Interleukin 7 (IL-7) stimulates the proliferation of B cell progenitors, thymocytes, and mature T cells through an interaction with a high affinity receptor (IL-7R) belonging to the hematopoietin receptor superfamily. We have further addressed the role of IL-7 and its receptor during B and T cell development by generating mice genetically deficient in IL-7R. Mutant mice display a profound reduction in thymic and peripheral lymphoid cellularity. Analyses of lymphoid progenitor populations in IL-7R-deficient mice define precisely those developmental stages affected by the mutation and reveal a critical role for IL-7R during early lymphoid development. Significantly, these studies indicate that the phase of thymocyte expansion occurring before the onset of T cell receptor gene rearrangement is critically dependent upon, and mediated by the high affinity receptor for IL-7.

The introduction of clonal assays and long-term culture systems has resulted in considerable progress in the understanding of the early events that control self-renewal and commitment to differentiation of pluripotent hematopoietic stem cells (PHSC). Relatively little is known about the factors that control the commitment of PHSC to the lymphoid lineages, especially the T cell lineage. In the present study, the expression of the proto-oncogene c-kit was used to isolate and study the capacity of highly purified day 14 colony-forming units-spleen (CFU-S) to reconstitute the thymus of sublethally irradiated Thy-1 congenic recipient mice. We demonstrate here that one c-kit positive (c-kitpos) stem cell upon intrathymic transfer can effectively reconstitute the thymus of a sublethally irradiated recipient. After a lag phase of 15 d, high levels of donor-derived thymocytes (Thy-1.1pos) could be detected until 65 d after transplantation in Thy-1.2pos host mice. Donor-derived cells were only detected in the lobe of the thymus in which cells were previously injected and not in the noninjected lobe. These data suggest that c-kitpos stem cells do not migrate from one lobe to another and that they do not re-seed the thymus after having migrated to the bone marrow. The level and duration of reconstitution was found to be cell dose dependent, suggesting that, over time, endogenous stem cells compete with donor stem cells for available sites in the thymus microenvironment. The data presented in this paper demonstrate that commitment of purified adult bone marrow-derived c-kitpos stem cells to the T cell differentiation pathway can occur in the thymus and does not have to happen in the bone marrow.

Low recovery and poor retroviral vector infection efficiency of hematopoietic stem cells has hindered application of gene therapy for disease affecting blood-forming tissues. Developmental restriction (or death) of stem cells during ex vivo infection has contributed to these difficulties. In these studies we report that the cytokine leukemia inhibitory factor (LIF) directly or indirectly supported the survival of hematopoietic stem cells during culture of bone marrow with vector-producing fibroblasts, resulting in efficient recovery of stem cells able to compete for engraftment in irradiated recipient animals. The infection efficiency of hematopoietic stem cells recovered from these cultures was approximately 80%; and all recipients (20/20) of the LIF-treated marrow were stably engrafted with the progeny of provirus-bearing stem cells. Expression of vector-encoded human adenosine deaminase (hADA) was detected in all recipients at levels averaging 15-50% of endogenous murine ADA in all their hematolymphoid tissues. Survival of stem cells in untreated cultures was approximately 10% of that observed from LIF-treated cultures, resulting in poor engraftment of recipient animals with transplanted cells. The infection efficiency of the few stem cells recovered from untreated cultures, however, was high (approximately 80%), suggesting that LIF did not have an effect on infection efficiency per se, but acted at the level of stem cell survival. Consistent with the poor engraftment observed in the control animals, expression of vector-encoded ADA was only approximately 4-20% of the endogenous levels. These results support the postulated role of LIF as a regulator of hematopoiesis and suggest that cytokine stimulation can positively affect inefficient retroviral vector transduction in hematopoietic stem cells.

CD45 antigens are protein tyrosine phosphatases. A possible link was evaluated between expression of CD45 antigens on human myeloid progenitor cells (MPC) (colony-forming unit-granulocyte/macrophage [CFU-GM], burst-forming unit-erythroid [BFU-E], and colony-forming unit-granulocyte/erythroid/macrophage/megakaryocyte [CFU-GEMM]) and regulation of MPC by colony-stimulating factors (CSF) (interleukin 3 [IL-3], GM-CSF, G-CSF, M-CSF, and erythropoietin [Epo]), a GM-CSF/IL-3 fusion protein, and mast cell growth factor (MGF; a c-kit ligand). Treatment of cells with antisense oligodeoxynucleotides (oligos) to exons 1 and 2, but not 4, 5, or 6, of the CD45 gene, or with monoclonal anti-CD45, significantly decreased CFU-GM colony formation stimulated with GM-CSF, IL-3, fusion protein, and GM-CSF + MGF, but not with G-CSF or M-CSF. It also decreased GM-CSF, IL-3, fusion protein, and MGF-enhanced Epo-dependent BFU-E and CFU-GEMM colony formation, but had little or no effect on BFU-E or CFU-GEMM colony formation stimulated by Epo alone. Similar results were obtained with unseparated or purified (greater than or equal to one of two cells being a MPC) bone marrow cells. Sorted populations of CD343+ HLA-DR+ marrow cells composed of 90% MPC were used to demonstrate capping of CD45 after crosslinking protocols. Also, a decreased percent of CD45+ cells and CD45 antigen density was noted after treatment of column-separated CD34+ cells with antisense oligos to exon 1 of the CD45 gene. These results demonstrate that CD45 cell surface antigens are linked to stimulation of early human MPC by IL-3, GM-CSF, a GM-CSF/IL-3 fusion protein, and MGF.

Pluripotent hematopoietic stem cells (PHSC) are very rare cells whose functional capabilities can only be analyzed indirectly. For a better understanding and possible manipulation of mechanisms that regulate self-renewal and commitment to differentiation of PHSC, it is necessary to purify these cells and to develop assays for their growth in vitro. In the present study, a rapid and simple, widely applicable procedure to highly purify day 14 spleen colony-forming cells (day 14 CFU-S) is described. Low density bone marrow cells (rho less than or equal to 1.078 g/cm3) were enriched by two successive light-activated cell sorting procedures. In the first sort, cells within a predetermined light scatter (blast cell) window that are wheat germ agglutinin/Texas Red (WGA/TxR) positive and mAb 15-1.4.1/fluorescein isothiocyanate negative (granulocyte-monocyte marker) were selected. In the second sort, cells were selected on the basis of retention of the supravital dye rhodamine 123 (Rh123). Cells that take up little Rh123 (Rh123 dull cells) and those that take up more Rh123 (Rh123 bright cells) were 237-fold and 132-fold enriched, respectively, for day 14 CFU-S. Both Rh123 fractions were cultured for various time periods in vitro in the presence of mast cell growth factor (MGF), with or without interleukin 3 (IL-3) or IL-1 alpha. Both Rh123 fractions proliferated in response to MGF alone as determined by a [3H]TdR assay or by counting nucleated cells present in the cultures over time. MGF also acted synergistically with both IL-3 and IL-1 alpha to promote stem cell proliferation. Stimulation of both Rh123 fractions with MGF alone did not result in a net increase of day 14 CFU-S. Stimulation with MGF + IL-3 or MGF + IL-alpha resulted in a 4.4- or 2.6-fold increase of day 14 CFU-S in the Rh123 dull fraction, and an 11.6-fold or 2.6-fold increase of day 14 CFU-S in the Rh123 bright fraction, respectively. The data presented in this paper indicate that in vitro MGF acts on primitive hematopoietic stem cells by itself and also is a potent synergistic factor in combination with IL-3 or IL-1 alpha.