Hemolysis inhibits humoral B-cell responses and modulates alloimmunization risk in patients with sickle cell disease

Abstract

Red blood cell alloimmunization remains a barrier for safe and effective transfusions in sickle cell disease (SCD), but the associated risk factors remain largely unknown. Intravascular hemolysis, a hallmark of SCD, results in the release of heme with potent immunomodulatory activity, although its effect on SCD humoral response, specifically alloimmunization, remains unclear. Here, we found that cell-free heme suppresses human B cell plasmablast/plasma cell differentiation by inhibiting the DOCK8/STAT3 signaling pathway, which is critical for B cell activation, as well as by upregulating heme oxygenase 1 (HO-1) through its enzymatic byproducts, carbon monoxide and biliverdin. Whereas non-alloimmunized SCD B cells were inhibited by exogenous heme, B cells from the alloimmunized group were non-responsive to heme inhibition and readily differentiated into plasma cells. Consistent with a differential B cell response to hemolysis, we found elevated B cell basal levels of DOCK8 and higher HO-1-mediated inhibition of activated B cells in non-alloimmunized compared to alloimmunized SCD patients. To overcome the alloimmunized B cell heme insensitivity, we screened several heme-binding molecules and identified quinine as a potent inhibitor of B cell activity, reversing the resistance to heme suppression in alloimmunized patients. B cell inhibition by quinine only occurred in the presence of heme and through HO-1 induction. Altogether, these data suggest that hemolysis can dampen the humoral B cell response and that B cell heme responsiveness maybe a determinant of alloimmunization risk in SCD. Quinine by restoring B cell heme sensitivity may have therapeutic potential to prevent and inhibit alloimmunization in SCD patients.

Impact of Hemin on Interleukin-21 Levels and Plasma Cells in Transfusion-Dependent Thalassemia with Positive and Negative Allo-Autoantibody

“…This indicates no relationship between plasma heme levels and the status of SCD patients with or without alloimmunization either before or after transfusion. 28 This further suggests that a transfusion carried out only once does not significantly change the heme level; whether repeated transfusions at a high frequency would affect the heme level and thereby affect the patient’s alloimmunization status is not certain.…”

“… 27 Heme inhibits the differentiation of B-cells into plasma cells by modulating the enzymatic activity of HO −1 in SCD patients. 28 It is not known whether heme affects IL-21 levels in thalassemia patients as in SCD patients, indicating that IL-21 plays a role in the differentiation of B-cells into plasma cells. Hemin was used for experimental studies as a heme preparation that is soluble in dimethyl sulfoxide and then diluted in a culture medium with a physiologically relevant pH.…”

Red cell transfusion and alloimmunization in sickle cell disease

“… 71 Furthermore, regulatory B cells from alloimmunized patients with SCD have a decreased ability to suppress monocyte activation. 72 Pal et al 73 demonstrated that hemolysis and cell-free heme typically suppress B cells and plasma cell differentiation, but alloimmunized patients with SCD had altered B-cell inhibition. Further mechanistic studies are required to elucidate the complex immunological pathways contributing to alloimmunization in SCD and to determine whether targeted reversal of immune dysregulation can reduce antibody formation.…”

Measurement of post‐transfusion red blood cell survival kinetics in sickle cell disease and β‐Thalassemia: A biotin label approach

“…The chronic inflammatory state associated with SCD and β-thalassemia, and the high incidence of alloimmunization, may result in reduced RCS of transfused RBCs. [5][6][7][8][9][10][11] Conversely, it may be hypothesized that RCS is prolonged in these diseases, due to the decreased RBC destruction capacity caused by functional asplenia or splenectomy, although this has previously not been shown in other disease states. 12,13 RCS studies in healthy volunteers have demonstrated that RCS kinetics are linear, with a total lifespan of transfused allogeneic RBCs up to 115 days.…”

“…It is likely that the RCS kinetics of transfused RBCs is different for SCD and β‐thalassemia compared to the RCS in healthy individuals. The chronic inflammatory state associated with SCD and β‐thalassemia, and the high incidence of alloimmunization, may result in reduced RCS of transfused RBCs 5–11 . Conversely, it may be hypothesized that RCS is prolonged in these diseases, due to the decreased RBC destruction capacity caused by functional asplenia or splenectomy, although this has previously not been shown in other disease states 12,13 .…”