Regulatory T Cells Exhibit Decreased Proliferation but Enhanced Suppression After Pulsing With...

American Journal of TransplantationWiley Periodicals Inc.

C© Copyright 2012 The American Society of Transplantationand the American Society of Transplant Surgeons

doi: 10.1111/j.1600-6143.2011.03963.x

Regulatory T Cells Exhibit Decreased Proliferationbut Enhanced Suppression After Pulsingwith Sirolimus

K. Singha, N. Kozyra, L. Stemporaa, A. D. Kirka,

C. P. Larsena, B. R. Blazarb and L. S. Keana,c,*

aThe Emory Transplant Center, Department of Surgery,Emory University School of Medicine, Atlanta, GAbDepartment of Pediatrics, Division of Blood and MarrowTransplantation, University of Minnesota, Minneapolis,MNcAflac Cancer Center and Blood Disorders Service,Children’s Healthcare of Atlanta, and Department ofPediatrics, Emory University School of Medicine, Atlanta,GA*Corresponding author: Leslie S. Kean,[email protected]

Although regulatory T cells (Tregs) suppress allo-immunity, difficulties in their large-scale productionand in maintaining their suppressive function afterexpansion have thus far limited their clinical appli-cability. Here we have used our nonhuman primatemodel to demonstrate that significant ex vivo Tregexpansion with potent suppressive capacity can beachieved and that Treg suppressive capacity can befurther enhanced by their exposure to a short pulse ofsirolimus. Both unpulsed and sirolimus-pulsed Tregs(SPTs) are capable of inhibiting proliferation of mul-tiple T-cell subpopulations, including CD4+ and CD8+T cells, as well as antigen-experienced CD28+CD95+memory and CD28−CD95+ effector subpopulations. Wefurther show that Tregs can be combined in vitro withCTLA4-Ig (belatacept) to lead to enhanced inhibitionof allo-proliferation. SPTs undergo less proliferation ina mixed lymphocyte reaction (MLR) when comparedwith unpulsed Tregs, suggesting that Treg-mediatedsuppression may be inversely related to their prolif-erative capacity. SPTs also display increased expres-sion of CD25 and CTLA4, implicating signaling throughthese molecules in their enhanced function. Our re-sults suggest that the creation of SPTs may provide anovel avenue to enhance Treg-based suppression ofallo-immunity, in a manner amenable to large-scaleex vivo expansion and combinatorial therapy withnovel, costimulation blockade-based immunosuppres-sion strategies.

Key words: immunosuppression, regulatory T cells,sirolimus

Abbreviations: BMT, bone marrow transplant; CTV,CellTrace Violet, GvHD, graft-versus-host disease;MLR, mixed lymphocyte reaction; SPTs, sirolimuspulsed Tregs; Tregs, regulatory T cells.

Received 13 September 2011, revised 09 December2011 and accepted for publication 18 December 2011

Introduction

While short-term success is now common after solid-organtransplantation, long-term results are still inadequate andinclude chronic rejection, as well as off-target toxicitiesof life-long immunosuppression. There is a growing re-alization that effective immunomodulation will likely re-quire the induction of active immune regulation as well asimmunosuppression, and that CD4+CD25+FoxP3+ regula-tory T cells (Tregs) may be key participants in this process.In murine models there is growing evidence to supportthe role of Tregs in both autoimmunity and transplantation(1–3). These data provide the rationale for the develop-ment of strategies whereby Tregs are used in conjunctionwith pharmacologic immunosuppression to downregulatealloreactivity.

Tregs comprise between 5% and 10% of the peripheralCD4+ T-cell pool (4) and develop either in the thymus (nat-ural Tregs, nTregs) (5,6) or are induced in the periphery(induced or iTregs) (7–9). Although both nTregs and iTregshave been implicated in regulating immune responses(6,10–17), there are concerns with the in vivo stability ofiTregs (18–20) due to the potential risk of their reversion to-ward an activated T effector phenotype. Adoptive transferof in vitro expanded nTregs has therefore moved the far-thest clinically, with the first phase I trials of this strategyfor GvHD prevention recently completed (21,22).

While these studies have documented the feasibility oftransfer of relatively low numbers of Tregs (∼3–4×106/kg)for GvHD prevention after BMT, many mechanistic andpractical questions about their production and delivery re-main. These include a determination of the dose depen-dence of Treg therapy in vivo, and a determination of theirpotency when combined with pharmacologic immunosup-pression. Of the standard immunosuppressive agents that

Singh et al.

have been combined with Tregs, perhaps the most de-tailed work has been accomplished with sirolimus. Studieshave shown that Tregs are preferentially able to retain theirsuppressive function in the presence of sirolimus (23,24).However, although Treg function persists, several studieshave demonstrated that prolonged sirolimus exposure cansubstantially inhibit Treg expansion (25,26).

To rigorously study the questions of Treg specificity andpotency during adoptive transfer, we have established anonhuman primate (NHP) model of Treg purification, ex-pansion and function (27). In this study, we use an in vitromodel of alloreactivity to provide the first evidence thatNHP Tregs can effectively inhibit both naive and memoryT-cell allo-proliferation, and that Tregs can combine withbelatacept to induce CD8-predominant suppression of allo-proliferation. In addition, we show that the potency of exvivo expanded Tregs can be significantly increased througha short pulse of sirolimus without compromising the abilityto highly expand these cells ex vivo. These observationsare expected to impact the development and implemen-tation of strategies for combinatorial Treg-based therapyduring both HSCT and solid-organ transplantation.

Materials and Methods

Animals

Rhesus macaques from the Yerkes National Primate Research Center or theNIAID-sponsored Rhesus macaque colony in Yemassee, South Carolina,were used in this study. All animals were treated in accordance with EmoryUniversity IACUC regulations.

Treg isolation

Peripheral blood lymphocytes (PBL) were purified from CPT tubes (BD Bio-sciences, Franklin Lakes, NJ, USA). RBCs were lysed (high-yield lyse, Invitro-gen, Carlsbad, CA, USA), the PBL washed with PBS, and then resuspendedat 107 cells/40 lL in MACS buffer (Miltenyi Biotec, Cologne, Germany).CD4+ T cells were purified aseptically by negative selection using the LDcolumn platform (Miltenyi Biotec). Cells were stained for CD4 (clone SK3,BD), CD25 (clone 4E3, Miltenyi Biotec) and CD127 (clone eBioRDR5, eBio-science, San Diego, CA, USA) and resuspended in FACS sorting buffer (PBS,2% fetal bovine serum, 25 mM HEPES buffer). CD4+CD25++CD127−/low

(Tregs) and CD4+CD25+/−CD127high (non-Tregs) were then purified flow cy-tometrically using aseptic technique. Treg purity was assessed by stainingfor CD4, CD25, CD127 and FoxP3 (clone PCH101, eBioscience).

Flow cytometry

On day 0 or day 21, 0.5–1×106 cells were stained for CD3 (clone SP34–2,BD), CD4, CD25, CD127, FoxP3 and CTLA-4 (clone BNI3, BD). Data wereacquired on an LSR II flow cytometer (BD Biosciences) and analyzed usingFlowJo cytometry analysis software (Treestar, Ashland, OR, USA). Thresh-olds for identifying positively staining cells were set with relevant isotypecontrol antibodies. These controls were critical, as small differences in thebinding of the isotype controls were noted between Treg and non-Treg cul-tures (Figure 1E), potentially due to cell-specific differences in nonspecificantibody binding after bead-based stimulation.

Ex vivo expansion of CD4+CD25++CD127−/low Tregs

Flow-sorted Tregs and non-Tregs were expanded by stimulating with anti-rhesus CD3 and anti-human CD28 coated microbeads (Miltenyi Biotec) at acell:bead ratio of 1:2 and culturing in X-vivo-15 media (Lonza) supplemented

with 5% human serum, 0.2% N-acetyl cysteine, 5 mM Hepes buffer, peni-cillin (100 IU/mL), streptomycin (100 lg/mL), gentamicin (20 lg/mL) andeither 2000 or 200 IU/mL of rhIL-2 (R&D Systems, Minneapolis, MN, USA)for Tregs or non-Tregs, respectively. Cultures were split and replenishedwith fresh media and rhIL-2 when the media became acidic (at a density of∼2–3×106 cells/mL). At days 7 and 14, cell numbers were counted and cul-tures restimulated as on day 0. Cells were recovered on day 21, magneticbeads removed with a magnetic column (Miltenyi Biotec) and their pheno-typic integrity assessed by staining for CD3, CD4, CD25, CD127 and FoxP3.In some cultures, 1–1000 nM of sirolimus was added at the time of eachstimulation. To create sirolimus pulsed Tregs (SPTs), Tregs were expandedin the absence of sirolimus until day 19, and then pulsed with 100 nM ofsirolimus (the standard dose used in human Treg cultures (24,25,28,29), forthe next 48 h. The cultures were then recovered, washed free of sirolimusand cryopreserved.

Suppression assay to measure the inhibitory activity of ex vivo

expanded Tregs

Treg-mediated suppression of allo-proliferation was assessed in an in vitroCFSE-MLR assay. 2×105 “responder” PBLs were labeled with CFSE as pre-viously described (27), and then either cultured without stimulation, or in thepresence of 4×105 irradiated allogeneic ‘stimulator’ PBLs in the absenceor presence of Tregs or non-Tregs. Treg cultures that were derived from thesame animal from which the responder PBLs were collected were referredto as “responder-specific” Tregs. MLRs were cultured for 5 days at 37◦Cin OpTmizer T-cell expansion media (Invitrogen) supplemented with 5%human serum, 2 mM glutamine, penicillin–streptomycin and gentamycin.On day 5, cells were stained for CD2, CD3, CD4, CD8, CD28, CD95 andFoxP3 and the proliferation of the responder T cells was assessed flowcytometrically by CFSE dilution. In some experiments, 200 lg/mL of be-latacept (Bristol–Myers Squibb) was also added. The gating strategy usedto assess proliferation is shown in Supporting Figure S1 and was as fol-lows: (1) Lymphocytes were identified with a forward-scatter (FSC) versusside-scatter (SSC) gate (2). T cells were identified using a CD3 versus FSCgate (3). The CD3 gate was further refined by gating on CD3+/CD2+ cells,which includes both memory and naive T-cell populations (30,31) (4). TheCFSE-labeled responder T cells were identified by applying a CFSE ver-sus CD2 gate, which facilitated the elimination of non-CFSE-labeled cellpopulations that could otherwise confound the interpretation of the data.This gate distinguishes the allo-proliferating CFSE-labeled cells from thenon-CFSE-labeled cells based on the fact that CD2 expression increasesduring proliferation. Thus, the threshold between the highly divided cells(with the lowest CFSE fluorescence) and the non-CFSE-labeled cells wasset based on their relative degree of CD2 fluorescence (5). A CD4 versusCD8 gate was then applied, and the CFSE fluorescence of the CD4+ andCD8+ subpopulations determined.

Allo-proliferation of Tregs

The allo-proliferation of Tregs was assessed with an MLR assay, usingTregs that were first stained with 5 lM CellTrace Violet (CTV, Invitrogen).The gating strategy was identical to that described above except that toestablish the threshold between unlabeled cells and highly divided CFSE-or CTV-labeled cells, control cultures were used that contained only CFSE-labeled responder cells (no CTV-labeled Tregs), or only CTV-labeled Tregs(no CFSE-labeled responders). In these cultures, the proliferating cells wereidentified by comparing either CFSE or CTV versus CD2 fluorescence, whichfacilitated the establishment of a gate that distinguished the nonlabeled cellpopulations from the allo-proliferating cells.

Statistical analysis

Data were analyzed by both paired and unpaired Student’s t-test. p valuesof ≤0.05 were considered statistically significant (∗p < 0.05, ∗∗p < 0.01,∗∗∗p < 0.001).

American Journal of Transplantationdoi: 10.1111/j.1600-6143.2011.03963.x

Regulatory T Cells and Transplant Tolerance

Results

Purification and expansion of rhesus macaque

CD4+CD25++CD127−/low Tregs

Rhesus macaque Tregs were purified by flow cytometry-based sorting of CD4+CD25++CD127−/low cells after ini-

tial column-based CD4 enrichment, using a FACSAria flow-based cell sorter (Figure 1A). This strategy resulted in suf-ficient yield (≥1×106 Tregs) and purity (>80%) of Tregsto proceed with expansion and functional characterization(Figure 1). As shown in Figure 1B, the vast majority ofsorted Tregs were CD25-positive, CD127-negative/low and

Figure 1: Isolation, ex vivo expansion and analysis of CD4+CD25++CD127−/lowTregs and CD4+CD25±CD127highnon-Tregs.

(A) Purification strategy for rhesus macaque Tregs: CD4+ T cells were first enriched from PBLs by depletion of non-CD4+ cells us-ing a NHP-specific CD4+ T-cell isolation kit. These “untouched” CD4+ T cells were stained for CD4, CD25 and CD127 and flow-sortedinto CD4+CD25++CD127−/low “Tregs” and CD4+CD25+/−CD127high “non-Tregs” on a BD FacsAria cell sorter. (B) The phenotype of theflow-sorted Tregs and non-Tregs was confirmed by staining for CD3, CD4, CD25, CD127 and FoxP3. The data shown are representativeof five independent experiments. Cells were first gated to identify CD3+/CD4+ cells, which were then queried for the level of expressionof CD25, CD127 and FoxP3. (C) The percentage of CD4+ T cells expressing CD25 and FoxP3 in putative Tregs (blue) and non-Tregs (red)after flow-based cell sorting (n = 5 separate Treg donors). Shown is the mean ± SEM. (D) Ex vivo expansion of Tregs and non-Tregs:flow-sorted Tregs and non-Tregs were stimulated with microbeads coated with anti-rhesus CD3 and anti-human CD28 antibodies at acell: bead ratio of 1:2 and cultured in X-vivo-15 medium supplemented with 5% human serum, antibiotics and 2000 IU/mL and 200 IU/mLof rhIL-2 for Tregs and non-Tregs respectively as described in the Materials and Methods section. Cells were restimulated on days 7 and14 and were recovered on day 21. Cell numbers were counted on days 7, 14 and 21. Shown are the average fold increases over baseline± SEM, n = 5 separate Treg donors. (E) The phenotype of the expanded Tregs and non-Tregs was confirmed by staining for CD3, CD4,CD25, CD127 and FoxP3 at day 21 of culture. The data shown are representative of five independent experiments. Cells were first gatedto identify CD3+/CD4+ cells, which were then queried for the level of expression of CD25, CD127 and FoxP3. (F) The percentage of CD4+T cells expressing CD25 and FoxP3 in expanded Tregs (blue) and non-Tregs (red) after 21 days of culture (n = 5 separate Treg donors).Shown is the mean ± SEM.


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Figure 1: Continued.

exhibited significant FoxP3 expression, unlike the sortednon-Tregs, which displayed significantly less CD25 andFoxP3 expression and significantly more CD127 expres-sion. Figure 1C shows the quantification of FoxP3 ex-pression between sorted Tregs and non-Tregs comparedto the isotype control, which documents that, similar towhat has been observed during the flow-based purifica-tion of human Tregs (21,26,28) prior to their expansion,64.3 ± 3.5% of the Treg isolates were FoxP3+ compared to2.8 ± 0.4% of the non-Treg isolates (p < 0.001). Figure 1Dshows the results of anti-CD3/CD28 bead-based expan-sion of both Treg (210–760-fold) and non-Treg (1200–2000-fold) cultures, and Figures 1E, F document that expandedTreg cultures maintained the CD25high/CD127low/FoxP3+

phenotype, with 90.2 ± 2.3% expressing FoxP3 af-ter 21 days in culture, compared to 18.3 ± 2.8%FoxP3 expression in the expanded non-Treg cultures(p < 0.001).

Only ex vivo expanded Tregs potently suppress

allo-proliferation of T effector cells

The suppressive capacity of both ex vivo expanded Tregsand non-Tregs was assessed using a CFSE-MLR prolif-

eration assay. As shown in Figure 2A, despite the factthat a small amount of FoxP3 expression was inducedin the non-Treg cultures (Figure 1F), these cells were notsuppressive: the addition of non-Tregs to MLR cultureswas noted to slightly enhance the allo-proliferation of bothCD4+ and CD8+ responder T cells, increasing the averagepercent proliferation of CD4+ cells from 9.4 ± 1.2% →22.1± 3.6% (n = 18, p = 0.004) and of CD8+ cells from40.6 ± 3.3% → to 50.5 ± 4.7% (n = 18, p = 0.01). Incontrast, the addition of Tregs to the MLR strongly sup-pressed the allo-proliferation of responder T cells, result-ing in 3.5 ± 0.4-fold inhibition of CD4+ proliferation and3.0 ± 0.4-fold inhibition of CD8+ proliferation (p < 0.001and p = 0.002, respectively, Figure 2B). Treg-mediated in-hibition was calculated by comparing the percentage ofresponder cells in Treg-containing MLRs that divided atleast once with the percentage of responder cells that haddivided when non-Tregs were added to the MLR. Treg-mediated suppression of allo-proliferation occurred pre-dominantly on highly proliferating cells. Thus, as shown inTable S1 and Figure S2, Tregs modestly inhibited the firstdivision cycle of responding CD4+ and CD8+ T cells (by 1.5-and 1.47-fold, respectively), but showed more substantial



inhibition of later stages of proliferation (inhibiting thesecond-sixth division cycle of responding CD4+ and CD8+

T cells by an average of 3.3 ± 0.1-fold and 0.3 ± 1.1-fold,respectively).

Tregs potently inhibited the accumulation of CD95+

T cells

Figure 2C and D documents Treg-mediated inhibition ofaccumulation of CD28+ and CD28− CD95+ cells in theMLR. Thus, in a standard MLR, significant accumulationof CD95+ memory phenotype cells (32) occurred after5 days in culture (Figure 2C). Whether this accumulationwas due to the selective expansion of memory cells that

were present in the CFSE-labeled responder T-cell pool, ordue to the conversion of naive T cells toward a memoryphenotype, could not be distinguished from these exper-iments. Nevertheless, when antigen-experienced mem-ory CD28+CD95+ or effector CD28−CD95+ cells wereanalyzed after 5 days of culture in the presence of exvivo expanded Tregs (Figure 2C and D), significant inhibi-tion of their accumulation was observed. Thus, when nor-malized against cultures to which non-Tregs were added,MLRs treated with expanded Tregs showed 3.0 ± 0.5-fold (p = 0.01) and 2.4 ± 0.5-fold (p = 0.029) inhibition ofCD28+CD95+ proliferation for CD4+ and CD8+ cells, re-spectively (Figure 2D, top row), and 2.9 ± 0.6 (p = 0.021)

Figure 2: Ex vivo expanded Tregs potently suppress allo-proliferation of T effector cells in an MLR assay. (A) CFSE-labeled responderT cells were allo-stimulated for 5 days with unlabeled antigen-presenting cells from MHC disparate donors in the absence or presenceof either ex vivo expanded Tregs or non-Tregs at a Treg/non-Treg:responder T-cell ratio of 1:1. Allo-proliferation of CD4+ and CD8+ T cellsover the 5-day period and its suppression by added Tregs was followed by CFSE dilution. The data shown are representative of nineindependent experiments. (B) Summary data showing paired analysis of MLR-based allo-proliferation (top panels) and the average foldinhibition of allo-proliferation (bottom panels) of CD4+ (left) and CD8+ T cells (right), in the presence of expanded non-Tregs and Tregs(n = 9 independent MLRs. These MLRs were performed using Treg cultures from five independent Treg donors. Treg cultures from thesedonors were assayed between 1 and 3 times in the MLR). Shown is the mean ± SEM. (C) CD28+CD95+ and CD28−CD95+ CD4+ andCD8+ T-cell subpopulations were identified flow cytometrically after the 5-day MLR and the suppression of their allo-proliferation by addedexpanded Tregs was determined by CFSE dilution of the respective responder populations. The data shown are representative of nineindependent experiments. (D) Summary data showing the relative allo-proliferation of CD4+CD28+CD95+ (upper left), CD4+CD28−CD95+(lower left), CD8+CD28+CD95+ (upper right) and CD8+CD28−CD95+ (lower right) T cells in an MLR assay in the presence of expandednon-Tregs and Tregs (n = 9 independent MLRs. These MLRs were performed using Treg cultures from five independent Treg donors.Treg cultures from these donors were assayed between 1 and 3 times in the MLR. Shown is the mean ± SEM). (E) Tregs suppressallo-proliferation of sorted naı̈ve and memory T-cell populations. CD3+CD28+CD95− naive T cells, CD3+CD28+CD95+ central memory Tcells and CD3+CD28−CD95+ effector/effector memory T cells were sorted flow cytometrically and then placed into a CFSE-MLR in theabsence or presence of ex vivo expanded Tregs or non-Tregs. Top row: CD28 and CD95 fluorescence before sorting (far left panel) andafter sorting of CD3+CD28+CD95−, CD3+CD28+CD95+ and CD3+CD28−CD95+ cells. Bottom three rows: CFSE fluorescence (shownas dot plots on the left and the same data as histograms on the right) in the presence of allo-stimulation plus non-Tregs or Tregs. (F)Combining Tregs with belatacept lead to enhanced inhibition of allo-proliferation. CFSE-labeled responder T cells were allo-stimulatedas in Figure 2A in the absence or presence of either belatacept (200 lg/mL), ex vivo expanded Tregs, or both. The allo-proliferation ofCD4+ (top two rows, shown as both dot plots and as histograms) and CD8+ T cells (bottom two rows, shown both as dot plots and ashistograms) and its suppression by added belatacept and/or Tregs was determined by CFSE dilution. The data shown are representativeof four independent experiments. (G) Summary data showing relative allo-proliferation of effector CD4+ T cells (left) and CD8+ T cells(right) in an MLR assay in the presence of belatacept, Tregs and both (n = 4 independent MLRs from two individual Treg donors). Shownis the mean ± SEM.


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and 2.7 ± 0.3-fold (p = 0.007) inhibition for CD28−CD95+

proliferation for CD4+ and CD8+ cells, respectively(Figure 2D, bottom row). Tregs were also able to inhibitthe proliferation of naive and memory T cells which werepurified flow cytometrically prior to placement in the MLR(Figure 2E). These results may have significant clinical rel-evance, given recent observations of costimulation block-ade resistant rejection mediated by CD28-negative mem-ory populations (30,31). They led us to investigate whetherTregs could effectively combine with belatacept to morecompletely suppress alloreactivity.

Tregs effectively combine with belatacept to inhibit

allo-proliferation

Figures 2F and G document that expanded Tregs couldindeed combine with CD28-directed costimulation block-ade to inhibit allo-proliferation. Thus, as shown in Figure2F and G, when Tregs alone were added to effector cells(at a 1:1 ratio) in the MLR, they resulted in 3.4 ± 0.1-foldinhibition of CD8+ proliferation and a 3.9 ± 0.5-fold inhibi-tion of CD4+ proliferation (p < 0.001 for CD8+ proliferationand p < 0.05 for CD4+ proliferation). While the amountof autologous T-cell proliferation (occurring in the absence




of allogeneic APCs) was too low to be able to determinean impact of belatacept (not shown), belatacept did clearlyinhibit allo-proliferation, with a greater salutary effect onCD4+ proliferation (7.1 ± 1.8-fold inhibition, p < 0.05) than

on CD8+ proliferation (2.0 ± 0.1-fold inhibition, p < 0.01).Importantly, the addition of ex vivo expanded nTregs tobelatacept had an additive suppressive effect in the MLR,significantly increasing the belatacept-mediated inhibition


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of CD8+ proliferation, resulting in 4.8 ± 0.2-fold inhibitionof proliferation (p < 0.01 when compared to either Tregsor belatacept alone). While Tregs had a less substantialimpact on belatacept-mediated inhibition of CD4+ prolif-

eration (8.7 ± 1.7-fold with belatacept + Tregs comparedto 7.1 ± 1.4-fold inhibition of proliferation with belataceptalone), in four independent experiments, the addition ofTregs to belatacept-containing cultures always resulted in



an incremental inhibition of CD4+ accumulation, which re-sulted in a statistically significant effect in paired analysis(p < 0.05). The CD8-directed additive effect of Tregs + be-latacept was also evident when lower Treg:Teffector cell ra-tios were used (Supporting Data, Figure S3 and S4), imply-ing that costimulation blockade with belatacept may be aclinically important partner for Treg-mediated cellular thera-pies. The inhibitory effect of belatacept on allo-proliferationwas likely not due to the induction of Tregs in the MLR,since no increase in FoxP3 expression on the responder Tcells was observed (Figure S5). This is similar to what hasbeen observed in transplant and autoimmunity patients(33–35) where no induction of Tregs has occurred in pa-tients treated with CTLA4-Ig.

Tregs proliferate in response to allogeneic stimulation

while simultaneously suppressing the

allo-proliferation of effector T cells

To quantify the impact that allo-stimulation made on theTregs themselves, we labeled the expanded Treg cultureswith CTV before adding them into MLR cultures containingeither allogeneic APCs alone or APCs plus CFSE-labeledresponder T cells. As shown in Figure 3A–G, expandedTregs were also capable of allo-specific proliferation(14.9 ± 2.8% proliferation in an MLR containing allogeneicAPCs vs. 0.4 ± 0.4% proliferation without allogeneic APCs,p = 0.003). Tregs proliferated similarly in response to al-logeneic APCs, whether in the presence or absence ofresponder T cells (Figure S6).

Figure 3: Tregs proliferate in response to allogeneic stimulation while simultaneously suppressing the proliferation

of effector T cells. Tregs were stained with the proliferation marker CellTrace Violet (CTV) as described in the Materi-als and Methods section, and then added to T effector cells stained with CFSE. Cultures were then allo-stimulated withAPCs for 5 days and the ability of the labeled Tregs to suppress allo-proliferation of effector T cells and to undergo pro-liferation was studied by simultaneously determining the dilution of CTV and CFSE dyes. (A) CFSE fluorescence from a ‘T-responders alone’ control in which CFSE-labeled responder T cells do not proliferate in the absence of allogeneic APCs.(B) CTV fluorescence from a ‘Tregs alone’ control in which labeled Tregs do not proliferate in the absence of allogeneic APCs. (C)CFSE and CTV fluorescence demonstrate the lack of proliferation of either responder T cells or Tregs in the absence of allogeneic APCs.(D) CFSE fluorescence from an allo-stimulated MLR showing significant proliferation of the CFSE-labeled responder T cells. (E) MLRconsisting of CFSE-labeled responder T cells, unlabeled stimulator APCs and CTV-labeled Tregs. The dilution of both CFSE (respondercells) and CTV (Tregs) fluorescence demonstrates proliferation of both responder T cells and Tregs in the presence of allogeneic APCs.Note that the proliferation of the CFSE-labeled responder T cells is reduced in the presence of Tregs (compare panel E to panels D andF). (F) MLR consisting of CFSE-labeled responder T cells, unlabeled stimulator APCs, and CTV labeled non-Tregs (no Tregs were added).The dilution of both CFSE (responder cells) and CTV (non-Tregs) fluorescence demonstrates proliferation of both responder T cells andnon-Tregs, but no inhibition of responder cell proliferation without added Tregs (compare panel F to panel E). (G) A summary of theproliferation of Tregs after auto- or allo-stimulation in MLRs in four independent experiments is shown (shown is the mean ± SEM).


Singh et al.

Significant inhibition of ex vivo Treg expansion by the

continuous presence of sirolimus

While several studies have suggested that sirolimus mayenrich bulk lymphocyte cultures for Tregs (26,36) this effectappears to be due to preferential survival of Tregs com-pared to conventional T cells, rather than to Treg-specificexpansion in sirolimus (26,37–39). In this study, we con-firmed that, as we have previously shown for human umbil-ical cord blood (40) and peripheral blood (41) nTregs, the in-clusion of continuous sirolimus in expanding NHP Treg andnon-Treg cultures strongly inhibited their anti-CD3/CD28mAb-mediated expansion (Figure S7). Thus, tonic exposureto sirolimus was counter-productive to our Treg expansionstrategy.

Sirolimus-pulsed Tregs (SPTs) demonstrate increased

suppressive capacity

Given that previous studies have suggested that sirolimusmay enhance Treg function (37,42,43), we determinedwhether short-term exposure to sirolimus could improveTreg potency while maintaining Treg yield. Thus, we firstexpanded nTregs for 19 days without sirolimus, and thenpulsed them with 100 nM of sirolimus (24,25,28,29) for48 h before Treg recovery and functional analysis. Asshown in Figures 4A–C, the sirolimus pulse more thandoubled the inhibitory capacity of expanded Treg cultures.Thus, sirolimus-pulsed Tregs (SPTs) exhibited a signifi-cantly enhanced suppressive capacity, which was predom-inantly directed against CD28+ CD4+ and CD8+ T cells. Asshown in Figure 4C, SPTs demonstrated 2.7 ± 0.3 and4.4 ± 0.7-fold inhibition of CD28+ CD4+ and CD8+ T cells,respectively (p < 0.01 compared to unpulsed Tregs). Thiseffect was dose dependent, as shown in the representa-tive example in Figure 4D.

The enhanced function of SPTs was accompanied by

alterations in their proliferative capacity and

phenotype

To determine the mechanisms by which SPTs exhibitedtheir increased inhibitory capacity, we determined theirphenotype and proliferative capacity using flow cytomet-ric analysis. As shown in Figure 5A and B, SPTs exhib-ited significantly less allo-proliferation than control Tregs,(p = 0.015), consistent with previous studies, which havedocumented an inverse relationship between Treg pro-liferation and function (44,45). In addition, as shown inFigure 5C, all of the proliferating cells in the SPT cultureswere FoxP3+, reinforcing the fact that the allo-proliferationthat we measured likely emanated from Tregs and notfrom the small number of FoxP3-negative cells present inthe ex vivo expanded cultures. In addition, flow cytometricanalysis demonstrated increased expression of both CD25(Figure 5D, p = 0.04) and CTLA4 (Figure 5E, p = 0.009)on SPTs compared to Tregs, consistent with the previ-ously documented role of CD25 and CTLA4 expression inTreg function (46,47). In contrast, expression levels of CD3,CD4, CD27, CD45RA, CD62L, CD127, CD179b, CD223,

CD279, GITR, MHC class II and phospho-Stat-5 did notshow any significant change in SPTs (data not shown), al-though FoxP3 expression was slightly decreased on SPTs(MFI = 557 ± 1243 for SPTs, 6464 ± 570 for Tregs,p = 0.03, data not shown).

Discussion

While murine studies have documented the ability ofboth nTregs and iTregs to downregulate allo-immunity(1–3,10,43) the broad translation of these observations tolarge animal models and to the clinic, especially for solid-organ transplantation, has yet to occur. However, the re-cent publication of the first phase I clinical trials using Tregs(which both employed Tregs during BMT as part of post-transplant GvHD immunoprophylaxis) (21,22) suggest thefeasibility of Treg-based approaches.

There have, historically, been significant barriers to thewide spread use of adoptive Treg immunotherapy. Thesehave included both the difficulty in producing these cellsin sufficient quantities for in vivo use and with the main-tenance of adequate suppressive function in massivelyexpanded Treg cultures. While Hippen et al. (12) haverecently reported a significant breakthrough in Treg expan-sion, their production remains technically challenging andextremely costly. Thus, designing strategies to increasethe potency of Tregs and identifying immunosuppressiveagents with which they can effectively combine (especiallyat lower nTreg doses) remains a key challenge for thefield.

In this manuscript, we use an in vitro model of primate allo-stimulation to document several mechanistic observationsthat may be critical for the translation of nTreg strategies tothe clinic. This model is strengthened by its ability to mea-sure the impact of Tregs on responder T-cell proliferation af-ter allogeneic stimulation, rather than after mitogenic (anti-CD3) antibody stimulation. Given that the mechanisms gov-erning CD3-stimulation and allo-stimulation are likely dis-tinct, and that allo-stimulation is expected to be the maindriver of the immune response after transplant, the abil-ity to interrogate mechanisms controlling allo-stimulationis expected to add to the predictive power of this modelwith respect to in vivo Treg function and potency.

Using this model we show that NHP Tregs are po-tent inhibitors of allo-proliferation of both CD28+ andCD28− memory T cells. This observation suggests thatnTregs may be good candidates for combination with cos-timulation blockade-based immunosuppression, given re-cent data suggesting that costimulation blockade-resistantrejection is often mediated by CD28-negative effec-tor/memory T cells (30,31,48,49). The observations madein this study with respect to Treg-mediated inhibition ofmemory T-cell allo-proliferation are quantitatively differ-ent than those documented by Yang et al. (50), which



Figure 4: Sirolimus-pulsed Tregs (SPTs) demonstrate increased suppressive capacity, predominantly against the proliferation

of CD28+CD95+ T cells. Flow-sorted Tregs were stimulated with anti-rhesus CD3 and anti-human CD28 coated microbeads and IL-2as described in the Materials and Methods section. On day 19, 100 nM of sirolimus was added to the cultures. Forty-eight hours latersirolimus-pulsed Tregs (SPTs) were recovered, washed free of sirolimus, beads removed and tested for their ability to suppress allo-proliferation in an MLR. (A) Representative data at a 1:1 Treg:responder T-cell ratio demonstrating the enhanced suppressive capacityof SPTs against CD4+ T-cell proliferation. Row (i) autologous responder T cells alone control (no allogeneic APCs added to the MLR).Row (ii) MLR with responder T cells + allogeneic APCs plus expanded non-Treg CD4+ T cells. Row (iii) MLR with responder T cells,+ allogeneic APCs plus expanded Tregs. Row (iv) MLR with responder T cells, + allogeneic APCs plus sirolimus-pulsed Tregs (SPTs).Columns show CFSE fluorescence after gating on the following T-cell subpopulations after the 5-day MLR: column 1: total CD4+ T cells;column 2: CD28+CD95+ CD4+ T cells; column 3: CD28−CD95+ CD4+ T cells. The data shown are representative of 11 individual MLRs.(B) Representative data at a 1:1 Treg:responder T-cell ratio demonstrating the enhanced suppressive capacity of SPTs against CD8+ T-cellproliferation. Row (i) autologous responder T cells alone control (no allogeneic APCs added to the MLR). Row (ii) MLR with responder Tcells + allogeneic APCs plus expanded non-Treg CD4+ T cells. Row (iii) MLR with responder T cells, + allogeneic APCs plus expandedTregs. Row (iv) MLR with responder T cells, + allogeneic APCs plus sirolimus-pulsed Tregs (SPTs). Columns show CFSE fluorescenceafter gating on the following T-cell subpopulations after the 5-day MLR: column 1: total CD8+ T cells; column 2: CD28+CD95+ CD8+ Tcells; column 3: CD28−CD95+ CD8+ T cells. The data shown are representative of eleven individual MLRs. (C) Summary data showingthe relative alloproliferation of total CD4+ T cells (upper left), CD4+CD28+CD95+ cells (upper middle), CD4+CD28−CD95+ cells (upperright), total CD8+ cells (lower left) CD8+CD28+CD95+ cells (lower middle) and CD8+CD28−CD95+cells (lower right), in an MLR assayin the presence of non-Tregs, unpulsed Tregs and SPTs (n = 11 independent MLRs. These MLRs were performed using SPT culturesfrom five independent Treg donors. SPT cultures from these donors were assayed between 1 and 3 times in the MLR.) The meanfold-inhibition ± SEM and p values for the comparison of Tregs versus SPTs were as follows. For total CD4+ T cells: Tregs: 3.1 ±0.28, SPTs: 4.5 ± 0.57, p = 0.006. For CD4+CD28+CD95+: Tregs: 1.9 ± 0.16, SPTs: 2.7 ± 0.3, p = 0.008. For CD4+CD28−CD95+:Tregs: 2.2 ± 0.21, SPTs: 2.9 ± 0.35, p = 0.02. For Total CD8+ T cells: Tregs: 2.9 ± 0.34, SPTs: 6.3 ± 0.79, p = 0.0001. ForCD8+CD28+CD95+: Tregs: 1.6 ± 0.12, SPTs: 4.4 ± 0.73, p = 0.003. For CD8+CD28−CD95+: Tregs: 9.9 ± 2.4, SPTs: 10.5 ± 2.4, p = 0.673.(D): Dose–response curve showing the degree of suppression when Tregs and SPTs were included in an MLR at the following ratiocompared to the responder T cells: 1:1; 1:2; 1:4; 1:8; 1:16; 1:32. Data are representative of two individual experiments.


Singh et al.


showed that in mice, while Tregs could inhibit in vitroallo-proliferation of both naive and memory T cells at highTreg:Teff ratios, Tregs were less potent at inhibiting mem-ory T-cell-driven allo-proliferation when present at lower ra-tios, and were unable to inhibit memory-driven allograft re-jection in vivo. While not yet proven experimentally, thesedifferences may reflect species-specific mechanistic dif-ferences in Treg function. Indeed, in humans, Tregs havebeen demonstrated to inhibit both a b and c d mem-ory cell function (51,52), suggesting that the breadth andstrength of the Treg response may differ between mice andprimates.

Our results also identify a one-step strategy that signif-icantly increased the in vitro potency of Tregs. The pro-duction of SPTs succeeded in improving the suppres-sive capacity of these cells while maintaining their abil-ity to be highly expanded in vitro prior to sirolimus ex-posure. Thus, while there are now substantial data fromthis study and others (12,26,53) demonstrating that con-tinuous exposure to sirolimus significantly impairs Tregexpansion, here we demonstrated that a short pulse ofsirolimus, which can be delivered after expansion, is suf-ficient to alter nTreg phenotype and function, resulting inimproved inhibitory capacity. Our results with SPTs do notyet distinguish whether exposure to sirolimus potentiatesthe functional competence of all of the Tregs in culture,or whether it selects for a highly active Treg subpopula-tion. This question remains an important area for futureinvestigation.

Our observation that nTreg-mediated suppression may beinversely related to their allo-proliferative capacity suggeststhat on a per-cell level, anergy may be mechanistically tiedto suppression, and mTOR inhibition, in its ability to poten-tiate anergy, may therefore potentiate the nTreg suppres-sive phenotype. These data are distinct from those recentlydescribed by Procaccini et al. (54), in which transient ex-posure of mouse and human Tregs to sirolimus enhancedtheir subsequent proliferation in response to CD3/CD28-mediated polyclonal stimulation, as part of an oscilla-tory response to mTOR signaling. However, our obser-vations using allo-stimulation rather than anti-CD3/CD28mAb-mediated stimulation are similar to those recently de-scribed by Wang et al. (55) who showed that in the settingof nonlymphopenic homeostatic and allo-stimulated pro-liferation, sirolimus significantly inhibited Treg replication.These results suggest that a delicate balance may be re-quired for the design of transplantation strategies basedon chronic sirolimus exposure, given that continuous expo-sure to sirolimus may actually result in the inhibition of Tregexpansion, despite potentially increasing their suppressivepotency.

These results also underscore the complexities associatedwith Treg function, expansion, and in vivo use, and supporttesting in large animal models such as the rhesus macaquein order to determine the optimal strategies to move for-ward to the clinic. These studies will allow us to rigorouslydetermine the ability of Tregs to proliferate in vivo, andthe impact that exposure of these cells to sirolimus (prior




to transfer or during ongoing immunosuppression), and toother conventional and novel immunosuppressive agents(including belatacept) may make on their expansion, sur-vival and function.

Acknowledgments

This work was supported by Yerkes, the National Primate Research Cen-ter Base Grant, #RR00165. CPL was supported by NIH grant #s 2U19AI051731, and 2P01 AI044644. LSK was supported by grant #s 5K08AI065822, 2U19 AI051731, 1R01 HL095791 and 2U24 RR018109, and by a

Burroughs Wellcome Fund Career Award in the Biomedical Sciences. A.D.K.was supported by JDRF 1-2008-594,1U01AI079223-01A1,5 U19 AI051731.B.R.B. was supported by NIH R01 AI34495, HL 56067, and P01 CA 067493.

Disclosure

The authors of this manuscript have no conflicts of inter-est to disclose as described by the American Journal ofTransplantation.


Singh et al.

Figure 5: The enhanced function of SPTs is accompanied by alterations in their proliferative capacity and phenotype. (A) SPTsare less proliferative than unpulsed Tregs in an allo-MLR. SPTs and control Tregs were stained with CTV dye and were added to an MLRin which effector T cells were stained with CFSE. Suppression of effector T-cell alloproliferation (not shown) and proliferation of SPTsand control Tregs were determined. Shown is a representative tracing of proliferation as measured by the dilution of CTV fluorescence,comparing unpulsed Tregs to SPTs. (B) Summary of the relative proliferation of SPTs compared to control Tregs from three independentexperiments. Shown is the mean ± SEM. (C) Both dividing and nondividing cells from the ex vivo expanded Treg cultures express FoxP3:SPTs were labeled with CTV and placed into the MLR. FoxP3 fluorescence was measured on the proliferating SPTs (CTVlow, green traces)and nonproliferating SPTs (CTVhigh, purple traces) as well as on the non-Tregs (black traces). (D) SPTs exhibit higher cell-surface expressionof CD25 compared to unpulsed Tregs. SPTs and control, unpulsed Tregs were stained for CD3, CD4 CD25 and intracellular FoxP3 andflow cytometry data were acquired on BD LSR II and analyzed by Flowjo. A representative histogram (left) and summary expressiondata (displayed as the mean fluorescence intensity (MFI) from five independent experiments (right) for CD25 fluorescence intensity areshown (shown is the mean ± SEM). (E) SPT cultures have more CTLA-4High Tregs compared to control, unpulsed Treg cultures. SPTs andcontrol unpulsed Tregs were stained for CD3, CD4, total CTLA-4 and intracellular FoxP3, and flow cytometry data were acquired on BDLSR II and analyzed by Flowjo. Left: representative density plots showing the number of Tregs in each culture that was CTLA-4High Right:summary data of CTLA-4High expression from four independent experiments are shown (shown is the mean ± SEM).



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Supporting Information

Additional supporting information may be found in the on-line version of this article.

Supporting Table 1. Treg- and SPT-mediated suppressionof CD4+ and CD8+ T cell allo-proliferation.

Supporting Figure S1: The gating strategy used to assessallo-proliferation. The following gating strategy was used toassess allo-proliferation of CFSE-labeled responder T cells:(1) Lymphocytes were identified with a forward-scatter(FSC) versus side-scatter (SSC) gate. (2) T cells were iden-tified using a CD3 versus FSC gate. (3) The CD3 gate wasfurther refined by gating on CD3+/CD2+ cells, which iden-tified both memory and naı̈ve populations, respectively.(4) The CFSE-labeled responder T cells were identified byapplying a CFSE vs CD2 gate, which facilitated the elimi-nation of non-CFSE-labeled cell populations that could oth-erwise confound the interpretation of the data. This gatedistinguishes the allo-proliferating CFSE-labeled cells fromthe non-CFSE labeled cells based on the fact that CD2 ex-pression increases during proliferation. Thus, the thresholdbetween the highly divided cells (with the lowest CFSE flu-orescence) and the non-CFSE-labeled cells was set basedon their relative degree of CD2 fluorescence. (5) A CD4 vsCD8 gate was then applied to the CFSE-labeled cells andthe CFSE fluorescence of the CD4+ and CD8+ subpopula-tions determined.

Supporting Figure S2: Tregs inhibit the accumulation ofcells in division cycles #2-6 more than they inhibit the ac-cumulation of cells in division cycle #1. The percent ofresponder T cells in the MLR that had divided between 1–6 times was determined in the presence and absence ofTregs using FlowJo flow cytometry analysis software. Thedegree of Treg-associated inhibition of CD4+ or CD8+ T cellaccumulation was then calculated. Black: Treg-associatedinhibition of accumulation of cells in division cycle #1. Red:Treg-associated inhibition of accumulation of cells in divi-sion cycles #2–6 (shown as the average +/− SEM).

Supporting Figure S3: Combining a sub-optimalTreg:Teffector ratio (1:8 Tregs: Teffectors) with belataceptleads to enhanced inhibition of alloproliferation. CFSE-labeled responder T cells were allo-stimulated in the ab-sence or presence of either belatacept (200 lg/ml), ex-vivoexpanded Tregs (1:8 Tregs: Effector ratio), or both. The allo-proliferation of CD4+ and CD8 T+ cells and its suppressionby added belatacept and/or Tregs was determined by CFSEdilution and is shown as both dot-plots and as histograms.The data shown is representative of four independent ex-periments.

Supporting Figure S4: Summary data for the impact ofa suboptimal Treg:Teffector ratio plus belatacept. This fig-ure shows the relative allo-proliferation of effector CD4 T+



cells (left) and CD8 T+ cells (right) in an MLR assay in thepresence of belatacept, Tregs (1:8 Tregs: effector ratio) orboth (n= 4). Shown is the mean +/− SEM.

Supporting Figure S5: Belatacept does not increaseFoxP3 expression on responder T cells. FoxP3 expressionwas quantified flow cytometrically on responder T cellsin an MLR. Shown is a representative example that doc-uments that the percent of responder T cells that wereFoxP3+ did not change in the presence of belatacept.

Supporting Figure S6: Tregs proliferate in response toallogeneic APCs alone. Shown is a representative exampleof the proliferation of Tregs in an MLR as measured by thedilution of CTV fluorescence. As shown in the Figure, Tregsproliferate in an MLR containing only Tregs and allogeneicAPCs, without the addition of responder T cells.

Supporting Figure S7: Significant inhibition of ex-vivo Tregexpansion when these cells are cultured in the contin-uous presence of sirolimus. Flow-sorted Tregs (left) andNon-Tregs (right) were stimulated with anti-rhesus-CD3and anti-human CD28 coated microbeads as described inMethods, in the continuous presence of increasing con-centrations of sirolimus. Cell numbers were counted onday 7, 14 and 21 and expressed as fold expansion. Thedata shown is representative of three independent experi-ments.

Please note: Wiley-Blackwell is not responsible for the con-tent or functionality of any supporting materials suppliedby the authors. Any queries (other than missing material)should be directed to the corresponding author for thearticle.


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