A Preclinical Canine Model of in Utero Hematopoietic Cell Transplantation–Induced Graft Vs. Host Disease.

In utero hematopoietic cell transplantation (IUHCT) offers the potential to achieve allo-engraftment and associated donor-specific tolerance (DST) without toxic conditioning. To evaluate IUHCT in a clinically relevant large animal model, we have developed the haploidentical canine model of IUHCT. Recent optimization of the model using a CD3 depleted graft has achieved consistent and stable levels of mixed hematopoietic chimerism (average > 10%) without graft versus host disease (GVHD). However, since GVHD represents the greatest theoretical risk of clinical IUHCT, it is important to identify the phenotype and requirements for IUHCT-induced GVHD in the canine model prior to clinical application. In the current study, we attempted to establish a threshold dose of CD3+ cells required for IUHCT-induced GVHD in the haploidentical canine model by increasing the absolute percentage of CD3+ cells in the donor inoculum and we characterized the GVHD phenotype observed.

43 fetuses of 9 pregnant bitches underwent ultrasound-guided intracardiac injection of maternal BM cells at 37.5–43 days gestational age (GA). In our large animal model, bone marrow yields, cellularity and litter size differ between dams. Due to these factors and because the targeted gestational age range coincides with an exponential growth phase for the dog fetus, standardization of cell dose per weight was not possible. Following MACS CD3 depletion, graft composition was standardized by adding back the CD3+ fraction to achieve donor CD3 content of 1%, 3%, 7.5%, and 16% (whole bone marrow [WBM] positive control) (Table 1). Live born puppies were monitored daily for signs of GVHD, including skin lesions, diarrhea, hematochezia, jaundice, weight loss and lethargy. Chimerism was measured by a PCR-based VNTR assay using commercially available primers.

Though prenatal survival of 75% was not significantly different than baseline (76%) and pups were not runted at birth, acute GVHD was observed postnatally in all ten live born recipients of either 7.5% CD3 cells or WBM, with onset ranging from 1–14 days of age. Recipients of grafts containing 1% and 3% CD3 cells showed no clinical evidence of GVHD.

Weight loss was the earliest and most consistent clinical feature of IUHCT-induced GVHD in our model. Various affected animals manifested additional symptoms in all GVHD target organs, including increased respiratory rate/effort, hepatic dysfunction, bloody diarrhea and skin lesions. The phenotype was severe and all ten ultimately required euthanasia between 2 and 21 days of age.

Necropsies performed during acute GVHD revealed significant erythematous and/or ulcerated skin change, hepatosplenomegaly, and punctate hemorrhage throughout the gastrointestinal tract and lung. Histology (H&E) demonstrated lymphocytic infiltrates and focal necrosis in target tissues as well as effacement of the dermal-epidermal junction and villous surfaces. Canalicular bile stasis was the most uniform and characteristic microscopic finding irrespective of hepatic functional status.

Chimerism levels in pups who survived to birth were compared based upon CD3 content of the donor graft (Table 1). Increasing CD3 percentage trended toward an increase in chimerism, though only the WBM group reached statistical significance (p=.043).

In the preclinical haploidentical canine model of IUHCT the threshold for induction of GVHD appears to be between 3% and 7.5% graft CD3⁺cell content. Interestingly, the absolute dose of CD3+ cells/kg fetal weight does not entirely correlate with the occurrence of GVHD, suggesting that the composition of the graft rather than the absolute T-cell content may determine the likelihood of GVHD. Given the complete absence of GVHD at a CD3 dose of 1% and the excellent engraftment achieved, it appears that there is a safe therapeutic index for achievement of chimerism without GVHD. When IUHCT-induced GVHD occurs, it is severe and clinically similar to postnatal GVHD. Notably, clinical GVHD only occurred after birth, suggesting suppressive mechanisms in utero. Additional studies are needed to further optimize the graft for support of engraftment without GVHD.

No relevant conflicts of interest to declare.