Donor hematopoietic cells from transgenic mice that express GFP are immunogenic in immunocompetent recipients.

OBJECTIVE: To study the immunogenicity of hematopoietic cells marked with green fluorescence protein (GFP) while avoiding the potentially confounding effects of viral gene transduction, marked cells from GFP+ transgenic mice were tracked after transplantation into unconditioned immunocompetent recipients. MATERIALS AND METHODS: Marrow was harvested from GFP+ transgenic mice that had been crossed onto a BALB/cByJ background. Unconditioned marrow transplantation involved infusion of sex-matched or sex-mismatched cells into female BALB/cByJ hosts. Engraftment and contribution to circulating nucleated blood cells were compared to recipients of donor cells that were not GFP-marked. Donor cells were detected by flow cytometry (GFP) and fluorescence in situ hybridization (FISH) for Y-chromosome sequences. RESULTS: Donor cells from mice of the same genetic background that did not express GFP were detected for more than four-weeks in unconditioned recipients. In contrast, GFP-marked cells in the blood peaked at one-week, declined to undetectable levels by two-weeks and were not detected in the marrow at sacrifice. In sex-mismatched studies, detection of male GFP+ donor cells by FISH yielded levels similar to those observed by flow cytometry, in contrast to the levels detected for many weeks in mice infused with male cells that did not express GFP. In immunocompetent recipients immunized with irradiated GFP-expressing cells, rechallenge with GFP+ cells resulted in the accelerated loss of donor cells. CONCLUSION: Donor marrow cells from GFP+ transgenic mice were lost after infusion into unconditioned immunocompetent mice and sensitization studies infer an immunologic mechanism. These results are similar to studies of virally transduced cells. Thus, infusion of cells with optimum engraftment potential could not compensate for the loss of donor cells due to immunogenicity.

Donor stem cells home to marrow efficiently and contribute to short- and long-term hematopoiesis after low-cell-dose unconditioned bone marrow transplantation.

OBJECTIVE: To study the homing of donor stem cells, their contribution to short- and long-term hematopoiesis, and their functional capacity in an unconditioned marrow transplant model using a clinically relevant low marrow cell dose. MATERIALS AND METHODS: A sex-mismatch model was used in which 20 x 10(6) male Balb/cByJ cells were infused into unconditioned female Balb/cByJ mice. Male donor cells were detected in blood and marrow by fluorescence in situ hybridization. Serial transplantation into irradiated secondary recipients was used to assay donor stem cells. Donor hematopoiesis was stimulated after transplant by treating recipients with either stem cell factor (SCF) or sublethal irradiation. RESULTS: Donor-derived male cells were detected in recipient blood (1.2-3.0%) and marrow (2.4-5.4%) for up to 27 weeks, indicating a contribution to short-term hematopoiesis. Male repopulating units approached theoretically expected values, suggesting that transplantation was efficient. Donor stem cell levels after serial transplantation were similar to results observed in primary recipients. Donor stem cell levels 24 hours after infusion (1.3 +/- 0.2% SE) were similar to levels at later time points, indicating that homing was efficient. Stimulation by either SCF or sublethal irradiation after transplant did not alter donor marrow chimerism, suggesting that donor stem cells responded to the stress similarly to endogenous cells. CONCLUSION: We conclude that in a clinically relevant low-cell-dose unconditioned transplant model, stem cells home to the marrow efficiently, contribute to both short- and long-term hematopoiesis, and, once engrafted, respond to stress effectively. These findings provide a rationale for the use of genetically modified stem cells in clinical protocols that omit intensive conditioning.