Alloreactivity following in utero transplantation of cytokine-stimulated hematopoietic stem cells: the role of recipient CD4(-) cells.
OBJECTIVE
We have previously reported immunity to donor antigens following in utero transplantation (IUT) of cytokine-stimulated allogeneic hematopoietic stem cells (sca(+)/lin(-)) (day 9 of gestation). Transplanted mice showed accelerated rejection of donor skin grafts and high anti-donor cytotoxic response, a finding not seen in the control mice. This was accompanied by an enhancement of Th1 over Th2 cytokine production and persistent donor microchimerism. In order to assess the role of the thymus in allograft rejection, prenatal transplants were performed under similar experimental conditions at a later gestational age, when the thymus is more developed (day 13).


METHODS
Cytokine-stimulated stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF)-purified sca(+)/lin(-) cells of C57BL/6 (H-2b, 1E(+)) background were injected into MHC-mismatched BALB/c (H-2d, 1E(-)) fetal mouse recipients at day 13 of gestation. Chimerism was determined by highly sensitive (0.001%) semiquantitative polymerase chain reaction (PCR). Mixed lymphocyte reaction (MLR) and cytotoxic T-cell assay (CTL) were used to evaluate tolerance vs immunity. Cytokine levels were quantified in MLR supernatants using ELISA assay. The percent of T cells was determined by flow cytometry (FACS) and CD4/CD8 ratio calculated. Postnatal boosts (transplants without conditioning) were performed at 6 months of age to enhance donor chimerism and test the degree of tolerance.


RESULTS
When assayed at 4 months of age, donor-type cells were not detected in the spleen or in the peripheral blood of BALB/c mice inoculated with C57BL/6 sca(+)/lin(-) cells. Transplanted but not control animals demonstrated high anti-donor MLR but not CTL responses. The increase of MLR reactivity was correlated with high levels of IL-2. Furthermore, transplanted mice showed higher resistance to postnatal boosts with allogeneic bone marrow (BM) cells, when compared to the control mice. The later resistance was accompanied by the expansion of host-type CD4 cells.


CONCLUSIONS
These data demonstrate that transplantation of cytokine-stimulated sca(+)/lin(-) allogeneic cells at 13 days of fetal development leads to the allosensitization, characterized by an enhancement of MLR alloreactivity and by the rejection of postnatal boosts (transplants without conditioning). Host-type CD4 cells might play a central role in this rejection. These findings indicate that the late injection of allogeneic cells may result in the development of allosensitization with subsequent donor graft rejection. Precise conditions for the development of tolerance must be established before prenatal transplants in humans with conditions other than SCID can be done.
Sefrioui
Hassan
H
Department of Medicine and Pediatrics, University of California, San Diego, USA.
Donahue
Jody
J
Srivastava
Anand Shanker
AS
Gilpin
Elizabeth
E
Lee
Tzong-Hae
TH
Carrier
Ewa
E
D000818:Animals; D015703:Antigens, CD / Q000032:analysis; D018952:Antigens, CD34 / Q000032:analysis; D016207:Cytokines / Q000032:analysis; D003602:Cytotoxicity, Immunologic; D005260:Female; D005865:Gestational Age; D016179:Granulocyte Colony-Stimulating Factor / Q000494:pharmacology; D018380:Hematopoietic Stem Cell Transplantation; D006801:Humans; D007694:Killer Cells, Natural / Q000276:immunology; D007959:Lymphocyte Culture Test, Mixed; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D011247:Pregnancy; D011994:Recombinant Proteins / Q000494:pharmacology; D019089:Stem Cell Factor / Q000494:pharmacology; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology
D015703:Antigens, CD; D018952:Antigens, CD34; D016207:Cytokines; D011994:Recombinant Proteins; D019089:Stem Cell Factor; D016179:Granulocyte Colony-Stimulating Factor