Altered Regulation of Immune Responses in Alloimmunized Chronically Transfused Patients with Sickle Cell Disease.

Abstract 637

Transfusion therapy is hampered by the development of alloantibodies to transfused red blood cells (RBCs). A recent mathematical model for RBC alloimmunization predicted that approximately 13% of the general population are antibody producers (responders) (Higgins and Sloan, 2008). However, immune molecular markers which may predict these transfusion responders have not been clearly demonstrated. We recently reported in a mouse model that key immune response regulators, CD4⁺ regulatory T cells (Tregs) have reduced activity in alloantibody responders compared to non-responders (Bao, 2009). (Bao, 2009). Here, we designed a study to determine whether alloimmunized humans have an altered immune response to red cell transfusions, similar to that seen in the mouse model. Peripheral Treg frequency and activity was studied in a cohort of 18 chronically transfused patients with sickle cell disease (SS), receiving either exchange transfusions (n=7) or simple transfusions (n=11). All had been receiving leukoreduced units, matched for Kell and Rh antigens on a roughly 4 weekly intervals for at least 2 years prior to the study. Nine patients were identified as having had a positive history of alloimmunization (responders), 6 on simple transfusions and 3 on exchange transfusions responders. We found no statistically significant differences in the Treg frequencies (Foxp3⁺CD25^hi in the CD4+ population) between alloimmunized and non-alloimmunized SS patients (p>0.1). However, Treg activity as measured by suppression of proliferation of autologous CD4⁺CD25^–cells at 1:4 and 1:16 ratios of Tregs: CD4⁺CD25^–cells was significantly lower in responders compared to non-responders (at 1:4 ratio, 38±5% alloimmunized versus 54±3% non-alloimmunized, p=0.02 and at 1:16 ratio, 10±6% versus 28±4%, p=0.03). To determine if there was a skewing of the T helper (Th) responses as a result of reduced Treg activity in responders, we analyzed the functional phenotype of peripheral CD4⁺ T cells by single-cell measurement of intracellular cytokines using flow cytometry. CD4⁺ T cells were classified as Th1, Th2 or Th17 by assessing their intracellular cytokine profile of IFN- γ, IL-4 and IL-17, respectively. Expression of secreted cytokines was also measured in stimulated cultured supernatants by ELISA. Following stimulation with PMA and ionomycin, we found higher proportion of IL-4 single positive Th2 cells as well as higher levels of secreted IL-4 in sorted populations of CD4⁺CD25⁻T cells from responders compared to non-responders receiving either simple or exchange transfusions (p=0.01), indicating that responder status is skewed towards a Th2 response. Although we did not find statistically significant differences in either secreted IL-17 or IFN- γ or in IL-17- or IFN- γ -expressing Th cells between alloimmunized and non-alloimmunized patients, our transfused SS cohort had significantly higher levels of secreted proinflammatory IL-17 and IFN- γ compared with normal healthy race-matched controls (n=7, p=0.03). These latter data are consistent with an underlying Th cytokine imbalance in chronically transfused SS patients, although it remains to be determined if the perturbations in cytokine balance is the result of chronic transfusions, or specific to SS patients. In summary, our data indicate that in chronically transfused SS patients, responders have compromised Treg activity. This may be responsible for the observed increase in Th2 responses, known to be associated with induction of antibody production. The mechanisms responsible for these differences in responders are under further study. Our findings suggest that Treg associated molecular markers may be used to predict in advance antibody producers to reduce alloimmunization-associated morbidity and mortality.