Lack of Immune Barrier to Allogeneic Hematopoietic Stem Cell Engraftment in T, B, and NK Cell Deficient (Rag2γ c^{−/ −}) Mice.

The way that allogeneic hematopoietic stem cells (HSCs) resist engraftment is not completely understood. Both natural killer cells (NK) and lymphocytes are thought to mediate the allograft barrier in donor/recipient pairs that are mismatched at the major histocompatibility complex (MHC). In addition, the clearing of niche “space” is thought to be required for donor cell engraftment. Here, we attempted to dissect the relative contribution of these host elements to hematopoietic resistance by using genetically defective mice as recipients of purified allogeneic HSCs. In prior studies we showed in different donor/recipient strain combinations that HSCs encounter greater resistance to engraftment as compared to unfractionated bone marrow, and that the relative resistance can be quantitated by titrating the numbers of HSCs needed to rescue lethally irradiated mice. Rescue of syngeneic or CD45 congenic recipients requires only ~200 HSCs, whereas higher HSC doses are required as the genetic disparity increases between donor/recipient pairs. In the present study, a highly resistant MHC-mismatched strain combination of AKR/J (H2^k) into C57BL/6 (H2^b) was selected. All lethally irradiated (950cGy) wild type B6 mice die of hematopoietic failure despite attempted rescue with 1000 AKR/J HSC. To test the contribution of host lymphocyte subsets on resistance B6.Rag2^{−/ −} (H2^b) mice lacking T and B cells, or B6.Rag2γ c^{−/ −} (H2^b) mice lacking T, B, and NK cells were used as recipients of titrated numbers (300, 1000, 3000, or 6000) of AKR/J HSC. No significant improvement in engraftment was observed in B6.Rag2^{−/ −} recipients as compared to B6.WT mice. However, an impressive difference was noted in the B6.Rag2γ c^{−/ −} mice, in which the immune barrier completely disappeared. A dose of 300 HSC rescued all irradiated B6.Rag2γ c^{−/ −} mice, and even 200 AKR HSC - an amount equivalent to the congenic dose-- rescued 100% of recipients. Chimerism analysis of all recipients at >1 year post-transplantation revealed 100% durable donor chimerism in all white cell lineages, including granulocytes. We then sought to determine if engraftment could be achieved in B6.Rag2γ c ^{−/ −} mice using non-myeloablative conditioning, or no radiation at all. B6.Rag2γ c ^{−/ −} recipients of 1000 AKR/J HSCs treated with 500 cGy, 400 cGy resulted in 100% donor engraftment, and 300cGy resulted in 65–70% donor engraftment. Furthermore, recipients that received no conditioning also engrafted since 10–20% of donor AKR/J granulocytes were detected. In contrast, B6.Rag2^{−/ −} mice that received the same radiation doses showed no evidence of engraftment. We studied by ex vivo bioluminescence imaging the trafficking of allogeneic FVB (H-2^q) HSC in irradiated versus unirradiated BALB/c.Rag2γ c ^{−/ −} recipients by using as donors FVB transgenic mice that express luciferase on the b-actin promoter. HSC were observed to enter the marrow space of irradiated mice within minutes following HSC infusion, whereas unirradiated mice demonstrated no luciferase signal until day +5 post-infusion. We conclude that Rag2γ c^{−/ −} mice have a profound reduction in the immune barrier to allogeneic HSC engraftment and in irradiated mice, HSC rapidly enter the marrow. In contrast, in unirradiated Rag2γ c^{−/ −} mice such trafficking is likely hindered by the endogenous HSC that occupy the niche space. We are currently comparing competitive engraftment of allogeneic versus congenic HSC in order to determine the role of MHC-matching between HSC and stromal elements, and to further understand the concept of niche space.