Ex Vivo Rapamycin Generates Th1/Tc1 or Th2/Tc2 Effector T Cells ...

Biology of Blood and Marrow Transplantation 12:905-918 (2006) 䊚 2006 American Society for Blood and Marrow Transplantation 1083-8791/06/1209-0001$32.00/0 doi:10.1016/j.bbmt.2006.05.014

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 Center for Cancer Research, National Institutes of Health, Experimental Transplantation and Immunology Branch, Bethesda, Maryland Correspondence and reprint requests: Daniel H. Fowler, MD, National Institutes of Health; 10 Center Drive, Building 10, CRC; 3 EAST Labs, 3-3330; Bethesda, MD, 20892 (e-mail: [email protected]). Received September 13, 2005; accepted May 26, 2006

ABSTRACT Rapamycin prevention of murine graft-versus-host disease (GVHD) is associated with a shift toward Th2- and Tc2-type cytokines. Recently, we found that use of rapamycin during ex vivo donor Th2 cell generation enhances the ability of adoptively transferred Th2 cells to prevent murine GVHD. In this study, using a method, without antigen-presenting cells, of T-cell expansion based on CD3,CD28 costimulation, we evaluated whether (1) rapamycin preferentially promotes the generation of Th2/Tc2 cells relative to Th1/Tc1 cells, (2) rapamycin-generated T-cell subsets induce cytokine skewing after allogeneic bone marrow transplantation (BMT), and (3) such in vivo cytokine skewing is sensitive to post-BMT rapamycin therapy. Contrary to our hypothesis, rapamycin did not preferentially promote Th2/Tc2 cell polarity, because rapamycin-generated Th1/Tc1 cells secreted type I cytokines (interleukin [IL]-2 and interferon-␥) did not secrete type II cytokines (IL-4, IL-5, IL-10, or IL-13) and mediated fasL-based cytolysis. Rapamycin influenced T-cell differentiation, because each of the Th1, Th2, Tc1, and Tc2 subsets generated in rapamycin had increased expression of the central-memory T-cell marker, L-selectin (CD62L). Rapamycin-generated Th1/Tc1 and Th2/Tc2 cells were not anergic but instead had increased expansion after costimulation in vitro, increased expansion in vivo after BMT, and maintained full capacity to skew toward type I or II cytokines after BMT, respectively; further, rapamycin-generated Th1/Tc1 cells mediated increased lethal GVHD relative to control Th1/Tc1 cells. Rapamycin therapy after BMT in recipients of rapamycin-generated Th1/Tc1 cells greatly reduced Th1/Tc1 cell number, greatly reduced type I cytokines, and reduced lethal GVHD; in marked contrast, rapamycin therapy in recipients of rapamycin-generated Th2/Tc2 cells nominally influenced the number of Th2/Tc2 cells in vivo and did not abrogate post-BMT type II cytokine skewing. In conclusion, ex vivo and in vivo usage of rapamycin may be used to modulate the post-BMT balance of Th1/Tc1 and Th2/Tc2 cell subsets. © 2006 American Society for Blood and Marrow Transplantation

KEY WORDS Rapamycin

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Th1/Th2

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Graft-versus-host disease

INTRODUCTION Rapamycin (sirolimus) has been shown to reduce murine graft-versus-host disease (GVHD) [1] and has recently demonstrated activity for the prevention [2,3] or therapy [4-6] of human GVHD. The molecular mechanism of rapamycin action involves drug binding to the immunophilin FKBP-12 [7] and subsequent inhibition of the mammalian target of rapamycin [8].

However, the cellular mechanisms of rapamycin action, particularly as they relate to allogeneic transplantation, are less well defined and appear to include modulation of antigen-presenting cells (APCs) for immune suppression by non–T-cell–mediated “indirect” effects [9], induction of regulatory cells that mediate immunosuppressive effects within GVHD target tissue [10], promotion of donor T-cell tolerance to host 905

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alloantigen during CD28 costimulation [11], and polarization after transplantation of T-cell subsets toward CD4⫹Th2 and CD8⫹Tc2 phenotypes [12,13]. Given this association of rapamycin therapy with type II cytokine polarization, we recently evaluated whether rapamycin might be used in vitro during donor CD4⫹Th2 cell generation to enhance Th2 cellbased GVHD prevention strategies [14]. In these experiments, we found that: (1) when using CD3, CD28 costimulation and interleukin (IL)-4 – based culture conditions, Th2 cell differentiation proceeded in the presence of high-dose rapamycin (10 ␮M); (2) relative to control Th2 cells, rapamycin-generated Th2 cells (Th2.rapa cells) more effectively upregulated type II cytokines, downregulated type I cytokines, and inhibited allospecific T-cell responses after bone marrow transplantation (BMT); and (3) Th2.rapa cells potently abrogated GVHD and graft-versus-tumor (GVT) effects through a mechanism dependent in part on Th2 cell IL-4 secretion. These findings indicate that rapamycin can be used exclusively in vitro to enhance Th2 cell therapy, thereby permitting an anti-GVHD effect of this drug to be used without potential for in vivo drug toxicity. In the present project, we further characterized the influence of rapamycin on T-cell subset generation in vitro and evaluated the effect of in vivo post-BMT rapamycin therapy on the post-BMT balance of rapamycingenerated, polarized, effector T cells. First, by using our established methods of T-cell polarization after APCfree costimulation, we tested whether the capacity to generate cytokine-polarized effector T cells in highdose rapamycin could be generalized from our prior observations relating to CD4⫹Th2 cells to the CD4⫹Th1, CD8⫹Tc1, and CD8⫹Tc2 subsets. Second, we evaluated whether rapamycin might influence polarized effector T-cell differentiation status into T “central memory” (TCM) cells that express the lymph node homing molecule L-selectin (CD62L) or T “effector memory” (TEM) cells that have reduced CD62L expression. Emerging data indicate that TCM status associates with enhanced in vivo effects after adoptive T-cell therapy, because unmanipulated CD62L-expressing allogeneic T cells mediate increased GVHD [15,16], CD62L-expressing allogeneic T regulatory cells have an increased capacity to prevent GVHD [17,18], and antigen-specific CD62Lexpressing syngeneic T cells mediate increased antitumor effects [19]. Third, we evaluated whether cytokine-polarized effector T cells generated in the presence of high-dose rapamycin maintain resistance to rapamycin in vivo after BMT. We reasoned that T-cell rapamycin resistance, which has been described in human CD8⫹ T cells [20], might allow further skewing in the post-BMT balance of Th1/Tc1 and Th2/Tc2 populations during rapamycin therapy.

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METHODS Animals

C57BL/6 ⫻ BALB/c F1 (CB6F1, H-2Kb/d), BALB/c (H-2Kd), C57BL/6 (B6, H-2Kb; Ly5.2), and Ly5.1 congenic (H-2Kb) mice were obtained from Frederick Cancer Research Facility (Frederick, Md). Thy1.1 congenic (H-2Kb) mice were obtained from Jackson Laboratory (Bar Harbor, Me). Mice were maintained in a specific pathogen-free facility at the National Institutes of Health and treated according to an approved animal protocol. Generation of Th1/Tc1 and Th2/Tc2 Subsets Using CD3 and CD28 Costimulation

Spleens were harvested and ACK lysed (Quality Biological, Inc, Gaithersburg, Md). B cells were depleted using goat anti-mouse immunoglobulin G bioparticles (Polysciences Inc, Warrington, Pa). Resultant splenic T cells were used for generation of Th1/ Tc1 or Th2/Tc2 cells. To generate antibody-coated beads, M450 Dynabeads (Dynal, Oslo, Norway) were incubated with anti-murine CD3 and CD28 monoclonal antibodies (mAbs; BD PharMingen, San Diego, Calif) in 0.1 M borate solution at 37°C overnight, as described previously [21,22]. CD3, CD28 mAb-coated beads were washed with phosphate buffered saline containing 3% fetal bovine serum (Gemini Bio-Products, Woodland, Calif), 5 mM ethylenediaminetetraacetic acid (Quality Biological, Inc), and 0.1% sodium azide (Sigma, St Louis, Mo) and resuspended at a bead concentration of 40 ⫻ 106 beads/mL. Costimulation was performed at a bead-to–T-cell ratio of 3:1. Complete media for T-cell culture contained RPMI 1640 (Mediatech, Herndon, Va), 10% fetal bovine serum (Gemini Bio-Products), penicillin-streptomycinglutamine (Life Technologies; Rockville, Md), nonessential amino acids (Life Technologies), and 2-mercaptoethanol (Sigma). Rapamycin (sirolimus) was purchased from LC Labs (Woburn, Mass). Media used to generate Th1/Tc1 cells consisted of recombinant human (rh) IL-2 (20 IU/mL; National Cancer Institute Repository, Rockville, Md), rhIL-7 (20 ng/ mL; Peprotech, Rocky Hill, NJ), recombinant murine (rm) IL-12 (10 ng/mL; Peprotech), anti-murine IL-4 mAb (clone 11B.11; 10 ␮g/mL; National Cancer Institute Repository), and 3.3 mM N-acetylcysteine (Bristol-Myers Squibb, Princeton, NJ). Medium used to generate Th2/Tc2 cells was supplemented with rhIL-2 (1000 IU/mL), rhIL-7 (20 ng/mL), rmIL-4 (1000 IU/mL; Peprotech), and Nacetyl-cysteine (3.3 mM). Cytokine-containing complete medium was added to cultures daily from days 2 to 6 to maintain cell concentration of 0.2-1.0 ⫻ 106 cells/mL; however, rmIL-12 was added only on day 0 of culture. Costimulated T cells were phenotyped by enzyme-linked immunosorbent assay

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Rapamycin-Generated Murine Th1/Tc1 and Th2/Tc2 Cells

(ELISA), cytotoxic T lymphocyte (CTL) assay, and fluorescence-activated cell sorting on day 6 of culture.

ties on gated CD4⫹ T cells. Five to ten thousand live events were acquired for analysis, with propidium iodide exclusion of dead cells.

Cytokine Secretion Profile by ELISA

Costimulated T cells were harvested, washed, and resuspended at 0.5 ⫻ 106 cells/mL with no stimulation or repeat costimulation. Supernatants were collected after 24 hours and cytokine content was evaluated by 2-site ELISA (IL-2 and IL-5: R&D Systems, Minneapoli, Minn; IL-4, IL-10, IL-13, and interferon-␥ [IFN-␥]: BioSource; Camarillo, Calif). CTL Assays

Costimulated T cells were evaluated for cytolytic capacity by standard 51Cr-release assays. Target cell lines used for CTL assays were provided by Dr P. Henkart (National Institutes of Health, Bethesda, Md); targets were labeled with 51Cr (Amersham; Buckinghamshire, England). For assays measuring granule-based killing, target cells were P815 cells coated with anti-murine CD3 mAb (clone 2C11; BD PharMingen); effector T cells were used without secondary stimulation. For fas-based killing, target cells were L1210-fas–transfected and nontransfected L1210 cells; effector T cells were stimulated with phorbol ester (PMA, 5 ng/mL; Sigma) and calcium ionophore (CI, 375 ng/mL; Sigma) for 24 hours before CTL assay. To inhibit exocytosis-based killing [23], fas-based CTL assays were performed in calcium-neutralized conditions (5 mM ethylene glycol bis (2-aminoethyl ether)-N,N,N=N=-tetraacetic acid (EGTA), 0.5 mMgCl2; Sigma). Supernatants were collected 8 hours after effector and target cell coincubation, transferred to LumaPlates (Packard, Meriden, Conn), and read (Microplate Scintillation and Luminescence Counter, Packard). Percent specific lysis was then calculated. Surface Phenotype of Th1/Tc1 and Th2/Tc2 Cells by Flow Cytometry

On days 0 and 6 of culture, T cells were harvested and brought to a final concentration of 0.5 ⫻ 106 cells/mL; T cells received no further stimulation or were stimulated for 24 hours with PMA/CI. Antibodies used included anti-murine CD4, CD8, CD62L, and CD44 conjugated to fluorescein isothiocyanate, phycoerythrin, or allophycocyanin (APC) (BD PharMingen). Three-color fluorescence-activated cell sorting was performed with FACSCalibur (BD Biosciences, San Jose, Calif) and CellQuest software (BD Biosciences). To determine CD62L expression on Th2 cells or rapamycin-generated Th2 cells at cell divisions 0 through 4, cultures were initiated with CD4⫹ T cells labeled with 1 ␮M 5(6)-carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes, Eugene, Ore); cell division number was calculated by successive 50% dilution in CFSE intensi-

Enumeration of T1 and T2 Cells In Vivo after Allogeneic BMT

To facilitate T-cell tracking after BMT, parental B6 T1 and T2 cells were generated from Thy1.1 and Ly5.1 donors, respectively, with or without rapamycin added to culture. In some experiments, to determine T-cell proliferation in vivo, polarized T cells were labeled with CFSE. CB6F1 hosts were lethally irradiated (1100 cGy; 137Cs gamma-radiation source; Gamma Cell 40, Atomic Energy of Canada, Ottawa, Canada) and reconstituted intravenously with parental B6 bone marrow cells (Ly5.2). To maintain consistency with our prior model [22], host mice also received breast cancer cells (intravenously; TS/A cells [24]; H-2d; 0.1 ⫻ 106 cells); however, determination of the potential influence of T1 versus T2 cells on an allogeneic GVT effect was not possible, because tumor cells were not consistently detected in vivo at day 5 after BMT. Bone marrow transplant was supplemented with a 1:1 ratio of Th1/Tc1 and Th2/Tc2 cells through separate intravenous injections (5 ⫻ 106 of each population); other control groups received 1 ⫻ 107 T cells of only Th1/Tc1 or Th2/Tc2 phenotype. Rapamycin therapy was administered (intraperitoneally) at a previously described dose of 1.5 mg · kg⫺1 · d⫺1 [13]; control mice received intraperitoneal injection of the drug vehicle, carboxy-methyl-cellulose (CMC; Sigma). On day 5 after BMT, spleen cell number was determined, and CD4⫹Th1, CD8⫹Tc1, CD4⫹Th2, and CD8⫹Tc2 populations were identified by flow cytometry using anti-CD4, anti-CD8, anti-CD45.1, and anti-CD90.1 (BD PharMingen). To analyze the effect of ex vivo or in vivo rapamycin on lethal GVHD, B6-into-BALB/c BMT was performed using 850 cGy of host irradiation followed by marrow infusion (1 ⫻ 107 cells/recipient) and separate infusion of ex vivo-generated control or rapamycinexposed Th1/Tc1 cells (1 ⫻ 107 Th1/Tc1 cells/recipient). Mice that underwent transplantation received in vivo rapamycin (1.5 mg · kg⫺1 · d⫺1 ⫻ 5 days) or control CMC drug vehicle and were then evaluated daily for survival; premorbid transplant recipients were euthanized. Determination of Post-transplant Cytokine Polarization

Splenic single-cell suspensions were obtained on day 5 after BMT. After determination of total spleen cell number, cells were adjusted to 0.5 ⫻ 106 cells/mL with no further stimulation or stimulation with antiCD3, anti-CD28 coated beads. After 24 hours, supernatants were collected and cytokine content was evaluated using 2-site ELISA.

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Statistics

Group comparisons were by paired Student t test; P ⬍ .05 was considered statistically significant. For survival analysis, Wilcoxon nonparametric rank-sum analysis was used. RESULTS Rapamycin Limits But Does Not Abrogate Th1/Tc1 and Th2/Tc2 Cell Expansion

T cells expanded approximately 100-fold over 6 days in culture after costimulation in Th1/Tc1 (T1) or Th2/Tc2 (T2) polarizing conditions in the absence of rapamycin (Figure 1A,B). Rapamycin inhibited T-cell expansion by approximately 1 log in T1 and T2 conditions at the lowest concentration tested (0.0008 ␮M); higher concentrations of rapamycin (up to 10 ␮M) resulted in further incremental reductions in T-cell yield. CD4⫹Th2 cells had reduced sensitivity to rapamycin inhibition, as evidenced by the

increased CD4:CD8 ratio in the T2 culture condition (Figure 1D). Th1/Tc1 and Th2/Tc2 Polarization Proceeds in the Presence of Rapamycin

Using this APC-free culture system, we next evaluated whether rapamycin inhibited Th1/Tc1 or Th2/ Tc2 cell polarization. T cells costimulated under type I polarizing conditions secreted the type I cytokines IL-2 and IFN-␥ concomitant with nominal or undetectable secretion of the type II cytokines IL-4, IL-5, IL-10, and IL-13 (Figure 2A); rapamycin, even at the highest dose tested (10 ␮M), reduced type I cytokine secretion but did not induce type II cytokine secretion. By comparison, T cells costimulated under type II polarizing conditions secreted high levels of type II cytokines but also secreted type I cytokines (Figure 2B); although rapamycin reduced the magnitude of type II cytokine secretion, IL-4 secretion was relatively spared, and type I cytokines were dramatically reduced.

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 (⽧) or with rapamycin at doses of 0.0008 ␮M (□), 0.004 ␮M ( ), 0.02 ␮M (⫻), 0.1 ␮M (ⴱ), and 10 ␮M (Œ). 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).

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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 ␮M, 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.

In sum, these data indicate that rapamycin modulated T-cell effector function by reducing the magnitude of cytokine secretion, but did not directly impair the capacity for T-cell polarization toward type I or II phenotypes.

in rapamycin partly reduced T-cell cytolytic capacity in a dose-dependent manner.

Cytolytic Function of Rapamycin-Generated Th1/Tc1 and Th2/Tc2 Cells

As discussed in the Introduction, one goal of our studies was to characterize the effect of rapamycin on polarized T-cell differentiation into central memory (TCM) versus effector memory (TEM) subsets as defined by the presence or absence of CD62L expression, respectively. Of note, in previous experiments by Blazar et al [13], in vivo rapamycin therapy was shown to increase post-BMT T-cell expression of CD62L. After costimulated T-cell expansion, all CD4⫹ and CD8⫹ T cells propagated with or without rapamycin in Th1/Tc1 and Th2/Tc2 conditions attained memory status, as defined by uniform expression of CD44 (data not shown). In the absence of rapamycin, CD62L was pref-

Similar to results that we and others previously obtained [22,25], the Th1/Tc1 population mediated potent fas-based cytolysis (Figure 2C, left panel) and limited killing by granule-mediated cytolysis (⬍20% specific lysis of P815 targets at a 30:1 effector:target ratio; data not shown). In contrast, the Th2/Tc2 population mediated potent granule-based killing (Figure 2C, right panel) and limited killing by fas-based cytolysis (⬍20% lysis of L1210-fas targets at a 30:1 effector:target ratio; data not shown). As Figure 2C illustrates, generation of Th1/Tc1 and Th2/Tc2 cells

Rapamycin Increases Th1, Th2, Tc1, and Tc2 Cell Expression of CD62L

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erentially expressed on expanded CD4⫹ T cells relative to CD8⫹ T cells and preferentially expressed on Th1type cells relative to Th2-type cells (Figure 3). In the presence of rapamycin, even at a relatively low concentration of 0.0008 ␮M, costimulated Th1, Th2, Tc1, and Tc2 cells each expressed greatly increased CD62L. This effect was particularly dramatic for Th2 and Tc2 cells, because these populations expressed only nominal CD62L in the absence of rapamycin. Further, rapamycin-generated T-cell subsets maintained significant expression of CD62L expression after further T-cell activation; in marked contrast, Th1, Tc1, Th2, and Tc2 cells generated without rapamycin were devoid of CD62L expression after restimulation. Rapamycin upregulation of CD62L was observed if data were expressed in terms of mean fluorescence intensity (MFI; Figure 3) or in terms of percentage of cells positive; relative to the control cultures generated without rapamycin, the addition of low-dose rapamycin (0.0008 ␮M) increased the percentage of cells expressing CD62L for the Th1 subset (from 79.4 ⫾

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2.3% to 93.1 ⫾ 2.3%, P ⬍ .05), the Tc1 subset (from 48.9 ⫾ 1.3% to 96.6 ⫾ 0.3%, P ⬍ .05), the Th2 subset (from 34.0 ⫾ 1.1% to 95.8 ⫾ 1.0%, P ⬍ .05), and the Tc2 subset (from 10.8 ⫾ 0.5% to 85.8 ⫾ 1.1%, P ⬍ .05). Similar increases in percentage of cells expressing CD62L were observed in the higher-dose rapamycin conditions (data not shown). Additional experiments using purified CD4⫹ Th2 cell cultures were performed to further characterize the effect of rapamycin on CD62L expression. CD4 cell expansion under control Th2 conditions resulted in a rapid downregulation of CD62L expression (Figure 4A; reduction from 52.2% CD4⫹CD62L⫹ cells on day 0 to 27.8 ⫾ 1.0% CD4⫹CD62L⫹ cells on day 2 of culture); in marked contrast, in the rapamycingenerated Th2 cell culture, 71.1 ⫾ 1.0% of cells were CD4⫹CD62L⫹ on day 2 of culture (increased relative to control Th2 culture, P ⬍ .0004). The percentage of Th2.rapa cells that expressed CD62L remained elevated relative to input CD4 cells and to control Th2 cells at days 4, 5, and 6 of culture. We reasoned that

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.

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the rapamycin-induced increase in CD62L might require T-cell division and thereby operate through a cell selection mechanism; to evaluate this possibility, input CD4⫹ T cells were labeled with CFSE and the MFI of CD62L expression was calculated for Th2 cell divisions 0 through 4 (Figure 4B). Consistent with the antiproliferative action of rapamycin, we observed that rapamycin-generated Th2 cells divided less than control Th2 cells did (day 2 of culture; 0 to 2 divisions versus up to 4 divisions). In addition, CD4⫹ T cells that had not undergone cell division in the rapamycincontaining Th2 cell culture condition had an increase in CD62L relative to input CD4 cells (increase in MFI from 144 to 1285 ⫾ 180). Further, rapamycin increased Th2 cell CD62L expression relative to control Th2 cell cultures for cells that had undergone 1 cell division (CD62L MFI: 1149 ⫾ 49 [Th2.rapa culture] versus 218 ⫾ 11 [Th2 culture], P ⬍ .0003) or 2 cell divisions (CD62L MFI: 674 ⫾ 20 [Th2.rapa culture] versus 174 ⫾ 14 [Th2 culture]; P ⬍ .0003). As such, the rapamycin-induced increase in CD62L expression occurred on a population and a per-cell basis and occurred independent of T-cell division. Rapamycin-Generated T Cells: Enhanced Expansion Upon Repeat Costimulation

In sum, these data indicate that rapamycin promoted the generation of polarized Th1/Tc1 and Th2/ Tc2 cells of central memory phenotype, as defined by increased CD44 expression, high-level expression of CD62L, and an intermediate level of cytokine and

cytolytic effector function. One functional characteristic of TCM cells is their increased expansion in response to CD28 costimulation relative to TEM cells, which are more reliant on alternative costimulatory pathways such as 4-1BBL [26]. To address this biology, Th1/Tc1 and Th2/Tc2 populations generated with or without rapamycin were harvested (day 6 of culture), and subjected to repeat CD28 costimulation in the absence of further rapamycin. Polarization in the presence of rapamycin yielded T cells with enhanced capacity for expansion upon repeat CD28 costimulation (Figure 5). Rapamycin-Generated T Cells: Enhanced Expansion After Allogeneic BMT

We next evaluated whether such rapamycin-generated T cells also had an increased capacity to induce T-cell cytokine polarization after allogeneic BMT. As described in Methods, to allow in vivo T-cell tracking, Th1/Tc1 and Th2/Tc2 cells were generated from CD45.1 and CD90.1 congenic donors, respectively. To evaluate the role of in vitro rapamycin to modulate the post-BMT balance of Th1/Tc1 and Th2/Tc2 populations, cohorts received Th1/Tc1 cells (generated with or without rapamycin) and Th2/Tc2 cells (generated with or without rapamycin) in a 1:1 ratio; control cohorts that received only Th1/Tc1 or only Th2/Tc2 cells expressed the anticipated type I or II cytokine profile, respectively, after BMT (data not shown). Rapamycin-generated Th1/Tc1 cells yielded increased numbers of CD4⫹Th1 and CD8⫹Tc1 cells

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).

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after BMT relative to Th1/Tc1 cells not generated in rapamycin (Figure 6A); importantly, post-BMT T cells in recipients of rapamycin-generated Th1/Tc1 cells had a type I phenotype, with significant IFN-␥ secretion concomitant with nominal secretion of IL-4 (Figure 6B, left panel). Similarly, rapamycin-generated Th2/Tc2 cells yielded increased numbers of CD4⫹Th2 cells and CD8⫹Tc2 cells after BMT relative to Th2/Tc2 cells not generated in rapamycin (Figure 6A); most strikingly, rapamycin-generated Th2/Tc2 cells also induced a marked shift toward type II cytokines after BMT, because T-cell capacity for IL-4 secretion was greatly increased and IFN-␥ secretion was reduced (Figure 6B, right panel). In sum, these data indicate that in vitro rapamycin increased the capacity of Th1/Tc1 cells to induce type I polarization after BMT and increased the capacity of Th2/Tc2 cells to induce type II polarization after BMT. In Vivo Rapamycin Therapy Downregulates Th1 and Tc1 Effector Cells After BMT

Further in vivo experiments were performed to evaluate the influence of rapamycin therapy on the post-BMT balance of effector Th1/Tc1 and Th2/Tc2 cells. In the data shown in Figure 6, we found that donor inocula consisting of a 1:1 cell mix of Th1/Tc1 cells generated in rapamycin and Th2/Tc2 cells generated in the absence of rapamycin induced a Th1/ Tc1 dominant response after BMT. Therefore, to study the effect of rapamycin on a Th1/Tc1 response, cohorts received this donor T-cell mix followed by in vivo therapy with rapamycin or CMC vehicle. Relative to administration of CMC vehicle, rapamycin therapy significantly reduced the post-BMT absolute number of CD4⫹Th1 cells and CD8⫹Tc1 cells (Figure 7A). The rapamycin-mediated reduction in Th1 and Tc1

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 ␮M. 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.

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cell number occurred in part through an antiproliferative mechanism, because rapamycin therapy significantly increased the post-BMT frequency of donor Th1/Tc1 cells that maintained expression of CFSE (Figure 7B; 12.5% CFSE⫹ Th1/Tc1 cells after CMC therapy versus 59.7% CFSE⫹ Th1/Tc1 cells after rapamycin therapy, P ⬍ .05). Rapamycin therapy nearly completely abrogated post-BMT T-cell capacity for IFN-␥ secretion; capacity for IL-2 secretion was also reduced, but this difference did not reach statistical significance. In the setting of this downregulation of Th1-type cytokines, there was no associated increase in secretion of the type II cytokines IL-4, IL-5, IL-10, or IL-13 (Figure 7C). As such, after adoptive transfer of rapamycin-generated Th1/Tc1 cells that expressed a TCM differentiation profile, in vivo rapamycin inhibited Th1 and Tc1 cell number after BMT and inhibited post-BMT type I cytokine secretion, but did not induce a type I to a type II shift in cytokine profile. Effector Th2 and Tc2 Cells Are Relatively Resistant to In Vivo Rapamycin Therapy

Cohorts were also included to assess whether rapamycin therapy modulated Th2/Tc2-driven in vivo responses. For this assessment, cohorts received a 1:1 donor cell mix that consisted of Th2/Tc2 cells generated in the presence of rapamycin and Th1/Tc1 cells generated in the absence of rapamycin, because this T-cell combination yielded a highly polarized type II cytokine secretion profile after BMT (Figure 6B). Relative to administration of drug vehicle, rapamycin modestly reduced the post-BMT absolute number of CD4⫹Th2 cells, and actually increased the number of CD8⫹Tc2 cells after BMT (Figure 8A). Further, rapamycin therapy yielded a relatively modest antiproliferative effect on the Th2/Tc2 population (Figure 8B; 9.9% CFSE⫹ Th2/Tc2 cells after CMC therapy versus 34.0% CFSE⫹ Th2/Tc2 cells after rapamycin therapy, P ⬍ .05). Rapamycin therapy did not significantly reduce type II cytokine polarization, because rapamycin resulted in a statistically significant reduction in post-BMT capacity for IFN-␥ secretion and did not abrogate IL-4 secretion capacity. However, rapamycin resulted in a statistically significant reduction in IL-13 secretion capacity; the capacity for secretion of the type II cytokines IL-5 and IL-10 was also reduced, but this difference did not reach statistical significance (Figure 8C). Of note, in this experiment, which involved administration of TEM-like Th1/Tc1 cells generated without rapamycin, in vivo rapamycin therapy did not reduce Th1 or Tc1 cell number and only modestly reduced post-BMT IFN-␥ secretion. In sum, these data indicate that Th2/Tc2 responses generated from TCM-like effectors were relatively resistant to in vivo rapamycin therapy.

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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 ␮M; 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-␥ 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.

Rapamycin-Generated Th1/Tc1 Cells Mediate Increased Lethal GVHD: Attenuation by In Vivo Rapamycin Therapy

Given the immunosuppressive nature of rapamycin and its known capacity to promote type II cytokine polarity in vivo, our observations that rapamycin-generated Th1/Tc1 cells had increased expansion and maintained a type I cytokine phenotype in vivo were not initially predicted. Given this new information, we hypothesized that rapamycin-generated Th1/Tc1 cells would generate increased GVHD relative to control Th1/Tc1 cells. Consistent with this hypothesis, we found that the median survival after fully major histocompatibility complex disparate BMT in recipients of rapamycin-generated Th1/Tc1 cells was only 7 days (Figure 9A); by comparison, median post-BMT survival in recipients of control Th1/Tc1 cells had not yet been met at 40 days after BMT (P ⬍ .027). Because rapamycin-generated Th1/Tc1 cells were

sensitive to inhibition by a short 5-day course of in vivo rapamycin therapy (Figure 7), we further hypothesized that the lethal GVHD mediated by this T-cell population might be attenuated by short-course rapamycin therapy after BMT. We found that rapamycin therapy attenuated lethal GVHD mediated by rapamycin-generated Th1/Tc1 cells, because median post-BMT survival increased from 7 to 31 days after BMT (Figure 9B; P ⫽ .022). In contrast, GVHD mediated by control Th1/Tc1 cells was unexpectedly increased by rapamycin therapy, because median postBMT survival was reduced from ⱖ40 to 7 days after BMT with rapamycin therapy (P ⫽ .024).

DISCUSSION Although the immunosuppressive effect of rapamycin has been recognized for decades, characteriza-

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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.

tion of the molecular and cellular mechanisms of rapamycin action is broadening and clinical application of rapamycin is expanding. In this study, we evaluated one putative cellular mechanism of rapamycin action (modulation of Th1/Th2 and Tc1/Tc2 cell subset differentiation) and one potential application of rapamycin (ex vivo and in vivo usage of rapamycin for modulation of the post-BMT balance of polarized T-cell subsets). Contrary to our hypothesis, we found that rapamycin did not differentially influence donor T-cell polarization toward polarized Th1/Tc1 and Th2/Tc2 cell subsets. That is, across a broad range of rapamycin concentrations, costimulation in the presence of IL-12 and neutralizing antibody to IL-4 yielded T cells with a Th1/Tc1 phenotype, as defined by the secretion of type I cytokines and the mediation of fas-based cytolysis. Our observation that in vitro rapamycin did not inhibit Th1/ Tc1 cell differentiation or cause a Th1/Tc1 to Th2/Tc2 shift stands in contrast to previous in vivo results, where rapamycin has been shown to promote a shift toward

cells of Th2 and Tc2 phenotypes [12,13]. Our findings indicate that the in vitro T-cell expansion method that we used, which includes APC-independent costimulation and exogenous IL-12, provides essential signals required for Th1/Tc1 cell differentiation that function independent of rapamycin. Such signals may not be provided in other models where APCs are operational, because rapamycin has been shown to inhibit APCs through mechanisms that include downregulation of costimulatory molecule expression [9] and IL-12/IL-18 cytokine pathways [27]. Because rapamycin does not appear to differentially modulate T-cell polarization pathways in a direct manner, the full diversity of Th1, Tc1, Th2, and Tc2 populations can be generated in rapamycin, thereby offering an opportunity to explore the potential utility of these populations via adoptive T-cell therapy. Of note, a recent study reported the generation of CD4⫹ T regulatory cells in rapamycin using an APC-free method [28], thereby providing another example of the diversity of T-cell phenotypes amenable to expansion in rapamycin.


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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.

Three aspects of our data suggest that rapamycin facilitates the generation of CD4⫹Th1/Th2 and CD8⫹Tc1/Tc2 cells with a central-memory effector phenotype. First, rapamycin greatly increased T-cell expression of CD62L, which is a marker for TCM cells [29]. Of note, the rapamycin-induced increase in CD62L was observed at a population level and a per-cell basis and did not require T-cell division; as such, “selection” for CD62L-expressing T cells during rapamycin exposure does not likely represent the primary mechanism for the observed CD62L increase. It is possible that reduced CD62L shedding and upregulation of mRNA transcription represent alternative mechanisms; regarding this latter possibility, we recently found that rapamycingenerated Th2 cells have greatly increased CD62L mRNA (data not shown). Our in vitro findings are consistent with the in vivo results of Blazar et al [13] who demonstrated that rapamycin prevention of murine GVHD was associated with increased donor T-cell expression of CD62L. Also consistent with

other studies, we found that, in the absence of rapamycin, Th1 cells expressed increased CD62L relative to Th2 cells [30,31]. Although we found that rapamycin upregulated CD62L on each T-cell subset evaluated, this effect was particularly prominent for the Th2 and Tc2 subsets, because these populations expressed nominal CD62L in the absence of rapamycin. Second, rapamycin-generated T cells expressed an intermediate level of T-cell effector function that is characteristic of TCM cells, including reduced magnitude of cytokine secretion, reduced secretion of “distal” type II cytokines such as IL-5, IL-10, and IL-13, and reduced cytolytic function [29]. Third, similar to other studies of adoptive T-cell transfer that have observed increased in vivo effects of CD62L-expressing TCM type cells [15-19], we found that each of the rapamycin-generated T-cell subsets had increased expansion after BMT and yielded a more marked polarization in the post-BMT cytokine profile. In recent experiments, we demonstrated that the

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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-methylcellulose 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.

increased capacity of rapamycin-generated T cells to modulate the post-BMT cytokine phenotype has important implications for allograft manipulation strategies, because Th2 cells generated in rapamycin were more effective than control Th2 cells for GVHD prevention [14]. The present data predict that use of in vitro rapamycin to generate Tc2, Th1, or Tc1 cells may enhance additional adoptive T-cell therapy applications. As an example, we recently showed that costimulated donor Tc2 cells abrogate murine graft rejection [32]; in light of the data demonstrating that Tc2 cells generated in rapamycin are present in increased numbers after BMT and contribute to enhanced type II polarization, we are currently testing the hypothesis that rapamycin-generated donor Tc2 cells might be particularly enriched in their capacity to

abrogate graft rejection. In addition, we found that allogeneic Th1/Tc1 cells generated in rapamycin mediated increased in vivo effects, because recipients of Th1/Tc1.rapa cells had increased lethal GVHD relative to recipients of control Th1/Tc1 cells. Further studies will be required to evaluate whether the enhanced in vivo function of rapamycin-generated Th1/ Tc1 cells might be harnessed for therapeutic purposes such as mediation of syngeneic GVT effects. Our findings also extend prior observations that support the existence of “rapamycin-resistant” T-cell populations, which have previously been identified within human CD8⫹ T cells that are promoted through increased strength of CD28 costimulation [33]. Although we found that rapamycin-resistant murine Th1, Th2, Tc1, and Tc2 cells could be generated


ex vivo in an APC-free system, we documented differential in vivo resistance to rapamycin, with effector Th1/Tc1 cells having increased sensitivity to rapamycin therapy relative to effector Th2/Tc2 cells. This finding suggests that the previously observed shift toward Th2/Tc2 responses with rapamycin therapy [13] is the result of a Th2/Tc2 “default” pathway, and not due to interconversion of Th1/Tc1 cells to a Th2/Tc2 phenotype. We speculate that this differential in vivo sensitivity to rapamycin may relate to an indirect pathway whereby rapamycin inhibits APC function, with subsequent preferential downregulation of Th1/Tc1 cell responses; current experiments in the laboratory seek to characterize the APC functional deficits induced by rapamycin after BMT. From an application standpoint, our results suggest that, for allograft modulation approaches aimed towards Th2/ Tc2 skewing, the combination of in vitro rapamycin for T-cell generation and post-BMT rapamycin therapy may preferentially limit Th1/Tc1 responses while preserving Th2/Tc2 responses. However, because rapamycin reduced Th1 and Tc1 cell number, inhibited post-BMT type I cytokine secretion, and attenuated lethal GVHD mediated by TCM-like Th1/Tc1.R cells but not TEM-like control Th1/Tc1 cells, in vivo rapamycin may inhibit only relatively early-stage effector T cells. In conclusion, rapamycin represents a unique immune modulation agent that does not differentially modulate T-cell cytokine phenotype in a direct manner. Rather, rapamycin is permissive for substantial Th1/Tc1 and Th2/Tc2 polarization, at least under conditions where polarization occurs in an APC-independent manner. Use of in vitro rapamycin during T-cell polarization may have significant application as an adoptive T-cell therapeutic approach, because the TCM cells generated through this method mediate enhanced in vivo effects. In vivo rapamycin therapy creates a supportive post-BMT milieu for Th2/Tc2 cell therapies, because such populations are relatively spared, whereas Th1/Tc1 cell responses are preferentially inhibited.

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