of May 8, 2018.This information is current as

T Cells+ and CD8+CD4Hyporesponsive Program in AlloreactiveGraft-versus-Host Disease by Inducing a

Specific Notch Inhibition Blocks−T Cell

Pavan Reddy, Philip D. King and Ivan MaillardShan, Ivy T. Tran, Ann Friedman, Timothy S. Blackwell, Ashley R. Sandy, Jooho Chung, Tomomi Toubai, Gloria T.

http://www.jimmunol.org/content/190/11/5818doi: 10.4049/jimmunol.12034522013;

2013; 190:5818-5828; Prepublished online 1 MayJ Immunol 

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2.DC1http://www.jimmunol.org/content/suppl/2013/05/01/jimmunol.120345

Referenceshttp://www.jimmunol.org/content/190/11/5818.full#ref-list-1

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The Journal of Immunology

T Cell–Specific Notch Inhibition Blocks Graft-versus-HostDisease by Inducing a Hyporesponsive Program inAlloreactive CD4+ and CD8+ T Cells

Ashley R. Sandy,*,† Jooho Chung,*,‡ Tomomi Toubai,x Gloria T. Shan,* Ivy T. Tran,*

Ann Friedman,*,{ Timothy S. Blackwell,k Pavan Reddy,x Philip D. King,# and

Ivan Maillard*,x,{

Graft-versus-host disease (GVHD) induced by donor-derived T cells remains the major limitation of allogeneic bone marrow trans-

plantation (allo-BMT). We previously reported that the pan-Notch inhibitor dominant-negative form of Mastermind-like 1

(DNMAML) markedly decreased the severity and mortality of acute GVHD mediated by CD4+ T cells in mice. To elucidate

the mechanisms of Notch action in GVHD and its role in CD8+ T cells, we studied the effects of Notch inhibition in alloreactive

CD4+ and CD8+ T cells using mouse models of allo-BMT. DNMAML blocked GVHD induced by either CD4+ or CD8+ T cells.

Both CD4+ and CD8+ Notch-deprived T cells had preserved expansion in lymphoid organs of recipients, but profoundly decreased

IFN-g production despite normal T-bet and enhanced Eomesodermin expression. Alloreactive DNMAML T cells exhibited

decreased Ras/MAPK and NF-kB activity upon ex vivo restimulation through the TCR. In addition, alloreactive T cells primed

in the absence of Notch signaling had increased expression of several negative regulators of T cell activation, including Dgka, Cblb,

and Pdcd1. DNMAML expression had modest effects on in vivo proliferation but preserved overall alloreactive T cell expansion

while enhancing accumulation of pre-existing natural regulatory T cells. Overall, DNMAML T cells acquired a hyporesponsive

phenotype that blocked cytokine production but maintained their expansion in irradiated allo-BMT recipients, as well as their

in vivo and ex vivo cytotoxic potential. Our results reveal parallel roles for Notch signaling in alloreactive CD4+ and CD8+ T cells

that differ from past reports of Notch action and highlight the therapeutic potential of Notch inhibition in GVHD. The Journal of

Immunology, 2013, 190: 5818–5828.

Notch signaling is a highly conserved cell-to-cell com-munication pathway with multiple functions in healthand disease (1). Notch receptors interact with Jagged or

Delta-like ligands, leading to proteolytic release of intracellular

Notch (ICN). ICN translocates into the nucleus to interact withCSL/RBP-Jk (encoded by Rbpj) and a Mastermind-like (MAML)transcriptional coactivator. ICN, CSL/RBP-Jk, and MAML as-semble a large complex mediating gene activation. In hemato-poiesis, Notch was first identified for its essential role during earlyT cell development (2). Other functions in B cell and myeloidlineages were subsequently reported (3–5). Moreover, emergingevidence indicates that Notch has important context-dependenteffects on mature T cell differentiation and function (6).Using genetic models of Notch inhibition, we discovered an

essential role of Notch signaling in CD4+ T cells mediating graft-versus-host disease (GVHD) after allogeneic bone marrow trans-plantation (allo-BMT) (7). GVHD is the most significant com-plication limiting the success of allo-BMT (8). Current strategiesto control GVHD rely on global immunosuppression. Thesestrategies are incompletely effective and decrease graft-versus-tumor activity. To improve allo-BMT outcome, it is essential toidentify new approaches that limit GVHD without eliminatingpotent anticancer effects. To inhibit Notch signaling downstreamof all Notch receptors, we expressed a Cre-inducible dominant-negative form of Mastermind-like 1 (DNMAML) under control ofthe ROSA26 locus. Upon Cd4-Cre expression, pan-Notch inhibi-tion was achieved in mature CD4+ and CD8+ T cells without in-terference with early stages of T cell development (7, 9, 10).DNMAML blocks the Notch transcriptional activation complexdownstream of all Notch receptors, with similar effects to thoseobserved in the absence of CSL/RBP-Jk (the DNA-binding tran-scription factor that mediates all the effects of canonical Notchsignaling). In mouse allo-BMT models, pan-Notch blockade indonor CD4+ T cells led to markedly reduced GVHD severity and

*Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109; †Graduate Pro-gram of Immunology, University of Michigan, Ann Arbor, MI 48109; ‡Graduate Pro-gram of Cellular and Molecular Biology, University of Michigan, Ann Arbor, MI48109; xDivision of Hematology-Oncology, Department of Internal Medicine, Univer-sity of Michigan, Ann Arbor, MI 48109; {Department of Cell and DevelopmentalBiology, University of Michigan, Ann Arbor, MI 48109; kDivision of Allergy, Pulmo-nary, and Critical Care Medicine, Department of Medicine, Vanderbilt UniversitySchool of Medicine, Nashville, TN 37232; and #Department of Microbiology andImmunology, University of Michigan, Ann Arbor, MI 48109

Received for publication December 18, 2012. Accepted for publication March 29,2013.

This work was supported by a Damon Runyon-Rachleff Innovation award (DRR-05A-09) and a Scholar Award from the American Society of Hematology and theNational Institutes of Health (RO1-AI091627) (to I.M.). Individual support includedNational Institutes of Health T32 training Grants AI007413-17 (to A.R.S.) andGM007315 (to J.C.), a Trainee Research Award from the American Society ofHematology (to G.T.S.), and a Miller Award for innovative immunology research(to A.R.S.). Flow cytometry was partially supported by University of MichiganCancer Center Grant P30-CA46592.

Address correspondence and reprint requests to Prof. Ivan Maillard, Life SciencesInstitute #6382A, 210 Washtenaw Avenue, University of Michigan, Ann Arbor, MI48109. E-mail address: imaillar@umich.edu

The online version of this article contains supplemental material.

Abbreviations used in this article: allo-BMT, allogeneic bone marrow transplantation;Dgk, diacylglycerol kinase; DNMAML, dominant-negative form of Mastermind-like1; FIR, Foxp3–internal ribosome entry site–RFP; GVHD, graft-versus-host disease;ICN, intracellular Notch; MAML, Mastermind-like; NGL, NF-kB–GFP–Luciferase;TCD BM, T cell–depleted bone marrow; Treg, regulatory T cell; WT, wild-type.

Copyright� 2013 by TheAmericanAssociation of Immunologists, Inc. 0022-1767/13/$16.00

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improved survival (7). Notch-deprived alloreactive CD4+ T cellshad decreased production of inflammatory cytokines, includingIFN-g, TNF-a, IL-17A, IL-4, and IL-2. Concomitantly, Notchinhibition led to increased accumulation of regulatory T cells(Tregs). However, Notch-deprived CD4+ alloreactive T cellswere capable of extensive proliferation, allowing for their en-hanced accumulation in lymphoid tissues. Despite reduced cyto-kine production, Notch-deprived CD4+ T cells retained potentcytotoxic potential in vivo. Thus, Notch signaling in T cells isa new attractive therapeutic target to control GVHD after allo-BMT.Both CD4+ and CD8+ T cells have pathogenic effects during

GVHD. Although we reported an essential function of Notch inCD4+ alloreactive T cells, no information has been available aboutNotch in CD8+ T cell–driven GVHD. Furthermore, it is unclear ifthe profound effects of Notch signaling in acute GVHD can beexplained by its previously reported effects in T cells or if newmechanisms are involved. Past work highlighted independent ef-fects of Notch signaling in CD4+ and CD8+ T cells. Notch wasshown to regulate Il4 and Gata3 expression during Th2 differen-tiation (9–12). In Th1 cells, pharmacological inhibitors anda Notch1 antisense strategy suggested that Notch controlled ex-pression of Tbx21, encoding T-bet, a transcription factor regulat-ing Ifng transcription (13). Notch signaling was also shown toinfluence Th17 and Treg differentiation, as well as CD4+ T celllongevity, at least in vitro (14–17). In CD8+ T cells, Notch wassuggested to act directly at the Eomes and Gzmb loci, with animpact on differentiation and function (18–20). However, thesefindings originate from heterogeneous experimental systems, dif-ferent immune contexts, and variable strategies to manipulateNotch signaling, including gain-of-function approaches and phar-macological inhibitors. These results can be confounded by off-target effects and may not reflect the physiological functions ofNotch in T cells.In this study, we investigated the cellular and molecular

mechanisms underlying the effects of Notch signaling in allo-reactive CD4+ and CD8+ T cells during GVHD. Our strategy reliedon in vivo priming of donor T cells in the presence or absence ofall canonical CSL/RBP-Jk and MAML-dependent Notch signalsspecifically in T cells, ensuring that T cells were exposed torelevant Notch ligands in the posttransplantation environment.Notch-deprived alloreactive CD4+ and CD8+ T cells shared aprofound defect in IFN-g production, suggesting parallel effectsof Notch in both T cell subsets. Decreased IFN-g was observeddespite preserved or enhanced expression of the transcriptionfactors T-bet and Eomesodermin, consistent with the absence ofa classical Th1 or effector CD8+ T cell differentiation defect.Notch-deprived alloreactive CD4+ and CD8+ T cells acquireda hyporesponsive phenotype with decreased Ras/MAPK and NF-kB signaling. Notch inhibition led to increased expression ofselected negative regulators of T cell activation. Some of thesecharacteristics have been observed in anergic T cells, suggestingthat Notch-inhibited CD4+ and CD8+ T cells acquire an anergy-like phenotype after allo-BMT, resulting in decreased productionof inflammatory cytokines. Despite these changes, Notch inhi-bition preserved alloreactive T cell expansion in vivo and onlyhad modest effects on their proliferative potential, while in-creasing expansion of pre-existing natural Tregs and preservinghigh cytotoxic potential. Altogether, our data demonstrate a novel,shared mechanism of Notch action in alloreactive CD4+ and CD8+

T cells during allo-BMT that differs from all previous reports ofNotch activity in T cells. Understanding these effects is essentialto harness the therapeutic benefits of Notch blockade to controlGVHD after allo-BMT.

Materials and MethodsMice

BALB/c (H-2d) and C57BL/6 (B6, H-2b, CD45.2+) mice were from Harlan(Indianapolis, IN); C57BL/6.Ptprca (B6-SJL, H-2b, CD45.1+) from the Na-tional Cancer Institute (Frederick, MD); and BALB/b (H-2b) and Foxp3–internal ribosome entry site–RFP (FIR) from The Jackson Laboratory (BarHarbor, ME) (21). NF-kB reporter mice (NF-kB–GFP–Luciferase [NGL])were described previously (22). B6.129S6-Tbx21tm1Glm/J mice were providedby Dr. B. Segal (University of Michigan, Ann Arbor, MI) (23); Eomesf/f 3Cd4-Cre mice by Dr. S. Reiner (Columbia University, New York, NY) (24);and Rbpj f/f mice by Dr. T. Honjo (Kyoto University, Kyoto, Japan) (4).ROSA26DNMAMLf mice (DNMAML) contain a Cre-inducible cassette encodingthe DNMAML-GFP pan-Notch inhibitor (9, 25). DNMAML and Rbpj f/f micewere crossed to Cd4-Cre mice. All mice were backcrossed to the B6 back-ground (more than eight generations). The University of Michigan Committeeon Use and Care of Animals approved all experiments.

Induction and assessment of GVHD

Mice underwent allo-BMT as described (7). T cell–depleted bone marrow(TCD BM) was prepared with anti-Thy1.2 Abs and complement (Cedar-lane Laboratories, Burlington, NC; .95% depletion). BALB/c recipientswere irradiated (850–900 rad, [137Cs]) 4 h before allo-BMT. We trans-planted donor B6-SJL TCD BM (4 to 5 3 106) with/without B6 spleno-cytes (5–10 3 106). Where indicated, CD4+ and CD8+ T cells wereMACS-purified (Miltenyi Biotec, Auburn, CA) and mixed before trans-plantation into irradiated BALB/b or BALB/c recipients. Clinical GVHDwas scored at least weekly.

Flow cytometry

The following Abs were from BioLegend (San Diego, CA): anti-CD4,CD8a, CD44, CD45.1, CD45.2, CD62L, TCR-b, CD3, H-2Kb, H-2Kd,IFN-g, IL-2, CD28 (37.51), CD152/Ctla-4 (UC10-4B9), and CD279/Pd-1(RMP1-30). Anti–T-bet (4B10), Eomesodermin (Dan11mag), and CD272/Btla (6F7) Abs were from eBioscience (San Diego, CA). For T cell re-stimulation, we used plate-bound anti-CD3 (145-2C11) and anti-CD28(37.51; BioLegend; 2.5 mg/ml) or PMA (50 ng/ml) and ionomycin (50ng/ml). Intracellular flow cytometry was performed per the manufacturer’sinstructions after addition of brefeldin A (.2 h) (BD Biosciences).Analysis/sorting was on the FACSCanto or FACSAria II/III (BD Bio-sciences). Dead cells were excluded with DAPI (Sigma-Aldrich, St. Louis,MO). Files were analyzed in FlowJo (Tree Star, San Carlos, CA).

Quantitative reverse-transcription PCR

RNA was isolated using the RNEasy MicroKit (Qiagen, Valencia, CA) orTRIzol (Invitrogen, Carlsbad, CA). cDNA was prepared with Superscript II(Invitrogen). Quantitative PCR was performed with TaqMan (Applied Bio-systems, Carlsbad, CA) or SYBR Green (Fisher Scientific, Rockford, IL) onMastercycler realplex (Eppendorf, Westbury, NY). Relative expression wascalculated using the DD threshold cycle method. Primers were from Primer-Bank (http://pga.mgh.harvard.edu/primerbank/) or Applied Biosystems.

SDS-PAGE and Western blotting

Naive (CD62LHighCD44Low) and alloreactive CD4+ and CD8+ T cells werenegatively selected using an anti-NK1.1/CD19/Gr-1/CD11b/CD11c mix-ture. T cells were incubated on ice with anti-CD3/CD28 Abs (0.2 mg/106

cells) followed by cross-linking with anti-Armenian hamster IgG (0.5 mg/106 cells; Jackson ImmunoResearch Laboratories, West Grove, PA) inRPMI 1640 at 37�C. Alternatively, T cells were activated with PMA(Sigma-Aldrich, 50 ng/ml) or DMSO at 37�C. Cells were lysed in Laemmlibuffer and 2-ME. Samples were run on 4–20% MiniTGX gels (Bio-Rad)and transferred to Immobilon-P membranes (GenHunter, Nashville, TN)(semidry transfer; Bio-Rad). Membranes were blocked in 10% FBS plusTBS-Tween (25 mM TrisBase [pH 8], 125 mM NaCl, and 0.05% Tween).Abs were from Cell Signaling Technology (Danvers, MA): anti-MEK1/2rabbit (47E6), p44/42 MAPK-Erk1/2 mouse (3A7), phospho-MEK1/2(Ser217/221) rabbit (41G9), and phospho-p44/42 MAPK-Erk1/2 (Thr202/Tyr204) (E10). Secondary Abs were peroxidase-conjugated goat anti-rabbitIgG (H+L) or donkey anti-mouse IgG (H+L) (Jackson ImmunoResearchLaboratories). Blots were developed with ECL Substrate (Thermo Scien-tific) and HyBlotCL (Denville Scientific, South Plainfield, NJ).

Intracellular cAMP assay

cAMP was measured using ELISA per the manufacturer’s instructions(Enzo LifeSciences, Farmingdale, NY).

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Luciferase assay

Sort-purified alloreactive T cells were restimulated with plate-boundanti-CD3/CD28 (BioLegend; 2.5 mg/ml) for 16 h. Luciferase activitywas measured using the Luciferase Assay System (Promega, Madison,WI) and read on a PerkinElmer Enspire2300 (PerkinElmer, Waltham,MA).

In vitro and in vivo proliferation assays

Splenocytes or CD4+ and CD8+ T cells were MACS-purified according tothe manufacturer’s instructions (Miltenyi Biotec) and labeled with 2.5 mMCFSE (Sigma-Aldrich). T cells were incubated for 3 d on anti-CD3/CD28 6recombinant human IL-2 (PeproTech). For BrdU pulse-chase experiments,splenocytes were transplanted into irradiated BALB/c mice. Starting at day 4posttransplantation, mice received three doses of BrdU (1 mg) i.p., 12 h apart.Four hours (day 5) or 3 d (day 8) after the last BrdU injection, spleens wereharvested for BrdU staining (BD Biosciences).

Serum cytokine analysis

Serum was collected on day 5 posttransplantation. Serum IFN-g levels weremeasured by mouse IFN-g duoset (R&D Systems, Minneapolis, MN) perthe manufacturer’s instructions (Immunology Core, University of Michi-gan Cancer Center).

In vivo cytotoxicity

Experiments were performed as previously described (7, 26). Briefly, le-thally irradiated (900 rad) BALB/c mice received TCD BM (53 106 cells)alone or with wild-type (WT) or DNMAML splenocytes (83 106). On day13, BALB/c recipients were challenged with a 1:1 mixture of CFSEHigh

BALB/c allogeneic targets and CFSELow B6 syngeneic control cells (107

each). After 18 h, elimination of BALB/c allogeneic targets was measuredby flow cytometry in the spleen.

[51Cr]release assay

Spleen and lymph nodes were harvested on day 8 posttransplantation.WT and DNMAML CD8+ T cells were purified by MACS according tothe manufacturer’s instructions (Miltenyi Biotec). A20 (H-2d) and P815(H-2d) cells were used as allogeneic targets, with EL4 (H-2b) as syn-geneic control targets. Tumor targets were labeled with 2 MBq Na2

51

CrO4 (PerkinElmer) for 2 h. After washing three times, labeled targetswere plated at 5 3 103 cells/well in U-bottom 96-well plates (Corning-Costar, Cambridge, MA). Splenocytes were added in triplicate wells atvarying E:T ratios and incubated for 5 h. [51Cr] activity in supernatantswas read in a LumaPlate (PerkinElmer) in an auto-g counter (PackardInstrument, Meriden, CT). Maximal and background release were de-termined by the addition of 2% Triton X-100 (Sigma-Aldrich) or mediaalone to targets, respectively. The percentage of specific lysis was cal-culated as 100 3 (sample count 2 background count)/(maximal count 2background count).

Statistical analysis

Comparison of two means was performed with two-tailed unpaired Studentt test. When less than five data points were available per group, we used theunpaired Mann–Whitney U test. Linear regression analysis was used forcomparison of cytotoxicity curves. Survival was compared using log-rankstatistics (Prism; GraphPad, La Jolla, CA).

ResultsNotch inhibition blocks acute GVHD mediated by CD4+ orCD8+ T cells

We previously reported an essential role for Notch in CD4+ T cellsduring acute GVHD in an MHC-mismatched allo-BMT model (B6anti-BALB/c) (7). To assess if alloreactive CD8+ T cells were alsosensitive to Notch signaling, we used ROSA26DNMAMLf 3 Cd4-Cre(DNMAML) mice as source of Notch-deprived CD4+ and CD8+

T cells. These mice express the DNMAML pan-Notch inhibi-tor in all mature CD4+ and CD8+ T cells. WT or DNMAMLB6 splenocytes were transplanted into irradiated BALB/c mice.Recipients were monitored for survival and GVHD severity. Allo-BMT recipients of B6 T cells died rapidly with severe GVHD (Fig.1A). In contrast, recipients of DNMAML CD4+ and CD8+ T cellssurvived as well as mice infused only with TCD BM (Fig. 1A). Wethen performed allo-BMT with purified CD4+ or CD8+ T cells.DNMAML expression blocked severe GVHD induced by CD4+

T cells (Fig. 1B). Purified CD8+ T cells also induced significantlethality posttransplantation with ,40% long-term survival (Fig.1C). In contrast, DNMAML CD8+ T cell recipients had .94%survival by day 100, similar to mice receiving no T cells (Fig. 1C).These data demonstrate that Notch is an essential regulator ofGVHD induced by either or both CD4+ and CD8+ T cells afterMHC-mismatched allo-BMT.To further investigate the role of Notch in CD4+ and CD8+

alloreactive T cells, we used the B6 anti-BALB/c model as wellas a minor histocompatibility Ag–mismatched model (B6 anti-BALB/b) in which lethal GVHD is mediated by CD4-dependentCD8+ T cells (27). Together, WT CD4+ and CD8+ T cells inducedsignificant lethality, with ,20% long-term survival in both models(Supplemental Fig. 1A, 1B). DNMAML CD4+ and CD8+ T cellrecipients achieved ∼80% survival (Supplemental Fig. 1A, 1B).When either CD4+ or CD8+ T cells expressed DNMAML, recip-ients were protected from severe GVHD with 80–90% of the micesurviving long-term (Supplemental Fig. 1A, 1B). These data in-

FIGURE 1. DNMAML inhibits GVHD mediated

by CD4+ and/or CD8+ T cells after MHC-mis-

matched bone marrow transplantation. Lethally ir-

radiated BALB/c mice (H-2d) were transplanted

with B6 TCD BM (5 3 106 cells) alone or with B6

(H-2b) splenocytes (A) containing CD4+ and CD8+

T cells from WT or DNMAML mice (10 3 106

cells; 16 mice/group) (p , 0.001 for WT versus

TCD and WT versus DNMAML survival). (B)

Purified B6 WT or DNMAML CD4+ T cells (2 3106 cells; 8–17 mice/group) (p , 0.001 for WT

versus TCD and WT versus DNMAML survival).

(C) Purified B6 WT or DNMAML CD8+ T cells

(5 3 106 cells; 13–17 mice/group) (p, 0.01 for WT

versus TCD; p , 0.001 for WT versus DNMAML

survival). Recipients were monitored over time for

survival and GVHD severity after transplantation

(clinical GVHD score, 0–10).

5820 NOTCH SIGNALING AND GRAFT-VERSUS-HOST DISEASE

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dicate that Notch is required in both CD4+ and CD8+ T cells tofully induce GVHD in MHC- and minor histocompatibility Ag–mismatched allo-BMT models.

Alloreactive DNMAML CD8+ T cells display intrinsic andCD4+ T cell–dependent defects in IFNg production

Notch inhibition preserves CD4+ T cell expansion after allo-BMT,but profoundly decreases IFN-g production (7). Because CD8+

T cells are a major source of IFN-g during GVHD, we assessedDNMAML CD8+ T cell expansion and IFN-g production. Aftertransplantation in the B6 anti-BALB/c model (Fig. 2A), we re-covered similar numbers of donor-derived WT and DNMAMLCD4+ and CD8+ T cells from lymphoid organs of allo-BMT re-cipients, indicating that Notch blockade did not prevent in vivoexpansion of these cells (Fig. 2B, 2C). In contrast, DNMAMLexpression markedly decreased IFN-g production by both allo-reactive CD4+ and CD8+ T cells (Fig. 2D), suggesting paralleleffects of Notch in these two subsets. These differences in IFN-gproduction could not be explained by increased apoptosis of

Notch-deprived T cells during the restimulation period (data notshown). To determine if changes in IFN-g production by Notch-deprived alloreactive T cells were biologically relevant, serumIFN-g levels were measured on day 5 after transplantation (Fig.2E). Serum IFN-g levels were markedly reduced in recipients ofDNMAML T cells. Because IFN-g production by CD8+ T cells isinfluenced by cell-intrinsic signals and CD4+ T cell help, westudied DNMAML CD8+ T cells in the presence of WT orDNMAML alloreactive CD4+ T cells (Fig. 2F). WT CD4+ andCD8+ T cells transplanted together produced abundant IFN-g. Inthe presence of WT CD4+ T cells, DNMAML expression in CD8+

T cells markedly decreased but did not abolish IFN-g production.When both DNMAML CD4+ and CD8+ T cells were infused,CD8+ T cells had little to no IFN-g production. Thus, Notchpromotes maximal IFN-g production by alloreactive CD8+ T cellsvia cell-intrinsic changes in the CD8+ compartment and effects onCD4+ T cell help.To verify that Notch signaling was potently inhibited in both

alloreactive DNMAML CD4+ and CD8+ T cells, we assessed

FIGURE 2. Alloreactive DNMAML CD4+ and

CD8+ T cells have preserved in vivo expansion but

markedly decreased IFN-g production. (A) Exper-

imental design: lethally irradiated BALB/c mice

were transplanted with combinations of WT or

DNMAML CD4+ and CD8+ B6 T cells. (B) Donor-

derived T cells were tracked by flow cytometry based

on expression of donor/host MHC class I molecules

and DNMAML-GFP. (C) Preserved expansion of

donor-derived H-2Kb+H-2Kd2 DNMAML (DN)

CD4+ and CD8+ T cells as compared with WT T cells

on day 5 posttransplantation (n = 3–5 mice/group, two

independent experiments). (D) Intracellular staining

for IFN-g in donor-derived H-2Kb+H-2Kd2 CD4+

and CD8+ spleen T cells after ex vivo anti-CD3/CD28

restimulation (n = 3–5 mice/group, representative of

more than three experiments). DNMAML inhibited

IFN-g production by CD4+ and CD8+ T cells. (E)

Serum was collected on day 5 after transplantation,

and IFN-g levels were measured by ELISA (n = 5

mice/group, two or more independent experiments).

(F) Mixed populations of WT or DNMAML CD4+

and CD8+ T cells (2 3 106 each) were transplanted

into lethally irradiated BALB/c recipients. IFN-g

production was measured by intracellular flow

cytometry on day 5 posttransplantation. DNMAML

blocked IFN-g production in CD8+ T cells both

through cell-intrinsic and CD4-dependent effects

(n = 3/group, representative of two experiments).

(G) Abundance of Dtx1 Notch target gene mRNA

in sort-purified alloreactive CD4+ and CD8+ T cells

on day 5 posttransplantation (n = 3 mice/group,

representative of more than three experiments).

*p , 0.05, **p , 0.01, ***p , 0.001.

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expression of the Notch target gene Dtx1 (Fig. 2G). Dtx1 tran-scripts were significantly reduced in both subsets of alloreactiveDNMAML T cells. Collectively, inhibition of Notch signaling inalloreactive T cells preserved their expansion but profoundly re-duced T cell–dependent IFN-g production and systemic IFN-glevels.

Notch inhibition blocks IFN-g production in alloreactiveCD4+ and CD8+ T cells despite preserved T-betand Eomesodermin expression

Notch was suggested to control the expression of Tbx21 (encodingT-bet) in CD4+ T cells and Eomes (encoding Eomesodermin)in CD8+ T cells (13, 18, 19). To assess if these mechanismsaccounted for decreased IFN-g production by Notch-deprivedCD4+ and CD8+ T cells after allo-BMT, we measured T-bet andEomesodermin levels at the peak of the effector response. Allo-reactive DNMAML CD4+ and CD8+ T cells had preserved Tbx21and increased Eomes transcripts as compared with WT T cells(Fig. 3A). We used flow cytometry to assess intracellular T-betand Eomesodermin and established Ab specificity by staining

alloreactive T cells from WT mice compared with Tbx212/2 andEomes f/f 3 Cd4-Cre mice (Fig. 3B). Consistent with mRNAfindings, alloreactive DNMAML CD4+ and CD8+ T cells hadpreserved intracellular T-bet and increased Eomesodermin (Fig.3C). As expected, we observed more Eomesodermin protein inCD8+ T cells than CD4+ T cells, consistent with accurate detec-tion of Eomesodermin (28). These data indicate that decreasedIFN-g production by DNMAML CD4+ and CD8+ T cells was notcaused by decreased Th1 and effector CD8+ T cell differentiationresulting from reduced expression of these master transcriptionfactors.PMA (a diacylglycerol analog) and ionomycin (a calcium

ionophore) are often used to elicit cytokine production to studyT cell differentiation. This strategy can reveal defective IFN-gproduction by Tbx212/2 T cells (29). In contrast, restimulation ofalloreactive DNMAML T cells with PMA/ionomycin restoredIFN-g production by both CD4+ (Fig. 3D) and CD8+ T cells (Fig.3E) close to levels observed in WT T cells. Partial rescue of IL-2production was also apparent (Supplemental Fig. 2). We con-firmed the effects of DNMAML-mediated Notch blockade on

FIGURE 3. Preserved T-bet and enhanced

Eomesodermin expression in alloreactive Notch-de-

prived CD4+ and CD8+ T cells, allowing restoration

of IFN-g production after treatment with PMA and

ionomycin. Lethally irradiated BALB/c mice (900

rad) were transplanted with B6-CD45.1 TCD BM

(53 106 cells) and splenocytes (103 106 cells) from

WT or DNMAML B6 mice. (A) Preserved Tbx21

mRNA (encoding T-bet) and enhanced Eomes tran-

scripts in DNMAML (DN) CD4+ and CD8+ T cells.

Donor-derived H-2Kb+H-2Kd2CD45.2+ CD4+ and

CD8+ T cells were sort-purified and subjected to

quantitative RT-PCR (day 14, n = 4 to 5 mice, rep-

resentative of three experiments). (B) Specific detec-

tion of T-bet and Eomesodermin in alloreactive

T cells with anti–T-bet (4B10) and anti-Eome-

sodermin (Dan11mag) Abs. Splenocytes from WT,

B6.129S6-Tbx21tm1Glm/J, or Eomes f/f 3 Cd4-Cre

mice were transplanted into irradiated BALB/c

recipients. Histograms show intracellular staining

with isotype control or specific Abs in donor-derived

H-2Kb+H-2Kd2CD45.2+ CD4+ or CD8+ T cells

(day 5) (n = 2). (C) Representative intracellular flow

cytometry plots and mean fluorescence intensity

(MFI) for T-bet and Eomesodermin expression in

alloreactive WT and DNMAML CD4+ and CD8+

T cells (day 14, n = 4 to 5 mice, representative of

four experiments). (D–G) At day 5 after transplan-

tation, spleen and lymph node cells were incubated

for 6 h with either anti-CD3/anti-CD28 (2.5 mg/ml

each) or PMA and ionomycin (P/I; 50 ng/ml and

500 ng/ml, respectively). Percent IFN-g+ cells as

measured by intracellular flow cytometry in WT and

DNMAML donor-derived CD4+ (D) and CD8+ (E)

T cells or WT and CSL/RBP-Jk–deficient donor-

derived CD4+ (F) and CD8+ (G) T cells (n = 5 mice/

group, representative of two or more experiments).

Representative flow cytometry plots are shown.

Numbers indicate the percentage of cells in each

quadrant. *p, 0.05, *** p, 0.001. KO, Knockout.

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IFN-g production using Rbpj2/2 T cells lacking CSL/RBP-Jk,a central component of the Notch transcriptional activation com-plex (Fig. 3F, 3G). Altogether, our observations were consistentwith the presence of functional T-bet and Eomesodermin capableof activating Ifng transcription in DNMAML or CSL/RBP-Jk–deficient alloreactive T cells, suggesting that PMA/ionomycinrestored activation of other pathways regulating cytokine pro-duction in these cells.

Notch-deprived alloreactive CD4+ and CD8+ T cells developblunted Ras/MAPK and NF-kB activation

One of the major pathways activated by PMA is Ras/MAPKsignaling, a key contributor to T cell cytokine gene transcription(30). To assess if alloreactive DNMAML T cells had decreasedRas/MAPK activation, we measured phosphorylation of Erk1/2and its upstream kinase Mek1. Notch-deprived CD4+ and CD8+

T cells were primed in vivo before sort purification and ex vivorestimulation through the TCR and CD28 coreceptor. Both allo-reactive DNMAML CD4+ and CD8+ T cells had a significant re-duction in Mek1 and Erk1/2 phosphorylation (Fig. 4A). Interestingly,naive CD62LHighCD44Low DNMAML CD4+ T cells only had

slightly decreased Erk1/2 phosphorylation, whereas naive DNMAMLCD8+ T cells activated Erk1/2 normally, suggesting that the ma-jority of the Ras/MAPK defect was acquired in vivo in the absenceof Notch signaling (Fig. 4B). Next, we assessed if PMA rescuedRas/MAPK activation in DNMAML alloreactive T cells. PMAinduced similar Erk2 phosphorylation in WT and DNMAMLT cells,indicating restoration of Ras/MAPK signaling in DNMAML T cells(Fig. 4C).NF-kB is another major pathway activated downstream of dia-

cylglycerol that can regulate Ifng transcription in T cells (31).To capture the overall NF-kB activity in Notch-deprived allo-reactive T cells, we bred DNMAML mice to transgenic NGLreporter mice and used F1 progeny as donors for allo-BMT(Supplemental Fig. 3) (22). On day 5 posttransplantation, allo-reactive NGL and NGL/DNMAML CD4+ and CD8+ T cells weresort-purified and restimulated with anti-CD3/CD28 Abs. DNMAMLCD4+ and CD8+ T cells had significantly reduced NF-kB/luciferaseactivity. Blunted signal transduction downstream of the TCR oc-curred in the absence of any changes in TCRb (Fig. 4D) or CD3ε(Fig. 4E) surface expression, although we observed a slight butsignificant decrease in CD28 expression (Fig. 4F). Altogether, both

FIGURE 4. Alloreactive Notch-deprived CD4+

and CD8+ T cells have an acquired defect in Ras/

MAPK pathway activation that is rescued by PMA.

(A) WT or DNMAML (DN) B6 splenocytes were

transplanted into lethally irradiated BALB/c recipi-

ents. On day 5, H-2Kb+H-2Kd2 alloreactive WT and

DNMAML CD4+ and CD8+ T cells were sort-pu-

rified and restimulated in vitro for 5–10 min at 37�C with anti-CD3/anti-CD28 and IgG cross link-

ing. Baseline activation was assessed by incubat-

ing cells at 37�C with IgG cross linker alone (0

min time point). Phosphorylated Erk1/2 and Mek1

were detected by Western blotting as compared

with total Erk1/2 and Mek1. (B) Naive CD62LHigh

CD44Low CD4+ and CD8+ T cells were sort-pu-

rified from WT and DNMAML mice. Mek1

phosphorylation was assessed after anti-CD3/CD28

restimulation. (C) Sort-purified, day 5 alloreactive

WT and DNMAML CD4+ or CD8+ T cells were

restimulated ex vivo with PMA for 59 (or DMSO as

negative control). In all experiments, the abundance

of phosphorylated proteins was measured by densi-

tometry relative to total protein levels. WT T cells

were set to 100% (n = 2–4 individual experiments,

six mice/group in each experiment). Cell-surface

CD3 (D), TCRb (E), and CD28 (F) levels were

assessed in alloreactive WT and DNMAML CD4+

and CD8+ T cells on day 5 posttransplantation.

Representative flow cytometry plots and mean fluo-

rescence intensity are shown. *p , 0.05, **p ,0.01.

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DNMAML CD4+ and CD8+ alloreactive T cells acquired a bluntedcapacity for Ras/MAPK and NF-kB activation downstream of TCR/CD28 signals after allo-BMT.

Alloreactive DNMAML CD4+ and CD8+ T cells haveincreased expression of multiple negative regulatorsof T cell activation

Decreased Ras/MAPK signaling leading to reduced cytokineproduction has been described in certain forms of T cell hypo-responsiveness or anergy (32, 33). Mechanistically, increasedexpression of diacylglycerol kinases (Dgk) such as Dgka andDgkz was linked to increased degradation of diacylglycerol intophosphatidic acid, resulting in blunted Erk1/Erk2 activation andcytokine production (34, 35). Interestingly, restimulated allo-reactive DNMAML CD4+ T cells had elevated levels of Dgka andDgkz mRNA, whereas DNMAML CD8+ T cells had increasedDgka and a trend for more Dgkz transcripts (Fig. 5A). This con-stellation of effects was reminiscent of T cell anergy. Therefore,we studied a panel of anergy-associated genes, starting with theNFAT-dependent genes Egr2/3 (36, 37). Egr3 but not Egr2 tran-scripts were increased in alloreactive DNMAML CD4+ and CD8+

T cells (Fig. 5B). We also observed increased expression ofRnf128 and Cblb, encoding Grail and Cbl-b, two E3 ubiquitinligases that function as negative regulators of T cell activation(38, 39). In contrast, Itch expression was not significantly changed(Fig. 5C). As coinhibitory receptors can also regulate alloreactiveT cell activation and function, we investigated expression of Btla,Ctla4, and Pdcd1 (encoding Pd-1) in DNMAML T cells. Although

Ctla4 expression was slightly decreased, alloreactive DNMAMLCD4+ T cells had increased Btla, and DNMAML CD8+ T cellselevated Pdcd1 mRNA (Fig. 5D). These mRNA changes corre-lated well to protein expression with decreased Ctla-4 and in-creased Blta and Pd-1 in alloreactive DNMAML CD4+ and CD8+

T cells (Fig. 5E). Finally, DNMAML alloreactive T cells had in-creased intracellular cAMP, a second messenger that providesnegative-feedback regulation of T cell activation (Fig. 5F) (40).Altogether, Notch-deprived alloreactive CD4+ and CD8+ T cells

acquired features of hyporesponsive T cells, including increasedexpression of several negative regulators of T cell activation, someof which are NFAT dependent. Importantly, naive DNMAMLCD4+ and CD8+ T cells expressed normal levels of these negativeregulators (except for Cblb and Itch, which were mildly elevatedin naive DNMAML CD4+ T cells) (Supplemental Fig. 4). Thesefindings suggest that Notch deprivation results in changes in naiveT cells that only become apparent after allo-BMT or that Notchinhibition has minimal influence on naive T cells, but profoundeffects on T cells in vivo during allo-BMT.

Notch inhibition modestly reduces in vivo proliferation ofalloreactive CD4+ and CD8+ T cells while enhancingexpansion of natural Tregs

DNMAML alloreactive T cells acquire increased levels of negativeregulators that have previously been associated with decreasedproliferation, at least using in vitro models of T cell anergy (41).However, in vitro and in vivo T cell proliferation are regulatedby different stimuli. To evaluate in detail the impact of Notch

FIGURE 5. Increased levels of Dgka mRNA and

other negative regulators of T cell activation in allo-

reactive DNMAML CD4+ and CD8+ T cells. H-2Kb+H-

2Kd2CD45.2+alloreactive WT or DNMAML T cells

were sort-purified at day 5 after transplantation.

Abundance of Dgka/Dgkz (A) and Egr2/Egr3 (B)

transcripts in anti-CD3/anti-CD28 restimulated allo-

reactive WT or DNMAML (DN) CD4+ and CD8+

T cells (n = 4 mice, representative of more than two

experiments). Expression of anergy-associated E3

ubiquitin ligases Cblb, Rnf128, and Itch (C) and ex-

pression of T cell coinhibitory receptors Btla, Pdcd1,

and Ctla4 (D) in sort-purified WT or DNMAML CD4+

and CD8+ T cells. Each symbol represents triplicate

quantitative RT-PCR data for cells purified from one

individual recipient (n = 5 mice, representative of two

experiments). (E) Cell-surface levels of T cell coin-

hibitory receptors in WT and DNMAML CD4+ and

CD8+ T cells (n = 5 mice/group, representative of two

experiments). Representative flow cytometry plots and

mean fluorescence intensity (MFI) are shown. (F) In-

creased intracellular cAMP in alloreactive DNMAML

CD4+ and CD8+ T cells relative to WT T cells on day 5

after transplantation. cAMP levels were very low in

naive cells irrespective of DNMAML expression (n = 3

experiments, six mice per group in each experiment).

*p , 0.05, **p , 0.01, ***p , 0.001.

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blockade on T cell proliferation in vivo after allo-BMT, we usedRbpj f/f 3 Cd4-Cre mice (Fig. 6). As DNMAMLT cells, CSL/RBP-Jk–deficient T cells fail to respond to Notch signals, but lackDNMAML-GFP fluorescence, allowing use of CFSE to track pro-liferation. At day 3 after allo-BMT, Notch-deficient T cells hadproliferated slightly less than WT T cells (Fig. 6A). However, byday 5, .99% of donor WT and CSL/RBP-Jk–deficient T cells hadproliferated for more than six to eight divisions (Fig. 6B). Thus,despite modestly reduced initial proliferation in the absence ofNotch signaling, Notch-deprived T cells accumulated to levelssimilar to WT T cells by day 5 after allo-BMT. After this initialburst, ex vivo restimulation of Notch-deprived alloreactive T cellsrevealed only modest cycling defects (Fig. 6C, 6D). Similar in vivoand in vitro results were observed when DNMAML T cells werelabeled with eFluor670 (data not shown). To assess ongoing in vivoproliferation, we performed a BrdU pulse-chase experiment 5–8 d after allo-BMT (Fig. 6E–G). CSL/RBP-Jk–deficient T cellsdemonstrated slightly reduced initial BrdU incorporation duringthe pulse phase (Fig. 6F). During chase, decreased loss of BrdU wasapparent in Notch-deprived T cells (Fig. 6G). Thus, Notch inhibi-tion modestly reduced in vivo proliferation of alloreactive CD4+ andCD8+ T cells, while preserving their massive initial expansion.Traditional immunosuppressants often prevent Treg expan-

sion, which can increase GVHD severity (42). If inhibition ofNotch signaling is to be used to prevent or treat GVHD, weneeded to assess the impact of Notch deprivation on Treg ex-pansion. Using FIR+ DNMAML mice, we observed increasedaccumulation of DNMAML as compared with WT Foxp3+ do-nor T cells (Fig. 7A) (21). Furthermore, depletion of Tregs fromthe donor DNMAML inoculum completely prevented Treg ex-pansion (Fig. 7B–D). These data indicate that rather than in-creasing conversion to induced Tregs, Notch blockade enhancedexpansion of pre-existing Tregs after allo-BMT.

Preserved antihost and antitumor cytotoxicity of alloreactiveNotch-deprived CD8+ T cells

Our previous work showed that alloreactive Notch-deprived CD4+

T cells had preserved cytotoxicity against allogeneic host andtumor cells after transplantation (7). To test the overall in vivocytotoxic potential of DNMAML B6 CD4+ and CD8+ T cells,BALB/c transplant recipients were challenged with a 1:1 mix ofCFSEHigh BALB/c and CFSELow B6-CD45.1 splenocytes. Cyto-toxicity against BALB/c targets was measured by flow cytometry(Fig. 8A). Although recipients receiving TCD BM could not elicitcytotoxicity against the BALB/c targets, mice receiving WT orDNMAML splenocytes showed high and similar cytotoxicityagainst CFSEHigh BALB/c targets (Fig. 8A).To determine if alloreactive purified DNMAML CD8+ T cells

could lyse allogeneic tumor cells, an in vitro cytotoxicity assaywas used. T cells were primed in vivo in lethally irradiated BALB/c recipients before assessing cytotoxicity ex vivo. Incubation ofalloreactive WT or DNMAML CD8+ T cells with 51Cr-labeledallogeneic A20 and P815 (H2kd) tumor cells showed efficientcytotoxicity, with preserved cytotoxicity against A20 cells andpreserved or only slightly reduced cytotoxicity against P815 cells(Fig. 8B). As expected, syngeneic EL4 (H2kb) tumor cells werenot killed (Fig. 8B). Collectively, these data demonstrate thatNotch inhibition in alloreactive CD8+ T cells preserved a highdegree of cytotoxic potential after transplantation.

DiscussionOur findings highlight a new, shared mechanism of Notch actionin alloreactive CD4+ and CD8+ T cells that differs from all pre-viously reported Notch functions in T cells. We used pan-Notchinhibition specifically in T cells to study the effects of Notchsignaling in CD4+ and CD8+ T cell differentiation and function in

FIGURE 6. Notch-deprived alloreactive CD4+ and

CD8+ T cells have a preserved initial proliferative

burst but subsequent reduced proliferation in vitro and

in vivo. CFSE-labeled T cells from WT or Rbpj f/f 3Cd4-Cre mice (RB knockout [KO], lacking all CSL/

RBP-Jk–mediated Notch signals) were transplanted

into lethally irradiated BALB/c recipients (900 rad).

Flow cytometry plots show CFSE dilution at day 3 (A)

and day 5 (B) in donor-derived H-2Kb+H-2Kd2 CD4+

and CD8+ T cells. WT or RBP-Jk KO T cells were

transplanted into lethally irradiated BALB/c recipients.

On day 5, purified WT and RBP-Jk KO CD4+ (C) or

CD8+ (D) T cells were CFSE-labeled and restimulated

in vitro for 3 d with anti-CD3/CD28 6 IL-2. Division

history of donor-derived H-2Kb+H-2Kd2 B6 T cells

was measured by flow cytometry (n = 6 to 7 mice/

group in each experiment, representative of more than

two experiments). (E) Design of BrdU pulse-chase

experiment. Lethally irradiated BALB/c recipients

were transplanted with splenocytes from WT or RBP-

Jk KO mice. Between days 4 and 5, transplant recip-

ients received three doses of BrdU 12 h apart. (F) Four

hours after the last BrdU injection, mice were eutha-

nized for day 5 BrdU incorporation analysis (pulse).

(G) At day 8 after transplantation, residual BrdU

content was assessed (chase). n = 5 mice/group in each

experiment, representative of two experiments. *p ,0.05.

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several models of acute GVHD. Notch was absolutely requiredin both CD4+ and CD8+ T cells to mediate lethal acute GVHD.Notch inhibition preserved in vivo T cell expansion, but led toprofoundly decreased IFN-g production by both T cell subsets.Decreased IFN-g production was not explained by an overalldefect in Th1 CD4+ or effector CD8+ T cell differentiation, asexpression of the master transcription factors T-bet and Eomeso-dermin was preserved or even enhanced in the absence of Notchsignaling. In contrast, analysis of signal transduction pathwaysdownstream of the TCR revealed defects in Ras/MAPK and NF-kB activation in Notch-deprived T cells, in addition to increased

expression of multiple negative regulators of T cell activation.These features of hyporesponsiveness were observed in vivo uponT cell activation without Notch signaling. Importantly, Notch in-hibition preserved the overall expansion of alloreactive T cells andefficient cytotoxic potential, while leading to increased accumula-tion of pre-existing natural Foxp3+ Tregs. This constellation of ef-fects led to beneficial immunomodulation, indicating that Notch isan attractive therapeutic target to control GVHD after allo-BMT.Past work in other immune contexts suggested that Notch could

directly regulate Tbx21 transcription in CD4+ T cells and Eomesexpression in CD8+ T cells (13, 18, 19). In contrast, Notch ap-peared dispensable for transcriptional activation of these genes inalloreactive T cells during GVHD (Fig. 3A–C) (7). It is possiblethat use of different experimental systems to manipulate Notchsignaling could underlie these discrepant observations. Othersreported that Notch receptors could have noncanonical effectsindependent of CSL/RBP-Jk and MAML, although the nature ofthese effects is not well defined (43). However, in alloreactiveT cells at least, the dominant effects of Notch are mediated bycanonical signaling and do not involve decreased Tbx21 and Eomesexpression. Alternatively, different signaling pathways may be re-quired to activate Tbx21 and Eomes transcription in distinct immuneresponses. After allo-BMT, T cell exposure to abundant alloantigensin a highly inflammatory environment may bypass the requirementfor Notch signaling to activate these genes. Moreover, an interestingfeature of Notch-deprived alloreactive T cells was increased Eomesexpression, a finding that was particularly apparent in CD8+ T cellsbut also detected in the CD4+ compartment. Although the regulationof Eomes expression during T cell differentiation is incompletelyunderstood, decreased IL-12 signaling and increased Foxo1 activitywere reported to enhance Eomes at the expense of Tbx21 expression(44, 45). The effects of Notch deprivation on Eomes could bemediated via interference with these pathways. Interestingly,increased Eomes expression was reported in memory CD8+ T cellsthat acquire an enhanced ability for long-term persistence, as op-posed to terminal differentiation (44, 46). This mechanism couldcontribute to the enhanced expansion and survival of Notch-deprivedalloreactive T cells after allo-BMT.In contrast to past findings suggesting that Notch regulates in-

dependent aspects of CD4+ and CD8+ T cell biology, our results

FIGURE 7. Notch inhibition enhances the accumu-

lation of natural Foxp3+ Tregs after allo-BMT. (A)

Detection of live Tregs on day 5 posttransplantation

using FIR mice crossed to ROSA26DNMAMLf 3 Cd4-

Cre mice, showing expanded Tregs among Notch-de-

prived alloreactive T cells. (B) To determine the origin

of the expanded Tregs, lethally irradiated BALB/c

recipients were transplanted with WT or DNMAML

CD4+ T cells with or without FIR+ Tregs and analyzed

on day 14 posttransplantation. (C) Postsort purity of

WT and DNMAML (DN) CD4+ T cell fractions after

depletion of FIR+ cells. (D) Frequency of donor-de-

rived FIR+ T cells as assessed by flow cytometry after

transplantation of WT or DNMAML CD4+ T cells,

including Tregs (+Treg), or depleted of Tregs (2Treg).

Day 14 alloreactive DNMAML Tregs were derived

from pre-existing Tregs. *p , 0.05, ***p , 0.001.

FIGURE 8. Alloreactive Notch-deprived CD8+ T cells maintain high

cytotoxic potential. (A) In vivo cytotoxicity assay. Transplanted BALB/c

recipient mice were challenged on day 14 with a 1:1 mixture of CFSE-

labeled allogeneic targets and control cells (CFSEHigh H-2Kd+ BALB/c

and CFSElow control H-2Kb+ B6-CD45.1 splenocytes, respectively). After

18 h, elimination of the BALB/c targets was assessed in the spleen by flow

cytometry. (B) BALB/c recipients were transplanted with TCD BM and

WT or DNMAML splenocytes (5 3 106 each). On day 8, alloreactive WT

and DNMAML and naive CD8+ T cells were MACS-purified and incu-

bated with 51Cr-labeled tumor cells at various E:T ratios for 5 h (represen-

tative of n = 3 independent experiments). A20 and P815 cells were allogeneic

targets (H2kd). EL4 cells were syngeneic controls (H2kb). **p , 0.01,

***p , 0.001.

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revealed similar effects of Notch in the CD4+ and CD8+ T cellcompartments during GVHD. Notch-deprived CD4+ and CD8+

T cells shared key functional properties and gene expression changesafter allo-BMT. During in vivo activation, DNMAML alloreactiveT cells acquired elevated levels of several negative regulators ofT cell activation, including genes for which activation is NFAT de-pendent. A prominent feature of Notch-deprived T cells wasdecreased activation of Ras/MAPK and NF-kB pathways. Ras/MAPK and NF-kB activation are controlled by diacylglycerol,whose levels are negatively regulated by the lipid kinasesDgka/z. Elevated Dgka and Dgkz expression in DNMAMLT cellsis reminiscent of past observations in models of T cell hypores-ponsiveness with decreased Ras/MAPK activation (34, 35). However,DNMAML T cells also acquired elevated expression of other nega-tive regulators of T cell activation, including E3 ubiquitin ligasesand coinhibitory receptors, in addition to increased intracellularcAMP. Thus, the functional properties of Notch-deprived allo-reactive T cells may not be explained entirely by decreased Ras-MAPK and NF-kB activation.Despite the profound effects of Notch inhibition on cytokine

production and a slight reduction in their initial proliferative burst,the overall accumulation of DNMAML T cells was preservedin vivo. These findings contrast with many other interventions thatdecrease expansion of alloreactive T cells in vivo. Interestingly,past work suggested that in vivo proliferation of alloreactive T cellswas independent of IL-2 and may be controlled by IL-15, at least inMHC-mismatched allo-BMT models (47). Furthermore, classicalmodels of T cell hyporesponsiveness and anergy have often beenexamined in vitro, a situation in which IL-2 production may playa critical role to support proliferation that does not reflect itseffects in vivo (41). Other possible mechanisms accounting for thepreserved or even enhanced in vivo expansion of DNMAMLalloreactive T cells include decreased activation-induced celldeath, for example, as a result of reduced exposure to IFN-g (48).In parallel to these effects on conventional alloreactive T cells,Notch inhibition markedly enhanced in vivo expansion of Foxp3+

Tregs without inhibiting effector T cell expansion, which wasexplained by increased expansion of pre-existing natural Tregspresent in the transplant inoculum. These findings may contributeto the protective effects of Notch inhibition in GVHD and contrastwith a shortcoming of many immunosuppressive strategies, in-cluding calcineurin inhibitors, which limit Treg proliferation (42).Alloreactive Notch-deprived CD8+ T cells had largely preserved

cytotoxicity after allo-BMT, similar to our previously reportedeffects of Notch deprivation in CD4+ T cells (7). An interestingfeature of alloreactive Notch-deprived T cells is their decreasedcytokine production but preserved cytotoxic potential. Dissocia-tion of cytokine production from cytotoxicity responses couldreflect differential sensitivity of these pathways to Notch inhibi-tion. Preserved or even enhanced T-bet and Eomesodermin ex-pression in Notch-deprived T cells could be responsible formaintaining their cytotoxic potential after allo-BMT. Prior workhas shown that T-bet and Eomesodermin are important for tran-scription of cytotoxic molecules (49). Collectively, Notch inhibi-tion in alloreactive CD4+ and CD8+ T cells preserved efficientcytotoxicity while minimizing GVHD.Altogether, our findings reveal a broad effect of Notch signaling

in CD4+ and CD8+ alloreactive T cells during GVHD. Notch-deprived alloreactive T cells acquired features previously associ-ated with hyporesponsiveness or anergy. However, this haddifferential effects on T cell effector functions in vivo in theposttransplantation environment, with profound inhibition of cy-tokine production but preserved T cell expansion, cytotoxic po-tential, and natural Treg accumulation. Notch-deprived T cells

maintained potent cytotoxic activity in vivo, suggesting a splitanergy phenotype (7, 50). Overall, Notch inhibition in T cellsinduced a unique combination of effects that potently inhibitedGVHD, highlighting the promise of this new therapeutic strategyafter allo-BMT.

AcknowledgmentsWe thank Dr. Tasuku Honjo (Kyoto University) for Rbpjf/f mice, Dr. Steve

Reiner (Columbia University) for Eomesf/f 3Cd4-Cremice, and Dr. Benjamin

Segal (University of Michigan) for Tbx212/2 mice.

DisclosuresThe authors have no financial conflicts of interest.

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