Rapamycin inhibits IL-4—induced dendritic cell maturation in vitro and dendritic cell mobilization and function in vivo by Holger Hackstein, Timucin Taner, Alan F. Zahorchak, Adrian E. Morelli, Alison J. Logar, Andre Gessner, and Angus W. Thomson Blood Volume 101(11):4457-4463 June 1, 2003 ©2003 by American Society of Hematology

The inhibitory effect of RAPA on DC maturation is IL-4 dependent and mediated via FKBP-12 binding. The inhibitory effect of RAPA on DC maturation is IL-4 dependent and mediated via FKBP-12 binding. BM-derived DCs were generated with GM-CSF with or without IL-4 and analyzed on day 7. (A-B) In the presence of IL-4, RAPA inhibited the cell surface expression of CD40, CD80, CD86, and MHC class II molecules and the allostimulatory activity of purified CD11c+ DCs, whereas FK506 exhibited no effect. Competition for RAPA's intracellular receptor FKBP12 by a molar excess of FK506 (panel A, second column from right) antagonized the inhibitory effects of RAPA on DC maturation. (C-E) In the absence of IL-4 (C-D), or in IL-4Rα—deficient mice (E), RAPA exerted no inhibitory effect on DC surface expression of CD40, CD80, CD86, MHC class II molecules, or T-cell allostimulatory acitivity. (A,C,E) Cells were gated on CD11c. The incidence of CD11c+ cells expressing the antigen of interest is indicated. Results show representative data from 10 (A), 3 (B,E), 5 (C), and 2 (D) similar experiments. Holger Hackstein et al. Blood 2003;101:4457-4463 ©2003 by American Society of Hematology

RAPA suppresses DC high-affinity IL-4R complex expression at the posttranscriptional level. RAPA suppresses DC high-affinity IL-4R complex expression at the posttranscriptional level. BM-derived DCs were generated with GM-CSF plus IL-4, purified by immunomagnetic bead sorting and subjected to RNase protection assay or directly analyzed by flow cytometry. (A) Comparative RNase protection assay analysis indicates no effect of RAPA on CD124 or CD132 mRNA expression. L32 and GAPDH represent internal controls. (B-C) Down-regulation of CD124 and CD132 cell surface expression by RAPA. Cells were gated on CD11c. The incidence of CD11c+ cells expressing the antigen of interest is indicated. Results show representative data of 3 (A-B) and 5 (C) experiments. Holger Hackstein et al. Blood 2003;101:4457-4463 ©2003 by American Society of Hematology

Analysis of RAPA effects on apoptosis in DCs Analysis of RAPA effects on apoptosis in DCs. RAPA inhibits apoptosis of LPS-stimulated DCs in an FKBP12-dependent manner but does not affect DC apoptosis when cells are cultured for an extended time in the absence of any stimuli (LPS, cytokines). Analysis of RAPA effects on apoptosis in DCs. RAPA inhibits apoptosis of LPS-stimulated DCs in an FKBP12-dependent manner but does not affect DC apoptosis when cells are cultured for an extended time in the absence of any stimuli (LPS, cytokines). BM-derived DCs were generated with GM-CSF plus IL-4 and washed, and equal numbers of cells were stimulated on day 7 with LPS only or cultured without any stimuli and analyzed after staining of phosphatidylserine translocation with FITC-annexin V in combination with the vital dye 7-AAD. (A) RAPA does not affect apoptosis and death when DCs are cultured for an extended time in the absence of any stimuli. (B-C) RAPA inhibits DC apoptosis and death in a dose-and time-dependent manner when DCs are stimulated with LPS. Competition for RAPA's intracellular receptor FKBP12 by a molar excess of FK506 blocks the inhibitory effect of RAPA on DC apoptosis and death. Doses indicate RAPA, unless stated otherwise. Cells were gated on CD11c. The incidence of cells in each quadrant is indicated. Apoptotic cells are stained annexin V+/7-AAD– and dead cells (late apoptotic or necrotic) are stained annexin V+/7-AAD+. Results show representative data from 3 (A) and 4 (B-C) experiments. Similar results were obtained with DCs generated in GM-CSF only. Holger Hackstein et al. Blood 2003;101:4457-4463 ©2003 by American Society of Hematology

DC generation in steady-state and dynamic conditions. DC generation in steady-state and dynamic conditions. In vivo administration of RAPA suppresses DC generation under (A) steady-state and (B-C) dynamic conditions and inhibits up-regulation of costimulatory molecules. (A-C) Effect of RAPA or drug vehicle on the number of CD11c+ DCs/tissue on day 10 (with or without Flt3L). Results are representative of 8 to 10 animals/treatment group. **P = .005 versus vehicle (A, normal spleen), P = .003 versus vehicle (B, Flt3L bone marrow), P = .002 versus vehicle (C, Flt3L spleen); 2-tailed Student t test. Bars indicate 95% confidence interval and mean (rectangle). (D-E) Effect of RAPA or drug vehicle on spleen weight and appearance (8 animals/treatment group) in Flt3L-treated animals on day 10. **P = .0004 versus vehicle, 2-tailed Student t test. Bars indicate 95% confidence interval and mean (rectangle). (F-G) Effect of in vivo RAPA or drug vehicle administration on costimulatory and MHC class II molecule up-regulation after ex vivo LPS stimulation. Cells were gated on CD11c. The median fluorescence intensity (MFI) (F) and relative MFI (G) of CD11c+ cells expressing the antigen of interest in comparison with cells from drug vehicle-treated control animals is indicated. (F) Typical data from one representative experiment on day 10 after start of treatment. (G) Each point represents a single experiment with 3 to 6 animals (with or without Flt3L) per treatment group after in vivo administration of RAPA (7-10 days) or vehicle. **P < .01 versus vehicle (Wilcoxon test). Holger Hackstein et al. Blood 2003;101:4457-4463 ©2003 by American Society of Hematology

In vivo administration of RAPA impairs DC T-cell stimulatory activity. In vivo administration of RAPA impairs DC T-cell stimulatory activity. (A) Allostimulatory activity of freshly isolated, immunomagnetic bead—purified B10 (H2Kb,IAb) DCs from animals (3/group) injected with RAPA (10 days) or drug vehicle (“vehicle DC”). Mean proliferative activity of fully allogeneic C3H (H2Kk, IAk) responder T cells in 72-hour MLRs is shown (± SD). (B-E) Adoptive transfer of freshly isolated splenic B10 DCs from animals (panel C, normal; panels D and E, Flt3L-treated) that were injected with RAPA (7 days) or vehicle into fully allogeneic C3H recipients. C3H mice received 5 × 105 magnetic bead—purified B10 DCs (intravenously). Ten days later, the mice were killed and splenic T cells restimulated with graded numbers of γ-irradiated donor splenocytes. (C-D) Mean proliferation of C3H responder T cells (3 animals/group) in 72-hour MLRs is shown (± SD). T cells from nonimmunized animals (“naive”), from animals given DCs from drug vehicle-injected controls, as well as syngeneic splenocytes (“syngeneic control”) were used as controls. (E) Effect of adoptive transfer of DCs on IFN-γ, IL-2, IL-4, and IL-10 production by recipient T cells after restimulation with donor alloantigen (splenocytes). Mean cytokine production of C3H responder T cells (3 animals/group) in a 72-hour MLR is shown (± SD). *P < .05 versus vehicle (2-tailed Student t test). Holger Hackstein et al. Blood 2003;101:4457-4463 ©2003 by American Society of Hematology

In vivo administration of RAPA promotes IL-4 hyporesponsiveness of DCs and suppresses TNF-α production. In vivo administration of RAPA promotes IL-4 hyporesponsiveness of DCs and suppresses TNF-α production. Animals were treated with RAPA (▴) or vehicle (plus Flt3L, 10 days; ▪). Splenic DCs were purified by density gradient centrifugation and immunomagnetic bead sorting and stimulated with LPS for 24 hours. (A-B) Titration of the effect of IL-4 (in the presence of LPS) on the production of bioactive IL-12p70. Mean IL-12p70 production (± 1 SD) by DCs from mice given RAPA for 7 days (A) and 10 days (B) versus drug vehicle-injected controls (3 animals/group). (C) Effect of RAPA on TNF-α production. Mean TNF-α production (± 1 SD) by DCs from animals that received RAPA for 10 days versus drug vehicle-injected controls (3 animals/group). *P < .05 versus vehicle (2-tailed Student t test). Holger Hackstein et al. Blood 2003;101:4457-4463 ©2003 by American Society of Hematology