Antibody-mediated signaling through PD-1 costimulates T cells and enhances CD28-dependent...

Antibody-mediated signaling through PD-1 costimulatesT cells and enhances CD28-dependent proliferation

Mar�a-Luisa del Rio*1, Giovanna Penuelas-Rivas1, Raul Dominguez-Perles1,Pablo Ramirez2, Pascual Parrilla2 and Jose-Ignacio Rodriguez-Barbosa*1

1 Unit of Transplantation Research, Arrixaca University Hospital, Murcia, Spain2 Liver Transplant Unit, Department of Surgery, Arrixaca University Hospital, Murcia,Spain

Programmed death-1 (PD-1, CD279) is a molecule expressed on activated T, B andmyeloid cells. The role of the interaction of PD-1 ligands (PD-L1 and PD-L2) with PD-1receptor and the type of signals (costimulatory or inhibitory) that are delivered is asubject of intense debate. Our study has characterized two monoclonal antibodies(mAb) against murine PD-1, termed clone 1H10 and clone 4F10, that recognizeddifferent epitopes from that of anti-PD-1, clone J43. We showed that neither of theminhibited anti-CD3-mediated proliferation, but 1H10 mAb induced direct T cellproliferation in the absence of any other stimulus. Moreover, PD-1 engagement with1H10 mAb costimulated anti-CD3-mediated proliferation and enhanced anti-CD3/CD28 proliferation on both CD4+ and CD8+ T cells in the low range of anti-CD3concentrations. Anti-PD-1-mediated proliferation induced with 1H10 mAb was alsoobserved in vivo on CD4+ and CD8+ T cells, when CFSE-labeled splenocytes wereadoptively transferred to irradiated syngeneic and allogeneic recipients. Overall, ourdata indicate that PD-1might not only deliver negative signals toTcells upon interactionthrough one of its ligands, PD-L1 as reported, but also could costimulate T cells,suggesting a dual potential functional activity of the extracellular domains of thisreceptor.

Introduction

A balance of positive and negative signals regulates Tcellactivation and proliferation, which is critical for themaintenance of peripheral tolerance. Two of the best-characterized pathways of T cell activation are CD80/CD28, CD86/CD28 and CD40/CD40L interactions.Binding of CD80/CD86 to CD28 delivers costimulatorypositive signals to T cells, while the engagement ofCD80/CD86 with CTLA-4 inhibits T cell activation [1].

CTLA-4 and CD28 bind to common ligands CD80 andCD86 and the presence of soluble CTLA-4.Ig fusionprotein abrogates costimulation through CD28. Thisoccurs because CTLA-4 binds with higher affinity toCD80 and CD86 than CD28. In addition, CD28 andCD40-deficient mice show impaired T and B cellresponses [2].

PD-1 gene was isolated by subtractive hybridizationof apoptosis-induced T cell hybridoma (2B4.11) and itsmRNA was up-regulated upon in vivo administration ofanti-CD3 in dying thymocytes [3]. The same authorssoon developed an mAb (clone J43) against murine PD-1 that immunoprecipitated a heavily glycosylatedmembrane protein of 50–55 kDa. Anti-PD-1 mAb (clone

Correspondence: Jose-Ignacio Rodriguez-Barbosa, Unit ofTransplantation Research, Experimental Surgery, ArrixacaUniversity Hospital, 30120-El Palmar, Murcia, SpainFax: +34-968-369364e-mail: [email protected]

Received 2/7/05Revised 30/8/05

Accepted 13/10/05

[DOI 10.1002/eji.200535232]

Key words:Costimulation� Programmed

death-1 (PD-1, CD279)� T cell proliferation

Abbreviations: BTLA: B and T lymphocyte attenuator �CHO: carcinomahamster ovarian � PD-1: programmed death-1

Eur. J. Immunol. 2005. 35: 3545–3560 Immunomodulation 3545

f 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji.de

* Present address: Institute of Immunology, Hannover MedicalSchool, 30625 Hannover, Germany

J43) has been used extensively in in vivo models ofimmunological relevance. It blocks the binding of mousePD-L1.Ig and PD-L2.Ig to PD-1-transfected cells. Thus, invivo antibody-mediated blockade of PD-1 receptor(clone J43) in autoimmune models, such as type 1autoimmune diabetes in NOD mice [4] and experi-mental autoimmune encephalomyelitis, accelerates notonly the onset and severity of the disease [5], but alsoexacerbates graft-versus-host disease [6], contact hy-persensitivity [7], and augments antiviral immunity [8].

Programmed death-1 (PD-1, CD279) is a newmember of the growing immunoglobulin superfamily[9–11] that it is expressed on activated T, B, thymocytesand myeloid cells [9, 12]. The cytoplasmic domain ofPD-1 exhibits an immunoreceptor tyrosine-based in-hibitory motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM) that appear to be responsiblefor the negative signaling by recruiting the src homology2-domain-containing tyrosine phosphatase, SHP-2, andthus attenuating the signal 1 responsible for theinitiation of the intracellular events [10]. The inhibitoryrole attributed to PD-1 has been established to a greatextent thanks to in vivo experiments with PD-1-deficientmice. The loss of negative regulation in PD-1-deficientmice in a C57BL/6 background developed lupus-likeautoimmune disease, arthritis or glomerulonephritiswith predominant IgG3 and C3 deposition in theglomeruli [13]. PD-1-deficient mice in BALB/c back-ground developed IgG autoantibodies reactive to cardiactroponin I, a 33-kDa protein expressed on cardiomyo-cytes and dilated cardiomyopathy [14, 15]. In bothinstances, the disease appeared late in life.

PD-1 ligands, PD-L1 and PD-L2 are B7 familymembers characterized by being able to display oneextracellular IgV and one IgC domain. Unlike CD80 andCD86, whose expression is mainly restricted to lymphoidcells, PD-L1 is expressed on non-lymphoid tissues as wellas on APC. PD-L2, however, is expressed exclusively onDC and to a lesser extent on activated macrophages [16,17]. Paradoxically, the engagement of PD-1 with itsligands, B7-H1 (PD-L1) and B7-DC (PD-L2) eitherdelivers negative signals to T cells and therefore inhibitsT cell activation in a similar manner to CTLA-4 [18–23]or costimulates T cells [24–27], depending on theconditions of the experimental setting.

To gain a further insight into the type of signals thatare delivered by PD-1 receptor, hybridomas secretingmAb against murine PD-1 were developed and thefunctional activity was studied in their capacity tomodulate T cell proliferation. Unexpectedly, among theantibodies developed, we only found either antibodiesthat did not deliver inhibitory or costimulatory signals toT cells or antibodies that directly stimulated T cellproliferation or enhanced it in the presence ofsuboptimal levels of anti-CD3 or anti-CD3/CD28. Our

data suggest that PD-1 may display a dual functionalactivity and serve not only as a receptor to delivernegative signals to T cells, but it may also potentiallytransmit costimulatory positive proliferative signals.This distinct functional activity may depend on theinteraction of different extracellular domains of PD-1with its ligands.

Results

Hybridomas secretingmAb (clone 4F10 and 1H10)recognizes murine PD-1 receptor

Two anti-murine PD-1 mAb (clone 4F10 and 1H10, bothrat IgG2a, kappa light chain) were produced andcharacterized in our study using PD-1-transfectedcarcinoma hamster ovarian (CHO) cell line (termedclone A6) as the immunizing antigen in Sprague-Drawley rats. The specificity of the mAb for PD-1 wasfirst demonstrated by flow cytometry. This demon-strated that both anti-PD-1 hybridomas specificallyrecognized clone A6. In addition to this, the resultswere negative when confronted with B and T lympho-cyte attenuator (BTLA)-transfected and empty plasmidtransfected CHO cell line (Fig. 1a). The specificity of theanti-PD-1 antibodies developed in this work along withanti-PD-1 (clone J43) was also confirmed in a sandwichELISA, for which anti-PD-1 mAb were used as captureantibodies. These antibodies were capable to specificallybind to the extracellular domain of a PD-1-murineIgG2a.Fc fusion protein (Fig. 1b), but failed to react withmurine IgG2a. Thus, the specificity of the anti-PD-1 mAbwas further confirmed.

Finally, the anti-PD-1 antibody reactivity was testedagainst resting and activated murine thymocytes andsplenocytes stimulated in vitro with PMA (10 ng/mL)and ionomycin (500 ng/mL) at days 0, 1, 2 and 3.Correlating with the reported changes in PD-1 expres-sion during activation [12], low level of PD-1 receptorexpression was observed on resting T cells. Thisexpression steadily increased after polyclonal T cellactivation (Fig. 1c).

Two novel antibodies recognizing PD-1 receptor havetherefore been reported in our study and representadditional tools for the study and functional character-ization of the PD-1 receptor.

Anti-PD-1 (clone 1H10 and 4F10) mAb recognizesdifferentepitopes fromthatofanti-PD-1 (clone J43)

We next examined whether the two anti-PD-1 mAbdescribed in our study recognized the same or distinctepitopes from that of the previously described anti-PD-1(clone J43) mAb [12].

Mar�a-Luisa del Rio et al. Eur. J. Immunol. 2005. 35: 3545–35603546


Figure 1. Hybridomas secreting mAb (clones 1H10 and 4F10) recognized specifically the PD-1 receptor. (a) The flow cytometryhistograms show the specificity of biotinylated rat anti-mouse PD-1 mAb (clones 4F10 and 1H10) against CHO cells transfectedwith PD-1 (clone A6) (dashed line), BTLA (dotted line) and empty plasmid (solid line). The reaction was developed using FITC-conjugated to streptavidin. (b) Sandwich ELISA showing that plate-bound anti-PD-1 mAb (4F10 mAb, white bars; 1H10 mAb,stripped bars; J43mAb, black bars) captured the PD-1-extracellular domain-mIgG2a.Fc fusion protein,whereas isotypematched ratIgG2a (gray bars) did not. In addition, none of the anti-PD-1 mAb recognized murine IgG2a or supernatant from mIgG2a.Fc-transfected CHO cell line. (c) PD-1 receptor expression is rapidly up-regulated upon T cell activation. Flow cytometry shows thekinetic course of in vitro PD-1 expression (from day 0 to day 3) on thymocytes and splenocytes after polyclonal activation in thepresence of PMA (10 ng/mL) and ionomycin (500 ng/mL). Biotinylated rat IgG2a or biotinylated hamster IgG isotype-matchedcontrol (solid line, filled histogram) and biotin-labeled anti-PD-1 mAb (1H10 mAb, dotted line; 4F10 mAb, dashed line; J43 mAb,hyphen-dotted line) were used to stain activated thymocytes and splenocytes. The reaction was developedwith FITC-conjugatedto streptavidin.



Figure 2. Anti-PD-1 mAb (clones 1H10 and 4F10) bind to different epitopes from that of anti-PD-1 (clone J43). Flow cytometrycompetition assays of anti-PD-1mAb for binding to PD-1-transfected CHO cell line (clone A6). (a) anti-PD-1mAb (clones 1H10 and4F10) recognized different epitopes from that of anti-PD-1 mAb (clone J43). Flow cytometry histograms display competition assaybetween a saturating concentration of unlabeled J43 and biotin-conjugated 1H10 mAb (upper panel) and between a saturatingconcentration of unlabeled J43 and biotin-conjugated 4F10 mAb (lower panel). Biotinylated anti-PD-1 mAb were developed byFITC-conjugated to streptavidin. (b) anti-PD-1 mAb (clones 1H10 and 4F10) competed each other for binding to PD-1-transfectedCHO cell line (clone A6). Flow cytometry histograms display competition assay between a saturating concentration of unlabeled4F10 mAb and biotin-conjugated 1H10 mAb (upper panel) and a saturating concentration of unlabeled 1H10 mAb and biotin-conjugated 4F10 mAb (lower panel). Biotinylated anti-PD-1 mAb were developed by FITC-conjugated to streptavidin.



A flow cytometry competition assay with PD-1-transfected CHO cell line and biotin-labeled, unlabeled4F10 and 1H10 mAb as well as unlabeled J43 was used.Thus, the addition of saturating amounts of unlabeledJ43 as competitor did not inhibit the binding of eitherbiotin-conjugated 1H10 mAb or biotin-conjugated 4F10mAb, indicating that the novel antibodies described inthis report reacted with different epitopes from that ofclone J43 (Fig. 2a). In contrast, the addition ofsaturating concentrations of unlabeled 4F10(5–10 lg/mL), used as competitor, inhibited the bindingof biotin-conjugated 1H10 mAb to PD-1 receptorexpressed on PD-1-transfected CHO cells (Fig. 2b).Similar competitive inhibition was observed whenbiotin-conjugated 4F10 mAb was used in the presenceof saturating concentrations of unlabeled 1H10 mAb(5–10 lg/mL) as competitor (Fig. 2b). These resultsindicate that 4F10 and 1H10 mAb may recognize thesame or overlapping epitopes on the extracellulardomains of the PD-1 receptor.

1H10 mAb-mediated PD-1 engagement inducesCD28-independent and CD28- dependent T cellactivation and proliferation

To examine whether anti-PD-1 mAb exhibited aproliferative effect on splenocytes, an in vitro directproliferative assay with anti-PD-1 mAb was used. The1H10 mAb induced a rapid activation and proliferationthat could be visualized in the cultures within 48 h ofincubation. The 1H10 mAb also induced clustering andincreased the size of splenocytes to a much greaterextent than that observed in cultures containing 4F10mAb, J43 mAb or rat IgG2a control (Fig. 3a). Interest-ingly, 1H10-mediated activation was accompanied byCD25 up-regulation on CD4+ T cells, being thepercentage of cells stained positive for anti-CD25 of65.7% compared to rat IgG2a (13.87%), 4F10 mAb

(19.24%) and J43 mAb (31.99%), whereas the percen-tage of positive cells labeled with 1H10 mAb on thesubset of CD8+ T cells was of 90.85% compared to ratIgG2a (4.08%); 4F10 mAb (10.80%) and J43 mAb(11.24%). This suggests that in the presence of IL-2, Tcells expressing CD25 may be sensitive to IL-2-mediatedproliferation (Fig. 3b). The 1H10 mAb also induced up-regulation of the early activation antigen CD69 in bothCD4+ and CD8+ T cells upon engagement with PD-1receptor (CD4+ T cells, 86.35% and CD8+ T cells,80.94%), whereas splenocytes treated with rat IgG2a,4F10 mAb and J43 mAb only showed basal expression ofCD69 (Fig. 3c). Overall, this indicates that PD-1signaling could induce rapid T cell activation.

To determine whether CD4+ and CD8+ T cells wereequally sensitive to direct 1H10 mAb-induced prolifera-tion and that the effects exhibited by this antibody werenot mediated through APC, magnetic bead affinitypurified CD4+ and CD8+ T cells were stimulated withsoluble anti-PD-1 mAb, anti-CD28 mAb and rat IgG2a

isotype control. As shown in Fig. 3, proliferation of CD4+

(Fig. 3d) and CD8+ (Fig. 3e) T cells was enhanced byapproximately tenfold in the presence of 1H10 mAb,compared to 4F10 mAb or rat IgG2a isotype control,which only displayed background proliferative activity.Remarkably, the addition of soluble 1H10 mAb alongwith anti-CD28 mAb enhanced proliferation of bothCD4+ and CD8+ T cells to a much greater extent thanany of them separately, either 1H10 mAb or anti-CD28mAb, as displayed in Fig. 3d and e. Therefore, 1H10mAbcan stimulate directly CD28-independent and CD28-dependent T cell growth.

1H10 mAb costimulates suboptimal TCRsignaling on both CD4+ and CD8+ T cells

To assess whether anti-PD-1 mAb could signal Tcells andtherefore inhibit or augment T cell proliferation, an in

Figure 3. Rapid T cell activation and proliferation upon engagement of PD-1 with 1H10 mAb (a) 1H10 mAb induced activation,clustering and increase in size of splenocytes. Splenocytes were cultured in vitrowith the following soluble mAb: rat IgG2a isotypecontrol, 4F10mAb, J43mAband 1H10mAb. All of themwereused at 0.25 lg/well. (b) CD25 up-regulation in bothCD4+ (upper panel)and CD8+ (lower panel) T cells 48 h after 1H10-mediated activation. Splenocytes from C57BL/6 mice were stimulated in vitrowithsoluble anti-PD-1 mAb (0.25 lg/well). T cells expressing CD25 marker were stained with PE-Cychrom-conjugated to anti-CD25mAb and analyzed by FACS on gatedCD4+TcRab+ andCD8+TcRab+. (c) CD69 up-regulationwas observed in both CD4+ (upper panel)andCD8+ (lower panel) T cells 48 h after signaling through PD-1with 1H10mAb. Splenocytes fromC57BL/6micewere stimulated invitrowith soluble anti-PD-1mAb (0.25 lg/well). T cells positive for CD69were stainedwith biotin-conjugated rat anti-mouse CD69and the reaction was developed with APC bound to streptavidin and analyzed by FACS on gated CD4+TcRab+ and CD8+TcRab+. (d)1H10 mAb interaction with PD-1 receptor induced direct CD4+ T cell activation and proliferation. The presence of anti-CD28 mAbincreased 1H10 mAb-mediated proliferation. Magnetic bead affinity purified CD4+ T cells from C57BL/6 mice were stimulated invitro with soluble anti-PD-1 mAb, anti-CD28 and rat IgG2a isotype control (0.25 lg/well) for 48 h. Proliferation was determined by[3H]thymidine incorporation in triplicate wells. The data are representative of five independent experiments. (e) 1H10 mAbinteraction with PD-1 receptor induced direct CD8+ T cell activation and proliferation. The presence of anti-CD28 mAb increased1H10 mAb-mediated proliferation. Magnetic bead affinity purified CD8+ T cells from C57BL/6 mice were stimulated in vitro withsoluble anti-PD-1 mAb, anti-CD28 and rat IgG2a isotype control (0.25 lg/well) for 48 h. Proliferation was determined by[3H]thymidine incorporation in triplicate wells. The data shown are representative of five independent experiments.



vitro anti-CD3-mediated proliferative assay was per-formed using decreasing concentrations of plate-boundanti-CD3 (ranging from 3 to 0.003 lg/well) in thepresence of soluble anti-PD-1 (clone 4F10 and 1H10),anti-CD28mAb and rat IgG2a isotype control at 0.25 lg/well.

Initially, an anti-CD3-mediated proliferation assaywas performed with C57BL/6 splenocytes to define theoptimal and suboptimal amount of anti-CD3, usingconcentrations ranging from 3 to 0.3 lg/well. Thus,concentrations of anti-CD3 of 0.05 and 0.005 lg/wellwere chosen as optimal and suboptimal, respectively. Tostudy whether there was a differential effect of 1H10mAb on CD4+ and CD8+ T cells, magnetic bead affinitypurified CD4+ and CD8+ Tcells were stimulated in vitrowith optimal and suboptimal concentrations of anti-CD3and anti-CD3/CD28.

As shown in Fig. 4, in vitro costimulation of purifiedCD4+ T cells with anti-CD3 (0.05 lg/well) in thepresence of anti-CD28 did not inhibit or enhanceproliferation upon the addition to the culture of eithersoluble 4F10 mAb, 1H10 mAb or isotype control(Fig. 4a). On the contrary, suboptimal anti-CD3-mediated proliferation (0.005 lg/well) costimulatedwith soluble anti-CD28 was greatly enhanced in thepresence of soluble 1H10 mAb. However, this prolifera-tion was neither inhibited nor enhanced by the otheranti-PD-1 mAb (clone 4F10) or the isotype control(Fig. 4b).

Similarly to CD4+ T cells, in vitro costimulation ofCD8+ T cells with optimal concentration of anti-CD3(0.05 lg/well) in the presence of anti-CD28 did notaugment CD8+ T cell proliferation when soluble 1H10mAb was added to the culture (Fig. 4c). However, undersuboptimal anti-CD3-mediated proliferation (0.005 lg/mL) or anti-CD3/CD28 costimulation, CD8+ T cellproliferation was notably increased in the presence ofsoluble anti-PD-1 mAb (clone 1H10), whereas the otheranti-PD-1 (clone 4F10) and the rat isotype control didnot affect T cell proliferation (Fig. 4d).

Overall, the detailed analysis of our data stronglysuggest that signaling through PD-1 with 1H10 mAb cancostimulate suboptimal anti-CD3-mediated prolifera-tion, and augment CD28-dependent CD4+ and CD8+ Tcell proliferation.

PD-1 triggering through 1H10 mAb boostedgreater IFN-c secretion on CD8+ than on CD4+ Tcells

Previous studies have shown that while human B7-H1.Igfusion protein stimulated the secretion of IL-10 andIFN-c, only negligible amounts of IL-2 and IL-4 weredetectable [25]. To assess whether the anti-PD-1 mAb(clone 1H10) stimulated the secretion of IL-2 and IFN-c

directly on T cells or could costimulate anti-CD3 or anti-CD3/CD28-mediated proliferation, the levels of thesecytokines were quantified.

The production of IFN-c was not affected on T cellsdirectly activated with 1H10 mAb alone (data notshown). IFN-c secretion was however significantlyaugmented in CD8+ T cells compared to CD4+ T cellswhen suboptimal doses of pre-coated anti-CD3 wereused (p < 0.0005) (Fig. 5a).

IFN-c secretion was also significantly enhanced insuboptimally anti-CD3 stimulated CD4+ T cells. Thesewere double-treated with soluble 1H10 mAb and anti-CD28 compared to rat isotype control (p < 0.0005) and1H10 mAb (p < 0.05) (Fig. 5b). Similarly to CD4+ Tcells, the increase in IFN-c production in CD8+ T cellswas only demonstrated after the combined addition ofboth soluble 1H10 mAb and anti-CD28 to suboptimallyanti-CD3-activated T cells when compared to 1H10 mAb(p < 0.0005), anti-CD28 (p < 0.0005) and rat isotypecontrol (p < 0.0005) (Fig. 5c).

IL-2 production was not, however, significantlyincreased in CD4+ T cells directly activated with 1H10mAb (Fig. 5d) or in CD4+ T cells stimulated with lowdoses of anti-CD3 and/or anti-CD28 (Fig. 5e).

These results indicate that CD28-dependent costi-mulation of T cells under suboptimal TCR signaling isboosted by signaling through PD-1. This results in thesecretion of IFN-c on both CD4+ and CD8+ T cells, butdoes not affect the secretion of IL-2 by CD4+ T cells.

In vivo 1H10 mAb administration mediatesantigen-dependent and independent T cellproliferation

To characterize further in vivo activation and expansionof individual CD4+ and CD8+ T cells mediated by anti-PD-1 mAb, an adoptive transfer experiment wasdesigned and CFSE dye was used to track T cell division.Thus, 30 � 106 of CFSE-labeled C57BL/6 splenocyteswere adoptively transferred i.v. to lethally irradiatedsyngeneic (C57BL/6) and allogeneic (BALB/c) recipi-ents. The number and the size of peaks of CD4+ andCD8+ T cell division were evaluated 60 h after adoptivetransfer.

As expected, CFSE-labeled CD4+ T cells did notproliferate in lethally irradiated syngeneic recipientsthat received either purified rat IgG control or 4F10mAbat 60 h after adoptive transfer. In contrast, 1H10 mAb-treated mice showed a marked CD4+ T cell proliferationand up to six peaks of T cell division were observed,indicating that 1H10 mAb directly stimulated syngeneicCD4+ T cells independently of the presence of TCRsignaling (Fig. 6a). When CFSE-labeled splenocyteswere transferred to allogeneic recipients, CD4+ T celldivision was much more significantly enhanced in 1H10



mAb-treated mice (up to eight peaks of T cell divisionwere observed) than in either purified rat IgG control or4F10-treated mice (Fig. 6b). Moreover, an increasednumber of cells in the less fluorescent peaks weredetected in 1H10 mAb-treated allogeneic recipients(Fig. 6b) compared to syngeneic recipients (Fig. 6a).This indicates that in the presence of alloantigen, 1H10mAb-treated mice show an increased CD4+ T celldivision rate compared to purified rat IgG control and4F10 mAb-treated mice. Therefore, in vivo administra-tion of 1H10 mAb and cross-linking of PD-1 receptor inthe presence of an allogeneic stimulus leads to anincrease of CD4+ T cell proliferation.

Similarly, CFSE-labeled C57BL/6 splenocytes adop-tively transferred to lethally irradiated syngeneicrecipients, showed that CD8+ T cells underwent threesmall peaks of proliferation in purified rat IgG controland in 4F10 mAb-treated mice, whereas in 1H10 mAb-treated mice, CD8+ T cell division was markedlyenhanced. Furthermore, the majority of cells underwentfour to five rounds of division (Fig. 6c). The rate of CD8+

T cell division was also slightly higher in 1H10 mAb-treated allogeneic recipients compared to purified ratIgG and 4F10 mAb-treated mice. Thus, 1H10 mAb-treated allogeneic recipients showed one more peak ofCD8+ T cell division (Fig. 6d) when compared to 1H10

Figure 4. CD28-dependent and independent T cell proliferation upon engagement of PD-1 with 1H10 mAb under suboptimal TCRsignaling. Affinity purified CD4+ T cells were stimulated in vitro with plate-bound (a) anti-CD3 (0.05 lg/well) and (b) anti-CD3(0.005 lg/well) in the presence of soluble anti-PD-1 mAb, anti-CD28 and rat isotype matched control at 0.25 lg/well. Arepresentative experiment of three with similar results is shown. Affinity purified CD8+ T cells were also stimulated in vitrowithplate-bound (c) anti-CD3 (0.05 lg/well) and (d) anti-CD3 (0.005 lg/well) in the presence of soluble anti-PD-1mAb, anti-CD28 and ratisotype matched control at 0.25 lg/well. A representative experiment of three with similar results is shown. Proliferation wasdetermined by [3H]thymidine incorporation in triplicate wells.



mAb-treated syngeneic recipients (Fig. 6c). This resultsuggests that in the presence of alloantigen, signalingthrough PD-1 receptor with 1H10mAb slightly increasesthe rate of CD8+ T cell division.

Discussion

The balance between activating and inhibiting signalsthat a T cell receives upon engagement with antigendefines the outcome of an immune response. PD-1 andBTLA are two members of the CD28 family that havebeen recently incorporated [9, 28, 29] into theincreasing number of molecules involved in T cellregulation.

This study has characterized two novel rat anti-murine IgG2a anti-PD-1 mAb that recognized distinct

epitopes from that of clone J43 and has furthermoreevaluated the in vitro and in vivo functional conse-quences of antibody-mediated engagement of PD-1receptor. J43 mAb is a blocking hamster IgG antibodythat does not deliver signals to T cells. In addition, its invivo activity has been claimed to derive from theblocking ability to prevent the binding of the PD-1ligands to PD-1 molecule [12]. Engagement of the PD-1receptor by 4F10 mAb reported in this work deliveredneither negative nor positive signals to T cells, compar-able to the results of a previous study dealing with anti-PD-1 mAb (clone J43) [12]. Therefore, these twoantibodies might act uniquely in preventing the bindingof PD-1 ligands to PD-1 receptor. In sharp contrast, thesecond anti-PD-1 mAb characterized in our work, clone1H10, was capable to directly activate and costimulate Tcell proliferation. It is also interesting to remark that

Figure 5. CD28-dependent costimulation of T cells synergizes with PD-1 engagement and stimulates greater secretion of IFN-c inCD8+ than in CD4+T cells. This, however, does not affect the secretion of IL-2 by CD4+ T cells. (a) IFN-c secretion was significantlyaugmented on CD8+ T cells compared to CD4+ T cells in the supernatant of low dose (0.005 lg/well) anti-CD3-stimulated CD4+ andCD8+ T cells in the presence of 1H10 and anti-CD28mAb (p< 0.0005). (b) IFN-c secretionwas significantly increased on CD4+ T cellswhen treatedwith 1H10mAband anti-CD28 (*) compared to rat IgG2a isotype (^) (p< 0.0005) and 1H10mAb (~) (p< 0.05). (c) IFN-csecretionwas also significantly augmented in CD8+ T cells stimulatedwith 1H10mAb and anti-CD28 (*) compared to rat IgG2a (^)(p< 0.0005), CD28 (&) (p< 0.0005), or 1H10mAb (~) (p< 0.0005). (d) IL-2 secretionwas neither significantly increased in CD4+ T cellsstimulated directly with 1H10 (~) or 1H10 plus anti-CD28 (*) mAb (e) nor when CD4+ T cells were stimulated with the sameantibodies in the low range of anti-CD3 concentration. All the soluble antibodies were added at 0.25 lg/well. Supernatants werecollected 48 h after T cell stimulation and the concentrations of IL-2 and IFN-c were determined by quantitative sandwich ELISA.Data depict one representative experiment of three with similar results.



Figure 6. In vivo administration of 1H10mAb induced greater T cell proliferation of CD4+ than CD8+ T cells. Increased rate of CD4+ Tcell division in lethally irradiated 1H10mAb-treated C57BL/6 syngeneic (a) and BALB/c allogeneic (b) recipients 60 h after adoptivetransfer of CFSE-labeled C57BL/6 splenocytes was observed compared to purified rat IgG control and 4F10 mAb groups. A slightincrease of CD8+ T cell division in lethally irradiated 1H10 mAb-treated syngeneic (c) and allogeneic (d) recipients 60 h afteradoptive transfer of CFSE-labeled splenocytes was observed compared to purified rat IgG and 4F10 mAb groups. CFSE-labeledlymphocyteswere gated on TcRab+CD4+ and TcRab+CD8+ and double positive cellswere analyzed in the FL-1 channel to assess thepeaks of division. Dead cells positive for propidium iodide were excluded from the analysis. The histograms shown are from twoindependent experiments with similar results.



although the two anti-PD-1 mAb (clone 1H10 and 4F10)characterized displayed mutual competition for bindingto PD-1 receptor, the biological consequences ofengaging PD-1 receptor with 4F10 mAb or 1H10 mAbwere completely distinct. Thus, the addition of soluble1H10 mAb costimulated anti-CD3 and anti-CD3/CD28-mediated proliferation, whereas 4F10mAb did not affectthis type of proliferative activity. Overall, despite the twoanti-PD-1mAb competing each other for binding to PD-1receptor, the data provided in this work supports thehypothesis that they may recognize overlapping, but notidentical epitopes, as the biological consequences oftriggering PD-1 receptor with one or the other arecompletely different. Another straightforward interpre-tation for the observed mutual competitive inhibition ofanti-PD-1 mAb developed in our work would be toaccept that the binding of one of the mAb to PD-1 wouldinterfere with the binding of the other mAb to the samemolecule and vice versa by simply steric hindrance. Thismay be because antigenic determinants recognized byclones 1H10 and 4F10 overlap and therefore are closelylocated on the three-dimensional native structure of PD-1 molecule. Additional experimental evidence support-ing the hypothesis that the two anti-PD-1 mAb describedin the present work recognized different epitopesoriginates from the observation that the reactivity of1H10 mAb in capture ELISA with a soluble form of theextracellular domain of PD-1-mIgG2a.Fc fusion proteinwas considerably reduced compared to the ability ofbinding of the others anti-PD-1 mAb (clones 4F10 andJ43). The data suggest that 1H10 mAb may recognize aconformational sensitive antigenic determinant.Although speculative, this epitope might be formed byAA sequences located far apart in the primary AAsequence. These sequences are brought close togetheron the surface of the native molecule by its folding inthree dimensions (assembled topographical epitope),whereas the conformational antigenic determinantrecognized by 4F10 mAb might only depend on three-dimensional structure of the native protein, and not ontwo different AA sequences brought together [30].

Rapid T cell activation and subsequent proliferationobserved after direct stimulation with 1H10 mAb ofpurified CD4+ and CD8+ Tcells was accompanied by up-regulation of the a chain of the IL-2 receptor, but nosignificant secretion of IL-2 was detected. Theseobservations are in agreement with previous dataclaiming that B7-H1.Ig costimulation only induced asmall production of IL-2 on human [25] and murine Tcells [31]. T cell receptor-mediated activation andcostimulation leads to IL-2 receptor up-regulation andIL-2 production, which in turn acts as an autocrine factorto support T cell proliferation. CD4+ Tcells appear to bethe major producers of IL-2, whereas CD8+ T cellsappear to be the major consumers of this cytokine [32].

IL-2 is often difficult to measure in culture because it iseasily consumed by the T cell population that producesit. The lack of IL-2 production observed in vitro inculture supernatant of purified CD4+ and CD8+ T cells,which were stimulated with a suboptimal concentrationof anti-CD3 and anti-CD28 costimulation and in thepresence of 1H10 mAb, contrasted with the highproliferative activity of T cells detected. To account forthis paradoxical observation, we hypothesize that PD-1costimulation may be able to promote Th1 and cytotoxicCD8+ T cell differentiation. The IL-2 produced wouldtherefore be consumed by either CD4+ Th1 or CD8+ PD-1-activated T cells. This argument is supported by theobservation that one of the PD-1 ligands, PD-L2,costimulates T cells. Consequently, PD-L2 KO micedisplayed reduced IFN-c production by CD4+ T cells,reduced IFN-c-dependent humoral immune responses,and diminished CTL responses [33].

The type of signals that are delivered by engagementof PD-1 receptor is the subject of enormous controversy.An abundant number of recent reports claimed that PD-1 delivers negative signals to T cells in vitro as well as invivo in several immunological processes [10, 28, 34–35].This experimental evidence has been gathered fromexperiments targeting PD-1 receptor with PD-1 ligandsfusion protein in transplantation [36] or by blocking PD-1 receptor with J43 mAb in autoimmune processes [11],GVH disease [6] and viral infection models [8]. PD-L1-deficient mice display markedly enhanced T cellproliferation [37] and autoimmunity is also acceleratedby blocking PD-L1 but not PD-L2 [4]. Despite all thecompelling evidence in favor of the fact that PD-1delivers negative signals toT cells, there are a number ofreports that claim the opposite. The first reports aimedat characterizing PD-L1 suggested that this ligandcostimulated both human [25] and murine [31] T cellsrather than inhibiting their function, either throughdirect engagement of PD-1 or acting on alternative notyet well-defined costimulatory receptors. In line withthese findings, PD-L1 has been reported to costimulate Tcell proliferation and cytokine secretion [25] andpreferentially enhance CD28-independent T-helper cellfunction [31]. In addition, this transgenic expression ofPD-L1 in islets accelerated the onset and course ofdiabetes in TCR transgenic mice [38]. Some authorshave even put forward a more challenging view of thecomplexity of PD-1 and PD-1 ligand interactionsuggesting that the costimulatory activity of PD-1ligands would be via receptors other than PD-1 [39].Thus, PD-L1 mutants that did not bind to PD-1 were ableto costimulate T cell proliferation and cytokine produc-tion with or without the expression of PD-1 on T cells orin the presence of soluble PD-1.Ig fusion protein [26].

Similarly to PD-L1, the type of signals delivered to Tcells by PD-L2 is also a subject of avid debate and



controversy. Only a few number of reports put forwardthat PD-L2 interaction with PD-1 receptor deliverednegative signals [20, 40]. The majority of experimentaldata reported so far came to the conclusion that PD-L2interaction with PD-1 triggers positive stimulatorysignals to T cells. PD-L2 deficient mice for exampleshowed diminished IFN-c production by CD4+ T cellsand reduced IFN-c -dependent humoral and CTLresponses [33]. PD-L2 also increased T cell proliferationand cytokine production in vitro under suboptimal TCRsignaling [24] and in vivo asthma models [41]. In linewith these observations, 1H10 mAb-mediated signalingthrough PD-1 induced a strong release of IFN-c on bothCD4+ and CD8+ T cells in a CD28-dependent fashion.

The data presented in this work is in agreement withthose reports claiming that PD-1 receptor may partici-pate in delivering positive costimulatory signals toTcells[25, 31, 33]. Thus, in vitro signaling through PD-1 withanti-PD-1 mAb (clone 1H10) costimulated CD3-mediated proliferation on both CD4+ and CD8+ T cellsin the presence of suboptimal concentrations of anti-CD3. This proliferation observed was further enhancedafter in vitro costimulation of T cells with anti-CD28.

CD8+ T cells divide more rapidly than CD4+ T cellswhen adoptively transferred to syngeneic irradiatedrecipients [42]. As expected, irradiation and homeo-static proliferation did not increase the CD4+ T celldivision rate in the 60 h assay used in our study neitherin control rat IgG nor in 4F10 mAb-treated syngeneicrecipient mice. However, three small peaks of divisionwere observed for CD8+ T cells when transferred tosyngeneic rat IgG control and 4F10 mAb-treatedrecipient mice. Both CD4+ and CD8+ Tcells proliferatedupon adoptive transfer to allogeneic recipient mice in ratIgG control and 4F10-treated mice (up to eight smallpeaks of T division were observed), however CD4+ Tcellproliferation was more prominent than CD8+ T cellproliferation in 1H10 mAb-treated allogeneic recipients.This indicates that in the presence of alloantigen, thecostimulation provided by PD-1 engagement demon-strated a more prominent T cell division rate and likelyCD4+ T cell differentiation than in CD8+ T cells.

In conclusion, our findings provide further experi-mental evidence to sustain the hypothesis that PD-1receptor might be a potential target to deliver positivecostimulatory signals to T cells.

Materials and methods

Mice and rats

Three-month-old female Sprague-Drawley rats, six to eightweek-old female BALB/c (H-2d) and C57BL/6 (H-2b) micewere used in our experiments. All rodents were purchasedfrom Charles River Laboratories (Spain) and the Central

Animal Facility of Murcia University and used for theimmunizations, in vivo, and in vitro experiments. Rodentswere housed under standard conditions with controlled light/dark cycles and had free access to water and rodent chow. Allexperiments with rodents were handled and cared for inaccordance with the European Guidelines for Animal Care andUse of Laboratory Animals.

Murine PD-1 PCR amplification, cloning, and celltransfection

Six to eight weeks-old C57BL/6 femalemice received 0.5 mL ofanti-CD3 mAb (clone 145–2C11, Armenian hamster IgG1)sterile tissue culture supernatant i.p. and thymocytes werecollected 2 days later. Total thymocyte RNA was extractedusing RNeasy Mini Kit (Qiagen, MA) and reverse transcriptionof the mRNA to cDNA was carried out with the GeneAmp RNAPCR Kit (Applied Biosystems). Nested PCR was performed forthe amplification of murine PD-1 using the external andinternal primers indicated in Table 1. Isolation of DNA, PCR,recovery of DNA fragments from agarose gels and agarose gelelectrophoresis were performed following standard techniquesas recommended by the commercial suppliers (Qiagen). Thecomplete murine PD-1 gene was amplified without the stopcodon, which was removed from the internal reverse primer.The internal forward primer was designed with a Kozaksequence before the start codon (Table 1). This PCR productwas then cloned in the eukaryotic expression vectorpcDNA3.1/V5-Hisf TOPOJ (Invitrogen, Life Technologies)following the instructions of themanufacturer. The sequencingof the amplified PCR product was performed at the DNASequencing Core Facility of “Sistemas Gen�micos” (Valencia,Spain) by using a capillary Beckman CEQ 2000 XL sequenceraccording to the manufacturer's instructions. The PD-1sequences obtained were analyzed and compared with thoseavailable at the GeneBank (NM_008798) using the BLASTcomputer program from the National Center for BiotechnologyInformation (www.ncbi.nlm.nih.gov/BLAST) to verify that theamplified murine PD-1 sequence was correct. The PD-1construct was then electroporated into competent E. coli(TOP 10 strain) and the plasmid DNA was purified usingendotoxin free Maxiprep kit (EndoFree Plasmid Maxi Kit,Qiagen) for the transfections.

Adherent CHO cell line was cultured in 6-well plates withcomplete RPMI 1640 (BioWhittaker, Cambrex, Belgium) untilcell growth reached 70% confluence. Before transfection, CHOcells were washed with additive free medium. The transfectionmixture composed of 2 lg of plasmid DNA containing the PD-1construct, 6 lL of lipofectamine (Invitrogen) and 200 lL ofOPTI-MEM (Biowhittaker) medium per well was added(Invitrogen) [43].

Transient transfectants were selected in the presence ofG418 (1 mg/mL) (Invitrogen) and two limited dilution cloningrounds were performed to isolate stable positive clones. Toselect those clones with the highest level of murine PD-1expression, an immunodot procedure using horseradishperoxidase (HRPO)-labeled anti-V5 mAb (Invitrogen, LifeTechnologies) was carried out.



Cloning and expression of PD-1 extracellular domain asa fusion protein

A 504-bp fragment of the extracellular domain of murine PD-1,excluding the signal peptide sequence, was PCR amplifiedusing oligonucleotides that appended unique Hind III andBamH I restriction sites onto the 50 and 30 ends. pcDNA 3.1plasmid containing the complete sequence of murine PD-1 wasused as a template (Table 1). The PCR PD-1 amplified sequencewas cloned into the pCR2.1 vector (Invitrogen), excised usingHind III and BamH I (New England Biolabs) restrictionenzymes and was cloned in frame in pSecTag2 Hygro variant bvector opened with the same set of enzymes. The ligationreaction was performed using T4 DNA ligase (Roche). Then,the region of the Fcc2a encoding the hinge, CH2, and CH3domains of the heavy chain was amplified by PCR usingnucleotides designed to append BamH I and Not I restrictionsites on the 50 and 30 ends using the murine Fcc2a fragmentcloned in pCR2.1 vector kindly provided by Dr. Terry Strom(Harvard University) as template. The PCR product wasdigested with BamH I and Not I restriction enzymes, purifiedby gel extraction kit (Qiagen) and finally ligated in frame nextto the PD-1 extracellular domain previously cloned inpSecTag2 Hygro variant b (Invitrogen). The correct openreading frame of the whole construct was confirmed by DNAsequencing.

The plasmid pSecTag2 Hygro encoding the extracellulardomain of PD-1 bound to the Fcc2 fragment was purified usingendotoxin-free Maxi-prep (Qiagen) and 2 lg/well were usedfor lipofectamine-mediated transfection of CHO cells seeded in6-well plates. The transfected CHO cell line was selected in the

presence of 1 mg/mL of Hygromycin (Roche) for a week.Subsequently, we carried out two limiting dilutions rounds toselect those cell lines with the greatest expression of theprotein of interest. A sandwich ELISA, using anti-murine IgG2a

capture antibody (clone R11–89, rat IgG1) and biotin-conjugated rat anti-mouse IgG2a detection antibody (cloneR19–15, rat IgG1), was used for the screening and selection ofhigh producing clones. HRPO-streptavidin was finally used todevelop the reaction. A similar sandwich ELISA was applied toconfirm the specificity of the anti-PD-1 mAb. Thus, anti-PD-1mAb characterized in our study were used as captureantibodies to confirm that they specifically recognized theextracellular domain of PD-1.mIgG2aFc fusion protein.

Immunization, fusion, and screening of the mAb

Three female Sprague-Drawley rats (8 weeks old) wereimmunized i.p. with 0.5 mL of a 1:1.2 mixture of 20 � 106

of murine PD-1-transfected CHO cells and Freund0s completeadjuvant (Sigma). Sixty days after the primary immunization,rats were boosted with 10 � 106 of murine PD-1 transfectedCHO cells suspended in sterile saline. These were consequentlyintravenously injected and the fusion was performed 3 dayslater [44, 45]. The fusion protocol has been describedpreviously [46]. In brief, 20�106 of non-Ig-secretingmyelomaP3 X63Ag8.653 cells [47] were fused with 50 � 106 ratsplenocytes in the presence of polyethylene glycol 1500(Sigma) in serum-free RPMI 1640. The fused cells were platedin ten 96-well plates (Costar, Cambridge, MA) and incubatedfor 24 h in complete RPMI 1640 medium containing 15% fetalcalf serum (Gibco), 2 mM glutamine (BioWhittaker), 1 mM

Table 1. Plasmids and primers used in this study



sodium pyruvate (Sigma), 10 mM HEPES (Sigma), 1 � MEMnonessential amino acids (Sigma), 100 U/mL of penicillin(BioWhittaker), 100 lg/mL of streptomycin and 5 � 10–5 M 2-mercaptoethanol (Sigma).

On the following day, hypoxanthine, aminopterin andthymidine (HAT) selective medium (Sigma) was added. After7 days of selection, HAT medium was substituted byhypoxanthine and thymidine (HT) medium. Finally, atday 16 post-fusion, culture supernatant was screened for thepresence of specific antibodies against murine PD-1 specificantibodies by flow cytometry. Hybridomas secreting antibodiesthat recognized the PD-1-transfected CHO cell line, but not thecontrol BTLA-transfected CHO and emptied plasmid trans-fected CHO cell line were selected for more detailed study .

Antibodies and flow cytometry

Hybridomas secreting anti-PD-1 mAb (clones 1H10 and 4F10)were gradually adapted in order for them to grow in serum-free Ultradoma (BioWhittaker) medium. The cells were thentransferred to BD cell line culture bottles (Becton Dickinson,Mountain View, CA) for large-scale production. The mAb-enriched culture supernatant was precipitated with ammo-nium sulfate, dialyzed against saline solution and bindingbuffer. The anti-PD-1 antibodies were purified by protein G-Sepharose affinity chromatography (Amersham Biosciences)and subsequently labeled with biotin (Sulfo-NHS-Biotin,Pierce) following manufacturer's instructions. Commercialbiotinylated anti-murine PD-1 (clone J43, Hamster IgG,eBiosciences) was also used as internal standard control tocompare the kinetics of PD-1 expression on thymocytes andsplenocytes with our anti-PD-1 mAb [12]. Streptavidin-conjugated FITC was then used to develop biotin-labeledanti-PD-1 mAb. These antibodies were used in the competitionassays and flow cytometry experiments. The isotype of theimmunoglobulin secreted by the anti-PD-1 mAb was deter-mined using a rat mAb isotyping kit (Serotec, UK).

The following azide-free/low endotoxin antibodies wereused in the proliferative assays: rat IgG2a isotype control (cloneR35–95), anti-CD3 (clone 2C11), anti-CD28 (clone 37.51) (allof them from PharMingen). Protein G affinity purified anti-PD-1 mAb (clones 1H10 and 4F10) were prepared in ourlaboratory as described above.

One-color flow cytometry was used for the screening of therat-mouse heterohybridomas secreting antibodies againstmurine PD-1. Tissue culture supernatants containing anti-murine PD-1 secreting hybridomas were collected at day 16post-fusion and incubated for 30 min with PD-1-transfectedCHO, BTLA-transfected CHO, empty plasmid-transfected CHOand non-transfected CHO cell line. Cells were subsequentlywashed once and incubated with an optimal dilution of a FITC-labeled goat F(ab0)2 anti-rat IgG (H+L) (Caltag, Burlingame,CA). After this period, cells were finally washed and acquiredin a FACScan cytometer (Becton Dickinson).

The kinetic course of PD-1 expression on CD4+ and CD8+ Tcells was followed after in vitro stimulation of thymocytes andsplenocytes with 10 ng/mL of PMA (Sigma) and 500 ng/mL ofionomycin (Sigma) at days 0, 1, 2 and 3.

To investigate whether 1H10-mediated proliferation wasaccompanied by IL-2 receptor (a chain) up-regulation on Tcells, splenocytes were activated in vitro with anti-PD-1 mAb(0.25 lg/well) and stained with phycoerythrin-Cychrom(PECy)-labeled anti-CD25 (clone PC61), biotinylated labeledanti-CD69 (clone H1.2F3) and PE-labeled anti-TCR b chain(clone H57). Ten thousand events of CD4+TcRab+ andCD8+TcRab+ double positive cells were gated and the levelof CD25 and CD69 expression was determined.

In order to track T cell division after adoptive transfer ofCFSE-labeled splenocytes, two-color flow cytometry wasperformed and the following fluorochrome-labeled mAb fromPharMingen were used: PE-labeled rat anti-mouse CD4 (cloneRM4–5) and PE-labeled rat anti-mouse CD8 (clone 53–6.7).Nonspecific FccR binding was blocked with saturated amountsof rat anti-mouse CD16/CD32 mAb and (clone 2.4G2) [48].Dead cells were excluded by propidium iodide. Flow cytometryacquisition was carried out on a FACScan cytometer and tenthousand gated events were collected from each sample. Dataanalysis was performed using WinList, version 5.0 (VeritySoftware House, Inc., Topsham, ME).

Competition assays

The competition assay for topographical epitope mapping is amethodology that allowed us to determine whether twoantibodies raised against certain molecule recognize the same,overlapping or distinct epitopes. For that purpose, all possiblepairs of biotin-labeled and unlabeled anti-murine PD-1 mAbwere assessed by flow cytometry. Thus, decreasing concentra-tions (ranging from 10 to 0.1 lg/mL) of unlabeled anti-PD-1(clone J43) were used as competitor antibody, which wasincubated with PD-1-transfected CHO cell line (clone A6).After 20 min of incubation, saturating amounts of biotiny-lated-4F10mAb and biotinylated-1H10 mAbwere added to thecell suspension and incubated 30 min on ice. Finally, the cellswere washed in Hank's balanced salt solution (HBSS) and thereaction was developed by adding FITC-conjugated tostreptavidin. A similar approach was used to determinewhether 4F10 mAb and 1H10 mAb recognized the same ordistinct epitopes.

T cell proliferation assays and analysis of cytokineproduction

Murine splenocytes from C57BL/6 mice were harvested andred cells were lysed in ammonium chloride potassium (ACK)lysing buffer (BioWhittaker). The cells were then resuspendedin complete RPMI 1640 medium supplemented with additivesand 10% v/v of FCS with low mitogenic activity. CD4+ andCD8+ T cells were purified by positive selection with magneticbeads (Miltenyi Biotech) (purity was higher than 90% by flowcytometry). For in vitro direct proliferation assay, splenocytes(2 � 105 cells/well) and purified CD4+ and CD8+ T cells (1 �105 cells/well) were cultured in U-bottom 96-well plates in thepresence of soluble anti-PD-1 mAb characterized in this work,as well as anti-CD28, and rat IgG2a isotype control, all of themat 0.25 lg/well.

To determine the suboptimal concentrations of anti-CD3-mediated proliferation, splenocytes (2 � 105 cells/well) were



stimulated in vitro with decreasing concentrations of pre-coated anti-CD3 (ranging from 3 to 0.003 lg/well) in thepresence of soluble anti-PD-1 mAb and rat IgG2a isotypecontrol at 0.25 lg/well.

Purified CD4+ and CD8+ T cells were incubated with twoconcentrations of plate-bound anti-CD3 (0.05 and 0.005 lg/well) in the presence of soluble anti-PD-1mAb, anti-CD28mAband rat isotype-matched control, all of them at 0.25 lg/well.

Plates were pulsed after 48 h of incubation with 1 lCi of[3H]thymidine per well. Sixteen hours later, the plates wereharvested using a Skatron collection system and thymidineincorporation counted on a Pharmacia LKB Betaplate. Themean and SD was calculated from four wells.

Supernatants were harvested immediately before [3H]thy-midine was added to the culture. IL-2 and IFN-cwere detectedin culture supernatants with an ELISA kit according to themanufacturer's instructions (R&D Systems, Minneapolis, MN).The limit of detection for IL-2 and IFN-cwas 31.2 and 18.8 pg/mL, respectively.

Adoptive transfer of CFSE-labeled splenocytes tosyngeneic and allogeneic recipients

In vivo CFSE proliferation assays for tracking of adoptivetransferred T cells have been described in previous studies[49,50]. Splenocytes were resuspended in HBSS at aconcentration of 1�107 cells/mL and labeled with 5 lM CFSE(Molecular Probes, Eugene, OR) for 15 minutes at 37� C in thedark. The labeling process was stopped by the addition ofcomplete RPMI 1640 10% FCS medium. The cells were thenresuspended in saline and injected i.v. into lethally irradiated(800 rad) syngeneic and allogeneic recipients that were treatedintraperitoneally with one dose of 2 mg of antibody per mouseof purified rat IgG, 4F10 mAb and 1H10 mAb at day 0. Sixtyhours after adoptive transfer, mice were euthanized andspleens and mesenteric lymph nodes were collected. Tomonitor CD4+ and CD8+ T cell proliferation, CFSE+CD4+

and CFSE+CD8+ were gated and the peaks of division weredocumented in FL-1 histograms.

Statistical analysis

Excel and Prophet Version 5.0 software were used to performthe statistical analyses. A Student's t-test was used to determinethe significance of differences in means. A p value of less than0.05 was considered significant.

Acknowledgements: We would like to thank Dr. RocioAlvarez (Head of the ImmunologyDepartment, ArrixacaUniversity Hospital) for facilitating our access to theflow cytometry and radioactive facility. This work hasbeen supported by a grant associated to FIS contract(Fondo de Investigaciones Sanitarias) # 01/3026 and theSpanish Transplantation National Network # C03/03.Mar�a Luisa del Rio and Raul Dominguez-Perles weresupported by a postdoctoral and predoctoral fellowshiprespectively from the “Fundaci�n S�neca”, MurciaRegion, Spain. We specially would like to thank Prof.Dr. Reinhold F�rster (Institute of Immunology, Han-

nover Medical School) for his help and advice and Dr.Mireia Guerau de Arellano (Joslin Institute for DiabetesResearch, Harvard Medical School) for her criticalreading of the manuscript and Miss Natalie Evans forassistance with the English language.

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