AbstractAbstract 4150 BackgroundHematopoietic stem cell transplant (HSCT) is the only known curative option for patients with severe congenital anemias including Sickle Cell Disease (SCD). Due to the higher risks of transplant-related mortality associated with myeloablative conditioning and the reversal of the SCD phenotype achieved with relatively low levels (as low as 11%) of stable donor chimerism, we have developed a non-myeloablative regimen for patients receiving matched sibling donor HSCT consisting of pre-transplant alemtuzumab, 300cGy total body irradiation, and post-transplant immunosuppression with sirolimus (rapamycin). This regimen has been successful in 23 of 26 patients to date. However, the relevant mechanisms that elicit and maintain immune tolerance in these patients remain unclear. We hypothesized that lymphocytes emerging early post-transplant would display a toleragenic or regulatory phenotype, characterized by the presence of T-regulatory cells (T-regs) and the absence of pro-inflammatory cytokines. MethodsFrozen mononuclear cell fractions were obtained from time points before or near day 60 or day 100 post-transplant from 11 patients enrolled on our transplant protocol, including one patient who experienced apparent graft rejection. 17-parameter flow cytometric analysis was performed after staining for various known cell surface determinants to characterize lymphocyte subsets and excluding non-viable cells. Using multiplex cytokine immunoassay, 67 analytes were measured on plasma obtained from these and additional patients, including two cases of graft rejection, to identify cytokines and chemokines present across a range of time points before and near day 100 post-transplant. ResultsFlow cytometric analyses reveal dramatically high proportion of T-regs (6.3%-32.8%) and T-helper-17 (Th17) (8.2–21.2%) lymphocytes present between 60 and 100 days post transplant. Not surprisingly, surface expression of the activation- and proliferation-associated markers CD38, CD39 and CD103 on CD4+ and CD8+ lymphocytes was highly elevated. Interestingly, the effector memory-associated Ig surface glycoprotein CD146 was highly expressed in engrafting patients despite the normal proportion of lymphocytes identified as effector memory cells (≥5%) based on CCR7 expression. While HLA-DR expression was high in most patients due to their active hematopoetic reconstitution, T lymphocytes in the rejecting patient, irrespective of subset, exhibit the lowest expression of HLA-DR compared to samples from similar time points in eventually-successful engrafters. Despite the limited time post-lymphodepletion, the majority of B cells present an IgM+IgD+ phenotype (>80% of all identified B cells), almost all of which are CD27− (naïve B cells). In the rejecting patient, this pattern is not observed and instead the major B cell population is IgG+IgD− (90%) and CD27+ (71% of IgD−), more similar to a memory phenotype.Luminex analysis at these time points reveals high expression of IL-17A in the successfully engrafting patients. Significant differences in the T-reg-associated cytokines IL-10 and IL-6 were not observed. Interestingly, expression of both T-helper-1-associated and T-helper-2-associated cytokines in patients who ultimately rejected their grafts were among the lowest of all samples in our analyses. Notably, rejecting patients exclusively expressed very high levels of the macrophage and T cell infiltration-associated cytokine CCL2 (MCP-1) in plasma around day 60 post-transplant. ConclusionAlthough T-regs and Th17 cells comprise a very high proportion of emerging T lymphocytes early post-transplant, the expression patterns of cellular activation markers, B cell markers, and cytokines may not be reflective of a classical toleragenic immune phenotype. These dichotomous observations have provided new insight into the kinetics of lymphoid reconstitution following nonmyeloablative conditioning and sirolimus-based immunosupporession and suggest targets for further investigation into both the long-term, stable mixed chimerism and the lack of GvHD observed in our patients. As additional transplants are performed in our SCD cohort, we also hope that this early time point analysis may assist in predicting outcomes before stable chimerism can be observed clinically. Disclosures:No relevant conflicts of interest to declare.