In this study, we have moved into a clinically applicable model of sex steroid ablation, chemical castration using leuprolide acetate, and have shown enhanced T cell reconstitution following allogeneic BMT. We built on previous studies done by ourselves and others, studying the effects of sex steroid ablation on immune reconstitution (15
), and demonstrated that chemical castration increases early hematopoietic progenitors in the BM as well as thymocyte subsets and splenic T cell numbers without exacerbating GVHD and maintaining GVT activity.
Previous studies have demonstrated that early hematopoietic progenitors are sex steroid sensitive. In this study, we also observed an increase, following chemical castration, in donor-derived LSK cells, CLPs, and myeloid progenitors following chemical castration. Although little is known about the effects of androgens on BM progenitors, several studies have addressed the effects of estrogen. Estrogen can act both directly on BM progenitor cells and indirectly via the BM stroma (4
), resulting in a decrease in lymphoid progenitor potential. This is thought to be due to a decrease in lymphoid-committed precursors in the Lin-
population, as well as a depletion of Lin-
population of cells that contain CLPs (4
). LSK cells are significantly decreased following 17β
) treatment of ERα
-replete mice (26
). Interestingly, more modest decreases are also seen in ERα
knockout mice, suggesting an ERα
-independent component. BM chimeras of ERα
-replete and knockout mice showed that the presence of ERα
on the hematopoietic cells was essential for the dramatic loss of LSK cells (26
Although the increase in myeloid progenitor cells did not lead to an increase in the number of donor-derived myeloid cells in the bone marrow or the periphery, we did observe a significant increase in donor-derived B cell numbers in the BM. This is in agreement with previous studies that have demonstrated that sex steroids, both androgens and estrogens, have a suppressive effect on B lymphopoiesis, while their withdrawal enhances B cell development (7
Thymic cellularity was significantly increased in leuprolide acetate-treated mice at all time points after allogeneic BMT. Interestingly, we observed differences in the thymus earlier than those seen in the BM, suggesting a direct effect of sex steroid ablation on the thymus. Sex steroid receptors are expressed on all thymocyte subsets (30
) as well as medullary and cortical thymic epithelial cells (31
). LHRH receptors are expressed on cells in the thymus (12
), although little is known about the cellular distribution. In this study, we used leuprolide acetate, an LHRH agonist, to chemically castrate BMT recipients; the leuprolide acetate may have direct effects on thymic reconstitution or indirect effects via the ablation of sex steroids.
Previous studies have documented that sex steroids effect both thymocytes and the thymic stroma. Surgical or chemical castration reverses and delays the onset of age-related thymic atrophy (8
), while the administration of sex steroids reverses these effects and also exacerbates normal thymic atrophy (9
). There is evidence to suggest that sex steroid ablation independently affects both the thymic stroma and the earliest T cell progenitors. Olsen et al. (31
) demonstrated that the presence of a functional androgen receptor on the thymic stroma is essential for androgen-dependent thymic atrophy to occur. Although Heng et al. (18
) demonstrated that early T cell progenitor (ETP) numbers were increased as early as 5 days after castration of 9-mo-old mice (18
), before any changes were observed in the thymic stroma (47
). Furthermore, castration of RAG-1-/-
mice resulted in an expansion of TN thymocyte subsets without any effect on thymic epithelial numbers (47
). The findings of Olsen et al. (31
) suggest a stromal-mediated effect of sex steroid ablation and the latter finding suggests that cross-talk between thymocytes and stromal cells may in fact be required for sex steroid-mediated thymic effects.
Interestingly, in this study, we were unable to identify ETPs in either group up to 42 days after allogeneic BMT. This finding is in agreement with an earlier study which demonstrated that early thymic reconstitution following BMT occurs in the absence of ETPs (48
). However, in this study, increases in TN subsets were observed as were increases in DP and mature SP thymocytes.
Although there has been much speculation as to the mechanism of sex steroid suppression of lymphopoiesis, there have been few studies that directly address this topic. TGF-β
production is decreased in the thymus and BM following androgen treatment, making this suppressive growth factor a major candidate for sex steroid-induced suppression of lymphopoiesis (49
). In aged mice, surgical castration did not appear to alter the mRNA expression of TGF-β
1, IL-7, or keratinocyte growth factor (KGF) (24
). While following autologous BMT, we found that TGF-β
1 was decreased in castrated mice in both thymic stromal cells and BM cells, while thymic stromal cell production of IL-6 was also decreased in castrated mice (15
). Castrated allogeneic BMT recipients had decreased TGF-β
1 and increased production of KGF in the BM, while no obvious changes were observed in the thymus (16
One of the most well-studied growth factors in the aging setting is the cytokine IL-7. The majority of studies find a decrease in thymic IL-7 production with age (51
), but treatment of very old mice with IL-7 does not result in increased thymic output (54
). Although intrathymic injection of IL-7-producing stromal cells enhances early thymocyte development (55
) and IL-7 treatment of old mice can reverse age-related increases in thymic apoptosis and enhance early T cell development (51
), IL-7 treatment does not reverse age-related involution (55
). Together, these finding suggest that a decline in IL-7 production with age may play a part in the dysfunction of age-related T cell development, but may not be involved in the involution process. IL-7 production is severely affected for an extended period following irradiation (56
), suggesting that it may not play a role in castration-enhanced immune reconstitution following HSC transplantation.
T cell reconstitution, both thymic and peripheral, was enhanced after syngeneic and allogeneic BMT and KGF treatment (57
). The same study showed that the thymi of KGF-treated HSC transplanted mice contained more IL-7 mRNA+
cells, suggesting a role for IL-7 in KGF-mediated enhanced immune reconstitution (57
). KGF treatment is also known to facilitate engraftment and enhance BM reconstitution following allogeneic BMT (58
). Therefore, the increase in KGF expression we observed in an earlier study (16
) may play a role in the enhanced immune reconstitution observed following allogeneic BMT and sex steroid ablation.
In this study, we have shown that the increased thymic reconstitution observed after allogeneic BMT and chemical castration leads to an increase in donor-derived splenic CD4+
T cells. The significant increase was observed only at the late time point of 42 days after BMT. This is not surprising, considering it takes as long as 28 days for a cell to mature from BM precursor to mature T cell (59
). The most striking increase was observed in the naive compartment of both CD4+
cells, suggesting an increase in thymic T cell production and export. Interestingly, there was an increase in memory populations as well. This may be due to the direct effects of leuprolide acetate on peripheral T cells that express LHRH receptors.
Using in vitro (proliferation assays and cytokine production) and in vivo (T cell-dependent B cell response to NP23-CGG) assays, we have demonstrated that the enhanced donor-derived T cell reconstitution observed following allogeneic BMT and leuprolide acetate treatment translates into a significant increase in T cell function.
Enhanced peripheral T cell function is an advantageous outcome in most settings. However, following allogeneic BMT, the exacerbation of GVHD as a consequence of therapies designed to enhance immune reconstitution is a grave concern. In this study, castration did not worsen GVHD and GVT activity remained intact, further supporting its potential use as an adjunct therapy following treatments that result in immunosuppression results.
The data presented in this study add significantly to a recent nonrandomized clinical study that compared leuprolide acetatetreated allogeneic and autologous BMT recipients to age-matched controls (61
). We have demonstrated, in a clinically relevant model of allogeneic BMT, that the enhanced peripheral T cell reconstitution is due to both increases in lymphoid-committed precursors as well as enhanced thymic regeneration. Furthermore, GVHD was not exacerbated and GVT activity was maintained. These data suggest that leuprolide acetate treatment may be a novel strategy to enhance posttransplant T cell reconstitution.