Fetal Immune Suppression as Adjunctive Therapy for In Utero Hematopoietic Stem Cell Transplantation in Nonhuman Primates
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《干细胞学杂志》
b Regional Primate Research Center, and
c Clinical Research Division, Fred Hutchinson Cancer Research Center, and Department of Pediatrics, University of Washington, Seattle, Washington, USA
Key Words. Non-human primate ? Fetal transplantation ? Immune suppression ? Adult stem cells ? Hematopoietic chimerism
Correspondence: Laurence E. Shields, M.D., Department of OB-GYN, Division of Perinatal Medicine, Box 356460, University of Washington, Seattle, Washington 98105–6460 USA. Telephone: 206-543-3714; Fax: 206-616-9479; e-mail: lshields@u.washington.edu
ABSTRACT
Many diseases that affect normal fetal hematopoietic and immune function result in fetal death ( thalassemia), severe central nervous system developmental abnormalities at birth (leukodystrophies), or lifelong morbidity (sickle cell disease and ? thalassemia) . Many of these disorders can be cured by postnatal bone marrow transplantation. Development of successful in utero hematopoietic stem-cell transplantation protocols would be advantageous, particularly for those disorders that result in fetal death or significant impairment at the time of birth. Naturally occurring models of in utero hematopoietic transplantation and preclinical animal models, primarily in fetal sheep, have suggested that clinical trials in the human fetus should be successful . More than 40 attempts of human fetal transplantation have been reported for a variety of different diseases. The theoretical advantages to fetal transplantation (immature immune system and expanding hematopoietic environment) have been used to describe the fetus as the perfect recipient for hematopoietic transplantation . Unfortunately, evidence of engraftment and disease improvement has been demonstrated only in fetuses with severe immunologic deficiencies .
Why attempts of in utero stem cell transplantation for fetuses with normal immune development have failed has not been clearly determined. However, available data have demonstrated that, even in the fetus, there are significant barriers to donor cell engraftment. It is likely that the barriers to achieving clinically relevant levels of engraftment prenatally are similar to those for postnatal stem cell transplantation—that is, graft failure from an inadequate number of donor stem cells, immune rejection of donor cells by the fetus, inadequate hematopoietic space for donor cell engraftment, or inferior competitiveness of donor cells relative to the autologous fetal hematopoietic stem cells.
Our group and others have shown that fetal recipients of adult CD34+-enriched or T cell–depleted marrow produces engraftment in nonhuman primates. Tolerance has also been demonstrated in these animals by reduced mixed lymphocyte culture responses and prolonged solid organ graft survival. Unfortunately, the levels of chimerism achieved in both of these models have been low and would not be expected to correct any of the target diseases for in utero hematopoietic therapy. We explored two methods of potentially increasing the level of chimerism achieved after in utero hematopoietic transplantation: (a) fetal immune suppression as an adjunctive to fetal transplantation and (b) post-natal infusion of donor hematopoietic progenitor/stem cells.
MATERIALS AND METHODS
A total of five fetuses treated with the combination of ATG and betamethasone were transplanted with haploidentical, allogeneic CD34+ cells enriched from bone marrow of the sire. The total CD34+ (3.7 x 109/kg) and CD2+ (1.6 x 107/kg) donor cell number was similar to another group of animals similarly transplanted that did not receive immunosuppression (Table 1) .
Two of the five animals treated with immune suppression were electively delivered prior to viability, and three animals were delivered at full-term gestation by elective cesarean section. The first of the preterm fetuses was electively delivered at 120 days (0.70 gestation) after routine ultrasound monitoring of the fetus demonstrated oligohydramnios and large echogenic kidneys. These findings were consistent with the infantile polycystic kidney disease, a lethal disorder that has been previously described in the macaque species . Pathological examination of the kidneys confirmed these findings and the absence of GVHD. The second preterm animal was delivered at 77 days (0.45 gestation) when fetal ascites was noted at the time of the scheduled third intrauterine injection. This fetus did not receive the third cell infusion and was delivered 3 days later after persistence of the ascites was noted. Pathologic examination did not identify any abnormalities of the abdominal structures, and there was no evidence of GVHD. Hematopoietic tissue from both of these animals was obtained, and demonstrated high levels of chimerism in fetal liver of the younger animal and in the marrow and cord blood of the older animal. Of the three animals delivered at term by cesarean section, one was euthanized at 19 months of age due to chronic osteomyelitis involving the right femur. The other two animals are alive and well at 14 and 16 months of age.
Influence of Fetal Immunosuppression on Initial Engraftment of Hematopoietic Progenitors
The two animals that were electively delivered prior to viability demonstrated high levels of chimerism within fetal liver, marrow, and peripheral blood (Table 2). In the younger fetus (0.45 gestation) 17% of fetal liver progenitors (CFCs) and 2.7% of all fetal liver cells were of donor origin, suggesting a high level of initial engraftment in that organ. Cells obtained from the marrow plated at the same time yielded no CFCs. The older fetus (0.70 gestation) demonstrated donor cells in both peripheral blood (34% CFCs) and marrow (43% CFCs and 0.15% total cells), but not in the fetal liver.
Table 2. Colony-forming cells (CFCs) from immune-suppressed preterm fetuses
In the three immunosuppressed animals delivered at term, initial evaluation of chimerism in the progenitor population (single CD34+ cell cultures) suggested that engraftment at birth was higher than that observed in the group of animals that did not receive immunosuppression (11.3% ± 2.7% and 5.1% ± 1.5%, respectively; p = .057) (Fig. 1, Table 3). Long-term follow-up of these animals, at 14, 16, and 19 months, respectively, demonstrated that the level of chimerism in both the marrow (p = .02) and FACS-purified CD34+ population (p = .01) was significantly higher in the immune-suppressed animals relative to controls. The proportion of donor-derived colonies formed from single CD34+ cells showed a trend toward higher levels in the immunosuppressed animals (12.0% ± 7.5% versus 5.0% ± 2.6%, respectively; p = .8) (Table 4). Nevertheless, the absolute number of donor cells in the marrow remained low.
Figure 1. Comparison of chimerism at birth in the hematopoietic progenitor population (single CD34+ cell cultures) in fetuses treated with (n = 3) and without (n = 7) immune suppression. The values are mean ± SEM (p = .06).
Table 3. Initial evaluation of chimerism in animals treated with immune suppression and delivered at terma
Table 4. Follow-up of chimerism (%) in marrow, progenitors, PBL, and FACS-purified PBL lineage cells
Influence of Fetal Immunosuppression on Peripheral Blood Chimerism
In addition to the higher levels of chimerism in both marrow and in marrow CD34+ progenitors in the immune-suppressed animals, there also was a trend toward higher levels of donor cells in the peripheral blood (p = .10) (Table 4). Even though the level of chimerism in peripheral blood was up to 10-fold higher in immune-suppressed than in control animals, the overall frequency of donor cells in peripheral blood was generally low (<1%) and would be unlikely to have clinical relevance. The one exception to this finding was in the CD13+ cell (5.2%) population in one animal (M01-088). This animal also had the highest level of progenitor cell chimerism. Unfortunately, at the time of that collection (19 months of age), this animal was euthanized for a bone lesion in one femur that was consistent with chronic osteomyelitis by pathological evaluation, and additional samples could not be obtained.
Influence of Postnatal Donor Cell Infusion on Chimerism
Three animals were treated postnatally with additional donor cell infusions to test the hypothesis that chimerism could be increased using this methodology . The first animal (M00-025) received CD34+-enriched cells (2.2 x 109/kg and 3.6 x 107/kg CD3+ cells) in utero and was in the cohort of animals we have previously reported . The other two animals were from the group of animals that was treated with in utero immune suppression. Animal K00-025 had an initial level of chimerism in the progenitor population of 6.0% at birth, which subsequently declined to 1.3% at 8 months of age. Tolerance, demonstrated by the absence of a mixed lymphocyte culture (MLC) response to the sire, was noted at 1, 4, and 7 months of age. This animal received three monthly infusions of T cell–depleted marrow cells. The average CD34+ cell dose per infusion was 7.3 x 107/kg (total = 21.9 x 107/kg and 3.6 x 106/kg CD2+ cells). Chimerism in the progenitor population had increased to 11.5% by 1 month after the first donor cell infusion. However, 18 months after the third infusion, the level of chimerism decreased to a level that was similar to the preboost level (2.4%). In addition, MLC responses were similar for both the dam and sire (donor). The other two animals received two infusions from growth factor–stimulated CD34+-enriched marrow cells. The first and second infusion was separated by 1 month. The total cell dose for each animal was 2 x 107/kg CD34+ and 1.0 x 105/kg CD2+. Five-fluorouracil (50 mg/kg), although not at a level that would produce myeloablation, was given 7 days prior to donor cell infusions in an attempt to reduce endogenous hematopoiesis. Because enriched donor cells would be antibody-coated, the animals were pretreated with a single course of prednisone (2 mg/kg) 24 hours prior to donor cell infusions. Although there was an increase in the level of chimerism after the first donor cell infusion, by 6 months after the second donor cell infusion, chimerism in the progenitor compartment and the peripheral blood was similar to that noted before reinfusion therapy (Fig. 2).
Figure 2. Data from pre- and postnatal booster therapy in animals M01-157 (top) and M01-207 (bottom). The data are from preinfusion, 1 month postinfusion, and 6 months after the second (last) infusion.
DISCUSSION
Shields LE, Lindton B, Andrews RG et al. Fetal hematopoietic stem cell transplantation: a challenge for the twenty-first century. J Hematother Stem Cell Res 2002;11:617–631.
Flake AW, Zanjani ED. In utero hematopoietic stem cell transplantation: ontogenic opportunities and biologic barriers. Blood 1999;94:2179–2191.
Jolly RD, Thompson KG, Murphy CE et al. Enzyme replacement therapy: an experiment of nature in a chimeric mannosidosis calf. Pediatr Res 1976;10:219–224.
Picus J, Aldrich WR, Letvin NL. A naturally occurring bone-marrow chimeric primate. Integrity of its immune system. Transplantation 1985;39:297–303.
Zanjani E, Ascensao JL, Flake AW et al. The fetus as an optimal donor and recipient of hemopoietic stem cells. Bone Marrow Transplant 1992;10:107–114.
Touraine JL, Roncarolo MG, Bacchetta R et al. Fetal liver transplantation: biology and clinical results. Bone Marrow Transplant 1993;1:119–122.
Westgren M, Ringden O, Bartmann P et al. Prenatal T-cell reconstruction after in utero transplantation with fetal liver cells in a patient with X-linked severe combined immunodeficiency. Am J Obstet Gynecol 2002;187:475–482.
Wengler GS, Lanfranchi A, Frusca T et al. In-utero transplantation of parental CD34 haematopoietic progenitor cells in a patient with X-linked severe combined immunodeficiency (SCIDXI). Lancet 1996;348:1484–1487.
Flake AW, Roncarolo MG, Puck JM et al. Treatment of X-linked severe combined immunodeficiency by in utero transplantation of paternal bone marrow. N Engl J Med 1996;335:1806–1810.
Shields LE, Gaur LK, Gough M et al. In utero hematopoietic stem cell transplantation in nonhuman primates: the role of T cells. STEM CELLS 2003;21:304–314.
Cowan MJ, Tarantal AF, Capper J et al. Long-term engraftment following in utero T cell-depleted parental marrow transplantation into fetal rhesus monkeys. Bone Marrow Transplant 1996;17:1157–1165.
Conrad S, Ha J, Lohr C et al. Ultrasound measurement of fetal growth in Macaca nemestrina. Am J Primatol 1995;36:15–00.
DeVito JL, Graham J, Sackett GP. Volumetric growth of the major brain divisions in fetal Macaca nemestrina. J Hirnforsch 1989;30:479–487.
Westgren M, Shields LE. In utero stem cell transplantation in humans. Ernst Schering Res Found Workshop 2001;33:197–221.
Shields LE, Bryant EM, Easterling TR et al. Fetal liver cell transplantation for the creation of lymphohematopoietic chimerism in fetal baboons. Am J Obstet Gynecol 1995;173:1157–1160.
Andrews RG, Torok-Storb B, Bernstein ID. Myeloid-associated differentiation antigens on stem cells and their progeny identified by monoclonal antibodies. Blood 1983;62:124–132.
Andrews RG, Singer JW, Bernstein ID. Precursors of colony-forming cells in humans can be distinguished from colony-forming cells by expression of the CD33 and CD34 antigens and light scatter properties. J Exp Med 1989;169:1721–1731.
Andrews RG, Peterson LJ, Morris J et al. Differential engraftment of genetically modified CD34(+) and CD34(–) hematopoietic cell subsets in lethally irradiated baboons. Exp Hematol 2000;28:508–518.
Reitsma MJ, Harrison MR, Pallavicini MG. Detection of a male-specific sequence in nonhuman primates through use of the polymerase chain reaction. Cytogenet Cell Genet 1993;64:213–216.
Horn PA, Topp MS, Morris JC et al. Highly efficient gene transfer into baboon marrow repopulating cells using GALV-pseudotype oncoretroviral vectors produced by human packaging cells. Blood 2002;100:3960–3967.
Allen MD, Weyhrich J, Gaur L et al. Prolonged allogeneic and xenogeneic microchimerism in unmatched primates without immunosuppression by intrathymic implantation of CD34+ donor marrow cells. Cell Immunol 1997;181:127–138.
Brent L, Linch DC, Rodeck CH et al. On the feasibility of inducing tolerance in man: a study in the cynomolgus monkey. Immunol Lett 1989;21:55–61.
Sakakibara I, Honjo S. Spontaneously occurring congenital polycystic kidney in a cynomolgus monkey (Macaca fascicularis). J Med Primatol 1990;19:501–506.
Baskin GB, Roberts JA, McAfee RD. Infantile polycystic renal disease in a rhesus monkey (Macaca mulatta). Lab Anim Sci 1981;31:181–183.
Zanjani ED, Ruthven A, Ruthven J et al. In utero hematopoietic stem cell transplantation results in donor specific tolerance and facilitates postnatal "boosting" of donor cell levels. Blood 1994;84:100a–0000.
Milner R, Shaaban A, Kim HB et al. Postnatal booster injections increase engraftment after in utero stem cell transplantation. J Surg Res 1999;83:44–47.
Carrier E, Lee TH, Busch MP et al. Induction of tolerance in nondefective mice after in utero transplantation of major histocompatibility complex-mismatched fetal hematopoietic stem cells. Blood 1995;86:4681–4690.
Donahue J, Gilpin E, Young D et al. Postnatal cytokines and boosts improve chimerism and hematological parameters in beta-thalassemic mice transplanted in utero. Transplantation 2001;71:1491–1494.
Touraine JL. In utero transplantation of fetal liver stem cells in humans. Blood Cells 1991;17:379–387.
Ek S, Ringden O, Markling L et al. Immunological capacity of human fetal liver cells. Bone Marrow Transplant 1994;14:9–14.
Lindton B, Markling L, Ringden O et al. Mixed lymphocyte culture of human fetal liver cells. Fetal Diagn Ther 2000;15:71–78.
Stites DP, Carr MC, Fudenberg HH. Ontogeny of cellular immunity in the human fetus: development of responses to phytohemagglutinin and to allogeneic cells. Cell Immunol 1974;11:257–271.
Almeida-Porada G, Flake AW, Glimp HA et al. Cotransplantation of stroma results in enhancement of engraftment and early expression of donor hematopoietic stem cells in utero. Exp Hematol 1999;27:1569–1575.
Hajdu K, Tanigawara S, McLean LK et al. In utero allogeneic hematopoietic stem cell transplantation to induce tolerance. Fetal Diagn Ther 1996;11:241–248.
Hayashi S, Peranteau WH, Shaaban AF et al. Complete allogeneic hematopoietic chimerism achieved by a combined strategy of in utero hematopoietic stem cell transplantation and postnatal donor lymphocyte infusion. Blood 2002;100:804–812.
Carrier E, Gilpin E, Lee TH et al. Microchimerism does not induce tolerance after in utero transplantation and may lead to the development of alloreactivity. J Lab Clin Med 2000;136:224–235.
Donahue J, Gilpin E, Lee TH et al. Microchimerism does not induce tolerance and sustains immunity after in utero transplantation. Transplantation 2001;71:359–368.
Yuh DD, Gandy KL, Hoyt G et al. Tolerance to cardiac allografts induced in utero with fetal liver cells. Circulation 1996;94(suppl II):304–307.
Chou SH, Chawla A, Lee TH et al. Increased engraftment and GVHD after in utero transplantation of MHC-mismatched bone marrow cells and CD80low, CD86(–) dendritic cells in a fetal mouse model. Transplantation 2001;72:1768–1776.
Bhattacharyya S, Chawla A, Smith K et al. Multilineage engraftment with minimal graft-versus-host disease following in utero transplantation of S-59 psoralen/ultraviolet a light-treated, sensitized T cells and adult T cell-depleted bone marrow in fetal mice. J Immunol 2002;169:6133–6140.
Crombleholme TM, Harrison MR, Zanjani ED. In utero transplantation of hematopoietic stem cells in sheep: the role of T cells in engraftment and graft-versus-host disease. J Pediatr Surg 1990;25:885–892.
Bambach BJ, Moser HW, Blakemore K et al. Engraftment following in utero bone marrow transplantation for globoid cell leukodystrophy. Bone Marrow Transplant 1997;19:399–402.
Almeida-Porada G, Porada CD, Tran N et al. Cotransplantation of human stromal cell progenitors into preimmune fetal sheep results in early appearance of human donor cells in circulation and boosts cell levels in bone marrow at later time points after transplantation. Blood 2000;95:3620–3627.
Sugiura K, Hisha H, Ishikawa J et al. Major histocompatibility complex restriction between hematopoietic stem cells and stromal cells in vitro. STEM CELLS 2001;19:46–58.
Bartholomew A, Sturgeon C, Siatskas M et al. Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo. Exp Hematol 2002;30:42–48.
Gotherstrom C, Ringden O,Westgren M et al. Immunomodulatory effects of human foetal liver-derived mesenchymal stem cells. Bone Marrow Transplant 2003;32:265–272.
Ruggeri L, Capanni M, Urbani E et al. Effectiveness of donor natural killer cell alloreactivity in mismatched hematopoietic transplants. Science 2002;295:2097–2100.
Ruggeri L, Capanni M, Martelli MF et al. Cellular therapy: exploiting NK cell alloreactivity in transplantation. Curr Opin Hematol 2001;8:355–359.(Laurence E. Shieldsa,b, L)
c Clinical Research Division, Fred Hutchinson Cancer Research Center, and Department of Pediatrics, University of Washington, Seattle, Washington, USA
Key Words. Non-human primate ? Fetal transplantation ? Immune suppression ? Adult stem cells ? Hematopoietic chimerism
Correspondence: Laurence E. Shields, M.D., Department of OB-GYN, Division of Perinatal Medicine, Box 356460, University of Washington, Seattle, Washington 98105–6460 USA. Telephone: 206-543-3714; Fax: 206-616-9479; e-mail: lshields@u.washington.edu
ABSTRACT
Many diseases that affect normal fetal hematopoietic and immune function result in fetal death ( thalassemia), severe central nervous system developmental abnormalities at birth (leukodystrophies), or lifelong morbidity (sickle cell disease and ? thalassemia) . Many of these disorders can be cured by postnatal bone marrow transplantation. Development of successful in utero hematopoietic stem-cell transplantation protocols would be advantageous, particularly for those disorders that result in fetal death or significant impairment at the time of birth. Naturally occurring models of in utero hematopoietic transplantation and preclinical animal models, primarily in fetal sheep, have suggested that clinical trials in the human fetus should be successful . More than 40 attempts of human fetal transplantation have been reported for a variety of different diseases. The theoretical advantages to fetal transplantation (immature immune system and expanding hematopoietic environment) have been used to describe the fetus as the perfect recipient for hematopoietic transplantation . Unfortunately, evidence of engraftment and disease improvement has been demonstrated only in fetuses with severe immunologic deficiencies .
Why attempts of in utero stem cell transplantation for fetuses with normal immune development have failed has not been clearly determined. However, available data have demonstrated that, even in the fetus, there are significant barriers to donor cell engraftment. It is likely that the barriers to achieving clinically relevant levels of engraftment prenatally are similar to those for postnatal stem cell transplantation—that is, graft failure from an inadequate number of donor stem cells, immune rejection of donor cells by the fetus, inadequate hematopoietic space for donor cell engraftment, or inferior competitiveness of donor cells relative to the autologous fetal hematopoietic stem cells.
Our group and others have shown that fetal recipients of adult CD34+-enriched or T cell–depleted marrow produces engraftment in nonhuman primates. Tolerance has also been demonstrated in these animals by reduced mixed lymphocyte culture responses and prolonged solid organ graft survival. Unfortunately, the levels of chimerism achieved in both of these models have been low and would not be expected to correct any of the target diseases for in utero hematopoietic therapy. We explored two methods of potentially increasing the level of chimerism achieved after in utero hematopoietic transplantation: (a) fetal immune suppression as an adjunctive to fetal transplantation and (b) post-natal infusion of donor hematopoietic progenitor/stem cells.
MATERIALS AND METHODS
A total of five fetuses treated with the combination of ATG and betamethasone were transplanted with haploidentical, allogeneic CD34+ cells enriched from bone marrow of the sire. The total CD34+ (3.7 x 109/kg) and CD2+ (1.6 x 107/kg) donor cell number was similar to another group of animals similarly transplanted that did not receive immunosuppression (Table 1) .
Two of the five animals treated with immune suppression were electively delivered prior to viability, and three animals were delivered at full-term gestation by elective cesarean section. The first of the preterm fetuses was electively delivered at 120 days (0.70 gestation) after routine ultrasound monitoring of the fetus demonstrated oligohydramnios and large echogenic kidneys. These findings were consistent with the infantile polycystic kidney disease, a lethal disorder that has been previously described in the macaque species . Pathological examination of the kidneys confirmed these findings and the absence of GVHD. The second preterm animal was delivered at 77 days (0.45 gestation) when fetal ascites was noted at the time of the scheduled third intrauterine injection. This fetus did not receive the third cell infusion and was delivered 3 days later after persistence of the ascites was noted. Pathologic examination did not identify any abnormalities of the abdominal structures, and there was no evidence of GVHD. Hematopoietic tissue from both of these animals was obtained, and demonstrated high levels of chimerism in fetal liver of the younger animal and in the marrow and cord blood of the older animal. Of the three animals delivered at term by cesarean section, one was euthanized at 19 months of age due to chronic osteomyelitis involving the right femur. The other two animals are alive and well at 14 and 16 months of age.
Influence of Fetal Immunosuppression on Initial Engraftment of Hematopoietic Progenitors
The two animals that were electively delivered prior to viability demonstrated high levels of chimerism within fetal liver, marrow, and peripheral blood (Table 2). In the younger fetus (0.45 gestation) 17% of fetal liver progenitors (CFCs) and 2.7% of all fetal liver cells were of donor origin, suggesting a high level of initial engraftment in that organ. Cells obtained from the marrow plated at the same time yielded no CFCs. The older fetus (0.70 gestation) demonstrated donor cells in both peripheral blood (34% CFCs) and marrow (43% CFCs and 0.15% total cells), but not in the fetal liver.
Table 2. Colony-forming cells (CFCs) from immune-suppressed preterm fetuses
In the three immunosuppressed animals delivered at term, initial evaluation of chimerism in the progenitor population (single CD34+ cell cultures) suggested that engraftment at birth was higher than that observed in the group of animals that did not receive immunosuppression (11.3% ± 2.7% and 5.1% ± 1.5%, respectively; p = .057) (Fig. 1, Table 3). Long-term follow-up of these animals, at 14, 16, and 19 months, respectively, demonstrated that the level of chimerism in both the marrow (p = .02) and FACS-purified CD34+ population (p = .01) was significantly higher in the immune-suppressed animals relative to controls. The proportion of donor-derived colonies formed from single CD34+ cells showed a trend toward higher levels in the immunosuppressed animals (12.0% ± 7.5% versus 5.0% ± 2.6%, respectively; p = .8) (Table 4). Nevertheless, the absolute number of donor cells in the marrow remained low.
Figure 1. Comparison of chimerism at birth in the hematopoietic progenitor population (single CD34+ cell cultures) in fetuses treated with (n = 3) and without (n = 7) immune suppression. The values are mean ± SEM (p = .06).
Table 3. Initial evaluation of chimerism in animals treated with immune suppression and delivered at terma
Table 4. Follow-up of chimerism (%) in marrow, progenitors, PBL, and FACS-purified PBL lineage cells
Influence of Fetal Immunosuppression on Peripheral Blood Chimerism
In addition to the higher levels of chimerism in both marrow and in marrow CD34+ progenitors in the immune-suppressed animals, there also was a trend toward higher levels of donor cells in the peripheral blood (p = .10) (Table 4). Even though the level of chimerism in peripheral blood was up to 10-fold higher in immune-suppressed than in control animals, the overall frequency of donor cells in peripheral blood was generally low (<1%) and would be unlikely to have clinical relevance. The one exception to this finding was in the CD13+ cell (5.2%) population in one animal (M01-088). This animal also had the highest level of progenitor cell chimerism. Unfortunately, at the time of that collection (19 months of age), this animal was euthanized for a bone lesion in one femur that was consistent with chronic osteomyelitis by pathological evaluation, and additional samples could not be obtained.
Influence of Postnatal Donor Cell Infusion on Chimerism
Three animals were treated postnatally with additional donor cell infusions to test the hypothesis that chimerism could be increased using this methodology . The first animal (M00-025) received CD34+-enriched cells (2.2 x 109/kg and 3.6 x 107/kg CD3+ cells) in utero and was in the cohort of animals we have previously reported . The other two animals were from the group of animals that was treated with in utero immune suppression. Animal K00-025 had an initial level of chimerism in the progenitor population of 6.0% at birth, which subsequently declined to 1.3% at 8 months of age. Tolerance, demonstrated by the absence of a mixed lymphocyte culture (MLC) response to the sire, was noted at 1, 4, and 7 months of age. This animal received three monthly infusions of T cell–depleted marrow cells. The average CD34+ cell dose per infusion was 7.3 x 107/kg (total = 21.9 x 107/kg and 3.6 x 106/kg CD2+ cells). Chimerism in the progenitor population had increased to 11.5% by 1 month after the first donor cell infusion. However, 18 months after the third infusion, the level of chimerism decreased to a level that was similar to the preboost level (2.4%). In addition, MLC responses were similar for both the dam and sire (donor). The other two animals received two infusions from growth factor–stimulated CD34+-enriched marrow cells. The first and second infusion was separated by 1 month. The total cell dose for each animal was 2 x 107/kg CD34+ and 1.0 x 105/kg CD2+. Five-fluorouracil (50 mg/kg), although not at a level that would produce myeloablation, was given 7 days prior to donor cell infusions in an attempt to reduce endogenous hematopoiesis. Because enriched donor cells would be antibody-coated, the animals were pretreated with a single course of prednisone (2 mg/kg) 24 hours prior to donor cell infusions. Although there was an increase in the level of chimerism after the first donor cell infusion, by 6 months after the second donor cell infusion, chimerism in the progenitor compartment and the peripheral blood was similar to that noted before reinfusion therapy (Fig. 2).
Figure 2. Data from pre- and postnatal booster therapy in animals M01-157 (top) and M01-207 (bottom). The data are from preinfusion, 1 month postinfusion, and 6 months after the second (last) infusion.
DISCUSSION
Shields LE, Lindton B, Andrews RG et al. Fetal hematopoietic stem cell transplantation: a challenge for the twenty-first century. J Hematother Stem Cell Res 2002;11:617–631.
Flake AW, Zanjani ED. In utero hematopoietic stem cell transplantation: ontogenic opportunities and biologic barriers. Blood 1999;94:2179–2191.
Jolly RD, Thompson KG, Murphy CE et al. Enzyme replacement therapy: an experiment of nature in a chimeric mannosidosis calf. Pediatr Res 1976;10:219–224.
Picus J, Aldrich WR, Letvin NL. A naturally occurring bone-marrow chimeric primate. Integrity of its immune system. Transplantation 1985;39:297–303.
Zanjani E, Ascensao JL, Flake AW et al. The fetus as an optimal donor and recipient of hemopoietic stem cells. Bone Marrow Transplant 1992;10:107–114.
Touraine JL, Roncarolo MG, Bacchetta R et al. Fetal liver transplantation: biology and clinical results. Bone Marrow Transplant 1993;1:119–122.
Westgren M, Ringden O, Bartmann P et al. Prenatal T-cell reconstruction after in utero transplantation with fetal liver cells in a patient with X-linked severe combined immunodeficiency. Am J Obstet Gynecol 2002;187:475–482.
Wengler GS, Lanfranchi A, Frusca T et al. In-utero transplantation of parental CD34 haematopoietic progenitor cells in a patient with X-linked severe combined immunodeficiency (SCIDXI). Lancet 1996;348:1484–1487.
Flake AW, Roncarolo MG, Puck JM et al. Treatment of X-linked severe combined immunodeficiency by in utero transplantation of paternal bone marrow. N Engl J Med 1996;335:1806–1810.
Shields LE, Gaur LK, Gough M et al. In utero hematopoietic stem cell transplantation in nonhuman primates: the role of T cells. STEM CELLS 2003;21:304–314.
Cowan MJ, Tarantal AF, Capper J et al. Long-term engraftment following in utero T cell-depleted parental marrow transplantation into fetal rhesus monkeys. Bone Marrow Transplant 1996;17:1157–1165.
Conrad S, Ha J, Lohr C et al. Ultrasound measurement of fetal growth in Macaca nemestrina. Am J Primatol 1995;36:15–00.
DeVito JL, Graham J, Sackett GP. Volumetric growth of the major brain divisions in fetal Macaca nemestrina. J Hirnforsch 1989;30:479–487.
Westgren M, Shields LE. In utero stem cell transplantation in humans. Ernst Schering Res Found Workshop 2001;33:197–221.
Shields LE, Bryant EM, Easterling TR et al. Fetal liver cell transplantation for the creation of lymphohematopoietic chimerism in fetal baboons. Am J Obstet Gynecol 1995;173:1157–1160.
Andrews RG, Torok-Storb B, Bernstein ID. Myeloid-associated differentiation antigens on stem cells and their progeny identified by monoclonal antibodies. Blood 1983;62:124–132.
Andrews RG, Singer JW, Bernstein ID. Precursors of colony-forming cells in humans can be distinguished from colony-forming cells by expression of the CD33 and CD34 antigens and light scatter properties. J Exp Med 1989;169:1721–1731.
Andrews RG, Peterson LJ, Morris J et al. Differential engraftment of genetically modified CD34(+) and CD34(–) hematopoietic cell subsets in lethally irradiated baboons. Exp Hematol 2000;28:508–518.
Reitsma MJ, Harrison MR, Pallavicini MG. Detection of a male-specific sequence in nonhuman primates through use of the polymerase chain reaction. Cytogenet Cell Genet 1993;64:213–216.
Horn PA, Topp MS, Morris JC et al. Highly efficient gene transfer into baboon marrow repopulating cells using GALV-pseudotype oncoretroviral vectors produced by human packaging cells. Blood 2002;100:3960–3967.
Allen MD, Weyhrich J, Gaur L et al. Prolonged allogeneic and xenogeneic microchimerism in unmatched primates without immunosuppression by intrathymic implantation of CD34+ donor marrow cells. Cell Immunol 1997;181:127–138.
Brent L, Linch DC, Rodeck CH et al. On the feasibility of inducing tolerance in man: a study in the cynomolgus monkey. Immunol Lett 1989;21:55–61.
Sakakibara I, Honjo S. Spontaneously occurring congenital polycystic kidney in a cynomolgus monkey (Macaca fascicularis). J Med Primatol 1990;19:501–506.
Baskin GB, Roberts JA, McAfee RD. Infantile polycystic renal disease in a rhesus monkey (Macaca mulatta). Lab Anim Sci 1981;31:181–183.
Zanjani ED, Ruthven A, Ruthven J et al. In utero hematopoietic stem cell transplantation results in donor specific tolerance and facilitates postnatal "boosting" of donor cell levels. Blood 1994;84:100a–0000.
Milner R, Shaaban A, Kim HB et al. Postnatal booster injections increase engraftment after in utero stem cell transplantation. J Surg Res 1999;83:44–47.
Carrier E, Lee TH, Busch MP et al. Induction of tolerance in nondefective mice after in utero transplantation of major histocompatibility complex-mismatched fetal hematopoietic stem cells. Blood 1995;86:4681–4690.
Donahue J, Gilpin E, Young D et al. Postnatal cytokines and boosts improve chimerism and hematological parameters in beta-thalassemic mice transplanted in utero. Transplantation 2001;71:1491–1494.
Touraine JL. In utero transplantation of fetal liver stem cells in humans. Blood Cells 1991;17:379–387.
Ek S, Ringden O, Markling L et al. Immunological capacity of human fetal liver cells. Bone Marrow Transplant 1994;14:9–14.
Lindton B, Markling L, Ringden O et al. Mixed lymphocyte culture of human fetal liver cells. Fetal Diagn Ther 2000;15:71–78.
Stites DP, Carr MC, Fudenberg HH. Ontogeny of cellular immunity in the human fetus: development of responses to phytohemagglutinin and to allogeneic cells. Cell Immunol 1974;11:257–271.
Almeida-Porada G, Flake AW, Glimp HA et al. Cotransplantation of stroma results in enhancement of engraftment and early expression of donor hematopoietic stem cells in utero. Exp Hematol 1999;27:1569–1575.
Hajdu K, Tanigawara S, McLean LK et al. In utero allogeneic hematopoietic stem cell transplantation to induce tolerance. Fetal Diagn Ther 1996;11:241–248.
Hayashi S, Peranteau WH, Shaaban AF et al. Complete allogeneic hematopoietic chimerism achieved by a combined strategy of in utero hematopoietic stem cell transplantation and postnatal donor lymphocyte infusion. Blood 2002;100:804–812.
Carrier E, Gilpin E, Lee TH et al. Microchimerism does not induce tolerance after in utero transplantation and may lead to the development of alloreactivity. J Lab Clin Med 2000;136:224–235.
Donahue J, Gilpin E, Lee TH et al. Microchimerism does not induce tolerance and sustains immunity after in utero transplantation. Transplantation 2001;71:359–368.
Yuh DD, Gandy KL, Hoyt G et al. Tolerance to cardiac allografts induced in utero with fetal liver cells. Circulation 1996;94(suppl II):304–307.
Chou SH, Chawla A, Lee TH et al. Increased engraftment and GVHD after in utero transplantation of MHC-mismatched bone marrow cells and CD80low, CD86(–) dendritic cells in a fetal mouse model. Transplantation 2001;72:1768–1776.
Bhattacharyya S, Chawla A, Smith K et al. Multilineage engraftment with minimal graft-versus-host disease following in utero transplantation of S-59 psoralen/ultraviolet a light-treated, sensitized T cells and adult T cell-depleted bone marrow in fetal mice. J Immunol 2002;169:6133–6140.
Crombleholme TM, Harrison MR, Zanjani ED. In utero transplantation of hematopoietic stem cells in sheep: the role of T cells in engraftment and graft-versus-host disease. J Pediatr Surg 1990;25:885–892.
Bambach BJ, Moser HW, Blakemore K et al. Engraftment following in utero bone marrow transplantation for globoid cell leukodystrophy. Bone Marrow Transplant 1997;19:399–402.
Almeida-Porada G, Porada CD, Tran N et al. Cotransplantation of human stromal cell progenitors into preimmune fetal sheep results in early appearance of human donor cells in circulation and boosts cell levels in bone marrow at later time points after transplantation. Blood 2000;95:3620–3627.
Sugiura K, Hisha H, Ishikawa J et al. Major histocompatibility complex restriction between hematopoietic stem cells and stromal cells in vitro. STEM CELLS 2001;19:46–58.
Bartholomew A, Sturgeon C, Siatskas M et al. Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo. Exp Hematol 2002;30:42–48.
Gotherstrom C, Ringden O,Westgren M et al. Immunomodulatory effects of human foetal liver-derived mesenchymal stem cells. Bone Marrow Transplant 2003;32:265–272.
Ruggeri L, Capanni M, Urbani E et al. Effectiveness of donor natural killer cell alloreactivity in mismatched hematopoietic transplants. Science 2002;295:2097–2100.
Ruggeri L, Capanni M, Martelli MF et al. Cellular therapy: exploiting NK cell alloreactivity in transplantation. Curr Opin Hematol 2001;8:355–359.(Laurence E. Shieldsa,b, L)