CD27 mediates interleukin-2-independent clonal expansion of the CD8؉ without effector differentiation

James M. Carr, Marlene J. Carrasco, James E. D. Thaventhiran, Paul J. Bambrough, Matthew Kraman, Alexander D. Edwards, Aymen Al-Shamkhani*, and Douglas T. Fearon†

Wellcome Trust Immunology Unit, Department of Medicine, University of Cambridge, Medical Research Council Centre, Hills Road, Cambridge CB2 2QH, United Kingdom

Contributed by Douglas T. Fearon, November 1, 2006 (sent for review September 5, 2006) The clonal expansion of antigen-specific CD8؉ T cells in response to and its ligation by CD70 promotes proliferation in vitro of microbial infections is essential for adaptive immunity. Although TCR-stimulated CD8ϩ T cells (12, 15). CD27-deficient mice IL-2 has been considered to be primarily responsible for this have impaired clonal expansion of virus-specific CD8ϩ T cells process, quantitatively normal expansion occurs in the absence of during primary infection with influenza (16) and secondary ؉ IL-2 receptor signaling. Here, we show that ligating CD27 on CD8 infection with lymphocytic choriomeningitis virus (17). Mice T cells that have been stimulated through the T cell receptor causes constitutively expressing CD70 on B cells develop a T cell their expansion in the absence of IL-2 by mediating two distinct proliferative disease in the absence of CD95 (18). As for CD137, cellular processes: enhancing cell cycling and promoting cell sur- it is expressed by activated but not naı¨ve CD8ϩ T cells and is vival by maintaining the expression of IL-7 receptor ␣. This path- ϩ ؉ required for normal expansion of CD8 T cells during secondary way for clonal expansion of the CD8 T cell is not associated with infections (19). Therefore, we have evaluated these two members ␥ the development of a capacity either for production of IFN- or for of the TNF receptor superfamily for their potential roles in cytotoxic T function and, therefore, is uncoupled from IL-2-independent CD8ϩ T cell expansion. differentiation. Furthermore, ligating CD27 increases the threshold concentration at which IL-2 induces IFN-␥-producing capability by Results the CD8؉ T cell, suggesting that CD27 signaling may suppress Loss of Function Studies Identify a CD70/CD27-Dependent Pathway of -effector differentiation. Finally, CD8؉ T cells that have been stim CD8؉ T Cell Proliferation Without Effector Differentiation. For a ulated by the TCR/CD27 pathway maintain their capacity for subsequent expansion and effector differentiation in response to model antigen-presenting cell, we took advantage of the A20 lymphoma line that expresses CD70, CD137 ligand 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 (CD137L), CD80, CD86, and CD32, the Fc RII, which enables -؉ cross-linking of TCR-bound, agonistic antibody. For the re contribute to the continuous generation of new effector CD8 T ϩ cells in persistent viral infections. sponding CD8 T cell, we used transgenic OT-I cells (20) from RAGϪ/Ϫ mice that are specific for the ovalbumin-derived pep- b CD70 ͉ granzyme B ͉ Interferon-␥ ͉ TNF receptor superfamily tide, OVA257–264 (SIINFEKL) complexed to H-2K (pMHC). We cultured purified, CD62Lhigh OT-I cells with mitomycin ␧ he clonal distribution of antigen receptors, which is the C-treated A20 cells in the presence of anti-CD3 , IL-7, and Thallmark of the adaptive immune system, must be linked to blocking antibodies to IL-2 and CD25 to suppress IL-2R signal- a mechanism for rapid clonal expansion in response to microbial ing. The roles of CD27, CD137, and CD28 were assessed by the antigens. Although this requirement relates to all types of addition of blocking antibodies to their ligands. After 45 h, viable , it is perhaps most stringent for the CD8ϩ T cell OT-I cells were counted and cultured for an additional 45 h with because one of its antimicrobial functions, the killing of infected fresh mitomycin C-treated A20 cells and the original conditions. cells, requires direct cell-cell contact. For three decades, IL-2 has In the absence of ligand-blocking antibodies, the number of OT-I been considered to be primarily responsible for the clonal cells expanded 16-fold over 90 h in a manner that depended on expansion of the CD8ϩ T cell (1–3). However, over the past 10 anti-CD3␧ (Fig. 1). Blocking antibodies to CD137L, CD80, and years, studies have shown that expansion of CD8ϩ T cells in CD86 were without effect, whereas anti-CD70 inhibited expan- primary viral infections can occur in the absence of IL-2 receptor sion by 70%. In the absence of anti-CD3␧, no cells were (IL-2R) signaling (4–8). Furthermore, prior IL-2R stimulation recovered at 90 h. Therefore, ligation of CD27, but not CD137 of CD8ϩ T cells has been shown to be necessary (8), unnecessary (7), or even detrimental (9) for their subsequent in vivo expan- sion in response to viral challenge. These different experimental Author contributions: J.M.C. and M.J.C. contributed equally to this work; J.M.C., M.J.C., outcomes reflect different experimental protocols and indicate J.E.D.T., P.J.B., and D.T.F. designed research; J.M.C., M.J.C., J.E.D.T., P.J.B., M.K., and A.D.E. ϩ performed research; A.A.-S. contributed new reagents/analytic tools; J.M.C., M.J.C., that the regulation of CD8 T cell clonal expansion and differ- J.E.D.T., P.J.B., M.K., A.D.E., and D.T.F. analyzed data; and J.M.C., M.J.C., and D.T.F. wrote entiation is still incompletely understood. the paper. In contrast to the many studies of the role of IL-2 in mediating The authors declare no conflict of interest. ϩ the proliferation and effector differentiation of the CD8 T cell, Freely available online through the PNAS open access option. little is known of the mechanism or biological role of the Abbreviations: CFSE, carboxyfluorescein succinimidyl ester; IL-2R, IL-2 receptor. IL-2-independent pathway. In an attempt to identify this path- *Present address: Tenovus Research Laboratory, Cancer Sciences Division, University of way, we focused our attention on the TNF receptor superfamily Southampton School of Medicine, Southampton General Hospital, Tremona Road, because of the example of -independent proliferation of Southampton SO15 5PA, United Kingdom. B cells stimulated through CD40 (10). Of the several members †To whom correspondence should be addressed. E-mail: [email protected]. ϩ of the TNF receptor superfamily expressed by CD8 T cells, This article contains supporting information online at www.pnas.org/cgi/content/full/ CD27 (11, 12) and CD137 (13, 14) are involved in cellular 0609706103/DC1. ϩ proliferation. CD27 is expressed by resting, naı¨ve CD8 T cells, © 2006 by The National Academy of Sciences of the USA

19454–19459 ͉ PNAS ͉ December 19, 2006 ͉ vol. 103 ͉ no. 51 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0609706104 Downloaded by guest on September 26, 2021 blocked required ligation of both TCR by anti-CD3␧ and CD27 by CD70 (Fig. 2 a and b). The absence of IL-7 caused a decrease in expansion of the TCR/CD27-stimulated OT-I cells (Fig. 2a, isotype Ϯ IL-7) but not of the TCR-stimulated cells (Fig. 2a, anti-CD70 Ϯ 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 CD8ϩ 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 coli- gating CD27 maintained the expression of IL-7R␣, even in the presence of IL-7, whereas activation through the TCR alone down-regulated IL-7R␣ (Fig. 2c). Therefore, costimulation by CD27 at least partially counteracts the known suppressive effects ␣ ϩ of IL-7 and TCR stimulation on IL-7R expression (21, 22). This Fig. 1. The TNF receptor superfamily members, CD27 and CD137, and CD8 enables IL-7 to promote the viability, but not proliferation, of the T cell expansion in the presence of blocking antibodies to IL-2 and CD25. OT-I ϩ ϩ TCR/CD27-stimulated CD8 T cell. Without being able to CD8 T cells were stimulated with mitomycin C-treated A20 cells in the presence or absence of 20 ng/ml anti-CD3␧, IL-7, and anti-IL-2/anti-CD25. respond to IL-7, the OT-I cells activated solely through the TCR Cultures contained the indicated blocking or isotype control antibodies. After cycle but die. ϩ 45 h, CD8ϩ T cells were restimulated by using the original culture conditions We compared the effector differentiation of OT-I CD8 T and then assessed for expansion at 90 h. Comparable results were obtained in cells that had been stimulated for 6 days with TCR ligation in the three similar experiments. presence of CD70 or IL-2. Relative to naı¨ve, unstimulated OT-I cells, cells that had proliferated in response to the TCR/IL-2R

ϩ pathway contained increased granzyme B, had down-regulated or CD28, promotes TCR-dependent expansion of CD8 T cells CD62L, were capable of synthesizing IFN-␥, and had CTL in the apparent absence of IL-2R stimulation. activity. In contrast, stimulation through the TCR/CD27 path- The role of IL-7 in this TCR- and CD27-dependent response way enhanced expression of CD62L, did not induce granzyme B, was examined by stimulating carboxyfluorescein succinimidyl caused only 25% of the cells to be capable of IFN-␥ production, ϩ ␧ ester (CFSE)-labeled OT-I CD8 T cells with anti-CD3 and and did not stimulate CTL differentiation [supporting informa- A20 cells in the presence or absence of this cytokine. Cell tion (SI) Fig. 7]. expansion and optimal cell cycling when IL-2R signaling was Determination of the Minimal Requirements for the TCR/CD27 Path- way. The antibody-blocking studies identify CD27 as an essential costimulatory receptor for mediating clonal expansion of the CD8ϩ 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 CD8ϩ T cell-only system by relying on presentation of pMHC by OT-I cells (23) in which the Il2 gene had been interrupted. IL-2Ϫ/Ϫ 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 CD8ϩ 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-2Ϫ/Ϫ OT-I cells expanded 5-fold and

1.5-fold by 82 h, whereas the absence of peptide or sCD70 caused IMMUNOLOGY 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-CD3␧ (Fig. 3b). In the absence of sCD70, the viability of OT-I cells stimulated with 0.1 nM OVA peptide was so com- promised that cell cycling was essentially absent (SI Fig. 8). Similar results were obtained when IL-2Ϫ/Ϫ OT-I cells were stimulated in the A20 system (SI Fig. 9). We also determined in this CD8ϩ T cell-only system whether the TCR/CD27 pathway causes effector differentiation by cul- Fig. 2. TCR/CD27-mediated clonal expansion and maintenance of IL-7R␣ Ϫ/Ϫ ϩ turing IL-2 OT-I cells with 1 nM OVA peptide, IL-7, and a expression. CFSE-labeled OT-I CD8 T cells were stimulated in the presence or range of sCD70 concentrations. There was a dose-dependent absence of IL-7 with A20 cells, anti-CD3␧ plus anti-IL-2/anti-CD25, and the ϩ indicated antibodies. At 90 h, CD8ϩ T cells were assessed for expansion (a), effect of sCD70 on the expansion of CD8 T cells (Fig. 4a) and, CFSE profiles (b), and IL-7R␣ expression (c). (b) Dotted histograms represent as with the A20 system, IL-7 was required for their optimal unstimulated cells. (c) Solid and dotted histograms represent staining with a expansion (Fig. 4a). Differentiation was then assessed at day 4 specific antibody and isotype control, respectively. Comparable results were with the OT-I cells that had received the optimal dose of 2.5 nM obtained in three similar experiments. sCD70 and compared with naı¨ve, unstimulated OT-I cells and

Carr et al. PNAS ͉ December 19, 2006 ͉ vol. 103 ͉ no. 51 ͉ 19455 Downloaded by guest on September 26, 2021 Fig. 3. TCR/CD27-mediated clonal expansion in an IL-2Ϫ/Ϫ CD8ϩ T cell-only system. IL-2Ϫ/Ϫ OT-I CD8ϩ T cells were cultured with variable concentrations of OVA peptide and either 2.5 nM sCD70 or anti-CD70. After 40 h, cells were restimulated with the original conditions plus IL-7 and then assessed at 82 h for expansion (a) and CFSE profiles (b) of unstimulated (dotted histogram) and stimulated (solid histogram) cells. Comparable results were obtained in three similar experiments.

OT-I cells that had been stimulated with 1 nM OVA peptide and IL-2 for 4 days. Although the TCR/CD27-stimulated cells in- creased their expression of CD44 to an even greater extent than did the TCR/IL-2R-stimulated cells, they resembled naı¨ve cells in their absence of IFN-␥ production and granzyme B expression Fig. 4. TCR/CD27 mediated clonal expansion without effector differentia- and in their maintenance of CD62L expression; CCR7 was tion. (a) IL-2Ϫ/Ϫ OT-I CD8ϩ T cells were cultured with 1 nM OVA peptide and partially down-regulated (Fig. 4b). The absence of IFN-␥ pro- anti-CD70 or variable concentrations of sCD70. After 45 h, cells were restim- duction was not caused by Tc2 differentiation, because the ulated with the original conditions, with or without IL-7, and then assessed for expansion at 90 h. (b) IL-2Ϫ/Ϫ CD8ϩ T cells were stimulated with 1 nM OVA TCR/CD27-stimulated cells did not produce IL-4 or IL-10 (SI ϩ peptide and 2.5 nM sCD70 or with 1 nM OVA peptide and IL-2 in the presence Fig. 10). CD8 T cells cultured in the presence of IL-2 acquired of anti-CD70. After 45 h, cells with sCD70 were restimulated with the original an effector phenotype, although there was only partial down- conditions plus IL-7, whereas cells with IL-2 were restimulated only by the regulation of CD62L (Fig. 4b). Therefore, the absence of addition of IL-2 and IL-7. Naı¨veand 90-h cultured CD8ϩ T cells were assessed effector differentiation is a characteristic of the TCR/CD27 for expression of selected and capacity for cytokine synthesis after pathway of CD8ϩ T cell clonal expansion. restimulation with OVA peptide (intracellular cytokine stain) (solid histogram, ϩ specific antibody; dotted histogram, isotype control). Numbers in histograms CD27-stimulated CD8 T cells are sensitive to CD95- represent the mean fluorescence intensity of staining with specific antibody. mediated fratricide in vivo (18). To determine whether avoiding Comparable results were obtained in three similar experiments. this process might improve the expansion of TCR/CD27- stimulated cells in vitro, we cultured variable numbers of Thy1.2ϩ OT-I cells alone or in the presence of sufficient Thy1.1ϩ, class can mediate marked, IL-2-independent expansion of pMHC- II-depleted splenocytes to achieve a total of 200,000 cells per specific CD8ϩ T cells in vitro. culture. Cells were stimulated by the addition of 10 nM OVA peptide and sCD70 in the presence of antibodies to IL-2, CD25, Signaling Through the TCR/CD27 Pathway and the Response of the and CD95L. After 3 days, cells were washed and recultured with CD8؉ T Cell to IL-2. Stimulation of the CD8ϩ T cell by the the original conditions for three more days. IL-7 was added at the TCR/CD27 pathway is a means for IL-2-independent clonal beginning in one experiment and on day 2 in a second experi- expansion without differentiation, but the potential presence of ment. Diluting OT-I cells with nonspecific splenocytes greatly IL-2 in vivo suggests that it may be necessary for the CD27- enhanced their expansion, whereas culturing a low number of stimulated CD8ϩ T cell to modify IL-2R signaling to avoid OT-I cells alone did not (Table 1). Expansion required sCD70. effector differentiation. To examine this possibility, IL-2Ϫ/Ϫ Although other effects of the splenocytes, such as providing OT-I cells were activated with 1 nM OVA peptide in the like type I IFN (24) that promote survival of CD8ϩ T presence or absence of 1.5 nM sCD70 and increasing concen- cells, may have had a role, this experiment suggests that when trations of IL-2. After 45 h, the cells were enumerated, washed, CD95-mediated fratricide is avoided, the TCR/CD27 pathway and resuspended in the original culture conditions with the

19456 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0609706104 Carr et al. Downloaded by guest on September 26, 2021 Table 1. In vitro expansion of TCR͞CD27-stimulated OT-I CD8؉ T on the expression of granzyme B induced by IL-2 (Fig. 5 c and cells admixed with nontransgenic splenocytes d). In contrast, sCD70 fully inhibited the ability of 1 ng/ml IL-2 ϩ ␥ Day 1: no. of Day 6: no. of to induce in CD8 T cells a capacity for TCR-induced IFN- Thy1.2ϩ OT-I Thy1.1ϩ Thy1.2ϩ OT-I Fold production and partially suppressed this effect at 2 ng/ml IL-2. ϩ cells splenocytes cells expansion Thus, costimulation of the CD8 T cell by CD27 alters IL-2R signaling in a manner that favors replication over effector 25,000 ϩ 219,128; 195,936 8.8; 7.8 differentiation. - 316,239; 141,922 12.6; 5.7 ϩ TCR/CD27-Stimulated CD8؉ T Cells as Progenitors of Effector Cells. To 50 ;43 249,280 ;214,758 5,000 - 78,678; 135,567 15.7; 27 determine whether CD8ϩ T cells that had been stimulated by the ϩ 500 153,229; 155,096 306; 310 TCR/CD27 pathway could serve as precursors of expanded - 2,604; 3,525 5.2; 7.0 ϩ ϩ numbers of effector CD8 T cells when physiologically stimu- 50 26,400; 49,823 528; 996 lated, 10,000 Thy1.2ϩ OT-I cells that had been cultured for 5 days - Too few to count in the presence of OVA peptide, sCD70, IL-7, and anti-IL-2/ ϩ The results of two independent experiments are shown. No OT-I cells were anti-CD25 were adoptively transferred to Thy1.1 C57BL/6 recovered on day 6 without sCD70. mice. One day later, the mice were infected with recombinant vaccinia expressing OVA , and 8 days after infection, the number and phenotype of H-2Kb-OVA tetramer-binding CD8ϩ addition of IL-7. After 90 h, the cells were counted and assessed splenocytes were assessed. No Thy1.2ϩ, CD8ϩ T cells could be for their content of granzyme B and TCR-mediated production detected in two uninfected mice, reflecting the small number of of IFN-␥. Costimulation by CD27 did not alter the threshold OT-I cells that had been transferred. In virus-challenged mice, concentration of IL-2 for promoting expansion of the OT-I cells, 1.2 Ϯ 0.6% (n ϭ 4; mean Ϯ 2 SD) of total CD8ϩ T cells were this being 1 ng/ml (Fig. 5 a and b). The possibly nonphysiological donor-derived, which was similar to the percent of recipient concentration of 20 ng/ml IL-2 was associated with contraction CD8ϩ T cells binding H-2Kb-OVA tetramer (1.0 Ϯ 1%; n ϭ 4). of the OT-I cells in the presence of sCD70, perhaps reflecting the Immediately before transfer, the TCR/CD27-stimulated OT-I effects of increased CD95 expression caused by CD27 signaling cells lacked a capacity for IFN-␥ synthesis, had no detectable (18) on IL-2-dependent, TCR-mediated activation-induced cell granzyme B, and were CD62L high (Fig. 6). After responding to death. Costimulation by CD27 did not have a consistent effect vaccinia/OVA in vivo, the OT-I cells had acquired an effector IMMUNOLOGY

Fig. 5. CD27 costimulation and suppression of IL-2-mediated effector differentiation of CD8ϩ T cells. IL-2Ϫ/Ϫ OT-I CD8ϩ 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 (a and c) or anti-CD70 (b and d). After 45 h, cells were restimulated with the original conditions plus IL-7 and then assessed at 90 h for expansion (a and b) and expression of granzyme B and capacity for IFN-␥ synthesis after restimulation with OVA peptide (c and d). (c and d Left) Solid and dotted histograms represent staining with specific antibody and isotype control, respectively. (c and d Right) Solid and dotted histograms represent specific IFN-␥ staining in cells restimulated or not with peptide, respectively. Numbers in histograms represent the mean fluorescence intensity of staining for granzyme B and for IFN-␥ in peptide-stimulated cells. Comparable results were obtained in two similar experiments.

Carr et al. PNAS ͉ December 19, 2006 ͉ vol. 103 ͉ no. 51 ͉ 19457 Downloaded by guest on September 26, 2021 This study raises the question of what role the TCR/CD27 pathway has in the biology of the CD8ϩ 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 CD8ϩ T cell that mediates secondary proliferative responses and the avoidance of replicative senescence. The central memory CD8ϩ T cell has a relatively undifferentiated phenotype, being CCR7ϩ, CD62Lhigh, IL-7R␣ϩ, and, at least for human central memory cells, lacking a capacity for producing IFN-␥ (29), all of which are characteristics of the TCR/CD27- stimulated CD8ϩ 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 CD8ϩ T cells that are capable of enhanced secondary expansion (8). The question of senescence is more relevant to the long term, ϩ ϩ continuous generation of new effector CD8 T cells during Fig. 6. TCR/CD27-stimulated CD8 T cells in vitro as progenitors of virally persistent viral infections than to intermittent clonal expansion induced effector cells in vivo. Thy1.2ϩ OT-I CD8ϩ T cells were stimulated with occurring with recurrent infections. It has been proposed that 1 nM OVA peptide and 0.9 nM sCD70-Ig in the presence of anti-IL-2/anti-CD25. ϩ After 45 and 90 h, cells were restimulated by using the original culture pMHC-stimulated CD8 T cells avoid replicative senescence by conditions plus IL-7. After 5 days, the CD8ϩ T cells were assessed for expression having a subset of relatively undifferentiated cells with a capa- of granzyme B and CD62L and for peptide-induced IFN-␥ synthesis, and 10,000 bility for self-renewal and that this subset develops by modifying of these cells were adoptively transferred to Thy1.1ϩ C57BL/6 recipients. The or avoiding signaling through IL-2R (30, 31). Replicative senes- following day, mice were infected with vaccinia-OVA. Eight days after infec- cence does occur in CD8ϩ T cells in persistent viral infections, ϩ ϩ tion, Thy1.2 , CD8 splenocytes were reassessed for differentiation (solid and senescence is associated with effector differentiation be- histogram, specific antibody; dotted histogram, isotype control). Numbers in cause senescent cells have TCR-mediated effector functions (9, histograms represent the mean fluorescence intensity of staining with specific 32–35). Although a recent report has suggested that recruitment antibody. Comparable results were obtained in two similar experiments. of new thymic emigrants can contribute to the CD8ϩ T cell response in persistent viral infections (36), the production of ϩ phenotype (Fig. 6). Thus, CD8ϩ T cells stimulated by the effector CD8 T cells by this means would become limiting as TCR/CD27 pathway retain the potential for further expansion thymic involution progressed with aging. The TCR/CD27 path- and effector differentiation. way for clonal expansion without effector differentiation of CD8ϩ T cells might provide an additional mechanism by which Discussion these cells cope with persistent viral infections. Finally, the present study may offer an improved means for the The present study finds that a TCR/CD27 pathway mediates the ϩ in vitro generation of CD8 T cells specific for virus- or expansion of the CD8ϩ T cell in the absence of signaling via the tumor-associated antigens for adoptive T cell therapy. Expansion IL-2R and without effector differentiation. Costimulation by of CD8ϩ T cells in vitro by repetitive stimulation in the presence CD27 promotes the two cellular responses that are required for of IL-2 causes loss of in vivo replicative function and diminished clonal expansion: cell cycling and enhanced survival (Figs. 1–4). effectiveness after adoptive transfer (9). The finding that the The latter is at least in part an indirect rather than direct effect, TCR/CD27 pathway can mediate significant clonal expansion in as had been proposed for CD27 (25), and is mediated by CD27 vitro (Table 1) suggests that it may be possible to develop counteracting suppression by TCR ligation of IL-7R␣ expression protocols for generating sufficient numbers of relatively undif- (Fig. 2). This enables the TCR/CD27-stimulated cell to respond ferentiated CD8ϩ T cells that will retain replicative function and to IL-7 selectively with enhanced viability, the cytokine having a capacity for effector differentiation after adoptive transfer no effect on cell cycling. This finding is interesting given the (Fig. 6). central importance of IL-7R␣ expression for maintaining the levels of clonally expanded CD8ϩ T cells in vivo (26, 27). Materials and Methods The aspect of the TCR/CD27 pathway that most clearly Mice, Reagents, Cell Lines, and Culture Conditions. Full details are distinguishes it from IL-2R-mediated cellular proliferation is the published in SI Materials and Methods. absence of effector differentiation (Figs. 4 and 6). Interestingly, the absence of full-effector differentiation also characterizes Soluble CD70 Constructs. CD70-Ig is a fusion protein between the IL-2-independent expansion of CD8ϩ T cells in vivo (4, 7). extracellular domain of murine CD70 and the Fc region of Further, stimulation through CD27 even diminishes effector human IgG1 (15). Soluble trimeric CD70 was constructed by differentiation in response to limited IL-2R signaling, as assessed fusing amino acids 41–195 of murine CD70, containing a sub- stitution of Cys to Ser at amino acid position 194, to a trimer- by IFN-␥ production (Fig. 5). Because this effect occurred ization domain. Molar concentrations of soluble CD70 proteins without altering the replicative response to IL-2, the inhibitory were calculated based on the molecular mass of the monomeric effect of CD27 is selective and at an undefined post-IL-2R step. form. See SI Materials and Methods for details. Thus, even if IL-2 is available, subsets of CD8ϩ T cells respond- ing to TCR/CD27 stimulation could expand without differenti- CD8؉ T Cell Purification. Naı¨ve CD8ϩ T cells were purified from ating to effector cells. The mechanism for this effect of CD27 the spleens of 2- to 4-month-old OT-I RAGϪ/Ϫ or IL-2Ϫ/Ϫ OT-I does not involve BCL6b, a transcriptional repressor that pro- RAGϪ/Ϫ mice. Spleens were homogenized and erythrocytes ϩ motes the secondary response of memory CD8 T cells (28) were lysed by using PUREGENE RBC lysis buffer. CD8ϩ T cells because, in contrast to CD27, BCL6b represses the proliferative were purified by negative selection with the Mouse CD8ϩ T Cell response to IL-2. Moreover, CD27 does not induce the expres- Isolation Kit. CD62Lhigh CD8ϩ T cells were isolated by positive sion of BCL6b (data not shown). selection with CD62L microbeads. The final population of naı¨ve

19458 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0609706104 Carr et al. Downloaded by guest on September 26, 2021 OT-I T cells was Ն98% CD8ϩ and Ն90% CD62Lhigh. For CD8ϩ Assays for CD8؉ T Cell Effector Differentiation. Cells were assessed T cell-only cultures, MHC class IIϩ cells also were removed for expression of selected proteins and capacity for cytokine before selection of CD62Lhigh cells by using a biotinylated synthesis. For analysis of cytokine production, cells were stim- anti-MHC class II antibody and antibiotin microbeads. Thy1.1ϩ, ulated with either 50 ng/ml phorbol 12-myristate 13-acetate and class II-depleted splenocytes were prepared by negative selec- 1 ␮M ionomycin or with 1 ␮M OVA peptide for 4 h at 37°C in tion with biotinylated anti-MHC class II, anti-CD11b, anti- the presence of either 2 ␮M monensin or 3 ␮g/ml brefeldin A. CD11c, anti-Ly6G antibodies and antibiotin microbeads. See SI Materials and Methods for staining protocols and analyses.

ϩ ϩ CFSE Labeling. See SI Materials and Methods for details. Adoptive Transfers. Naı¨ve Thy1.2 OT-I CD8 T cells from the spleens of female mice were stimulated by using OVA peptide Stimulation of Primary CD8؉ T Cells. Purified naı¨ve OT-I CD8ϩ T and sCD70-Ig in the presence of anti-IL-2 and anti-CD25; IL-7 cells were activated at 1 ϫ 105 cells in 200 ␮l of complete medium was added after the initial 45 h of culture. After 5 days, viable per well of a 96-well, flat bottomed tissue culture plate, using cells were isolated via density gradient centrifugation by using anti-CD3␧ and 2 ϫ 105 mitomycin C-treated A20 cells (see SI Ficoll-Paque PLUS, and 1 ϫ 104 cells were i.v. coinjected into female Thy1.1ϩ C57BL/6 recipients with 2 ϫ 106 CFSE-labeled Materials and Methods for treatment procedure). Blocking an- ϩ tibodies, or matched isotype controls, were included where total splenocytes from Thy1.1 C57BL/6 mice, to allow evalu- indicated at a final concentration of 5 ␮g/ml. Anti-CD95L (10 ation of the efficacy of the transfer. The following day, mice were ϫ 6 ␮g/ml) also was included where indicated. infected i.p. with 5 10 pfu vaccinia expressing the ovalbumin To activate CD8ϩ T cells by self-presentation of OVA peptide, protein. Eight days later, splenocytes were stained with peridinin purified naı¨ve IL-2Ϫ/Ϫ OT-I CD8ϩ T cells were cultured at 2 ϫ chlorophyll protein-conjugated anti-CD8, allophycocyanin- 105 cells in 200 ␮l of complete medium with or without OVA conjugated anti-Thy1.2 and phycoerythrin-conjugated b peptide in the presence of either sCD70 and 5 ␮g/ml isotype SIINFEKL/H-2K tetramers and analyzed by flow cytometry. control antibody, or with anti-CD70. Recombinant mouse IL-7 and IL-2 also were included where We thank Alan Rickinson and for their suggestions. This work was supported by grants from the Wellcome Trust, National indicated at 2 and 20 ng/ml, respectively, unless otherwise stated. ϩ Institutes of Health, and Medical Research Council (MRC) (to D.T.F.), The number of viable CD8 T cells was determined by flow the Wellcome Trust (to J.E.D.T.), the Simms Scholarship of the Uni- cytometry with counting beads, and cells were washed and versity of Cambridge (to J.M.C.), the MRC (to M.J.C.), and the James recultured as indicated. Baird Fund of the University of Cambridge (to P.B.).

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