The remarkable ability of HSCs to sustain multilineage hematopoiesis for the lifetime of an individual constitutes the foundation for their routine use in a range of clinical applications, including the treatment of primary immunodeficiencies (41
), malignancies (43
), as conditioners for transplantation tolerance of tissue or organ grafts from the donors (46
), and as a method to reverse some types of autoimmunity (47
). The success of such therapies relies on the ability of HSCs to home to unique niches leading to sustained multilineage hematopoiesis. The studies presented here have quantified the number of these HSC niches that are available for engraftment at any given point in unconditioned animals as ~0.1–1.0% of all HSC niches. Assuming a total adult murine bone marrow cellularity of 5 × 108
) and an endogenous HSC frequency of 0.01% (), the number of open HSC niches can be estimated to be 50–500. This is strikingly similar to the number of HSCs estimated to be in circulation at any given point (16
). The data suggest that HSCs that circulate normally have exited and left vacant their previous HSC niche. Thus, a constant exchange may be occurring between endogenous HSCs under normal circumstances, perhaps to maintain hematopoietic balance between and within each bone marrow compartment. In support of this, we have found little difference in the granulocyte chimerism rates between experiments when a single transplant of HSCs is provided in doses ranging from 800 to 4,000 cells. Although previous reports have suggested that the cell doses of transplanted bone marrow correlate with total chimerism linearly, such data at most show replacement of bone marrow and mature cells in bulk and do not reflect replacement of HSCs, which represent only 0.01% of unfractionated marrow (9
). When HSCs are repetitively transplanted, however, we have observed increases in granulocyte chimerism ( vs. ). Thus, occupation of available HSC niches after transplantation of an excess of exogenous HSCs, which remain in circulation for ~1–5 min after transplantation (16
), does not preclude additional niches from becoming available subsequently. Conceivably, continuous transfusion of low numbers of HSCs would be superior to singly administered boluses, as the rate of niche emptying and filling is high. Because there does not appear to be an obvious increase in granulocyte chimerism with time or cell dose above a threshold level, the data also suggest that transplanted HSCs must find their way rapidly to an appropriate niche and cannot recirculate indefinitely in search of empty niches without the loss of hematopoietic potential.
The ability of transplanted HSCs to self-renew for the lifetime of the organism ensures a constant production of normal lymphoid cells through each developmental stage. In the genetic mutants used in our work, host lymphocyte development is blocked or perturbed at defined developmental stages. At each developmental stage or thereafter, wild-type donor cells have a competitive advantage and can opportunistically expand or accumulate to ultimately give rise to large numbers of normal mature lymphocytes. Several factors have been implicated in the expansion of the early B cell and thymocyte lineages, including IL-7 (24
), stem cell factor (50
), Flt3 ligand (22
), and recently, various Wnt/Frizzled pathways (52
). In the case of γc−/−
animals, the pro–B-B population appears to have defects in IL-7–dependent expansion, providing a proliferative advantage to wild-type donor cells at these stages (24
mice likely reconstitute more slowly because their lymphocytes can develop normally through the pro–B cell as well as DN3 thymocyte stages and occupy the appropriate stromal microenvironments (31
). However, because RAG2−/−
lymphocytes cannot advance past these stages (57
), small numbers of developing donor-derived cells can expand and accumulate without competition at the pre–B as well as DN4 thymocyte cell stages and all subsequent developmental steps. Although it is possible that the donor LT-HSCs will only persist for finite periods of time because of the considerable demand imposed by the ~250,000-fold expansion to the lymphocyte stage, we have observed no meaningful decline in granulocyte or lymphocyte chimerism at any time point up to 30 wk after transplantation of primary recipients. Nonetheless, because we have observed some declines in HSC potency after secondary transplantation ( vs. ), in clinical settings it would be advisable to keep donor LT-HSCs stored in the event that grafts do not persist indefinitely. Additionally, the low levels of granulocyte chimerism achieved from a single transplant are unlikely to be clinically useful for patients suffering from myeloid deficiencies.
We also show conclusively that stable engraftment within these rare niches by minor histocompatibility–mismatched HSCs is tightly regulated by host CD4+ T cells. HSCs from CD45.1 mice cannot productively engraft unirradiated congenic CD45.2 mice, yet they routinely engraft the genetically unreactive F1 strain (CD45.1 × CD45.2). To our knowledge, the only antigenic difference between these strains is the CD45 allele, which is normally considered to be a relatively innocuous congenic marker. Similarly, HSCs isolated from GFP-transgenic mice backcrossed to the C57BL/Ka genetic background cannot productively engraft wild-type C57BL/Ka mice. The only antigenic difference between these strains to our knowledge is the GFP gene product. These experiments prove that very small antigenic differences lead to a complete rejection of donor HSC grafts in the absence of cytoreductive conditioning.
Encouragingly, however, the elimination of CD4+
T cell function allows for the functional and sustained engraftment of HSCs with minor histocompatibility mismatches in our system. Xu et al. (17
) have shown that transient antibody-mediated depletion of host αβ T cells in mice enhances engraftment of donor bone marrow with minor histocompatibility mismatches. Spitzer et al. (58
) have shown that conditioning haploidentical patients with a depleting α-CD2 antibody along with low-dose cytoreductive treatments before bone marrow transplantation allows, at the minimum, transient multilineage engraftment. Consistent with this, we demonstrate that transient antibody-mediated CD4+
T cell depletion alone is sufficient to allow short-term engraftment of wild-type donor HSCs and restoration of B cells in a mouse model of non-SCID. More complete CD4+
T cell depletions and/or better methods to increase donor-derived thymic dendritic cell contribution might allow for lasting donor hematopoiesis.
Even in the absence of inherited genetic mutations, both mice and humans develop diminished immune capacity with age. This progressive loss of immune function has recently been attributed to HSC-intrinsic defects in differentiation to lymphoid-primed progenitors (29
). Because we have demonstrated that a very small number of properly functioning HSCs can mask the defects in a much larger pool of HSCs, it is tempting to speculate that age-related immune defects don't become readily apparent until nearly all fully “young” HSCs are exhausted. The reintroduction of fully multipotent HSCs, perhaps obtained as an autologous sample earlier in life, might significantly delay age-related immune decline.
Numerous studies have shown how common conditioning treatments used before bone marrow transplantation, such as irradiation, cyclophosphamide, and busulfan, can cause serious side effects, including lowered platelet counts, infertility, and secondary malignancies (59
). When these cytotoxic therapies are used to treat hematologic malignancies, the side effects must unfortunately be tolerated as a byproduct of necessary chemotherapy. The necessity of such conditioning treatments for hematopoietic deficiencies before HSC transplantation, however, should be reconsidered. Although it is true that available niche space is low under normal conditions, we show that transplantation of modest numbers of highly purified HSCs can engraft the few niches that are available and correct lymphoid deficiencies. Thus, niche space is not an absolute limiting factor to HSC-mediated correction of B, T, or NK cell deficiencies.
Unlike the myeloablative regimens almost always performed on non-SCID immunodeficient patients, SCID patients who receive MHC-matched CD34-enriched or T cell–depleted bone marrow grafts generally do not receive cytoreductive conditioning before transplantation (37
). However, it has been suggested that HSC engraftment does not occur in these patients (61
). Because many of these patients show poor B cell lymphopoiesis and lose T cell counts with time, it has been proposed that the lymphoid correction occurs as a result of engraftment of short-lived progenitor cells, which along with mature cells constitute the vast majority of transplanted cells, rather than HSCs with full hematopoietic and self-renewal potential (62
). A careful examination of the data, however, shows that ~0.8% of CD34+
cells in an unconditioned SCID patient who received a bone marrow transplant are not of host origin (61
). Although the authors, understandably, did not consider this level of engraftment meaningful, our results suggest that small numbers of HSCs have engrafted in these patients and that eventual T cell loss may be a reflection of HSC exhaustion rather than an initial failure to engraft. This hypothesis is reinforced by suggestions that the process of physiological HSC circulation seems to be conserved between mice and humans (16
). Alternatively, the lack of B and T lymphopoiesis has correlated well with graft-versus-host disease (GVHD) in previous studies (66
). In MHC-matched settings, bone marrow grafts are often transplanted without manipulation, whereas in HLA-mismatched settings, T cell depletions or CD34 enrichments of donor marrow can still leave up to 104
T cells/kg (37
). Our results show that even in MHC-matched congenic mouse model systems, immune responses can recognize and reject very slightly mismatched cells, suggesting that it is very likely that GVH responses occur in all patients that receive any mature T cells as part of their nonautologous graft. Although GVHD may not be classified as clinically significant or obviously symptomatic in all cases, subtle GVH effects on B and T lymphopoiesis might still occur. Thus, the use of purified HSC transplants, which do not cause GVHD (30
), may potentially avoid poor B lymphopoiesis. In our mouse model system, we have observed sustained B and T lymphopoiesis for the duration of our experiments after purified HSC transplantation.
The mechanism by which transplanted HSCs correct hematopoietic deficiencies in our unconditioned recipients is applicable to the correction of many types of both SCID and non-SCID immunodeficiencies, but these studies at the same time clearly demonstrate that very subtle minor histocompatibility differences can mediate the rejection of HSC grafts when host T lymphocytes are present. Our data suggest that transplantation of purified HSCs, in combination with highly specific lymphoablative treatments when necessary, can correct lymphoid deficiencies in immunodeficient patients without the undesired side effects, such as toxic conditioning and GVHD, often associated with current conditioning and transplantation regimens. Future experiments will determine if the same strategy can be applied to the correction of myeloid deficiencies.