64±10.87×106 and WT: 31.54±15.52×106 for B220+; Hax1−/−: 3.71±0.77×106 and WT: 2.55±1.05×106 for T1; Hax1−/−: 6.91±3.61×106 and WT: 4.73±2.23×106 for T2; Hax1−/−: 5.89±2.89×106 and WT: 4.53±2.39×106 for mature B cells; Hax1−/−: 2.92±1.84×106 and WT: 2.34±1.16×106 for MZ B cells). Our data clearly demonstrate that Hax1−/− LSK cells in a Hax1+/+ environment were able to fully reconstitute the lethally irradiated hosts. To further investigate the reason for the massive B lymphocyte deficiency, we investigated Protein Tyrosine Kinase inhibitor the expression of CXCR4 and BAFFR on splenic B cells. CXCR4 is expressed on hematopoietic precursors 22 as well as on centroblasts within the germinal centre

18. CXCR4-expressing cells migrate towards CXCL12, expressed by stromal cells and germinal center dark zone compartments. Thus, an impaired CXCR4 expression would severely impede normal B-cell development. Alternatively, signals through the BAFFR have a significant role in promoting B-cell survival and homeostatic proliferation 23. For real time analysis, we isolated total splenocytes of four 10-wk-old WT and Hax1−/− mice and enriched for B lymphocytes using magnetic cell sorting. Both the CXCR4 and the BAFFR Selleckchem Afatinib amplification showed prominent amplification products. Most interestingly, CXCR4 expression

in HAX1-deficient B cells was decreased by around 70% compared to WT cells. BAFFR expression was slightly, but not significantly, decreased in HAX1-deficient B cells (Fig. 7A). However, the decreased expression had no effect on the formation of follicular structures. No differences in the distribution of B- Adenosine and T-cell areas, as stained by CD3 and B220, were detectable (Fig. 7B). Because of the fact that the transfer of Hax1−/− bone marrow cells into a HAX1+ environment gave rise to normal levels of B220+ cells and functional B-cell subsets, we conclude that the severely decreased

CXCR4 expression on HAX1-deficient B cells is not solely responsible for the described B-cell loss in Hax−/− mice. Previously, we described HAX1 as interaction partner of membrane bound IgE (mIgE) 24. From that point of view, it would have been of most interest to analyse IgE responses on a Hax1-deficient background. However, the short lifespan of Hax1−/− mice impeded a direct analysis. Therefore, we focused on the detailed investigation of the biological function of HAX1 during lymphocyte development. Hax1−/− mice are characterized by a severely diminished cellularity of lymphoid tissues accompanied by a significant reduction of B and Tlymphocytes. Recently, Chao et al. 25 reported on the role of HAX1 with a similar approach. Our results demonstrate that the developmental impairment is not restricted to specific developmental stages. We observed reduced numbers of B cells from the pro-pre B-cell stage in the bone marrow to mature stages in the spleen. The analysis of splenic subpopulations clearly demonstrated a continuation of the developmental defects for T1 and T2 B cells 26, 27.