Biology of Blood and Marrow Transplantation 13:530-542 (2007) ᮊ 2007 American Society for Blood and Marrow Transplantation 1083-8791/07/1305-0001$32.00/0 doi:10.1016/j.bbmt.2007.01.071 Elevation of Intracellular Cyclic AMP in Alloreactive CD4؉ T Cells Induces Alloantigen-Specific Tolerance That Can Prevent GVHD Lethality In Vivo Matthew J O’Shaughnessy,1 Zong-Ming Chen,1 Irene Gramaglia,1 Patricia A Taylor,1 Angela Panoskaltsis-Mortari,1 Christine Vogtenhuber,1 Ed Palmer,2 Thomas Grader-Beck,3 Vassiliki A Boussiotis,3 and Bruce R Blazar1 University of Minnesota Cancer Center and Department of Pediatrics, Division of Bone Marrow Transplantation, Minneapolis, Minnesota; 2Laboratory of Transplantation Immunology and Nephrology, Department of Research, University Hospital-Basel, CH-4031 Basel, Switzerland; 3Department of Adult Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts Correspondence and reprint requests: Bruce Blazar, MD, University of Minnesota Cancer Center and Department of Pediatrics, Division of Bone Marrow Transplantation, Minneapolis, MN 55455 (e-mail: blaza001@umn.edu) Received October 23, 2006; accepted January 11, 2007 ABSTRACT Cyclic AMP (cAMP) is an important negative regulator of T cell activation, and an increased level of cAMP is associated with T cell hyporesponsiveness in vitro We sought to determine whether elevating intracellular cAMP levels ex vivo in alloreactive T cells during primary mixed lymphocyte reactions (MLR) is sufficient to induce alloantigen-specific tolerance and prevent graft-versus-host disease (GVHD) Primary MLRs were treated with exogenous 8Br-cAMP and IBMX, a compound that increases intracellular cAMP levels by inhibition of phosphodiesterases T cell proliferation and IL-2 responsiveness in the treated primary MLR cultures were greatly reduced, and viable T cells recovered on day also had impaired responses to restimulation with alloantigen compared to control-treated cells, but without an impairment to nonspecific mitogens Labeling experiments showed that cAMP/IBMX inhibited alloreactive T cell proliferation by limiting the number of cell divisions, increasing susceptibility to apoptosis, and rendering nondeleted alloreactive T cells hyporesponsive to alloantigen restimulation cAMP/IBMX-treated CD4؉ T cells had a markedly reduced capacity for GVHD lethality in major histocompatibility complex class II disparate recipients, but maintained the capacity to mediate other CD4؉ T cell responses in vivo Thus, our results provide the first preclinical evidence of using cAMP-elevating pharmaceutical reagents to achieve long-term alloantigen-specific T cell tolerance that is sufficient to prevent GVHD © 2007 American Society for Blood and Marrow Transplantation KEY WORDS GVHD ● Tolerance ● T cells ● Mice INTRODUCTION Cyclic AMP (cAMP) is a second messenger utilized by cells to regulate a variety of cellular functions Elevation of cAMP levels can stimulate proliferation of various cell types, including epithelial cells, hepatocytes, keratinocytes, and pancreatic islet  cells [1] In contrast, proliferation of fibroblasts, smooth muscle, neuronal, and lymphoid cells are known to be inhibited by cAMP [2,3] In T lymphocytes, cAMP serves as an important negative regulator; elevated cAMP levels are associated with inhibition of activa530 ● Transgenic ● Immune response tion and differentiation induced by signaling through the T cell receptor (TCR) [4,5] In humans, cAMP levels in T cells from HIV-infected patients and individuals with common variable immunodeficiency are elevated, which renders these T cells hyporesponsive to mitogenic stimulation [6,7] It has also been shown that anergic T cell clones have increased levels of intracellular cAMP associated with the elevation of p27kip1 levels and the inhibition of antigen-specific proliferation and IL-2 production [8] The inhibitory function of cAMP is mediated primarily through cAMP-dependent protein kinase A cAMP Induces CD4؉ Alloantigen-Specific Tolerance type I (PKAI) [9] This isoenzyme has been shown to colocalize with the TCR during activation [10], and may activate C-terminal Src kinase, leading to inhibition of Lck-mediated TCR chain phosphorylation [11] cAMP-dependent PKAI can also mediate its negative regulatory function via the formation of a complex between the cAMP response element binding protein and the cAMP response element modulator, which serves as an inhibitor for IL-2 gene transcription [12] Elevated levels of cAMP have also been implicated in the inhibition of protein kinase C, which may lead to the dysfunction of Ras pathways that are critical for T cell activation and IL-2 production [13] cAMP can also interfere with cell cycle progression by increasing p27kip1 activity and decreasing cyclin D and cyclin E expression, which eventually leads to deactivation of cyclin-dependent kinase (cdk)-2 and cdk4 and cell cycle arrest at the G1 phase [8,14] Intracellular levels of cAMP are controlled by types of enzymes: adenyl cyclases, which use adenosine triphosphate as a substrate to synthesize cAMP, and the cAMP-specific phosphodiesterases (PDEs), which catalytically hydrolyze cAMP to biologically inactive adenosine 5=-monophosphate [15,16] Clinically approved reagents are being used to regulate the functions of these enzymes in vivo, and therefore easily alter intracellular cAMP levels Current clinical uses of PDE inhibitors include the treatment of asthma and chronic obstructive pulmonary disease [17,18] Given the inhibitory nature of cAMP in T cell proliferation and differentiation, we sought to examine the feasibility of using these pharmacologic reagents to elevate intracellular cAMP in alloreactive T cells during primary mixed lymphocyte reactions (MLRs) to induce alloantigen-specific T cell tolerance for graft-versus-host disease (GVHD) prevention In these studies, 8Br-cAMP, a cell-permeable cAMP analog, and isobutyl-methylxanthine (IBMX), a compound that prevents the degradation of intracellular cAMP by inhibiting PDEs [19], were used to treat primary MLR cultures containing purified CD4ϩ T cells as responders and irradiated major histocompatibility complex (MHC) class II disparate splenocytes as stimulators Our data demonstrated that combined, Br-cAMP and IBMX-treated alloreactive T cells were hyporesponsive to alloantigenic stimulator cells, which was associated with an impairment in cell cycle progression and increased susceptibility to apoptosis When treated T cells obtained from primary MLR cultures were provided CD3 and CD28 signals, they failed to activate the Ras pathway as measured by phosphorylation of ERK1 and ERK2 MAP kinases and to produce cytokines including IL-2 In vivo, Br-cAMP/IBMX-treated cells had a substantially reduced capacity to cause GVHD lethality when infused into MHC class II disparate recipients However, 531 treated cells retained the capacity to mediate other CD4ϩ T cell responses similar to control cultured cells Taken together, our data are consistent with a model where 8Br-cAMP/IBMX treatment induces alloantigen-specific T cell hyporesponsiveness during a primary MLR, which has potential for clinical application for prevention of GVHD and other T cellmediated immune disorders MATERIALS AND METHODS Mice B6.C.H2bm12/KhEg (termed bm12) mice and B6 Rag-1Ϫ/Ϫ mice were purchased from The Jackson Laboratory (Bar Harbor, ME) C57BL/6 (B6, CD45.2ϩ) and B6.Ly5.2/Cr (CD45.1ϩ) mice were purchased from the National Institutes of Health (Bethesda, MD) B6 OT-II TCR transgenic mice were generated as described [20] and provided by Dr Marc Jenkins (University of Minnesota, Minneapolis, MN) with permission from Dr William Heath (The Walter and Eliza Hall Institute, Victoria, Australia) 3BBM74 TCR (B6 anti-bm12) transgenic mice were generated as described [21] All mice were housed in a specific pathogen-free facility in microisolator cages according to NIH guidelines Mouse studies have been reviewed and approved by the University of Minnesota Institutional Animal Care and Use Committee In Vitro MLR Cultures CD4ϩ T cells were isolated from lymph node preparations as described [22] Purity of the preparation was routinely Ն95% CD4ϩ T cells as determined by FACS Responder CD4ϩ T cells were mixed with irradiated (30 Gy), T- and NK-cell depleted bm12 splenic stimulators, which were prepared as described [22] Cells were cultured at 37°C and 10% CO2 for days at 0.5 ϫ 106/mL in 24-well plates (Costar, Acton, MA) in DMEM (BioWhittaker, Walkersville, MD) containing 10% FBS (Hyclone, Logan, UT), 50 mM 2-ME (Sigma, St Louis, MO), 10 mM HEPES buffer, mM sodium pyruvate (Life Technologies, Grand Island, NY), amino acid supplements (1.5 mM Lglutamine, L-arginine L-asparagine), (Sigma), and antibiotics (100 U/mL penicillin; 100 mg/mL streptomycin) (Sigma) IBMX (BIOMOL Research Labs, Plymouth Meeting, PA) and 8Br-cAMP (8-bromoadenosine 3=5=-cyclic monophosphate) (Sigma) were dissolved in DMSO and added to cultures at final concentrations of 25 M and 50 M, respectively To monitor primary responses, 96-well round-bottom microtiter plates (Costar) were set up to contain 105 responders and stimulators per well To monitor secondary responses, ϫ 104 washed, viable responders and 105 irradiated (30 Gy) bm12 stimulators were 532 plated in the presence or absence of IL-2 (100 U/mL) (Amgen, Thousand Oaks, CA) In some experiments, anti-CD3 plus anti-CD28 mAb-coated microspheres [23] (Dr Carl June, Philadelphia, PA) were used to stimulate recovered T cells Microtiter wells were pulsed with tritiated thymidine (1 Ci/well) (Amersham Life Sciences, Buckinghamshire, United Kingdom) on the indicated days for 16 to 18 hours prior to harvesting and counted in the absence of scintillation fluid on a -plate reader (Packard Instrument Company, Meriden, CT) and analyzed in triplicate Cytokine Analysis Mixed lymphocyte reaction supernatants were obtained for cytokine analysis and analyzed using a multiplex assay kit (R & D Systems, Minneapolis, MN) and a Luminex analyzer (Austin, TX) according to the manufacturer’s specifications Sensitivity of the assay was 2-3 pg/mL CFSE Staining Freshly purified CD4ϩ T cells were stained with M carboxyfluorescein diacetate succinimidyl ester (CFSE) (Molecular Probes, Eugene, OR) at a concentration of 2.5 ϫ 106/mL in PBS at room temperature for minutes with shaking Labeling was stopped by addition of FCS to a final concentration of 10%, followed by washing 2ϫ with cold 2% FCS in PBS Calculations for responder frequency and proliferative capacity were done as described [24] Flow Cytometry CD4ϩ T cells were identified with either PerCPor allophycocyanin-conjugated anti-CD4 (RM4-5) mAb CD25 expression was determined by staining with PE-conjugated anti-CD25 (PC61) mAb In some experiments, nonalloreactive OT-II and alloreactive 3BBM74 transgenic CD4ϩ T cells were identified based on positive staining with PE-conjugated antiV5 TCR mAb and anti-V8 TCR mAb, respectively All mAbs were from Pharmingen (San Diego, CA), and staining was done according to manufacturer’s protocol Determination of cell viability was performed by labeling cells with FITC- or allophycocyanin-conjugated Annexin V (Pharmingen) and/or propidium iodide according to manufacturer’s protocol All flow cytometric experiments were done using a FACSCalibur (Becton Dickinson, San Jose, CA) MACS Separation 3BBM74 TCR Tg T cells were separated from congenic B6 CD4ϩ T cells (CD45.1) from day cultures by staining with PE-conjugated mAb for the congenic marker CD45.1 (Pharmingen), followed by rounds of anti-PE microbeads (Miltenyi, Auburn, CA), and application to MACS cell separation col- M J O’Shaughnessy et al umns (Miltenyi Biotec) The purity of 3BBM74 TCR Tg cells in the negative selection fraction was Ն92% Cell Cycle Analysis Freshly purified B6 CD4ϩ T cells were stimulated with plate-bound anti-CD3 (1 g/mL) and soluble anti-CD28 (5 g/mL) at million cells/mL in 24-well culture plates The cells were washed and stained with FITC conjugated anti-CD4 mAb (Pharmingen), washed again, and permeabilized with 0.1% Triton X-100 for minutes on ice Propidium iodide (50 g/mL) was added to the tubes and the samples analyzed on a FACSCalibur for DNA content In Vitro Activation of T Cells and Immunoblotting T cells isolated from MLR cultures were resuspended at 107 cells/mL and incubated with anti-CD3ε (hybridoma 145-2C11, hamster IgG1, provided by Dr Jeffrey Bluestone, San Franscisco, CA) (1 g/mL) and anti-CD28 (hybridoma 37.51, hamster IgG1, provided by Dr James Allison, Berkeley, CA) (5 g/mL) at 4°C for 20 min, washed, and incubated with goat antihamster IgG (10 g/mL) at 37°C for indicated times, and cell lysates were prepared as described [25] Equal amounts of protein were analyzed by 10% SDS-PAGE on microgels, transferred to nitrocellulose membranes, and immunoblotted with mAb for pERK1/2 (Santa Cruz Biotechnology, Santa Cruz, CA) Blots were stripped and reblotted with mAb for ERK1/2 (Upstate Biotechnology, Lake Placid, NY) Immunodetection was performed by incubation with horseradish peroxidase-conjugated anti-Ig (1:5000) (Promega, Madison, WI) as indicated by the host origin of the primary antibody and detected by autoradiography Measurement of GVHD Lethality and In Vivo Responses bm12 recipients were sublethally irradiated by exposing mice to Gy total body irradiation (TBI) from a 137Cesium source at a dose rate of 85 cGy/ Four to hours later, 105 freshly isolated B6 CD4ϩ T cells or day cultured cells were injected into the lateral tail vein To measure contact hypersensitivity, 106 freshly isolated B6 CD4ϩ T cells or day cultured cells were injected into B6.Rag-1Ϫ/Ϫ mice After 30 days, mice were sensitized with 0.15 mL 3% 4-ethoxymethylene-2-phenyloxazolone (oxazolone) (Sigma) in ethanol:acetone (3:1) on shaved thoracic and abdominal skin and then rechallenged days later on the right ear with 0.025 mL 1% oxazolone in acetone:olive oil (4:1) to induce a contact hypersensitivity (CH) response [26] Seventy-two hours after challenge, swelling in the right ear was cAMP Induces CD4؉ Alloantigen-Specific Tolerance 533 measured with a digital microcaliper (Fowler, Newton, MA) Statistical Analysis Survival data were analyzed by life-table methods, and actuarial survival rates are shown Group comparisons were made by log-rank test statistics For all other data, statistical significance was determined using Student’s t-test and P Ͻ 05 was considered significant RESULTS Br-cAMP and IBMX Treatment Inhibits T Cell Proliferation and IL-2 Responsiveness in Primary MLR Cultures Because activation of the cAMP pathway has been shown to be a negative regulator of T cell activation, we sought to determine whether the activation of this pathway was sufficient to downregulate T cell proliferation in primary MLR cultures Highly purified CD4ϩ T cells were incubated for days with irradiated, T cell-depleted MHC class II disparate splenic stimulators in the presence of 8Br-cAMP, a cell permeable cAMP analog At a concentration of 50 M Br-cAMP, T cell proliferation was reduced by 50% at time of peak proliferation (day 7), compared to vehicle-treated control cultures (Figure 1A, P Ͻ 004) Although viable cells recovered on day of 8BrcAMP-versus vehicle-treated cultures showed a delayed response to restimulation with alloantigen (peak proliferation at day for 8Br-cAMP-treated versus day for control-treated), peak proliferation upon restimulation was comparable to control cultures (data not shown) Because 8Br-cAMP could be catabolized by endogenous phosphodiesterases in treated cells thereby leading to suboptimal T cell hyporesponsiveness, we also tested the effects of IBMX to inhibit endogenous PDE activity IBMX independently reduced T cell proliferation in primary MLR cultures, but inhibition was reproducibly greater when it was added together with 8Br-cAMP As shown in Figure 1A, 50 M 8Br-cAMP and 25 M IBMX inhibited proliferation in a primary MLR culture by Ͼ90% compared to vehicle-treated control cultures (mean inhibition at day in separate experiments was 92 Ϯ 3%, P Ͻ 0002) Analysis of culture supernatants indicated that cAMP/IBMX-treated primary cultures had significantly less IL-2 present than control culture supernatants (Figure 1B, P Ͻ 05) To determine if suboptimal IL-2 production was responsible for the impaired proliferation observed in cAMP/IBMX-treated primary MLR cultures, some cAMP/IBMX-treated cultures were supplemented with exogenous IL-2 (100 U/mL) Addition of exogenous IL-2 only partially reversed the Figure Inhibition of T cell proliferation and IL-2 production by Br-cAMP and IBMX (A) Primary MLR cultures consisted of B6 CD4ϩ T cells and bm12 splenic stimulators in the presence or absence of 8Br-cAMP (50 M) and/or IBMX (25 M) All P-values Ͻ.05 versus vehicle except cAMP-treated on days and (B) Primary MLR supernatants were analyzed in triplicate for soluble IL-2 on days 3, 5, and of the culture Mean concentration Ϯ SEM is shown (C) Some cultures were supplemented with 100 U/mL exogenous IL-2 at initiation of culture IL-2 increases proliferation of cAMP/IBMX-treated MLR cultures (P Ͻ 02) For all data points versus vehicle, P Ͻ 03 On the y-axis are mean cpm Ϯ SEM On the x-axis are days in primary culture Shown is of representative experiments cAMP/IBMX inhibition of T cells (P Ͻ 02 versus cAMP/IBMX-treated), indicating that a defect in IL-2 production alone was not responsible for the observed 534 inhibited proliferation of the cAMP/IBMX-treated cells (Figure 1C) Alternatively, the elevated intracellular cAMP levels might have reduced the ability of T cells to respond to IL-2 Consistent with the latter explanation, flow cytometric analysis of day MLR cells showed that whereas at least 50% of controlcultured cells expressed CD25, the ␣ subunit of the IL-2 receptor, Ͻ20% of cAMP/IBMX-treated cells expressed CD25 in all experiments (see Figure 2D), suggesting that the failure to upregulate CD25 may M J O’Shaughnessy et al contribute, at least in part, to the mechanism of cAMP/IBMX induced hyporesponsiveness in the primary MLR Secondary Alloresponses of Br-cAMP/IBMX-Treated Cells Are Inhibited Due to Decreased IL-2 Production To study secondary in vitro responses, vehicle or cAMP/IBMX-treated T cells were recovered on day Figure Secondary hyporesponsiveness of 8Br-cAMP/IBMX-treated MLR cultures is specific to alloantigen (A) Day vehicle-treated or Br-cAMP/IBMX-treated MLR cultures were washed free of treatments and restimulated with fresh bm12 splenic stimulators (105 per well) (P Ͻ 05 for cAMP/IBMX-treated cultures versus control at days and 4) (B) IL-2 levels in cell-free supernatants collected from nonsupplemented secondary MLR cultures were determined (C) Experiments were performed as in (A) with the addition of exogenous IL-2 (100 U/mL) (P Ͻ 03 for cAMP/IBMX versus control at days and 6) (D) Surface expression of CD25 was measured on days 0, 2, 4, and of some secondary MLR cultures On the y-axis is percent of CD4ϩ T cells in the designated culture that express CD25 (P Ͻ 002 for cAMP/IBMX-treated versus control at all time points) (E) MLR cultured cells were restimulated with anti-CD3 mAb and anti-CD28 mAb-coated beads (105 per well) to assess proliferative responses (P Ͻ 002 for cAMP/IBMX-treated versus control on days and 6) On the y-axis are mean cpm Ϯ SEM; on the x-axis is days of secondary culture (F) CD25 expression and (G) IL-2 production were also measured One of representative experiments is shown cAMP Induces CD4؉ Alloantigen-Specific Tolerance of a primary MLR, thoroughly washed, and equal numbers of viable T cells were plated and restimulated with bm12 splenic stimulators Prior to washing and replating, the viability of recovered cells was 54 Ϯ 20% (range ϭ 34%-86%) for vehicle-treated cells and 22 Ϯ 16% (range ϭ 6%-48%) for cAMP/IBMXtreated cells (P ϭ 02 versus vehicle-treated) as determined by Annexin V/PI staining As shown in Figure 2A, vehicle-treated cells responded vigorously to restimulation with alloantigen, whereas peak responses of cAMP/IBMX-treated cells were reduced by 80%90% (P Ͻ 05), despite the fact that equal numbers of viable cells were plated in both secondary cultures Analysis of culture supernatants for the presence of soluble cytokines indicated that IL-2 was produced by cAMP/IBMX-treated cells, but levels were reduced by Ͼ70% (Figure 2B) To determine if inhibited secondary MLR responses resulted from decreased IL-2 production, we supplemented secondary cultures with exogenous IL-2 (100 U/mL) cAMP/IBMX-tolerized cells showed minimal response to IL-2 supplementation at an early time point (day 2) of secondary culture, but responded vigorously at later time points (days 4-6) when the culture contained exogenous IL-2 (Figure 2C) Increased proliferation in response to alloantigen in the presence of exogenous IL-2 corresponded to the time when cAMP/IBMX-tolerized cells became activated and upregulated CD25 expression from baseline levels of input cells in primary MLR culture (range ϭ 10%-15%) to Ͼ60% by day of secondary MLR culture (Figure 2D) Together, these data indicate that cAMP/IBMX-treated T cells initially are hyporesponsive to alloantigenic restimulation, associated with low CD25 expression and IL-2 production at early time points of secondary MLR culture, in contrast to control-treated cells, which had evidence of a primed response to alloantigen restimulation To determine whether the T cells were globally inhibited in their function, we chose anti-CD3 mAb ϩ anti-CD28 mAb-coated microbeads to ensure a sufficient mitogenic stimulus to all T cells to determine the response of the entire culture These mAbs provided a potent stimulus for proliferation of both control cultured and cAMP/IBMX-cultured cells (Figure 2E) Compared to control-treated cells, cAMP/ IBMX-treated cells had evidence of a modestly lower proliferation response on day that was equivalent by day of stimulation and exceeded that of control-treated cells on day In comparison to alloantigen stimulation, cAMP/IBMX-treated cells that responded to anti-CD3 ϩ anti-CD28 mAbs upregulated CD25 expression earlier (Figure 2F) and produced higher levels of IL-2 (Figure 2G) Thus, cAMP/IBMX treatment renders alloreactive T cells hyporesponsive to restimulation with alloantigen, but the remaining T cells still have the capacity to mount a proliferative 535 response when stimulated through the TCR in a nonspecific manner with anti-CD3 and anti-CD28 mAbs These data indicate that cAMP/IBMX-treated CD4ϩ T cells are not permanently and globally disabled Br-cAMP/IBMX Treatment Limits Proliferative Capacity of Alloreactive T Cells To better understand the mechanisms responsible for alloantigen-specific T cell hyporesponsiveness induced by 8Br-cAMP/IBMX, purified CD4ϩ T cells from 3BBM74 TCR transgenic mice were used as a source of alloantigen-specific T cells These transgenic T cells are specifically reactive to bm12 alloantigen, and are easily identified in flow cytometric analysis by their characteristic TCR To directly assess responses of nonalloreactive T cells, we used purified CD4ϩ T cells from OT-II TCR transgenic mice that are specifically reactive to OVAp rather than to the bm12 stimulators To monitor cell division of these transgenic clones, they were labeled with CFSE and added to a primary MLR so that they each represented 10% of the total CD4ϩ sample As shown in Figure 3, OT-II cells largely remain undivided in both the vehicle-treated and cAMP/IBMX-treated groups In contrast, a significant fraction of 3BBM74 T cells had undergone multiple cell divisions in both groups (Table and Figure 3) Quantitative analysis indicated that by day of culture an average of 8% of naïve vehicle-treated OT-II T cells and 2% of naïve cAMP/ IBMX-treated OT-II cells had undergone at least one cell division (Table 1) In contrast, an average of 59% and 55% of naïve alloreactive 3BBM74 TCR transgenic T cells from vehicle-treated and cAMP/IBMXtreated groups, respectively, responded to stimulation by undergoing cell division On average, each alloreactive cell that divided gave rise to 17.1 daughter cells in the control group, but only 6.9 daughter cells in the cAMP/IBMX-treated group Thus, cAMP/ IBMX treatment of MLR cultures controls alloreactive cell proliferation, at least in part by limiting the proliferative capacity of activated alloreactive T cells, but not by limiting the frequency of responding alloreactive cells Activated Cells Are Sensitized to Apoptosis by Br-cAMP/IBMX Treatment, But Surviving Cells Are Anergic It was possible that increased sensitization of activated cells to apoptosis might contribute to the limited net proliferation of alloreactive T cells, thereby accounting at least in part for the reduced number of daughter cells in the treated versus control MLR cultures To determine whether cAMP/IBMX treatment induces apoptosis in proliferating T cells, we examined apoptosis as measured by Annexin V binding and cell division as measured by CFSE dilution of CFSE- 536 M J O’Shaughnessy et al Figure 8Br-cAMP/IBMX limits cell division of alloreactive CD4ϩ T cells Alloreactive transgenic (3BBM74) and nonalloreactive transgenic (OT-II) CD4ϩ T cells were labeled with CFSE and added to primary MLR cultures of B6 CD4ϩ T cells and bm12 splenic stimulators Transgenic cells were identified by TCR V usage, and CFSE dilution was measured on day in control cultures (light line) and Br-cAMP/IBMX-treated cultures (heavy line) Day control is shaded histogram Gates for each cell division are as indicated One of representative experiments is shown labeled B6 CD4ϩ T cells that were stimulated with anti-CD3 ϩ anti-CD28 mAbs in the presence or absence of cAMP/IBMX treatment As shown in Figure 4A, Ͻ9% of total CD4ϩ T cells in the vehicle-treated culture were apoptotic In contrast, cAMP/IBMX treatment resulted in Annexin V binding in 20% of total CD4ϩ T cells As the number of cell divisions increased for both groups the fraction of apoptotic cells also increased, but at each stage, a larger fraction of cAMP/IBMX-treated cells were apoptotic (Figure 4B) We conclude from this experiment that cAMP/ IBMX treatment increases susceptibility to apoptosis that is especially prominent as activated T cells accumulate division events To further assess whether sensitization to apoptosis by cAMP/IBMX treatment was associated with antigen stimulation, we analyzed MLR cultured and Table 8Br-cAMP/IBMX Limits Proliferative Capacity of Alloreactive T Cells Frequency of Proliferation Vehicle cAMP ؉ IBMX Proliferative Capacity OT-II 3BBM74 OT-II 3BBM74 8% 2% 59% 55% 5.1 3.0 17.1 6.9 CFSE-labeled alloreactive (3BBM74 TCR Tg) and nonalloreactive (OT-II Tg) CD4ϩ T cells were cultured with B6 CD4ϩ T cells and bm12 splenic stimulators in the presence or absence of cAMP/IBMX On day 4, V8ϩ (3BBM74) or V5ϩ (OT-II) CD4ϩ T cells were analyzed for CFSE dilution by flow cytometry Frequency of proliferation is the proportion of input 3BBM74 or OT-II CD4ϩ T cells that responded to stimulation by dividing Proliferative capacity is the mean number of daughter cells generated per naïve 3BBM74 or OT-II CD4ϩ T cell that divided Data is from of representative experiments CFSE-labeled 3BBM74 TCR transgenic CD4ϩ T cells for Annexin V binding As shown in Figure 4C, only 16% of control-cultured 3BBM74 alloantigenspecific cells were antigen-responsive (CFSElo quadrants) and bound Annexin V by day In contrast, 30% of 3BBM74 CD4ϩ T cells in the cAMP/IBMXtreated MLR were CFSElo and bound Annexin V, which represents almost one-half of total antigenresponders Similar results were obtained when the bulk population of nontransgenic B6 CD4ϩ T cells that also contain alloreactive T cells were analyzed (14% for control and 24% for cAMP/IBMX) Thus, cAMP/IBMX inhibited the division of both alloreactive polyclonal and alloantigen-specific TCR transgenic CD4ϩ T cells and, in addition, increased apoptosis in both cell populations at all numbers of cell division, similar to data shown in Figure 4B As a consequence of increased apoptosis, overall cell viability was reduced in cAMP/IBMX cultures as discussed above This overall reduction in cell viability is likely accounted for by the presence of fewer viable alloreactive T cells at the end of culture To assess the function of alloreactive T cells that were not deleted and did not undergo apoptosis after the primary MLR tolerization period, we studied the secondary response of purified alloreactive TCR transgenic cells Purified 3BBM74 T cells, separated from polyclonal T cells using CD45 alleleic determinants, were obtained from day control or cAMP/ IBMX-treated cultures and then restimulated with either bm12 splenic stimulators or anti-CD3 ϩ antiCD28 coated microbeads Figure 5A shows that the response of equal numbers of viable cAMP/IBMXtreated alloreactive transgenic cells is reduced by 50% versus control at day of restimulation, and by Ͼ75% cAMP Induces CD4؉ Alloantigen-Specific Tolerance 537 Because purified cAMP/IBMX-treated alloreactive T cells are hyporesponsive to restimulation with alloantigens but not to stimulation via CD3 and CD28 signals, we conclude that alloantigen— but not global—T cell hyporesponsiveness is induced by cAMP/IBMX treatment in alloreactive T cells that survive the tolerization procedure Br-cAMP/IBMX Treatment of Stimulated T Cells Inhibits Cell Cycle Progression To further probe the nature of the defect in proliferative capacity of antigen stimulated cAMP/IBMXtreated cells, we performed a basic cell cycle analysis based on propidium iodide staining of cellular DNA content Because a primary MLR culture cannot provide synchronized antigenic stimulation (data not shown), we used anti-CD3 ϩ anti-CD28 stimulation as a model of T cell activation for this analysis Two Figure 8Br-cAMP/IBMX treatment increases apoptosis in activated CD4ϩ T cells (A) B6 CD4ϩ T cells were labeled with CFSE and stimulated with anti-CD3 ϩ anti-CD28 coated microbeads in the presence or absence of cAMP/IBMX On day of culture, CFSE dilution and Annexin V binding were assessed Cells in the CFSEhi quadrants had not undergone cell division (B) Annexin V binding was determined for each discrete population of daughter cells from 0-6 cell divisions (C) B6 CD4ϩ T cells were combined with alloreactive 3BBM74 TCR transgenic CD4ϩ T cells, labeled with CFSE, and stimulated with irradiated T cell-depleted bm12 stimulators in the presence or absence of cAMP/IBMX On day of the culture, transgenic T cells were identified by flow cytometry with CD4 and V8 mAbs, and analyzed for CFSE dilution and Annexin V staining One of representative experiments is shown at days and of restimulation (P Ͻ 005 at all time points) The peak response to stimulation with antiCD3/anti-CD28 coated beads was comparable for both groups, although the peak response was delayed for cAMP/IBMX-treated alloreactive cells (Figure 5B) Figure Alloreactive T cells purified from 8Br-cAMP/IBMXtreated MLR are hyporesponsive to alloantigen 3BBM74 TCR Tg T cells (CD45.2) were cultured with congenic (CD45.1) wild-type CD4ϩ T cells in control and 8Br-cAMP/IBMX-treated MLRs for days, separated by MACS, and restimulated with (A) bm12 splenocytes or (B) anti-CD3/anti-CD28 mAb-coated beads (105 per well) Proliferation of 105 of control or 8Br-cAMP/IBMXtreated 3BBM74 Tg T cells was measured on days 2, 4, and of the culture P Ͻ 007 for cAMP/IBMX-treated versus control at all time points 538 M J O’Shaughnessy et al Defective TCR ؉ CD28-Mediated ERK1/2 Activation in T Cells Treated with Br-cAMP/IBMX Table 8Br-cAMP/IBMX Treatment of anti-CD3 ϩ anti-CD28 mAb Stimulated CD4ϩ T Cells Results in Decreased Progression Through the Cell Cycle cAMP ؉ IBMX Vehicle Expt Day Expt Day 2N 2N 11 6 82 65 48 30 46 10 11 29 86 75 42 14 29 18 95 47 35 46 47 31 94 89 66 4 B6 CD4ϩ T cells were stimulated by plate bound anti-CD3 mAb (1 g/ml) and soluble anti-CD28 mAb (5 g/ml) At 24, 72, and 120 hours, cells were subjected to cell cycle analysis Percentage of apoptotic (Ͻ2N), non-cycling (2N) or cycling (Ͼ2N) cells was determined for CD4ϩ T cells stimulated in the presence of vehicle or 8Br-cAMP/IBMX Data from two experiments are shown replicate experiments are shown in Table By day of stimulation, 30%-46% of CD4ϩ T cells in the absence of cAMP/IBMX treatment had more than diploid DNA content (Ͼ2 N), indicating that they were at the S-phase of the cell cycle At the same time point, only 4%-14% of cAMP/IBMX-treated cells were in the S-phase, whereas 75%-89% remained in the G0/G1 phase At day 5, significantly fewer cells from the cAMP/IBMX group were in the S-phase compared to control (3%-29% versus 46%-47%) Notably, by day 5, the percentage of apoptotic CD4ϩ T cells (less than diploid DNA content) in the cAMP/ IBMX-treated group was 29%-31%, compared to 6%-18% of control cultured CD4ϩ T cells, whereas the percentage of cells in G0/G1 was comparable for both groups Taken together, these data indicate that cAMP/IBMX treatment can decrease the number of T-cells that respond to TCR stimulation by progressing to the S-phase of the cell cycle, and instead, may result in cell death Previous studies have shown that an altered pattern of proximal biochemical events is induced when anergic T cells are activated via their TCR even in the presence of signals delivered via the CD28 costimulatory pathway [27], including impaired activation of mitogen-activated protein kinase (MAPK)/extracellular-regulated kinase (ERK) components of the Ras pathway Because 8Br-cAMP/ IBMX treatment rendered alloreactive T cells incapable of proliferation during primary and secondary MLR, we examined the status of ERK activation in cultured cells from a B6 anti-bm12 MLR after crosslinking of TCR/CD3 and CD28 with the relevant antibodies As shown in Figure 6, activation of ERK1/2 was completely inhibited in T cells that were treated with 8Br-cAMP/IBMX during primary culture then restimulated for or minutes Impairment of the MAPK/ERK pathway by cAMP/ IBMX treatment may result in the observed defects in IL-2 production and cell cycle progression as we have observed in other types of alloantigen-specific tolerance [8,28] Br-cAMP/IBMX-Treated Cells Have Reduced Capacity to Mediate Lethal GVHD To determine whether in vitro hyporesponsiveness to alloantigen induced by 8Br-cAMP/IBMX treatment would be preserved in vivo, we used a murine model of GVHD induction where transfer of 105 naïve B6 CD4ϩ T cells uniformly results in GVHD lethality in sublethally irradiated bm12 recipients [22] The infusion of 105 naïve B6 CD4ϩ T cells resulted in GVHDinduced bone marrow aplasia and lethality within 30 days after transfer of cells in all control mice (n ϭ 12) (Figure 7A) Likewise, infusion of 105 B6 CD4ϩ T cells recovered from an 8-day control-cultured MLR Figure Defective ERK activation in T cells recovered from 8Br-cAMP/IBMX-treated primary MLR culture T cells were recovered on day from B6 anti-bm12 primary MLR cultures with or without cAMP/IBMX treatment Subsequently, cells were incubated in vitro with anti-CD3 and anti-CD28 antibodies followed by crosslinking with rabbit antihamster Ig for 0, 1, or minutes Cell lysates were prepared and analyzed by SDS-PAGE followed by immunoblot with anti-phosphoERK1/2 mAb Blots were stripped and reprobed with ERK1/2 mAb cAMP Induces CD4؉ Alloantigen-Specific Tolerance 539 Figure 8Br-cAMP/IBMX-treated MLR cells have a significant reduction in GVHD lethality but retain CD4ϩ T cell function in vivo (A) Sublethally irradiated bm12 mice received 105 naïve (n ϭ 12), day vehicle-cultured (n ϭ 15), or day 8Br-cAMP/IBMX-cultured cells (n ϭ 23) Nineteen of 23 mice that received 8Br-cAMP/IBMX-cultured cells survived long-term (60 days) without clinical symptoms of GVHD (P Ͻ 001 versus controls) Survival is indicated on the y-axis; days posttransfer of cells is on the x-axis (B) B6.Rag-1Ϫ/Ϫ mice received no cells (n ϭ 5) or 106 day vehicle-cultured (n ϭ 4) or day 8Br-cAMP/IBMX-cultured cells (n ϭ 6) After 30 days, mice were sensitized with 3% oxazolone on thoracic and abdominal skin, and challenged with 1% oxazolone days later on the right ear Thickness of the pinna was measured immediately before and 72 hours postchallenge Ear swelling (mean Ϯ SEM) resulting from oxazolone challenge is shown Also shown is ear swelling on wild-type B6 mice that were either unprimed (n ϭ 2) or primed on thoracic and abdominal skin with oxazolone (n ϭ 3) prior to challenge on the right ear Ear swelling was significant for mice that received transferred cells versus unprimed B6 mice or RagϪ/Ϫ mice (P Ͻ 05), whereas no significant differences were measured for RagϪ/Ϫ mice that received cAMP/IBMX-treated cells versus primed B6 mice or RagϪ/Ϫ mice that received vehicle-treated cells (P ϭ 57 and P ϭ 59, respectively) resulted in uniform lethality of all recipients (n ϭ 15) In contrast, administration of 105 B6 CD4ϩ T cells from an day 8Br-cAMP/IBMX-treated MLR resulted in significant (P Ͻ 001) long-term survival (Ͼ60 days) in 19 of 23 recipients Taken together, these results indicate that ex vivo treatment of alloreactive T cells with 8Br-cAMP/IBMX was sufficient to reduce alloreactive T cell responses and to significantly reduce lethal GVHD in MHC Class II disparate recipients CH Response Is Mediated by Adoptively Transferred 8Br-cAMP/IBMX-Treated Cells Although cAMP/IBMX-treated cells cause significantly less GVHD than fresh or control-cultured cells, the ability of cAMP/IBMX-treated cells to respond to nonalloantigen stimulation in vivo would provide evidence that this tolerization strategy is not globally inhibitory to T cell responses but rather is preferential for alloreactive T cell responses in vivo 540 Thus, we transferred 106 day vehicle-cultured MLR cells, 106 day cAMP/IBMX-cultured MLR cells, or no cells to B6.Rag-1Ϫ/Ϫ recipient mice so that the only responding T cell population was from the adoptive transfer After a 30-day period to allow T cell repopulation to occur, recipient mice were sensitized to 3% oxazolone on thoracic and abdominal skin and then rechallenged days later with 1% oxazolone on the right ear to induce a CH response Because T cell activation and homing to the site of inflammation are necessary steps for a CH response, measurement of ear swelling in the challenged ear provides evidence that the adoptively transferred T cells could respond in vivo to a neo-antigen The results of this experiment, shown in Figure 7B, indicate that mice that received either day control-cultured MLR cells or day cAMP/IBMX-cultured cells exhibited similar amounts of ear swelling when rechallenged with oxazolone (P ϭ 57) The amount of ear swelling is also equivalent to sensitized wild-type B6 mice with an intact immune system (P ϭ 59) Wild-type B6 mice that were not sensitized to oxazolone and B6.Rag1Ϫ/Ϫ mice that received no adoptively transferred T cells but were sensitized with oxazolone exhibited minimal ear swelling when challenged with oxazolone (P Ͻ 05 versus transferred groups) Together, these data indicate that the response that occurred in B6.Rag-1Ϫ/Ϫ mice receiving adoptively transferred cAMP/IBMX-treated cells resulted from a functional in vivo response to oxazolone challenge that resulted in equivalent CD4ϩ T cell responses as controltreated cells in vivo Thus, despite having significantly reduced capacity to mediate GVHD lethality, cAMP/ IBMX-treated cells have the ability to mount functional immune responses in vivo, which indicates that the ex vivo cAMP/IBMX tolerization procedure preferentially inhibits alloreactive T cell responses in vivo DISCUSSION Cyclic AMP is a known physiologic inhibitor of T cell activation Although elevation of intracellular cAMP has been shown to induce T cell hyporesponsiveness in vitro [8,29], it has not been formally proved that this method could achieve immune tolerance that is sufficient for prevention and treatment of T cellmediated diseases such as autoimmune disorders, graft rejection, and GVHD following bone marrow transplantation In this paper, we showed that increasing the intracellular cAMP level in alloreactive T cells using 8Br-cAMP and IBMX during a primary MLR could induce alloantigen specific T cell hyporesponsiveness that was sufficient to significantly reduce the capacity of CD4ϩ T cells to induce GVHD without globally impairing CD4ϩ T cell responses to other stimuli (oxazolone) in vivo These data provide strong evidence that suggests that pharmacologic regulation M J O’Shaughnessy et al of intracellular cAMP levels in pathogenic T cells could be an appropriate therapeutic target for the modulation of unwanted immune responses Although an elevated level of intracellular cAMP is uniformly suppressive to a polyclonal T cell population, our data indicate that hyporesponsiveness only occurs in T cells that are antigen stimulated at the time of pharmacologically induced elevations in intracellular cAMP We conclude this based on the results of in vitro experiments that showed cAMP/IBMXtreated cells recovered from a primary MLR were hyporesponsive to restimulation with alloantigen, but capable of intact responses to nonspecific TCR stimulation Responses to nonspecific TCR stimulation were intact at but not days after removal from cAMP/IBMX containing cultures Thus, we favor the explanation that the hyporesponsiveness of the cAMP/ IBMX-treated cells in secondary culture resulted from antigen-specific tolerance or deletion without a substantial induction of hyporesponsiveness in the remaining nontolerized T cells The relatively higher response of control cultured cells to alloantigen repriming and more modest increase in early responses to anti-CD3 ϩ anti-CD28 mAbs may represent the kinetic differences that would be expected to be seen between cultures that contained a frequency of antigen-primed T cells in contrast to the cAMP/IBMXtreated cells that were comprised primarily of nonalloreactive, and hence primarily naïve, T cells The failure of IL-2 supplementation to restore the alloantigen responses of cAMP/IBMX in secondary MLR cultures indicates that IL-2 production is not responsible for this defective response The correlation of CD25 antigen expression levels with the response kinetics of alloantigen as well as anti-CD3 mAb ϩ anti-CD28 mAb induced stimulation may reflect the conversion of antigen nonprimed, “naïve” cAMP/ IBMX-treated cells to antigen-primed, “stimulated” CD4ϩ T cells Therefore, the kinetics of cAMP/ IBMX-treated cells and CD25 upregulation in secondary culture would lag behind control cells that have already been stimulated in primary culture As mentioned previously, at least major mechanisms, anergy and deletion of alloreactive T cells, may account for the slow response of cAMP/IBMX-treated cultured cells to alloantigen restimulation in vitro and in vivo [30] To date, it has not been clear whether an elevated cAMP level in T cells during antigen-stimulation could result in cell death However, it is known that cAMP can mediate apoptosis in some tumor cells [31] macrophages and pro-B cells [32] Our findings indicate that alloreactive T cells in cAMP/IBMXtreated MLRs had increased apoptosis, particularly in alloreactive cells that had undergone cell division Thus, induction of apoptosis in alloreactive T cells likely contributes to the observed in vitro and in vivo hyporesponsiveness cAMP Induces CD4؉ Alloantigen-Specific Tolerance Although apoptosis limits the frequency of surviving alloreactive T cells, a fraction of alloreactive cells survive the cAMP/IBMX treatment Purification of surviving Tg alloreactive cells indicated that an anergic phenotype was induced in these cells It is known that failure of cell cycle progression from the G1 to S-phase during TCR stimulation can lead to T cell anergy [33] It was previously shown that an elevated level of cAMP can interfere with cell cycle progression, possibly via a cAMP mediated increase of p27kip1 expression [8,34] p27kip1 is a cdk inhibitor whose function is to regulate G1 to S-phase transition of the cell cycle [35] In our experiments, 8Br-cAMP/IBMX treatment impaired cell cycle progression, resulting in decreased frequency of cells in the S-phase Another hallmark for anergic T cells is that they are viable but not proliferate upon antigen stimulation or produce IL-2 [36] In our experiments, we showed that T cells recovered from a 8Br-cAMP/IBMX-treated primary MLR culture did not proliferate and produced significantly reduced IL-2 upon alloantigen restimulation Interestingly, this secondary alloantigen-specific hyporesponsiveness was reversible in the presence of exogenous IL-2 at time periods of CD25 upregulation, indicating that defective IL-2 production was present in 8Br-cAMP/IBMX-treated T cells, and might be responsible in part for their unresponsiveness to alloantigen restimulation Alternatively, the slower kinetics of CD25 upregulation in cAMP/ IBMX-treated T cells was simply reflective of the resting state of the nonalloreactive T cells that dominated the cAMP/IBMX and not the control culture We have previously reported using the same culture system and anti-CD154 mAb to induce tolerance and inhibit GVHD capacity of adoptively transferred T cells in vivo, exogenous IL-2 rescued both the primary and secondary MLR cultures from T cell hyporesponsiveness, in contrast to cAMP/IBMX, in which only the secondary alloantigen responses could be rescued [37,38] Despite the restorative effects of exogenous IL-2 at 50 IU/mL on in vitro proliferation of alloreactive T cells exposed to anti-CD154 mAb in primary MLR cultures, GVHD was not able to be readily generated in vivo in sublethally irradiated bm12 mice given adoptively transferred CD4ϩ T cells obtained from anti-CD154 mAb-treated MLR cultures even when recipients were given high doses of exogenous IL-2 for weeks posttransfer [37] With respect to the IL-2 production defect in cAMP/ IBMX-treated cells, elevated intracellular cAMP level could lead to IL-2 production failure in T cells via several biochemical pathways, including inhibition of the PKC pathway, which eventually leads to dysfunction of MAPK/ERK pathways and failure of IL-2 production [13,34,39] Whether any persistent signaling pathway defects may be responsible for the IL-2 production defect in the 8Br-cAMP/IBMX-treated T 541 cells even at a normalized intracellular cAMP level in secondary cultures remains to be determined Alternatively, cAMP/IBMX treatment may inhibit CD25 upregulation per se, and when 8Br-cAMP and IBMX are no longer present (at the time of secondary stimulation) CD25 upregulation can occur, which allows exogenous IL-2 to restore proliferation The ability to selectively eliminate alloreactive responses while preserving in vivo immune function of donor T cells is an important discovery that could be of significant value in many clinical settings This feature is particularly suitable for ex vivo tolerance applications [30] because of the ability to regulate antigen access, and hence stimulation, in this setting For example, one could use the method to specifically target GVHD causing alloreactive T cells but not potential viral-specific or tumor-specific T cells In addition to GVHD prophylaxis, the targeted antigen specificity provided by an ex vivo tolerance approach could be advantageous for delayed lymphocyte infusion treatment of bone marrow transplant patients with relapsed leukemia or severe viral infections Because antileukemia and antiviral responses often require CD8ϩ T cell function, future studies will be required to explore the effect of cAMP elevation on CD8ϩ T cellmediated alloresponses in vitro and in vivo In summary, we have shown that elevation of 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