Evaluation of a Conditioning Regimen of Post-Transplant Cyclophosphamide and Sirolimus with or without CTLA4-Ig in a Mismatched Murine Model

Abstract

Introduction Hematopoietic stem cell transplant (HSCT) is the only curative option available for patients living with sickle cell disease (SCD). Stable mixed chimerism is sufficient to reverse the sickle phenotype. The current conditioning regimen of post-transplant cyclophosphamide (PT-Cy) and sirolimus (Sir) has proven to synergistically induce stable mixed chimerism. 1 The mechanism of tolerance is not fully understood. Previous studies demonstrated that a combination of Sir and abatacept (CTLA4-Ig) promotes tolerance and prolongs allograft survival. 2 3 Regulatory cells and cytokines, such as regulatory T cells and IL-10, respectively, have shown to play an important role in tolerance induction. Objective To improve the conditioning regimen and expound upon the mechanism of tolerance within our non-myeloablative, haploidentical HSCT regimen. Methods Female Balb/c donors and male C57BL/6 recipients were used in a mismatched murine model. Recipient mice were conditioned with 200 cGy total body irradiation and PT-Cy and Sir with or without CTLA4-Ig. Recipient and donor mice deficient in IL-10 were used to examine the role of IL-10 in transplant tolerance. Donor chimerism levels of recipients which were wild type (WT) or IL-10 deficient were compared after being transplanted with either WT or IL-10 deficient donor bone marrow cells. Results Mice conditioned with PT-Cy and Sir with or without CTLA4-Ig did not show a significant difference in donor chimerism levels at all PT time points tested through 16 weeks (p>0.05, Figure 1). Further, subset populations consisting of CD4 and CD8 lymphocytes, CD19 B cells, and CD11b macrophages resulted in no significant difference between groups. Recipient mice which were deficient in IL-10 and transplanted with bone marrow cells from IL-10 deficient donors engrafted. In addition, donor chimerism levels measured in IL-10 deficient recipients were significantly lower in comparison to WT recipients at 4 weeks PT (9.17±3.4% vs. 36.42±7.2%, p 0.05). This suggests that IL-10 plays a role during the early stage of tolerance induction. Conclusion A conditioning regimen of PT-Cy and Sir with or without CTLA4-Ig yields similar chimeric results. While IL-10 may act as a tolerogenic cytokine during early engraftment, our findings also suggest there are other compensatory mechanisms that induce tolerance and therefore maintain engraftment.