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CUTTING EDGE Cutting Edge: A Critical Role for CD70 in CD8 T Cell Priming by CD40-Licensed APCs1 Vadim Y. Taraban, Tania F. Rowley, and Aymen Al-Shamkhani2 The CD154/CD40 interaction is an important pathway of CD4 T cell help for CD8 T cell responses. In this study, we address the role of CD70, a member of the TNF superfamily and the ligand for the T cell costimulatory receptor CD27, in CD40-mediated priming of CD8 T cells. Using an agonistic anti-CD40 mAb to mimic the CD154/CD40 interaction we demonstrate that the priming of OT-I TCR transgenic or endogenous mouse OVA-specific CD8 T cells is critically dependent on CD70/CD27 interaction. CD70 blockade inhibited CD40-mediated clonal expansion of CD8 T cells and reduced the number of memory CD8 T cells generated. Furthermore, CD70 blockade during the initial priming of CD8 T cells inhibited the ability of memory CD8 T cells to expand in response to a second encounter with Ag. Our data indicate that CD70 expression on APCs plays a key role in CD40dependent CD8 T cell responses. The Journal of Immunology, 2004, 173: 6542– 6546.
T
he nature of the molecular interactions between T cells and APCs is a critical determinant of the ability of T cells to respond effectively to pathogens and to maintain self-tolerance (1–9). For example, T cells primed by mature dendritic cells (DCs)3 that express high levels of MHC and costimulatory molecules proliferate and survive more efficiently than T cells stimulated by immature DCs (8). Furthermore, it has been shown that presentation of Ag to T cells in the steady state by immature DCs can result in tolerance (7, 9). Maturation of DCs is stimulated by microbial infection as a result of specific recognition of pathogen-associated molecular patterns, as well as by CD40 signaling (1, 2, 10). The interaction between CD154 on activated CD4 T cells and CD40 on DCs is thought to represent an important pathway by which CD4 T cells provide help for CD8 T cell responses (2–5, 9, 11). In support of this, administration of agonistic anti-CD40 mAbs that mimic CD4 T cell help has been shown to prevent CD8 T cell tolerance induced by administration of soluble protein, peptides, Ag-loaded dying cells, or tumor cells (6, 7, 12, 13). The mechanism by which CD40 activation, or licensing of DCs allows effective priming of CD8 T cells is not fully known. CD40 sig-
Cancer Sciences Division, Tenovus Research Laboratory, University of Southampton School of Medicine, Southampton General Hospital, Southampton, United Kingdom Received for publication June 7, 2004. Accepted for publication October 1, 2004. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Copyright © 2004 by The American Association of Immunologists, Inc.
naling enhances expression of the costimulatory molecules CD80, CD86, CD70, 4-1BB ligand, and OX40 ligand on the surface of APCs (1, 14 –16). Several lines of evidence indicate that the up-regulation of CD80 and CD86, and consequently engagement of the CD28 pathway, is not sufficient to account for the stimulation of CD8 T cells by CD40 signaling. Thus, strong CD8 T cell responses were elicited by administration of a CD8 T cell peptide epitope together with the agonistic antiCD40 mAb in CD28-deficient mice (6). The ability of agonistic anti-CD40 mAb to enhance the expansion and survival of CD8 T cells following administration of superantigen was only partly reduced by blocking the interaction between CD80/86 and CD28 (17). In addition, it has been shown that long-term acceptance of skin and cardiac allografts requires blocking of the CD40 and CD28 pathways, demonstrating that these pathways can function independently of each other (18). In this study, we address the role of CD70 (14), a member of the TNF superfamily and the ligand for the costimulatory receptor CD27 (19, 20), in CD40-dependent CD8 T cell responses.
Materials and Methods Abs, reagents, and cells The anti-CD40 (3/23), anti-A31 lymphoma Id (Mc39-16) (both rat IgG2a), and anti-Fc␥ II and III receptors mAbs (2.4G2) were provided by Prof. M. Glennie (University of Southampton, Southampton, U.K.). PE-labeled H-2Kb OVA peptide 257–264 (OVAp) tetramers were obtained from Proimmune (Oxford, U.K.). The anti-CD70 mAb (IgG2a) was generated by immunizing LOU rats with soluble recombinant CD70 protein (sCD70) (20) and splenocytes from immunized rats were fused with NS1 myeloma cells. Bone marrowderived DCs (BM-DCs) were prepared by culturing bone marrow cells in IL-4 (5 ng/ml) and GM-CSF (5 ng/ml) for 6 days. In some cultures, soluble recombinant Fc-CD154 (10 g/ml) was added 24 or 48 h before cell harvesting.
Mice and in vivo experiments OVA-specific H-2Kb-restricted TCR transgenic T cells (1 ⫻ 106) from OT-I mice were transferred by i.v. injection into sex-matched C57BL/6 recipients. After 1 or 2 days, T cells were primed by i.p. administration of OVA (5 mg) in combination with: anti-CD40 and control anti-A31 lymphoma Id mAb (500 g of each), or anti-CD40 and anti-CD70 mAb (500 g of each). The next day mice received an additional injection of control anti-A31 lymphoma Id mAb, or anti-CD70 mAb (500 g). CD4 T cell depletion (⬎99% efficient) was conducted by i.p. injection of anti-CD4 mAbs (2 ⫻ 1 mg of YTA 3.1.2 and 2 ⫻ 0.5 mg of YTS 191.2). Secondary stimulation was conducted by i.v. injection of OVAp (20 nmol). Endogenous OVA-specific CD8 T cell responses were examined in C57BL/6 mice using the above immunization protocol. Animal experiments were conducted according to the U.K. Home Office license guidelines and approved by the University of Southampton’s ethical committee. 1
This study was supported by grants from Cancer Research U.K. and Tenovus.
2
Address correspondence and reprint requests to Dr. Aymen Al-Shamkhani, Tenovus Research Laboratory, Southampton General Hospital, Tremona Road, Southampton SO16 6YD, U.K. E-mail address:
[email protected] 3 Abbreviations used in this paper: DC, dendritic cell; sCD70, soluble CD70; OVAp, OVA peptide 257–264; BM-DC, bone marrow-derived DC.
0022-1767/04/$02.00
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Flow cytometry For tracking Ag-specific T cells, blood samples (50 l), or lymph node/spleen cells were stained with PE-labeled H-2Kb OVAp tetramers and allophycocyanin-labeled anti-CD8␣ (20). Expression of CD70 on BM-DCs was determined using biotinylated mAb in the presence of anti-Fc␥ II and III receptors mAb (2.4G2).
CFSE dilution analysis OT-I T cells were labeled with CFSE (Molecular Probes, Leiden, The Netherlands) as described previously (20). One million cells were then administered by i.v. injection to C57BL/6 mice. Twenty-four hours later, mice were immunized as above, or left unstimulated. The level of CFSE in CD8⫹ tetramer⫹ cells was analyzed by flow cytometry.
In vivo cytotoxicity assay C57BL/6 splenocytes, unpulsed or pulsed with OVAp (1 M) for 90 min at 37°C, were labeled with low (0.5 M) and high (10 M) concentrations of CFSE, respectively, and coinjected in a 1:1 ratio (2.6 ⫻ 107 total cells) into mice. Sixteen hours later, spleens were removed and the ratio of CFSElow/CFSEhigh cells was determined by flow cytometry. The CFSElow/CFSEhigh cell ratio in naive mice defined the 0% lysis level.
Results Expression of CD70 on DCs
The anti-CD70 mAb, generated by immunization with sCD70, bound to COS-7 cells transfected with membranebound CD70 but did not bind to untransfected cells (data not shown). This mAb did not stain resting CD11c⫹ BM-DCs, however, binding was observed upon their stimulation via CD40 (Fig. 1). The binding of the anti-CD70 mAb to CD40stimulated BM-DCs was abolished by incubation with sCD70, thus confirming the specificity of the binding (Fig. 1). CD40-stimulated expansion of CD8 OT-I T cells is blocked by antiCD70 mAb
Initially we showed that the anti-CD70 mAb was capable of blocking the interaction between sCD70 and membrane-expressed CD27 in vitro (data not shown). Having demonstrated that the anti-CD70 mAb is a blocking reagent, we next examined the effects of blocking the CD70/CD27 interaction on CD40-driven expansion of CD8 OT-I T cells in vivo. Lefranc¸ois et al. (12) have previously shown that administration of agonistic anti-CD40 mAb prevents the induction of CD8 T cell tolerance resulting from injection of soluble OVA. Furthermore, they showed that the agonistic anti-CD40 mAb mediated its effects indirectly by activation of APCs and in the absence of MHC class II, suggesting that CD4 T cells are not required for this response (12). Consistent with their findings, we show here that injection of agonistic anti-CD40 mAb together with OVA enhances the expansion and survival of adoptively transferred CD8 OT-I T cells compared with administration of OVA alone (Fig. 2A). When animals were injected with agonistic anti-CD40 mAb and anti-CD70 mAb, the
FIGURE 1. Expression of CD70 on BM-DCs. BM-DCs were left unstimulated, or stimulated via CD40 for 24 – 48 h. Histogram plots showing the binding of isotype control mAb (dotted line) or anti-CD70 mAb (bold line). The binding of anti-CD70 mAb to cells was blocked by preincubation with sCD70 (solid thin line).
FIGURE 2. CD70 blockade inhibits CD40-mediated expansion of OT-I T cells during the primary response. A, OT-I T cells were enumerated in samples (n ⫽ 3) of peripheral blood from mice immunized with OVA (Ag) and different combinations of mAbs. B, CD70 blockade of CD40-mediated expansion of OT-I T cells is independent of CD4 T cells. Following adoptive transfer of OT-I T cells, some mice were depleted of CD4 T cells (⫺CD4) before priming with OVA and anti-CD40 with or without anti-CD70 mAb. OT-I T cells were enumerated in samples of peripheral blood (n ⫽ 3). The data shown are from the peak of the primary response (day 4). C, Representative histogram plots of CFSE-labeled OT-I T cells obtained from lymph nodes of unstimulated mice, or mice primed 72 h earlier with OVA (Ag), OVA with anti-CD40 and control mAb (Ag⫹␣CD40), or OVA with anti-CD40 and anti-CD70 mAb (Ag⫹␣CD40⫹␣CD70). The data are representative of three experiments.
clonal expansion of CD8 OT-I T cells at the peak of the response was substantially reduced (3- to 4-fold) (Fig. 2A). Furthermore, ⬃3-fold fewer CD8 OT-I T cells survived after the contraction phase (day 14) in animals that received anti-CD70 mAb together with the agonistic anti-CD40 mAb, compared with the group of animals that received agonistic anti-CD40 mAb and control IgG (Fig. 2A). Similarly, the anti-CD70 mAb blocked the clonal expansion of CD8 OT-I T cells triggered by immunization with agonistic anti-CD40 mAb and OVAp, the antigenic peptide epitope recognized by CD8 OT-I T cells (data not shown). In addition, CD70 blockade inhibited the CD40-driven expansion of OT-I T cells in the absence of CD4 T cells (Fig. 2B). Injection of anti-CD70 mAb without agonistic anti-CD40 mAb did not affect the CD8 T cell response to OVA (data not shown). Together, these results suggest that
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CUTTING EDGE: CD70 REQUIREMENT IN CD40-DEPENDENT CD8 T CELL RESPONSES
CD40-induced expression of CD70 on APCs directly stimulates activated CD8 T cells via CD27 signaling. To address whether CD70 blockade affects CD8 OT-I T cell proliferation in vivo, we adoptively transferred CFSE-labeled cells into mice and examined their proliferation by CFSE dilution analysis. Seventy-two hours after injection of OVA and agonistic anti-CD40 mAb, the majority (70%) of OT-I T cells had divided seven to eight times (Fig. 2C). In contrast, OT-I T cells that had undergone seven to eight divisions represented only a minority (30%) of the total OT- I T cells in animals that received Ag alone, or Ag with anti-CD40 and anti-CD70 mAbs (Fig. 2C). This indicates that the CD70/CD27 interaction is required for optimal CD8 T cell proliferation in vivo. The effect of CD70 blockade during the primary response on the function of memory CD8 OT-I T cells
Approximately 3-fold fewer memory CD8 OT-I T cells were generated when the CD70/CD27 interaction was blocked during the primary response (Fig. 3A; day 0 before rechallenge and Fig. 3C). In addition to these quantitative effects we sought to address whether CD70 blockade during the primary response affects the quality of the memory CD8 T cells generated. Therefore, we examined the ability of memory CD8 OT-I T cells to expand upon secondary encounter with Ag. Groups of animals that had been previously injected with OVA, OVA with agonistic anti-CD40 mAb and control IgG, or OVA with agonistic anti-CD40 mAb and anti-CD70 mAb, were rechallenged 40 days later with OVAp. Memory CD8 OT-I T cells generated by immunization with OVA and agonistic anti-CD40 mAb expanded by 9-fold, whereas those generated in the absence of CD70/CD27 interaction responded poorly, increasing by only 2-fold (Fig. 3A). Under certain conditions CD8 T cells display split anergy; they have lytic activity but do not proliferate in response to Ag (21). Therefore, we investigated whether blockade of CD70 also affected the differentiation of naive CD8 OT-I T cells into CTLs. The cytolytic activities of CD8 OT-I T cells were examined 45 days after priming using an in vivo assay of specific killing. No target cell killing was detected in mice that received OVA alone, whereas in mice that received OVA with agonistic anti-CD40 mAb and control IgG, or OVA with agonistic anti-CD40 mAb and anti-CD70 mAb, target cells were killed efficiently (Fig. 3B). The slightly lower level of target cell killing in animals injected with agonistic anti-CD40 mAb and anti-CD70 mAb compared with the group that received agonistic anti-CD40 mAb and control IgG is consistent with the lower number of surviving OT-I T cells in the former group (Fig. 3C). Furthermore, using an ex vivo cytotoxicity assay and equivalent cell numbers, we did not detect differences in the lytic activity of OT-I T cells isolated from animals 11 days after priming with OVA and anti-CD40 mAb, or OVA and antiCD40/anti-CD70 mAbs (data not shown). Thus, the CD70/ CD27 interaction during priming appears to be dispensable for the differentiation of CD8 OT-I T cells into cytotoxic cells, but essential for their ability to expand upon rechallenge with the Ag. CD70 is required for CD40-mediated priming of endogenous OVAspecific CD8 T cells
The observed dependence of CD40-mediated priming of CD8 T cells on CD70 could have been influenced by the relatively high frequency of Ag-specific cells following adoptive transfer of OT-I T cells. Therefore, we examined the consequence of
FIGURE 3. CD70/CD27 interaction during priming of OT-I T cells is essential for their expansion during secondary responses, but not for their cytolytic activity. A, Groups of mice (n ⫽ 3) were primed with OVA and different combinations of mAbs and 40 days later rechallenged with OVAp (day 0). OT-I T cells were enumerated in samples of peripheral blood (n ⫽ 3). The data are representative of three experiments. B, In vivo killing of unpulsed (CFSElow) and OVAp-pulsed (CFSEhigh) target cells 45 days after primary challenge with OVA and different combinations of mAbs. Representative histogram plots are shown from three similar experiments. The percentage of specific killing is also indicated (n ⫽ 3). C, The percentage of surviving OT-I T cells in peripheral blood 45 days after primary challenge. The data are representative of three experiments.
CD70 blockade on the response of endogenous OVA-specific CD8 T cells. As with OT-I T cells, the CD40-mediated expansion of endogenous OVA-specific CD8 T cells was profoundly inhibited by administration of anti-CD70 mAb (Fig. 4A). Furthermore, whereas animals primed with OVA and anti-CD40 mAb generated strong secondary responses, those primed with OVA alone or OVA with anti-CD40 and anti-CD70 generated weak responses upon antigenic rechallenge (Fig. 4B). We also addressed the possibility that the anti-CD70 mAb could have mediated its effects by depletion of CD70-expressing cells. We took advantage of the fact that CD40 triggering activates B cells and stimulates their expression of CD70 (14). In contrast with the effects of the anti-CD70 mAb on CD8 T cell priming, CD40-triggered B cell expansion was not affected by the
The Journal of Immunology
FIGURE 4. CD70 is required for CD40-mediated priming of endogenous OVA-specific CD8 T cells. A, Endogenous OVA-specific CD8 T cells were enumerated in samples (n ⫽ 3) of peripheral blood from mice immunized with OVA (Ag) and different combinations of mAbs. B, Fifteen days after the initial challenge with Ag, mice were rechallenged i.p. with OVAp (20 nmol) and antiCD40 mAb (500 g). OVA-specific CD8 T cells were enumerated in samples (n ⫽ 3) of peripheral blood by H-2Kb OVAp tetramer staining. The data are representative of three experiments.
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with Ag is initiated at the priming stage (22–24). In addition, we show for the first time that the CD27 signaling pathway constitutes an important part of this instructional programming of CD8 T cells. The results presented in this study support a progressive model of T cell differentiation (8). Thus, we demonstrate that the ability of naive CD8 T cells to gain cytolytic function and differentiate into cells capable of expanding rapidly following secondary encounter with Ag are governed by different activation thresholds. A number of studies have shown that CD4 T cell help for CD8 T cell responses occurs indirectly via CD40-mediated activation of DCs (2, 5, 9, 11). Recently, an alternative mechanism of CD4 T cell help for CD8 T cells has been proposed. Bourgeois et al. (25) showed that CD4 T cell help for CD8 T cell responses against the male histocompatibility-Y Ag is mediated directly via CD40 on CD8 T cells. However, the contribution of this pathway to CD8 T cell responses in our model is unlikely. Using the same in vivo model that we have used in this study, it was shown previously that the effect of the agonistic anti-CD40 mAb was completely abolished when wild-type CD8 T cells were transferred into CD40-deficient hosts (12). Taken together, our results are consistent with the CD40-licensing model of APCs by CD4 T cells and imply a role for CD70 expression on APCs in CD4 T cell help for CD8 T cell responses.
Acknowledgments anti-CD70 mAb (data not shown). This indicates that the antiCD70 mAb does not deplete CD70-expressing cells in vivo.
Discussion Our data provide the first direct evidence for an important functional role for CD70 in CD40-mediated priming of CD8 T cells in vivo. In this study, we demonstrate that CD70 blockade inhibits the CD40-mediated expansion of CD8 T cells during the primary response to OVA (Figs. 2 and 4A). Furthermore, fewer CD8 memory T cells are generated in the absence of the CD70/CD27 interaction (Fig. 3C). Our results are consistent with previous studies demonstrating reduced CD8 T cell responses to influenza virus in CD27-deficient mice (19). Optimal CD8 T cell responses to influenza virus require CD40 (11), however, influenza virus is capable of activating DCs directly (2, 10), thus enhancing their ability to stimulate CD8 T cells. It remains unclear which of these pathways is affected by the lack of CD27 costimulation in the influenza infection model. The results of our study using soluble Ag clearly show that CD70/CD27 interaction is critical for CD40-mediated priming of CD8 T cells. Whether CD70 also plays a role in CD8 T cell priming by virus-matured DCs needs further investigation. By blocking the CD70/CD27 interaction only during the primary response we demonstrated an important role for this interaction in the generation of fully competent memory CD8 T cells. Thus, blockade of CD70 in the primary response resulted in the generation of CD8 T cells capable of lysing target cells (Fig. 3B and data not shown), but unable to expand efficiently in response to a secondary antigenic challenge (Fig. 3A). Our results are in agreement with recent findings demonstrating that the program of differentiation of naive CD8 T cells into cells that respond effectively upon secondary encounter
We thank Martin Glennie and Tom MacDonald for helpful discussions.
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