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Introduction: We have created a non-human primate (NHP) model of GvHD. Here we determined whether mTOR inhibition with sirolimus and CD28:CD80/86 costimulation blockade with belatacept could combine synergistically to prevent this disease.
Methods: Rhesus macaques were irradiated (9.6 Gy), and then transplanted with GCSF-mobilized PBSC from a haploidentical donor. Recipients were treated with either sirolimus alone, belatacept alone or combination therapy. Clinical GvHD was monitored using an NHP grading scale and multiparameter flow cytometric analysis (MFC) was performed.
Results: Untreated controls (n = 5) developed rapid, severe aGvHD and succumbed rapidly (MST = 7 days). Treatment with either sirolimus or belatacept alone partially protected against GvHD. Sirolimus-treated recipients (n = 6) developed predominantly GI disease and had an MST of 14 days (Figure 1A). Recipients treated with belatacept alone (n = 3) developed primarily liver aGvHD and had an MST of 11 days. In striking contrast, recipients treated with combined sirolimus + belatacept (n = 5) demonstrated neither uncontrolled diarrhea nor hyperbilirubinemia at the timed terminal analysis (1 month post-transplant).
We used MFC to measure the immunologic consequences of sirolimus and belatacept on T cell proliferation (Ki-67) and cytotoxity (granzyme B). While untreated aGvHD was associated with rampant CD8+ proliferation (with 83% Ki-67+ CD8+ T cells vs 4.7% pre-transplant), sirolimus or belatacept monotherapy partially controlled proliferation (35% and 65% Ki-67+ with sirolimus or belatacept respectively). Combined sirolimus + belatacept dramatically reduced proliferation (to 8%, favorably comparing with 13% Ki-67+ using tacrolimus/MTX).
Sirolimus and belatacept also partially controlled T cell cytotoxicity: While untreated aGvHD was associated with excessive CD8+ granzyme B expression (82% granzyme Bvery high vs 0.3% pre-transplant) sirolimus or belatacept monotherapy partially controlled cytotoxicity (8% and 35 % granzyme Bvery high with sirolimus or belatacept respectively). Combination therapy dramatically reduced granzyme Bvery high expression, to 1.5%, favorably comparing with 4% using CNI/MTX.
The ability of sirolimus, belatacept, or the combination to control Ki-67 and Granzyme B expression closely correlated with survival (Figure 1B,C), and significant co-expression of granzyme B in the Ki-67+ cells was observed (Figure 1D), suggesting that dual-positive Ki-67/Granzyme B cells may mark a pathogenic population, amenable to tracking in the peripheral blood.
Implications: These results show, for the first time, that sirolimus and belatacept can combine synergistically to control primate aGvHD. They also identify CD8+/Ki-67+/Granzyme Bvery high dual-positive T cells as a potentially sensitive biomarker of GvHD pathogenesis, amenable to monitoring longitudinally in the blood.
