Inhibitory Receptor, gp49B1, Is Co-Expressed with c-Kit and Regulates Hematopoiesis during Development

During development, hematopoietic stem cells (HSCs) undergo dramatic expansion in fetal liver, and then migrate to spleen and bone marrow afterwards. Mouse HSCs are enriched in lineage^- Sca-1⁺ c-Kit⁺ (LSK) cells and defined by their ability to reconstitute the hematopoietic system of lethally irradiated recipients. Although c-Kit is required for HSCs function, heterogeneous c-Kit expression represents functionally distinct subsets of HSCs and progenitor cells. Recently, we demonstrated the one of inhibitory leukocyte immunoglobulin-like receptors, LILRB2, and its mouse homolog, PIRB, are expressed in HSCs. Besides PIRB, the gp49B1 is the only other member of mouse LILRB family. The function of gp49B1 in hematopoiesis is not known.

Here we demonstrated that gp49B1 is not expressed by LSK cells of adult mice but is expressed on neonatal bone marrow and spleen LSK cells. Two distinct populations of neonatal LSK cells can be identified based on c-Kit expression. In neonatal bone marrow, 96% of c-Kit^hi LSK cells are gp49B1⁺ whereas only 3% of c-Kit^lo LSK cells express gp49B1. Similarly, 99% of c-Kit^hi but only 9% of c-Kit^lo LSK cells are gp49B1⁺ in neonatal spleen. The gp49B1⁺ LSK cells showed 4.2-folds higher expression level of c-Kit than that of the gp49B1^- LSK cells. Because c-Kit is required for hematopoietic progenitor or HSC (HSPC) function, we sought to test whether gp49B1 has regulatory effects on HSC activity. Neonatal splenic gp49B1^- LSK cells produced 26-folds more colonies than gp49⁺ LSK cells after 7 day in methylcellulose media. To compare their reconstitution abilities, we injected 1,000 sorted neonatal splenic gp49B1⁺ or gp49B1^- LSK cells into lethally irradiated 8 weeks-old C57BL6 mice. All mice transplanted by gp49B1⁺ LSK cells died within 2 weeks post-transplantation, whereas all gp49B1^- LSK cells transplanted survived. These results suggest gp49B1^- LSK cells, which have less c-Kit expression, are enriched for HSC activity. To further confirm it, 500 sorted gp49B1⁺ or gp49B1^- LSK cells (CD45.2⁺) were transplanted with 100,000 competitor bone marrow cells (CD45.1⁺) into lethally irradiated congenic recipients (CD45.1⁺). Mice transplanted with gp49B1^- LSK cells exhibited increasing peripheral blood donor CD45 chimerism levels from 3 to 18 weeks after transplant (14.7%~68.2%); but gp49B1⁺ LSK cells transplanted mice only have modest chimerism levels (<2%; exception of 1 out of 7 mice has 13% at 18 weeks). Interestingly, neonatal splenic gp49B1⁺ LSK cells exhibited a lineage bias compared to gp49B1^- LSK cells after transplantation (B cell: 2% vs. 30.1%, p<0.01; T cell: 61.3% vs. 17.4%, p<0.05; and Myeloid cell: 42.8% vs. 60.2%, p=0.32). Consistently, c-Kit^hi LSK cells of which over 96% are gp49B1⁺ showed much less HSPC activities comparing with c-Kit^lo LSK cells in colony formation (40-folds less), non-competitive transplantation (all died in 2 weeks vs. all survived after transplantation), and competitive transplantation (donor CD45 chimerism: 0.04~0.34% vs. 5.8~33.6%, from 6 to 20 weeks).

We continued to study the function of gp49B1's function using gp49b1 deficient mice. We found that c-Kit^hi LSK cells were increased in gp49B1-deficient mouse (0.18% vs. 0.14%), whereas KO c-Kit^hi LSK% decreased (0.09% vs. 0.14%). The c-Kit^hi LSK cells of gp49B1-deficient mouse also exhibited low repopulation potential. While the same number of WT and KO LSK cells had comparable repopulation abilities, five hundred gp49b1-deficient c-Kit^lo LSK cells exhibited a greater reconstitution capacity (65.3% vs. 33.6%) than wild-type c-Kit^lo LSK cells. These results suggest that gp49B1 may regulate the repopulation of primitive neonatal hematopoietic cells.

Together, our results demonstrate that the gp49B1 is co-expressed with high level of c-Kit in hematopoietic progenitor cells of neonatal mouse, and it regulates maturation, repopulation, and differentiation of hematopoietic cells during development.