Rapamycin Is Highly Effective in a Mouse Model of Immune-Mediated Bone Marrow Failure By Mechanisms Distinct from Cyclosporine
Authors: Feng Xingmin , Lin Zenghua , Desierto Marie , Keyvanfar Keyvan , Malide Daniela , Chen Jichun , Young Neal S. 
Published: October 2019

Abstract
Acquired aplastic anemia (AA) is bone marrow (BM) failure characterized by pancytopenia and marrow hypocellularity, in most patients due to immune attack by T cells that target hematopoietic stem and progenitor cells. Most patients respond to immunosuppressive therapy, but relapse, especially on withdrawal of cyclosporine A (CsA), occurs frequently (Scheinberg P, Am J Hematol., 2014). Rapamycin has been successful in some human autoimmune diseases and in mouse models of autoimmunity; rapamycin also appears to induce tolerance, as for example in the organ transplant setting. We have developed murine models of BM failure; animals can be salvaged by biologics and drugs that are effective in humans with AA. One purpose of these models is to test potential new therapies. We have compared rapamycin with customary immunosuppression by CsA. Infusion of lymph node cells from C57BL6 (B6) donor mice into CByB6F1 (F1) recipient mice (MHC-mismatched) induced massive BM destruction by activated T cells. Treatment with rapamycin (2 mg/kg/day, starting 1 hour post lymphocyte injection and continued for 2 weeks, n=9) effectively ameliorated pancytopenia and improved BM cellularity, better than did maximal dosing with CsA (50 mg/kg/day, starting 1 hour post lymphocyte injection, continued for 5 days, n=8) (Fig 1A). Rapamycin eliminated most BM-infiltrating CD8+ T cells, while CsA had less effect on CD8+ T cells than did rapamycin. Elimination of BM infiltrated T cells and restoration of megakaryocytes by rapamycin was visualized by confocal microscopy using whole-mounts of sternum, for which donor B6 lymph node cells were replaced with B6-DsRed lymph node cells. Plasma cytokines were measured by Luminex: IFNg, TNFa, IL-2, MIP1b, RANTES, sCD137 (all p < 0.001) were increased in BM failure mice compared with the control animals, indicating an inflammatory environment in AA. Rapamycin reduced these cytokines (p < 0.001) but increased Th2 cytokines such as IL-4 and IL-10 (p < 0.001) levels. CsA only decreased sCD137, reversely it even increased IFNg levels. Transcriptome analysis using pooled FACS-sorted CD4+ and CD8+ T cells from BM focusing on genes related to T cell functions revealed that rapamycin suppressed expression of Icam1, and Tnfsf14 in CD8+ T cells, and Cd27, Lgals3, Il10ra, Itga1, Tbx21, Gzmb, Tnfsf14 and Cd70 in CD4+ T cells, but increased Il-4, Il-2ra, and Tnfrsf8 expression in CD4+ T cells compared with AA mice. CsA suppressed Lgals3 in CD8+ T cells and Cd70 in CD4+ T cells, suggesting differential mechanisms of action by these two immunosuppressive drugs. All untreated AA mice (n=6) died within 3 weeks post lymphocyte infusion, while all mice treated with rapamycin for 2 weeks (n=8) survived until study termination at 7 weeks; similar results were obtained when we tested delayed treatment with rapamycin (starting 3 days post lymphocyte injection and continued for 10 days, n=8) in BM failure mice; but brief exposure to rapamycin, for only 5 days from 1 hour post lymphocyte infusion (n=8), could not rescue mice, suggesting a requirement for sustained administration. In contrast, all animals treated with CsA (n=6) died within 5 weeks (Fig 1B). We also tested the effect of rapamycin on antigen-specific T cells in another BM failure model induced by infusion of lymphocytes from B6 donor mice into C.B10-H2b /LilMcd recipient mice (MHC-matched but minor antigen-mismatched, n=10), in which BM destruction is mediated by H60-specific cytotoxic T cells (CTL) (Chen J, JI, 2007). Similar results were observed. Flow cytometry revealed massive expansion of H60-specific CTL in BM of untreated AA mice, rapamycin eliminated BM CD8+ T cell infiltration. CsA decreased BM CD8+ T cells, but had much weaker effect on H60 CTLs (Fig 1C). In summary, rapamycin is effective in treatment of AA murine models, which holds implications in the application in immune-mediated pathophysiologies in the laboratory and in the clinic. Compared with CsA, rapamycin suppressed expression of T cell activation genes more broadly, increased Th2 cytokines, eliminated antigen-specific T cells, and had better survival rate in animal BM failure model, supporting a clinical trial of rapamycin to prevent relapse and induce tolerance in patients with AA, many of whom are dependent on CsA administration for support of blood counts but at risk of CsA nephrotoxicity.

Disclosures
No relevant conflicts of interest to declare.