IUHCT offers the potential to treat a large number of hematologic genetic disorders without the myeloablative conditioning regimens currently used for haploidentical or mismatched postnatal HSCT transplant protocols. Early success in the sheep model suggested that significant levels of allogeneic engraftment could be relatively easily achieved by IUHCT. However, with the exception of immunodeficiency disorders38-40
, this proved not to be the case in humans, and other animal species, where early clinical and experimental studies resulted in limited or no detectable engraftment. Subsequent experimental studies in mice have confirmed that there are both competitive and immunologic barriers to engraftment after IUHCT.37,41,42
Nevertheless, these barriers can be overcome, at least in a percentage of animals, to achieve low levels of mixed hematopoietic chimerism across full MHC barriers, with associated DST. There appears to be a threshold of chimerism required for consistent association of DST in the murine model of around 1%.6
Chimerism levels below this threshold result in inconsistent tolerance, with only a fraction of animals accepting skin grafts or demonstrating enhancement of chimerism after postnatal same donor transplants.3-6
In mice, animals that are tolerant after IUHCT, can be transplanted after birth utilizing a variety of minimal conditioning strategies with enhancement of donor chimerism to levels that would be therapeutic for most target disorders.2,5,6
With this potential clinical strategy in mind, we wished to develop a pre-clinical model where techniques developed in the murine model could be assessed for their applicability to human disease.
In this study, we were able to perform IUHCT in the CLAD model using clinically applicable methodology with similar perinatal mortality to that seen following the natural breeding of dogs.43
IUHCT using haploidentical donors resulted in low levels of mixed hematopoietic chimerism that remained stable for over 18 months. The low levels of chimerism achieved in both CLAD and CLAD carrier dogs reflect the highly competitive engraftment milieu in the canine fetus, which appears to be analogous to that in humans. Remarkably, all dogs that could be analyzed with sensitive methodology (i.e. CD18 or SrY Q-PCR) were engrafted after IUHCT. In the animals where lineage analysis could be performed, donor chimerism was multilineage and durable, supporting the engraftment of hematopoietic stem cells at the time of IUHCT. Consistent with previous postnatal studies in the CLAD model, we demonstrate that even the low levels of engraftment achieved after IUHCT, are adequate to phenotypically ameliorate or correct the disease.
Perhaps the most important observation of this study, was the ability of IUHCT to induce DST in two of the chimeric animals, as evidenced by the ability to enhance engraftment after birth using a non-myeloablative conditioning regimen with sustained high levels of donor chimerism thereafter. This was in distinct contrast to what would be expected for haploidentical transplants using the same conditioning regimen in the absence of tolerance induction by IUHCT. To assess the presence of DST we used CFSE MLR. The one dog, Baku, that demonstrated definitively decreased donor specific reactivity also was successfully boosted, whereas the one dog that demonstrated definitive reactivity to donor cells and also underwent a postnatal transplant (Duke) rapidly lost donor chimerism. The remainder of the dogs who were assessed by MLR had intermediate results. Each of these dogs demonstrated an initial rise in chimerism levels with subsequent loss of engraftment by 14 weeks post HSCT. This pattern of engraftment was distinct from the complete absence of chimerism seen in non-IUHCT control dogs, in which donor chimerism could not be detected at any time after HSCT. These data support our premise that IUHCT was associated with DST, at least in a subset of recipients. However, due to the low number of dogs transplanted in this study, and limitations in the assessment of tolerance in the canine model, we have not absolutely proven that DST resulted from the IUHCT.
Our data in the canine model can be compared and contrasted to results in the murine model. While levels of engraftment in chimeric dogs were generally lower than what can be achieved in mice, the frequency of engraftment appears at least as high, and perhaps higher in the dog, than in the mouse. Sustained chimerism is observed in only around 30% of mice after allogeneic IUHCT, whereas in this study every dog in which sensitive detection methodology could be applied after IUHCT demonstrated sustained chimerism. The canine model appears remarkably similar to the murine model with respect to the correlation of level of chimerism with the apparent association of DST. In the murine model, only approximately 60% of animals with less than 1% chimerism will demonstrate enhancement of chimerism with a Busulfan conditioned same donor HSCT, whereas 100% of animals with greater than 1% chimerism can be boosted.6
In this study where levels of chimerism appear to be equal to or less than 1%, 2 of 6 total dogs and 2 of 5 CLAD carriers that underwent postnatal same donor transplants experienced a sustained increase in levels of donor chimerism. Thus, we would anticipate, based on our murine data, that the achievement of levels of donor chimerism even slightly higher than those in the current study should result in the consistent association of tolerance and allow all recipients of IUHCT to be boosted to potentially therapeutic levels of chimerism after birth.
The primary clinical risk of IUHCT would be anticipated to be GVHD. While the numbers of dogs transplanted in this initial experience were too small to make confident statements regarding the risks of IUHCT in a large animal system, it was encouraging that despite the intentional addition of significant numbers of T-cells, GVHD was not observed. Our protocol for T-cell reconstitution in this study was adapted from studies in the pig model of IUHCT, where multilineage chimerism with DST was achieved after BM was T-cell depleted and unprocessed whole BM was added back to achieve a T-cell concentration of 1.5%44,45
. This was in contrast to the strictly lymphoid chimerism that resulted from add back of isolated CD3+ cells at the same CD3+ dose suggesting that other populations in BM besides T-cells may be important in facilitation of HSC engraftment after IUHCT. Earlier studies in the sheep and primate models of IUHCT have utilized similar doses of T-cells to support engraftment.46,47
In the current study, T cell doses as high as 7.8 × 106
cells/kg estimated fetal weight were administered without evidence of GVHD but the upper limit of T-cell dose and the safety profile of T-cell dosing with or without other cell populations remains to be defined, Further studies in the canine model are required prior to any conclusions regarding the safety of various T-cell doses after IUHCT can be made.
The results of the current study compare favorably and extend upon the results of Blakemore et al.34,48
who were the first to assess allogeneic IUHCT using haploidentical donor cells in the normal canine model. Similar to our study, they found an overall perinatal mortality rate of 31%. Analysis for donor cell chimerism was performed at a single, short-term time point in the neonatal period on harvested hematopoietic tissues. Their results suggest the presence of low levels (0.01% to <2%) of donor cell chimerism in some of the tissues of some recipients. However, they were unable to demonstrate any evidence of DST following IUHCT by in vitro
MLR. Interestingly, there did not appear to be a relationship between T-cell dose and levels of chimerism, and no GVHD was observed, even at high T-cell doses. Beyond the observations that low level chimerism can be achieved without GVHD, direct comparisons between our data and that of Blakemore et al. are difficult due to methodologic differences in the donor cell preparations, donor cell detection methodology, and particularly because of the major difference in length of follow up.
In the current study, we use a protocol of Busulfan conditioning with a short course of post-transplant immunosuppression for GVHD prophylaxis. This regimen has been shown to result in successful engraftment in matched littermate CLAD dogs.27
However, this regimen, as observed in our control animals, and in previous postnatal experience (personal communication Dennis Hickstein) does not allow for successful parental haploidentical engraftment in recipients that did not receive IUHCT. In this study, two of the recipients of an IUHCT demonstrated successful long-term enhancement of engraftment following same donor postnatal haploidentical HSCT. Engraftment levels in these recipients remained stable at 35-45% donor cell chimerism up to the last time point of analysis. The remaining four recipients of IUHCT all experienced an initial rise in chimerism, but subsequently lost measurable engraftment by 14 weeks post transplant. Although the initial rise in engraftment level was encouraging, and distinct from the engraftment pattern seen in the two control dogs that did not undergo IUHCT, we believe the loss of chimerism seen in these recipients reflects an immune rejection. Due to technical limitations of quantifying chimerism in the canine model we were unable to determine the original presence or levels of engraftment in 4 of the 6 recipients of IUHCT that received a postnatal transplant. The sensitivity of the assay used to detect chimerism is 5%, and thus we know that engraftment, if present, occurred at chimerism levels less than 5% in these dogs. Additionally, it is reasonable to assume, based on IUHCT engraftment levels seen in Billie, Ella, Duke, and Bonnie, that chimerism levels were <1%. Thus, it is not surprising that all animals did not show evidence of tolerance. Finally, we believe that the temporary enhancement of chimerism in those dogs in which engraftment following IUHCT could not be determined supports the presence of chimerism, as experience with IUHCT has documented that no tolerance occurs without concomitant donor cell chimerism.
While this study is a valuable first step toward optimization of IUHCT for clinical application, much more work needs to be done. Basic questions such as the optimal timing of transplantation and correlation with specific stages of immune and hematopoietic development require further investigation. Although many questions remain, this study provides encouraging evidence that IUHCT can result in successful engraftment and induction of DST in a preclinical large animal model. While we have demonstrated that the low levels of engraftment achieved by IUHCT can reverse the lethal phenotype of CLAD, we consider the most exciting aspect of the current study to be the apparent finding that IUHCT can in some circumstances induce DST and that this tolerance can provide a platform for postnatal minimal conditioning HSCT with enhancement of donor cell engraftment to clinically relevant levels. We feel that if further enhancement of engraftment in this model with consistent tolerance induction can be achieved, than appropriate clinical trials of IUHCT for the treatment of genetic disorders that can be prenatally diagnosed and treated by mixed hematopoietic chimerism, such as the hemoglobinopathies and selected immunodeficiency disorders could be initiated.