Hematopietic Stem Cells with Defects in the NF-κB Alternative Pathway Show a Deficient Repopulation Capacity

Abstract 1269

Hematopoietic stem cells (HSCs) maintain the production of all blood cells through the lifespan of an organism, and regenerate the whole hematopoietic system after stressful episodes such as high dose chemotherapy or upon transplantation. The functions of HSCs in these 2 situations, steady-state and under stress, are controlled by a variety of molecules, which may provide different contribution to each process. We investigated whether the NF-kB alternative pathway might have a role in HSCs functions, using mice deficient for two components of this pathway: NF-kB-inducing kinase (NIK) or p52. The activation of NIK is generally known as the alternative (or non-canonical) NF-kB pathway, and drives the post-translational processing of p100 to mature p52, which results in the translocation to the nucleus of p52-containing complexes such as p52/RelB. Apart from the already reported defects in B-cell maturation, both NIK- and p52-deficient mice did not present major disturbances in blood cells numbers. The absolute numbers of marrow cells were not different among the knocked-out and the wild-type mice. We first studied the compartment of marrow cells known to be enriched for HSCs, either lineage-depleted Sca1-positive ckit-positive cells (LSK), or CD150 positive CD48 negative cells. The proportions of marrow cells with the immunophenotype of HSCs in either NIK-deficient or p52-deficient mice were similar to those in control mice. The amount of clonogeneic progenitor cells in the marrow was assessed in standard CFU-GM cultures, and gave no differences in output in any of the mice studied. We set up in vitro liquid cultures with murine stem cell factor and human interleukin-11, and determined the cellular production weekly. Cultures started with NIK-deficient marrow cells produced significantly less numbers of cells and CFU-GM, compared with those started with wild type marrow. This deficit in hematopoietic capacity was further confirmed in a more stringent assay of HSC function, the in vivo competitive repopulation assay. Equal numbers of lineage-depleted (Lin-) CD45.2 marrow cells of either NIK-deficient, p52-deficient or wild-type mice, were mixed with Lin- CD45.1 marrow cells of syngeneic mice, and transplanted into lethally irradiated CD45.1 recipients. Four months after transplant, the chimeric status and the hematopoietic lineage repopulation of CD45.2 cells was assessed in peripheral blood (PB). NIK- or p52-deficient HSCs repopulated the B-, T- and myeloid-lineages but at significantly lower levels when compared to wild type HSCs. Total donor CD45.2 cells and total CD45.2 LSK cells were also significantly lower in the marrows of mice transplanted with NIK- or p52-deficient HSCs versus those of controls. We used the marrows of the repopulated mice for secondary transplants, and confirmed the defect in the repopulating capacity of NIK- and p52-deficient HSCs. Our results suggest that the NF-kB alternative pathway plays a role in the function of HSCs, and this role may be important under stress conditions.