Deficient thymopoiesis and retarded recovery of naive CD4(+) T cells are important determinants of insufficient immune-competence following hematopoietic stem cell transplantation (HSCT). Although keratinocyte growth factor (KGF) may protect the thymic epithelium, stem cell factor (SCF) is involved in early thymopoiesis. We evaluated whether KGF alone or combined with SCF would affect thymopoiesis and hematologic recovery following myeloablative autologous HSCT into rhesus macaques. Purpose-bred adult rhesus macaques received 10(6) autologous CD34(+)-selected mononuclear bone marrow cells (BMC)/kg after 9 Gy myeloablative conditioning. Animals were treated with phosphate-buffered saline (PBS) (n = 2), KGF alone (n = 2), or KGF combined with SCF (n = 2). KGF-treated animals showed accelerated hematologic recovery, improved thymopoiesis, and enhanced naive T-cell recovery following transplantation. Improved T cell recovery was not associated with protection against cytomegalovirus reactivation nor with improved antibody response to tetanus toxoid vaccination. Animals treated with KGF and SCF experienced severe adverse events that precluded evaluation of thymopoiesis and T cell recovery. Collectively, our data confirm that KGF may enhance thymopoiesis.
Deficient thymopoiesis is a pivotal determinant of impaired immune competence following hematopoietic stem cell transplantation (HSCT). Stem cell factor (SCF) is essentially involved in early thymopoiesis. We evaluated whether SCF administration would improve recovery of thymopoiesis following HSCT in immunodeficient mice receiving: 1) bone marrow (BM) transplantation of congenic mice; or 2) human fetal liver HSCT in the human immune system mouse model. Following murine BM transplantation, SCF significantly enhanced thymopoiesis and peripheral T cell recovery in lymph nodes and spleen. SCF did not affect BM lymphoid progenitor recovery and/or expansion. Median thymic cellularity increased from 0.9 in PBS- to 266 × 104/thymus in SCF-treated mice (p = 0.05). Following human HSCT in human immune system mice, higher thymic cellularity was observed in SCF-treated mice. Double-negative and early double-positive thymocyte subsets increased, but especially late double-positive, CD4 single-positive, and CD8 single-positive thymocyte subsets were significantly enhanced (p < 0.05). These results show that exogenous supply of SCF may significantly improve murine and human posttransplant thymopoiesis, for which the effect is probably exerted by directly promoting T cell development intrathymically rather than by enhanced entry of prethymically expanded lymphoid progenitors.
Leucine-rich repeat-containing G protein-coupled receptor (Lgr)5 is a marker for epithelial stem cells in the adult intestine of mice. Lgr5 transcripts have also been detected in the developing murine thymus, leading to speculation that Lgr5 is a marker for the longsought stem cell of the thymus. To address the nature of the Lgr5-expressing thymic epithelial cells (TECs), we used Lgr5-GFP reporter mice. We show that epithelial cells expressing Lgr5 protein are present in the fetal thymus during a specific developmental window yet are no longer detectable at birth. To analyze the function of the Lgr5 protein during thymus development, we generated Lgr5 −/− mice. These experiments unequivocally show that thymus development is not perturbed in the absence of Lgr5, that all TEC subsets develop in Lgr5 −/− mice and that T cells are produced in the expected ratios. Finally, by using an inducible lineage tracing system to track the progeny of Lgr5 + fetal TECs in vivo, we demonstrated that Lgr5 + fetal TECs have no detectable progeny in the later fetal thymus. In sum, we show that presence of the Lgr5 protein is not a prerequisite for proper thymus organogenesis. Keywords:Fetal thymus r Lgr5 r Lineage tracing r Thymic epithelial cells IntroductionThymic epithelial cells (TECs) form a 3D network that is essential for the proper proliferation, differentiation, and selection of developing thymocytes. Epithelial derived factors include growth factors, differentiation signals, and self-antigens expressed via MHC class I (MHCI) and MHC class II (MHCII) (reviewed in [1]). Presentation of self-antigens on TECs [2-5], the exact timing, affinity, and cofactors for the physiological contact between TECs and developing T cells (reviewed in [6]) is fundamental for proper T-cell development.The murine thymus originates from the third pharyngeal pouch at day E9.5 of embryonic development and is solely derived from the endoderm [7]. Specification of the thymus involves the sequential upregulation of important transcription factors (Hoxa3, Eya1, Rae2, chordin, and BMP; (reviewed in [8]) eventually leading to the expression of the thymic-specific transcription Correspondence: Prof. Jan J. Cornelissen e-mail: J.Cornelissen@erasmusmc.nl factor Foxn1 [9,10]. From E11.5 onwards, the first precursor T cells migrate into the thymic anlage and noncanonical NF-κB signaling becomes important for full differentiation of the medullary microenvironment, culminating in the upregulation of autoimmune regulator (Aire) [11][12][13] that enables medullary TECs to express self-antigens [2,3]. In the adult thymus cross-talk remains important, as the process of differentiation but also maintenance of medullary TECs, via ligation of RANK and CD40 by ligands expressed on thymocytes [11,12,14].Mature cortical and medullary TEC originate from a common thymic epithelial progenitor cell (TEPC) [15,16]. Although full differentiation of mature TECs from a clonal precursor population has been demonstrated, the precise phenotypical characterization of that precursor...
3725 Deficient thymopoiesis, due to epithelial injury by chemo- and/or radiotherapy, age-associated thymic involution and by graft-versus-host disease, is an important determinant of the impaired immune competence following allogeneic transplantation. Therefore, strategies to improve thymopoiesis are considered pivotal to improve T cell recovery and immune competence after transplantation. As SCF is a cytokine produced by thymic stroma and its receptor, c-kit, is expressed by the earliest thymocytes, we evaluated whether SCF administration would improve thymic recovery following stem cell transplantation in immuno-deficient mice receiving: (1) a T-cell depleted bone marrow (BM) graft of congenic mice, or (2) a CD34+CD38low-selected xenogenic hematopoietic stem cell (HSC) graft of human fetal liver origin (HIS) mice model). In the mouse-mouse model, 10–12 week old rag-1−/− mice were 3 Gy irradiated (137Cs -source) and received 2×105 T-cell depleted C57Bl/6 (CD45.1) congeneic bone marrow cells intravenously. Recipient mice received either PBS or recombinat rat SCF (Amgen, USA, 100μg/kg per injection) by subcutaneous injection 3 times a week from day 1 until the end of the experiment. In this model, SCF enhanced thymopoiesis and peripheral T-cell recovery. BM lymphoid progenitor recovery was not affected. Median thymic cellularity increased from 0.9 in PBS- to 266 × 104/thymus in SCF-treated mice (p=0.05), which increase was similarly distributed over the thymocyte subsets of double negative (DN), double positive (DP), and CD4+, CD8+ single positive thymocytes. Next, we assessed whether SCF-induced improved thymic recovery also translated into improved T-cell recovery in the periphery. Absolute numbers of donor-derived newly developed CD4+ and CD8+ T-cells were quantified in peripheral blood, spleen and lymph nodes at weeks 4 and 6 post-transplantation. T-cell numbers were low at 4 weeks after transplantation and did not differ between PBS or SCF treated animals. However, at 6 weeks T-cell numbers were significantly increased in spleen and lymph nodes of SCF treated animals (p<0.05). Next, we studied the effect of recombinant human (rh) SCF in our HIS mouse model. In short, newborn (days 3–7) Rag-2−/−gc−/− mice were 3.5 Gy irradiated and transplanted with 5–10 × 104 CD34+CD38low human fetal liver (FL) cells intra-hepatically. CD34+CD38low cells were isolated via a two step procedure. CD34+− FL cells were isolated using a CD34 human progenitor cell-isolation kit and further sorted as CD38low using a FACS Aria (BD biosciences). In HIS mice, PBS or rhSCF (Amgen, USA) 100μg/kg per injection) was administered intraperitoneally (i.p.) 3 times weekly as of day 14 following transplantation. Similar to the murine BMT model, a higher thymic cellularity was observed in SCF treated mice (Fig. 1) in the HIS model. DN and early DP thymocyte subsets were enhanced, albeit not significantly. In contrast, lateDP, CD4SP and CD8SP thymocyte subset recovery was significantly enhanced in thymi of SCF-treated HIS mice. As the HIS model remains a hybrid human–mouse system with limited cytokine cross reactivity and in which MHC-HLA mismatch compromises peripheral T-cell survival (Legrand et al J IMMUNOL 2009), the model did not allow us to study the effect of SCF on T-cells in the peripheral lymphoid organs. Figure 1: SCF improves thymic recovery following human SCT in an HIS mouse model. Newborn rag-2−/− γc−/− mice were 3.5 Gy irradiated and received 5–10 × 104 CD34+CD38low human fetal liver cells intra-hepatically and were treated with PBS or SCF. At indicated times post transplantation, thymi were harvested analyzed for the human thymocyte subsets: TN (CD3-CD4-CD8-), early DP (CD3- CD4+CD8+), late DP (CD3+CD4+CD8+), CD4SP and CD8SP (CD3+CD4-CD8+). Figure 1:. SCF improves thymic recovery following human SCT in an HIS mouse model. Newborn rag-2−/− γc−/− mice were 3.5 Gy irradiated and received 5–10 × 104 CD34+CD38low human fetal liver cells intra-hepatically and were treated with PBS or SCF. At indicated times post transplantation, thymi were harvested analyzed for the human thymocyte subsets: TN (CD3-CD4-CD8-), early DP (CD3- CD4+CD8+), late DP (CD3+CD4+CD8+), CD4SP and CD8SP (CD3+CD4-CD8+). Collectively, these results show that SCF may significantly improve post-transplant thymopoiesis after both experimental murine BMT and human fetal liver HSC transplantation in HIS mice. While peripheral T-cell recovery was significantly enhanced in the murine BMT model, it remains to be established whether improved human thymopoiesis will also translate into better peripheral T-cell recovery and enhanced immune competence towards opportunistic infections as well as a better recovery of regulatory T cells. These studies now set the stage for studying SCF, either alone or combined with other thymopoietic cytokines, in a larger pre-clinical animal model. Disclosures: No relevant conflicts of interest to declare.
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