FOXN1 recombinant protein enhances T‐cell regeneration after hematopoietic stem cell transplantation in mice

Abstract

A prolonged period of T-cell recovery is the major challenge in hematopoietic stem cell transplantation (HSCT). Thymic epithelial cells (TECs) are the major component of the thymic microenvironment for T-cell generation. However, TECs undergo degeneration over time. FOXN1 plays a critical role in TEC development and is required to maintain adult TECs for thymopoiesis. To investigate the potential application of FOXN1, we have cloned and expressed recombinant FOXN1 protein (rFOXN1) that was fused with cellpenetrating peptides. We show here that the rFOXN1 protein can translocate from the cell surface into the cytoplasm and nucleus. Administration of rFOXN1 into both congenic and allogeneic HSCT recipient mice increased the number of TECs, resulting in enhanced thymopoiesis that led to an increased number of functional T cells in the periphery. The increased number of TECs is due to the enhanced survival and proliferation of TECs. Our results suggest that rFOXN1 has the potential to be used in enhancing T-cell regeneration in patients following HSCT.Keywords: FOXN1 r Hematopoietic stem cell transplantation r T-cell generation r Thymic epithelial cells r Thymus Additional supporting information may be found in the online version of this article at the publisher's web-site IntroductionHSCT is widely used in the treatment of hematopoietic and nonhematopoietic diseases. However, this procedure often suffers from a long period of T-cell recovery, which contributes to increased incidences of infection and relapses of cancer [1][2][3][4][5][6]. Therefore, strategies to enhance T-cell regeneration after HSCT would be greatly beneficial.Correspondence: Dr. Laijun Lai e-mail: laijun.lai@uconn.edu T-cell development occurs primarily in the thymus, and is critically dependent on the thymic microenvironment, in which TECs are the major component. The importance of TECs in thymocyte development has been highlighted by the fact that abnormal TEC development results in immunodeficiency and autoimmunity [7,8]. It is known that TECs undergo a qualitative and quantitative loss over time [8][9][10]. In addition, radiation, chemotherapy, immunosuppressive drugs, and infections, etc. may injure the TECs. Therefore, strategies to restore TECs should lead to enhanced T-cell regeneration and adaptive immunity.FOXN1 is a member of the winged helix/forkhead box transcription factor family. It is generally acknowledged that FOXN1 is a pivotal regulator for TEC development [11][12][13][14][15][16][17][18][19]. Mice homozygous for loss-of-function mutation in FOXN1 display the 'nude' phenotype (FOXN1 nu/nu ), which is characterized by congenital athymia and hairlessness. A mutation in the human FOXN1 gene also results in a nude phenotype [20,21]. FOXN1 is expressed in fetal thymus and postnatal TECs and its expression is progressively down-regulated with aging [15,16,[22][23][24][25]. FOXN1 is required not only for TEC development in fetal thymus, but also for maintenance of the postnatal thymus [15,16,23,24,26]. The reduced expression or indu...

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“…Mice were anesthetized and injected with 5 × 10 4 cells in 10–20 μl PBS into the thymus posterior to the upper sternum using a 26–28 gauge needle as described ( 49 ).…”

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“…Importantly, thymic expression of both Bmpr1a and Bmpr2 were identified on TEC populations, with a higher expression of the non-redundant Bmpr2 on cTECs ( 85 ); consistent with BMP4-induced expression of FOXN1 and its downstream target delta-like 4 (DLL4) specific to cTECs ( 85 , 87 ). Although the importance for FOXN1 and DLL4 for the development of TECs and thymocytes, respectively, has been well studied ( 88 , 89 ), recent findings have also highlighted their importance for thymic regeneration, with intrathymic concentration of DLL4 profoundly impacting on thymic size ( 90 ), and reports suggesting that the induction of FOXN1 can counteract age-associated thymic involution ( 91 ), acute damage ( 85 ), and thymic damage post-transplantation ( 92 ). While much of the role of BMP4 seems to be mediated by induction of the FOXN1/DLL4 axis, given the requirement for BMP4 in in vitro differentiation of TECs from multipotent progenitors ( 93 – 95 ), it is possible that an alternate mechanism may be by stimulating bipotent progenitors present in the adult thymus ( 96 – 99 ).…”

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“…Some of the mice were injected intrathymically (i.t.) with mESC-TEPs (EpCAM1 + cells, 5 × 10 4 ) or mESC-control cells (EpCAM1 − cells, 5 × 10 4 ) on day 0 with a procedure as described [37].…”

“…Single-cell suspensions of the thymus, spleen, graft, and mESC-derived cells were stained with the fluorochrome-conjugated Abs as described [32, 33, 37, 38]. For intracellular staining, the cells were first permeabilized with a BD Cytofix/Cytoperm solution for 20 min at 4 °C.…”