Letters to the Editor

In several rodent models spleen allotransplantation has been shown to be associated with donorspecific tolerance to allo-antigens [1]. To study the tolerogenic capacity of the spleen in a systematic manner in large animals with known major histocompatibility (MHC) barriers, we have developed an allogeneic spleen transplantation (Tx) model in MHC-defined miniature swine [2]. One of the goals of investigating spleen Tx as a potential for tolerance induction was its extrapolation to xenoTx. In our allogeneic spleen Tx experiments, we have observed that all recipients develop hematopoietic cell chimerism in the blood. Across full MHC barriers, when recipients were treated with 100 cGy of whole body irradiation, 700 cGy thymic irradiation, and a 45-day course of cyclosporine, multilineage chimerism was present in the blood and lymphoid tissues, and in some cases resulted in engraftment of donor cells in recipient bone marrow (BM). This was associated with in vitro donor-specific hyporesponsiveness in mixed leukocyte reaction (MLR) and cell-mediated lympholysis (CML) assays. Long-term survivors demonstrated evidence for donor-specific regulatory T-cell activity, as peripheral blood mononuclear cells from these pigs contained a cell population that was able to suppress anti-donor responses of cells from naı̈ve recipient MHCmatched pigs in suppressor CML and suppressor MLR assays (F.J.M.F. Dor, unpublished data). We have performed xenogeneic pig spleen Tx in two baboons, in the first of which we established that the surgical technique used in our pig alloTx model was also feasible in the pig-to-baboonmodel. The second baboon (B221) also received donorspecific BM Tx 4 h later. We here report our observations in B221 and compare them with those in a baboon (B219) that received BM Tx only (with no spleen Tx). Both baboons were conditioned with a fairly complex regimen based on the pig-to-baboon BM Tx regimen reported previously [3,4]. Both donor pigs were miniature swine generated by nuclear transfer/embryo transfer with greatly reduced expression of a1,3-galactosyltransferase RNA, associated with reduced levels of Gala1–3Gal (Gal) when compared with wild-type swine; some Gal was detectable by flow cytometry on hematopoietic cells. No measures were taken to deplete natural anti-Gal antibodies, but complement depletion was performed using cobra venom factor, which was administered for the first 21 days. Spleen Tx was carried out in B221 in the abdomen using the previously-described technique [2]. BM was harvested from the donor pigs and prepared for infusion into the baboon as previously described [5]. BM cells (1.33 and 0.8 10/ kg in B221 and B219, respectively) were infused intravenously without untoward effect (in B221 4 h after spleen Tx). Blood was drawn at intervals to determine the extent of hematopoietic cell chimerism by flow cytometry. The maximum percentage of pig cells (using a ‘pan-pig’ antibody) in the blood of B221 was 16.8% (2 h after spleen Tx), but only 1.9 % in B219 (on day 2) (Fig. 1], and the duration of pig cell macrochimerism was 12 days in B221 compared with only 5 days in B219. In B221, the absolute number of pig cells was 689/ll after spleen Tx and 876/ll after BM Tx, respectively (Fig. 2] (2 h after spleen Tx and BM Tx); in contrast, in B219 the peak absolute number of pig cells 2 h after the BM Tx was 85/ll (Fig. 2). In both cases, loss of chimerism was most likely related to phagocytosis of pig cells by host macrophages [3,6].