Effet de la surexpression du gène Hoxb4 sur la prolifération homéostatique des cellules T mémoires

Les cellules T mémoires (Tm) protègent l’organisme contre les réinfections de pathogènes qu’il a déjà combattu. Les Tm possèdent plusieurs propriétés en commun avec les cellules souches hématopoïétiques (CSH), notamment la capacité de se différencier, de s’auto-renouveler et de maintenir une population relativement constante au sein de l’organisme via des mécanismes homéostatiques. Il a été démontré que Hoxb4, un membre de la famille des facteurs de transcription Hox, était capable d’induire l’expansion des CSH in vivo et in vitro de façon rapide. Au vu de ces parallèles, nous avons posé l’hypothèse que la surexpression de Hoxb4 pourrait induire l’expansion de populations de Tm. Nous avons analysé les populations de Tm et lymphocytes T naïfs (Tn) dans les organes lymphoïdes de souris transgéniques surexprimant Hoxb4 et les avons comparées à des souris de type sauvage (wt). Alors que la fréquence des cellules T matures Hoxb4 diminuait avec l’âge, leur phénotype ainsi que leur viabilité demeuraient inchangés. Ensuite, nous avons procédé à des transplantations en compétition de Tm (CD4+CD44hi) Hoxb4 et wt chez des hôtes dépourvus de lymphocytes T (CD3-/-) dans le but d’évaluer leur contribution à la reconstitution du compartiment T après 2 mois. Au final, les Tm wt avait contribué un peu plus que les Tm Hoxb4 à la reconstitution (~60%). Des analyses fonctionnelles et phénotypiques ont montré que les Tm Hoxb4 possédaient une fonctionnalité normale, mais se distinguaient des Tm wt par la présence d’une faible population qui présentait un phénotype « mémoire central » (Tcm), conférant habituellement une longévité accrue. Les cellules des ganglions lymphatiques totaux des hôtes furent transplantées de façon sérielle chez trois générations de nouveaux hôtes. Le phénotype Tcm observés chez les Tm Hoxb4 était récapitulé chez les hôtes secondaires uniquement. Les ratios sont demeurés en faveur des Tm wt lors des deux transplantations suivantes, mais les Tm Hoxb4 ont commencé à montrer un avantage compétitif chez certains hôtes quaternaires. Une transplantation en compétition à court terme de Tm Hoxb4 et wt marqués avec un marqueur cytoplasmique ont démontré la présence chez les Tm Hoxb4 seulement d’une faible population CD62Lhi proliférant lentement. Ainsi, l’expansion préférentielle de Tcm CD4 par le biais d’une sélection ou d’une différenciation induite par la surexpression de Hoxb4 pourrait potentiellement leur permettre de maintenir un état de quiescence leur permettant de persister plus longtemps suite à des transplantations sérielles.Memory T cells (Tm) protect the organism against reinfection from pathogens they’ve already encountered. Tm share characteristics with hematopoietic stem cells (HSC), such as the capacity to differentiate, self-renew and maintain a relatively constant population via homeostatic mechanisms. Hoxb4, a member of the Hox genes family of transcription factors, has been shown to expand HSCs rapidly in vivo and in vitro. Thus, drawing from these parallels we hypothesise that Hoxb4 overexpression could lead to expansion of Tm populations. Tm and naïve T cell (Tn) populations were analysed in the lymphoid organs of young and aged transgenic mice overexpressing Hoxb4 in comparison with wild type (wt) mice. While the frequencies of mature Hoxb4 T cells in lymphoid organs seemed to decline with age, the phenotype or the cell viability remained unaffected. Next, CD4+CD44hi Hoxb4 Tm were transferred into T cell deficient (CD3-/-) hosts in competition with wt CD4+CD44hi Tm and evaluated for their contribution to T cell reconstitution after 2 months. Engraftment of wt Tm in secondary lymphoid organs was slightly higher than Hoxb4 Tm (~60%). Functional assays and phenotypic analysis showed that Hoxb4 Tm exhibited normal functionality, but in contrast to wt Tm, a fraction of Hoxb4 Tm exhibited a more central memory (Tcm) phenotype, indicative of a longer lifespan. Total lymph nodes from hosts were serially re-transplanted for three generations. The Tcm phenotype of the Hoxb4 Tm present in the primary hosts was recapitulated in the secondary but not in the tertiary hosts. The ratios remained in favor of the wt Tm after two subsequent expansion rounds, but Hoxb4 Tm showed a competitive advantage over wt Tm in some quaternary hosts. Cell tracking of a short term transplantation of Hoxb4 and wt Tm in competition exposed a small population of CD62Lhi cells displaying slow proliferation in the Hoxb4 Tm only. Thus preferential CD4+ Tcm expansion by selection or differentiation could potentially allow Hoxb4 Tm to persist longer following serial transplantations due to a more quiescent state

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

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

Change of naïve and MP T cell populations in <i>Hoxb4</i> transgenic and wt mice with age.

<p>Scatter plots showing the percentage of (<b>A</b>) CD4 and (<b>B</b>) CD8 T cells that are naïve (CD44^lo/CD62L^hi), MP (CD44^hi) or are a subpopulation of MP T cells (CD44^hi/Ly6C^hi) for LN, Spl and BM derived from individual <i>Hoxb4</i> transgenic and wt (n = 6–8) age matched mice. Young mice are between 3–4 months and old mice are all older than 28 months. Naïve and MP populations change significantly with age, but not between <i>Hoxb4</i> and wt mice. *P<0.05, 2-tailed Student ttest. MP = memory phenotype, Wt = wild type, 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

Percentage of T cell populations in hematopoietic organs of young adult mice.

<p>Note that no significant differences were observed between T cell populations of <i>Hoxb4</i> and wild type mice. 1-tailed Student ttest, comparing <i>Hoxb4</i> vs. wild type mice. LN = Lymph node; BM = bone marrow.</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

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