HAX1 deficiency: Impact on lymphopoiesis and B‐cell development

Abstract

HAX1 was originally described as HS1-associated protein with a suggested function in receptor-mediated apoptotic and proliferative responses of lymphoid cells. Recent publications refer to a complex and multifunctional role of this protein. To investigate the in vivo function of HAX1 (HS1-associated protein X1) in B cells, we generated a Hax1-deficient mouse strain. Targeted deletion of Hax1 resulted in premature death around the age of 12 wk accompanied by a severe reduction of lymphocytes in spleen, thymus and bone marrow. In the bone marrow, all B-cell populations were lost comparably. In the spleen, B220 1 cells were reduced by almost 70%. However, as investigated by adoptive transfer experiments, this impairment is not exclusively B-cell intrinsic and we hypothesize that a HAX1-deficient environment cannot sufficiently provide the essential factors for proper lymphocyte development, trafficking and survival. Hax1 À/À B cells show a significantly reduced expression of CXCR4, which might have an influence on the observed defects in B-cell development.

HAX1 deletion impairs BCR internalization and leads to delayed BCR-mediated apoptosis

“…Hax1 -/-bone marrow cells show a decreased number of B220 1 cells HAX1 deletion dramatically decreased the number of bone marrow cells, 8,29 among which the overall cell type distribution was only marginally affected (i.e., a trend toward a decreased percentage of lymphocytes) ( Figure 1a). However, within the lymphocyte population, we found that the percentage of B220 1 cells decreased significantly (Figure 1b), which suggests impaired lymphopoiesis throughout the development of B220 1 cells.…”

“…28 We generated a mouse model in which we deleted exons 2 and 3 of HAX1 on chromosome 3 and abolished the production of both splice variants; this model was used to demonstrate the crucial function of HAX1 in B cell development and in hematopoietic stem cell homeostasis maintenance. 29 Additionally, as described in detail by Chao et al, 8 HAX1 deletion led to death at the age of 12 weeks; this premature death was caused by a loss of motor coordination, leading to a failure to drink and eat. Here, we aimed to explain the reduced number of B cells in more detail, and we asked whether the general viability of the bone marrow B progenitor cells and of the splenic B cells might be affected by the proposed anti-apoptotic function of HAX1.…”

“…The BALB/c Hax1 -/-mouse strain was generated by PecklSchmid et al 29 Breeding and maintenance were conducted in the animal facility at the University of Salzburg according to the institutional and national guidelines for animal care and use.…”

“…The original human IgE plasmid (dEMPD) was kindly provided by Facundo Batista. 29,31 Antibodies For the in vitro stimulation of splenic B cells and bone marrow cells, we used goat anti-mouse IgM (Southern Biotechnology, 1020-01) antibody. For BCR internalization experiments, we used rat anti-mouse IgM-FITC (BD Pharmingen, R6-60-2) and goat anti-mouse IgM-pHrodo (Southern Biotechnology, 1020-01) antibodies.…”

“…Complete HAX1 cloned into the pHIS-parallel 2 vector 29,30 was used for the production of recombinant HAX1 protein. Protein expression was induced in the E.coli strain BL21 at a density of OD 600 5 0.8 by adding 0.75 M IPTG (stock: 1 M) and incubating at 26uC overnight.…”