Allogeneic bone marrow transplantation (BMT) generally requires conditioning of the recipient with cytoreductive treatments to prevent immunological rejection of the graft. Because these regimens can be associated with serious side effects (1
), preparative treatments are often omitted for the treatment of diseases such as severe combined immunodeficiency (SCID), as these patients are incapable of rejecting donor grafts (2
). Nevertheless, although large numbers of B and T lymphocytes are at least transiently generated, the levels of donor HSC engraftment are usually less than 1% following transplantation into unconditioned SCID recipients (3
). Although several studies have concluded that transplanted HSCs can easily replace endogenous HSCs without conditioning (7
), earlier studies suggested that the availability of niches is a limiting factor to transplantation (9
). Thus, we reasoned that donor HSC engraftment might be limited by the occupancy of appropriate niches by endogenous HSCs, and that the development of reagents that specifically remove host HSCs could lead to safer transplantation-based therapies for hematological and non-hematological disorders than those currently in use.
To address whether endogenous HSCs can be replaced by transplanted HSCs in a linear dose-dependent manner, unconditioned Rag2−/−γc−/− mice (10
) were transplanted with varying numbers of c-kit+
HSCs from GFP+ mice (12
). Peripheral blood granulocyte chimerism was measured at 16 weeks post-transplant, which has been previously shown to accurately reflect donor HSC chimerism in this system (3
). Donor granulocyte chimerism increased measurably in doses of between 10 and 250 transplanted HSCs, but transplantation of more than 250 cells led to at most modest increases in chimerism (). Transplantation of 18,000 HSCs, representing approximately 70% of the total number of HSCs in an adult mouse (17
), led to a mean chimerism of only 3% (, top panel). Similar results were obtained through transplantation of unfractionated bone marrow (supporting online text and Fig. S1
). These data suggest that without conditioning, HSC engraftment is limited by the number of saturable niches that are empty at the time of transplant or become empty as the transplanted cells still survive.
Available HSC niches can be saturated with donor HSCs
To determine the specificity of these niches, we competitively transplanted unconditioned Rag2−/−γc−/− (CD45.2) mice with 1000 CD45.1 HSCs along with 100,000 GFP+
progenitor cells, which are the immediate progeny of HSCs (16
). No significant difference in donor HSC chimerism was observed in these mice relative to recipients that received 1000 HSCs alone (, lower right panel), suggesting that HSCs and their immediate progeny use distinct niches to maintain function.
To determine whether the specific elimination of host HSCs would allow for high levels of donor HSC engraftment, we compared a number of different candidate HSC-depleting monoclonal antibodies, including α-Sca1 (13
), α-integrin α4 (20
), and α-ESAM1 (21
), but ultimately selected ACK2, an antibody known to recognize and antagonize c-kit (22
), the receptor for stem cell factor (SCF) (23
). We reasoned that if the ACK2 antibody were capable of depleting endogenous HSCs, residual antibody in the serum of mice would also deplete transplanted donor HSCs. To determine the kinetics of antibody clearance in vivo
, we administered ACK2 intravenously to Rag2−/−γc−/− mice and tested the serum for the presence of antibody (12
). ACK2 remained in the serum until five days post-injection; however no ACK2 was detected by 7–8 days after injection (). All mice survived the ACK2 treatment with no obvious signs of distress (supporting online text, Table S1, and Fig. S2
). At this time point, a ~99% decrease in the number of HSCs () was observed, and comparable levels of depletion were observed in B cell deficient and wild type mice (supplemental text and Fig. S3
). To further confirm HSC depletion, at 9 days post-ACK2 treatment, 200,000 bone marrow cells were transplanted into lethally irradiated recipients (12
). Transplantation of BM from ACK2-treated animals led to >90% lower engraftment relative to controls (), thus confirming that ACK2 depletes functional HSCs from the BM.
ACK2 treatment depletes HSCs in vivo
To determine the mobilizing effects of ACK2 treatment, the entire splenocyte populations of ACK2-conditioned animals were transplanted into lethally irradiated recipients (12
). The donor chimerism resulting from ACK2-treated splenocytes was reduced by >95% relative to controls (). These data indicate that ACK2-treatment does not induce HSC depletion from the BM by mobilizing HSCs to the spleen. Moreover, we were unable to detect HSCs in the liver 9 days after ACK2 treatment by flow cytometry (data not shown). Mobilization was only observed during the recovery phase, well after ACK2 had already cleared from the serum (Fig. S4
To determine the mechanism by which ACK2 depletes HSCs, we compared the effects of ACK2 treatment to that of 2B8, another c-kit monoclonal antibody of the same IgG2b isotype. 2B8 treatment did not decrease functional HSC numbers in vivo
, as transplantation of bone marrow cells from mice treated with 2B8 resulted in levels of engraftment similar to those of controls (). Next, we cultured purified HSCs in the presence of ACK2 and found that it completely inhibited SCF-dependent proliferation, but not thrombopoietin (TPO)-mediated proliferation () (12
). These data, along with the data that distinct c-kit-expressing progenitors are depleted at differential rates (supporting online text and Fig. S5
), suggest that ACK2 does not deplete through Fc-mediated functions. Rather, consistent with studies using c-kit mutant mice (24
) (supporting online text), the complete inhibition of c-kit signaling by ACK2 () can deplete HSCs while the partial inhibition by 2B8 cannot.
By 23 days post-treatment, HSC cell surface profiles (Fig. S4
) and numbers () had returned to near normal levels. These data indicate that ACK2 causes a significant but transient depletion of host HSCs and results in a short window during which ACK2-treated animals might be receptive to donor HSC transplantation. To test whether the ablation of host HSCs could improve the efficiency of donor HSC engraftment, RAG2−/− (CD45.1) mice were conditioned with ACK2 and transplanted with 5000 wild type CD45.2 HSCs after serum clearance of ACK2. The mean donor granulocyte chimerism at 37 weeks post transplant was 16.1%, reflecting a >10-fold increase over control recipients (). These engrafted HSCs also gave rise to peripheral B and T cells ().
ACK2 treatment enhances HSC engraftment
We also analyzed BM HSC chimerism directly at this late time point to confirm the increase in engraftment, and found that it correlated well with peripheral blood granulocyte chimerism (). Secondary transplants of donor HSCs, re-isolated from primary recipients led to multi-lineage engraftment for at least 16 weeks post-transplant (), confirming that transplanted HSCs regain their normal cell surface phenotype and cell cycle status (Fig. S6
) by at least 7–9 months post-transplant in ACK2-treated animals (12
These data did not distinguish whether ACK2 treatment increased niche space or whether, as in unconditioned animals, only a small fraction of transplanted HSCs initially engrafted but then competitively expanded. If niche space had truly been freed, HSC chimerism would be expected to increase linearly with transplanted cell dose. To distinguish between these alternatives, ACK2-conditioned RAG2−/−γc−/− (CD45.2) recipient mice were transplanted with varying doses of CD45.1 LT-HSCs. Donor engraftment increased linearly with transplanted HSC dose in 2 independent experiments at both early and late timepoints (), consistent with the emptying of HSC niches by ACK2 treatment. Strikingly, donor chimerism values of up to 90% were achieved at the 35,000 HSC dose (), which, upon extrapolation to humans, is a clinically obtainable number (supporting online text and Fig. S7
Donor chimerism increases with transplanted HSC cell number in ACK2-treated mice
Allogeneic BMT is used routinely for a number of clinical purposes, such as for the treatment of SCID (26
). Here, we provide evidence that in the absence of conditioning, donor HSC engraftment is limited by the occupancy of appropriate niches by host HSCs. These data offer an explanation for the poor donor HSC engraftment observed in unconditioned SCID patients (4
), which in turn may be responsible for the low levels of donor B lymphopoiesis and the finite duration of T cell production (6
We demonstrate above that administration of ACK2 in vivo
leads to the rapid but transient depletion of host HSCs and subsequent transplantation of highly purified HSCs leads to donor chimerism levels of up to 90%. When coupled with highly specific immunosuppressive depleting antibodies, shown to be effective in both mice (29
) and humans (30
) as transplantation conditioners, the use of HSC-specific depleting antibodies may be an attractive alternative to conventional methods of conditioning, which carry significant health risks (1
), and may thus increase the utility of allogeneic BMT for both hematological and non-hematological disorders.