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Transplantation and AutoimmunityResponses in Human Models ofCells Durably Control Pathogenic T Cell Clinical-Grade Multipotent Adult Progenitor

Raber, Robert Deans and Timothy I. M. TreeTimothy Allsopp, Anthony E. Ting, Sarah Busch, AmyAnia Skowera, Robin R. Knight, Jef Pinxteren, Bart Vaes, James L. Reading, Jennie H. M. Yang, Shereen Sabbah,

ol.1202710http://www.jimmunol.org/content/early/2013/03/31/jimmun

published online 1 April 2013J Immunol 

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The Journal of Immunology

Clinical-Grade Multipotent Adult Progenitor Cells DurablyControl Pathogenic T Cell Responses in Human Models ofTransplantation and Autoimmunity

James L. Reading,* Jennie H. M. Yang,* Shereen Sabbah,* Ania Skowera,*

Robin R. Knight,* Jef Pinxteren,† Bart Vaes,† Timothy Allsopp,‡ Anthony E. Ting,x

Sarah Busch,x Amy Raber,x Robert Deans,x and Timothy I. M. Tree*,{

A major goal of immunotherapy remains the control of pathogenic T cell responses that drive autoimmunity and allograft

rejection. Adherent progenitor cells, including mesenchymal stromal cells (MSCs) and multipotent adult progenitor cells

(MAPCs), represent attractive immunomodulatory cell therapy candidates currently active in clinical trials. MAPCs can be

distinguished fromMSCs on the basis of cellular phenotype, size, transcriptional profile, and expansion capacity. However, despite

their ongoing evaluation in autoimmune and allogeneic solid organ transplantation settings, data supporting the immune regu-

latory potential of clinical-grade MAPCs are limited. In this study, we used allogeneic islet transplantation as a model indication to

assess the ability of clinical-grade MAPCs to control T cell responses that drive immunopathology in human autoimmune disease

and allograft rejection. MAPCs suppressed T cell proliferation and Th1 and Th17 cytokine production while increasing secretion

of IL-10 and were able to suppress effector functions of bona fide autoreactive T cells from individuals with type 1 diabetes

mellitus, including killing of human islets. Furthermore, MAPCs favored the proliferation of regulatory T cells during homeo-

static expansion driven by g-chain cytokines and exerted a durable, yet reversible, control of T cell function. MAPC suppression

required licensing and proceeded via IDO-mediated tryptophan catabolism. Therefore, the common immune modulatory char-

acteristics of clinical-grade MAPCs shown in this study suggest that they can be regarded as an alternative source of adult

progenitor cells with similar clinical usefulness to MSCs. Taken collectively, these findings may guide the successful deployment of

both MSCs and MAPCs for the amelioration of human autoimmunity and allograft rejection. The Journal of Immunology, 2013,

190: 000–000.

Amajor goal of immunotherapy remains the control ofpathogenic T cell responses that drive autoimmunity andallograft rejection. Although systemic immune suppres-

sion provides an indispensable means of controlling otherwisehazardous T cell responses, long-term use of these agents is un-desirable because of malignancies, toxic drug interactions, andopportunistic infection. Cellular therapies represent an emergingclass of alternative treatment options with the capacity to providecontext-dependent and site-specific immunomodulation. Adherentstem and progenitor cells, which can be isolated from a variety oftissues and expanded ex vivo, are one candidate cell therapy

currently active in clinical trials; a prototype is the mesenchymalstromal cell (MSC). Isolation and culture conditions can influence

properties of this class of cell, notably with respect to expansion

capacity and progression to terminal differentiation. Independent

of tissue source, cells within this class share immunomodula-

tory properties that are well documented in recent reviews (1–3).

Multipotent adult progenitor cells (MAPCs) are a population of

adherent progenitor cells isolated from adult bone marrow that

meet the International Society for Cellular Therapy criteria ap-

plied to MSC as a prototype for adherent stem and progenitor cells

(i.e., expressing CD73 and CD105, negative for MHC class II and

CD45, and capable of differentiation to mesenchymal lineages) (4,

5). Compared with standard MSC culture conditions, MAPCs are

isolated using hypoxic conditions in media supplemented with

growth factors (epidermal growth factor and platelet-derived

growth factor) and kept at subconfluent culture density (6–8).

These conditions maintain expression of telomerase, leading to an

increase in expansion capacity prior to senescence, a finding that

is consistent with the reported use of forced telomerase expression

in standard MSC culture (9). This may be advantageous in a

translational context, because it enables clinical manufacturing

through the creation of a master cell bank and production of

uniform clinical doses without the use of multiple donors.

Clinical-grade MAPCs have been isolated using these conditions

and are currently active in phase II clinical development as an

“off-the-shelf” infused cell product for ulcerative colitis and is-

chemic stroke (10). A single master cell bank has been sufficient

to support these and previous phase I studies in acute myocardial

*Department of Immunobiology, King’s College London, Guy’s Hospital, LondonSE1 9RT, United Kingdom; †ReGenesys, Bioincubator Leuven, 3001 Leuven, Bel-gium; ‡Neusentis Regenerative Medicine, Pfizer Ltd., Great Abington, CambridgeCB21 6GP, United Kingdom; xAthersys, Inc., Cleveland, OH 44115; and {NationalInstitutes of Health Research Biomedical Research Centre at Guy’s and St. Thomas’National Health Service Foundation Trust and King’s College London, London SE19RT, United Kingdom

Received for publication September 27, 2012. Accepted for publication March 4,2013.

Address correspondence and reprint requests to Dr. Timothy I.M. Tree, Departmentof Immunobiology, King’s College London, Third Floor, Borough Wing, Guy’sHospital, London SE1 9RT, U.K. E-mail address: Timothy.Tree@kcl.ac.uk

The online version of this article contains supplemental material.

Abbreviations used in this article: AIT, allogeneic islet transplantation; DC, dendriticcell; HA, hemagglutinin; HP, homeostatic proliferation; ICS, intracellular cytokinestaining; MAPC, multipotent adult progenitor cell; MSC, mesenchymal stromal cell;1-MT, 1-methyl tryptophan; PD, population doubling; PPI, preproinsulin; T1D, type1 diabetes mellitus; Teff, effector T cell; Treg, regulatory T cell.

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

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infarction and allogeneic bone marrow transplant in which alldoses were safe and well tolerated (10, 11). However, despite theirongoing evaluation in autoimmune and allogeneic solid organ–transplantation settings, data supporting the immune regulatorypotential of clinical-grade MAPCs are limited. An understandingof the potency, breadth, and mechanism of immune regulationconferred by these cells is essential to design parameters andinfer the findings of future clinical trials.The objectives of this study were two-fold. First, we sought

to ascertain whether clinical-grade MAPCs display the immuno-modulatory properties and mechanisms that are common to MSCs.Second, we wished to test the suitability of clinical-grade MAPCs forthe treatment of human autoimmune disease and allograft rejectionusing human in vitro model systems. Therefore, we evaluated thepotential for clinical-grade MAPCs to suppress physiological, Ag-mediated T cell responses that drive pathology in human autoim-munity and allograft rejection. To conduct this study, we used themodel indication of allogeneic islet transplantation (AIT), in whichgraft attrition is governed by several well-defined pathologicalcheckpoints, including both allospecific and anti-self T cellresponses (12). These include Ag-driven proliferation of allospe-cific and autoreactive T cells, production of pathogenic Th1 andTh17 cytokines (e.g., IFN-g, TNF-a, and IL-17), destruction oftarget tissue, cytokine-driven (homeostatic) proliferation of T cells,and recurrence of allo- or autoimmune responses. Given that themajority of these checkpoints are common to autoimmunity andallograft rejection, these findings may lead to new translationalstrategies using these adult progenitor cells or new biologics ordrugs to advance this clinical setting.

Materials and MethodsMSC and MAPC cell culture

MultiStem clinical-grade MAPCs, generated by Athersys, were isolated fromdonor bone marrow aspirate from a 21-y-old white male. This was processedaccording to previously described methods (7). Briefly, human MAPCs wereisolated from a single bone marrow aspirate, obtained with consent froma healthy donor, and cultured in fibronectin-coated plastic tissue cultureflasks. Cell cultures were maintained under low oxygen tension in a hu-midified atmosphere of 5% CO2. Cells were cultured to subconfluencein MultiStem culture media (low-glucose DMEM [Life TechnologiesInvitrogen] supplemented with FBS [Atlas], ITS liquid media supplement[Sigma], MCDB [Sigma], platelet-derived growth factor [R&D Systems],epidermal growth factor [R&D Systems], dexamethasone [Sigma], peni-cillin/streptomycin [Life Technologies Invitrogen], 2-Phospho-L-ascorbicacid [Sigma], and linoleic acid–albumin [Sigma]). Cells were passagedevery 3–4 d and harvested using trypsin/EDTA (Life TechnologiesInvitrogen). Flow cytometric analysis of surface-expressed Ags confirmedthat MultiStem cells used in this study were a homogenous population.The cells were positive (.90%) for CD49c and CD90 and negative (,5%)for MHC class II and CD45 (all Abs were from BD Biosciences). Cells werecryopreserved in PLASMA-LYTE A (Baxter) with DMSO and human se-rum albumin. Immediately prior to their use in in vitro assays, MultiStemcells were thawed and washed twice in complete culture medium. HumanMSCs were isolated on plastic tissue culture flasks from the same bonemarrow aspirate as theMAPCs. Cell cultures were expanded in a humidifiedatmosphere of 5% under normal oxygen tension. MSCs were grown toconfluence in MSCGM Mesenchymal Stem Cell Growth Medium (Lonza)before passaging. Cells were counted at every passage, and populationdoublings (PDs) were calculated based on the number of cells initiallyseeded (Ci) and the number of cells harvested (Ch) using the followingequation: PDh = PDi + log2 (Ch/Ci).

Responder and stimulator cell culture

Following informed consent, PBMCs were isolated from fresh blood ofhealthy volunteers, therapeutically bled individuals with primary hemo-chromatosis, and individuals with long-standing type 1 diabetes mellitus(T1D; .3 y postdiagnosis) by density-gradient centrifugation using Lym-phoprep (Axis-Shield) and cryopreserved as previously described (13). Allcells were labeled with CFSE or the Violet proliferation kit (MolecularProbes, Invitrogen), and dead cells were excluded with 7-aminoactinomycin

D (Calbiochem) or Fixable Live/Dead stains (Molecular Probes, Invitrogen).Enzira vaccine preparation (Pfizer) was used as a source of recombinanthemagglutinin (HA) and human T cell expander beads (Dynal, Invitrogen)as a source of anti-CD3, anti-CD28–coated beads. Monocyte-derived den-dritic cells (DCs) were generated from PBMCs by incubation for 6 d withIL-4 and GM-CSF (R&D Systems), as previously described (13). To assessmodulation of islet-specific T cell responses, PBMCs from individuals withT1D were first assessed for proliferation in response to a panel of previouslydescribed islet peptides (14), and cocultures with MAPCs were subse-quently performed with selected peptides known to elicit a proliferativeresponse in that individual. The cytotoxic human T cell clone 1E6 specificfor preproinsulin (PPI)15–24 was isolated from an individual newly diag-nosed with T1D, as previously described, and used as indicated in the figurelegends (15). Assays were set up using 1 3 105 PBMCs/well in round-bottom 96-well plates (Corning) at 37˚C, 5%CO2. MAPCs and stimuli wereadded at 0 h at the ratios indicated or as described in the figure legends.

Flow cytometry

Fluorochrome-labeled Abs used in these studies were anti–CD3-FITC,anti–CD8-allophycocyanin-H7, anti–CD56-allophycocyanin, anti–CD25-PE, anti–HLA-DR–PE, anti–TNF-a–allophycocyanin, anti–FOXP3–AlexaFluor 647 (BD Biosciences); anti–CD3-PE-Cy7, anti–CD4–Pacific Blue,anti–CD19–Pacific Blue, anti–CD45RA–Alexa Fluor 700, anti–IL-17–PE,anti–IL-10–Brilliant violet 421 (BioLegend); anti–IFN-g–PerCP–Cy5.5,anti–Ki67-FITC (eBioscience); and anti–CD14-PE-Cy5 (Beckman Coulter).Intracellular cytokine staining (ICS) was performed using the intracellularstaining kit (BioLegend) or FOXP3 Fix/Perm staining set (eBioscience),according to themanufacturers’ instructions. Flow cytometry acquisitionwasperformed on a BD FACSCanto II using FACSDiva software v6.0 (BD) andanalyzed using FlowJo v 7.6.3 software (TreeStar). Untouched CD4+ CFSEhi

cells from cell cultures were identified using the untouched CD4 T cellisolation kit (MACS; Miltenyi Biotec) and streptavidin-PE (BioLegend),according to the manufacturers’ instructions, and isolated by cell sorting ona BD FACSAria. To characterize theMAPC andMSC phenotype, cells wereanalyzed using the following anti-human Abs: CD13-allophycocyanin,CD34-FITC, CD44-FITC, CD45-FITC, CD73-PE, CD90-allophycocyanin,CD105-allophycocyanin, HLA-I–allophycocyanin, and HLA-II–PE (allfrom BD Biosciences). Labeled cells were analyzed using MACSQuantwith MACSQuantify software (Miltenyi Biotec).

Cytometric bead array

Cell culture supernatants were frozen at 280˚C and analyzed using aMilliplex MAP kit and Luminex FLEXMAP 3D instrumentation (Millipore),the Enspire platform (PerkinElmer), or FlowCytomix (eBiosciences), ac-cording to the manufacturers’ instructions.

Islet cell cytotoxicity assays

Human islets were isolated and cultured as described previously (15).Cytotoxicity was analyzed by a nonradioactive Europium TDA cytotox-icity assay using DELFIA Technology (PerkinElmer), according to themanufacturers’ instructions, as previously described (16, 17).

IDO blocking and Transwell experiments

IDO-inhibition assays were carried out as indicated in the figure legendsusing frozen, sterile filtered stocks of 1-methyl-l-tryptophan and L-tryp-tophan (Sigma) prepared in 0.5 M HCl and adjusted to pH 7.4 usingNaOH. Transwell cultures used 24-well plates with a 1-mM pore size insert(Corning).

Statistical analysis

Analysis of paired data was completed using one-tailed (to test hypothesesstating directional change) or two-tailed (to test hypotheses where changesmay occur bidirectionally) Wilcoxon signed-rank tests (not normally dis-tributed) or paired Student t tests (normally distributed). Unpaired groupswere analyzed by an unpaired Student t test. All tests were performed,including a 95% confidence interval, using GraphPad Prism Software v5.0.

ResultsMAPCs can be functionally and phenotypically distinguishedfrom MSCs

MSCs and MAPCs used in this study were characterized by flowcytometry and met the release criteria described in Fig. 1A. Inagreement with previous publications, MAPCs displayed en-

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hanced replicative potential (Fig. 1B) and exhibited significantlyhigher levels of telomerase activity compared with autologousMSCs (Fig. 1C).

MAPCs suppress T cell proliferation

Islet allograft rejection is associated with in vitro proliferativeT cell responses to both allo- and autoantigen, suggesting thatregulation of this immunopathological checkpoint may promotegraft survival (18). To confirm that MAPCs suppress T cell pro-liferation, we stimulated CFSE-labeled PBMCs in the presenceof third-party clinical-grade MAPCs or MSCs derived from thesame individual and measured cell division by CFSE dye dilutionin gated T cell populations. T cells were initially stimulated withanti-CD3, anti-CD28–coated beads (hereafter anti-CD3/28) (Fig.2A–C). However, because stimulation using immobilized Absmay not accurately emulate the complexity and nature of Ag-specific responses, we also examined proliferation in responseto Ags used to model allogeneic transplantation (third-party DCs)(Fig. 2D–F) and the effector memory responses observed in au-toimmunity (influenza HA; hereafter HA) (Fig. 2G–I). Althoughthere was considerable interindividual variation in the level ofproliferation observed for each stimulus, MAPCs and MSCs sig-nificantly inhibited proliferation of both CD4 and CD8 T cellsunder all conditions. There was no significant difference in thelevel of suppression by MSCs compared with MAPCs for any ofthe culture conditions. MAPCs were nonimmunogenic in unstim-ulated cocultures (Supplemental Fig. 1A) and suppressed T cellproliferation in a dose-dependent manner (Supplemental Fig.1B–E). Furthermore, multiple batches of MAPCs demonstratedsimilar levels of suppression, irrespective of donor origin or lot(Supplemental Fig. 1F), whereas suppression was not observed

with HUVECs (Supplemental Fig. 1G) or MAPCs fixed with PFAor formaldehyde (data not shown). To confirm the mechanism bywhich MAPCs suppressed T cell proliferation, CFSE dilutionwas measured within viable CD3+ cells of anti-CD3/28–stimu-lated PBMCs in the bottom chamber of a Transwell system.MAPCs were then seeded in the upper chamber in the presenceor absence of resting or stimulated PBMCs. The presence ofMAPCs in the upper chamber resulted in low levels of suppres-sion compared with contact. This was augmented by the presenceof unstimulated PBMCs and further enhanced when MAPCs werein contact with stimulated PBMCs, achieving a level of sup-pression that was not statistically significantly different from thatobserved under contact conditions (Fig. 3A). Therefore, MAPC-mediated T cell suppression can occur via soluble mediator(s)that are produced in response to either secreted or cell-associatedmolecules expressed by proximal, activated PBMCs. Because thecatabolic enzyme IDO has been implicated in MSC-mediatedT cell suppression, we next blocked the IDO pathway with thechemical inhibitor 1-methyl tryptophan (1-MT) or supplementedMAPC–PBMC cocultures with L-tryptophan to compensate forIDO-mediated tryptophan catabolism. Both 1-MT and L-trypto-phan were sufficient to reverse the majority of MAPC-mediatedsuppression, suggesting that IDO-mediated tryptophan catabo-lism is largely accountable for MAPC-mediated T cell suppres-sion in anti-CD3/28–stimulated cultures (Fig. 3B). In line withthese findings, both rIFN-g and supernatant of stimulated PBMCcultures were sufficient to induce IDO1 gene expression inMAPCs .100,000-fold (data not shown). Suppression of T cellproliferation did not result in increased levels of T cell apoptosisor necrosis and was independent of PGE2 synthesis (Supple-mental Fig. 2).

FIGURE 1. Phenotypic and expansion potential differences between MAPCs and MSCs. (A) Criteria that are used for flow cytometry analysis to es-

tablish the MAPC phenotype. (B) Growth curves representing PDs of MAPCs from three donors and MSCs derived from the same donors. (C) Telomerase

activity of MAPCs and MSCs from one donor, represented in relative units. Results are representative of at least three independent experiments. *p , 0.05.

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MAPCs suppress production of pathogenic but not protectivecytokines

The elicitation of pathogenic T cell responses requires the presenceof antigraft and antiself T cell pools, as well as a bias in effectorfunction. For example, islet-specific CD4 T cells found in indi-viduals with newly diagnosed T1D primarily secrete proin-flammatory cytokines (e.g., IFN-g), and recent data also suggesta role for islet-specific Th17 CD4 T cells in the augmentation ofb cell apoptosis in T1D (19). In contrast, islet-specific responsesin healthy individuals are dominated by IL-10 production fromcells with a potent regulatory phenotype (14, 20). Likewise, graftdestruction in AIT is associated with increased frequencies ofproinflammatory alloantigen-specific T cells and the loss of IL-10production (21–23). Thus, redressing the balance of pathogenic(IFN-g/IL-17) and protective (IL-10) cytokines during responsesto allo- or autoantigen is a desirable property for interventions

targeting islet allograft rejection. Therefore, we examined the

potential for MAPCs to regulate effector and regulatory cytokine

production in our Ag-driven (DC and HA) models of allogeneic

and effector memory responses. Analysis of PBMC coculture

supernatants revealed that IFN-g was significantly suppressed by

MAPCs and MSCs, whereas IL-10 production was increased un-

der both stimulation conditions and to a greater extent by MSCs

(Fig. 4A, 4B). However, because we did not reproducibly observe

detectable levels of IL-17 in culture supernatants, we also chose

to measure pathogenic cytokine production by ICS following

restimulation with PMA and ionomycin after the initial culture

with DCs or HA. Gating on CFSElo cells enabled us to assess cyto-

kine production in responding, Ag-specific T cells. In this assay

system, both DC- and HA-specific IFN-g and IL-17 production

was significantly suppressed, with similar potency observed for

MAPCs and MSCs (Fig. 4C–F). IFN-g and TNF-a secretion was

FIGURE 2. MAPCs suppress T cell proliferation. CFSE-labeled PBMCs were stimulated with 1:500 anti-CD3/28–coated beads (A–C), 1:100 third-party

DCs (D–F), or 50 ng/ml HA (G–I) in the presence or absence of 1:4 MAPCs/PBMCs or MSCs/PBMCs, and proliferation was measured in gated viable

CD3+ lymphocytes by dye-dilution using flow cytometry. FACS plots from representative experiments displaying CFSE dilution within CD3+ viable

lymphocytes of PBMCs stimulated with anti-CD3/28 (A), DCs (D), or HA (G) in the absence or presence of 1:4 MAPCs or MSCs. Proliferation of CD4 (B,

E, H) and CD8 (C, F, I) T cells from five donors (four donors for HA) in the presence or absence of 1:4 MSCs or MAPCs. Horizontal lines represent the

mean, and error bars represent the SEM. *p , 0.05, **p , 0.01. ns, Not significant.

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also suppressed in CD8 T cells, whereas similar results were ob-tained using MSCs and MAPCs at lower ratios (1:10) of progenitorcells/PBMCs (data not shown). Therefore, like MSCs, MAPCssuppress pathogenic Th1 and Th17 cytokine production during Ag-driven T cell responses while enhancing IL-10.

MAPCs suppress proliferation and effector function ofislet-specific autoreactive T cells from individuals with T1D

Autoreactive T cell responses of individuals with T1D (such asthose receiving islet allografts) have been reported to exhibit re-sistance to regulatory T cell (Treg)-mediated suppression (24, 25).Therefore, we examined whether MAPCs could suppress prolif-eration of islet-specific CD4 T cells from individuals with T1D.MAPCs significantly suppressed proliferation of autoreactive ef-fector T cells in response to their natural autoantigenic peptides(Fig. 5A–C); however, because of the low frequency and magni-tude of these responses, we were unable to measure cytokineproduction in this system. Therefore, we also examined the abilityof MAPCs to modulate cytokine production and cytolytic effector

function of highly activated autoreactive CTLs using a PPI-specific CTL clone (1E6) that exhibits a TNF-a–dependent cy-totoxicity of human islets (15). MAPCs potently inhibited cyto-kine production (Fig. 5D, 5E) and, at higher ratios, reduced theability of the clone to kill human islets (Fig. 5F). These resultsindicate that MAPCs can suppress proliferation, cytokine secre-tion, and killing of bona fide autoreactive T cells from individualswith T1D, potentially leading to increased survival of target tissue.

MAPCs suppress g-chain cytokine–dependent proliferation ofT cells

Immune suppression regimens to prevent allograft rejection involvelymphodepleting induction therapy and the subsequent use ofagents to target T cell proliferation. Lymphocyte reconstitutionfollowing induction therapy occurs via homeostatic proliferation(HP) of spared, pre-existing T cell pools in a process that requiresg-chain cytokines (typically IL-7 and IL-15). Although the grad-ual recline of immune competence is desirable, studies showedthat the rate of reconstitution and composition of repopulatingT cells influence allograft survival, with CD4 CD25hi FOXP3+

Tregs and effector T cells corresponding with graft protection ordestruction, respectively (26–29). In AIT, HP can lead to resur-gence of autoreactive T cells, whereas the appearance of FOXP3+

T cells can be favored by the use of specific induction therapies(30, 31). Therefore, we assessed the ability of MAPCs to modulateT cell proliferation in PBMC populations stimulated by g-chaincytokines (IL-2, IL-7, or IL-15) and assessed Ki67 expression asa surrogate marker of proliferation. MAPCs significantly reducedKi67 expression in CD4 and CD8 T cells in response to all cy-tokines, with the exception of CD8 cells stimulated with IL-2, inwhich low levels of Ki67 induction were observed (Fig. 6A, 6B).Subgating analyses revealed that MAPCs preferentially suppressedKi67 induction in effector T cell (Teff) populations (CD4+CD25+

FOXP32) compared with Tregs (CD4+CD25hiFOXP3+) in IL-2– and IL-15–stimulated cultures, but not in IL-7–stimulatedcultures (Fig. 6C, 6D), suggesting that MAPCs may selectivelyfavor the outgrowth of Tregs in vitro. No differences were ob-served in the suppression of HP by MAPCs compared with au-tologous MSCs, confirming that modulation of cytokine-drivenproliferation is a common feature of MSCs and MAPCs (Sup-plemental Fig. 3).

MAPCs exert durable T cell suppression

We next sought to examine whether coculture with MAPCs resultedin durable suppression that persisted when MAPCs were no longerpresent. Given the reported short t1/2 of MSCs in vivo, this is a keyquestion that has implications for repeat-dosing regimens and dis-cerning therapeutic mechanisms of progenitor cells in vivo (32,33). We reasoned that, if MAPCs imposed a durable suppressionof proliferation, exposure to MAPCs during primary stimulationwould result in reduced proliferation during secondary restim-ulation, even when the MAPCs were no longer present. We testedthis by sorting both “resting” CFSEhi cells from PBMCs previ-ously cultured in the presence or absence of MAPCs and “sup-pressed” CFSEhi cells from cultures stimulated with DCs in thepresence of MAPCs (Fig. 7A). Resting CD4 T cells that werepreviously exposed to MAPCs proliferated significantly less inresponse to DC stimulation than did resting cells previously cul-tured in the absence of MAPCs (Fig. 7B). Furthermore, “sup-pressed” CD4 T cells that were isolated from DC+MAPC culturesexhibited significantly lower levels of proliferation compared withboth resting cells and resting cells exposed to MAPCs. Addition ofexogenous IL-2 during secondary restimulation was sufficient torescue the durable suppression observed in MAPC-exposed CD4

FIGURE 3. MAPCs suppress T cell proliferation via soluble factor(s),

including IDO, following short-range licensing. (A) PBMCs were stimu-

lated with 1:500 anti-CD3/28 beads in the presence or absence of 1:4

MAPCs (contact). Alternatively, MAPCs were inserted in the upper

chamber of a Transwell system either alone or in the presence of 1:1

PBMCs that were either resting or stimulated with 1:100 anti-CD3/28

beads. Suppression of CD4+ T cell proliferation was calculated relative to

contact-mediated suppression (set to 100% for each experiment) according

to CFSE dilution. (B) PBMCs were stimulated with 1:10 anti-CD3/28

beads, and CD4+ T cell proliferation was determined according to CFSE

dilution in the presence or absence of 1:3 MAPCs/PBMCs after 5 d, with

or without 1 mM 1-MT or 100 mg/ml L-tryptophan (TRP). Error bars

represent SEM from three independent experiments. *p , 0.05, **p ,0.001, ****p , 0.0001.

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T cells. Although IL-2 seemed to be more effective at restoring theproliferation of cells isolated from the “resting” (+MAPC) com-pared with the “suppressed” (DC+MAPC) culture in individual ex-periments (Fig. 7A), this was not statistically significant overall(Fig. 7B). Cells from all cultures proliferated vigorously and to anequivalent level in response to restimulation with anti-CD3/28.Similar results were seen when using an effector-memory modelof stimulation (Supplemental Fig. 4A, 4B). Therefore, the fate ofT cells previously exposed to MAPCs is a durable, yet reversible,suppression of Ag-driven proliferation that persists in the absenceof MAPCs.

DiscussionImmunomodulatory progenitor cells, such as MSCs and MAPCs,continue to hold much promise for the treatment of T cell–drivenimmunopathology. Human MAPCs can be distinguished fromhuman MSCs on the basis of cell surface phenotype, cell size,transcriptional profile, cytokine production, differentiation poten-tial and replicative capacity (6, 10, 34, 35). Previous work showedthat murine MAPCs exert therapeutic immunomodulatory effectsin the context of graft-versus-host disease prophylaxis, in MLR

systems in vitro, and that human MAPCs prevented inflammationin a murine model of stroke, supporting a role for these cells intreating human immune-mediated diseases (36–38).This study demonstrates that, despite their enhanced expansion

potential and phenotypic differences, clinical-grade MAPCs ex-hibit similar immunomodulatory characteristics and potency toMSCs, downregulating T cell proliferation and functioning, in partvia IDO-mediated tryptophan catabolism (39). This is in agree-ment with a recent report illustrating that human MAPCs arenonimmunogenic and suppress T cell proliferation in an IDO-dependent and PGE2-independent manner (40). In addition, ourTranswell system reveals that maximal suppression is achievablesolely by secreted factors, provided that MAPCs are in closeproximity to activated PBMCs. This result aligns with severalreports highlighting that MSC-mediated suppression can occur viathe IDO pathway downstream of licensing signals (e.g., IFN-g)and supports the notion that MAPCs would be most suppressive inthe context of an inflammatory environment/ongoing immune re-sponse (41). Consistent with this, rodent MAPCs were shown toexhibit efficacy in the context of graft-versus-host disease onlywhen present at sites of allopriming (36). Although implicated in

FIGURE 4. MAPCs suppress pathogenic T cell cytokine production. CFSE-labeled PBMCs were stimulated with 1:100 third-party DCs (A, C, E) or 50

ng/ml HA (B, D, F) in the presence or absence of MSCs or MAPCs at a ratio of 1:4 for 6 d. (A and B) Supernatants were analyzed by cytometric bead array,

and data are expressed in pg/ml. Following the initial coculture as above, PBMCs were restimulated with PMA (10 ng/ml) and ionomycin (1 mg/ml) for 5 h

in the presence of brefeldin A and monensin, and ICS was performed. (C and D) FACS plots from representative experiments displaying IFN-g expression

within gated CD3+ CD4+ viable lymphocytes of PBMCs restimulated in the presence or absence of MAPCs or MSCs. (E and F) Production of IFN-g (left

panel) and IL-17 (right panel) within CFSElo CD4+ T cells. Horizontal lines represent the mean, and error bars represent the SEM. Results are repre-

sentative of at least two independent experiments. *p , 0.05, **p , 0.01. ns, Not significant.

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MSC-mediated T cell suppression, we did not observe a role forPGE2 in MAPC-mediated T cell suppression under any stimula-tion conditions. This may be due to intrinsic differences betweenMAPCs and MSCs or reflect differences in assay methodology(e.g., responder cell type analyzed or readout of proliferation).In agreement with previous reports using MSCs, we demonstrate

that clinical-grade MAPCs potently suppress Th1 signature cyto-kines while enhancing the production of IL-10. It is not clear whetherthe elevated levels of IL-10 seen in MSC compared with MAPCcocultures emanate from differences in IL-10 being produced by theprogenitor cell types directly or results indirectly from differencesin their modulation of other leukocytes. However, at lower ratios(i.e., 1:10), differences in IL-10 induction between MAPCs andMSCs were not apparent (data not shown). Although IL-10 inductionand Th1 cytokine suppression are regarded as consensus propertiesfor MSCs, suppression of Th17 cells remains more contentious, withdiscordant reports proposing that MSCs can enhance or inhibit Th17expansion (42–48). It was suggested that this may reflect the in-fluence of a network of interdependent variables, such as donor, celltype, licensing, T cell activation status, level of contact, and balancein Th17-enhancing (i.e., TGF-b/IL-6) versus inhibitory (e.g., IDO,PGE2) factors (3, 41). Although, overall, we observed suppressionof IL-17 production, we identified a small proportion of individuals

(one of seven donors tested) in whom MAPC coculture led to in-creased IL-17 expression following both DC and HA stimulation,despite the suppression of IFN-g under the same conditions (Sup-plemental Fig. 4C, 4D). Thus, this result reaffirms that interdonorvariation may account for some of the discrepancies observed inprevious reports examining IL-17.In agreement with Zanone et al. (49), we found that MAPCs can

suppress both proliferation and cytokine production from bonafide autoreactive T cells from individuals with T1D. Our studyalso reveals that MAPCs can attenuate the killing potential of ahighly activated autoreactive CTL clone, demonstrating that aprogenitor cell therapy may be capable of preserving human targettissue from cytotoxic destruction.In agreement with a previous report using MSCs, our HP data

imply that MAPCs and MSCs attenuate, but do not abrogate,cytokine-driven T cell proliferation. Therefore, this may facilitategradual lymphocyte reconstitution while preventing graft attrition,which is associated with rapid T cell repopulation (50). We ad-ditionally demonstrated that MAPCs preferentially suppressedTeffs compared with Tregs during cytokine-driven proliferation,reaffirming similar findings with MSCs in Ag-driven systems (41).Although these results remain encouraging, further in vivo in-vestigation is required to fully discern the mechanism by which

FIGURE 5. MAPCs suppress proliferation, cytokine production, and cytotoxicity of islet-specific autoreactive T cells. (A) FACS plots showing gated

viable CD4+ lymphocytes from G157, a T1D patient, stimulated with vehicle (DMSO), pentavalent Ag (PED), or IA2 709–736 (peptide derived from islet

autoantigen, Insulinoma-associated Ag 2) for 6 d in the absence or presence of MAPCs at a ratio of 1:2. (B) Bar graph displaying proliferation of CD4

T cells from individual G157, as described above; error bars indicate the SEM of independently established biological duplicates. (C) Proliferation of CD4

T cells from individuals with T1D stimulated with islet peptides in the presence or absence of 1:2 MAPCs as described above (n = 3 individuals studied in

two independent experiments). (D) Representative FACS plot of PPI-specific CD8 CTL clone 1E6 stimulated with peptide-pulsed, HLA-A*02, irradiated

PBMCs at a ratio of 1:3 (clone/PBMC) for 24 h in the absence or presence of 1:4 MAPCs. TNF-a production was measured by ICS. Plots show gated viable

CTLs. (E) Bar graph showing data from a single experiment representative of four experiments completed measuring TNF-a in the CTL clone, as described

above. (F) Human islets were labeled with Europium dye and pulsed with PPI15–23 peptide. CTL clone was added at a ratio of 15:1 in the presence or

absence of MAPCs or HUVECs at the ratios indicated for 4 h. Specific killing was calculated as previously described; error bars indicate the SEM of

independently established biological duplicates. *p , 0.01.

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MAPCs and MSCs suppress HP and to comprehensively under-stand the selective effects seen on Tregs and Teffs, including thedifferential responses to IL-2/IL-15 versus IL-7.The potential for MSCs to impose durable suppression has been

investigated in MLR systems, often with conflicting results that havebeen attributed to differences in assay design (51–53). In our in-vestigation, we assessed responses of suppressed cells in the ab-sence of the potentially confounding effects of other cell types fromthe primary culture, allowing us to examine the long-term, CD4T cell–intrinsic effects of MAPC suppression. Our results suggestthat suppression of CD4 T cell proliferation by MAPCs impartsa cell-intrinsic long-term effect that may be bypassed when usingartificial stimulation and, therefore, likely is dependent on theprocess of physiological Ag presentation or costimulation. Of rel-evance, rodent MAPCs were shown to induce CTLA-4 expressionon responding T cells in MLR assays, which abrogates APC-mediated costimulation but is circumvented using immobilizedAbs (36). An equally plausible explanation is that long-term effectsmay be overcome by signal strength, either directly through en-hanced signaling or indirectly by inducing de novo synthesis ofendogenous IL-2. The latter of these explanations is consistent withthe reversal of durable suppression that we observed using exoge-nous IL-2, which remains compatible with that possibility thatMAPCs cause IDO-induced T cell anergy (53, 54). These data mayexplain, in part, the paradoxical longevity of immune modulation,

despite the short half-life of infused MSCs in vivo, and observa-tions from clinical trials that repeat dosing of MSCs and MAPCsdoes not incrementally improve therapeutic benefit (6, 33).Taken collectively, the data suggest that MAPCs, like MSCs,

may be effective in regulating T cell responses associated withallograft rejection and autoimmunity, such as those that contributeto failure of AIT. Further preclinical investigations will substantiatewhether the novel findings of direct protection of target tissue fromCTL destruction, preferential HP of Tregs, and durable suppressiontranslate to therapeutic benefit in vivo. In AIT, such effects maysynergize with the established potential for MSCs to prevent b cellapoptosis, induce b cell neogenesis, and promote revasculariza-tion or organization of islet morphology (55). This class of ad-herent stem and progenitor cells continues to display therapeuticefficacy in a range of clinical indications and represents a prom-ising alternative or adjunct therapy for pharmacological immunesuppression.

DisclosuresT.I.M.T. is in receipt of an unrestricted strategic research agreement funded

by Athersys, Inc. A.E.T., S.B., and R.D. are employees of Athersys, Inc. R.D.

holds shares in Athersys, Inc. J.P. is an employee of Regensys, a wholly

owned subsidiary of Athersys, Inc. T.A. is an employee of Neusentis Regen-

erative Medicine (Pfizer Ltd.). The other authors have no financial conflicts

of interest.

FIGURE 6. MAPCs preferentially suppress proliferation of Teffs versus Tregs during HP driven by g-chain cytokines. PBMCs were stimulated with IL-2

(50 IU/ml), IL-7 (50 ng/ml), or IL-15 (50 ng/ml) for 72 h in the presence or absence of MAPCs, as indicated, and Ki67 induction was measured in viable

CD3+ lymphocytes. (A) Representative FACS plot displaying Ki67 versus CD4 in cells treated with IL-2 (left panels), IL-7 (center panels), or IL-15 (right

panels) in the absence (upper panels) or presence (lower panels) of 1:4 MAPCs. (B) Ki67 induction following IL-2 (left panels), IL-7 (center panels), or IL-

15 (right panels) treatment in CD4 (upper panels) or CD8 (lower panels) in the presence or absence of 1:4 MAPCs. (C) FACS plots showing subgating of

CD25- and FOXP3-expressing Treg and Teff populations within viable CD3+ CD4+ lymphocytes and Ki67 expression of either subset in the presence or

absence of MAPCs. (D) Paired analysis of Ki67 suppression by MAPCs within gated Treg and Teff populations in the presence of IL-2, IL-7, or IL-15. Data

points represent independent biological duplicates of three donors. Results are representative of at least two independent experiments. *p , 0.05.

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FIGURE 7. MAPCs impose a legacy of durable suppression during alloreactive T cell responses that is rescued via IL-2 or stimulation with immobilized

Abs. (A) FACS plots and experimental design of secondary restimulation experiments. PBMCs were not activated (NA), not activated in the presence of

MAPCs (NA+MAPC), stimulated with 1:40 third-party DCs (DC), or stimulated with DCs in the presence of MAPC (DC+MAPC) for 6 d. PBMC cultures

were then analyzed for CFSE dilution on a FACS sorter, and untouched, viable CFSEhi CD4+ T cells were sorted from each condition. Sorted CD4+ T cells

were restimulated with no activation (NA) or third-party DCs at different ratios in the absence (DC) or presence (DC+IL-2) of 50 IU/ml IL-2 or stimulated

with 1:10 anti-CD3/28. All FACS plots show CFSE dilution (x-axis) versus FSC-H in gated viable CD4+ T cells. (B) Bar graph displaying proliferation of

viable, CD4+ T cells upon secondary restimulation with the stimuli denoted on the x-axis. Cells were sorted from each of the primary stimulation conditions

indicated in the legend. Error bars represent the SD of two independent experiments. Data are representative of three independent experiments using allo-

DC stimulation. ***p, 0.001.

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