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Tolerance to kidney allotransplants (KTx) without chronic immunosuppressive therapy is achievable in several preclinical primate models, many of which show a requirement for donor bone marrow (DBM) induced chimerism. When initiated on KTx day 0, anti-CD3 immunotoxin (IT) induces stable tolerance without DBM if the proinflammatory INF-y and TNF-a responses are prevented by peritransplant (peri-tx) treatment with NF-kB inhibitors 15-Deoxyspergualin (DSG) and methylprednisolone (MP). To determined if chimeric cells might be seeded from the KTx, we examined recipients given peri-tx IT, DSG and MP for evidence of chimerism and acquired specific unresponsiveness.
Methods: 16 rhesus monkey received a KTx with immunosuppression as follows: for the 1st 3 days, IT at 150 ug/ kg total dose, MP tapered from 7 mg/kg/day, and DSG at 2.5 mg/kg/day with the DSG continued up to 14 days. All were followed for >_ 3 months. 8 received only the KTx and 8 received additional donor CD34+ cells. All had multiple mismatches for donor MHC Class II DRB alleles and shared 1 donor DRB-1 allele. Chimerism was performed by PCR-SSP for mismatched DRB alleles. Frequency (f) changes in alloreactive PBL CTLp were assayed in limiting dilution cultures containing rhIL-2 and donor (D) or indifferent 3rd party (I) stimulator cells, and f was estimated by x2 minimization. Recipients over 250 days were challenged with D & I skin allografts
Results: Overall, 56% of recipients had stable, long term KTx function (mean Cr =0.9 +_ 0.3) and no rejection at a median 245 days with the longest survivor in good health at 513 days. Graft loss to rejection was 12.5% and 25% with and without D-CD34+ cell infusion, respectively. A mortality of 37.5% was from mixed unrelated and iatrogeneic causes without rejection, lymphomas or definable infections. At a 0.002% sensitivity level for chimerism detection. 15/16 recipients each tested on multiple (3-12) occasions in >I00 assays exhibited no chimerism in blood or skin at any time; 1/16 had PBL chimerism in the 1st week. 1 had chimerism only in lymph nodes, liver & spleen, and 1 showed only thymic chimerism. 75% had a 0.6-O.9 log10 specific reduction in anti-D CTLp f without significant change in the f of anti-l CTLp. Challenge D skin grafts were accepted for 4-5 weeks while I grafts rejected in 6-9 days,
Conclusions: Despite a lack of demonstrable chimerism, even after donor CD34+ cell infusion, the majority of recipients developed evidence of acquired specific tolerance. This protocol promotes development of a robust tolerance mechanism that appears to be independent of peripheral chimerism.