LRF Maintains HSC Homeostasis by Preventing Lymphoid-Primed LT-HSCs From Excessive Differentiation

Abstract 44

Hematopoietic stem cells (HSCs) are the most primitive cells in the hematopoietic system and are under tight regulation for self-renewal and differentiation. Notch signals are essential for the emergence of definitive hematopoiesis in embryos and are critical regulators for lymphoid lineage fate determination. However, their role in adult HSC function is currently under debate.

LRF (Leukemia/ Lymphoma Related Factor, also known as Zbtb7a/pokemon) is a transcriptional factor that plays a key role in lymphoid lineage fate determination, erythroid terminal differentiation and germinal center B cell proliferation. LRF loss at HSC/progenitor levels led to excessive differentiation of HSCs into T cells in the BM at the expense of B cell development. Concomitantly, the numbers of LT-HSCs (CD34⁻CD150⁺CD48⁻Flt3⁻IL7Rα⁻Lin⁻Sca-1⁺c-Kit⁺) were significantly reduced in LRF^flox/floxMx1-Cre+ mice one month after LRF inactivation. Reduced LT-HSC numbers in LRF^flox/floxMx1-Cre+ mice were almost completely rescued by the genetic loss of Notch1 (LRF^flox/floxNotch1^flox/floxMx1-Cre+) or anti-DLL4 antibody treatment, suggesting that the reduction in LT-HSCs numbers was caused by Notch1/DLL4-mediated mechanisms. Furthermore, immunohistochemical (IHC) analysis demonstrated a dramatic increase in DLL4 protein levels in BM hematopoietic cells in LRF^flox/floxMx1-Cre+ mice, although the precise mechanisms for this remain unknown. To determine LRF function in HSCs, highly enriched 50 LT-HSCs were transplanted to lethally-irradiated CD45.1⁺ congenic recipient mice and contributions of donor-derived cells (CD45.2⁺) in recipients' peripheral blood (PB) had been examined over 4 months after transplant. Compared to wild-type (WT) cells, LRF-deficient LT-HSCs barely contributed to lymphoid development (both T and B) in the recipients, while myeloid reconstitutions were largely unaffected.

Using antibodies raised against the ligand binding domains of Notch1 and Notch2, we found that Notch proteins are expressed in a gradient at the most primitive CD34⁻LT-HSCs in adult BM. The CD34⁻LT-HSCs expressing Notch1 at high levels (Notch1^high LT-HSCs) were susceptible to LRF inactivation and disappeared upon LRF inactivation. Only Notch1^low LT-HSCs remained in the BM of LRF^flox/floxMx1-Cre+ mice after pIpC injections. To elucidate the qualitative difference between Notch1^high and Notch1^low LT-HSCs in normal hematopoiesis, we analyzed cell cycle status, gene expression profiles and in vitro colony forming capacities at the single cell level. We found that the Notch1^high LT-HSCs were in more active cell cycle as compared to Notch1^low fractions. Single cell q-PCR analysis demonstrated Notch1^low LT-HSCs express stem cell-related genes (e.g. Gata2, Mpl and Runx1) at higher levels compared to Notch1^high LT-HSCs. Furthermore, Notch1^high LT-HSCs reconstituted hematopoietic system more quickly compared to the Notch1^low cells when transplanted to lethally-irradiated recipient mice. There is no difference in colony forming capacities between two LT-HSC subtypes. Since Notch1^lowLT-HSCs gave rise to Notch1^highLT-HSCs (and vice versa) in recipients' BM, these two LT-HSC fractions are likely to be interchangeable.

Taken together, our data suggest that the LT-HSCs expressing Notch1 at high levels (Notch1^high LT-HSCs) are “lymphoid-primed”, which are susceptible to LRF loss. We propose a model in which LRF acts as a safeguard to prevent lymphoid-primed LT-HSCs from excessive T-cell differentiation in the BM micro-environment. Our study sheds a new light on the regulatory mechanisms regulating the balance between HSC self-renewal and lymphoid differentiation.