suggesting particular importance for inhibition of E proteins, Myb, and/or Gfi1 (growth factor independence 1) in diversion. However, Notch signaling only protects against diversion of cells that have undergone T lineage specification after Thy-1 and CD25 up-regulation. The results imply that in T cell precursors, Notch/Delta signaling normally acts to modulate and channel PU.1 transcriptional activities during the stages from T lineage specification until commitment

Expression of the inhibitory Ly49E receptor is not critically involved in the immune response against cutaneous, pulmonary or liver tumours

Natural killer (NK) lymphocytes are part of the innate immune system and are important in immune protection against tumourigenesis. NK cells display a broad repertoire of activating and inhibitory cell surface receptors that regulate NK cell activity. The Ly49 family of NK receptors is composed of several members that recognize major histocompatibility complex class I (MHC-I) or MHC-I-related molecules. Ly49E is a unique inhibitory member, being triggered by the non-MHC-I-related protein urokinase plasminogen activator (uPA) in contrast to the known MHC-I-triggering of the other inhibitory Ly49 receptors. Ly49E also has an uncommon expression pattern on NK cells, including high expression on liver DX5-NK cells. Furthermore, Ly49E is the only Ly49 member expressed by epidermal gamma delta T cells. As gamma delta T cells and/or NK cells have been shown to be involved in the regulation of cutaneous, pulmonary and liver malignancies, and as uPA is involved in tumourigenesis, we investigated the role of the inhibitory Ly49E receptor in the anti-tumour immune response. We demonstrate that, although Ly49E is highly expressed on epidermal gamma delta T cells and liver NK cells, this receptor does not play a major role in the control of skin tumour formation or in lung and liver tumour development

Ly49E Expression on CD8αα-expressing intestinal intraepithelial lymphocytes plays no detectable role in the development and progression of experimentally induced inflammatory bowel diseases

The Ly49E NK receptor is a unique inhibitory receptor, presenting with a high degree of conservation among mouse strains and expression on both NK cells and intraepithelial-localised T cells. Amongst intraepithelial-localised T cells, the Ly49E receptor is abundantly expressed on CD8 alpha alpha-expressing innate-like intestinal intraepithelial lymphocytes (iIELs), which contribute to front-line defense at the mucosal barrier. Inflammatory bowel diseases (IBDs), encompassing Crohn's disease and ulcerative colitis, have previously been suggested to have an autoreactive origin and to evolve from a dysbalance between regulatory and effector functions in the intestinal immune system. Here, we made use of Ly49E-deficient mice to characterize the role of Ly49E receptor expression on CD8 alpha alpha-expressing iIELs in the development and progression of IBD. For this purpose we used the dextran sodium sulphate (DSS)- and trinitrobenzenesulfonic-acid (TNBS)-induced colitis models, and the TNF Delta ARE ileitis model. We show that Ly49E is expressed on a high proportion of CD8 alpha alpha-positive iIELs, with higher expression in the colon as compared to the small intestine. However, Ly49E expression on small intestinal and colonic iIELs does not influence the development or progression of inflammatory bowel diseases

In vitro human embryonic stem cell hematopoiesis mimics MYB-independent yolk sac hematopoiesis

Although hematopoietic precursor activity can be generated in vitro from human embryonic stem cells, there is no solid evidence for the appearance of multipotent, self-renewing and transplantable hematopoietic stem cells. This could be due to short half-life of hematopoietic stem cells in culture or, alternatively, human embryonic stem cellinitiated hematopoiesis may be hematopoietic stem cell-independent, similar to yolk sac hematopoiesis, generating multipotent progenitors with limited expansion capacity. Since a MYB was reported to be an excellent marker for hematopoietic stem cell-dependent hematopoiesis, we generated a MYB-eGFP reporter human embryonic stem cell line to study formation of hematopoietic progenitor cells in vitro. We found CD34(+) hemogenic endothelial cells rounding up and developing into CD43(+) hematopoietic cells without expression of MYB-eGFP. MYB-eGFP+ cells appeared relatively late in embryoid body cultures as CD34(+) CD43(+) CD45(-/lo) cells. These MYB-eGFP(+) cells were CD33 positive, proliferated in IL-3 containing media and hematopoietic differentiation was restricted to the granulocytic lineage. In agreement with data obtained on murine Myb(-/-) embryonic stem cells, bright eGFP expression was observed in a subpopulation of cells, during directed myeloid differentiation, which again belonged to the granulocytic lineage. In contrast, CD14(+) macrophage cells were consistently eGFP-and were derived from eGFPprecursors only. In summary, no evidence was obtained for in vitro generation of MYB+ hematopoietic stem cells during embryoid body cultures. The observed MYB expression appeared late in culture and was confined to the granulocytic lineage

A murine intestinal intraepithelial NKp46-negative innate lymphoid cell population characterized by group 1 properties

The Ly49E receptor is preferentially expressed on murine innate-like lymphocytes, such as epidermal V gamma 3 T cells, intestinal intraepithelial CD8 alpha alpha(+) T lymphocytes, and CD49a(+) liver natural killer (NK) cells. As the latter have recently been shown to be distinct from conventional NK cells and have innate lymphoid cell type 1 (ILC1) properties, we investigated Ly49E expression on intestinal ILC populations. Here, we show that Ly49E expression is very low on known ILC populations, but it can be used to define a previously unrecognized intraepithelial innate lymphoid population. This Ly49E-positive population is negative for NKp46 and CD8 alpha alpha, expresses CD49a and CD103, and requires T-bet expression and IL-15 signaling for differentiation and/or survival. Transcriptome analysis reveals a group 1 ILC gene profile, different from NK cells, iCD8 alpha cells, and intraepithelial ILC1. Importantly, NKp46(-)CD8 alpha alpha(-)Ly49E(+) cells produce interferon (IFN)-gamma, suggesting that this previously unrecognized population may contribute to Th1-mediated immunity

HES1 and HES4 have non-redundant roles downstream of Notch during early human T-cell development

In both mouse and human, Notch1 activation is the main initial driver to induce T-cell development in hematopoietic progenitor cells. The initiation of this developmental process coincides with Notch1-dependent repression of differentiation towards other hematopoietic lineages. Although well described in mice, the role of the individual Notch1 target genes during these hematopoietic developmental choices is still unclear in human, particularly for HES4 since no orthologous gene is present in the mouse. Here, we investigated the functional capacity of the Notch1 target genes HES1 and HES4 to modulate human Notch1-dependent hematopoietic lineage decisions and their requirement during early T-cell development. We show that both genes are upregulated in a Notch-dependent manner during early T-cell development and that HES1 acts as a repressor of differentiation by maintaining a quiescent stem cell signature in CD34(+) hematopoietic progenitor cells. While HES4 can also inhibit natural killer and myeloid cell development like HES1, it acts differently on the T- versus B-cell lineage choice. Surprisingly, HES4 is incapable of repressing B-cell development, the most sensitive hematopoietic lineage with respect to Notch-mediated repression. In contrast to HES1, HES4 promotes initiation of early T-cell development, but ectopic expression of HES4, or HES1 and HES4 combined, is insufficient to induce T-lineage differentiation. Importantly, knockdown of HES1 or HES4 significantly reduces human T-cell development. Overall, we show that the Notch1 target genes HES1 and HES4 have non-redundant roles during early human T-cell development which may relate to differences in mediating Notch-dependent human hematopoietic lineage decisions