High incidence of xenogenic bone marrow engraftment in pig-to-baboon intra-bone bone marrow transplantation

Abstract

Previous attempts of α-1,3-galactocyltransferase knockout (GalTKO) pig bone marrow (BM) transplantation (Tx) into baboons have demonstrated a loss of macro-chimerism within 24 h in most cases. In order to achieve improved engraftment with persistence of peripheral chimerism, we have developed a new strategy of intra-bone BM (IBBM) Tx. Six baboons received GalTKO BM cells, with one-half of the cells transplanted into the bilateral tibiae directly and the remaining cells injected intravenously (IBBM/BM-Tx) with a conditioning immunosuppressive regimen. In order to assess immune responses induced by the combined IBBM/BM-Tx, three recipients received donor SLA-matched GalTKO kidneys in the peri-operative period of IBBM/BM-Tx (Group 1), and the others received kidneys 2 months after IBBM/BM-Tx (Group 2). Peripheral macro-chimerism was continuously detectable for up to 13 days (mean 7.7 days; range 3-13) post-IBBM/BM-Tx and in three animals, macro-chimerism reappeared at days 10, 14 and 21. Pig CFUs, indicating porcine progenitor cell engraftment, were detected in the host BM in four of six recipients on days 14, 15, 19 and 28. In addition, anti-pig unresponsiveness was observed by in vitro assays. GalTKO/pCMV-kidneys survived for extended periods (47 and 60 days). This strategy may provide a potent adjunct for inducing xenogeneic tolerance through BM-Tx.

Keywords: animal models: nonhuman primate; bone marrow/hematopoietic stem cell transplantation; tolerance: experimental; translational research/science; xenoantigen; xenotransplantation.

Figure 1

(A) Peripheral macro-chimerism after IBBM/BM-Tx. (B) Facs profiles of peripheral macrochimerism. (a) B331 (Group 1), (b) B344 (Group 2), (c) Positive control (normal pig blood), (d) Negative control (naive baboon blood). Pink squares showed pig cells (pan-pig antigen positive cells) in baboon recipient.

Figure 2. Bone marrow macro- and micro-chimerism after IBBM/BM-Tx

(A) Pan-pig positive cells were detected in right tibia and right iliac crest from the bone marrow aspiration samples on POD15 (B344). (B) Positive bands of porcine cytochrome b were detected in the tibia (3) and sternum bone (4) of B324, and in the right tibia (5) and right iliac crest (6) of B296. 1 and 2 were positive control.

Figure 3. Mixed lymphocyte reaction (MLR)

Pretransplant MLR showed a robust proliferative response to GalTKO pig and allogeneic cell stimulators. These animals showed general hyporesponsiveness until around 30 days after IBBM/BM-Tx. However, allogeneic responses began to recover while stimulation by GalTKO DD pig cells (BM donor type) remained very low. B336 and B344 showed hyporesponsiveness against kidney donor (the different haplotype GalTKO HH pig) cells on POD139 and POD55, respectively (asterisk shows response against haplotype HH).

Figure 4. ELISPOT assay for INFγ

Many spots could be seen in the wells stimulated by allogeneic cells (C and D), human cells (F) and PHA (G) on POD183 (B336), while the spots were few in the well stimulated by GalTKO DD pig cells (E). This was similar to what was observed in wells stimulated by media (A) and autologous PBMC (B).

Figure 5

(A) Changes in pig IgG antibody against GalTKO pig cells in sera of recipients after IBBM/BM-Tx assessed by flow cytometry. No animals developed anti-pig antibodies. (B) Histology (HE and PAS staining) of the kidney grafts. Kidney samples were taken at day 20 for B318 (primary graft), at day 19 for B324 and day 264 (60 days after 2nd K-Tx) for B336.