IL-10 Is a Novel Ligand for Hematopoietic Stem Cell Self-Renewal.

IL-10 has been known to play a major role in modulating inflammatory and immune response. However, the anemia sign in targeted disruption of IL-10 (IL-10KO) and supportive effects of IL-10 during in-vitro culture of hematopoietic progenitor cells implicated potential role of IL-10 for hematopoietic function.

To delineate the physiological significance of IL-10 for normal hematopoiesis, first, we undertook study to examine the various hematopoietic compartments in IL-10 KO mice in comparison to wild type (WT) counterparts.

In the phenotypic analysis, the bone marrow cells (BMC) of IL-10 KO mice showed modest hypocellularity including TER119+ cells, but no difference were observed in the assay for in-vitro colony forming cells (CFC) (3 Exp.).

In contrast, the number of more primitive hematopoietic cells as defined by long term culture (LTC-IC) was 2-fold lower in the BMCs of IL-10 KO mice compared to WT, and further decrease (7-fold) were observed in those IL-10 KO mice that had enterocolitis (2 Exp). Similarly, when equivalent numbers of BMCs from IL-10 KO or WT were transplanted into irradiated congeneic recipient (Pep3b-Ly5.1), IL-10 KO BMC showed 2-fold lower level of engraftment (88% vs 44%) than WT over the span of post-transplantation 3 to 12 weeks (2 exp, n=7 ea,). In the limiting dilution analysis to measure the HSC contents, the frequency of competitive repopulating unit (CRU) in the IL-10 KO BMC was also two fold lower than WT (1/2164 vs. 1/5931, respectively) with further decreasse in CRU frequency being observed for those from IL-10 KO with enterocolitis (< 1/17380), suggesting that IL-10 KO mice has decreased content of hematopoietic stem cell (HSC).

To exclude possible indirect biological effects in IL-10 KO mice, and see if IL-10 have direct effect on quantity of HSCs, normal 5-FU prestimulated BMCs were cultured for 5 days on stromal cells that had been retrovirally transduced with IL-10 or control vector (MIG) along with TPO, SCF and FL. When the cells were transplanted into the recipients, cells cultured in the IL-10 secreting stroma exhibited significantly higher level engraftment compared to those cultured in the control stroma (23% vs 54% for PB, 16% vs 49% for BM engraftment, respectively, n=4). Secondary transplantation of these primary recipients at post-transplantation 9 month showed that the BM cells grown in IL-10 secreting stroma had 663 donor-derived CRUs, while those transplanted with control group had 215 donor-derived CRUs, a 3-fold higher CRU contents in the presence of IL-10. In further studies to see if IL-10 could be a direct ligand for primitive HSCs, first, expression of IL-10 receptor on those cells were confirmed by RT-PCR. Next, purified HSCs (c-kit+Sca-1+Lin-) without accessory cells were cultured in the stroma-free condition with or without exogeneous addition of IL-10 for 5 days, and transplanted into recipients in limiting dilution. The result showed that CRU frequency of cells cultured with IL-10 was 3-fold higher than those cultured in the absence of IL-10 (1/525 vs. 1/1465), thus suggesting that IL-10 is a direct ligand for HSC self-renewal. These results may implicate significance of IL-10 for pathogenesis and treatment of autoimmune diseases by stem cell transplantation as well as for improved ex-vivo expansion of HSCs.