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DependentSupereffector CD8 T Cells That Become IL-7Pathway to Induce Production of CD134 Costimulation Couples the CD137

VellaCroft, Byoung S. Kwon, Robert S. Mittler and Anthony T. Seung-Joo Lee, Robert J. Rossi, Sun-Kyeong Lee, Michael

http://www.jimmunol.org/content/179/4/2203doi: 10.4049/jimmunol.179.4.2203

2007; 179:2203-2214; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/179/4/2203.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2007 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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CD134 Costimulation Couples the CD137 Pathway to InduceProduction of Supereffector CD8 T Cells That Become IL-7Dependent1

Seung-Joo Lee,* Robert J. Rossi,* Sun-Kyeong Lee,† Michael Croft,‡ Byoung S. Kwon,§

Robert S. Mittler,¶ and Anthony T. Vella2*

The TNFR superfamily members 4-1BB (CD137) and OX40 (CD134) are costimulatory molecules that potently boost CD8 andCD4 T cell responses. Concomitant therapeutic administration of agonist anti-CD137 and -CD134 mAbs mediates rejection ofestablished tumors and fosters powerful CD8 T cell responses. To reveal the mechanism, the role of CD137 expression by specificCD8 T cells was determined to be essential for optimal clonal expansion and accumulation of effector cells. Nonetheless, dualcostimulation induced production of supereffector CD8 T cells when either the specific T cells or the host alone bore CD137.Perhaps surprisingly, the total absence of CD137 prevented anti-CD134 augmentation of supereffector differentiation demon-strating an unappreciated link between these related pathways. Ultimately, it was reasoned that these powerful dual costimulatoryresponses involved common � family members, and we show substantial increases of CD25 and IL-7R�-chain expression by thespecific CD8 T cells. To investigate this further, it was shown that IL-7 mediated T cell accumulation, but importantly, a gradualand preferential effect of survival was directed toward supereffector CD8 T cells. In fact, a clear enhancement of effector differ-entiation was demonstrated to be proportional to the increasing amount of IL-7R� expression by the specific CD8 T cells.Therefore, dual costimulation through CD137 and CD134 drives production and survival of supereffector CD8 T cells through adistinct IL-7-dependent pathway. The Journal of Immunology, 2007, 179: 2203–2214.

T he role of TNFR superfamily members for T cell costimu-lation has received a great deal of attention, especially intheir ability to potently stimulate effector T cells, impact

tolerance, and deliver long-term survival signals to specific T cells(1). In their own right, signaling through CD137 (4-1BB) andCD134 (OX40) have been shown to support very similar responsesalthough not identical. CD134 is expressed early after CD4 T cellactivation and mediates induction of effector T cell differentiation(2–4). Recent reports have also shown that CD134 can costimulateT regulatory cells (5), influence bystanding cells, and also facilitateaccumulation of effector T cells in pulmonary tissue (6, 7). Inmuch the same way, stimulation of CD137 induces very similareffects in populations of peptide-specific CD8 T cells (1, 8, 9). Inparticular, CD137-stimulated T cells display enhanced cytotoxicityand cytokine production (1, 8, 10), while at the same time endow-ing CD8 T cells to persist for long periods of time (9, 11–13), aswell as developing suppressor function (14–19). Nevertheless,both costimulatory pathways can overlap in their effects on CD4and CD8 T cell subpopulations (10, 12, 20–22).

Previously, we demonstrated that certain combinations of dualcostimulation such as combined activation through CD137 andCD134 induced a profound effect on the generation and quality ofpeptide-specific CD8 T cells (23). Stimulation of both costimula-tors, in a protocol that mimicked immunotherapy (24), inducedCD8 T cell clonal expansion and effector T cell generation, whichwas accentuated in a mouse tumor cell model. Similar data usinga wider variety of experimental murine tumor models was alsoreported (25). Additionally, recent data expanded upon these re-sults by demonstrating that concomitant immunotherapy with anti-DR5, -CD137, and -CD40 mAbs were able to eradicate pre-estab-lished tumors (26). Finally, dual costimulation induced by CD80and CD137L was shown to enhance CD8 T cell responses in lym-phocyte populations from long-term infected HIV� donors (27).Therefore, these data collectively suggest that combined stimula-tion of costimulatory pathways can lead to efficacious immuno-therapy of tumors in humans as well as having the potential forvaccine development.

Although the potency of dual costimulation is notable, themechanism explaining why two costimulators can be synergistic isunclear. This is particularly interesting for CD137 and CD134,where surface expression on T cells is relatively similar (28, 29),and both molecules use comparable, but perhaps not identical, sig-naling pathways (1, 30, 31). A significant difference, however, isthat in general CD134 predominantly stimulates CD4 over CD8 Tcells (2, 32–35), and the opposite is the case for CD137 (8, 35–38).Furthermore, CD137 is widely expressed on many different celltypes including dendritic cells (DCs)3 (36, 39–44), but based on

*Department of Immunology and †Division of Endocrinology, Department of Med-icine, University of Connecticut Health Center, Farmington, CT 06030; ‡Division ofMolecular Immunology, La Jolla Institute for Allergy and Immunology, La Jolla, CA92121; §Immunomodulation Research Center, University of Ulsan, Ulsan, South Ko-rea; and ¶Department of Surgery and Emory Vaccine Center, Emory UniversitySchool of Medicine, Atlanta, GA 30329

Received for publication February 28, 2007. Accepted for publication June 11, 2007.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by National Institutes of Health Grants R01 AI42858 andAI52108 (to A.T.V.).2 Address correspondence and reprint requests to Dr. Anthony T. Vella, Departmentof Immunology, University of Connecticut Health Center, 263 Farmington Avenue,Farmington, CT 06030.

3 Abbreviations used in this paper: DC, dendritic cell; �c, common �; PLN, peripherallymph node; MLN, mesenteric lymph node; WT, wild type.

Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00

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current data, CD134 does not appear to be as pervasively ex-pressed (3, 4, 45). In this report, some of these concepts werereinforced and extended using the OT-I CD8 T cell transfer modelinterfaced with immunotherapeutic use of agonist anti-CD137 and-CD134 mAbs. It is shown that an optimal dual costimulatory ef-fect through CD137 and CD134 required CD137 expression on thespecific CD8 T cells. Specifically, effector differentiation was stillretained when T cells were CD137 deficient, but accumulation wassubstantially reduced in lymphoid and peripheral sites. Second,CD134 costimulation was largely ineffectual at inducing superpro-duction of effector cytokines in the absence of the CD137 pathway.These results suggested that dual costimulation induced effectson both the specific T cells and host APCs, and revealed anunappreciated interdependent link of CD137 function forCD134 costimulation. Delving deeper, we detected a preferen-tial increase of the common � (�c) cytokine receptors CD25 andIL-7R� (CD127) on the dual costimulated CD8 T cells vs theindividually costimulated cells. Purified CD8 T cells respondedto IL-7 by surviving better in an overnight culture, but moreimportantly they acquired supereffector function. An in vivotest of this latter notion demonstrated that although IL-7 wasimportant for overall T cell accumulation, it played a crucialrole for the survival of supereffector T cells.

Thus, the TNFR members CD137 and CD134 impart condition-ing on specific CD8 T cells to use the �c cytokine family membersfor optimal effector differentiation and survival. These character-istics further support the approach of using the TNFR superfamilymembers to promote T cell costimulation to eradicate tumors inhumans, and for vaccine development.

Materials and MethodsMice

C57BL/6 mice were purchased from the National Cancer Institute (Fred-erick, MD) or The Jackson Laboratory. IL-7�/� mice were provided byDrs. L. Puddington (46) and L. Lefrancois (47) (University of ConnecticutHealth Center, Farmington, CT). The OT-I, OT-I RAG�/�, CD137�/�

OT-1, and CD137�/� (48) transgenic mice were bred by our laboratory.All mice were maintained in the animal facility at the University of Con-necticut Health Center in accordance with National Institutes of Healthguidelines.

Injection schedules and immunization procedure

In all experiments, 1–2 � 106 cells from the lymph nodes and spleen ofeither OT-I, OT-I RAG�/�, or CD137�/� OT-I transgenic mice were adop-tively transferred into C57BL/6 or various kinds of knockout mice by thei.v. route. The following day, 100 �g of SIINFEKL peptide (InvitrogenLife Technologies) in PBS was injected i.p. The anti-CD137 mAb is anagonist rat mAb specific for murine 4-1BB (3H3 rat hybridoma; Ref. 8),and the agonistic anti-CD134 is a rat mAb specific for murine OX40(OX86 hybridoma; Ref. 45). Each mAb was purified from respective hy-bridoma supernatant using a protein G (Invitrogen Life Technologies).Mice were given doses of 25 �g of anti-CD137 and/or 50 �g of anti-CD134 Abs at the same time as SIINFEKL peptide. The doses of Abs weredetermined by titration experiments based on the effect of these Abs on Tcells in vivo, and in Fig. 1, a one-fifth dose of Abs, which was 5 �g ofanti-CD137 and 10 �g of anti-CD134, was used to show the potency ofsynergy of dual costimulation.

Cell processing

Spleen, peripheral lymph nodes (PLNs; inguinal, axillary, brachial), andmesenteric lymph node (MLN) were crushed through nylon mesh cellstrainers (Falcon; BD Biosciences). Spleen was treated with ammoniumchloride to lyse RBC. After several washes, the cells were counted using aZ1 particle counter (Beckman Coulter). PBL was collected from tail veinin the presence of heparin and then treated with ammonium chloride. Liverand lung lymphocytes were obtained as described (49). Liver tissue wasperfused and crushed through a cell strainer. The pelleted cells were iso-lated from 35% Percoll (Sigma-Aldrich) gradient and then treated withammonium chloride. Lung tissue was perfused and cut into pieces. Thesecells were incubated at 37°C in the presence of 1.3 mM EDTA for 30 min

and then in collagenase solution (Invitrogen Life Technologies) for 1 h.After being crushed through a cell strainer, cells were resuspended in 44%Percoll (Amersham Biosciences) and layered above a 67% Percoll cushion.After centrifugation, cells were isolated from the interface and washed.

In vitro culturing

In Fig. 5, day 2.5 spleens were treated with collagenase (Roche) at 37°C for30 min. One hundred microliters of 0.1 mM EDTA was added to inactivatethe activity of collagenase. Day 4 spleens were directly applied to a cellstrainer. Splenocytes were crushed through a cell strainer and run througha nylon wool column (PerkinElmer Life and Analytical Sciences) to re-move APCs. Cells were incubated with biotin-, PE-, or FITC-conjugatedanti-CD45.1 mAb (eBioscience), and then labeled with anti-biotin, or anti-PE, or anti-FITC microbead (Miltenyi Biotec) according to the manufac-turer’s recommendation. CD45.1� cells were selected by MACS columns(Miltenyi Biotec). After washing, purity of CD45.1� OT-I was measuredby flow cytometry and was typically 80–95% CD45.1� cells. The purifiedOT-I T cells were resuspended in CTM (MEM containing amino acids,salts, antibiotics, and FBS), and 100,000 cells were placed into a well of a96-well plate and cultured at 37°C. For in vitro experiment, recombinantmouse IL-7 (PeproTech) was added at 10 ng/ml at the beginning of theculture.

Cell staining and flow cytometry

The following mAbs were purchased from eBioscience: anti-CD45.1, anti-CD45.2, anti-CD8, anti-CD25, anti-IFN-�, anti-TNF, and control rat IgG1.The following mAbs were purchased from BD Biosciences: anti-CD127and control rat IgG2a.

Cells were stained with primary Abs in the presence of a blocking so-lution containing 5% normal mouse serum (Sigma-Aldrich), 10 �g/ml hu-man gamma-globulin (Sigma-Aldrich), and 0.1% sodium azide in culturesupernatant from the 2.4.G.2 hybridoma (anti-FcR; Ref. 50) for 30 min onice and then washed in wash buffer (3% FBS and 0.1% sodium azide inBSS). For CFSE staining, cells were isolated, pelleted, and resuspended inTC solution (MEM, amino acids, and antibiotics). The cells were labeledwith 15 �M CFSE (Molecular Probes), incubated at 37°C for 10 min, andlabeling was stopped with cold CTM.

For intracellular cytokine staining, 1 � 106 splenocytes, or peripheralblood cells were cultured with 1 �g of brefeldin A (Calbiochem) in thepresence of 1 �g of SIINFEKL peptide at 37°C for 5 h. For overnight-cultured cells (Fig. 5), naive splenocytes were provided into a well at thebeginning of restimulation. The cells were stained with either allophyco-cyanin-conjugated anti-CD45.1, anti-CD8, or anti-CD127 mAbs on ice for30 min, and after a couple washes, the cells were fixed with 2% parafor-maldehyde in BSS. The cells were placed in permeabilization buffer(0.25% saponin in wash buffer) and then incubated with anti-IFN-�, anti-TNF, or an isotype control rat IgG1 for 20 min at room temperature.

ResultsEffects of dual costimulation on specific CD8 T cells

To study the robust effect of dual costimulation in vivo, a low doseof agonist anti-CD137 and -CD134 was tested in an OT-I adoptivetransfer model. OT-I CD8 T cells were transferred into C57BL/6mice, immunized as in Materials and Methods, and tracked byCD45.1 congenic marker staining. On days 2.5–10.5, the fre-quency of donor OT-I CD8 T cells from peripheral blood wasanalyzed by flow cytometry (Fig. 1A). At the peak of immuneresponse (day 4.5), dual costimulation led to massive clonal ex-pansion of OT-I T cells compared with the single costimulationgroups. For example, there was a �2.9-fold increase in the fre-quency of dual costimulated specific CD8 T cells in blood at thistime point. On day 10.5, spleen, PLNs, and MLN were analyzedfor the presence of OT-I CD8 T cells (Fig. 1B). Dual costimulationprofoundly enhanced the accumulation of OT-I T cells in all tis-sues compared with the other single costimulation treatments. Forinstance, dual costimulation induced a �7-fold increase in the per-centages and a �12.6-fold increase in the numbers of OT-I CD8 Tcells in spleen. Collectively, these data show a remarkable syner-gism through CD137 and CD134 dual costimulation by enhancingthe frequency and number of Ag-specific CD8 T cells without theuse of known adjuvants.

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Next, we tested effectiveness of effector function in thesegroups. Day 4.5 peripheral blood cells were restimulated in vitrowith SIINFEKL peptide for �5 h and then assayed for intracellularcytokine production. Approximately 65% of dual costimulatedOT-I T cells made TNF (Fig. 1C, see upper panel) and �80%synthesized IFN-� (data not shown). Compared with the singlecostimulator treatments, there was a 2-fold increase in the percent-age of TNF-producing OT-I T cells, and no substantial increase inIFN-�-producing OT-I T cells in the dual costimulated group. Ef-fector function of day 10.5 dual costimulated OT-I T cells inspleen induced �96% IFN-� production and around 85% for TNF(Fig. 1D, upper panel). Although there was little difference in thepercentage of OT-I T cells producing effector cytokines, there was,however, a massive increase from spleen, when normalized for theabsolute numbers of OT-I effector cells (Fig. 1D, lower panel).Dual costimulation generated an 18- and 16-fold increase in TNFand IFN-�, respectively, over anti-CD137 alone. The fact that thenumber of cytokine producers was much greater than the percent-

age of producers suggests that dual costimulation has a muchgreater effect on the expansion of effector T cells, rather thanjust impacting their effector differentiation process. Thus, dualcostimulation generates excellent quality of effector T cells thataccumulated in huge numbers.

Next, we characterized the role of CD137 signaling directly onT cells during clonal expansion and accumulation of effector cells.To test this notion, one group of C57BL/6 mice received wild-type(WT) OT-I spleen cells, and the other CD137-deficient(CD137�/�) OT-I cells. The following day, both groups were im-munized with dual costimulation plus peptide, and on days 2.5 to10.5, peripheral blood cells were analyzed for the presence of OT-IT cells by flow cytometry (Fig. 2A). The data show a substantialdecrease of OT-I T cell clonal expansion in blood in the absenceof CD137 expression on CD8 T cells, and the same is true whenthe host fails to express CD137. On day 4.5, dual costimulationgenerated �18% of CD137�/� OT-I CD8 T cells, which was a�2.5-fold decrease compared with WT OT-I. Therefore, clonal

FIGURE 1. Dual costimulation synergistically stimulates clonal expansion of peptide-specific effector CD8 T cells. OT-I Rag�/� splenocytes wereadoptively transferred into C57BL/6 mice. The next day, recipient mice were treated with SIINFEKL peptide plus 5 �g of anti-CD137 (gray-filledcircle); SIINFEKL peptide plus 10 �g of anti-CD134 (�); or SIINFEKL peptide plus both Abs (f). A, On days 2.5 to 10.5, the percentages of OT-IT cells from peripheral blood were analyzed by flow cytometry (left). Shown are the mean percentages � SEM of CD45.1�CD8� cells. Datacontained a total of at least five mice in each group from three independent experiments. B, OT-I T cells from spleen, PLNs, and MLN were examinedon day 10.5, and the mean percentages (upper panel) and absolute numbers (lower panel) � SEM of CD45.1� CD8� cells (right). Each groupcontained at least five mice from three independent experiments. C, Day 4.5 peripheral blood cells were restimulated with SIINFEKL peptide for�5 h and then stained for the presence of intracellular cytokine. Zebra plots are representatives of each group, and the numbers indicate thepercentages of OT-I T cells producing TNF (upper). Lower panel, The mean percentages � SEM of OT-I T cells producing TNF (left) and IFN-�(right) are shown. Data are pooled from three separate experiments with a total of seven mice in each group. D, Days 10.5 and 12.5 spleen cellswere restimulated with SIINFEKL peptide for �5 h and then stained for intracellular TNF (left) and IFN-� (right) level. The graphs show thepercentages (upper) and numbers (lower) � SEM of CD45.1� cells producing IFN-� and TNF. Data are combined from three different experimentsfor a total of at least six mice in each group. The arrows indicate the fold difference of cytokine production between dual costimulation treatmentgroup and anti-CD137 group.

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expansion of specific CD8 T cells was dependent upon directCD137 signaling, because the WT host possesses CD137 on DCsand other cells of the immune system (39–44). For example, ex-pression of CD137 on the host was also important for expansionbecause there was a 1.8-fold reduction in blood on day 4.5 whenthe host did not express CD137 while the OT-I T cells were WT.This was also the case for OT-I T cells in various tissues on day4.5. The data show reduced accumulation of specific CD137�/�

OT-I CD8 T cells in lymphoid and nonlymphoid sites. For exam-ple, after dual costimulation, the lack of CD137 expression onOT-I T cells led to an �7-fold decrease in the numbers of OT-I Tcells in spleen and MLN and �4-fold decrease in liver. A smallerreduction was also detected when the CD137�/� host received WTOT-I cells, 1.7-fold less in spleen and 1.6-fold less in liver (datanot shown). In general, these responses are much better than a ratIgG control response in a WT OT-I to WT recipient as we detect

about a 10- and 60-fold decrease in spleen on days 4 and 5, re-spectively, after rat IgG compared with dual costimulation (datanot shown). Thus, these results suggest that direct CD137 signal-ing on CD8 T cells is crucial for the accumulation of specific CD8T cells and this trend was also observed on day 10.5 (data not shown).Taken together, these data show that CD137 expression on specificCD8 T cells and host is required for optimal T cell clonal expansion,but they may not necessarily relate to effector function.

Next, we asked whether effector function was also affected bythe absence of CD137 expression on CD8 T cells. Day 4.5 spleen(Fig. 2, C and D) and liver (Fig. 2E) cells taken from mice thatreceived either WT or CD137�/� OT-I cells were restimulated invitro, and IFN-� and TNF production examined by intracellularcytokine staining. There was no difference in the percentages ofOT-I T cells producing IFN-� or TNF between these groups sug-gesting that effector function was undamaged in the absence of

FIGURE 2. Direct CD137 expression by CD8 T cells is required for clonal expression, not for effector function. OT-I (f) or CD137�/� OT-I(gray-filled circle) splenocytes were adoptively transferred into C57BL/6 mice, and OT-I cells into CD137�/� OT-I (light gray-filled triangle). Thenext day, all recipient mice were immunized with SIINFEKL peptide plus dual costimulation. A, On days 2.5–10.5, blood OT-I CD8 T cells wasanalyzed by staining for the congenic marker (CD45.1� for WT OT-I or CD45.2� for CD137�/� OT-I) CD8� double-positive cells. The meanpercentages � SEM of OT-I cells are shown. Each group contained at least 13 mice per group from six different experiments. B, On day 4.5, spleen,MLN, PLNs, and liver cells were isolated, and the presence of OT-I T cells in each tissue was evaluated. Bar graphs represent the mean numbers �SEM pooled from five separate experiments with a total of at least 13 mice in each group. C–E, Day 4.5 spleen and liver cells were restimulatedin vitro and then assayed for intracellular cytokine production. C, Representative dot plots are shown from each group, and the numbers indicatethe percentages of spleen OT-I T cells producing IFN-� and TNF. D, Shown are the mean percentages (upper panel) and numbers (lower panel) �SEM of spleen OT-I T cells staining positive for IFN-� (left) or TNF (right). All data are combined from three different experiments for a total ofat least 10 mice per group. E, Bar graphs represent the mean percentages (upper) or numbers (lower) � SEM of liver OT-I T cells producing IFN-�and TNF. These are pooled from three independent experiments with a total of at least eight mice in each group. The arrows show the fold reductionin the CD137�/� OT-I transfer group compared with the WT OT-I group.

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direct CD137 signaling on T cells. However, when normalized forthe actual number of OT-I T cells producing effector cytokine, itwas profoundly reduced in CD137�/� OT-I transfer group. Therewas an 11.3-fold decrease in IFN-� positive cells and an 11.6-fold

decrease in TNF-producing OT-I T cells (Fig. 2D). This trend wassimilar in the frequency and absolute numbers of OT-I effectorcells when liver was examined, a site where effector CD8 T cellsare known to reside (Fig. 2E) (51, 52). In slight contrast, a smaller

FIGURE 3. CD134 stimulation induces supereffector T cells through dependency of CD137 expression on either CD8 T cells or host. A, WT OT-I splenocyteswere adoptively transferred into CD137�/� mice (group I), and spleen cells from CD137�/� OT-I transgenic mice were transferred into either C57BL/6 (groupII) or CD137�/� (group III) mice. On day 0, recipient mice received SIINFEKL peptide plus anti-CD134 Abs, and rat IgG was injected into another portion ofrecipient mice in each group as a control. After 60 h, spleen cells were restimulated in vitro and then analyzed for IFN-� production. The dot plot in the lowerleft panel is representative of IFN-� production by splenocytes from the anti-CD134-treated group I mice, and then these IFN-�� CD8� cells are gated. Six dotplots in the lower right panel show representatives of superproducers gated from IFN-��CD8� double-positive cells from each treatment. The superproducers weredetermined by setting an analysis region of the anti-CD134-treated IFN-��CD8� cells from group I at �10% of the top IFN-�-producing cells and then applyingthis exact region to all the samples. B, Bar graphs show the mean percentages (left) and numbers (right) � SEM of IFN-� superproducers within the CD8�IFN-��

population, and these represent combined data from four separate experiments with a total of at least four mice in each group.

FIGURE 4. Dual costimulation increases CD25 and IL-7R� expression on specific CD8 T cells. OT-I Rag�/� splenocytes were adoptivelytransferred into C57BL/6 mice. On day 0, recipient mice were given SIINFEKL peptide with anti-CD137 (gray-filled circle); SIINFEKL peptide withanti-CD134 (�); or SIINFEKL peptide with dual costimulation (f). On days 3–5, spleen cells were analyzed for the presence of OT-I cells positivefor CD25 (A and B) or IL-7R� (C and D). Analysis region from the isotype control staining in each mouse was set at �2% to determine thepercentage of CD25- or IL-7R�-positive cells within CD45.1� cells. A, Shown are representative zebra plots from day 4 spleen cells examiningCD25 expression. The numbers listed indicate the percentages of CD45.1� cells expressing CD25. B, Shown are the mean percentages � SEM ofCD45.1� cells positive for CD25 expression. C, Zebra plots are representative of day 4 splenic OT-I T cells to analyze for IL-7R� expression. Thenumbers represent CD45.1� cells expressing IL-7R�. D, Data show the mean percentages � SEM of CD45.1� cells positive for IL-7R� expression.These data are pooled from four separate experiments for a total of at least four mice per group.

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1.7-fold reduction was seen for the number of IFN-� producers forspleen and liver when WT OT-I were transferred into CD137�/�

mice (data not shown). Thus, clonal expansion of peptide-specificCD8 T cells through dual costimulation depends on direct CD137signaling, but differentiation into effector CD8 T cells was inde-pendent of direct CD137 activation.

Supereffector function driven by CD134 signaling is dependenton CD137 expression

To test the role of CD134 signaling on the CD8 T cell response,WT OT-I spleen cells were adoptively transferred into CD137�/�

mice (group I); CD137�/� OT-I splenocytes into C57BL/6 mice(group II); and CD137�/� OT-I spleen cells into CD137�/� mice(group III) (Fig. 3A). All recipient mice were immunized withSIINFEKL peptide plus either rat IgG control or anti-CD134 mAb.Spleen cells were isolated 2.5 days later, restimulated in vitro,and evaluated for IFN-� synthesis. Because CD134 endowsCD8 T cells with effector function (22, 29, 53, 54), we reasonedthat CD134 costimulation would induce the production of su-pereffectors as we have recently demonstrated using CD4 Tcells (55). The proportion of IFN-� positive cells within the

OT-I CD8 population was analyzed for superproducers (Fig.3A, lower panel). In group I, anti-CD134 treatment boosted theIFN-�-producing population compared with control IgG (Fig.3B). This showed that CD137 on accessory cells was not im-portant for CD134 costimulation of CD8 T cells. Group IIshowed a �3-fold increase indicating that CD137 on the spe-cific CD8 T cells was also indispensable. In group III, neitherthe accessory cells nor the specific CD8 T cell bore CD137 (Fig.3A, bottom schematic), which profoundly inhibited the CD134immune response (Fig. 3B). Also, results from WT OT-I trans-fer into WT were very similar to the results from groups I andII, although these mice were assayed at day 3 instead of day 2.5(data not shown). These data show that in a system where CD4T cell help is unnecessary for CD8 effector production, CD134costimulation depends on CD137 expression on either the spe-cific CD8 T cells or accessory cells.

We also examined CFSE dilution of the transferred OT-I Tcells and detected no substantial differences between the groupsregardless of whether the mice received anti-CD134 (data notshown). Based on numbers of OT-I T cells at this time point, wedetected an increase in groups I and II, compared with group III,

FIGURE 5. During overnight culture, IL-7 increases survival of OT-I effector T cells. A, C57BL/6 mice received splenocytes from OT-I Rag�/�

mice, and the next day, treated with SIINFEKL peptide plus control IgG (light gray-filled square); SIINFEKL peptide plus anti-CD137 (gray-filledsquare); SIINFEKL peptide plus anti-CD134 (�); or SIINFEKL peptide plus dual costimulation (f). On days 2.5 or 4, spleens were removed, andCD45.1� cells isolated by MACS columns. Purified CD45.1� cells (105) were placed into a well of a 96-well plate and cultured with either mediumalone or 10 ng/ml recombinant murine IL-7. The following day, cells were restimulated with naive spleen cells and SIINFEKL peptide forintracellular cytokine production. B, Histograms show representative staining of IFN-� within viable CD45.1� population from day 2.5 (upper panel)and day 4 (lower panel) spleen cells. The gray tinted line indicates the isotype control staining and the thick black line represents IFN-� expressionlevel in CD45.1� gated cells. The superproducer was determined by setting an analysis region on dual costimulated CD45.1� cells at �10% of topIFN-�-producing cells and applying this exact region to all the samples. Numbers indicate viable IFN-� superproducers in each group. Thesehistograms are representative of three independent experiments. C, Shown are the mean percentages � SEM of surviving IFN-� (left) and TNF(right) superproducers within CD45.1� population (from B, lower panel). The percentages were calculated on superproducers from live CD45.1�

cells.

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but these were relatively minor differences as were the effects ofanti-CD134 (data not shown). Importantly, day 2.5 is not anoptimal time point for measuring clonal expansion so these re-sults were not surprising; however, it is critical to note that latertime points such day 5 are difficult to interpret because theCFSE is completely diluted out preventing any distinction be-tween expansion of endogenous T cells with that of the trans-ferred cells.

Dual costimulation induces higher CD25 and IL-7R� expressionon OT-I T cells

To study whether any survival cytokine was involved in the clonalexpansion through dual costimulation, the expression level of sev-eral cytokine receptors in the �c chain cytokine family such asCD25 and IL-7R� (CD127) was evaluated. Thus, OT-I-recipientC57BL/6 mice were immunized (SIINFEKL peptide with eitherrat IgG control, anti-CD137, -CD134, or dual costimulation), andon days 3–5, spleen cells were analyzed for the percentages ofOT-I T cells positive for CD25 (Fig. 4, A and B) and IL-7R� (Fig.4, C and D) expression. In all cases, CD25 expression on OT-I Tcells gradually decreased between days 3 to 5 (Fig. 4B), whereasIL-7R� expression progressively increased (Fig. 4D). However,dual costimulated OT-I T cells reproducibly contained a greaterproportion of CD25- and IL-7R�-expressing cells on days 3 and 4compared with the single costimulation treatments.

To test the idea that these cytokines were responsible for theaccumulation of dual costimulated effector CD8 T cells, OT-I-recipient mice were immunized in vivo as in Fig. 4. CD45.1� cellswere purified from spleen on either days 2.5 or 4, cultured over-night either in the absence or presence of IL-7, and then restimu-lated with peptide to measure effector cytokine synthesis (Fig. 5A).Fig. 5B shows that dual costimulated OT-I T cells generated moreIFN-� superproducers in the presence of IL-7 compared with thesingle costimulators, and this is also the case in the absence ofIL-7. Importantly, when normalized for the percentage of surviv-ing superproducers (by multiply the percentage of viable OT-I Tcells by the percent of superproducers), day 4 dual costimulatedOT-I T cells contained a higher frequency of surviving superef-fector T cells whether or not IL-7 was present (Fig. 5C). Survivalcytokine IL-7 was able to enhance accumulation of the superef-fector subpopulation regardless of any kind of costimulation, andthis was the case for IL-2 as well (data not shown). Taken together,the data implies that survival cytokines such as IL-7 are likely tobe involved in the action of dual costimulation.

Thus, it was hypothesized that IL-7 might play a critical role inthe mechanism of dual costimulation. Therefore, OT-I splenocyteswere adoptively transferred into either C57BL/6, IL-7�/�, orIL-7�/�, immunized with dual costimulation and peptide, andon days 2.5–10.5, the presence of OT-I CD8 T cells detected inperipheral blood by flow cytometry (Fig. 6A). There was nosignificant difference in the magnitude of clonal expansion ofOT-I T cells in the absence or presence of IL-7 suggesting thatclonal expansion of dual costimulated OT-I CD8 T cells wasindependent of IL-7. On day 10.5, however, the frequency ofOT-I T cells in blood was 1.7-fold lower in the IL-7�/� groupcompared with control C57BL/6 mice. This was consistent withthe percentages of OT-I CD8 T cells in spleen, lung, and liver.For example, there was a 2-fold decrease in IL-7�/� mice com-pared with the control group (Fig. 6B, left panels); however,there were equivalent percentages in peripheral LN (data notshown). The absolute numbers of splenic OT-I T cells weredramatically reduced in the absence of IL-7, where there was a14-fold reduction (Fig. 6B, right panel), but this may be due tothe reduced cellularity in lymphoid tissue in IL-7�/� mice.

Nonetheless, we still observed a �2-fold reduction of the ab-solute numbers of OT-I T cells in lung and liver (Fig. 6B, rightpanel), which was comparable to the reduction in percentages(Fig. 6B, left panel). Together, these data suggest that IL-7 didnot influence clonal expansion but had impacted accumulationof dual costimulated OT-I T cells in peripheral tissue andspleen.

To answer the question of whether the absence of IL-7 had aneffect on effector function of dual costimulated OT-I T cells, day10.5 spleen and liver cells taken from either control, IL-7�/�, orIL-7�/� mice were restimulated in vitro and analyzed for intra-cellular cytokine production. Liver (data not shown) and spleen

FIGURE 6. Dual costimulation induces clonal expansion indepen-dently of IL-7. Splenocytes from OT-I Rag�/� mice were transferredinto either C57BL/6 (f), IL-7 heterozygotes (�/�; gray-filled triangle),or IL-7�/� mice (hatch-filled diamond). The following day, all the re-cipient mice were treated with SIINFEKL peptide plus dual costimu-lation. A, On days 2.5–10.5, the mean percentages � SEM of CD45.1�

CD8� double-positive cells in peripheral blood were analyzed. Datashown are combined from six independent experiments for a total of atleast 16 mice in C57BL/6 and IL-7�/� group, and 6 IL-7�/� mice fromtwo experiments. B, On day 10.5 in spleen, lung, and liver, CD45.1�

CD8� double-positive OT-I T cells were calculated. The mean percent-ages (left) and numbers (right) � SEM of CD45.1�CD8� cells areindicated. Each group had at least 9 mice combined from at least fourexperiments.

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(Fig. 7A, upper panel) OT-I T cells from the IL-7�/� group gen-erated fewer effector T cell producers, and the cells from heterozy-gous mice showed intermediated responses. Importantly, the per-centages of superproducers in both spleen (Fig. 7A, lower panel)and liver (Fig. 7B, upper panel) were profoundly reduced in IL-7�/� mice, where there was a �3-fold decrease compared withcontrol mice. The absolute numbers of splenic OT-I superproduc-ers were �16-fold lower in the absence of IL-7 (data not shown),which may be more of an outcome as the reduced cellularity inlymphoid tissues in these knockout mice as discussed earlier. Inliver where cellularity was comparable to percentage (Fig. 6B),there was an 8-fold reduction in the numbers of IFN-� superpro-ducers, and a 6-fold decrease in TNF superproducers (Fig. 7B,lower panel). Second, we tested whether the lack of IL-7 aug-mented an increase of negative to low effector cytokine producers.The IL-7�/� group showed a 2-fold increase in the percentage ofnegative to low effector cytokine producers in the IL-7�/� groupcompared with WT (Fig. 7C, spleen, and Fig. 7D, liver). The ab-

solute numbers of IFN-� or TNF negative to low producers in liverwere about the same in the IL-7�/� compared with WT (Fig. 7D,lower panel). Thus, unavailability of IL-7 resulted in accumulationof the negative to low effector OT-I T cell population. Collectively,the data suggest that IL-7-mediated survival after dual costimula-tion through CD137 and CD134 preferentially acts on superpro-ducing effector CD8 T cells.

To further test the idea that IL-7-mediated survival acts on su-perproducers, day 10.5 peripheral blood cells were restimulated invitro and then assayed for intracellular cytokine production (Fig.8A). IFN-� TNF double superproducers were reduced in IL-7�/�

mice. When compared between day 4.5 and 10.5 blood OT-I Tcells, very little decrease in frequency of double superproducersfrom IL-7�/� mice on day 4.5, but a 3-fold reduction of doublesuperproducers on day 10.5 was observed (Fig. 8B). These datasuggest the increasing importance of IL-7 for survival of superef-fector CD8 T cells was based on elapsed time, rather than an all-or-nothing effect.

FIGURE 7. In the absence of IL-7,effector function of dual costimulatedCD8 T cells is reduced. Adoptivetransfer of OT-I Rag�/� splenocytesinto either control (C57BL/6 and IL-7�/�), or IL-7�/� mice was con-ducted, and the next day, mice wereimmunized with SIINFEKL peptideplus dual costimulation. On day 10.5,spleen (A and C) or liver (B and D)cells were restimulated with peptidein vitro. A, Splenic CD45.1� cellswere analyzed for intracellular IFN-�or TNF level. Upper panel, Represen-tative histograms of IFN-� and TNFproduction by gated CD45.1� cells.The gray histogram is the isotypecontrol and the thick black lines rep-resent cytokine staining. The numbersshow IFN-� or TNF superproducersby setting an analysis region onCD45.1� cells at �10% of the top cy-tokine producer and keeping the exactregion for all the samples. Lowerpanel, Individual percentages ofIFN-� or TNF producers and super-producers in splenic CD45.1� cellsare quantitated in scatter plots. Dataare combined from six different ex-periments. B, Liver OT-I effector Tcells were examined as in A, and arefrom five separate experiments. Thenumbers of liver OT-I superproducersappeared in the lower scatter plot. Cand D, Splenic (C) and liver (D) OT-Ieffector T cells were also analyzed fornegative to low (Neg/low) IFN-� orTNF producers. The negative to lowproducers were determined by settingan analysis region at �30% of thelowest cytokine producer and keepingthe exact region for all the samples.The lines mark the average valuesand the arrow indicates the fold re-duction in the IL-7�/� group com-pared with control.

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To test whether WT DCs were able to increase effector func-tion in the IL-7�/� group, OT-I T cells on day 12.5 or 14.5 wereisolated from either control or IL-7�/� mice, and restimulatedin vitro in the presence of WT APCs (Table I). Although theeffector cytokine producing ability was comparable between thecontrol and IL-7�/� group, the frequency of double superpro-

ducers was substantially reduced in OT-I T cells taken fromIL-7�/� mice even in the presence of WT APCs. This impliesthat after dual costimulation, accumulation of supereffector Tcells is defective in the absence of IL-7 or that OT-I T cells areconditioned to differentiate into superproducers in the presenceof IL-7.

Moreover, it was examined whether a proportion of IL-7R�-expressing OT-I T cells had different effector cytokine productionprofiles. Day 8.5 peripheral blood cells from dual costimulatedOT-I recipient C57BL/6 mice were assayed for IL-7R� expressionand IFN-� production upon restimulation (Fig. 8C). IL-7R�high

expressing OT-I T cells synthesized 82% of IFN-�, whereas theIL-7R� mixed population generated 26% of IFN-� producers andthe negative population produced very little IFN-�. We also ob-served similar results at different time points, where higher IL-7R�expression tracked with more IFN-� production (data not shown).Taken together, our data suggest that dual costimulation enhancesIL-7R� expression on Ag-specific CD8 T cells, leading to survivalof OT-I T cells, especially supereffector T cells.

DiscussionPreviously, it was demonstrated that simultaneous administrationof agonist anti-CD137 and -CD134 mAbs substantially enhancedCD8 effector T cell function leading to rapid rejection of murinetumors, but the mechanism of dual costimulation remained un-known (23). Here, it is shown that CD137 was critical for op-timal T cell clonal expansion and CD134 triggered supereffectorfunction, which was also dependent upon CD137 signaling. Incombination, the CD137 and CD134 costimulatory pathwaysfacilitated effector T cell survival with coincident induction ofIL-7R�, but exogenous IL-7 was essential for accumulation ofthese supereffector CD8 T cells. Thus, this model shows aninterdependent bridge between the TNFR and the �c chain cy-tokine families.

To characterize the effect of dual costimulation by CD137 andCD134 on CD8 T cells, we examined whether the dose of theanti-CD137 and -CD134 mAbs dictated the synergistic action ofdual costimulation on OT-I T cells. Our data persuasively demon-strated that a low dose of dual costimulation synergistically in-creased clonal expansion of OT-I CD8 T cells. These resultsshowed that dual costimulation induced a bona fide synergisticsignal between the two different costimulatory pathways. Second,

FIGURE 8. Optimal dual costimulation relies on IL-7 for continual ac-cumulation of supereffector OT-I T cells. OT-I Rag�/� cells were trans-ferred into either control (C57BL/6) or IL-7�/� mice, and immunized withSIINFEKL peptide plus dual costimulation. A, Day 10.5 peripheral bloodcells were analyzed for intracellular IFN-� and TNF production upon re-stimulation. Upper panel, Histograms of viable CD45.1� cells. Lowerpanel, Zebra plots represent IFN-� and TNF production of OT-I T cellsgated from the upper panel. The gates and numbers indicate double su-perproducers by setting an analysis region at �20% of the top IFN-��TNF� double-positive cells and keeping the same region for all thesamples. B, Individual double superproducers from day 4.5 (Left) and day10.5 (Right) peripheral blood cells were quantitated in scatter plots, anddata are pooled from at least three separate experiments. The lines repre-sent the average value, and the arrows indicate the fold reduction in theIL-7�/� group compared with control. C, Day 8.5 peripheral blood cellsfrom control mice were evaluated for intracellular IFN-� production. Up-per panel, Histogram of IL-7R� expression by gated CD45.1� cells in thethick black line. The gray line is the isotype control. Based on IL-7R�expression level, CD45.1� cells are divided into three groups (Hi, Mixed,and Negative). Lower panel, IFN-� production level of OT-I T cells gatedfrom the upper panel is shown in zebra plot. The numbers indicate therelative percentages of CD45.1� cells producing IFN-�. This experimentwas performed in five different time points.

Table I. Added APCs do not rescue recall of OT-I T cells that havebeen conditioned in IL-7�/� micea

Expt. GroupIFN-�

ProducerTNF

Producer

IFN-� TNFDouble

Superproducer

1 WT 94.5 � 0.4 97.0 � 0.6 14.7 � 4.2(Day 14.5) IL-7�/� 81.0 � 2.8 91.6 � 0.8 4.7 � 0.52 WT 88.8 � 1.0 89.2 � 0.9 9.0 � 1.2(Day 14.5) IL-7�/� 79.7 � 1.3 85.1 � 0.1 3.3 � 1.23 WT 93.4 � 0.9 94.3 � 1.0 22.3 � 3.4(Day 12.5) IL-7�/� 76.9 � 4.4 79.1 � 6.6 13.4 � 4.54 WT 95.0 � 0.4 87.9 � 3.7 12.3 � 1.6(Day 12.5) IL-7�/� 93.9 � 0.4 93.6 � 2.6 7.7 � 0.8

a OT-I spleen cells were adoptively transferred into either C57BL/6 (WT) or IL-7�/� mice. The next day, all recipient mice were injected with SIINFEKL peptideplus dual costimulation. On day 14.5 (expt. 1 and 2), OT-I T cells were purified fromspleen using MACS columns with anti-CD45.1 Abs. On day 12.5 (expt. 3 and 4),peripheral blood cells were run through a nylon wool column. Afterward, the day 14.5or 12.5 cells were placed into a well of a 96-well plate containing naive splenocytesfrom C57BL/6. They were restimulated in vitro with peptide for �5 h and assayed forintracellular cytokine. The data are the mean percentages � SEM, and each experi-ment has at least two mice per group.

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dual costimulation profoundly enhanced cumulative CD8 effectorfunction compared with single costimulators (Fig. 1D). Perhaps,this is the reason that dual costimulation effectively retarded tumorgrowth (23, 25), and permitted expansion of OT-I T cells aftertransfer of just 1000 cells (23).

Because CD137 is expressed on activated T cells, DCs, andother cells of the immune system (5, 36, 39–44, 56), the role ofdirect CD137 expression on CD8 T cells was unclear. Therefore,we tested this notion and demonstrated that CD137�/� OT-I Tcells expanded poorly compared with WT OT-I T cells, but unex-pectedly the effector function of CD137�/� OT-I T cells was com-mensurate with that of WT OT-I T cells (Fig. 2, C–E). Based onnumerous studies that CD137 stimulation enhances CD8 effectorfunction (8, 10, 28, 57), our data implied that CD137 signaling onhost accessory cells was able to compensate for the lack of CD137expression on the activated T cells. Second, when CD137�/� micewere used as recipients and immunized, effector function of WTOT-I T cells was also optimal (data not shown). Thus, in the pres-ence of CD137 expression on either CD8 T cells or host, CD8 Tcells were able to differentiate into effectors. This suggests thatdifferentiation of effector CD8 T cells may be mediated throughfactors such as cytokines produced from either CD137 stimulated-CD8 T cells or -APCs. Perhaps the APCs produce important proin-flammatory cytokines that can provide help to the responding Tcells for effector differentiation. This is borne out in recent workclearly demonstrating that CD137 stimulation on DCs can inducesecretion of IL-6 and IL-12 (58, 59). Second, this idea is consistentwith our results showing that the CD137-deficient host supportedweaker responses than a WT host (Fig. 2A). Ultimately, however,our data did show that direct CD137 activation on CD8 T cells wascritical for cumulative enhancement of effector function rather thanimproved effector differentiation.

An interesting contrast was recently observed by Lee et al. (29)showing that CD137�/� OT-I effector T cells exceeded expan-sion by the WT OT-I T cells after in vivo priming with OVA-expressing adenovirus. Thus, different model systems, wherethe combination of strength/duration of Ag stimulation, cyto-kine milieu, or engagement of other signaling pathways such asTLR stimulation (60), can dramatically influence the effect ofCD137 costimulation. A more specific difference is that oursystem used enforced costimulation, such as that for tumor im-munotherapy (8, 24, 26), with SIINFEKL peptide that limitsCD4 T cell help. Therefore, the role of CD134 in our dualcostimulation system may be different from that in an infectionmodel, because our system has few CD4 T cell targets com-pared with CD8. Consistent with this possibility are severalstudies demonstrating that CD134 can also directly enhanceCD8 T cell responses (22, 29, 53, 54). Thus, to clarify the roleof CD134 on CD8 T cells, we stimulated production of CD8effectors through CD134 activation, but surprisingly this waspositively regulated by CD137 signaling (Fig. 3). Taken to-gether, our data suggest that CD134 costimulation of CD8 Tcells is linked to the function of the CD137 pathway. Thus, dualcostimulation may promote CD137-driven expansion withCD134-induced effector function of specific CD8 T cells, sug-gesting a dichotomous role between CD137 and CD134 as re-cently proposed (29).

Clonal expansion and effector differentiation are mechanicallytied to T cell survival. Previously, dual costimulation was shown toenhance in vitro survival of activated CD8 T cells compared withsingle costimulators (23), and this evidence suggested that sur-vivability was requisite for the action of dual costimulation.Consistent with this notion, dual costimulation induced IL-7R�expression on OT-I T cells (Fig. 4), a cytokine receptor pathway

known to enhance survival of naive and memory T cells (61–63). Exogenous IL-7 enhanced accumulation of supereffector Tcells (Fig. 5), suggesting that IL-7 may be involved in the actionof dual costimulation. This hypothesis was tested in vivo bytransferring OT-I cells into IL-7�/� mice and stimulating themwith peptide in the presence of dual costimulation. There was adecrease in survival (Fig. 6), but it appeared to be a subpopu-lation rather than a general decline. This idea was consistentwith earlier results demonstrating a survival dependency of IL-7for memory T cells (61– 63). Nevertheless, upon close exami-nation, we found that effector function of dual costimulatedOT-I T cells in IL-7�/� mice was profoundly reduced, espe-cially the superproducers (Fig. 7A).

This new data led us to speculate that IL-7-mediated survivalwas distinctly critical for accumulation of supereffector cells,which is an important therapeutic outcome by CD137 and CD134dual costimulation (23). Accumulation of effector cells in nonlym-phoid tissues such as liver is pivotal for controlling infection, andis a destination whereby primary and memory effector CD8 T cellsmigrate to and from lymphoid tissues (51, 52). In IL-7�/� mice,there was a substantial decrease in the frequency and numbers ofsuperproducers in liver (Fig. 7B), implying an important role forIL-7 in inducing the accumulation of this subpopulation in non-lymphoid tissue. This evidence was supported by a reciprocal testshowing that after transfer and immunization, in IL-7�/� mice,there was an increase in the proportion and numbers of negative tolow cytokine producers in liver as well as in spleen (Fig. 7, C andD). Thus, IL-7 depravation had a preferential effect on diminishingthe accumulation of superproducers. Accordingly, the literaturehas shown that the IL-7R� is down-regulated after activation, butreturns on memory T cells (61, 64), and our idea was that the effectof IL-7 on dual costimulated superproducers would be greaterwhen IL-7R� expression was restored at a later time. In fact, itwas shown that as the amount of IL-7R� expression increased,the ability to produce IFN-� in CD8 effector T cells was sub-stantially greater (Fig. 8). These data may explain earlier stud-ies demonstrating that IL-7 can be an effective adjuvant by im-proving the immune response of CD8 T cells in patients withcancer, AIDS, or old age (65–70).

The intracellular molecular mechanism of how dual costimula-tion enhanced clonal expansion and increased accumulation of ef-fector CD8 T cells is currently unclear, but is at least predicated ona heightened survival signaling program. Specifically, dual co-stimulation may increase growth factor or IL-2-driven clonal ex-pansion, followed by intensified IL-7 survival signaling leading toaccumulation of supereffector cells. Because both costimulatorypathways function through TRAFs, dual costimulation may ulti-mately depend on an enhanced loading, or poising of NF-�B com-ponents (30, 31, 71).

In summary, our results demonstrate how CD8 T cell super-effector responses develop after dual costimulation. This wasbased on CD137 and CD134 augmenting IL-7 signaling on CD8T cells driving accumulation of supereffectors. Ultimately, thisstudy may provide a protocol for rapid induction of CD8 ef-fector responses in the absence of functional T cell help, suchas that during AIDS.

DisclosuresThe authors have no financial conflict of interest.

References1. Watts, T. H. 2005. TNF/TNFR family members in costimulation of T cell re-

sponses. Annu. Rev. Immunol. 23: 23–68.

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2. Gramaglia, I., A. D. Weinberg, M. Lemon, and M. Croft. 1998. OX-40 ligand: apotent costimulatory molecule for sustaining primary CD4 T cell responses.J. Immunol. 161: 6510–6517.

3. Weinberg, A. D., A. T. Vella, and M. Croft. 1998. OX-40: life beyond the effectorT cell stage. Semin. Immunol. 10: 471–480.

4. Sugamura, K., N. Ishii, and A. D. Weinberg. 2004. Therapeutic targeting of theeffector T-cell co-stimulatory molecule OX40. Nat. Rev. Immunol. 4: 420–431.

5. Valzasina, B., C. Guiducci, H. Dislich, N. Killeen, A. D. Weinberg, andM. P. Colombo. 2005. Triggering of OX40 (CD134) on CD4�CD25� T cellsblocks their inhibitory activity: a novel regulatory role for OX40 and its com-parison with GITR. Blood 105: 2845.

6. Hoshino, A., Y. Tanaka, H. Akiba, Y. Asakura, Y. Mita, T. Sakurai, A. Takaoka,S. Nakaike, N. Ishii, K. Sugamura, et al. 2003. Critical role for OX40 ligand inthe development of pathogenic Th2 cells in a murine model of asthma. Eur.J. Immunol. 33: 861–869.

7. Salek-Ardakani, S., J. Song, B. S. Halteman, A. G. Jember, H. Akiba, H. Yagita,and M. Croft. 2003. OX40 (CD134) controls memory T helper 2 cells that drivelung inflammation. J. Exp. Med. 198: 315–324.

8. Shuford, W. W., K. Klussman, D. D. Tritchler, D. T. Loo, J. Chalupny,A. W. Siadak, T. J. Brown, J. Emswiler, H. Raecho, C. P. Larsen, et al. 1997.4-1BB costimulatory signals preferentially induce CD8� T cell proliferation andlead to the amplification in vivo of cytotoxic T cell responses. J. Exp. Med. 186:47–55.

9. Takahashi, C., R. S. Mittler, and A. T. Vella. 1999. Cutting edge: 4-1BB is a bonafide CD8 T cell survival signal. J. Immunol. 162: 5037–5040.

10. Cooper, D., P. Bansal-Pakala, and M. Croft. 2002. 4-1BB (CD137) controls theclonal expansion and survival of CD8 T cells in vivo but does not contribute tothe development of cytotoxicity. Eur. J. Immunol. 32: 521–529.

11. Hurtado, J. C., Y. J. Kim, and B. S. Kwon. 1997. Signals through 4-1BB arecostimulatory to previously activated splenic T cells and inhibit activation-in-duced cell death. J. Immunol. 158: 2600–2609.

12. Cannons, J. L., P. Lau, B. Ghumman, M. A. DeBenedette, H. Yagita,K. Okumura, and T. H. Watts. 2001. 4-1BB ligand induces cell division, sustainssurvival, and enhances effector function of CD4 and CD8 T cells with similarefficacy. J. Immunol. 167: 1313–1324.

13. Lee, H. W., S. J. Park, B. K. Choi, H. H. Kim, K. O. Nam, and B. S. Kwon. 2002.4-1BB promotes the survival of CD8� T lymphocytes by increasing expressionof Bcl-xL and Bfl-1. J. Immunol. 169: 4882–4888.

14. Sun, Y., X. Lin, H. M. Chen, Q. Wu, S. K. Subudhi, L. Chen, and Y. X. Fu. 2002.Administration of agonistic anti-4-1BB monoclonal antibody leads to the ame-lioration of experimental autoimmune encephalomyelitis. J. Immunol. 168:1457–1465.

15. Myers, L., C. Takahashi, R. S. Mittler, R. J. Rossi, and A. T. Vella. 2003. EffectorCD8 T cells possess suppressor function after 4-1BB and Toll-like receptor trig-gering. Proc. Natl. Acad. Sci. USA 100: 5348–5353.

16. Myers, L., M. Croft, B. S. Kwon, R. S. Mittler, and A. T. Vella. 2005. Peptide-specific CD8 T regulatory cells use IFN-� to elaborate TGF-�-based suppression.J. Immunol. 174: 7625–7632.

17. Lee, S. W., A. T. Vella, B. S. Kwon, and M. Croft. 2005. Enhanced CD4 T cellresponsiveness in the absence of 4-1BB. J. Immunol. 174: 6803–6808.

18. Menoret, A., L. M. Myers, S. J. Lee, R. S. Mittler, R. J. Rossi, and A. T. Vella.2006. TGFb protein processing and activity through TCR triggering of primaryCD8� T regulatory cells. J. Immunol. 177: 6091–6097.

19. Vinay, D. S., K. Cha, and B. S. Kwon. 2006. Dual immunoregulatory pathwaysof 4-1BB signaling. J. Mol. Med. 84: 726–736.

20. Gramaglia, I., D. Cooper, K. T. Miner, B. S. Kwon, and M. Croft. 2000. Co-stimulation of antigen-specific CD4 T cells by 4-1BB ligand. Eur. J. Immunol.30: 392–402.

21. De Smedt, T., J. Smith, P. Baum, W. Fanslow, E. Butz, and C. Maliszewski.2002. OX40 costimulation enhances the development of T cell responses inducedby dendritic cells in vivo. J. Immunol. 168: 661–670.

22. Bansal-Pakala, P., B. S. Halteman, M. H. Cheng, and M. Croft. 2004. Costimu-lation of CD8 T cell responses by OX40. J. Immunol. 172: 4821–4825.

23. Lee, S. J., L. Myers, G. Muralimohan, J. Dai, Y. Qiao, Z. Li, R. S. Mittler, andA. T. Vella. 2004. 4-1BB and OX40 dual costimulation synergistically stimulateprimary specific CD8 T cells for robust effector function. J. Immunol. 173:3002–3012.

24. Melero, I., W. W. Shuford, S. A. Newby, A. Aruffo, J. A. Ledbetter,K. E. Hellstrom, R. S. Mittler, and L. Chen. 1997. Monoclonal antibodies againstthe 4-1BB T-cell activation molecule eradicate established tumors. Nat. Med. 3:682–685.

25. Cuadros, C., A. L. Dominguez, P. L. Lollini, M. Croft, R. S. Mittler,P. Borgstrom, and J. Lustgarten. 2005. Vaccination with dendritic cells pulsedwith apoptotic tumors in combination with anti-OX40 and anti-4-1BB monoclo-nal antibodies induces T cell-mediated protective immunity in Her-2/neu trans-genic mice. Int. J. Cancer 116: 934–943.

26. Uno, T., K. Takeda, Y. Kojima, H. Yoshizawa, H. Akiba, R. S. Mittler, F. Gejyo,K. Okumura, H. Yagita, and M. J. Smyth. 2006. Eradication of established tumorsin mice by a combination antibody-based therapy. Nat. Med. 12: 693–698.

27. Bukczynski, J., T. Wen, C. Wang, N. Christie, J. P. Routy, M. R. Boulassel,C. M. Kovacs, K. S. Macdonald, M. Ostrowski, R. P. Sekaly, et al. 2005. En-hancement of HIV-specific CD8 T cell responses by dual costimulation withCD80 and CD137L. J. Immunol. 175: 6378–6389.

28. Taraban, V. Y., T. F. Rowley, L. O’Brien, H. T. Chan, L. E. Haswell,M. H. Green, A. L. Tutt, M. J. Glennie, and A. al-Shamkhani. 2002. Expressionand costimulatory effects of the TNF receptor superfamily members CD134

(OX40) and CD137 (4-1BB), and their role in the generation of anti-tumor im-mune responses. Eur. J. Immunol. 32: 3617–3627.

29. Lee, S. W., Y. Park, A. Song, H. Cheroutre, B. S. Kwon, and M. Croft. 2006.Functional dichotomy between OX40 and 4-1BB in modulating effector CD8 Tcell responses. J. Immunol. 177: 4464–4472.

30. Arch, R. H., and C. B. Thompson. 1998. 4-1BB and OX40 are members of atumor necrosis factor (TNF)-nerve growth factor receptor subfamily that bindTNF receptor-associated factors and activate nuclear factor �B. Mol. Cell Biol.18: 558–565.

31. Dempsey, P. W., S. E. Doyle, J. Q. He, and G. Cheng. 2003. The signalingadaptors and pathways activated by TNF superfamily. Cytokine Growth FactorRev. 14: 193–209.

32. Chen, A. I., A. J. McAdam, J. E. Buhlmann, S. Scott, M. L. Lupher,E. A. Greenfield, P. R. Baum, W. C. Fanslow, D. M. Calderhead, G. J. Freeman,and A. H. Sharpe. 1999. OX40-ligand has a critical costimulatory role in dendriticcell: T cell interactions. Immunity 11: 689–698.

33. Kopf, M., C. Ruedl, N. Schmitz, A. Gallimore, K. Lefrang, B. Ecabert,B. Odermatt, and M. F. Bachmann. 1999. OX40-deficient mice are defective inTh cell proliferation but are competent in generating B cell and CTL responsesafter virus infection. Immunity 11: 699–708.

34. Gramaglia, I., A. Jember, S. D. Pippig, A. D. Weinberg, N. Killeen, and M. Croft.2000. The OX40 costimulatory receptor determines the development of CD4memory by regulating primary clonal expansion. J. Immunol. 165: 3043–3050.

35. Dawicki, W., E. M. Bertram, A. H. Sharpe, and T. H. Watts. 2004. 4-1BB andOX40 act independently to facilitate robust CD8 and CD4 recall responses. J. Im-munol. 173: 5944–5951.

36. Pollok, K. E., Y. J. Kim, Z. Zhou, J. C. Hurtado, K. K. Kim, R. T. Pickard, andB. S. Kwon. 1993. Inducible T cell antigen 4-1BB: analysis of expression andfunction. J. Immunol. 150: 771–781.

37. Halstead, E. S., Y. M. Mueller, J. D. Altman, and P. D. Katsikis. 2002. In vivostimulation of CD137 broadens primary antiviral CD8� T cell responses. Nat.Immunol. 3: 536–541.

38. Dawicki, W., and T. H. Watts. 2004. Expression and function of 4-1BB duringCD4 versus CD8 T cell responses in vivo. Eur. J. Immunol. 34: 743–751.

39. Wilcox, R. A., A. I. Chapoval, K. S. Gorski, M. Otsuji, T. Shin, D. B. Flies,K. Tamada, R. S. Mittler, H. Tsuchiya, D. M. Pardoll, and L. Chen. 2002. Cuttingedge: expression of functional CD137 receptor by dendritic cells. J. Immunol.168: 4262–4267.

40. Futagawa, T., H. Akiba, T. Kodama, K. Takeda, Y. Hosoda, H. Yagita, andK. Okumura. 2002. Expression and function of 4-1BB and 4-1BB ligand onmurine dendritic cells. Int. Immunol. 14: 275–286.

41. Wilcox, R. A., K. Tamada, S. E. Strome, and L. Chen. 2002. Signaling throughNK cell-associated CD137 promotes both helper function for CD8� cytolytic Tcells and responsiveness to IL-2 but not cytolytic activity. J. Immunol. 169:4230–4236.

42. Melero, I., J. V. Johnston, W. W. Shufford, R. S. Mittler, and L. Chen. 1998.NK1.1 cells express 4-1BB (CDw137) costimulatory molecule and are requiredfor tumor immunity elicited by anti-4-1BB monoclonal antibodies. Cell. Immu-nol. 190: 167–172.

43. Zheng, G., B. Wang, and A. Chen. 2004. The 4-1BB costimulation augments theproliferation of CD4�CD25� regulatory T cells. J. Immunol. 173: 2428–2434.

44. Nishimoto, H., S. W. Lee, H. Hong, K. G. Potter, M. Maeda-Yamamoto,T. Kinoshita, Y. Kawakami, R. S. Mittler, B. S. Kwon, C. F. Ware, et al. 2005.Costimulation of mast cells by 4-1BB, a member of the tumor necrosis factorreceptor superfamily, with the high-affinity IgE receptor. Blood 106: 4241–4248.

45. al-Shamkhani, A., M. L. Birkeland, M. Puklavec, M. H. Brown, W. James, andA. N. Barclay. 1996. OX40 is differentially expressed on activated rat and mouseT cells and is the sole receptor for the OX40 ligand. Eur. J. Immunol. 26:1695–1699.

46. Lee, S. K., J. F. Kalinowski, S. L. Jastrzebski, L. Puddington, and J. A. Lorenzo.2003. Interleukin-7 is a direct inhibitor of in vitro osteoclastogenesis. Endocri-nology 144: 3524–3531.

47. Klonowski, K. D., K. J. Williams, A. L. Marzo, and L. Lefrancois. 2006. Cuttingedge: IL-7-independent regulation of IL-7 receptor � expression and memoryCD8 T cell development. J. Immunol. 177: 4247–4251.

48. Kwon, B. S., J. C. Hurtado, Z. H. Lee, K. B. Kwack, S. K. Seo, B. K. Choi,B. H. Koller, G. Wolisi, H. E. Broxmeyer, and D. S. Vinay. 2002. Immuneresponses in 4-1BB (CD137)-deficient mice. J. Immunol. 168: 5483–5490.

49. Masopust, D., V. Vezys, A. L. Marzo, and L. Lefrancois. 2001. Preferentiallocalization of effector memory cells in nonlymphoid tissue. Science 291:2413–2417.

50. Unkeless, J. C. 1979. Characterization of a monoclonal antibody directed againstmouse macrophage and lymphocyte Fc receptors. J. Exp. Med. 150: 580–596.

51. Sallusto, F., D. Lenig, R. Forster, M. Lipp, and A. Lanzavecchia. 1999. Twosubsets of memory T lymphocytes with distinct homing potentials and effectorfunctions. Nature 401: 708–712.

52. Reinhardt, R. L., A. Khoruts, R. Merica, T. Zell, and M. K. Jenkins. 2001. Vi-sualizing the generation of memory CD4 T cells in the whole body. Nature 401:101–105.

53. Song, A., X. Tang, K. M. Harms, and M. Croft. 2005. OX40 and Bcl-xL promotethe persistence of CD8 T cells to recall tumor-associated antigen. J. Immunol.175: 3534–3541.

54. Ruby, C. E., W. L. Redmond, D. Haley, and A. D. Weinberg. 2007. Anti-OX40stimulation in vivo enhances CD8� memory T cell survival and significantlyincreases recall responses. Eur. J. Immunol. 37: 157–166.

2213The Journal of Immunology

by guest on Decem

ber 24, 2018http://w

ww

.jimm

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ownloaded from

55. Maxwell, J. R., R. Yadav, R. J. Rossi, C. E. Ruby, A. D. Weinberg, H. L. Aguila,and A. T. Vella. 2006. IL-18 bridges innate and adaptive immunity throughIFN-� and the CD134 pathway. J. Immunol. 177: 234–245.

56. Vinay, D. S., B. K. Choi, J. S. Bae, W. Y. Kim, B. M. Gebhardt, and B. S. Kwon.2004. CD137-deficient mice have reduced NK/NKT cell numbers and function,are resistant to lipopolysaccharide-induced shock syndromes, and have lowerIL-4 responses. J. Immunol. 173: 4218–4229.

57. Tan, J. T., J. K. Whitmire, R. Ahmed, T. C. Pearson, and C. P. Larsen. 1999.4-1BB ligand, a member of the TNF family, is important for the generation ofantiviral CD8 T cell responses. J. Immunol. 163: 4859–4868.

58. Futagawa, T., H. Akiba, T. Kodama, K. Takeda, Y. Hosoda, H. Yagita, andK. Okumura. 2002. Expression and function of 4-1BB and 4-1BB ligand onmurine dendritic cells. Int. Immunol. 14: 275–286.

59. Wilcox, R. A., A. I. Chapoval, K. S. Gorski, M. Otsuji, T. Shin, D. B. Flies,K. Tamada, R. S. Mittler, H. Tsuchiya, D. M. Pardoll, and L. Chen. 2002. Cuttingedge: expression of functional CD137 receptor by dendritic cells. J. Immunol.168: 4262–4267.

60. Myers, L., S. W. Lee, R. J. Rossi, L. Lefrancois, B. S. Kwon, R. S. Mittler,M. Croft, and A. T. Vella. 2006. Combined CD137 (4-1BB) and adjuvant therapygenerates a developing pool of peptide-specific CD8 memory T cells. Int. Im-munol. 18: 325–333.

61. Schluns, K. S., W. C. Kieper, S. C. Jameson, and L. Lefrancois. 2000. Interleu-kin-7 mediates the homeostasis of naive and memory CD8 T cells in vivo. Nat.Immunol. 1: 426–432.

62. Schluns, K. S., and L. Lefrancois. 2003. Cytokine control of memory T-celldevelopment and survival. Nat. Rev. Immunol. 3: 269–279.

63. Ma, A., R. Koka, and P. Burkett. 2006. Diverse functions of IL-2: IL-15, and IL-7in lymphoid homeostasis. Annu. Rev. Immunol. 24: 657–679.

64. Kaech, S. M., J. T. Tan, E. J. Wherry, B. T. Konieczny, C. D. Surh, andR. Ahmed. 2003. Selective expression of the interleukin 7 receptor identifieseffector CD8 T cells that give rise to long-lived memory cells. Nat. Immunol. 4:1191–1198.

65. Aoki, T., K. Tashiro, S. Miyatake, T. Kinashi, T. Nakano, Y. Oda, H. Kikuchi,and T. Honjo. 1992. Expression of murine interleukin 7 in a murine glioma cellline results in reduced tumorigenicity in vivo. Proc. Natl. Acad. Sci. USA 89:3850–3854.

66. Sica, D., P. Rayman, J. Stanley, M. Edinger, R. R. Tubbs, E. Klein, R. Bukowski,and J. H. Finke. 1993. Interleukin 7 enhances the proliferation and effector func-tion of tumor-infiltrating lymphocytes from renal-cell carcinoma. Int. J. Cancer53: 941–947.

67. Kim, T. S., S. W. Chung, and S. Y. Hwang. 2000. Augmentation of antitumorimmunity by genetically engineered fibroblast cells to express both B7.1 andinterleukin-7. Vaccine 18: 2886–2894.

68. Melchionda, F., T. J. Fry, M. J. Milliron, M. A. McKirdy, Y. Tagaya, andC. L. Mackall. 2005. Adjuvant IL-7 or IL-15 overcomes immunodominance andimproves survival of the CD8� memory cell pool. J. Clin. Invest. 115:1177–1187.

69. Carini, C., and M. Essex. 1994. Interleukin 2-independent interleukin 7 activityenhances cytotoxic immune response of HIV-1-infected individuals. AIDS Res.Hum. Retroviruses 10: 121–130.

70. Kim, H.-R., M. S. Hong, J. M. Dan, and I. Kang. 2006. Altered IL-7Ra expres-sion with aging and the potential implications of IL-7 therapy on CD8� T-cellimmune responses. Blood 107: 2855–2862.

71. Jang, I. K., Z. H. Lee, Y. J. Kim, S. H. Kim, and B. S. Kwon. 1998. Human4-1BB (CD137) signals are mediated by TRAF2 and activate nuclear factor-�B.Biochem. Biophys. Res. Commun. 242: 613–620.

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