Sirolimus and GvHD Control: the Rhesus Macaque GvHD Model Reveals That Sirolimus Preferentially Inhibits T Cell Proliferation While Permitting Breakthrough T cell Activation After MHC-Mismatched Hematopoietic Stem Cell Transplantation

Given the emerging importance of sirolimus as a therapuetic for graft-versus host disease (GvHD), it is critical to rigorously define the mechanisms by which this agent impacts T cell immunity after hematopoietic stem cell transplantation (HSCT). Therefore, we have used our novel rhesus macaque model of haploidentical HSCT and GVHD to probe the mechanisms of sirolimus-mediated GvHD prevention when given as a monotherapy. The insights gained from this study will facilitate the rational design of sirolimus-containing combinatorial therapies to maximize immunosuppressive efficacy.

Transplant recipients were prepared with 8Gy total body irradiation and were then infused with MHC-mismatched donor leukopheresis products(n=3, avg. 6.5×10⁸ TNC/kg, 3.4×10⁷ total T cells/kg). Recipients received sirolimus monotherapy (serum troughs 5–15 ng/mL) alone as post-transplant immunosuppresson. Clinical GvHD was monitored according to our standard primate GvHD scoring system and flow cytometric analysis was performed to determine the immune phenotype of sirolimus-treated recipients compared to a cohort of recipients (n= 3) that were given no GvHD immunoprophylaxis.

Sirolimus modestly prolonged survival after MHC-mismatched HSCT compared to no immunosuppression (>19 days versus 6.5 days in the untreated cohort, with GvHD confirmed histopathologically at the time of necropsy). We found that sirolimus significantly inhibited lymphocyte proliferation in transplant recipients: The ALC remained suppressed post-transplant (eg ALC of 0.46 × 10⁶/mL on day 15 post-transplant versus 4.3 × 10⁶/mL pre-transplant, with recovery of other leukocytes: WBC=5.1 × 10⁶/mL, ANC=2.6 × 10⁶/mL). These results suggest that sirolimus can have a profound impact on lymphocyte proliferation, inhibiting GvHD-associated lymphocyte expansion by as much as 200–300-fold compared to untreated controls. Sirolimus had a similar impact on CD4+ and CD8+ subpopulation expansion. Thus, while CD4+ T cells and CD8+ T cells expanded by as much as 300-fold and 2000-fold, respectively, without sirolimus, the expansion of these cells was significantly blunted with sirolimus, with maximal expansion of CD4+ and CD8+ T cells being 4- and 3.6-fold, respectively compared to the post-transplant nadir. Sirolimus-treated recipients also better controlled the upregulation of the proliferation marker Ki-67 on CD4+ or CD8+ T cells. Thus, while untreated recipients upregulated Ki-67 expression by as much as 10-fold after engraftment, (with >80-98% T cells expressing high levels of Ki-67 post-transplant versus 5–10% pre-transplant) sirolimus-treated recipients better controlled Ki-67 expression (17-40% Ki-67-high CD4+ and CD8+ T cells post-transplant).

While the impact of sirolimus on T cell proliferation was profound, it failed to completely inhibit activation of T cells, as measured by both Granzyme B and CD127 expression. Thus, when effector CD4+ and CD8+ T cell cytotoxic potential was measured by determining expression levels of granzyme B, we found that sirolimus could not downregulate this key component of immune function and GvHD-mediated target organ damage: Granzyme B expression in both CD4+ and CD8+ CD28-/CD95+ effector T cells was unchanged despite sirolimus monotherapy. Down-regulation of CD127 expression, which identifies activated CD8+ T cells in both humans and rhesus macaques, also demonstrated resistance to sirolimus treatment. Thus, while a cohort of recipients that were treated with combined costimulation blockade and sirolimus maintained stable CD127 levels post-transplant, and untreated animals demonstrated total loss of CD127, up to 60% of CD8+ T cells in sirolimus-treated recipients down-regulated CD127, consistent with breakthrough activation of these cells despite mTOR inhibition.

These results indicate that while the predominant effect of sirolimus during GvHD prophylaxis is its striking ability to inhibit T cell proliferation, sirolimus-based immunosuppression spares some cellular signaling pathways which control T cell activation. These results imply that therapies that are combined with sirolimus during multimodal GvHD prophylaxis should be directed at inhibiting T cell activation rather than proliferation, in order to target non-redundant pathways of alloimmune activation during GvHD control.

No relevant conflicts of interest to declare.