Ex Vivo Rapamycin Generates Th1/Tc1 or Th2/Tc2 Effector T Cells With Enhanced In Vivo Function and Differential Sensitivity to Post-transplant Rapamycin Therapy  Unsu Jung, Jason E. Foley, Andreas A. Erdmann, Yoko Toda, Todd Borenstein, Jacopo Mariotti, Daniel H. Fowler  Biology of Blood and Marrow Transplantation  Volume 12, Issue 9, Pages 905-918 (September 2006) DOI: 10.1016/j.bbmt.2006.05.014 Copyright © 2006 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 1 Effect of rapamycin (Rapa) on T-cell expansion under type I and II polarizing conditions. Splenic T cells were costimulated with anti-CD3, anti-CD28–coated beads in the presence of IL-12 and anti–IL-4 (type I condition, T1) or in the presence of IL-4 (type II condition, T2). Fold increases in T-cell number are shown for T1 (A) and T2 (B) cultures without rapamycin (♦) or with rapamycin at doses of 0.0008 μM (□), 0.004 μM (▵), 0.02 μM (×), 0.1 μM (∗), and 10 μM (○). Results shown are mean values of 5 replicates for each culture condition that were generated from separate donors and run in parallel; each T1 and T2 culture in rapamycin had reduced expansion relative to culture without rapamycin (*P < .05). On day 7, cultures were tested by flow cytometry to determine the effect of rapamycin on the CD4:CD8 ratio under T1 (C) and T2 (D) conditions. Results shown are mean ± SEM of 5 replicates for each culture condition that were generated from separate donors and run in parallel; rapamycin increased the CD4:CD8 ratio compared with control cultures not receiving rapamycin (*P < .05). Biology of Blood and Marrow Transplantation 2006 12, 905-918DOI: (10.1016/j.bbmt.2006.05.014) Copyright © 2006 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 2 Cytokine secretion profile and cytolytic function of T1 and T2 effectors generated with or without rapamycin. On day 6, T1 (A) and T2 (B) cultures were harvested, washed, and resuspended at 0.5 × 106 cells/mL with no stimulation (results not shown) or with repeat costimulation (results shown). The 24-hour supernatants were evaluated by ELISA for cytokine content (reduced cytokine level compared with control culture without rapamycin, *P < .05). Rapamycin dose level “0” refers to no rapamycin added, and dose levels 1, 2, 3, 4, and 5 reflect rapamycin concentrations of 0.0008, 0.004, 0.02, 0.1, and 10 μM, respectively. C. To evaluate fas-based cytolytic function (left panel), T1 cells were stimulated with PMA/CI for 24 hours and coincubated with 51Cr-labeled L1210-fas targets in calcium-neutralized conditions; to evaluate granule-dependent killing (right panel) T2 cells were coincubated in a heteroconjugate assay with 51Cr-labeled P815 targets in calcium-replete conditions. All values shown for cytokine and cytolytic assays are mean ± SEM of 5 replicates for each culture condition that were generated from separate donors and run in parallel. E:T = effector:target. Biology of Blood and Marrow Transplantation 2006 12, 905-918DOI: (10.1016/j.bbmt.2006.05.014) Copyright © 2006 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 3 Rapamycin augments Th1, Th2, Tc1, and Tc2 cell expressions of CD62L. CD4+ and CD8+ T cells were costimulated without or with rapamycin (at the concentrations shown) under T1 or T2 polarizing conditions. On day 6, T cells were incubated overnight without further stimulation or with PMA/CI stimulation. On day 7, T cells were then evaluated by flow cytometry for CD4, CD8, and CD62L expression; results for T cells not restimulated are shown as open bars and those for restimulated T cells are shown as solid bars. Results are expressed as change in CD4 or CD8 cell expression of CD62L relative to isotype controls (mean fluorescence intensity, MFI). Results for CD4+ and CD8+ cells expanded in the T1 condition are shown in the upper left and right panels, respectively; results for CD4+ and CD8+ cells expanded in the T2 condition are shown in the lower left and right panels, respectively. CD62L expression is increased relative to control cells generated without rapamycin (*P < .05); values are mean ± SEM of 5 replicates for each culture condition that were generated from separate donors and run in parallel. Biology of Blood and Marrow Transplantation 2006 12, 905-918DOI: (10.1016/j.bbmt.2006.05.014) Copyright © 2006 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 4 Rapamycin increases CD62L expression independent of T-cell division. CD4+ T cells were purified and stained with CFSE dye, and baseline expression of CD62L was determined by flow cytometry and expressed in terms of (A) percentage of positive cells and (B) mean fluorescence intensity (MFI). A. Four separate cultures of control Th2 cells (Th2) or rapamycin-generated Th2 cells (Th2.R) were established, and the total percentage of Th2 cells expressing CD62L was monitored on days 2, 4, 5, and 6 of culture (results shown are mean ± SEM of 4 cultures; increased percentage of CD62L+ cells in the Th2.R condition relative to the Th2 condition, *P < .0001). B. On day 2 of culture, the CFSE-labeled cells in the Th2 and Th2.R conditions were determined by flow cytometric dye dilution analysis to have not undergone cell division or to have divided 1, 2, 3, or 4 times; CD4 cells were gated based on cell division, and the MFI of CD62L expression was determined (results are mean ± SEM of 4 cultures; increased CD62L expression in Th2.R cells versus Th2 cells at same cell division number, *P < .0001). Biology of Blood and Marrow Transplantation 2006 12, 905-918DOI: (10.1016/j.bbmt.2006.05.014) Copyright © 2006 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 5 Rapamycin (Rapa)-generated Th1/Tc1 and Th2/Tc2 populations have increased expansion in response to repeat costimulation. From day 0 to day 6 of culture, Th1/Tc1 or Th2/Tc2 cells were generated by costimulation in T1 (A) or T2 (B) polarizing conditions without or with rapamycin at concentrations of 0.1 or 10 μM. On day 6 of culture, T cells from each of these 6 conditions were washed and restimulated in the absence of further rapamycin; T-cell expansion was then monitored from day 6 to day 13 of culture. Each rapamycin generated culture had increased expansion relative to the relevant control culture not generated in rapamycin (*P < .05); results represent mean values of 3 independent experiments. Biology of Blood and Marrow Transplantation 2006 12, 905-918DOI: (10.1016/j.bbmt.2006.05.014) Copyright © 2006 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 6 Rapamycin-generated Th1/Tc1 and Th2/Tc2 cells have increased expansion in vivo after allogeneic BMT. Th1/Tc1 and Th2/Tc2 cells were generated with (10 μM; T1.R and T2.R) or without (T1 and T2) rapamycin. To allow cell tracking in vivo after cell mixing experiments, T1 or T1.R cells were generated from CD45.1 donors, whereas T2 or T2.R cells were generated from CD90.1 donors. The expanded parental T cells were injected intravenously into lethally irradiated CB6F1 hosts; cohorts shown received a 1:1 cell mix of T1 and T2 cells, T1.R and T2 cells, or T1 and T2.R cells (each population, 5 × 106 cells injected). A. On day 5 after BMT, the absolute number of CD4+Th1, CD4+Th2, CD8+Tc1, and CD8+Tc2 cells was calculated by total spleen cell counts and flow cytometry. B. For post-BMT cytokine phenotype, spleen cells were adjusted to 0.5 × 106 cells/mL and costimulated for 24 hours; the supernatants were tested for IFN-γ and IL-4 content by ELISA. Each result is the mean ± SEM of 5 subjects per cohort; *P < .05, statistically significant difference compared with cohort receiving T1 and T2 cells generated without rapamycin. Biology of Blood and Marrow Transplantation 2006 12, 905-918DOI: (10.1016/j.bbmt.2006.05.014) Copyright © 2006 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 7 Rapamycin (Rapa) therapy inhibits Th1/Tc1 cells in vivo after BMT. To study the effect of rapamycin on a Th1/Tc1-driven response, donor T-cell inocula consisted of rapamycin-generated Th1/Tc1 cells and Th2/Tc2 cells generated without rapamycin. T cells were administered at a 1:1 ratio (5 × 106 of each population) into lethally irradiated CB6F1 hosts; after BMT, recipients of this T-cell mix received CMC vehicle or rapamycin (1.5 mg·kg−1·d−1 × 5 days). On day 5 after BMT, the number of Th1, Th2, Tc1, and Tc2 cells was calculated by total spleen cell counts and flow cytometry (A), and the percentage of Th1/Tc1 cells retaining CFSE dye was measured (B). C. For post-BMT cytokine phenotype, splenic single cells were harvested at day 5 after BMT and costimulated; after 24 hours, supernatants were collected and cytokine content was evaluated by ELISA. Results are mean ± SEM of 5 subjects per cohort; there was a statistically significant difference between CMC and Rapa cohorts (*P < .05). Similar results were observed in a second experiment. Biology of Blood and Marrow Transplantation 2006 12, 905-918DOI: (10.1016/j.bbmt.2006.05.014) Copyright © 2006 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 8 Effector Th2/Tc2 cells are relatively resistant to rapamycin (Rapa) therapy after BMT. To study the effect of rapamycin on a Th2/Tc2-driven response, donor T-cell inocula consisted of rapamycin-generated Th2/Tc2 cells and Th1/Tc1 cells generated without rapamycin. T cells were administered at a 1:1 ratio (5 × 106 of each population) into lethally irradiated CB6F1 hosts; after BMT, recipients of this T-cell mix received CMC vehicle or rapamycin (1.5 mg·kg−1·d−1 × 5 days). On day 5 after BMT, the number of Th1, Th2, Tc1, and Tc2 cells was calculated by total spleen cell counts and flow cytometry (A), and the percentage of Th2/Tc2 cells retaining CFSE dye was measured (B). C. For post-BMT cytokine phenotype, splenic single cells were harvested at day 5 after BMT and costimulated; after 24 hours, supernatants were collected and cytokine content was evaluated by ELISA. Results are means ± SEM of 5 per cohort; the difference between the CMC and Rapa cohorts was statistically significant (*P < .05). Similar results were observed in a second experiment. Biology of Blood and Marrow Transplantation 2006 12, 905-918DOI: (10.1016/j.bbmt.2006.05.014) Copyright © 2006 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 9 Rapamycin-generated Th1/Tc1 cells mediate increased lethal GVHD: attenuation by in vivo short-course rapamycin therapy. B6-into-BALB/c BMT was performed using 850 cGy of host irradiation followed by marrow infusion alone (BMT; 1 × 107 cells/recipient; n = 10) or in combination with a separate infusion of ex vivo generated control Th1/Tc1 cells (Th1/Tc1; 1 × 107 cells/recipient, n = 10) or rapamycin-exposed Th1/Tc1 cells (Th1/Tc1.R; 1 × 107 cells/recipient, n = 20); each of these cohorts was administered the carboxy-methyl-cellulose drug vehicle (intraperitoneally) for the first 5 days after BMT. A. Survival results for these 3 cohorts are shown (*P = .027, reduced survival in Th1/Tc1.R cohort versus Th1/Tc1 cohort). B. Additional cohorts in this experiment received the Th1/Tc1.R cells and 5 days of post-BMT rapamycin therapy (Th1/Tc1.R + Rapamycin Tx; left panel; rapamycin dose of 1.5 mg·kg−1·d−1; n = 20) or the Th1/Tc1 cells and post-BMT rapamycin therapy (Th1/Tc1 + Rapamycin Tx; right panel; n = 10). *P = .022, rapamycin therapy reduced lethal GVHD in Th1/Tc1.R recipients; **P = .024, rapamycin therapy increased lethal GVHD in Th1/Tc1 recipients. Biology of Blood and Marrow Transplantation 2006 12, 905-918DOI: (10.1016/j.bbmt.2006.05.014) Copyright © 2006 American Society for Blood and Marrow Transplantation Terms and Conditions