Karina Yazdanbakhsh

Red blood cell alloimmunization in sickle cell disease: pathophysiology, risk factors, and transfusion management.

Red blood cell transfusions have reduced morbidity and mortality for patients with sickle cell disease. Transfusions can lead to erythrocyte alloimmunization, however, with serious complications for the patient including life-threatening delayed hemolytic transfusion reactions and difficulty in finding compatible units, which can cause transfusion delays. In this review, we discuss the risk factors associated with alloimmunization with emphasis on possible mechanisms that can trigger delayed hemolytic transfusion reactions in sickle cell disease, and we describe the challenges in transfusion management of these patients, including opportunities and emerging approaches for minimizing this life-threatening complication.

Defective circulating CD25 regulatory T cells in patients with chronic immune thrombocytopenic purpura.

Immune thrombocytopenic purpura (ITP) is characterized by the presence of antiplatelet autoantibodies as a result of loss of tolerance. CD4+CD25+ regulatory T cells (Tregs) are important for maintenance of peripheral tolerance. Decreased levels of peripheral Tregs in patients with ITP have been reported. To test whether inefficient production or reduced immunosuppressive activity of Tregs contributes to loss of tolerance in patients with chronic ITP, we investigated the frequency and function of their circulating CD4+CD25(hi) Tregs. We found a com-parable frequency of circulating CD4+CD25(hi)Foxp3+ Tregs in patients and controls (n = 16, P > .05). However, sorted CD4+CD25(hi) cells from patients with chronic ITP (n = 13) had a 2-fold reduction of in vitro immunosuppressive activity compared with controls (n = 10, P < .05). The impaired suppression was specific to Tregs as shown by cross-mixing experiments with T cells from controls. These data suggest that functional defects in Tregs contribute to breakdown of self-tolerance in patients with chronic ITP.

Improved regulatory T cell activity in patients with chronic immune thrombocytopenia treated with thrombopoietic agents

Immune thrombocytopenia (ITP) is an autoantibody-mediated bleeding disorder with both accelerated platelet destruction and impaired platelet production. We and others have described impaired regulatory CD4(+)CD25(hi) T cells (Treg) numbers and/or suppressive function in ITP patients. Clinical trials using thrombopoietic agents to stimulate platelet production have shown favorable outcomes in ITP patients, but information on the immunologic responses of treated patients are lacking. We studied the immunologic profile of chronic ITP patients before (n = 10) and during treatment with thrombopoietin receptor (TPO-R) agonists (n = 9). Treg activity, as measured by suppression of proliferation of autologous CD4(+) CD25(-) cells, was improved in patients on treatment (P < .05), and the improvement correlated with reduction in interleukin-2-producing CD4(+) cells, consistent with dampening of immune responses. There was a concomitant increase in total circulating transforming growth factor-β1 (TGF-β1) levels (P = .002) in patients on treatment, and the levels of TGF-β1 correlated with the degree of improvement in platelet counts (r = .8, P = .0002). This suggests that platelets in patients on TPO-R treatment may play a role in improving Treg function, either directly or indirectly by enhanced release of TGF-β1 as a result of greater platelet turnover. In conclusion, our findings suggest that thrombopoietic agents in patients with ITP have profound effects to restore immune tolerance.

Defective regulatory B-cell compartment in patients with immune thrombocytopenia

B lymphocytes producing antiplatelet autoantibodies play a major role in autoimmune thrombocytopenia (ITP). However, certain B cells, including the human CD19(+)CD24(hi)CD38(hi) subpopulation, possess regulatory functions mediated partly by IL-10. In a cohort of chronic ITP patients with low platelet counts who consisted of patients off treatment, we found a lower frequency of CD19(+)CD24(hi)CD38(hi) in the peripheral compartment of nonsplenectomized patients (P = .03). IL-10 expression after activation was decreased in all ITP circulating CD19(+) subpopulations (P < .03), and inhibition of monocyte TNF-α expression by activated B cells was reduced in patients with platelet numbers of < 50 × 10(9) cells/L (P = .001), indicating that regulatory B cells of patients with ITP are functionally impaired in their ability to dampen monocyte activation. Interestingly, in nonsplenectomized patients whose platelet counts were elevated after treatment with thrombopoietic agents, the frequency of CD19(+)CD24(hi)CD38(hi) B cells was increased compared with those before treatment (P = .02). Altogether, these data indicate a compromised regulatory B-cell compartment as an additional defect in immune regulation in patients with chronic ITP that may be restored in responders to thrombopoietic treatment.

Molecular mechanisms that lead to reduced expression of Duffy antigens.

BACKGROUND: In the Duffy blood group system, the null phenotype Fy(a–b–) has been classically associated with a mutated GATA box, while the Fyx phenotype weak Fyb is associated with Arg89Cys and Ala100Thr mutations. This report assesses the prevalence of the Duffy GATA box and the Fyx‐associated mutations in white and African American (black) donors and investigates the molecular mechanism underlying the Fyx phenotype.

Immune regulation in chronically transfused allo-antibody responder and nonresponder patients with sickle cell disease and β-thalassemia major.

Red blood cell alloimmunization is a major complication of transfusion therapy. Host immune markers that can predict antibody responders remain poorly described. As regulatory T cells (Tregs) play a role in alloimmunization in mouse models, we analyzed the Treg compartment of a cohort of chronically transfused patients with sickle cell disease (SCD, n = 22) and β‐thalassemia major (n = 8) with and without alloantibodies. We found reduced Treg activity in alloantibody responders compared with nonresponders as seen in mice. Higher circulating anti‐inflammatory IL‐10 levels and lower IFN‐γ levels were detected in non‐alloimmunized SCD patients. Stimulated sorted CD4+ cells from half of the alloimmunized patients had increased frequency of IL‐4 expression compared with nonresponders, indicating a skewed T helper (Th) 2 humoral immune response in a subgroup of antibody responders. All patients had increased Th17 responses, suggesting an underlying inflammatory state. Although small, our study indicates an altered immunoregulatory state in alloantibody responders which may help future identification of potential molecular risk factors for alloimmunization. Am. J. Hematol. 2011.

CD16+ monocytes control T-cell subset development in immune thrombocytopenia.

Immune thrombocytopenia (ITP) results from decreased platelet production and accelerated platelet destruction. Impaired CD4(+) regulatory T-cell (Treg) compartment and skewed Th1 and possibly Th17 responses have been described in ITP patients. The trigger for aberrant T-cell polarization remains unknown. Because monocytes have a critical role in development and polarization of T-cell subsets, we explored the contribution of monocyte subsets in control of Treg and Th development in patients with ITP. Unlike circulating classic CD14(hi)CD16(-) subpopulation, the CD16(+) monocyte subset was expanded in ITP patients with low platelet counts on thrombopoietic agents and positively correlated with T-cell CD4(+)IFN-γ(+) levels, but negatively with circulating CD4(+)CD25(hi)Foxp3(+) and IL-17(+) Th cells. Using a coculture model, we found that CD16(+) ITP monocytes promoted the expansion of IFN-γ(+)CD4(+) cells and concomitantly inhibited the proliferation of Tregs and IL-17(+) Th cells. Th-1-polarizing cytokine IL-12, secreted after direct contact of patient T-cell and CD16(+) monocytes, was responsible for the inhibitory effect on Treg and IL-17(+)CD4(+) cell proliferation. Our findings are consistent with ITP CD16(+) monocytes promoting Th1 development, which in turn negatively regulates IL-17 and Treg induction. This underscores the critical role of CD16(+) monocytes in the generation of potentially pathogenic Th responses in ITP.

Molecular insights into blood groups and implications for blood transfusion.

&NA; In recent years technologic advances have led to the understanding of the molecular genetics of many blood group antigens. This review highlights the molecular basis of selected antigens and describes implications for transfusion medicine. The knowledge can be used in the following clinical settings to genotype a patient: 1) to identify a fetus at risk for hemolytic disease of the newborn 2) to genotype patients who have been recently transfused or whose erythrocytes have a positive direct antiglobulin test 3) to determine which phenotypically Fy(b‐) patients have the FY*B gene, 4) to genotype when an antigen has a depressed expression on erythrocytes, 5) to mass screen for anitgen‐negative donors, 6) to resolve blood group A, B, and D typing discrepancies, 7) to determine the zygosity of RhD, 8) to determine the origin of engrafted leukocytes in a stem cell recipient, 9) to determine the origin of lymphocytes in a patient with graft‐versus‐disease, 10) for tissue typing, 11) for paternity testing, and 12) for forensic testing.

Therapeutic Hemoglobin Levels after Gene Transfer in β-Thalassemia Mice and in Hematopoietic Cells of β-Thalassemia and Sickle Cells Disease Patients

Preclinical and clinical studies demonstrate the feasibility of treating β-thalassemia and Sickle Cell Disease (SCD) by lentiviral-mediated transfer of the human β-globin gene. However, previous studies have not addressed whether the ability of lentiviral vectors to increase hemoglobin synthesis might vary in different patients. We generated lentiviral vectors carrying the human β-globin gene with and without an ankyrin insulator and compared their ability to induce hemoglobin synthesis in vitro and in thalassemic mice. We found that insertion of an ankyrin insulator leads to higher, potentially therapeutic levels of human β-globin through a novel mechanism that links the rate of transcription of the transgenic β-globin mRNA during erythroid differentiation with polysomal binding and efficient translation, as reported here for the first time. We also established a preclinical assay to test the ability of this novel vector to synthesize adult hemoglobin in erythroid precursors and in CD34+ cells isolated from patients affected by β-thalassemia and SCD. Among the thalassemic patients, we identified a subset of specimens in which hemoglobin production can be achieved using fewer copies of the vector integrated than in others. In SCD specimens the treatment with AnkT9W ameliorates erythropoiesis by increasing adult hemoglobin (Hb A) and concurrently reducing the sickling tetramer (Hb S). Our results suggest two major findings. First, we discovered that for the purpose of expressing the β-globin gene the ankyrin element is particularly suitable. Second, our analysis of a large group of specimens from β-thalassemic and SCD patients indicates that clinical trials could benefit from a simple test to predict the relationship between the number of vector copies integrated and the total amount of hemoglobin produced in the erythroid cells of prospective patients. This approach would provide vital information to select the best candidates for these clinical trials, before patients undergo myeloablation and bone marrow transplant.

Regulatory T-cell status in red cell alloimmunized responder and nonresponder mice.

Red blood cell alloimmunization remains a major complication for transfusion-dependent patients, but immune factors governing risk for alloimmunization are unknown. We hypothesized that CD4+ regulatory T cells (Tregs), which we have shown control the rate and the frequency of red blood cell alloimmunization in mouse models, may dictate responder/nonresponder status. Using a transfusion regimen in which more than 50% of mice develop alloantibodies to human glycophorin A antigen, we found reduced in vitro and in vivo Treg-suppressive activity in responders compared with nonresponders that was the result of impaired Treg suppressor function. Moreover, responders were prone to developing additional alloantibodies to strong immunogens, whereas nonresponders were resistant to alloimmunization. Altogether, our data raise the possibility that Treg activity may be used as a marker for identifying responder/nonresponder status in transfusion recipients.