Rôle des gènes HOX du paralogue 4 dans l'autorenouvellement des cellules souches et progéniteurs hématopoïétiques

La transplantation de cellules souches hématopoïétiques (CSH) est un traitement couramment utilisé pour traiter plusieurs types de maladies hématologiques telles que les leucémies. Par contre, une limite importante de ce type de traitement est la quantité restreinte de CSH disponibles pour la transplantation. Il importe donc de trouver des moyens pour expandre efficacement ces cellules ex vivo tout en préservant leurs propriétés. Le gène HOXB4 est présentement un candidat très prometteur pour atteindre cet objectif. Il a en effet été montré que HOXB4 est capable d’expandre les CSH in vivo et in vitro sans mener au développement de leucémie. Le gène HOXC4, qui appartient au même paralogue est aussi en mesure d’expandre les cellules hématopoïétiques primitives suggérant un rôle commun pour les gènes HOX du paralogue 4 dans l’autorenouvellement des CSH. Le gène HOXA4 est dix fois plus exprimé que le gène HOXB4 dans des CSH du foie fœtal au moment de leur principale expansion. De plus, les CSH mutantes pour Hoxa4, contrairement aux CSH mutantes pour Hoxb4, sont incapables de reconstituer un receveur irradié lorsqu’elles sont transplantées en condition de compétition. HOXA4 pourrait donc jouer un rôle plus important que les autres gènes du paralogue 4 pour l’expansion des CSH au niveau physiologique. Nous avons donc posé l’hypothèse que HOXA4 est capable d’expandre des CSH de façon plus importante que HOXB4. Les résultats obtenues dans le cadre de ce projet de recherche ont montré que la surexpression de HOXA4 était capable d’expandre les CSH et les progéniteurs hématopoïétiques primitifs dans le même ordre que ce qui est connu pour HOXB4. Des cultures et des essais de transplantation en situation de compétition ont confirmé la capacité égale des CSH surexprimant HOXA4 et HOXB4 de proliférer et de reconstituer les receveurs irradiés à long terme. Par contre, nous avons observé une meilleure reconstitution périphérique à court terme par les CSH HOXA4+ par rapport aux CSH HOXB4+, associée à une meilleure reconstitution lymphoïde. Nous avons aussi comparé les niveaux d’expression de gènes cibles potentiels dans des CSH surexprimant HOXA4 ou HOXB4 et observer que plusieurs gènes importants pour la fonction des CSH était régulé positivement suite à leur surexpression, notamment plusieurs gènes impliqués dans les voies de signalisation Notch et Wnt, tels que des récepteurs et ligands. Les gènes HOX du paralogue 4 pourraient donc réguler la communication entre les CSH et leur microenvironnement via ces voies de signalisation majeures et ainsi réguler leur autorenouvellement. La modulation de différents gènes codant pour des facteurs de transcription et des molécules impliquées dans la pluripotence suggère également que HOXA4 et HOXB4 utilisent des mécanismes intrinsèques et extrinsèques pour réguler leur potentiel d’autorenouvellement. Ces connaissances pourront ainsi être utilisées pour optimiser les protocoles d’expansion ex vivo des CSH dans un but thérapeutique.Transplantation of hematopoietic stem cells (HSC) is a treatment commonly used to treat several types of hematological diseases such as leukemia. However, a major limitation of this type of treatment is the limited number of HSC available for transplantation. It is therefore important to develop ways to expand these cells ex vivo. The HOXB4 gene is a promising candidate for achieving this goal. It has indeed been shown that HOXB4 is able to expand HSC in vivo and in vitro without inducing leukemia. HOXC4, which belongs to the same paralog group, is also able to expand primitive hematopoietic cell suggesting a common role for paralog 4 HOX genes in the self-renewal of HSC. HOXA4 is ten times more expressed in fetal liver HSC during their primary expansion. Furthermore, Hoxa4 mutant HSC, unlike Hoxb4 mutant HSC, are unable to reconstitute an irradiated recipient when transplanted in competition. Therefore, HOXA4 could play a more important role than other paralog 4 genes for HSC expansion at the physiological level and we hypothesized that HOXA4 can expand HSC more efficiently than HOXB4. The results obtained during this research project showed that the overexpression of HOXA4 expand HSC and primitive hematopoietic progenitors in the same order as HOXB4. Direct competitive culture and transplantation assays confirmed the equal capacity of HSC overexpressing HOXA4 and HOXB4 to proliferate and engraft at long-term. However, we observed a better short-term peripheral reconstitution by HOXA4+ HSC compared to HOXB4+ HSC, which was associated with a better lymphoid reconstitution. We also compared the expression levels of potential target genes in HSC overexpressing HOXA4 or HOXB4 and observed that many genes important for HSC function were upregulated following their overexpression, including several genes involved in the Notch and Wnt signaling pathway. These included both receptors as well as ligands, indicating that HOX4 genes might regulate the communication of primitive HSCs with their environment through these major signaling pathways and promote self-renewal. In addition, modulation of genes coding for transcription factors and molecules known for their function in pluripotency suggest that HOXA4 and HOXB4 have both intrinsic and extrinsic potential to control self renewal potential. This knowledge can then be further explored and used to optimize ex vivo HSC expansion protocols for clinical purposes

Hoxa cluster genes determine the proliferative activity of adult mouse hematopoietic stem and progenitor cells

Determination of defined roles for endogenous homeobox (Hox) genes in adult hematopoietic stem and progenitor cell (HSPC) activity has been hampered by a combination of embryonic defects and functional redundancy. Here we show that conditional homozygous deletion of the Hoxa cluster (Hoxa−/−) results in a marked reduction of adult HSPC activity, both in vitro and in vivo. Specifically, proliferation of Hoxa−/− HSPCs is reduced compared with wild-type (WT) cells in vitro and they are less competitive in vivo. Notably, the loss of Hoxa genes had little impact on HSPC differentiation. Comparative RNA sequencing analyses of Hoxa−/− and WT hematopoietic stem cells (CD150+/CD48−/Lineage−/c-kit+/Sca-1+) identified a large number of differentially expressed genes, three of which (Nr4a3, Col1a1, and Hnf4a) showed >10-fold reduced levels. Engineered overexpression of Hoxa9 in Hoxa−/− HSPCs resulted in partial phenotypic rescue in vivo with associated recovery in expression of a large proportion of deregulated genes. Together, these results provide definitive evidence linking Hoxa gene expression to proliferation of adult HSPCs

Hoxa9 collaborates with E2A-PBX1 in mouse B cell leukemia in association with Flt3 activation and decrease of B cell gene expression

Background: The fusion protein E2A-PBX1 induces pediatric B cell leukemia in human. Previously, we reported oncogenic interactions between homeobox (Hox) genes and E2A-PBX1 in murine T cell leukemia. A proviral insertional mutagenesis screen with our E2A-PBX1 B cell leukemia mouse model identified Hoxa genes as potential collaborators to E2A-PBX1. Here we studied whether Hoxa9 could enhance E2A-PBX1 leukemogenesis. Results: We show that Hoxa9 confers a proliferative advantage to E2A-PBX1 B cells. Transplantation experiments with E2A-PBX1 transgenic B cells overexpressing Hoxa9 isolated from bone marrow chimeras showed that Hoxa9 accelerates the generation of E2A-PBX1 B cell leukemia, but Hoxa9 is unable to transform B cells alone. Quantitative-reverse transcriptase polymerase chain reaction analysis demonstrated a strong repression of B cell specific genes in these E2A-PBX1/Hoxa9 leukemias in addition to Flt3 activation, indicating inhibition of B cell differentiation in combination with enhanced proliferation. Overexpression of Hoxa9 in established E2A-PBX1 mouse leukemic B cells resulted in a growth advantage in vitro, which was also characterized by an enhanced expression of Flt3. Conclusions: we show for the first time that Hoxa9 collaborates with E2A-PBX1 in the oncogenic transformation of B cells in a mouse model that involves Flt3 signaling, which is potentially relevant to human disease

CD5 levels define functionally heterogeneous populations of naïve human CD4+ T cells

Studies in murine models show that subthreshold TCR interactions with self-peptide are required for thymic development and peripheral survival of naïve T cells. Recently, differences in the strength of tonic TCR interactions with self-peptide, as read-out by cell surface levels of CD5, were associated with distinct effector potentials among sorted populations of T cells in mice. However, whether CD5 can also be used to parse functional heterogeneity among human T cells is less clear. Our study demonstrates that CD5 levels correlate with TCR signal strength in human naïve CD4+ T cells. Further, we describe a relationship between CD5 levels on naïve human CD4+ T cells and binding affinity to foreign peptide, in addition to a predominance of CD5hi T cells in the memory compartment. Differences in gene expression and biases in cytokine production potential between CD5lo and CD5hi naïve human CD4+ T cells are consistent with observations in mice. Together, these data validate the use of CD5 surface levels as a marker of heterogeneity among human naïve CD4+ T cells with important implications for the identification of functionally biased T- cell populations that can be exploited to improve the efficacy of adoptive cell therapies

Hoxb4 overexpression in CD4 memory phenotype T cells increases the central memory population upon homeostatic proliferation.

Memory T cell populations allow a rapid immune response to pathogens that have been previously encountered and thus form the basis of success in vaccinations. However, the molecular pathways underlying the development and maintenance of these cells are only starting to be unveiled. Memory T cells have the capacity to self renew as do hematopoietic stem cells, and overlapping gene expression profiles suggested that these cells might use the same self-renewal pathways. The transcription factor Hoxb4 has been shown to promote self-renewal divisions of hematopoietic stem cells resulting in an expansion of these cells. In this study we investigated whether overexpression of Hoxb4 could provide an advantage to CD4 memory phenotype T cells in engrafting the niche of T cell deficient mice following adoptive transfer. Competitive transplantation experiments demonstrated that CD4 memory phenotype T cells derived from mice transgenic for Hoxb4 contributed overall less to the repopulation of the lymphoid organs than wild type CD4 memory phenotype T cells after two months. These proportions were relatively maintained following serial transplantation in secondary and tertiary mice. Interestingly, a significantly higher percentage of the Hoxb4 CD4 memory phenotype T cell population expressed the CD62L and Ly6C surface markers, characteristic for central memory T cells, after homeostatic proliferation. Thus Hoxb4 favours the maintenance and increase of the CD4 central memory phenotype T cell population. These cells are more stem cell like and might eventually lead to an advantage of Hoxb4 T cells after subjecting the cells to additional rounds of proliferation

Medium-term competitive homeostatic proliferations (60 days) of <i>Hoxb4</i> transgenic and wt CD4 MP T cells.

<p>(<b>A</b>) FACS profile showing fractions of <i>Hoxb4</i> and wt cells to donor derived MP T population in lymph node (LN). (<b>B</b>) Stacked bar graphs indicating the average contributions of <i>Hoxb4</i> and wt cells in LN, Spl and BM measured in three independent experiments; n = 9. (<b>C</b>) FACS profiles for the expression of typical memory T cell surface markers on <i>Hoxb4</i> and wt MP T cells in the BM. (<b>D</b>) Average subpopulations of <i>Hoxb4</i> and wt MP T cells expressing the indicated surface markers in LN (upper panel), Spl (middle panel) and BM (lower panel). (<b>E</b>) Percentage of <i>Hoxb4</i> and wt MP T cells (gated on CD44^hi) positive for indicated cytokines (n = 3–6). *P<0.05, 2-tailed Student ttest. MP = memory phenotype, wt = wild type, LN = lymph node, Spl = spleen and BM = bone marrow, TNF = tumor necrosis factor; IL-2 = interleukine-2; IFN = interferon.</p

Long-term competitive homeostatic proliferations (180 days) of <i>Hoxb4</i> transgenic and wt CD4 cells.

<p>(<b>A</b>) Scheme of serial transplantations. 10×10⁶ cells of the LNs of primary hosts that received a transplant composed of equal doses of <i>Hoxb4</i> and wt MP T cells were serially transplanted into secondary and tertiary hosts with a 60 days interval. (<b>B</b>) Compilation of <i>Hoxb4</i> and wt fractions of donor derived cells in LN, Spl and BM of secondary (n = 6) and tertiary hosts (n = 4) from two independent experiments. (<b>C</b>) Bar graphs showing the average percentage of cells positive for CD62L and Ly6C within the <i>Hoxb4</i> or wt memory T populations. *P<0.05, 2-tailed Student ttest. Wt = wild type, MP = memory phenotype, LN = lymph node, Spl = spleen and BM = bone marrow.</p

Total donor cells contribution to hematopoietic organs and the proportion of <i>Hoxb4</i> versus wild type cells.

<p>LN = Lymph node; BM = bone marrow, wt = wild type.</p

Competitive short-term homeostatic proliferations (7 days) of <i>Hoxb4</i> transgenic and wt CD4 MP T cells.

<p>(<b>A</b>) Scheme of the experimental approach. CD4 MP T cells are sorted from CellTrace™ Violet (CTV) labelled cells isolated from LN and Spl of <i>Hoxb4</i> (CD45.1/2) and congenic wt (CD45.1) mice. Cells of both genotypes are transplanted in a 1∶1 ratio in CD3ε^−/− (CD45.2) mice. (<b>B</b>) FACS profiles showing <i>Hoxb4</i> and wt fractions to donor derived CD4 MP T population (CD45.1) in LN (left panel). Representative FACS profiles for CD62L and CTV on <i>Hoxb4</i> and wt populations. Loss of CTV tracer indicates that most cells are dividing rapidly (right panels). (<b>C</b>) Average contribution (%) of <i>Hoxb4</i> and wt cells to donor derived MP T cells in LN, Spl and BM (n = 3). (<b>D</b>) Percentage of CD62L^hi MP T cells in <i>Hoxb4</i> and wt population found in lymphoid organs. *P<0.05; paired 2-tailed Student ttest. Wt = wild type, MP = memory phenotype, LN = lymph node, Spl = spleen and BM = bone marrow.</p