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Proc Natl Acad Sci U S A. Dec 19, 2006; 103(51): 19454–19459.
Published online Dec 11, 2006. doi:  10.1073/pnas.0609706104
PMCID: PMC1697827

CD27 mediates interleukin-2-independent clonal expansion of the CD8+ T cell without effector differentiation


The clonal expansion of antigen-specific CD8+ T cells in response to microbial infections is essential for adaptive immunity. Although IL-2 has been considered to be primarily responsible for this process, quantitatively normal expansion occurs in the absence of IL-2 receptor signaling. Here, we show that ligating CD27 on CD8+ T cells that have been stimulated through the T cell receptor causes their expansion in the absence of IL-2 by mediating two distinct cellular processes: enhancing cell cycling and promoting cell survival by maintaining the expression of IL-7 receptor α. This pathway for clonal expansion of the CD8+ T cell is not associated with the development of a capacity either for production of IFN-γ or for cytotoxic T lymphocyte function and, therefore, is uncoupled from differentiation. Furthermore, ligating CD27 increases the threshold concentration at which IL-2 induces IFN-γ-producing capability by the CD8+ T cell, suggesting that CD27 signaling may suppress effector differentiation. Finally, CD8+ T cells that have been stimulated by the TCR/CD27 pathway maintain their capacity for subsequent expansion and effector differentiation in response to a viral challenge in vivo. Thus, the TCR/CD27 pathway enables the CD8+ T cell to replicate by a process of self-renewal, which may contribute to the continuous generation of new effector CD8+ T cells in persistent viral infections.

Keywords: CD70, granzyme B, Interferon-γ, TNF receptor superfamily

The clonal distribution of antigen receptors, which is the hallmark of the adaptive immune system, must be linked to a mechanism for rapid clonal expansion in response to microbial antigens. Although this requirement relates to all types of lymphocytes, it is perhaps most stringent for the CD8+ T cell because one of its antimicrobial functions, the killing of infected cells, requires direct cell-cell contact. For three decades, IL-2 has been considered to be primarily responsible for the clonal expansion of the CD8+ T cell (13). However, over the past 10 years, studies have shown that expansion of CD8+ T cells in primary viral infections can occur in the absence of IL-2 receptor (IL-2R) signaling (48). Furthermore, prior IL-2R stimulation of CD8+ T cells has been shown to be necessary (8), unnecessary (7), or even detrimental (9) for their subsequent in vivo expansion in response to viral challenge. These different experimental outcomes reflect different experimental protocols and indicate that the regulation of CD8+ T cell clonal expansion and differentiation is still incompletely understood.

In contrast to the many studies of the role of IL-2 in mediating the proliferation and effector differentiation of the CD8+ T cell, little is known of the mechanism or biological role of the IL-2-independent pathway. In an attempt to identify this pathway, we focused our attention on the TNF receptor superfamily because of the example of cytokine-independent proliferation of B cells stimulated through CD40 (10). Of the several members of the TNF receptor superfamily expressed by CD8+ T cells, CD27 (11, 12) and CD137 (13, 14) are involved in cellular proliferation. CD27 is expressed by resting, naïve CD8+ T cells, and its ligation by CD70 promotes proliferation in vitro of TCR-stimulated CD8+ T cells (12, 15). CD27-deficient mice have impaired clonal expansion of virus-specific CD8+ T cells during primary infection with influenza (16) and secondary infection with lymphocytic choriomeningitis virus (17). Mice constitutively expressing CD70 on B cells develop a T cell proliferative disease in the absence of CD95 (18). As for CD137, it is expressed by activated but not naïve CD8+ T cells and is required for normal expansion of CD8+ T cells during secondary infections (19). Therefore, we have evaluated these two members of the TNF receptor superfamily for their potential roles in IL-2-independent CD8+ T cell expansion.


Loss of Function Studies Identify a CD70/CD27-Dependent Pathway of CD8+ T Cell Proliferation Without Effector Differentiation.

For a model antigen-presenting cell, we took advantage of the A20 B cell lymphoma line that expresses CD70, CD137 ligand (CD137L), CD80, CD86, and CD32, the FcγRII, which enables cross-linking of TCR-bound, agonistic antibody. For the responding CD8+ T cell, we used transgenic OT-I cells (20) from RAG−/− mice that are specific for the ovalbumin-derived peptide, OVA257–264 (SIINFEKL) complexed to H-2Kb (pMHC). We cultured purified, CD62Lhigh OT-I cells with mitomycin C-treated A20 cells in the presence of anti-CD3ε, IL-7, and blocking antibodies to IL-2 and CD25 to suppress IL-2R signaling. The roles of CD27, CD137, and CD28 were assessed by the addition of blocking antibodies to their ligands. After 45 h, viable OT-I cells were counted and cultured for an additional 45 h with fresh mitomycin C-treated A20 cells and the original conditions. In the absence of ligand-blocking antibodies, the number of OT-I cells expanded 16-fold over 90 h in a manner that depended on anti-CD3ε (Fig. 1). Blocking antibodies to CD137L, CD80, and CD86 were without effect, whereas anti-CD70 inhibited expansion by 70%. In the absence of anti-CD3ε, no cells were recovered at 90 h. Therefore, ligation of CD27, but not CD137 or CD28, promotes TCR-dependent expansion of CD8+ T cells in the apparent absence of IL-2R stimulation.

Fig. 1.
The TNF receptor superfamily members, CD27 and CD137, and CD8+ T cell expansion in the presence of blocking antibodies to IL-2 and CD25. OT-I CD8+ T cells were stimulated with mitomycin C-treated A20 cells in the presence or absence of 20 ng/ml anti-CD3ε, ...

The role of IL-7 in this TCR- and CD27-dependent response was examined by stimulating carboxyfluorescein succinimidyl ester (CFSE)-labeled OT-I CD8+ T cells with anti-CD3ε and A20 cells in the presence or absence of this cytokine. Cell expansion and optimal cell cycling when IL-2R signaling was blocked required ligation of both TCR by anti-CD3ε and CD27 by CD70 (Fig. 2a and b). The absence of IL-7 caused a decrease in expansion of the TCR/CD27-stimulated OT-I cells (Fig. 2a, isotype ± IL-7) but not of the TCR-stimulated cells (Fig. 2a, anti-CD70 ± IL-7). IL-7 did not affect cell cycling in either stimulatory condition (Fig. 2b). Therefore, IL-7 enhances the expansion of TCR/CD27-stimulated CD8+ T cells by promoting their viability, not by driving cell cycling. An explanation for this selective effect of IL-7 was suggested by the finding that coligating CD27 maintained the expression of IL-7Rα, even in the presence of IL-7, whereas activation through the TCR alone down-regulated IL-7Rα (Fig. 2c). Therefore, costimulation by CD27 at least partially counteracts the known suppressive effects of IL-7 and TCR stimulation on IL-7Rα expression (21, 22). This enables IL-7 to promote the viability, but not proliferation, of the TCR/CD27-stimulated CD8+ T cell. Without being able to respond to IL-7, the OT-I cells activated solely through the TCR cycle but die.

Fig. 2.
TCR/CD27-mediated clonal expansion and maintenance of IL-7Rα expression. CFSE-labeled OT-I CD8+ T cells were stimulated in the presence or absence of IL-7 with A20 cells, anti-CD3ε plus anti-IL-2/anti-CD25, and the indicated antibodies. ...

We compared the effector differentiation of OT-I CD8+ T cells that had been stimulated for 6 days with TCR ligation in the presence of CD70 or IL-2. Relative to naïve, unstimulated OT-I cells, cells that had proliferated in response to the TCR/IL-2R pathway contained increased granzyme B, had down-regulated CD62L, were capable of synthesizing IFN-γ, and had CTL activity. In contrast, stimulation through the TCR/CD27 pathway enhanced expression of CD62L, did not induce granzyme B, caused only 25% of the cells to be capable of IFN-γ production, and did not stimulate CTL differentiation [supporting information (SI) Fig. 7].

Determination of the Minimal Requirements for the TCR/CD27 Pathway.

The antibody-blocking studies identify CD27 as an essential costimulatory receptor for mediating clonal expansion of the CD8+ T cell, but they cannot exclude the possibility that ligands other than CD80, CD86, and CD137L on A20 cells may be involved. Furthermore, the use of neutralizing antibodies to IL-2 and CD25 do not definitively exclude a contribution of IL-2R signaling. To confirm a requirement for signaling only through the TCR and CD27, we developed a CD8+ T cell-only system by relying on presentation of pMHC by OT-I cells (23) in which the Il2 gene had been interrupted. IL-2−/− OT-I cells were cultured with 1 nM, 0.1 nM, or no OVA peptide, and CD70 was provided in a soluble recombinant form, sCD70. In cultures lacking sCD70, anti-CD70 was added to exclude potential effects of CD70 expressed by activated CD8+ T cells (12). After 40 h, cells were washed and recultured under the original conditions with the addition of IL-7 and readdition of OVA peptide, which maintained optimal responsiveness of the cells to ligation of CD27. In the presence of sCD70 and 1 nM and 0.1 nM OVA peptide, respectively, the IL-2−/− OT-I cells expanded 5-fold and 1.5-fold by 82 h, whereas the absence of peptide or sCD70 caused few cells to be recovered at this time (Fig. 3a). The capacity of CD27 to promote cell cycling was more evident with the physiological stimulus of pMHC than had been apparent with anti-CD3ε (Fig. 3b). In the absence of sCD70, the viability of OT-I cells stimulated with 0.1 nM OVA peptide was so compromised that cell cycling was essentially absent (SI Fig. 8). Similar results were obtained when IL-2−/− OT-I cells were stimulated in the A20 system (SI Fig. 9).

Fig. 3.
TCR/CD27-mediated clonal expansion in an IL-2−/− CD8+ T cell-only system. IL-2−/− OT-I CD8+ T cells were cultured with variable concentrations of OVA peptide and either 2.5 nM sCD70 or anti-CD70. After 40 h, cells were ...

We also determined in this CD8+ T cell-only system whether the TCR/CD27 pathway causes effector differentiation by culturing IL-2−/− OT-I cells with 1 nM OVA peptide, IL-7, and a range of sCD70 concentrations. There was a dose-dependent effect of sCD70 on the expansion of CD8+ T cells (Fig. 4a) and, as with the A20 system, IL-7 was required for their optimal expansion (Fig. 4a). Differentiation was then assessed at day 4 with the OT-I cells that had received the optimal dose of 2.5 nM sCD70 and compared with naïve, unstimulated OT-I cells and OT-I cells that had been stimulated with 1 nM OVA peptide and IL-2 for 4 days. Although the TCR/CD27-stimulated cells increased their expression of CD44 to an even greater extent than did the TCR/IL-2R-stimulated cells, they resembled naïve cells in their absence of IFN-γ production and granzyme B expression and in their maintenance of CD62L expression; CCR7 was partially down-regulated (Fig. 4b). The absence of IFN-γ production was not caused by Tc2 differentiation, because the TCR/CD27-stimulated cells did not produce IL-4 or IL-10 (SI Fig. 10). CD8+ T cells cultured in the presence of IL-2 acquired an effector phenotype, although there was only partial down-regulation of CD62L (Fig. 4b). Therefore, the absence of effector differentiation is a characteristic of the TCR/CD27 pathway of CD8+ T cell clonal expansion.

Fig. 4.
TCR/CD27 mediated clonal expansion without effector differentiation. (a) IL-2−/− OT-I CD8+ T cells were cultured with 1 nM OVA peptide and anti-CD70 or variable concentrations of sCD70. After 45 h, cells were restimulated with the original ...

CD27-stimulated CD8+ T cells are sensitive to CD95-mediated fratricide in vivo (18). To determine whether avoiding this process might improve the expansion of TCR/CD27-stimulated cells in vitro, we cultured variable numbers of Thy1.2+ OT-I cells alone or in the presence of sufficient Thy1.1+, class II-depleted splenocytes to achieve a total of 200,000 cells per culture. Cells were stimulated by the addition of 10 nM OVA peptide and sCD70 in the presence of antibodies to IL-2, CD25, and CD95L. After 3 days, cells were washed and recultured with the original conditions for three more days. IL-7 was added at the beginning in one experiment and on day 2 in a second experiment. Diluting OT-I cells with nonspecific splenocytes greatly enhanced their expansion, whereas culturing a low number of OT-I cells alone did not (Table 1). Expansion required sCD70. Although other effects of the splenocytes, such as providing cytokines like type I IFN (24) that promote survival of CD8+ T cells, may have had a role, this experiment suggests that when CD95-mediated fratricide is avoided, the TCR/CD27 pathway can mediate marked, IL-2-independent expansion of pMHC-specific CD8+ T cells in vitro.

Table 1.
In vitro expansion of TCR/CD27-stimulated OT-I CD8+ T cells admixed with nontransgenic splenocytes

Signaling Through the TCR/CD27 Pathway and the Response of the CD8+ T Cell to IL-2.

Stimulation of the CD8+ T cell by the TCR/CD27 pathway is a means for IL-2-independent clonal expansion without differentiation, but the potential presence of IL-2 in vivo suggests that it may be necessary for the CD27-stimulated CD8+ T cell to modify IL-2R signaling to avoid effector differentiation. To examine this possibility, IL-2−/− OT-I cells were activated with 1 nM OVA peptide in the presence or absence of 1.5 nM sCD70 and increasing concentrations of IL-2. After 45 h, the cells were enumerated, washed, and resuspended in the original culture conditions with the addition of IL-7. After 90 h, the cells were counted and assessed for their content of granzyme B and TCR-mediated production of IFN-γ. Costimulation by CD27 did not alter the threshold concentration of IL-2 for promoting expansion of the OT-I cells, this being 1 ng/ml (Fig. 5a and b). The possibly nonphysiological concentration of 20 ng/ml IL-2 was associated with contraction of the OT-I cells in the presence of sCD70, perhaps reflecting the effects of increased CD95 expression caused by CD27 signaling (18) on IL-2-dependent, TCR-mediated activation-induced cell death. Costimulation by CD27 did not have a consistent effect on the expression of granzyme B induced by IL-2 (Fig. 5 c and d). In contrast, sCD70 fully inhibited the ability of 1 ng/ml IL-2 to induce in CD8+ T cells a capacity for TCR-induced IFN-γ production and partially suppressed this effect at 2 ng/ml IL-2. Thus, costimulation of the CD8+ T cell by CD27 alters IL-2R signaling in a manner that favors replication over effector differentiation.

Fig. 5.
CD27 costimulation and suppression of IL-2-mediated effector differentiation of CD8+ T cells. IL-2−/− OT-I CD8+ T cells were stimulated with 1 nM OVA peptide and increasing concentrations of IL-2 in the presence of either 1.5 nM sCD70-Ig ...

TCR/CD27-Stimulated CD8+ T Cells as Progenitors of Effector Cells.

To determine whether CD8+ T cells that had been stimulated by the TCR/CD27 pathway could serve as precursors of expanded numbers of effector CD8+ T cells when physiologically stimulated, 10,000 Thy1.2+ OT-I cells that had been cultured for 5 days in the presence of OVA peptide, sCD70, IL-7, and anti-IL-2/anti-CD25 were adoptively transferred to Thy1.1+ C57BL/6 mice. One day later, the mice were infected with recombinant vaccinia expressing OVA protein, and 8 days after infection, the number and phenotype of H-2Kb-OVA tetramer-binding CD8+ splenocytes were assessed. No Thy1.2+, CD8+ T cells could be detected in two uninfected mice, reflecting the small number of OT-I cells that had been transferred. In virus-challenged mice, 1.2 ± 0.6% (n = 4; mean ± 2 SD) of total CD8+ T cells were donor-derived, which was similar to the percent of recipient CD8+ T cells binding H-2Kb-OVA tetramer (1.0 ± 1%; n = 4). Immediately before transfer, the TCR/CD27-stimulated OT-I cells lacked a capacity for IFN-γ synthesis, had no detectable granzyme B, and were CD62L high (Fig. 6). After responding to vaccinia/OVA in vivo, the OT-I cells had acquired an effector phenotype (Fig. 6). Thus, CD8+ T cells stimulated by the TCR/CD27 pathway retain the potential for further expansion and effector differentiation.

Fig. 6.
TCR/CD27-stimulated CD8+ T cells in vitro as progenitors of virally induced effector cells in vivo. Thy1.2+ OT-I CD8+ T cells were stimulated with 1 nM OVA peptide and 0.9 nM sCD70-Ig in the presence of anti-IL-2/anti-CD25. After 45 and 90 h, cells were ...


The present study finds that a TCR/CD27 pathway mediates the expansion of the CD8+ T cell in the absence of signaling via the IL-2R and without effector differentiation. Costimulation by CD27 promotes the two cellular responses that are required for clonal expansion: cell cycling and enhanced survival (Figs. 114). The latter is at least in part an indirect rather than direct effect, as had been proposed for CD27 (25), and is mediated by CD27 counteracting suppression by TCR ligation of IL-7Rα expression (Fig. 2). This enables the TCR/CD27-stimulated cell to respond to IL-7 selectively with enhanced viability, the cytokine having no effect on cell cycling. This finding is interesting given the central importance of IL-7Rα expression for maintaining the levels of clonally expanded CD8+ T cells in vivo (26, 27).

The aspect of the TCR/CD27 pathway that most clearly distinguishes it from IL-2R-mediated cellular proliferation is the absence of effector differentiation (Figs. 4 and and6).6). Interestingly, the absence of full-effector differentiation also characterizes IL-2-independent expansion of CD8+ T cells in vivo (4, 7). Further, stimulation through CD27 even diminishes effector differentiation in response to limited IL-2R signaling, as assessed by IFN-γ production (Fig. 5). Because this effect occurred without altering the replicative response to IL-2, the inhibitory effect of CD27 is selective and at an undefined post-IL-2R step. Thus, even if IL-2 is available, subsets of CD8+ T cells responding to TCR/CD27 stimulation could expand without differentiating to effector cells. The mechanism for this effect of CD27 does not involve BCL6b, a transcriptional repressor that promotes the secondary response of memory CD8+ T cells (28) because, in contrast to CD27, BCL6b represses the proliferative response to IL-2. Moreover, CD27 does not induce the expression of BCL6b (data not shown).

This study raises the question of what role the TCR/CD27 pathway has in the biology of the CD8+ T cell. Its unique characteristic, relative to the TCR/IL-2R pathway, is absence of effector differentiation, and two responses may depend on such a pathway of clonal expansion: the generation of the central memory CD8+ T cell that mediates secondary proliferative responses and the avoidance of replicative senescence. The central memory CD8+ T cell has a relatively undifferentiated phenotype, being CCR7+, CD62Lhigh, IL-7Rα+, and, at least for human central memory cells, lacking a capacity for producing IFN-γ (29), all of which are characteristics of the TCR/CD27-stimulated CD8+ T cell. The modification, as opposed to total inhibition, of IL-2R signaling by CD27 stimulation to favor proliferation over acquisition of effector function may be related to the IL-2-dependent development of memory CD8+ T cells that are capable of enhanced secondary expansion (8).

The question of senescence is more relevant to the long term, continuous generation of new effector CD8+ T cells during persistent viral infections than to intermittent clonal expansion occurring with recurrent infections. It has been proposed that pMHC-stimulated CD8+ T cells avoid replicative senescence by having a subset of relatively undifferentiated cells with a capability for self-renewal and that this subset develops by modifying or avoiding signaling through IL-2R (30, 31). Replicative senescence does occur in CD8+ T cells in persistent viral infections, and senescence is associated with effector differentiation because senescent cells have TCR-mediated effector functions (9, 3235). Although a recent report has suggested that recruitment of new thymic emigrants can contribute to the CD8+ T cell response in persistent viral infections (36), the production of effector CD8+ T cells by this means would become limiting as thymic involution progressed with aging. The TCR/CD27 pathway for clonal expansion without effector differentiation of CD8+ T cells might provide an additional mechanism by which these cells cope with persistent viral infections.

Finally, the present study may offer an improved means for the in vitro generation of CD8+ T cells specific for virus- or tumor-associated antigens for adoptive T cell therapy. Expansion of CD8+ T cells in vitro by repetitive stimulation in the presence of IL-2 causes loss of in vivo replicative function and diminished effectiveness after adoptive transfer (9). The finding that the TCR/CD27 pathway can mediate significant clonal expansion in vitro (Table 1) suggests that it may be possible to develop protocols for generating sufficient numbers of relatively undifferentiated CD8+ T cells that will retain replicative function and a capacity for effector differentiation after adoptive transfer (Fig. 6).

Materials and Methods

Mice, Reagents, Cell Lines, and Culture Conditions.

Full details are published in SI Materials and Methods.

Soluble CD70 Constructs.

CD70-Ig is a fusion protein between the extracellular domain of murine CD70 and the Fc region of human IgG1 (15). Soluble trimeric CD70 was constructed by fusing amino acids 41–195 of murine CD70, containing a substitution of Cys to Ser at amino acid position 194, to a trimerization domain. Molar concentrations of soluble CD70 proteins were calculated based on the molecular mass of the monomeric form. See SI Materials and Methods for details.

CD8+ T Cell Purification.

Naïve CD8+ T cells were purified from the spleens of 2- to 4-month-old OT-I RAG−/− or IL-2−/− OT-I RAG−/− mice. Spleens were homogenized and erythrocytes were lysed by using PUREGENE RBC lysis buffer. CD8+ T cells were purified by negative selection with the Mouse CD8+ T Cell Isolation Kit. CD62Lhigh CD8+ T cells were isolated by positive selection with CD62L microbeads. The final population of naïve OT-I T cells was ≥98% CD8+ and ≥90% CD62Lhigh. For CD8+ T cell-only cultures, MHC class II+ cells also were removed before selection of CD62Lhigh cells by using a biotinylated anti-MHC class II antibody and antibiotin microbeads. Thy1.1+, class II-depleted splenocytes were prepared by negative selection with biotinylated anti-MHC class II, anti-CD11b, anti-CD11c, anti-Ly6G antibodies and antibiotin microbeads.

CFSE Labeling.

See SI Materials and Methods for details.

Stimulation of Primary CD8+ T Cells.

Purified naïve OT-I CD8+ T cells were activated at 1 × 105 cells in 200 μl of complete medium per well of a 96-well, flat bottomed tissue culture plate, using anti-CD3ε and 2 × 105 mitomycin C-treated A20 cells (see SI Materials and Methods for treatment procedure). Blocking antibodies, or matched isotype controls, were included where indicated at a final concentration of 5 μg/ml. Anti-CD95L (10 μg/ml) also was included where indicated.

To activate CD8+ T cells by self-presentation of OVA peptide, purified naïve IL-2−/− OT-I CD8+ T cells were cultured at 2 × 105 cells in 200 μl of complete medium with or without OVA peptide in the presence of either sCD70 and 5 μg/ml isotype control antibody, or with anti-CD70.

Recombinant mouse IL-7 and IL-2 also were included where indicated at 2 and 20 ng/ml, respectively, unless otherwise stated. The number of viable CD8+ T cells was determined by flow cytometry with counting beads, and cells were washed and recultured as indicated.

Assays for CD8+ T Cell Effector Differentiation.

Cells were assessed for expression of selected proteins and capacity for cytokine synthesis. For analysis of cytokine production, cells were stimulated with either 50 ng/ml phorbol 12-myristate 13-acetate and 1 μM ionomycin or with 1 μM OVA peptide for 4 h at 37°C in the presence of either 2 μM monensin or 3 μg/ml brefeldin A. See SI Materials and Methods for staining protocols and analyses.

Adoptive Transfers.

Naïve Thy1.2+ OT-I CD8+ T cells from the spleens of female mice were stimulated by using OVA peptide and sCD70-Ig in the presence of anti-IL-2 and anti-CD25; IL-7 was added after the initial 45 h of culture. After 5 days, viable cells were isolated via density gradient centrifugation by using Ficoll-Paque PLUS, and 1 × 104 cells were i.v. coinjected into female Thy1.1+ C57BL/6 recipients with 2 × 106 CFSE-labeled total splenocytes from Thy1.1+ C57BL/6 mice, to allow evaluation of the efficacy of the transfer. The following day, mice were infected i.p. with 5 × 106 pfu vaccinia expressing the ovalbumin protein. Eight days later, splenocytes were stained with peridinin chlorophyll protein-conjugated anti-CD8, allophycocyanin-conjugated anti-Thy1.2 and phycoerythrin-conjugated SIINFEKL/H-2Kb tetramers and analyzed by flow cytometry.

Supplementary Material

Supporting Information:


We thank Alan Rickinson and Jannie Borst for their suggestions. This work was supported by grants from the Wellcome Trust, National Institutes of Health, and Medical Research Council (MRC) (to D.T.F.), the Wellcome Trust (to J.E.D.T.), the Simms Scholarship of the University of Cambridge (to J.M.C.), the MRC (to M.J.C.), and the James Baird Fund of the University of Cambridge (to P.B.).


carboxyfluorescein succinimidyl ester
IL-2 receptor.


The authors declare no conflict of interest.

This article contains supporting information online at www.pnas.org/cgi/content/full/0609706104/DC1.


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