Abstract

In order to understand the mechanisms underlying the increased rates of stem cell transplant rejection in patients with sickle cell disease (SCD) we have developed a mouse model of transplant rejection utilizing the Berkeley SCD mouse. We find that Berkeley SCD mice exhibit a significantly higher rate of transplant rejection when transplanted with fully allogeneic bone marrow from an SJL donor after non-myeloablative conditioning and peri-transplant T cell costimulation blockade. Thus, while control C57BL/6 (BL/6) mice or hemizygous litter-mates exhibit 80–85% engraftment rates, only 20% of Berkeley SCD mice engraft with donor marrow. Transplant rejection in the SCD mice is mediated by CD8+ and/or NK1.1+ cells, as depletion with antibodies directed at either of these cell-surface molecules increases engraftment rates to 75–90%. To dissect the mechanistic underpinnings of these observations, we performed a multiparameter phenotypic and functional flow-cytometric analysis of leukocytes from the peripheral blood, lymph nodes and spleen from SCD mice, and compared them to the two non-SCD controls. We observed that SCD mice displayed striking phenotypic differences compared to normal mice, but that their hemizygous litter-mates were indistinguishable from the BL/6 controls, underscoring the sickle-specific nature of our observations. SCD mice showed minimal phenotypic variation in the peripheral blood, but the spleens and lymph nodes from SCD mice were highly abnormal. Sickle mice exhibited ∼10-fold more splenic NK and NK-T cells than either the BL/6 or hemizygous controls. These cells produced large quantities of both TNF-α and IFN-γ, potentially contributing to the inflammatory milleu that is associated with SCD. Furthermore, while absolute numbers of splenic T cells were not different in sickle mice compared to controls, their phenotype was skewed toward a highly activated state. Thus, while many (40%) splenic memory T cells from normal controls exhibited a Central Memory (Tcm) phenotype, there were fewer Tcm cells in SCD mice (6%), and a skewing of the memory repertoire towards a more activated Intermediate Memory phenotype (71% in SCD mice compared to 25% in controls). Finally, in ELISpot analysis, lymph node cells from naive sickle mice, having never been previously exposed to allo-antigen, were able to secrete IFN-γ when briefly co-cultured with donor SJL stimulators, a phenomenon not observed with either control BL/6 or hemizygous controls, again, pointing to the increased inflammatory milieu and potential for rejection in the sickle mice. In summary, we have observed evidence for a multi-faceted increase in immune activation in SCD mice, which correlates with their increased transplant rejection. These results point both toward possible therapeutic targets aimed at decreasing SCD-mediated transplant rejection and toward future studies in SCD patients, in order to determine whether similar mechanisms of immune activation also occur in the patient population.

Author notes

Disclosure: No relevant conflicts of interest to declare.