Weili Bao

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.

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.

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.

Hemin Controls T Cell Polarization in Sickle Cell Alloimmunization

Patients with sickle cell disease (SCD) often require transfusions to treat and prevent worsening anemia and other SCD complications. However, transfusions can trigger alloimmunization against transfused RBCs with serious clinical sequelae. Risk factors for alloimmunization in SCD remain poorly understood. We recently reported altered regulatory T cell (Treg) and Th responses with higher circulating Th1 (IFN-γ+) cytokines in chronically transfused SCD patients with alloantibodies as compared with those without alloantibodies. Because monocytes play a critical role in polarization of T cell subsets and participate in clearance of transfused RBCs, we tested the hypothesis that in response to the RBC breakdown product hemin, monocyte control of T cell polarization will differ between alloimmunized and non-alloimmunized SCD patients. Exogenous hemin induced Treg polarization in purified T cell/monocyte cocultures from healthy volunteers through the monocyte anti-inflammatory heme-degrading enzyme heme oxygenase-1. Importantly, hemin primarily through its effect on CD16+ monocytes induced an anti-inflammatory (higher Treg/lower Th1) polarization state in the non-alloimmunized SCD group, whereas it had little effect in the alloimmunized group. Non-alloimmunized SCD CD16+ monocytes expressed higher basal levels of heme oxygenase-1. Furthermore, IL-12, which contributed to a proinflammatory polarization state (low Treg/high Th1) in SCD, was dampened in hemin-treated stimulated monocytes from non-alloimmunized SCD patients, but not in the alloimmunized group. These data suggest that unlike alloimmunized patients, non-alloimmunized SCD CD16+ monocytes in response to transfused RBC breakdown products promote an anti-inflammatory state that is less conducive to alloimmunization.

Regulatory B‐cell compartment in transfused alloimmunized and non‐alloimmunized patients with sickle cell disease

Transfusion therapy is a life‐sustaining treatment for patients with sickle cell disease (SCD), but can cause serious complications including alloimmunization. We previously reported diminished regulatory T cells (Tregs) and skewed Th2 responses in alloimmunized SCD patients. We hypothesized that the B cell regulatory (Breg) compartment, which controls Treg and Th differentiation, may also be compromised in allosensitized SCD patients. Phenotypically, we did not find differences in the frequency or numbers of CD24hiCD38hi and CD24hiCD27+ B cell subsets, both previously identified as human Bregs, between alloimmunized and non‐alloimmunized SCD patients on regular transfusions. However, at the functional level, CD19+ B cells from alloimmunized SCD patients expressed lower levels of IL‐10 following stimulation as compared with non‐alloimmunized patients (P < 0.05), and had reduced ability in inhibiting autologous CD14+ monocyte TNF‐α expression (P < 0.05). These findings suggest that Bregs from alloimmunized and non‐alloimmunized SCD patients differ in their ability to produce IL‐10 and dampen monocyte activation, all consistent with an altered immunoregulatory state in alloimmunized SCD patients. Am. J. Hematol. 88:736–740, 2013.

Immune Dysregulation in Immune Thrombocytopenia

Immune thrombocytopenia (ITP) is a bleeding disorder characterized by low platelet counts due to decreased platelet production as well as increased platelet destruction by autoimmune mechanisms. A shift toward Th1 and possibly Th17 cells together with impaired regulatory compartment, including T-regulatory (Tregs) and B-regulatory (Bregs) cells, have been reported, suggesting a generalized immune dysregulation in ITP. Interestingly, several treatments including the use of thrombopoietic agents appear to be associated with improvement in the regulatory compartment. Understanding how Th1/Th17/Treg differentiation and expansion are controlled is central to uncovering how autoimmunity may be sustained in chronic ITP and reversed following response to therapy. In this review, we will summarize the recent findings on the state of the Breg and Treg compartments in ITP, the role of monocyte subsets in the control of Th/Treg expansion, and our working model of how the regulatory compartment may impact response to treatment and the means by which this information may guide therapy in ITP patients in the future.

Immunoregulatory Effects of Stored Red Blood Cells

Some clinical studies have identified potential adverse patient outcomes associated with RBC storage length. This may in part be due to the release of potentially hazardous bioactive products that accumulate during storage and are delivered at high concentrations during transfusion. In this situation, a proinflammatory tissue microenvironment may be established that can alter immunoregulatory mechanisms. This review highlights some of the potential immunomodulatory effects of stored RBCs that may be responsible for adverse transfusion reactions.

Immunologic characterization suggests reduced alloimmunization in a murine model of thalassemia intermedia.

Transfusion therapy remains a mainstay of treatment for patients with thalassemia major and to a lesser extent for the less anemic patients with thalassemia intermedia. We have previously reported a role for regulatory T cells (Tregs) in the control of antibody responses in wild‐type C57BL/6 (WT) mice exposed to allogeneic red blood cell transfusions. As an initial step to study and characterize immune regulation in thalassemias, we performed an immunologic cell–type characterization of C57BL/6 Hbbth‐1/Hbbth‐1 mouse model of thalassemia intermedia (Thal) in steady state as well as after transfusions with allogeneic blood.