* Authors on the Steering Committee contributed equally to the oversight of the study, including study design and maintaining the quality of study conduct. CONTRIBUTORS Owen O'Connor, Barbara Pro, Tim Illidge and Lorenz Trumper formed the ECHELON-2 steering committee and contributed equally to the oversight of the study, including study design and maintaining the quality of study conduct.

Keratinocyte growth factor (KGF) is a member of the fibroblast growth factor family that mediates epithelial cell proliferation and differentiation in a variety of tissues, including the thymus. We studied the role of KGF in T-cell development with KGF ؊/؊ mice and demonstrated that thymic cellularity and the distribution of thymocyte subsets among KGF ؊/؊ , wildtype (WT), and KGF ؉/؊ mice were similar. However, KGF ؊/؊ mice are more vulnerable to sublethal irradiation (450 cGy), and a significant decrease was found in thymic cellularity after irradiation. Defective thymopoiesis and peripheral T-cell reconstitution were found in KGF ؊/؊ recipients of syngeneic or allogeneic bone marrow transplant, but using KGF ؊/؊ mice as a donor did not affect T-cell development after transplantation. Despite causing an early developmental block in the thymus, administration of KGF to young and old mice enhanced thymopoiesis. Exogenous KGF also accelerated thymic recovery after irradiation, cyclophosphamide, and dexamethasone treatment. IntroductionKeratinocyte growth factor (KGF) is a 28-kDa fibroblast growth factor family member (FGF-7) that mediates epithelial cell proliferation and differentiation in a variety of tissues, including the gut (gut epithelial cells), skin (keratinocytes), and thymus (thymic epithelial cells). [1][2][3] KGF is produced by mesenchymal cells and has a paracrine effect on epithelial cells 4,5 ; it binds FGFR2IIIb, a splice variant of FGF receptor 2, expressed predominantly on these cell types. FGFR2IIIb is activated by 4 known ligands: FGF-1, FGF3, 7 The heterogeneous stromal cell compartment of the thymus includes both cortical and medullary epithelial cells, as well as mesenchymal cells (including fibroblasts). Mesenchymal cells produce fibroblast growth factors and support thymocyte development, especially in cortical areas (reviewed in Anderson and Jenkinson 8 ). Jenkinson et al 9 reported that mesenchymal cells regulate the proliferation of thymic epithelial cells via the production of KGF (FGF-7) and fibroblast growth factor-10 (FGF-10) during fetal development, but the role of mesenchymal cells in regulating the composition of thymic stroma in the neonatal and postnatal period is unclear.Erikson et al 10 have demonstrated that KGF and FGFR2IIIb signaling can affect the development and function of thymic epithelium (TE). In the adult thymus, mature ␣␤ ϩ thymocytes are capable of producing KGF, which leads to the expansion of thymic medullary epithelial cells. 10 However, KGF expression is not detectable in the triple negative (CD3 Ϫ CD4 Ϫ CD8 Ϫ ) thymocyte precursors. 10 In contrast, peripheral ␣␤ Ϫ T cells do not secrete KGF, even in epithelial tissues that comprise the skin, intestine, and vagina. However, ␥␦ Ϫ T cells in epithelial tissues do produce KGF and may also regulate epithelial cell growth. 11 KGF can function as a growth factor for epithelial protection and repair, is found in a variety of tissues (extensively reviewed by Finch and Rubin 12 ), and is up-regulated after various ...

Immunoincompetence after allogeneic hematopoietic stem cell transplantation (HSCT) affects in particular the T-cell lineage and is associated with an increased risk for infections, graft failure and malignant relapse. To generate large numbers of T-cell precursors for adoptive therapy, we cultured mouse hematopoietic stem cells (HSCs) in vitro on OP9 mouse stromal cells expressing the Notch-1 ligand Delta-like-1 (OP9-DL1). We infused these cells, together with T-cell-depleted mouse bone marrow or purified HSCs, into lethally irradiated allogeneic recipients and determined their effect on T-cell reconstitution after transplantation. Recipients of OP9-DL1-derived T-cell precursors showed increased thymic cellularity and substantially improved donor T-cell chimerism (versus recipients of bone marrow or HSCs only). OP9-DL1-derived T-cell precursors gave rise to host-tolerant CD4+ and CD8+ populations with normal T-cell antigen receptor repertoires, cytokine secretion and proliferative responses to antigen. Administration of OP9-DL1-derived T-cell precursors increased resistance to infection with Listeria monocytogenes and mediated significant graft-versus-tumor (GVT) activity but not graft-versus-host disease (GVHD). We conclude that the adoptive transfer of OP9-DL1-derived T-cell precursors markedly enhances T-cell reconstitution after transplantation, resulting in GVT activity without GVHD.

When cell populations are incubated with the DNA-binding dye Hoechst 33342 and subjected to flow cytometry analysis for Hoechst 33342 emissions, active efflux of the dye by the ABCG2/BCRP1 transporter causes certain cells to appear as a segregated cohort, known as a side population (SP). Stem cells from several tissues have been shown to possess the SP phenotype. As the lack of specific surface markers has hindered the isolation and subsequent biochemical characterization of epithelial stem cells this study sought to determine the existence of SP cells and expression of ABCG2 in the epithelia of the ocular surface and evaluate whether such SP cells had features associated with epithelial stem cells. Human and rabbit limbal-corneal and conjunctival epithelial cells were incubated with Hoechst 33342, and analyzed and sorted by flow cytometry. Sorted cells were subjected to several tests to determine whether the isolated SP cells displayed features consistent with the stem cell phenotype. Side populations amounting to <1% of total cells, which were sensitive to the ABCG2-inhibitor fumitremorgin C, were found in the conjunctival and limbal epithelia, but were absent from the stem cell-free corneal epithelium. Immunohistochemistry was used to establish the spatial expression pattern of ABCG2. The antigen was detected in clusters of conjunctival and limbal epithelia basal cells but was not present in the corneal epithelium. SP cells were characterized by extremely low light side scattering and contained a high percentage of cells that: showed slow cycling prior to tissue collection; exhibited an initial delay in proliferation after culturing; and displayed clonogenic capacity and resistance to phorbol-induced differentiation; all features that are consistent with a stem cell phenotype.

Lymphocyte Peyer patch adhesion molecule (LPAM) or alpha(4)beta(7) integrin is expressed on lymphocytes and is responsible for T-cell homing into gut-associated lymphoid tissues through its binding to mucosal addressin cell adhesion molecule (MAdCAM), which is present on high endothelial venules of mucosal lymphoid organs. We found in murine allogeneic bone marrow transplantation (BMT) models that recipients of alpha(4)beta(7)(-) donor T cells had significantly less graft-versus-host disease (GVHD) morbidity and mortality compared with recipients of alpha(4)beta(7)(+) donor T cells. A kinetic posttransplantation analysis of lymphocytes in the intestines and mesenteric lymph nodes demonstrated a delayed invasion of lower numbers of alpha(4)beta(7)(+) T cells in recipients of alpha(4)beta(7)(-) T cells compared with recipients of alpha(4)beta(7)(+) T cells. Histopathologic analysis of GVHD target organs revealed that recipients of alpha(4)beta(7)(-) T cells developed less GVHD of the intestines and liver, whereas there was no difference in cutaneous and thymic GVHD between recipients of alpha(4)beta(7)(-) or alpha(4)beta(7)(+) T cells. Finally, we found that in vivo GVT activity of alpha(4)beta(7)(-) donor T cells was preserved. We conclude that the alpha(4)beta(7) integrin is important for the invasion of alloreactive donor T cells into the gut and the subsequent development of intestinal GVHD and overall GVHD morbidity and mortality.

Prolonged immunodeficiency after allogeneic bone marrow transplantation (BMT) causes significant morbidity and mortality from infection. This study examined in murine models the effects of interleukin-7 (IL-7) given to young and middle-aged (9-month-old) recipients of major histocompatibility complex (MHC)-matched or -mismatched allogeneic BMT. Although administration of IL-7 from day 0 to 14 after syngeneic BMT promoted lymphoid reconstitution, this regimen was ineffective after allogeneic BMT. However, IL-7 administration from day 14 (or 21) to 27 after allogeneic BMT accelerated restoration of the major lymphoid cell populations even in middle-aged recipients. This IntroductionAll recipients of an allogeneic bone marrow transplant (BMT) are subject to a varying period of T and B lymphocytopenia, which is associated with an increased susceptibility to infections, such as cytomegalovirus, herpes simplex virus, varicella zoster virus, fungi, and encapsulated bacteria as well as Epstein-Barr virusinduced lymphoproliferative disease. The severity and duration of this immunoincompetence is determined by a number of variables, including previous chemotherapy or radiation therapy, a lack of sustained transfer of donor immunity, recapitulation of immunologic ontology, thymic involution after puberty, donor/host histoincompatibility, graft-versus-host disease (GVHD), and graft rejection (reviewed in Parkman and Weinberg 1 ). An inverse relationship between transplant recipient age and capacity to generate T lymphocytes (especially CD4 ϩ T cells) has been found in several studies. [2][3][4][5][6][7][8] Regeneration of T cells after allogeneic BMT can occur by at least 3 mechanisms: (1) a thymus-dependent pathway that involves positive and negative selection and represents a recapitulation of fetal T-cell ontogeny, (2) a thymus-independent pathway that is limited to the generation of small numbers of T cells from the bone marrow (BM) and intestines, [9][10][11][12] and (3) a thymus-independent pathway that involves the peripheral expansion of mature donor T cells that were transferred with the stem cell graft. [13][14][15][16][17][18] Data from human and murine studies demonstrate that T-cell reconstitution in older recipients of transplants depends more on the extrathymic pathways and results in slower reconstitution with fewer T cells (especially naive CD4 ϩ T cells) and a diminished T-cell repertoire. [13][14][15][16]18,19 Human interleukin-7 (IL-7) is a 25-kd glycoprotein that is secreted by fetal liver cells and stromal cells in the BM and thymus. [20][21][22] The IL-7 receptor (IL-7R) consists of the IL-7R␣ chain (CD127) 23 and the common cytokine receptor ␥ chain 24,25 and is expressed on early thymocytes, activated T cells, pre-B cells, and BM macrophages. 23 IL-7 is a nonredundant cytokine for T-and B-cell development and mice deficient for IL-7 or IL-7R␣ or mice treated with neutralizing anti-IL-7 antibodies all display severely impaired B-and T-cell development. 26,27 In humans, a defect in the IL-7R␣ chain 2...

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In allogeneic bone marrow transplantation (BMT) donor T cells are primarily responsible for antihost activity, resulting in graft-versus-host disease (GVHD), and for antileukemia activity, resulting in the graft-versus-leukemia (GVL) effect. The relative contributions of the Fas ligand (FasL) and perforin cytotoxic pathways in GVHD and GVL activity were studied by using FasL-defective or perforin-deficient donor T cells in murine parent 3 F1 models for allogeneic bone marrow transplantation. It was found that FasL-defective B6.gld donor T cells display diminished GVHD activity but have intact GVL activity. In contrast, perforin-deficient B6.pfp ؊/؊ donor T cells have intact GVHD activity but display diminished GVL activity. Splenic T cells from recipients of B6.gld or B6.pfp ؊/؊ T cells had identical proliferative and cytokine responses to host antigens; however, splenic T cells from recipients of B6.pfp ؊/؊ T cells had no cytolytic activity against leukemia cells in a cytotoxicity assay. In experiments with selected CD4 ؉ or CD8 ؉ donor T cells, the FasL pathway was important for GVHD activity by both CD4 ؉ and CD8 ؉ T cells, whereas the perforin pathway was required for CD8-mediated GVL activity.These data demonstrate in a murine model for allogeneic bone marrow transplantation that donor T cells mediate GVHD activity primarily through the FasL effector pathway and GVL activity through the perforin pathway. This suggests that donor T cells make differential use of cytolytic pathways and that the specific blockade of one cytotoxic pathway may be used to prevent GVHD without interfering with GVL activity. IntroductionAllogeneic bone marrow transplantation (BMT) is an important therapeutic modality for a variety of diseases, including hematologic malignancies. The therapeutic benefits of allogeneic BMT are not only derived from the high dose of chemoradiation but also from a graft-versus-leukemia (GVL) effect. [1][2][3] Clinical evidence for a GVL effect comes from studies demonstrating an increased relapse rate after BMT from an identical twin, autologous BMT, and T-cell depletion of the allogeneic bone marrow (BM) graft (reviewed in Truitt and Johnson 3 and Antin 4 ). Most studies indicate that the GVL effect is primarily mediated by allogeneic donor T cells, which recognize either leukemia-specific antigens or alloantigens expressed on normal and malignant cells. [5][6][7][8] Graft-versus-host disease (GVHD) remains the single most important complication of allogeneic BMT and is defined as a progressive systemic illness with immunosuppression, cachexia, and specific target organ disease of the skin, liver, and intestines. 9 Although the complex pathophysiology of acute GVHD involves the conditioning regimen (radiation or chemotherapy), cytokines, nitric oxide, and non-T effector cells (reviewed in Krenger et al 10 ), the cytolytic activity of donor T cells is essential for the development of GVHD activity.Cytolytic activity of cytotoxic T lymphocytes (CTL) is primarily mediated through 2 effector mechanisms: ...