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In β-thalassemia, the mechanism driving ineffective erythropoiesis (IE) is insufficiently understood. We analyzed mice affected by β-thalassemia and observed, unexpectedly, a relatively small increase in apoptosis of their erythroid cells compared with healthy mice. Therefore, we sought to determine whether IE could also be characterized by limited erythroid cell differentiation. In thalassemic mice, we observed that a greater than normal percentage of erythroid cells was in S-phase, exhibiting an erythroblast-like morphology. Thalassemic cells were associated with expression of cell cycle–promoting genes such as EpoR, Jak2, Cyclin-A, Cdk2, and Ki-67 and the antiapoptotic protein Bcl-XL. The cells also differentiated less than normal erythroid ones in vitro. To investigate whether Jak2 could be responsible for the limited cell differentiation, we administered a Jak2 inhibitor, TG101209, to healthy and thalassemic mice. Exposure to TG101209 dramatically decreased the spleen size but also affected anemia. Although our data do not exclude a role for apoptosis in IE, we propose that expansion of the erythroid pool followed by limited cell differentiation exacerbates IE in thalassemia. In addition, these results suggest that use of Jak2 inhibitors has the potential to profoundly change the management of this disorder.

Background. Prolonged immunodeficiency after allogeneic bone marrow transplantation (allo BMT) results in significant morbidity and mortality from infection. Previous studies in murine syngeneic BMT models have demonstrated that posttransplantation insulin-like growth factor (IGF)-I administration could enhance immune reconstitution. Methods. To analyze the effects of IGF-I on immune reconstitution and graft-versus-host disease (GVHD) after allo BMT, we used murine models for MHC-matched and -mismatched allo BMT. Young (3-month-old) recipient mice received 4 mg/kg per day of human IGF-I from days 14 to 28 by continuous subcutaneous administration. Results. IGF-I administration resulted in increased thymic precursor populations (triple negative-2 and triple negative-3) as determined on day 28 but had no effect on overall thymic cellularity. In the periphery, the numbers of donor-derived splenic CD3+ T cells were increased and these cells had an improved proliferative response to mitogen stimulation. IGF-I treatment also significantly increased the numbers of pro-, pre-, and mature B cells and myeloid cell populations in the spleens of allo BMT recipients on day 28. The administration of IGF-I in combination with interleukin 7 had a remarkable additive effect on B-cell, but not on T-cell, lymphopoiesis. Finally, we tested the effects of IGF-I administration on the development of GVHD in three different MHC-matched and -mismatched models and found no changes in GVHD morbidity and mortality. Conclusion. IGF-I administration can enhance lymphoid and myeloid reconstitution after allo BMT without aggravating GVHD.

Prolonged immunodeficiency after allogeneic bone marrow transplantation (allo BMT) results in significant morbidity and mortality from infection. Previous studies in murine syngeneic BMT models have demonstrated that posttransplantation insulin-like growth factor (IGF)-I administration could enhance immune reconstitution.To analyze the effects of IGF-I on immune reconstitution and graft-versus-host disease (GVHD) after allo BMT, we used murine models for MHC-matched and -mismatched allo BMT. Young (3-month-old) recipient mice received 4 mg/kg per day of human IGF-I from days 14 to 28 by continuous subcutaneous administration.IGF-I administration resulted in increased thymic precursor populations (triple negative-2 and triple negative-3) as determined on day 28 but had no effect on overall thymic cellularity. In the periphery, the numbers of donor-derived splenic CD3+ T cells were increased and these cells had an improved proliferative response to mitogen stimulation. IGF-I treatment also significantly increased the numbers of pro-, pre-, and mature B cells and myeloid cell populations in the spleens of allo BMT recipients on day 28. The administration of IGF-I in combination with interleukin 7 had a remarkable additive effect on B-cell, but not on T-cell, lymphopoiesis. Finally, we tested the effects of IGF-I administration on the development of GVHD in three different MHC-matched and -mismatched models and found no changes in GVHD morbidity and mortality.IGF-I administration can enhance lymphoid and myeloid reconstitution after allo BMT without aggravating GVHD.

β-Thalassemia intermedia and major are characterized by ineffective erythropoiesis (IE), requiring sporadic or chronic blood transfusions, respectively. Some of the major consequences of IE are extra-medullary hematopoiesis (EMH), splenomegaly and systemic iron overload mediated by transfusion therapy and down-regulation of hepcidin. Using mouse models of β-thalassemia intermedia (th3/+) and major (th3/th3), and human specimens we investigated IE in this disorder. Th3/+ and th3/th3 erythroid cells were analyzed with respect to rates of apoptosis and degrees of cell proliferation and differentiation. We found that there was both a relative and absolute expansion of the immature erythroid progenitor cell fraction in thalassemic mice compared to cells in the final stages of differentiation. Further investigation of the thalassemic erythroid cells in vivo and in vitro indicated that a larger number of the thalassemic cells are associated with the phosphorylated form of the Jak2 protein kinase than in normal mice. In fact, their proliferation was prevented by TG101209, a Jak2 inhibitor. Similar compounds are currently utilized or being considered for use in the treatment of myeloproliferative diseases such as polycythemia vera. In order to assess the potential of Jak2 inhibitors in limiting IE in β-thalassemia, we administered TG101209 to th3/+ mice. We found that both 10 and 18 days of treatment were sufficient to dramatically reduce the spleen size and the percentage of immature erythroid progenitors therein compared with administration of a placebo. However, these changes were associated with decreasing hemoglobin levels (Blood, 2008, 112:875–85). We speculated that this problem could be overcome by administration of blood transfusions during treatment, an extension of current management in thalassemia as noted above. To test this hypothesis, we administered TG101209 to th3/th3 mice in conjunction with regular blood transfusions. Administration of TG101209 to transfused th3/+ mice is in progress. Our preliminary data on th3/th3 mice indicates that simultaneous administration of TG101209 and transfused blood not only reverses splenomegaly, but also results in higher Hb levels (N ≥ 3). The increased hemoglobin levels observed, compared to those in mice treated with transfusion therapy alone, suggest that the use of Jak2 inhibitors may reduce the amount of blood per transfusion and/or the rate of transfusion required in thalassemia. Since the increased iron absorption in thalassemic mice is a direct consequence of IE, treatment with TG101209 could also be beneficial in ameliorating this process. According to several observations, suppression of erythropoiesis should lead to increased Hamp1 expression in the presence of iron overload. Therefore, under conditions of Jak2 inhibition, Hamp1 transcription is expected to increase. To test this hypothesis we are currently analyzing organ iron levels and the expression of iron-related genes in drug-treated mice. In conclusion, although our study does not exclude a role for apoptosis in the IE of β-thalassemia, we have demonstrated that increased cell proliferation and limited cell differentiation play a significant role in this process. Moreover, we show for the first time that a Jak2 inhibitor is effective in decreasing the spleen size of thalassemic mice. This could represent a completely new approach to the treatment of splenomegaly in β-thalassemia patients, perhaps coupled with blood transfusion. That administration of a Jak2 inhibitor reverses splenomegaly and also ameliorates the degree of iron overload could provide an opportunity to gain new insight into the dynamic processes of iron absorption and erythropoiesis in this pathological condition.

Ineffective erythropoiesis (IE) in β-thalassemia has been attributed to erythroid cell death mediated by apoptosis or hemolysis during the maturation process. Historically, ferrokinetic studies in this disease suggested that 60%–80% of erythroid precursors die in the marrow or extramedullary sites. However, several observations have challenged this view. First, the number of apoptotic erythroid cells in patients is low compared to net expansion of the erythroid cell pool. Second, hemolytic markers in β-thalassemic patients are normal or only slightly increased, unless additional pathological conditions appear. Third, our most recent study (Blood, Gardenghi et al, 2007 Jun 1) demonstrated that GI iron absorption in β-thalassemia is increased by the dysregulation of genes such as hepcidin and ferroportin that control iron absorption, resulting in iron levels that exceed the amount required for erythropoiesis. We have undertaken a detailed investigation using cohorts of mice (n>30 per genotype) with β-thalassemia intermedia (th3/+) and major (th3/th3). Using these models, we have previously shown that the severity of anemia (as low as 1 g/dL) inversely correlates with the total number of nucleated erythroid cells (»100 fold compared to wild-type (wt) mice). Cytological analysis has clearly shown that thalassemic spleen specimens were comprised of a homogeneous pre-erythroblastic population. In contrast, the percentage of apoptotic cells and the level of hemolytic markers, such as bilirubin and lactic acid dehydrogenase, slightly increased or were not different compared to wt mice. While not excluding a role for apoptosis, our observations suggest that control of the cell cycle and maturation of erythroid precursors play an important role in IE. We then explored whether the erythroid cell cycle was dysregulated in our model system. We found that erythropoietin (Epo) levels were raised in thalassemic animals by as much as three orders of magnitude. Binding of Epo to its receptor (EpoR), activates antiapoptotic and cell cycle promoting genes, through activation of Jak2 and Stat5. By Western blot we demonstrated up-regulation of EpoR, Stat5 and the antiapoptotic protein BclXL, as well as that of proliferation promoting genes, such as CycA and Cdk2, in purified thalassemic erythroid cells compared to those of wt animals. This data was confirmed by staining both wt and thalassemic liver and spleen sections using the proliferation markers Ki67 and Mcm3, by clonogenic assay and by analysis of the percentage of erythroid cells in S-phase after BrdU injection. In the latter case, we observed 22%, 30% and 44% BrdU+ cells from wt, th3/+ and th3/th3 mice, respectively. In addition, freshly purified thalassemic erythroid cells proliferate faster in vitro than normal cells, a phenomenon blocked by AG490, a Jak2 inhibitor. Significantly, we have been able to reproduce results from our animal studies in humans, comparing normal and thalassemic blood and spleen specimens. In conclusion, we propose that IE in β-thalassemia is likely to be the result of altered cell proliferation and impaired cell differentiation, which in turn limit apoptosis, thereby mimicking tumor-like behavior.

Ineffective erythropoiesis (IE) in β-thalassemia is described as increased expansion of erythroid progenitor cells in combination with accelerated apoptosis and intramedullary hemolysis. However, evidence for this assumption is not particularly strong. In this study we evaluated the kinetics of red blood cell proliferation and survival in thalassemic mice that exhibit levels of anemia consistent with thalassemia intermedia (th3/+) and major (th3/th3), as we described previously. Th3/+ mice show anemia and increased reticulocyte counts (8.9±1.1 g/dL and 17.7±2.6x105/ul compared to 14.9±1.1 g/dL and 2.6±0.4x105/ul in +/+), whereas th3/th3 mice show more severe anemia than th3/+ mice and reduced production of reticulocytes (3.0±1.2 g/dL and 1.8±0.7x105/ul). As soon as two months of age, EPO levels in thalassemic mice are significantly increase over normal mice, 10 times in th3/+ and up to three orders of magnitude in th3/th3 mice. The total number of nucleated erythroid cells (spleen + bone marrow) increased from 3.3±0.9x108 in +/+ to 1.2±0.5x109 (or 3.6 folds) in th3/+ and 1.6±0.6x109 (or 4.8 folds) in th3/th3 mice (N=5 per group), whereas the level of apoptotic cells increased only 2 to 3 folds in percentage in th3/+ and th3/th3, respectively, as observed using the apoptotic Annexin-V and erythroid specific Ter119 markers (9.9±5.0, 14.3±2.5, 24.2±6.7 in BM and 10.6±3.5, 14.8±6.5, 25.4±6.2 in spleen of +/+, th3/+ and th3/th3, respectively; n=3 per genotype). This data was confirmed by TUNEL, cleaved caspase-3/7 and by immunostaining assays. Bilirubin and LDH levels were not different between thalassemic and +/+ mice. Altogether these observations indicated that in thalassemia there is a disproportion between number of proliferating and dying cells with a net increase of erythroid cells. Furthermore, microarray analysis on erythroid cells indicated increased expression of cell cycle promoting genes such as Ki67, Mcm3, Cyclin-A, CDK2 and BclXL (2 to 6 folds compared to +/+ mice, n=5 per genotype). This data was confirmed by Q-PCR, Western blot, immunostaining, clonogenic assay and by analysis of the percentage of erythroid cells in S-phase after in vivo BrdU injection (22, 30 and 44% BrdU+ in +/+, th3/+ and th3/th3, respectively). On the other hand, in th3/th3, which show more apoptosis than th3/+ mice, the cyclin-dependent kinase inhibitor p21 was upregulated both at the RNA (50-folds) and protein level. P53 was also analyzed in th3/th3 mice, showing no expression. In order to investigate the function of p21 in thalassemic erythroid cells, its expression was analyzed on purified erythroid cells isolated from th3/th3 that were transfused (10.4±0.4 g/dL of Hb) or thalassemic mice showing different levels of anemia (6.2±0.2 and 2.1±0.8 g/dL of Hb, respectively; N=3 per each group). We observed that the level of p21 increased with anemia and the severity of the pathology. However, in th3/th3 mice that were injected with BrdU, immunostaining analysis indicated that a large amount of p21+ erythroid cells were also BrdU+. In conclusion, we propose that erythropoiesis in β-thalassemia is characterized by enhanced expression of cell cycle promoting and survival factors that are able to overcome or mitigate p21 cell cycle block and, probably, apoptosis.

Inducible costimulator (ICOS) is a member of the B7 family that is expressed on activated and memory T cells and is involved in the regulation of TH1 and TH2 effector cytokine production after CD3/TCR stimulation. Studies with ICOS inhibition or ICOS−/− recipients have demonstrated prolonged allograft survival after heart or liver transplantation in animal models. To study the role of ICOS expression on alloreactive T cells in graft-versus-host disease (GVHD), we used allogeneic MHC class I and II disparate hematopoietic stem cell transplantation (allo-HSCT) models. We first analyzed the expression of ICOS by transferring CFSE-labeled donor T cells into irradiated allogeneic recipients and observed an increased expression of ICOS on alloreactive T cells compared to non-alloreactive T cells. We then studied B6-ICOS−/− alloreactive T cells and found intact proliferation in vivo (as determined by adoptive transfer of CFSE labeled T cells and donor T cell numbers in the spleen of allo-HSCT recipients), intact cytotoxicity, intact up regulation of activation markers, but decreased IFN-γ production in vitro. We then performed GVHD experiments in two models with full MHC class I and II disparity and observed significantly less GVHD morbidity and mortality in recipients of ICOS−/− donor T cells. Furthermore, histopathological analysis demonstrated less GVHD in all target organs (skin, liver, small bowel and large bowel) of recipients of ICOS−/− splenic T cells compared to recipients of wild type T cells. We harvested target organs (spleen, thymus, liver and gut) on days 7, 14, and 21 to examine donor T cell content (naïve and activated T cells) and found no significant difference in the total T cell numbers and subpopulations. Interestingly, in GVHD/graft-versus-tumor (GVT) experiments, ICOS−/− donor T cells displayed intact GVT activity, while their GVH activity was diminished. We then tested the levels of IFN-γ in the sera of mice undergoing GVHD and observed decreased serum levels in recipients of B6-ICOS−/− T cells. In conclusion, alloreactive ICOS−/− donor T cells display less GVHD morbidity and mortality due to decreased IFN-γ production, while proliferation, infiltration and GVT activity remain intact. These data suggests that strategies to inhibit ICOS could be useful for the prevention and/or treatment of GVHD in recipients of an allo-HSCT.