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T-lymphocyte depletion of bone marrow grafts compromises engraftment, suggesting a facilitating mechanism provided by the T cells that has been shown to associate with CD8+but not CD4+T cells. Explanations for this phenomenon have focused on immune targeting of residual host cells or cytokine production. We provide evidence for an alternative mechanism based on cooperative effects on cell motility. We observed that engraftment of CD34+cells in a β2-microglobulin–deficient nonobese diabetic/severe combined immunodeficiency (β2m−/−NOD/SCID) mouse model paralleled clinical observations in humans, with an enhancing effect noted from the addition of CD8+cells but not CD4+cells. This correlated with CD8+augmentation of CD34+cell homing to the bone marrow in vivo and CD8+cell–associated increases of CD34+cell transmigration through a bone marrow endothelial cell line in vitro. The cooperative interaction was not sensitive to brefeldin A inhibition of protein secretion. However, cytochalasin D–induced inhibition of CD8+cytoskeletal rearrangements abrogated CD34+transendothelial migration and impaired CD34+cell homing in vivo. CD8+cells did not migrate in tandem with CD34+cells or alter endothelial barrier integrity; rather, they affected phosphotyrosine-mediated signaling in CD34+cells in response to the chemokine stromal derived factor-1α (SDF-1α). These data demonstrate cell-cell cooperativity between different cell types in mediating chemotactic events and provide one potential explanation for the clinically observed effect of CD8+cells on bone marrow transplantation. This modification of cell migration by neighboring cells provides broad possibilities for combinatorial effects between cells of different types to influence cell localization.

T-lymphocyte depletion of bone marrow grafts compromises engraftment, suggesting a facilitating mechanism provided by the T cells that has been shown to associate with CD8+ but not CD4+ T cells. Explanations for this phenomenon have focused on immune targeting of residual host cells or cytokine production. We provide evidence for an alternative mechanism based on cooperative effects on cell motility. We observed that engraftment of CD34+ cells in a β2-microglobulin–deficient nonobese diabetic/severe combined immunodeficiency (β2m−/− NOD/SCID) mouse model paralleled clinical observations in humans, with an enhancing effect noted from the addition of CD8+ cells but not CD4+ cells. This correlated with CD8+ augmentation of CD34+cell homing to the bone marrow in vivo and CD8+cell–associated increases of CD34+ cell transmigration through a bone marrow endothelial cell line in vitro. The cooperative interaction was not sensitive to brefeldin A inhibition of protein secretion. However, cytochalasin D–induced inhibition of CD8+ cytoskeletal rearrangements abrogated CD34+ transendothelial migration and impaired CD34+ cell homing in vivo. CD8+ cells did not migrate in tandem with CD34+ cells or alter endothelial barrier integrity; rather, they affected phosphotyrosine-mediated signaling in CD34+ cells in response to the chemokine stromal derived factor-1α (SDF-1α). These data demonstrate cell-cell cooperativity between different cell types in mediating chemotactic events and provide one potential explanation for the clinically observed effect of CD8+ cells on bone marrow transplantation. This modification of cell migration by neighboring cells provides broad possibilities for combinatorial effects between cells of different types to influence cell localization.

During development, hematopoietic stem cells (HSCs) translocate from the fetal liver to the bone marrow, which remains the site of hematopoiesis throughout adulthood. In the bone marrow the HSCs are located at the endosteal surface, where the osteoblasts are a key component of the stem cell niche. The exogenous signals that specifically direct HSCs to the bone marrow have been thought to include stimulation of the chemokine receptor CXCR4 by its cognate ligand stromal derived factor-1α (SDF-1α or CXCL12). However, experiments in which CXCR4−/− fetal liver hematopoietic cells were transplanted into wild-type hosts demonstrated efficient engraftment of the HSCs in the bone marrow. In addition, treatment of HSCs with inhibitors of Gαi-coupled signaling, which blocks transmigration towards SDF-1αin vitro, does not affect bone marrow homing and engraftment in vivo. Therefore, we examined whether Gsα-coupled mechanisms play a key role in the engraftment of the HSCs in the bone marrow environment. Utilizing an inducible-conditional knockout of Gsα, we found that deletion of the gene in hematopoietic bone marrow cells did not affect their ability to perform in the in vitro primitive CFU-C or LTC-IC assay systems. However, Gsα−/− cells were unable to establish effective hematopoiesis in the bone marrow microenvironment in vivo in a competitive repopulation assay (41.1% contribution from wild-type cells versus 1.4% from knockout cells). These effects were not due to an inability of the cells to function in the bone marrow in vivo as deletion of Gsα following establishment of hematopoiesis had no effects on the HSCs. Examining the ability of the HSCs to home to the bone marrow, though, demonstrated that deletion of Gsα resulted in a marked impairment of the ability of the stem cells to localize to the marrow space (approximately 9-fold reduction in the level of primitive cell homing). Furthermore, treatment of BM MNCs with an activator of Gsα augmented the cells homing and thus engraftment potential. These studies demonstrate that Gsα is critical to the localization of HSCs to the bone marrow. Which receptors utilize this pathway in this context remains unknown. However, Gsα represents a previously unrecognized signaling pathway for homing and engraftment of HSCs to bone marrow. Pharmacologic activation of Gsα in HSC ex vivo prior to transplantation offers a potential method for enhancing stem cell engraftment efficiency.

During development, hematopoietic stem cells (HSCs) translocate from the fetal liver to the bone marrow, which remains the site of hematopoiesis throughout adulthood. In the bone marrow the HSCs are located at the endosteal surface, where the osteoblasts are a key component of the stem cell niche. The exogenous signals that specifically direct HSCs to the bone marrow have been thought to include stimulation of the chemokine receptor CXCR4 by its cognate ligand stromal derived factor-1α (SDF-1α or CXCL12). However, experiments in which CXCR4−/−fetal liver hematopoietic cells were transplanted into wild-type hosts demonstrated efficient engraftment of the HSCs in the bone marrow. In addition, treatment of HSCs with inhibitors of Gαi-coupled signaling, which blocks transmigration towards SDF-1αin vitro, does not affect bone marrow homing and engraftment in vivo. Therefore, we examined whether Gsα-coupled mechanisms play a key role in the engraftment of the HSCs in the bone marrow environment. Utilizing an inducible-conditional knockout of Gsα, we found that deletion of the gene in hematopoietic bone marrow cells did not affect their ability to perform in the in vitroprimitive CFU-C or LTC-IC assay systems. However, Gsα−/−cells were unable to establish effective hematopoiesis in the bone marrow microenvironment in vivoin a competitive repopulation assay (41.1% contribution from wild-type cells versus 1.4% from knockout cells). These effects were not due to an inability of the cells to function in the bone marrow in vivoas deletion of Gsα following establishment of hematopoiesis had no effects on the HSCs. Examining the ability of the HSCs to home to the bone marrow, though, demonstrated that deletion of Gsα resulted in a marked impairment of the ability of the stem cells to localize to the marrow space (approximately 9-fold reduction in the level of primitive cell homing). Furthermore, treatment of BM MNCs with an activator of Gsα augmented the cells homing and thus engraftment potential. These studies demonstrate that Gsα is critical to the localization of HSCs to the bone marrow. Which receptors utilize this pathway in this context remains unknown. However, Gsα represents a previously unrecognized signaling pathway for homing and engraftment of HSCs to bone marrow. Pharmacologic activation of Gsα in HSC ex vivoprior to transplantation offers a potential method for enhancing stem cell engraftment efficiency.