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Ex Vivo Rapamycin Generates Apoptosis-Resistant Donor Th2 Cells That Persist In Vivo and Prevent Hemopoietic Stem Cell Graft Rejection¹

Jacopo Mariotti^2,*, Jason Foley^*, Unsu Jung^*, Todd Borenstein^*, Nermina Kantardzic^*, Soo Han^*, Joshua T. Hanson^*, Elaine Wong^*, Nicole Buxhoeveden^*, Jane B. Trepel, Antonio Tito Fojo, William Telford^* and Daniel H. Fowler^*

^* Experimental Transplantation and Immunology Branch and Medical Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892

Because ex vivo rapamycin generates murine Th2 cells that prevent Graft-versus-host disease more potently than control Th2 cells, we hypothesized that rapamycin would generate Th2/Tc2 cells (Th2/Tc2.R cells) that abrogate fully MHC-disparate hemopoietic stem cell rejection more effectively than control Th2/Tc2 cells. In a B6-into-BALB/c graft rejection model, donor Th2/Tc2.R cells were indeed enriched in their capacity to prevent rejection; importantly, highly purified CD4⁺ Th2.R cells were also highly efficacious for preventing rejection. Rapamycin-generated Th2/Tc2 cells were less likely to die after adoptive transfer, accumulated in vivo at advanced proliferative cycles, and were present in 10-fold higher numbers than control Th2/Tc2 cells. Th2.R cells had a multifaceted, apoptosis-resistant phenotype, including: 1) reduced apoptosis after staurosporine addition, serum starvation, or CD3/CD28 costimulation; 2) reduced activation of caspases 3 and 9; and 3) increased anti-apoptotic Bcl-x_L expression and reduced proapoptotic Bim and Bid expression. Using host-versus-graft reactivity as an immune correlate of graft rejection, we found that the in vivo efficacy of Th2/Tc2.R cells 1) did not require Th2/Tc2.R cell expression of IL-4, IL-10, perforin, or Fas ligand; 2) could not be reversed by IL-2, IL-7, or IL-15 posttransplant therapy; and 3) was intact after therapy with Th2.R cells relatively devoid of Foxp3 expression. We conclude that ex vivo rapamycin generates Th2 cells that are resistant to apoptosis, persist in vivo, and effectively prevent rejection by a mechanism that may be distinct from previously described graft-facilitating T cells.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by the Center for Cancer Research, National Cancer Institute, an Intramural Research Program.

² Address correspondence and reprint requests to Dr. Jacopo Mariotti, Experimental Transplantation and Immunology Branch, National Cancer Institute, National Institutes of Health, Clinical Research Center, 3-East Labs, 3-3330, Bethesda, MD 20892. E-mail address: marriottj{at}mail.nih.gov

³ Abbreviations used in this paper: HSCT, hemopoietic stem cell transplantation; GVHD, graft-versus-host disease; Treg, regulatory; mTOR, mammalian target of rapamycin; HSC, hemopoietic stem cell; HVGR, host-versus-graft reactivity; FasL, Fas ligand; rh, recombinant human; rm, recombinant mouse; CM, conditioned medium; SP, side population; DC, dendritic cell; PARP, poly(ADP-ribose) polymerase; WT, wild type; GSK, glycogen synthase kinase; KO, knockout; TPO, thrombopoietin; SCT, stem cell transplant; HT, host T.