Altered Heme-Mediated Modulation of Dendritic Cell Function in Sickle Cell Alloimmunization

Red blood cell alloimmunization can be a life-threatening complication for patients with sickle cell disease (SCD) receiving therapeutic transfusions. However, it remains unknown why only some (20-60%) SCD patients develop alloantibodies whereas others do not. Because of ongoing hemolysis in SCD, we have investigated the effects of toxic hemin on T cell responses of patients with SCD on chronic transfusion therapy. We found impaired monocyte control of proinflammatory T cells in response to hemin in alloimmunized SCD patients, partly due to defective levels of monocyte heme-oxygenase-1 (HO-1), an anti-inflammatory heme degrading enzyme that catabolizes heme into iron, biliverdin and carbon monoxide (CO). Monocytes are precursors of dendritic cells (DCs), which represent the antigen presenting cells most able to initiate and regulate immune responses. However, little is known about the functional properties of DCs in SCD patients. We therefore hypothesized that DCs of alloimmunized SCD patients would also have impaired response to hemin. Monocyte-derived DCs (moDCs) from healthy donor controls (n=11) and a cohort of SCD patients (aged 15-30 on chronic transfusion therapy every 3-4 weeks for at least two years using C,E,K phenotyped-matched, leukodepleted units) grouped either as "non-alloimmunized" (no history of antibody production, n=6), versus "alloimmunized" (with a history of having produced at least one alloantibody, n=7) were matured in the absence or presence of hemin (5uM and 20uM) with TLR agonists: LPS/IFNg (TLR4 agonist+STAT1 activation) or R848 (TLR7/8 agonist). Hemin-exposed mature and immature (no TLR agonist) moDCs were then assayed for HO-1 expression, cytokine production, co-stimulation, and T cell priming. Interestingly, upon hemin exposure, immature moDCs from healthy donors and non-alloimmunized patients upregulated more HO-1 (1056±206 fold; mean fluorescent intensity (MFI): 12283±1818 at 20uM hemin) than alloimmunized patients (fold increase 494±49; MFI: 7422±959, p + T cells (ie. priming), we performed mixed lymphocyte reactions: whereas hemin inhibited (roughly 30%) moDC-mediated priming of pro-inflammatory IFNg secreting T H 1 cells in healthy donors and non-alloimmunized patients (%CD4 + IFNg + without hemin 58%±9% with 20uM hemin 41%±8%, p=0.005), T H 1 cell responses were not suppressed in the alloimmunized group (without hemin: 52%±10% with 20uM hemin: 54%±10%, p=0.4). In summary, hemin-exposed moDCs from healthy donors and non-alloimmunized SCD patients suppress CD83 upregulation and inhibit ensuing T H 1 cell responses, possibly through HO-1 induction and subsequent production of CO. In contrast, CD83 upregulation and proinflammatory T cell priming are not inhibited by hemin in alloimmunized patients, probably representing a mechanism by which heme-associated inflammation is maintained in alloimmunized SCD, thereby increasing their risk of alloimmunization. Deciphering this mechanism not only offers a potential biomarker for alloimmunization, but also opens the way to designing innovative treatments for these patients. Disclosures No relevant conflicts of interest to declare.