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engraftment was observed in all recipients prepared with 4.5 Gy TBI but not
in those prepared with 2 Gy TBI. When both methotrexate and cyclosporin
were given after HCT, 3 of 5 recipients (80%) prepared with 2 Gy TBI had
sustained mixed chimerism. When mycophenolate mofetil and cyclosporin
were given after HCT, 11 of 12 recipients (92%) prepared with 2 Gy TBI had
sustained mixed chimerism. These observations played an indispensable
role leading to the development of non-myeloablative conditioning regi-
mens for HCT in humans, as summarized above.
Future directions
Experiments by Luznik et al.
[161]
illustrated key concepts and highlighted
new directions in methods to prevent rejection and establish engraftment
after allogeneic HCT. Their approach was based on the administration of
high-dose cyclophosphamide on day 3 after HCT. At this time after HCT,
donor T cells that recognize recipient alloantigens are proliferating rapidly,
and recipient T cells that recognize donor alloantigens are proliferating like-
wise. Donor and recipient cells that do not recognize alloantigens remain
relatively quiescent, since their proliferation is driven solely by homeostatic
mechanisms associated with lymphopenia. Rapidly proliferating T cells are
susceptible to high-dose cyclophosphamide, whereas quiescent cells are
resistant.
114
Engraftment could not be established in MHC-mismatched recipients pre-
pared with 2 Gy TBI followed by administration of high-dose cyclophos-
phamide on day 3 after HCT with 2 × 10
7
T-cell-depleted marrow cells or
in recipients prepared with the combination of fludarabine and 2 Gy TBI in
the absence of high-dose cyclophosphamide on day 3 after HCT. Low level
donor chimerism was observed in 7 of 10 non-irradiated recipients given
fludarabine before HCT and treated with high-dose cyclophosphamide on
day 3 after HCT. In recipients prepared with the combination of fludarabine
and 2 Gy TBI followed by administration of high-dose cyclophosphamide
on day 3 after HCT with 2 × 10
7
T-cell-depleted marrow cells, high-level
mixed chimerism was observed in 10 of 10 recipients.
Taken together, these results demonstrate that pretransplant conditioning
with fludarabine and post-transplant administration of high-dose cyclo-
phosphamide were both necessary in order to attain adequate immuno-
suppression to prevent rejection, but this combination had little effect on
hematopoietic cells in the recipient. Thus, the graft containing 2 × 10
7
donor
marrow cells could not compete with recipient stem cells that occupied
hematopoietic niches in recipients that were not treated with TBI. Addi-
tional experiments showed that the level of chimerism was directly propor-
tional to TBI exposures between 50 and 200 cGy. Increasing the size of the
graft from 5 × 10
6
to 10 × 10
6
marrow cells and increasing the TBI exposure
from 100 cGy to 200 cGy had similar effects on the level of donor chime-
rism. These results suggest that the primary effect of TBI in this model is to
reduce the number of recipient hematopoietic stem cells that compete with
donor stem cells for occupancy of niches in the marrow.
These observations have led to the development of protocols that enable
consistent engraftment after HCT with HLA-haploidentical donors
[162]
.
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