A chromosome 16 quantitative trait locus regulates allogeneic bone marrow engraftment in nonmyeloablated mice

Abstract

Identifying genes that regulate bone marrow (BM) engraftment may reveal molecular targets for overcoming engraftment barriers. To achieve this aim, we applied a forward genetic approach in a mouse model of nonmyeloablative BM transplantation. We evaluated engraftment of allogeneic and syngeneic BM in BALB.K and B10.BR recipients. This allowed us to partition engraftment resistance into its intermediate phenotypes, which are firstly the immune-mediated resistance and secondly the nonimmune rejection of donor BM cells. We observed that BALB.K and B10.BR mice differed with regard to each of these resistance mechanisms, thereby providing evidence that both are under genetic control. We then generated a segregating backcross ( IntroductionUnderstanding engraftment barriers is central to successful allogeneic hematopoietic cell transplantation. These barriers are difficult to study because multiple cell types, molecular pathways, and mechanisms act in concert. Both host natural killer (NK) cells and T cells, for example, can mediate resistance to engraftment. 1,2 Elimination of donor hematopoietic cells by these immune cell populations likely requires cytolytic mechanisms, such as granzyme and perforin. However, key cytolytic pathways have not been identified, because neutralization of each singly, 3,4 or even several simultaneously, 5,6 fails to eliminate engraftment barriers. In addition, nonimmune elements related to bone marrow (BM) "space" and hematopoietic stem cell (HSC) niche interactions likely contribute to engraftment barriers 7,8 and further confound efforts to study their underlying biology.Genetic studies of disease and physiologic traits may provide key insights into molecular mechanisms and lead to novel therapy. 9,10 With regard to hematopoietic engraftment barriers, it is established that polymorphisms of genes encoding histocompatibility antigens are causal in activating cellular responses that mediate graft rejection. 11 Other than this understanding, however, little is known about the genetic regulation of engraftment resistance. We addressed this problem by applying a forward genetic approach using a new mouse model of nonmyeloablative BM transplantation. We first characterized donor chimerism, tolerance, and immune resistance mechanisms to show that our model shares all features of nonmyeloablative allogeneic BM engraftment in rodents. We next compared allogeneic and syngeneic BM engraftment to further model, respectively, immune-mediated resistance and nonimmune rejection of hematopoietic cells. From a genetic perspective, these resistance mechanisms can be said to represent the 2 intermediate phenotypes that constitute the overall BM rejection trait. We provide evidence that both are under genetic control by demonstrating strain-specific variation, between BALB.K and B10.BR mice, in these engraftment attributes. We then used a segregating backcross (BC) generated from these 2 strains for genetic linkage analysis and identified a novel quantitative trait locus (QTL) on proximal chromosome 16, ...

Fine mapping of the Bmgr5 quantitative trait locus for allogeneic bone marrow engraftment in mice

“…As shown in Fig. 1, and consistent with prior observations (Cao et al 2009b), parental BALB.K mice uniformly engrafted with AKR/J BM (23/23, 100%) when conditioned with TBI 400 cGy but not with TBI 300 cGy. TBI 400 cGy was not permissive for AKR/J BM engraftment in B10.BR mice (0/14, 0%), however, which required TBI 500 cGy for reliable engraftment.…”

“…The B10.BR line was purchased from the Jackson Laboratory (Stock #004804, Bar Harbor, MN). Donor BM harvesting, transplantation, and evaluation for post-transplant donor blood chimerism was performed as previously described (Cao et al 2009b). …”

“…SNP genotyping was performed by PCR of tail tip DNA for high-resolution melt curve analysis on a LightCycler 480 Instrument (Roche Applied Science, Indianapolis, IN) as previously described (Cao et al 2009b). In 23 of the 43 chromosome 16 SNP markers used in this study, SNP genotyping was also performed by high-resolution melt curve analysis using unlabeled 3′-phosphorylated oligonucleotide probes as described elsewhere (Bramwell et al 2012; Montgomery et al 2007).…”

“…Marker map position has been updated using the Standard Genetic Map for the Laboratory Mouse (Cox et al 2009). An additional 117 SNP and 86 simple sequence length polymorphism (SSLP) markers evenly distributed across the remaining autosomal chromosomes, used before and listed previously (Cao et al 2009a; Cao et al 2009b), were used to select against residual background heterozygosity. Verification of consomic line construction was performed using the high-density 620K Affymetrix Mouse Diversity Genotyping Array (Affymetrix, Santa Clara, CA) via a service provided by the Jackson Laboratory (JAX Mouse Diversity Genotyping Array Service, Bar Harbor, MN).…”

“…We previously identified a BM engraftment quantitative trait locus (QTL), termed Bmgr5 (Cao et al 2009b), in a major histocompatibility complex (MHC)-matched mouse model of allogeneic BM transplantation. In this model, BALB.K mice are susceptible whereas B10.BR mice are resistant to engraftment when conditioned with non-myeloablative total body irradiation (TBI) and transplanted with AKR/J BM.…”